Download the role of earthworm gut-associated microorganisms

THE ROLE OF EARTHWORM GUT-ASSOCIATED MICROORGANISMS
IN THE FATE OF PRIONS IN SOIL
Von der Fakultät für Lebenswissenschaften
der Technischen Universität Carolo-Wilhelmina
zu Braunschweig
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
(Dr. rer. nat.)
genehmigte
Dissertation
von Taras Jur’evič Nechitaylo
aus Krasnodar, Russland
2
Acknowledgement
I would like to thank Prof. Dr. Kenneth N. Timmis for his guidance in the work and help. I thank
Peter N. Golyshin for patience and strong support on this way. Many thanks to my other colleagues,
which also taught me and made the life in the lab and studies easy: Manuel Ferrer, Alex Neef,
Angelika Arnscheidt, Olga Golyshina, Tanja Chernikova, Christoph Gertler, Agnes Waliczek,
Britta Scheithauer, Julia Sabirova, Oleg Kotsurbenko, and other wonderful labmates. I am also
grateful to Michail Yakimov and Vitor Martins dos Santos for useful discussions and suggestions.
I am very obliged to my family: my parents and my brother, my parents on low and of course to my
wife, which made all of their best to support me.
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Summary.....................................................………………………………………………... 5
1. Introduction...........................................................................................................……... 7
Prion diseases: early hypotheses...………...………………..........…......…......……….. 7
The basics of the prion concept………………………………………………….……... 8
Putative prion dissemination pathways………………………………………….……... 10
Earthworms: a putative factor of the dissemination of TSE infectivity in soil?.………. 11
Objectives of the study…………………………………………………………………. 16
2. Materials and Methods.............................…......................................................……….. 17
2.1 Sampling and general experimental design..................................................………. 17
2.2 Fluorescence in situ Hybridization (FISH)………..……………………….………. 18
2.2.1 FISH with soil, intestine, and casts samples…………………………….……... 18
Isolation of cells from environmental samples…………………………….……….. 18
Fluorescence in situ Hybridization procedure……………………………………… 18
2.2.2 Design of group-specific nucleotide probe and the FISH with the earthworm
tissue……………………………………………………………………………. 19
2.3 rRNA and rRNA gene amplifications...……………………………………………. 20
2.3.1 Constructing of 16S rRNA clone libraries…………………………….……….. 20
Total DNA/RNA isolation and reverse transcription………………………………. 20
PCR-amplification………………………………………………………………….. 20
Constructing of clone libraries…………………………………………….………... 20
Sequencing of cloned 16 rDNA and phylogenetic analysis……………….……….. 21
2.2.2 Taxon-specific Single Strand Conformation Polymorphism (SSCP)
analysis………………………………………………………………………….. 21
Design of taxon-specific 16S rRNA gene primers…………………………………. 21
SSCP: total DNA/RNA isolation and PCR…………………………………………. 23
Preparation of ssDNA and gel electrophoresis……………………………….…….. 23
Isolation and PCR amplification of DNA fragments from polyacrylamide gels…… 23
Sequencing of 16 rDNA……………………………………………………………. 23
Phylogenetic analysis……………………………………………………………….. 24
Chimera checking and constructing of phylogenetic trees…………………………. 24
2.4 Isolation and identification of pure microbial cultures................................……….. 24
Isolation of bacteria and fungi with serial plate dilution method………….……….. 24
Identification of microbial isolates...............................................................……….. 25
Preparing cultures for the PCR amplification………………………………………. 25
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PCR amplification of the 16S rRNA genes of the isolates…………………………. 25
Sequencing of amplicons and analysis of sequence data………………….………... 26
2.5 RecPrP proteolysis…………………………………………………………………. 26
2.5.1 Recombinant protein synthesis………………………………………………. 26
2.5.2 RecPrP proteolytic assay……………………………………………….……. 27
2.5.2.1 PrP proteolytic assay of pure isolates............................................................ 27
2.4.2.2 Effect of earthworms and gut microbiota on recPrP retaining……….……. 27
Extraction of recPrP from the soddy-podzolic soil and earthworm cast………… 27
Aqueous extracts assay........................................................................................... 28
2.5.2.3 Western blot analysis..................................................................................... 29
3. Results…………………………………………………………………………………… 31
3.1 Effect of earthworm gut environment on microbial community of soil…………… 31
3.1.1 Preliminary studies of microbial community changes in the substrate upon
passage through the earthworm gut………………………….…….…………... 31
3.1.1.1 Characterization of the microbial population with FISH………….…….. 31
3.1.1.2 Clone libraries…………………………………………………………… 35
3.1.2 Bacteria of class Mollicutes in the earthworm tissues…………………………. 39
3.1.3 FISH and SSCP analysis of microbial communities in the substratum used for
prion proteolytic assay………………………………………….……………… 43
3.1.3.1 Characterization of the microbial population with FISH…………..……. 43
3.1.2.2 SSCP analysis...................................................................................……. 45
3.2 Recombinant prion proteolysis assays.............................................................…….. 58
3.2.1 Proteolytic activity of pure isolates……………………………………………. 58
3.2.2 Effect of earthworms and gut microbiota on recPrP retaining………………… 64
Unspecific proteolytic activity………………………………………………… 64
Proteolysis of recPrP…………………………………………………….…….. 66
4. Discussion………………………………………………………………………………... 69
4.1 Effect of earthworm gut environment upon the microbial community……………. 69
4.2 Proteolytic activity of the soil and earthworm-modified microbial
communities……………………………………………………………………………. 77
5. General conclusions……………………………………………………………………... 82
References………………………………………………………………………………….. 83
Supplementary materials………………………………………….……………….……… 94
Curriculum Vitae………………………………………………………………………….. 100
Summary
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Summary
The earthworm-associated microbial communities were studied for their ability to degrade a
recombinant PrP used as a model of the agent of transmissible spongiform encephalopathy (TSE).
Initially the microbial compositions of substrata (soddy-podzolic soil and horse manure compost)
and their changes upon the passage through the guts of earthworms (species Lumbricus terrestris,
Aporrectodea caliginosa, and Eisenia fetida) and the bacterial composition of the earthworm gut
environment were studied using rRNA-based techniques, fluorescence in situ hybridization (FISH)
and PCR-based approaches (cloning and single strand conformation polymorphism (SSCP)
analyses).
In the most cases the number of physiologically active bacteria, i.e. those hybridized with universal
FISH probe, was slightly higher in the earthworm casts in comparison to substrata. Bacterial
populations of substrata were undergoing severe alterations upon transit through the earthworm gut
depending mainly of the initial microbial composition presented in the substrata, in contrast to that,
earthworm species-specific effects on the bacterial population composition were not detected.
Certain common regularities of microbial population modification upon passage were noticed.
Clone libraries of substrata (soddy-podzolic soil and compost) and earthworm-derived systems (gut
and cast) revealed a high diversity of microorganisms. Phylum Proteobacteria was the most diverse
among the others; CFB was the second numerous taxon. Except of the bacteria, the eukarya (fungi
(Ascomycota), algae (Chlorophyta), Colpodae (Protozoa), Monocystidae (Alveolata), and
roundworm (Nematoda)) were detected in the substrata and earthworm-derived systems.
Application the SSCP analysis with universal and newly designed taxon-specific primers targeting
α-,
β-
and
γ-Proteobacteria,
Myxococccales
(δ-Proteobacteria),
CFB
group,
Bacilli,
Verrucomicrobia, and Planctomycetes identified the bacterial groups sensitive to, resistant against,
and promoted by earthworm gut environment whereas significant differences between rRNA gene
and rRNA pools were observed for all bacterial taxa except of CFB bacterial group.
Novel family ‘Lumbricoplasmataceae’ within the class Mollicutes (Firmicutes) was proposed after
detection in the gut and cast clone libraries of a monophyletic cluster of sequences and in situ
detection with specific oligonucleotide probe by FISH analysis in the earthworm tissues.
Our data suggests the bacteria from the group CFB are promoted by the gut environment, the
bacteria of family ‘Lumbricoplasmataceae’ are obligate earthworm-associated organisms, for
Gammaproteobacteria and Bacilli gut environment is hostile, although bacteria of the genus
Pseudomonas,
family
of
unclassified
Sphingomonadaceae
(Alphaproteobacteria),
and
Summary
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Alcaligenes faecalis (Betaproteobacteria) could be designated as gut-resistant component of
community.
Pure microbial cultures and water-soluble content from the soil and earthworm casts of L. terrestris
and A. caliginosa were elucidated for their abilities to digest recombinant prion. Up to 20% of
bacterial species were able to digest recPrP; Gammaproteobacteria, Actinobacteria, and Bacilli
were the taxa with the biggest potential to deplete the recPrP. Most of studied fungal isolates did
perform the recPrP digestion.
RecPrP was demonstrated to be depleted in vitro in aqueous extracts of the soil and the cast within
2-6 days. Non-specific proteolytic activity strongly increased from soil substratum to the cast
through the trypsin- and chymotrypsin-like proteases released by the earthworm. However, the
passage through the gut did not promote any enhanced recPrP digestion, which lasted under given
conditions in vitro within 2-6 days. Thus, under applied conditions the microbial-earthworm gut
systems do not produce proteases de novo, which notably affect the prion proteolysis.
1. Introduction
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1. Introduction
Prion diseases: early hypotheses
The group of prion diseases also known as transmissible spongiform encephalopathy (TSE)
includes the most well-known human prion diseases (kuru, Creutzfeldt-Jakob disease (CJD), and its
variants (familial, sporadic, latrogenic, fatal familial insomnia and the new variant of CJD (vCJD)
(Will et al. (1996)) and bovine spongiform encephalopathy (BSE) of cattle and scrapie of sheep and
goats. Besides, this kind of prion disease was recognized in deer and elk species in North America
and named chronic wasting disease (CWD); in greater kudu (Kirkwood et al., 1993); in zoological
ruminants and non-human primates (Bons et al., 1999); feline spongiform encephalopathy of
zoological and domestic cats (FSE) (Pearson et al., 1992), and in other predator mammalians:
transmissible mink encephalopathy (TME) (Marsh and Hadlow, 1992). On the basis of following
analyses it was suggested that these apparently novel TSEs including vCJD had the same origin –
BSE (Collinge et al., 1996; Collinge, 1999). All of these variants of the prion diseases cause a
progressive degeneration of the central nerve system ending in inevitable death.
The transmissibility of the TSEs was accidentally demonstrated in 1937, when the population of
Scottish sheep was inoculated against the common virus with extract of the brain tissue
unknowingly derived from a scrapie animal. In humans, kuru was emerged at the beginning of the
1900s among the cannibalistic tribes of New Guinea, reached epidemic proportions in the mid1950s and disappeared progressively in the latter half of the century to complete absence at the end
of the 1990s. The transmissibility of kuru to monkeys was demonstrated in the 1960s (Gajdusek et
al., 1966, Gibbs et al., 1968). The CWD was first noticed at the late 1960s in Colorado wildlife
research facility and later was identified as spongiform encefalopathy-forming disease according to
the histological studies (William and Yang, 1980).
The exact nature of the transmissible pathogen has been debated since the mid-1960s. The
incubation period of prion diseases is unusually long (up to several years) and the agent was
initially thought to be a slow virus (Cho, 1976). Further research, however, has emphasized that the
agent responsible for scrapie was very resistant to UV and ionizing radiation,i.e. against the
treatments that normally destroy nucleic acids (Alper et al., 1967). The other hypothesis, so called
"virino hypothesis", suggested the presence of an agent-specific nucleic acid enveloped in a hostspecified protein (Fig. 1A-c). This concept was proposed to explain the lack of an immune response
by the host along with the strain variation (Kimberlin, 1982). The virus and virino hypotheses have
apparently lost their importance (though have not been neglected completely) since many studies
1. Introduction
8
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conducted in numerouslaboratories could not identify either TSE-specific or TSE-associated nucleic
acids, or nucleic acid that could encode a small protein (Riesner et al., 1993).
The basics of the prion concept
A dominating current hypothesis is the ‘protein-only’ (or ‘prion’) hypothesis (Griffith, 1967),
whereas a hypothetical protein is believed to comprise the entire infective particle (Fig. 1A-b). Soon
after publication of the prion hypothesis, the PrP protein was discovered (Bolton et al., 1982).
Another experimental evidence of the hypothesis was the demonstration that PrP and TSE
infectivity were co-purified (Diringer et al. 1983) and it was suggested that TSE infections are
caused by an infectious protein, PrP (McKinley et al., 1983).
The DNA encoding PrP was detected, sequenced and the prion was recognized as a host
glycoprotein with unknown function (Oesch et al. 1985). The primary structure of the mature PrP
protein comprises approximately 210 amino acids. It has two N-glycosylation sites (Oesch et al.,
1985) and a C-terminal glycophosphoinositol (GPI) anchor (Stahl et al., 1990b). PrP mRNA proved
to be the product of a single host gene, which is present in the brain of uninfected animals and is
constitutively expressed by many cell types. One distinguishes two PrP forms according to the their
biochemical properties. The physiologically occurring PrPC fraction attached with GPI to the outer
surface of the plasma membrane (Fig. 1A-a); the PrPC can be glycosylated on one or both of two
asparagine residues with a variety of glycans; soluble in detergents (Meyer et al., 1986) and
released from the surface of tissue culture cells by phosphoinositol phospholipase C (PIPLC) (Stahl
et al., 1990a). PrPC is suggested to be the normal form of the protein, present in both uninfected and
infected tissues. An isoform named PrPSc almost invariably detected in TSE-infected tissues and
cells is not released by PIPLC (Stahl et al., 1990a). Both normal and scrapie isoforms of PrP
encoded by the same gene Prnp (Basler et al., 1986) and it has been proposed that the normal prion
PrPC converses itself into ‘multipling’ infectious agent PrPSc (Prusiner, 1991).
There is however no evidence of structural differences between the normal PrPC and isoform PrPSc
(Stahl et al., 1993). The normal PrPC has three α-helices and one small region of β-sheet, while
abnormal isoform PrPSc has a higher degree of β-sheet (Riek et al. 1996). These structural changes
cause the alterations in biochemical properties, such as protease resistance, and capability to form
larger-order aggregates. The PrPC fraction is protease-sensitive. The PrPSc fraction detected only in
TSE-affected tissues is partially protease-resistant (Meyer et al., 1986).
It should be noted that ‘protease resistance’ is rather relative definition: some forms of PrP are more
resistant to treatment with proteinase K (PK) than PrPC but are nonetheless non-infectious (Post et
al. 1998; Appel et al., 1999).
1. Introduction
9
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A
B
Figure 1. A: Models for the propagation of the TSE agent (prion). a) In a normal cell, PrPC (yellow square) is
synthesized, transported to the cell surface and eventually internalized. b) The protein-only model postulates that the
infectious entity, the prion, is congruent with an isoform of PrP, here designated as PrPSc (blue circle). Exogenous PrPSc
causes catalytic conversion of PrPC to PrPSc, either at the cell surface or after internalization. c)The virino model
postulates that the infectious agent consists of a TSE-specific nucleic acid associated with or packaged in PrPSc. The
hypothetical nucleic acid is replicated in the cell and associates with PrPC, which is thereby converted to PrPSc. B:
Models for the conversion of PrPC to PrPSc. a) The refolding model. The conformational change iskinetically
controlled, a high activation energy barrier preventing spontaneous conversion at detectable rates. Interaction with
exogenously introduced PrPSc (blue circle) causes PrPC (yellow square) to undergo an induced conformational change to
yield PrPSc. This reaction could be facilitated by an enzyme or chaperone. In the case of certain mutations in PrPC,
spontaneous conversion to PrPSc can occur as a rare event, explaining why familial Creutzfeldt–Jacob disease (CJD) or
Gerstmann–Sträussler–Sheinker syndrome (GSS) arise spontaneously, albeit late in life. Sporadic CJD (sCJD) might
arise when an extremely rare event (occurring in about one in a million individuals per year) leads to spontaneous
conversion of PrPC to PrPSc. b) The seeding model. PrPC (yellow square) and PrPSc (or a PrPSc-like molecule; shown as
a blue circle) are in equilibrium, with PrPC strongly favoured. PrPSc is only stabilized when it adds onto a crystal-like
seed or aggregate of PrPSc. Seed formation is rare; however, once a seed is present, monomer addition ensues rapidly.
To explain exponential conversion rates, aggregates must be continuously fragmented, generating increasing surfaces
for accretion (Weissmann, 2004).
1. Introduction
10
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The mechanism of self-propagating alteration of PrPC to pathogenic scrapie form PrPSc within the
proposed protein-only hypothesis is unknown. Two models for the conversion of PrPC to PrPSc were
suggested. The first one, named refolding model, proposes the PrPC is undergoing modification
under the influence of a PrPSc molecule (Prusiner, 1991). The second one, named seeding model
suggests that both normal and abnormal PrP isofoms are present in some equilibrium with the
strong prevalence of PrPC. This balance is being broken in case the "seeds" of PrPSc come into the
game upon infection (Orgel, 1996). Once the seeds occur, the oligomer formation and modification
of PrPC to PrPSc ensues rapidly.
Although the prion-only hypothesis is broadly accepted in the scientific community, it has certain
weaknesses. As it was mentioned above, some strains appeared to be resistance without being
infectious, but in some cases the infectivity is being propagated in the absence of detectable PrP
resistance to proteolysis (PrPres) (Lasmezas et al., 1997). Scrapie and other TSEs have a different
'strains' characterized by variable incubation periods, clinical features, and neuropathology. They
could have distinct abilities to catalyze PrP conversion and could selectively target different brain
regions, producing the diversity of clinical symptoms and neuropathological alterations
characteristic of prion strains. Small quantities of nucleic acids were detected in infectious samples
and PrPres interacts with high affinity with nucleic acids, especially RNA, which could help to
catalyze the conversion of PrPC into PrPres in vitro. All those controversies together make the prion
hypothesis doubtful (Soto and Castillo, 2004).
Putative prion dissemination pathways
Above arguments unambiguously suggest the important role of PrP in prion diseases. The next
essential event in the epidemiology of TSE is the infectivity of its agent, i.e. prion. The transmission
of a TSE from one species to another is far less efficient than within the same species and even
sometimes impossible, which hence resulted in a concept of a "species barrier". However, the
events in zoological collections in Great Britain and France lead to conclusion that the "species
barrier"-crossing is possible. Animals with diagnosed TSEs were effectively infected with
contaminated foodstuff (Sigurdson and Miller, 2003). The possible exception was greater kudu
infected by horizontal spread among animals in a manner similar to scrapie and CWD (Kirkwood et
al., 1993). Thus, prion disease is mainly acquired through oral infection and then spread from the
peripheral to the central nervous system (CNS) (Weissmann, 2004).
The environmental risks of the TSE infection agent have recently being studied. The contaminated
soil can become a potential reservoir of TSE infectivity as a result of (i) accidental dispersion from
storage plants of meat and bone meal, (ii) incorporation of meat and bone meal in fertilizers, (iii)
1. Introduction
11
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spreading of effluents of slaughter-houses, rendering plants and gelatin industry, (iv) possible
natural contamination of pasture soils by grazing herds and (v) burial of carcasses of contaminated
animals. Environmental sources of contaminated with TSE infection agent (particularly, CWD)
represent potential obstacles to control in natural and captive settings. Under experimental
conditions, mule deer (Odocoileus hemionus) became infected in paddocks containing naturally
infected deer, in paddocks where infected deer carcasses were decomposed for approx. 2 years and
the paddocks where infected deer had last resided more than 2 years earlier (Miller et al., 2004).
The exact mechanisms for CWD transmission in excreta-contaminated paddocks is uncertain, but
foraging and soil consumption seems to be most probable, while the fate of PrP in the soil was
unclear (Miller et al., 2004).
Earthworms: a putative factor of the dissemination of TSE infectivity in soil?
Soil and soil minerals serve as a reservoir of TSE infectivity (Brown and Gajdusek, 1991). Recent
investigation used ovine recPrP and mica (a phyllosilicate, has similar physicochemical surface
properties to soil clays) considered as a model of negatively charged mineral surfaces have shown
strong adsorption of recPrP to the surface of mica. It was suggested that molecules, which are
adsorbed never leave the surface even after a very short residence time at the interface and the
mobility of the proteins at the interface was very low. Negative surfaces such as mica are able to
concentrate protein, and possibly to change protein conformation (Vasina et al., 2005). Though that
seems to be unlikely that interaction of normal prions (PrPC) with soil clay surfaces could induce a
change of conformation leading to the pathogenic form of prions (PrPSc) (Revault et al., 2005).
PrPSc was adsorbed to montmorillonite and kaolinite, quartz, and four whole soil samples and
preserve its infectivity (Johnson et al., 2006). Thus, soil micro- and mesoorganisms (soil
invertebrates) could greatly affect the distribution or decontamination of PrP.
Among the soil invertebrates, earthworms play an essential role in carbon turnover, soil formation,
participating in cellulose degradation and humus accumulation. In the upper soil horizon the
earthworms generate mosaic microzone by their activity (Brown, 1995; Devliegher and Verstraete,
1997; Römbke et al., 2005; Tiunov and Kuznetsova, 2000). The earthworms do (i) penetrate soil by
burrow activity hence increasing aeration; (ii) transfer soil and organic matter by casting; (iii)
humiliate organic material as a first step in organic matter breakdown (including cattle feces in
meadows); (iv) change the diversity and improve the activity of the microbial community by
selective feeding and provide feces rich in nutrients. Bearing in mind large numbers of these
animals in the soil one may consider their significant effect on microbial population of soil.
1. Introduction
12
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Figure 2. Schematic representation of the functional relationships between earthworms and their
external environment (Römbke et al., 2005).
First of all, earthworm gut performs a unique environment subsystem of soil environment. The
earthworm gut has stable conditions different from surrounding environment: permanent anoxia, the
pool of free amino acids, organic acids, alcohols, sugars and of hydrogen, the products of organic
oligo- and polymer degradation (Karsten, and Drake, 1995; Horn et al., 2003). The gut mucosa
could be used as an environment and nutrition by microorganisms and gut-derived enzymes of
earthworms could affect the microbes on the soil particles (Brown, 1995). On the other hand, the
earthworm is migrating in the soil and thus soil (or another substrate as leaf litter or manure
composts) flows through earthworm gut being affected by this system.
One aspect of environmental significance of earthworms discovered recently was the extensive
N2O production by microorganisms in the earthworm gut (Karsten and Drake, 1995, 1997; Matthies
et al., 1999; Ihssen et al., 2003; Horn et al., 2003, 2006). The numbers of fermentative anaerobes
and microbes that used nitrate as a terminal electron acceptor were approximately 2 orders of
magnitude higher in the earthworm gut than in the soil from which the earthworms originated. In
the gut of the earthworms Aporrectodea caliginosa and Eisenia fetida the microbial composition
was changed towards the increasing numbers of spore non-forming bacteria and decreasing
numbers of spore-forming bacteria, which also resulted in enhanced levels of nitrogen fixation
(Tereschenko and Naplekova, 2002). A number of novel N2O-producing species of bacteria from
genera Dechloromonas (Betaproteobacteria), Flavobacterium (Cytophaga-Flavobacteria group of
1. Introduction
13
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Bacteroidetes), and Paenibacillus (class Bacilli) were isolated from the gut of Aporrectodea
caliginosa (Horn et al., 2005).
Microorganisms in the earthworm gut and the effect of the gut environment on microbial
populations were extensively studied in the past with the classical methods of microbiology. The
extensive data on the cultured organisms inhabiting the earthworm-associated ecosystems one
should consider with a certain salt grain due to the well-known disadvantages and biases of the
plating/culturing techniques. Nevertheless some regularities could be noticed.
The first point, the augmentation with some groups of bacteria, which occurred upon transit through
the gut. This phenomenon was described for L. terrestris by Daniel and Anderson (1992). Besides,
the number of the living bacterial cells estimated by epifluorescence microscopy method, in general
correlated with data from plating method (Kristufek et al., 1992).
The ratio of microbes capable of growth under obligatory anaerobic conditions to those capable of
growth aerobically was higher in the worm intestine than in the soil (Karsten, Drake, 1995). The
total number of platable aerobic and facultatively anaerobic bacteria was 7x106 g-1 dry gut content
in the foregut, but it increased to 1.6x107 and 2,9x107 in the midgut and hindgut of
Lumbricus rubellus. The increasing number of anaerobic bacteria correlated well to the anaerobic
conditions deteced in the worm gut.
Actinomycetes were constantly isolated from the gut of different earthworm species and consdered
to be an abundant bacterial group in the microbial community of the earthworm gut. Their number
increased in the foregut of A. caliginosa (Kristufek et al., 1992). The actinomycetes of genera
Streptomyces (including S. diastsatochromogenes, S. nogalater) and Micromonospora being a
dominant in the gut of the earthworms L. rubellus and Octolasion montanum produced antibiotics
acting against Bacillus subtilus and Sachharomyces serevisiae (Kristufek et al., 1993). The
actinobacteria (S. lipmanii, S. olivaceus, S. antibioticus) together with Vibrio-type bacteria were
considered as pre-dominating organisms in the earthworms E. lucens (Contreras, 1980).
Simultaneously, certain bacteria decreased their numbers upon passage: the Gammaproteobacteria,
E. coli BJ18 and P. putida MM1 and MM11, decreased their numbers upon passage, while the
transit did not affect Aeromonas hydrophila DMU115 or Enterobacter cloacae A107 (Pedersen and
Hendriksen, 1993).
Plant pathogen fungus Fusarium oxysporum was able to survive upon passage through the gut of
earthworm Pheretima sp. though the earthworms caused a decline of total cell number of the plant
pathogen in soil but expanded its distribution in the soil (Toyota and Kimura, 1994). Other authors
detected that numbers of micromycetes in the guts of L. rubellus and A. caliginosa were relatively
stable (Kristufek et al., 1992).
1. Introduction
14
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Microflora of intestinal guts of Lumbricus terrestris and Octolasion cyaneum was extensively
studied with electron microscopy (Jolly et al., 1993), however no clear message could be taken for.
Application of the molecular tools allowed a more accurate estimation of performances of separate
microbial groups. Microbial populations in soil and casts of the earthworm Lumbricus rubellus
were examined through cultivation and 16S rDNA sequence analysis of the clone libraries. The
clones related to the Acidobacteria, Cytophagales, Chloroflexi, and Gammaproteobacteria were
detected in the libraries. Among the isolates, Aeromonas spp. were dominating, although these
bacteria were not isolated from the soil, besides, the other Gammaproteobacteria were found to be
quite abundant among clones (49%). The soil isolates were mostly Actinobacteria (53%),
Firmicutes (16%), and Gammaproteobacteria (19%). Isolates obtained from the sides of earthworm
burrows were not different from soil isolates. Diversity indices for the collections of isolates along
with rRNA gene libraries indicated that the species richness and evenness were decreased in the
casts compared to their levels in the soil. These results were consistent with a model where a large
portion of the microbial population from the soil passes through the gastrointestinal tract of the
earthworm remains unchanged, while representatives of some phyla increase their abundance
(Furlong et al., 2002).
Filamentous fungi in the gut of L. terrestris estimated with fluorescence image analysis were found
mainly disrupted before arriving in the intestine. Remaining hyphae in the foregut were completely
digested during passage through the gut. Spores of fungi were not detected throughout the study.
Numbers of bacteria usually increased from fore- to hindgut. This increase did not correlate to
contents of organic material and only partially due to a multiplication of bacterial cells. Numbers of
dividing cells accounted in total for approximately 12% of all bacteria, increasing from fore- to
hindgut. (Schönholzer et al., 1999). Following estimation of changes of microbial community
structure upon passage through the digestive tract of L. terrestris showed significantly reduction of
bacterial populations belonging to the α-, β- and γ-classes of Proteobacteria. Populations of the δsubdivision of Proteobacteria and the Cytophaga–Flavobacterium cluster of the CFB phylum
increased in cast. These results suggest a large impact of passage through the digestive tract of L.
terrestris on bacterial community structure (Schönholzer et al., 2002).
The work of Egert and colleagues (2004) addressed bacterial and archaeal community structures in
soil, gut, and fresh casts of L. terrestris using terminal-restriction fragment length polymorphism
(T-RFLP) analysis of 16S rRNA gene fragments. Ecological indices of community diversity and
similarity, calculated from the T-RFLP profiles revealed only small differences between the
bacterial and archaeal communities in soil, gut, and fresh casts under both feeding conditions,
especially in comparison to other soil-feeding invertebrates. Since no dominant gut-specific OTUs
1. Introduction
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were detected, the existence of an abundant indigenous earthworm microbial community is in the
opinion of the authors rather doubtful, at least in the midgut region of L. terrestris (Egert et. al.,
2004).
The bacteria associated with the intestine and casts of another earthworm, L. rubellus, were
examined with 16S rRNA gene clone libraries, and fluorescence in situ hybridization (FISH) by
Singleton and co-authors (2003). Bacterial libraries constructed from washed earthworm intestine
tissues contained several phylotypes, which were rare or absent in the cast libraries. The specific
phylotypes present depended on the date of sampling and included representatives of the
Acidobacteria, Firmicutes, Betaproteobacteria, and one phylogenetically deep, unclassified group.
Juvenile earthworms collected subsequently contained three of the four phylotypes observed in the
intestine clone libraries. The Firmicutes phylotype was examined by FISH and was found to be a
short rod that was represented only a small fraction of the total population of the juvenile samples.
These results suggested that the microbial community tightly associated with the intestine was not
diverse and was represented rather by Gammaproteobacteria (Singleton et al., 2003).
Bacteria were detected also inside the earthworm bodies. They populated the nephridia of
earthworms (L. terrestris, A. tuberculata, O. lacteum, and E. fetida) and were studied recently with
FISH and 16S rDNA sequence analyses. 16S rRNA gene sequences of the symbionts formed a
monophyletic cluster within the genus Acidovorax. Similarity between symbiont sequences from
different host species was 95.5–97.6%, whereas similarity between symbiont sequences from
individual animals of the same species was >99%. Bacteria of the genus Acidovorax were dominant
in the nephridia according to the FISH analysis performed with Acidovorax-specific oligonucleotide
probe. Thus, these bacteria were suggested to be an earthworm symbiont, which could play a role in
protein degradation during nitrogen excretion by earthworms (Schramm et. al. 2003). Davidson and
Stahl (2006) who performed curing experiments, FISH analysis with Acidovorax-specific probes,
and 16S rRNA gene sequence analysis, provided the evidence, that the egg capsules of E. fetida
contain high numbers of the bacterial symbiont and that nephridia of juveniles are colonized during
development within the egg capsule.
One of the major problems of previous studies was that some of the authors have used analyses
based on the direct enumeration (e.g. through FISH) alone (Schönholzer et al., 2002, Fisher et al.,
1995), the others used solely the PCR amplification of 16S rDNA and sequencing analysis of the
derived libraries (Furlong et al., 2002, Egert et. al., 2004). However, the higher stability of DNA in
environments in many cases does not allow to adequately concluding on the microbial community
composition. Still, compared to the well-studied termite gut microflora, the intestinal microbiota of
the earthworm remains poorly understood. There is no data available about regularity of alteration
1. Introduction
16
________________________________________________________________________________
of soil microbial community upon its passage through the gut of different earthworm species and
the main factors (microbial community of substratum or earthworm gut environment) affecting
these alterations.
To sum up, currently there is no solid data on the major patterns (if any) on the alterations of
microbial community from initial substratum passing through the gut. This basic knowledge is
absolutely necessary if one aims at the analysis of the worm-associated microbiome as a potential
factor influencing PrP in the soil. Consequently, no studies were conducted up to date to determine
the potential of the microorganisms, both, autochtonous or passing through the gut, of the worm (or
other soil invertebrates), to influence the fate of infectious agents, such as PrP.
The present work was a part of the European Union Project ‘Biotic and Abiotic Mechanisms of TSE
Infectivity Retention and Dissemination in Soil’ (QLRT-2001-02493) especially dedicated to study
the possible pathways of the prion dissemination in soil or soil-associated systems. A number of
ecological and epidemiological studies aimed at the understanding the mechanisms of interaction of
model prion (ovine recombinant prion, recPrP) with soil clay minerals and organic compaunds
(humus), migration of TSE infectivity through the soil column; effect of soil microorganisms and
microbes associated with soil invertebrates on the prion exposed, and the role of necrophagous
insects in the dissemination of TSE infectivity from soil-buried carcasses.
Objectives of the study
The aim of this investigation was to estimate the potential role of microorganisms associated with
earthworm intestine in the rPrP fate in the soil. Two major tasks of the present study were:
•
To characterize the microbial community in the earthworm gut: (i) to monitor the population
changes of different taxonomic groups in substrata passing through the earthworms guts and
define their common regularities; (ii) to estimate the composition of microbial communities
and (iii) to define the putative gut-associated microorganisms.
•
To determine the potential activity of microorganisms for recPrP proteolysis: (i) proteolytic
activity of pure bacterial and fungal isolates; (ii) proteolysis of recPrP by soil microbial
community and upon passage through the earthworm gut by intestine-augmented/stimulated
microbes.
2. Materials and methods
17
________________________________________________________________________________
2. Materials and Methods
2.1 Sampling and general experimental design
Three earthworm species were used in the study, Lumbricus terrestris and Aporrectodea caliginosa,
typically populating soddy-podzolic arable soil and Eisenia fetida inhabiting horse manure compost.
Soddy-podzolic soil and the earthworms were collected from the top ploughed horizon (0-20 cm)
under crop rotation at Ecological Soil Station of Moscow Lomonosov State University
(Solnechnogorskiy district, Moscow region, Russia). Horse manure compost and earthworms E.
fetida were taken at Chashnikovo Biological Station (Solnechnogorskiy district, Moscow region,
Russia).
Three groups of earthworms were analyzed: (1) three earthworm species (L. terrestris represented
by 3 animals, 6 individual animals of A. caliginosa and 5 of E. fetida) with respective substrata)
were collected at the autumn 2002; (2) 10 earthworms A. caliginosa and soil were taken at the
spring 2003; and (3) 30 exemplars of L. terrestris and 50 exemplars of A. caliginosa with the soil
were collected in the autumn 2004. Groups (1) and (2) were used in the preliminary experiments for
studying the most common regularity in changes of microbial community passing through the gut
of the earthworms. The group (3) was used for more detailed investigation of both intestine
bacterial populations and for rPrP proteolytic activity of microbial populations from the soil and
earthworm sources. Different species of the animals from the first group were held together to
estimate the effect of individual features of species on changes of soil bacterial community upon
passage through the worm guts. The earthworms from the third group were kept separately each
from other. The first two groups were kept for two weeks at 15° C, while the third group was kept
for three month before the experiment at same temperature. During this time earthworms were fed
with nonsterile oak leaf litter.
Pure microbial cultures were also using for 16S and 18S rRNA gene sequence analysis and we
performed the identification of the pure cultures of microorganisms isolated by our colleagues from
the Department of Soil Biology of the Moscow Lomonosov State University (Moscow, Russia)
from the earthworms, soil and worm excrements. The isolates were consequently scored with
various proteolytic assays.
The culture-independent rRNA-based techniques (FISH and RT-PCR) were applied for
investigating biodiversity of the soil, compost, earthworm intestine, and casts. Taxon-specific SSCP
analysis was performed for a rapid study of microbial communities. Clone libraries were
2. Materials and methods
18
________________________________________________________________________________
constructed to roughly estimate bacterial composition and FISH analysis was applied for a direct
enumeration of microorganisms from different taxonomic groups.
2.2 Fluorescence in situ Hybridization (FISH)
2.2.1 FISH with soil, intestine, and casts samples
Isolation of microbial cells from environmental samples
Substrates (soddy-podzolic soil and compost) 2 g each were fixed overnight at 4° C in 10 ml of
freshly prepared 4 % paraformaldehyde/PBS (137 mM NaCl; 2,7 mM KCl; 10 mM Na2HPO4; 2
mM KH2PO4) solution. Then samples were homogenized with vortexing for 5 min. and centrifuged
at 800 g for 1 min. Microorganisms were collected from supernatant by separation cells from soil
particles via Nycodenz gradient as described previously (Berry et al., 2003). Cells were washed
twice with PBS and stored in PBS/ethanol (1/1) solution at –20° C.
Casts were collected by keeping earthworms on the wet sterile filter paper for 6 hours at 15° C and
then earthworms were placed back into the soil/compost substratum. Collected fresh casts were
fixed and treated as the soil samples above.
The guts were dissected, the gut content and empty guts were fixed separately in 4%
paraformaldehyde in PBS overnight at 4° C. Microorganisms from the gut walls were washed off
with PBS (1,5 ml) for 2 min and vortexing in Matrix tube (Fast RNA Spin Kit for soil, Qbiogene).
Big particles of gut were excluded from suspension by centrifugation for 1 min at 200 g. Cells from
supernatant were collected with centrifugation for 4 min at 10000 g and stored in PBS/ethanol (1/1
vol/vol) solution at –20° C.
Fluorescence in situ Hybridization procedure
The aliquots (5 or 10 µl) of the samples were hybridized with Cy3-labeled oligonucleotide probes
EUB338 (5’-GCTGCCTCCCGTAGGAGT-3’) to specifically detect most of Bacteria (Amann
et. al., 1990); ALF968 (5’-GGTAAGGTTCTGCGCGTT-3’) for Alphaproteobacteria excluding
Rickettsiales (Neef, 1997); BET42a (5’-GCCTTCCCACTTCGTTT-3’) for Betaproteobacteria and
GAM42a (5’-GCCTTCCCACATCGTTT-3’) for Gammaproteobacteria (Manz et al., 1992);
DELTA495a (5'-AGTTAGCCGGTGCTTCCT-3') for most of Deltaproteobacteria (Loy et. al.,
2002); CF319a (5’-CCGTMTTACCGCGGCTGCTGGCA-3’) for Cytophaga-Flavobacteriumgroup of the Bacteroidetes (Manz et al., 1996); EUB3338 II (5'-GCAGCCACCCGTAGGTGT-3')
for Planctomycetes and EUB3338 III (5'- CTGCCACCCGTAGGTGT-3') for Verrucomicrobia
(Daims H. et al., 1999); LGC354A (5'-TGGAAGATTCCCTACTGC-3’) for Firmicutes (Gram-
2. Materials and methods
19
________________________________________________________________________________
positive
bacteria
with
low
G+C
content)
(Meier
et. al.,
1999 );
HGC69A
(5-
TATAGTTACCACCGCCGT-3) for Actinobacteria (Gram-positive bacteria with high G+C
content) (Roller et. al., 1994); PF2 (5’-CTCTGGCTTCACCCTATTC-3’) for all yeasts (Kempf
et. al., 2000); ARCH915 (5’-GTGCTCCCCCGCCAATTCCT-3’) for Archaea (Stahl and Amann,
1991).
Details
of
these
oligonucleotide
probes
are
available
at
ProbeBase
URL,
http://www.microbial-ecology.net/probebase/ (Loy et. al., 2003).
2.2.2 Design of group-specific nucleotide probe and the FISH with the earthworm tissues
The specific nucleotide probe was designed to detect Mollicutes-like organisms (MLO) whose
sequences were found in the clone libraries from earthworm gut content and casts. These sequences
were aligned together with other related sequences including those from class Mollicutes available
from the GenBank database (Benson et al., 2003) using ClustalW software (Thompson et al., 1994).
Alignment files were processed with BioEdit software to design primer specific for target bacterial
group.
Such
specific
oligonucleotide
probe
was
named
LUM1225
(5’-
GCTTACTGTCACCAGTTT-3’) (corresponding to its position in the 16S rRNA gene in
M. pulmonis (AF125582)). The specificity of the primers was checked against the RDPII database
(Cole et al., 2005) using Probe Match software. The nucleotide probe labeled with 5’AlexaFluor546 was synthesized and delivered by Invitrogen (Germany).
Preparation of the tissues and hybridization were done according to the previously published data
(Boye et al., 2001; Amand et al., 2005). The earthworms were narcotized with ethyl ester. Middle
body parts were dissected and fixed in the 10% buffered formalin for 24 hrs at 4° C, with following
washing in PBS. Tissues were embedded in paraffin, cut into 10 µm-thick sections, and mounted on
slides prepared with silane (Sigma-Aldrich). The tissue sections were dewaxed in 100% xylene for
10 min and washed in absolute ethanol. All sections were allowed to air-dry before hybridization.
Hybridization was performed in 10µl of hybridization buffer (900 mM NaCl, 20 mM Tris (pH 8,0),
0,01% SDS, and 35% formamide) with final concentration of probe 5 ng/µl. The slides were
incubated at 44° C for 6h in humid chamber. After hybridization the slides were washed with the
buffer consisted of 225 mM NaCl, 5 mM EDTA, 0,01% SDS, and 20 mM Tris (pH 8,0) for 20 min
at 46° C. The slides were rinsed with distilled water, air-dried in the dark, and finally amended with
Citifluor antifading agent. The slides were examined with Axioskop 40 epifluorescence microscope
(Zeiss, Germany) and Axiovert 100 TV laser confocal microscope (Zeiss, Germany).
2. Materials and methods
20
________________________________________________________________________________
2.3 rRNA and rRNA gene amplifications
2.3.1 Constructing of 16S rRNA clone libraries
Total DNA/RNA isolation and reverse transcription
Total DNA was extracted from the soil and cast samples with FastDNA SPIN Kit for Soil
(Qbiogene, Germany) quantified spectrophotometrically and was directly utilized as the template
for PCR.
Total RNA was extracted from the samples with the Fast RNA Spin Blue Kit (for soil) (Qbiogene,
Germany). Quantification of isolated total RNA was also done with BioPhotometer (Eppendorf,
Germany). RNA was treated with DNase I (Invitrogen, Germany) according to the manufacturers
protocol. The amount of RNA used for reverse transcription was 1µg per reaction (20 µl) for each
sample. Single-strand DNA was obtained using Super ScriptTM First Strand Synthesis System for
RT-PCR (Invitrogen, Germany) with the universal primer for 16S rRNA R1492 (5’CGGYTACCTTGTTACGACTT-3’).
PCR-amplification
Amplification of single strand DNA obtained with reverse transcription was done with 16S rDNA
specific primers F530 (5’-TCCGTGCCAGCAGCCGCCG-3’) and R1492. Amplification was done
in 20µl reaction with Taq DNA Polymerase, recombinant (Invitrogen, Gemany) and original
reagents according to the basic PCR protocol. Amount of ssDNA template was 30-50 ng/reaction.
Reaction mixtures were subjected to the following thermal cycling parameters: 30 cycles of 96° C
for 1 min, 45° C for 1 min, 72° C for 2 min, followed by a final extension at 72° C for 10 min.
Achieved PCR products were purified by QIAEX II Gel Extraction Kit (QIAGEN, Germany) from
the 0,8% agarose gel.
Constructing of clone libraries
The purified PCR products were ligated into plasmid vector pCRII-TOPO with following
transformation into the electrocompetent cells E. coli (TOPO 10) by electroporation (U, 1,8 kV; R,
200 Om) (TOPO TA Cloning kit, Invitrogen, Germany). Colonies were blue/white screened on LB
agar with 50µg/ml kanamycin and 25µl/ml X-Gal (Promega, Germany). Randomly chosen clones
were transferred into 96-well plates and further analyzed.
2. Materials and methods
21
________________________________________________________________________________
Sequencing of cloned 16 rDNA and phylogenetic analysis
Bacterial clones were grown in the 96-well microtiter plates with 100 µl of LB medium with
kanamycin (50 µg/ml) at 37° C overnight. Bacteria were pelleted by centrifugation (1000 g for 3
min at 4° C), washed once with 1x PBS and resuspended in the PCR-lysis solution A without
proteinase K (67 mM Tris-Cl (pH 8,8); 16 mM NH4SO4; 5 µM β-mercaptoethanol; 6,7 mM MgCl2;
6,7 µM EDTA (pH 8,0) (Sambrook, Russel, 2002) and heated at 95° C for 10 min.
PCR amplification was done with above bacterial lysate (1µl), Taq DNA Polymerase, recombinant
(Invitrogen) and original reagents according to the basic PCR protocol using primers M13 forward
(5’-GACGTTGTAAAACGACGGCCAG-3’)
and
M13
reverse
(5’-
GAGGAAACAGCTATGACCATG-3’) in 20µl of final volume with thermal/time conditions
described above. PCR products were purified with MinElute 96 UF PCR Purification Kit (Qiagen,
Germany).
The monodirectional sequencing was proceeded with reverse primer R1492 according to the
protocol for BigDye Terminator v1.1 Cycle Sequencing Kit from Applied Biosystems (USA):
Ready Reaction Premix (2,5x) 4µl; BigDye Sequencing Buffer (5,0x) 2µl; primer 10pmol; clear
PCR product 1µl; deionized water up to 20µl) followed by 25 cycles of 96° C for 20 s, 50° C for 20
s, 60° C for 240 s, followed by at 4° C hold.
Obtained sequences were analyzed against the GenBank database using BLAST alignment software
(http://www.ncbi.nlm.nih.gov/BLAST/) (Altschul et al., 1997).
2.3.2 Taxon-specific Single Strand Conformation Polymorphism (SSCP) analysis
Design of taxon-specific 16S rRNA gene primers.
Sequences (minimum of 1300 bp long) of described bacterial species of Proteobacteria (α, β, γ, δclasses), CFB group, Planctomycetes, Verrucomicrobia, Actinobacteria (HGC), and class Bacilli
available from the GenBank database (Benson et al., 2003) and related to those detected with
culturing and RT-PCR approaches were selected and aligned with outgroups sequences using
ClustalW software (Thompson et al., 1994). Alignment files were processed with BioEdit software
to design primers, which were supposed to be consent with taxon-conserved regions, 18-25 bp-long
and be situated between 400 and 500 each from other. Specificity of the primers was checked with
PrimeRose
software
(available
at
the
URL:
http://www.cf.ac.uk/biosi/research/biosoft/Primrose/index.html) against constructed databases,
including 16S rDNA sequences of isolated bacterial strains and RT-PCR obtained from the
analysed ecosystems. Additionally primers were screened against the RDPII database (Cole et al.,
2. Materials and methods
22
________________________________________________________________________________
2005) using Probe Match software. Less specific primer in the set carried phosphate group on 5’terminus.
Specificity of the primers for annealing was determined using temperature gradient PCR in
thermocycler Eppendorf 5341 (Germany). Reactions were performed in 20µl of final volume using
recombinant Taq DNA Polymerase (Qiagen, Germany) and original reagents according to the basic
PCR protocol. Bacteria were lysed in PCR-lysis solution A without proteinase K (67 mM Tris-Cl
(pH 8,8); 16 mM NH4SO4; 5 µM β-mercaptoethanol; 6,7 mM MgCl2 ; 6,7 µM EDTA (pH 8,0)
(Sambrook, Russel, 2002) and heated at 95° C for 5 min were used as DNA template. 16S cDNA
was used as template for Verrucomicrobia, Planctomycetes and Myxococcales.
Reaction mixtures were subjected to the following thermal cycling parameters: 30 cycles of
denaturation at 96° C for 1 min, annealing with temperature gradient (first frame at 45-55°, and
second frame at 55-65° C) for 1 min, and extension at 72 °C for 1 min, followed by a final
extension at 72 °C for 7 min. Outgrouped bacterial species were used as a negative control. The
quality of PCR products was verified through 2% agarose gel electrophoresis.
Table 1. Sets of taxon-specific SSCP primers and their annealing temperatures
Target group
1
Name
2
α -proteobacteria ALF F822p
ALF R1234
Primer
Sequence
5’-CCACGCCGTAAACKATGA-3’
5’-CSYGTAAGGGCCATGAGG-3’
β -proteobacteria BET F750p
5’-GACGCTCAKGCACGAAAGCGT-3’
BET R1227
5’-TGACGTGTGWAGCCCCACCYA-3’
γ -proteobacteria GAM F538p
50
412
45
477
50
503
48
462
50
417
48
485
48
459
50
485
5’-RAGGGTGCAAGCGTTAAT-3’
GAM R1041 5’-YNNNGTTCCCGAAGGC-3’
Myxococcales
DEL F972p
5’-CGCAGAACCTTACCTGGK-3’
(δ-proteobacteria)
DEL R1434
5’-GACTTCTGGAGCAAYYG-3’
CFB
CFB F522
5’-TYAYTGGGTTTAAAGGGT-3’
CFB R939p
5’-TAAGGTTCCTCGCGTANCA-3’
Verrucomicrobia VER F901p
5’-AGCGGTGGAGTATGTGGC-3’
VER R1209
5’-GCATTGTAGTACGTGTGC-3’
PLA F949p
5’-GCGMARAACCTTATCC-3’
PLA R1408
5’-CCNCNCTTTSGTGGCT-3’
Bacilli
BAC F348p
5’-CAGCAGTAGGGAATCTTC-3’
(Firmicutes)
BAC R833
5’-ATGARTGCTARGTGTTAG-3’
Planctomycetes
Annealing3 Expected
T, °C
fragment,
bp3
2. Materials and methods
23
________________________________________________________________________________
1
positions in the target groups: Alphaproteobacteria (B. diminuta X87274); Betaproteobacteria
(A. faecalis AF155147); Gammaproteobacteria (E. coli NC004431); δ-proteobacteria (M. fulvus
AJ233917); CFB (F. jonsoniae AB078043); Verrucomicrobia (V. spinosum X90515);
Planctomycetes (P. limnophilus X62911); Bacilli (B. subtilis AY030331); 2 p – indicates the
phosphate group;
3
annealing temperature calculated for the primer set;
SSCP: total DNA/RNA isolation and PCR
Total DNA and RNA were extracted from the soil and cast samples as it was described above.
Amplification with taxon-specific primers (RNA was previously undergone reverse transcription)
was performed at annealing temperature illustrated in Table 1. Additionally, universal primers
Com1 (5’-CAGCAGCCGCGGTAATAC-3’) and Com2-Ph (5’-CCGTCAATTCCTTTGAGTTT3’) were used (Schweiger and Tebbe, 1998). Amplification was done with serial dilutions of
template. Obtained PCR products were purified with QIAEX II Gel Extraction Kit (QIAGEN,
Germany) from the 2% agarose gel.
Preparation of ssDNA and gel electrophoresis
Pure PCR products were treated with λ−exonuclease (Fermentas, Germany) and consequent SSCP
analysis was performed as it was previously described (Schwieger and Tebbe, 1998) on 21 cm-long
0,6X MDE gels (Cambrex, Germany) with glycerol (5%). The electrophoresis was performed in a
Pharmacia Multiphor II apparatus (Pharmacia, Germany) at 400 V for 14 hours at 20° C. Gels were
silver stained (Bassam et al. 1991) and dried at room temperature.
Isolation and PCR amplification of DNA fragments from polyacrylamide gels.
Single bands detected in polyacrylamide gels after silver staining were cut out with disposable
scalpel blades. Gel slices were transferred to 96-well microtiter plate containing 20 µl of elution
buffer (10mM Tris-Base, 5mM KCl, 1,5mM MgCl x 6H2O, 0.1% Triton X100, pH 9,0) and boiled
at 95° C for 10 min. Reamplification with corresponding primers was done under conditions
described above with 1 µl supernatant as a template after spinning down the samples. PCR products
were purified with MinElute 96 UF PCR Purification Kit (Qiagen, Germany).
Sequencing of 16 rDNA
The monodirectional sequencing was done with corresponding downstream primers according to
the protocol for BigDye Terminator v1.1 Cycle Sequencing Kit from Applied Biosystems (USA):
Ready Reaction Premix (2,5x) 4µl; BigDye Sequencing Buffer (5,0x) 2µl; primer 10 pmol; clear
2. Materials and methods
24
________________________________________________________________________________
PCR product 1µl; deionized water up to 20 µl) followed by 25 cycles of 96° C for 20 s, 50° C for 20
s, 60° C for 240 s, followed by 10 min-hold at 4° C.
In case the sequencing reaction was unclear, the PCR product was cloned with TOPO TA Cloning
Kit (Invitrogen, Germany) and sequenced from the pCRII plasmid with M13F or M13R
oligonucleotides as described above.
Obtained
sequences
were
analysed
using
BLAST
alignment
software
(http://www.ncbi.nlm.nih.gov/BLAST/) (Altschul et al., 1997), Ribosomal Database Project II
(RDP)
(http://rdp.cme.msu.edu/html/) (Cole et al., 2005).
Phylogenetic analysis
Phylogenetic analysis was then performed using the program package Phylip (Felsenstein, 2001) as
described
by
Yakimov
and
colleagues
(2005),
and
ClustalW
online
tool
(http://www.ebi.ac.uk/clustalw/index.html) (Thompson et al., 1994) with sequences loaded from
NCBI database (http://www.ncbi.nlm.nih.gov/) (Altschul et al., 1997).
Chimera checking and constructing of phylogenetic trees
All cloned insertions with identity less than 95% to already described organisms were analyzed with
CHIMERA_CHECK
program
at
the
Ribosomal
Database
Project
II
(RDP-II)
http://35.8.164.52/cgis/chimera.cgi?su=SSU (Cole et al., 2005). The sequences suspected to be
chimeric were excluded from analysis.
2.4 Isolation and identification of pure microbial cultures
Isolation of bacteria and fungi with serial plate dilution method
Bacteria and fungi were isolated from soil, compost, gut, and excrements of the earthworms on the
glucose-peptone-yeast agar (glucose, 1 g; peptone, 2 g; yeast extract, 1 g; casein hydrolyzate, 1g;
KH2PO4, 0,5 g; K2HPO4, 0,5, g; Difco agar, 15 g; distilled H2O up to 1000 ml; pH 7,2). Fungal
growth was inhibited by nystatin or cycloheximide and bacterial growth was limited by adding the
streptomycin sulfate. The isolation of microorganisms was done by plate dilution method and by
placing of particles of soil, gut, and excrements directly on the surface of the solid medium. For
homogenization of samples and desorbing of microorganisms from mineral/organic particles the
homogenizer DIAX 900 (Heidolph) was used. Fifty mg of dry compost or excrements or freshly
obtained gut tissue (dry weight around 10-50 mg) was placed into 500 ml of sterile tap water (for
2. Materials and methods
25
________________________________________________________________________________
bacteria) or into 10 ml for fungi. The samples were homogenized at 8000 rpm for 30 sec in 10 ml of
sterile water. Aliquots (20-400 µl) were spreaded by spatula on the surface of the agar medium. The
Petri dishes were incubated for 10-28 days at room temperature (18-20° C).
Microbial strains in several replicates were isolated from each group of colonies that had similar
cultural and morphological features on the slants with same medium (for fungi also – on malt-agar)
and maintained at 4°C. For fungi, relative abundance of isolates of different species/genera from
total isolates from gut or soil on the plates was calculated.
Identification of microbial isolates
The isolates were identified by the sequence analysis of 16S rRNA (bacteria) and the ribosomal
intergenic space D1-D2 region (fungi).
Preparing cultures for the PCR amplification
Bacteria were grown in 96-well microtiter plates with 100µl of LB medium (tryptone, 10 g; yeast
extract, 5 g; NaCl, 10 g; distilled H2O up to 1000 ml) at 30° C for 2 days. Consequently the cells
were pelleted by centrifugation (Heraeus Sepatech Omnifuge 2,0 RS; 500g at 100 C for 15min) and
washed once with PBS (NaCl, 137 mM; KCl, 2,7 mM; Na2HPO4, 10 mM; KH2PO4, 2 mM). Cells
were resuspended and boiled in 100µl of PCR-lysis solution A (Tris-Cl (pH 8,8), 67 mM; NH4SO4,
16 mM; β-mercaptoethanol, 5 µM; MgCl2, 6,7 mM; EDTA (pH 8,0), 6,7 µM; SDS, 1,7 µM)
(Sambrook, Russel, 2002) for 10 min.
Fungal isolates were grown in 96 well-microtiter plates with 50µl of Czapek medium (sucrose, 30,0
g; KNO3, 2,0 g; K2HPO4, 1,0 g; MgSO4*7H2O, 0,5 g; KCl, 0,5 g; FeSO4*7H2O, 0,01 g; yeast
extract, 2,0 g; peptone, 5,0 g; distilled H2O up to 1000 ml) in each well, at 30° C for 2 days. Fungal
biomass was collected with centrifugation as described above, washed with PBS and treated with
lyticase in buffer Y (sorbitol, 1M; EDTA 0,1M; pH 7,4) at 37° C for 30 min. Then biomass was
washed with PBS from buffer Y and lyticase and resuspended in 100µl of PCR-lysis buffer A with
proteinase K, incubated at 55° C for 2 hrs, and boiled at 95° C for 10 min.
PCR amplification of the 16S rRNA genes of the isolates
Reaction mixes were set up in a PCR hood in a room separated from that used to extract DNA and
the amplification and post-PCR room in order to minimize contamination. Reaction mixes (total, 20
µl) were set up as follows: deionized H2O 6 µl; Q-solution 4 µl; 20 mM solution of four dNTPs (pH
8,0) 4µl; PCR Buffer (10x; contains 15 mM MgCl2) 2,0 µl; 1,0 U of Taq DNA polymerase
(Amplitaq; Perkin Elmer); 6*10-3 nmol each primers of the 16S rDNA (for bacteria) primers F27
2. Materials and methods
26
________________________________________________________________________________
forward
(5’-AGAGTTTGATCMTGGCTCAG-3’)
and
R1492
reverse
(5’-
TACGGYTACCTTGTTACGACTT-3’) and 18S rDNA (for fungi) primer NL-1 forward (5’GCATATCAATAAGCGGAGGAAAA-3’) and NL-4 reverse (5’-GGTCCGTGTTTCAAGACGG3’) and 1 µl of DNA template.
The reaction mixtures were subjected to the following thermal cycling parameters in a Perkin Elmer
9600 thermocycler: 30 cycles of 96 °C for 1 min, 45 °C for 1 min, 72 °C for 2 min, followed by a
final extension at 72 °C for 10 min. After amplification the aliquots (4 µl) were removed from each
reaction mixture and examined by electrophoresis (150 V, 25 min) in gels composed of 0,8 % (w/v)
agarose (Gibco, UK) in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.3),
stained with ethidium bromide (5 µg/100 ml). Gels were visualized under UV illumination using a
gel image analysis system (VVP Products, UK).
Sequencing of amplicons and analysis of sequence data
PCR products were cleaned by MinElute 96 UF PCR purification kit (QIAGEN, Germany) prior to
sequencing, particularly to remove dNTPs, polymerases, salts, and primers. The monodirectional
sequencing was proceeded with one corresponding primer according to the protocol for BigDye
Terminator v1.1 Cycle Sequencing Kit from Applied Biosystems (Ready Reaction Premix (2,5x)
4µl; BigDye Sequencing Buffer (5,0x) 2µl; primer 10pmol; clear PCR product 2µl; deionized water
up to 20µl) followed by 25 cycles of 96 °C for 20 s, 50 °C for 20 s, 60 °C for 240 s, followed by a
4 °C hold. The resulted 16S and 18S rDNA sequences were compared with those stored in the
GenBank Data system using BLAST alignment software (http://www.ncbi.nlm.nih.gov/BLAST/)
(Altschul et al., 1997), and Ribosomal Database Project II (RDP) (http://rdp.cme.msu.edu/html/)
(Cole et al., 2005).
2.5 RecPrP proteolysis
2.5.1 Recombinant protein synthesis
Ovine ARQ genetic variant of the prion protein (94% homology with the bovine PrP (Rezaei et al.,
2002) used for the experiments was synthesized and delivered by our colleagues Dr. Human Rezaei
and Mrs. Peggy Rigou from INRA (Institut National de la Recherche Agronomique, Unité de
Virologie et Immunologie Moléculaires, Jouy-en-Josas, France). The recombinant PrP (without
GPI; cloned in pET 22+) was expressed in inclusion bodies in E. coli BL21 DE3 strain according to
the previous protocol (Rezaei et al., 2000). After lysis, sonication and solubilization of the inclusion
2. Materials and methods
27
________________________________________________________________________________
bodies in urea, purification of the protein was performed on a Ni Sepharose column using the
propensity of the N-terminal octarepeat region to chelate transition metals. Refolding of the protein
was achieved on the column by heterogeneous phase renaturation simultaneously to purification,
favoring intra-molecular rather than inter-molecular disulfide bond formation. After elution by 1 M
imidazole, the purified protein was recovered in the desired buffer by passage on a G25 desalting
column. This procedure leads to a high degree of purification, as checked by SDS-PAGE and
Western Blot and to a strictly monomeric alpha-rich protein.
2.5.2 RecPrP proteolytic assay
2.5.2.1 PrP proteolytic assay of pure isolates
Among more than 1500 bacterial and 400 fungal cultures isolated from the soddy-podzolic soil,
horse manure compost, gut content and cast of earthworm (Aporrectodea caliginosa, Lumbricus
terrestris and Eisenia fetida) randomly chosen strains (1 strain from each specie) were investigated
for rPrP proteolysis. Medium M9 (Sambrook, Russel, 2002) (11µl) with minor modification
(without glucose; peptone – 0,5 g/l; gelatin – 0,5 g/l) was inoculated with pure microbial cultures
and 40 ng of recPrP. After 3 day incubating recPrP digestion was checked by Western Blot analysis.
2.5.2.2 Effect of earthworms and gut microbiota on recPrP retaining
Extraction of recPrP from the soddy-podzolic soil and earthworm cast
In the preliminary experiments, two protocols were applied to extract recPrP (100 ng) from soil
(1 g).
The first method, elaborated by Dr. Robert Somerville, (Institute of Animal Health,
Neuropathogenesis Unit, University of Edinburgh, United Kingdom) proposed to treat 1 g recPrPcontaminated soil/sand sample with 1 ml of the extraction buffer (100 mM Na2HPO4-1% sarkosyl).
Samples were thoroughly mixed on a vortex mixer and were attached to a horizontal shaker for
vigorous shaking at 600 rpm for 2 h. After 2 h, samples were mixed briefly on the vortex mixer, and
then spun down at 6000 g for 15 min. The supernatant was then carefully poured off into another 2
ml tube, and a sub-sample (10µL) was taken for analysis by SDS-PAGE and Western blotting.
The second method was based on the electroelution of recPrP from the soil (Rigou et al., 2006).
Negatively charged recPrP protein was extracted from soil in an assembly of used tubes filled with
acrylamide gel flanked with agarose plugs and covered at the edges with a dialysis membrane. The
2. Materials and methods
28
________________________________________________________________________________
tubes were imbedded into the agarose stacked on the tray for a standard horizontal electrophoresis
unit. The protein eluted in this device was subjected to Western blot analysis.
The mixture of protease Inhibitor Cocktails Sets (II+III) (Calbiochem) (Tab. 2) was added into the
control samples (20 µl/g soil) before extraction with both protocols.
Table 2. Composition of Protease Inhibitor Cocktail Sets (Calbiochem)
Compound
Target protease
Protease Inhibitor
Protease Inhibitor
Cocktail Set II
Cocktail Set III
AEBSF, hydrochloride
Serine protease
+
+
Bestatine
Aminopeptidase B and
Leucine Aminopeptidase
+
+
E-64, Protease inhibitor
Cysteine protease
+
+
EDTA, disodium
Metalloproteases
+
–
Pepstatine A
Aspartic protease
+
+
Aprotinin
Trypsin, chymotrypsin,
coagulation factors involved
in the prephase of blood
clotting tissue and
leukocytic proteinases,
kallikrein, plasmin
–
+
Leupeptine, hemisulfate
Trypsin-like proteases and
cysteine proteases
–
+
Aqueous extracts assay
The aqueous extracts were prepared from the soil and earthworm cast to determine enzymatic
proteolytic activity of the water phase. Samples (approx. 0,5 g) were homogenized by vortex in
water (50% w/v) and centrifuged at 11000 g for 5 min. Supernatant was filtered through the
membrane filter (0,22 µm, Millipore, USA) into the clean tube. General amount of proteins in the
solutions were determined with the Bradford dye-binding Kit (Bio-Rad, USA) as described above.
Protease Inhibitor Cocktail Sets II (recommended for use with bacterial cell extracts) and III
(recommended for use with mammalian cell and tissue extracts) (Calbiochem) were used to
determine the role of proteases derived from bacteria and eucarya in unspecific and recPrP
proteolytic activity (Tab. 2). There were in total four experimental setups for aqueous extract with
2. Materials and methods
29
________________________________________________________________________________
recPrP: (1) without Inhibitor Cocktails; (2) with Inhibitor Cocktail Sets (II+III); (3) with Inhibitor
Cocktail Set II; (4) with Inhibitor Cocktail Set III. Protease Inhibitors (1µl of each cocktails and
their equal (vol./vol.) mixture (sets II+III)) were added to correspond setup.
Unspecific proteolytic activity of aqueous extracts was checked with Protease ScreeningTM Kit
(GenoTech, USA) at 15° C for 12 hrs.
RecPrP proteolysis was performed in 12 µl of aqueous extract suspension (total reaction volume)
included 40 ng of recPrP and approximately equal amount of proteins in each sample. Sampling
was done after 0,2; 1; 2, 6 and 12 hrs as well as after 1, 2, 3, 4, and 6 days of incubation at 15° C.
Total sample volume was analyzed with Western Blot analysis for remained PrP amount.
Control digestion of recPrP (40 ng in 12 µl of total reaction volume) was done with modified
trypsin (0,8 ng/µl) in the supplemented buffer (New England Biolabs, Germany).
2.5.2.3 Western blot analysis
Western blot analysis was performed as described previously (Thackray et al., 2004). The total
sample volume from each experiment was loaded on the SDS-PAGE (12% vol/vol) and after
electrophoresis subsequently blotted to the nitrocellulose membranes (PerkinElmer) in transfer
buffer (25 mM Tris, 192 mM glycine, 10% isopropanol) at 400 mA (constant) for 1 h as was
previously described (Baron et al., 1999) with blotter (Peqlab, Germany). Membranes were blocked
with TBS-T (10 mM Tris-Cl pH 7.8, 100 mM NaCl, 0.05 % Tween 20) with 5 % skimmed milk
(Thackray et al., 2004) and subsequently incubated with primary mouse antibodies PrP248 for Nterminus (residue numbers 60-95 according to ovine sequence) and VRQ14 for the interhelix loop
(residue numbers 194–199 according to ovine sequence) (Rezaei et al., 2005) at the concentration 5
µg/ml for 1 h at room temperature (Fig. 3).
2. Materials and methods
30
________________________________________________________________________________
Figure 3. Localization of antibody epitopes on the 3D structure of ovine recPrP (Rezaei et al.,
2005).
This procedure was followed by the incubation with goat anti-mouse IgG (H+L) horseradish
peroxidase conjugate (at 1/1000) (Molecular Probes, Germany). All antibody dilutions were done in
1 % non-fat milk in TBS-T. Recombinant PrP bands were detected on an X-ray film X-OMAT
Kodak, (USA) after treatment with by enhanced chemiluminescence reagent (ECL) (Pierce, USA).
The lowest detectable amount of recPrP by the analysis was ~10 ng per lane.
3. Results
31
________________________________________________________________________________
3. Results
3.1 Effect of earthworm gut environment on microbial community of soil
3.1.1 Preliminary studies of changes of microbial community in the substrate upon passage
through the earthworm gut
3.1.1.1 Characterization of the microbial population with FISH
Changes microbial community composition through the passage of the gut of autumncollected L. terrestris
Alphaproteobacteria were a dominant group in the soil and gut samples but their total number
became 10-times lower in the cast (Fig. 4A). Relative numbers of Betaproteobacteria and
Firmicutes were high in the soil, shifted down in the gut content, and almost restored in the
excrements. CFB, Gammaproteobacteria, and Actinobacteria were augmented during passage
although amount of γ-proteobacteria in the gut content was low (Fig. 4A). Yeast extraordinary
mounted their numbers in the gut contents, but they were detected neither in the soil, nor in the cast
(Fig. 4A). Single cells of Deltaproteobacteria were observed in the soil and cast samples. Archaea
were not detected in any source. Bacteria related to CFB group, Firmicutes, and β- and γproteobacteria were more numerous in the gut-wall samples in compare with gut content (Fig. 4A).
Ratio of bacteria hybridized with to the Bacteria-specific probe (EUB338) to those stained with
DAPI (EUB338/DAPI) was lower in the soddy-podzolic soil in comparison with excrement (0,20
and 0,29 respectively) and even lower in the gut (gut wall sample 0,19; gut content sample 0,17).
Changes in microbial community composition through the passage of the gut of A. caliginosa
Autumn-collected A. caliginosa
Passage through the gut significantly decreased numbers of Alphaproteobacteria (Fig. 4B) in
contrast to Betaproteobacteria who exhibited an opposite behavior. Index of the former grew up
significantly in the gut content and was not changed notably in the cast (Fig. 4B).
Gammaproteobacteria as Firmicutes reduced their numbers in the gut content and restored their
relative densities in the excrements. Changes of CFB and Actinobacteria were similar – increasing
in the gut content and slight decreasing in the excrements (Fig. 4B). Composition of microbial
3. Results
32
________________________________________________________________________________
community on the gut walls was different from that one of the gut content. Only Betaproteobacteria
had similar abundances as in the gut contents (Fig. 4B). Numbers of Actinobacteria, CFB, and
Alphaproteobacteria were lower in the gut-wall samples. Opposite results were noticed for
Firmicutes and Gammaproteobacteria. The differences were most remarkable for Firmicutes (Fig.
4B).
Spring-collected A. caliginosa
We did not evaluate the gut wall microbial community composition in this earthworm group. While
Alphaproteobacteria gradually increased the numbers in the gut contents and excrements, CFB and
yeasts increased their densities in the gut but decreased in the cast. Passage through the gut caused
strong reduction of Betaproteobacteria and Gammaproteobacteria already in the gut and in the
excrements the indices were the same (Fig 4C). Firmicutes declined in the gut content and restored
their numbers in the cast. Quantity of Actinobacteria and Archaea was pretty stable in all samples
(Fig. 4C). Separate single cells of Deltaproteobacteria were observed in each sample.
Ratio EUB338/DAPI in the autumn- and spring-colleted soddy-podzolic soil was comparable (0,20
and 0,18) and in the cast samples this index was also higher (0,30 and 0,23 autumn- and springcolleted earthworms respectively), fluctuating in the gut content samples (0,16 and 0,27).
Changes in microbial community composition through the passage of the gut of E. fetida
Unlike in the soddy-podzolic soil and in the earthworms populating that substratum, EUB338/DAPI
ratio was much higher in the compost and in the cast of earthworm E. fetida (0,42 and 0,35
respectively), this ratio was the lowest in the gut content (0,15).
One of the most notable events was the 8-fold increase of numbers of CFB bacteria in the gut
content in comparison with the compost and a consecutive two-fold reduction of their numbers in
the cast. Significant growth of the population of Actinobacteria and Alphaproteobacteria took place
as well (Alphaproteobacteria became a dominant group) with a simultaneous strong reduction of
Beta- and Gammaproteobacteria (Fig. 4D). Yeasts and Archaea were detected in the compost in
quite low concentration (Fig. 4D). Few cells of Deltaproteobacteria and 28 fragments of mycelia
and just 2 yeast cells were observed in the gut content among approximately 1000 total cells
counted in 30 in total microscope fields. Numbers of Actinobacteria and Betaproteobacteria in the
gut-wall samples were twice and three-fold higher than in gut content, respectively. In contrast to
that, Alphaproteobacteria had two-fold lower proportion on the gut walls. Gammaproteobacteria
3. Results
33
________________________________________________________________________________
and Firmicutes were not detected in the community of gut wall. The CFB had similar abundance in
the gut-wall and gut-content samples (Fig. 4D).
A
100%
1
90%
17
16
80%
6
5
5
9
70%
60%
18
11
5
11
9
19
40%
30%
41
34
31
10%
0%
soddy-podzolic soil
100%
D
gut walls
1
2
17
90%
22
6
5
5
80%
70%
60%
25
50%
gut contents
11
27
70%
60%
50%
27
2
30%
39
40%
4
0%
C
100%
90%
19
80%
7
9
70%
41
20%
19
10%
3
16
3
40
gut walls
4
7
11
9
3
6
13
9
3
3
2
3
16
excrements
25
38
40%
25
gut contents
11
6
5
11
11
50%
29
23
11
15
10%
13
4
8
6
18
31
20%
30
21
24
27
18
25
18
4
60%
30%
30%
32
27
compost
excrements
37
9
20%
10%
38
5
40%
19
21
7
1
80%
12
9
100%
90%
8
20%
B
15
8
16
25
50%
13
37
23
0%
0%
soddy-podzolic soil
Alphaproteobacteria
gut wall
; Betaproteobacteria
gut contents
excrements
; Gammaproteobacteria ; CFB ; Actinobacteria
soddy-podzolic soil
gut contents
; Firmicutes ; Archaea
excrement
; Yeasts
Figure 4. Changes of relative abundance of majortaxonomic groups of microorganisms in substratum passing through the gut. A: autumn-collected
soddy-podzolic soil and L. terrestris; B: autumn-collected soddy-podzolic soil and A. calliginosa; C: spring-collected soddy-podzolic soil and A.
calliginosa; D: horse manure compost and E. fetida.
3 Results
35
________________________________________________________________________________
3.1.1.2 Clone libraries
In total, 305 unique bacterial clones were identified in 12 libraries. Except of bacteria we detected
the unspecifically amplified 12S mitochondrial and 18S rRNA genes of earthworms and other
eukaryotic organisms in gut content and cast libraries.
Proteobacteria had a highest numbers among all bacterial clones (54%). The most numerous
phylum was Gammaproteobacteria: 83 clones (27%) from 7 orders. Alphaproteobacteria (14%)
were represented by 7 and Betaproteobacteria (11%) by 5 orders. Deltaproteobacteria were minor
in the clone libraries (5 clones, 2%). CFB were the second numerous group of bacteria (61 clones,
20%) represented by Flavobacteria (detected mostly in the gut content and cast), Sphingobacteria
(detected mostly in soils and compost) classes. Among Actinobacteria (25 clones, 8%) clones from
Micrococcineae suborder were the most frequent. Firmicutes (34 clones, 8%) were detected in each
library, but class Mollicutes was found only in gut and cast of earthworms; class Bacilli (clones
linked to Bacillus, Paenibacillus, Brevibacillus, Aeurinibacillus, Geobacillus and Anoxybacillus
genera) was the common in the majority of libraries except of gut wall library of L. terrestris;
Clostridia (Ruminococcus genus) were the only class of Firmicutes identified in horse manure
compost and autumn-collected A. cliginosa cast library (related to Desulfotomaculum genus).
Acidobacteria (Fibrobacteres/Acidobacteria group), Myxococcales, and Cloroflexi were found only
in gut content or cast libraries. Verrucomicrobia and Planctomycetes were found in all sources but
they were more abundant and identical to each other in the gut content and cast libraries (Suppl.
Fig. 34).
Clones of some bacterial taxa clearly affiliated their taxonomic placement distanced from known
species. The clusters were formed by clones equally distant from Succinivibrionaceae and
Legionellaceae (Gammaproteobacteria) in compost, gut content of E. fetida and cast of
L. terrestris. Other bacterial clones were considered as “unclassified” and apparently belonged to
Acidimicrobidae and Actinobacteridae subclasses (Actinobacteria) with unclear affiliation (Suppl.
Fig. 37, 39). Some clones from Sphingobacteriales family also formed a cluster together with a
symbiont sf. Flavobacterium of Tetraponera binghami (AF459795) as closest hit (van Borm et al.,
2002).
Some identical OTUs were detected in the substrata (compost or soil) and in gut libraries of
earthworm inhabiting these substrata, or in the gut and cast libraries, but none were found in all
three samples, substratum, gut, and cast.
3. Results
36
________________________________________________________________________________
Autumn-collected soddy-podzolic soil
Proteobacteria were dominated class of microorganisms making up to 78 % among 28 clones.
Among Gammaproteobacteria (13 OTUs) a half of the clones was related to bacteria of family
Xantomonadaceae; other numerous clones were from family Pseudomonadaceae (including genus
Pseudomonas) (Suppl. Fig. 37). Alphaproteobacteria (6 OTUs) were represented by organisms
from
orders
Rhizobiales,
Betaproteobacteria
(3
Sphingomonadales,
OTUs)
were
and
corresponding
Rhodospirillales
to
families
(Suppl.
Fig.
35).
Alcaligenaceae
and
Burkholderiaceae, and bacteria of CFB (3 OTUs) related to classes Flavobacteria and
Sphingobacteria (Suppl. Fig. 36, 38). Verrucomicrobia, Planctomycetes, and Firmicutes (class
Bacilli) were present in the library only as single clones (Suppl. Fig. 34, 39).
Autumn-collected L. terrestris
Microbial community composition in this variant was more similar between the gut content and cast
in contrast to soil and gut-wall libraries. But affiliation of OTUs was stronger between gut-content
and cast libraries. The same families as in the soddy-podzolic soil but without Sphingomonadales
were represented by Alphaproteobacteria (6 OTUs) in the gut-wall library. Betaproteobacteria
were detected in each library by single clones related to family Commamonadaceae (3 OTUs)
(Suppl. Fig. 36). Gammaproteobacteria were represented by clones from families Aeromonadaceae
(3 OTUs) and Schevanellaceae (1 OTU) in the gut wall, and by families Legionellaceae (1 OTU)
and Pseudomonadaceae (2 OTUs) in another libraries. Five pairs of OTUs from Flavobacterium
genus (CFB), 4 pairs of OTU from Planctomycetes and 1 pair of OTU from Verrucomicrobia were
represented in the gut content and cast libraries (Suppl. Fig. 34, 38). Actinobacteria were detected
in a single OTU (Microbacterium genus) in the gut-wall and two matching OTU (Nocardioides
genus) in the gut content and cast; another clone of Mycobacterium genus was discovered in the
cast library (Suppl. Fig. 39). Three clones of Bacilli (class Firmicutes) linked to the genus Bacillus
were found in the gut content and cast libraries.
We also detected a single OTU (2 clones) closely related to roundworms of genus Rhabditis
(Nematoda) (Fig. 5) (18S rDNA sequence identity 98%). Eukaryotes from family Monocystidae
(Apicomplexa; 1 OTU, 3 clones) were also present in the library; sequence identity of 18S rDNA to
closest hit (Monocystis agilis AF213515) was 90%.
3. Results
37
________________________________________________________________________________
Figure 5. Roundworms of genus Rhabditis (Nematoda) found alive in the soddy-podtolic soil and
the cast of L. terrestris: magnification, ×8; bar, 1cm.
Autumn-collected A. caliginosa
The clone libraries from A. caliginosa-derived environments did not show a clear consistency
between the libraries in contrast to L. terrestris. CFB were more abundant in the gut content library.
Numbers of OTUs from orders Flavobacteriales and Sphingomonadales were comparable (6 and 5
respectively) (Suppl. Fig. 38). Firmicutes were also more frequent in the gut content library (4
OTUs from class Bacilli), than in soil or cast (1 OTU from class Clostridia) (Suppl. Fig. 39). In
contrast to CFB and Firmicutes, compositions of Alpha-, Beta-, and Gammaproteobacteria were
more inconsistent in the cast (4, 3, and 4 different taxa in each subclass respectively) and soil
libraries than in the gut content (1, 1, and 1 taxon from each subclass respectively) (Suppl. Fig. 3537). Actinobacteria of suborders Micrococcineae (6 OTUs) and Propionibacterineae (1 OTU) were
detected only in the cast library (Suppl. Fig. 39) A single clone represented Verrucomicrobia and
Chloroflexi (class Thermomicrobia) in the cast library (Suppl. Fig. 34).
Spring-collected soddy-podzolic soil and A. caliginosa
The clones derived from CFB were abundant in the soil and gut-content libraries and scarce in the
cast library. All clones in soil library were matching the bacteria from order Sphingomonadales;
most of the clones had a high affinity to symbiont sf. Flavobacterium of Tetraponera binghami
(AF459795). There were 3 OTUs in the gut content library similar to those from the libraries of
autumn-collected A. caliginosa and related to genera Flavobacterium and one OTU to Flexibacter.
Bacteria of CFB in the cast were detected in single OTU close to symbiont sf. Flavobacterium of
Tetraponera binghami (AF459795) (Suppl. Fig 38). Proteobacteria were not very numerous in the
soil library and represented by 2 OTUs from Alphaproteobacteria, 3 OTUs from
3. Results
38
________________________________________________________________________________
Betaproteobacteria, and single OTU from Gammaproteobacteria (Suppl. Fig. 35-37). This class of
bacteria was more regularly present in cast than in gut content: 3 and 7 OTUs belonged to
Alphaproteobacteria were found, correspondingly. Betaproteobacteria (3 OTUs) were detected
only in the cast library. Gammaproteobacteria were also seldom in both, gut content library (2
OTUs) and in the cast library (3 OTUs). One OTU from order of Desulfomonadales and 2 OTUs
from orders of Desulfomonadales and Myxococcales (Deltaproteobacteria) were recognized in the
gut content and cast libraries respectively (Suppl. Fig. 34). Firmicutes from genera Geobacillus and
Brevibacillus (3 OTUs, class Bacilli) and genus Desulfotomaculum (2 OTUs, class Clostridia)
occured in the soil library. 2 OTUs of genus Geobacillus, class Bacilli was found in the cast library.
No Firmicutes were detected in the gut content library. Actinobacteria were lacking in the soil but
present in the cast (3 OTUs) and frequent in the gut content libraries (7 OTUs) (Suppl. Fig. 39).
Horse manure compost and E. fetida
Proteobacteria were the most numerous group in the gut content and cast libraries.
Alphaproteobacteria were detected only in compost library (3 OTUs). Two OTUs represented
Betaproteobacteria in the compost library and 1 OTU in the cast library. Gammaproteobacteria
were the most numerous class with highest diversity in the compost library (8 OTUs from 5
families including Legionellaceae). Diversity was lower in the gut content (6 OTUs from families
Legionellaceae and Pseudomonadaceae), and lowest in the cast library (2 OTUs from
Aeromonadaceae and Enterobacteriaceae) (Suppl. Fig. 37). Single OTU related to the genus
Geobacter (Deltaproteobacteria) was detected in the compost library. (Suppl. Fig. 34). CFB of
classes Sphingobacteria and Flavobacteria (8 and 3 OTUs respectively) were enriched in the
compost library. Bacteria from the class Sphingobacteria were detected in both libraries (2 OTUs in
gut content and 1 OTU in the cast). Bacteria linked to the genus Flavobacteria appeared only in the
cast (2 OTUs) (Suppl. Fig. 38). Single OTU of Planctomycetes and Actinobacteria (from
unclassified cluster) were detected in the compost library (Suppl. Fig. 34, 39). Single OTU from
Actinobacteria recognized in the cast library was related to Acidomicrobium ferrooxidans (subclass
Acidimicrobidae). Firmicutes in the compost library were affiliated to genus Ruminococcus of class
Clostridia (4 OTUs). All Firmicutes detected in the gut content library were related to Mollicutes (4
OTUs) (data not shown), and the single OTU found in cast library was close to Bacillus sphaericus
(Suppl. Fig. 39).
Apart from bacteria, we amplified 18S rDNA from eucarya of family Chlorophyceae (1 OTU) in
the cast library (closest hit was Chlamydomonas pitschmannii (U70789); sequence identity <90%).
3. Results
39
________________________________________________________________________________
3.1.2 Bacteria of class Mollicutes in the earthworm tissues
Apart from other microbes, bacteria belonged to class Mollicutes (Firmicutes) were detected in the
clone libraries of cast and gut content of E. fetida and L. terrestris. The clones were abundant in the
libraries, affiliated together in the cluster, and had low sequence similarity (83%) to the closest hit
(Mycoplasma pulmonis AF125582) (Fig. 6); the clones from the same origin had varied sequence
identity each to other (92-99%). Mollicutes-like organisms (MLO) have been reported to be present
by a single clone in the intestine library of L. rubellus (Singleton et al., 2003) but its sequence
identity both to our clones was low (best hit ~80%) as well as to other described Mollicutes.
Significant evolutionary distance of novel discovered bacterial clones from the other bacteria of the
class Mollicutes, source of isolation (earthworms of family Lumbricidae), and well-known host
specificity of Mollicutes make the bacteria from novel discovered cluster to be thought a new
Lt-EX-G9
Lt-GC-G5
Lt-EX-E12
Ef-GC-D1
cluster ‘Lumbricoplasma’
Lt-EX-E10
Ef-GC-A3
Ef-GC-A6
Lt-GC-E6
Ef-GC-H1
Mycoplasma anseris AF125584
Mycoplasma spumans AF125587
Mycoplasma indiense AF125593
Mycoplasma agassizii AF060821
Mycoplasma pulmonis AF125582
Mycoplasma adleri U67943
Mycoplasma caviae AF221111
Mycoplasmatales
Mycoplasma felis U09787
Ureaplasma cati D78649
Ureaplasma felinum D78651
Ureaplasma canigenitalium D78648
Ureaplasma diversum D78650
Ureaplasma gallorale U62937
Ureaplasma urealyticum L08642
uncultured earthworm intestine bacterium AY154648
Mycoplasma mycoides subsp. Mycoydes U26050
Mycoplasma yeatsii U67946
Mesoplasma corruscae AY168929
Entomoplasma freundtii AF03695
Spiroplasma citri M23942
Entomoplasmatales
Spiroplasma culicicola AY189129
Spiroplasma helicoides AY189132
Anaeroplasma bactoclasticum M25049
Anaeroplasma varium M23934
Anaeroplasma abactoclasticum M25050
Anaeroplasmatales
Erysipelothrix inopinata AJ550617
Erysipelothrix rhusiopathiae M23728
0.1
taxonomy group, named cluster ‘Lumbricoplasma’ candidates.
Figure 6. Phylogenetic distribution of the clones (~ 1000 bp length ) from class Mollicutes in the
samples of gut content (GC) and casts (EX) of two earthworm species: L. terrestris (Lt); E. fetida
(Ef).
On the baseis of sequence analysis of ‘Lumbricoplasma’ cluster, the ‘Lumbricoplasma’-specific 5’labelled AlexaFluor546 nucleotide probe LUM1225 was synthesized by Invitrogen (Germany). The
3. Results
40
________________________________________________________________________________
localization of the bacteria from cluster ‘Lumbricoplasma’ was studied using FISH analysis. The
microscopic analysis has turned out the nodules localization of the rod-shaped bacteria mostly in the
ring and longitudinal muscles and coelom tissues but also in the gut tissues and outer epidermis
(Fig. 7-10).
A
B
C
D
Figure 7. Bacteria of cluster ‘Lumbricoplasma’ and their tissue specimen in the ring and
longitudinal muscles of the earthworm L. terrestris visualized with ‘Lumbricoplasma’-specific
nucleotide probe using fluorescent microscopy. A, B: magnification, ×200; bar, 120 µm. C, D:
magnification, ×630; bar, 20 µm.
3. Results
41
________________________________________________________________________________
A
B
C
D
E
F
Figure 8. Bacteria of cluster ‘Lumbricoplasma’ and their tissue specimen in the earthworm
A. caliginosa visualized with ‘Lumbricoplasma’-specific nucleotide analysis using fluorescent
microscopy. Ring muscles (A): magnification, ×200; bar, 150 µm. Longitudinal muscles (B):
magnification, ×200; bar, 150 µm. Different parts of gut wall: (C) magnification, ×400; bar, 40 µm;
(D) magnification, ×400; bar, 40 µm; in the same field (E) magnification, ×400; bar, 40 µm;
magnification, ×630; bar, 20 µm (F);
A
B
C
D
E
F
Figure 9. Bacteria of cluster ‘Lumbricoplasma’ and their tissue specimen in the earthworm E. fetida
visualized with ‘Lumbricoplasma’-specific nucleotide probe analysis using fluorescent microscopy.
Coelom (A, B): magnification, ×200; bar, 60 µm. Gut wall (C): magnification, ×400; bar, 30 µm;
3. Results
42
________________________________________________________________________________
Longitudinal muscles: magnification, ×400; bar, 30 µm (D); magnification, ×400; bar, 30 µm (D),
in the same field magnification, ×630; bar, 10 µm (E).
A
B
C
D
Figure 10. Bacteria of cluster ‘Lumbricoplasma’ visualized with ‘Lumbricoplasma’-specific
nucleotide probe in the longitudinal muscles of E. fetida using laser confocal microscopy. A-D:
magnification, ×1000; bar, 1 µm.
3. Results
43
________________________________________________________________________________
3.1.3 FISH and SSCP analysis of microbial communities in the substratum used for prion
proteolytic assay
For recPrP proteolysis assay the samples of soddy-podzolic soil and earthworm casts collected in
the spring 2005 were used. Initially they were examined with FISH and SSCP analyses to reveal the
microbial community composition in these particular samples.
Preliminary studies of microbial communities with FISH revealed gradually changes of microbiota
upon gut passage; this is why only initial substratum and cast (and not the gut content) were chosen
for the forthcoming analysis.
3.1.3.1 Characterization of the microbial population with FISH
Bacterial population changes upon passage through the gut of L. terrestris
Alpha-, Betaproteobacteria, and CFB bacteria were dominant in the bacterial soil population and
had comparable number each to other. The rest bacterial groups comprised in total 11%. Bacteria of
CFB group upon passage increased their relative numbers almost twice and reached 50% mark.
Proportion of Alpha-, Gammaproteobacteria, and LGC bacteria decreased two-fold (14, 2, 1%
respectively), whlie Betaproteobacteria and Actinobacteria kept the same or almost the same
numbers (Actinobacteria had 3 and 4% in soil and cast respectively) (Fig. 11).
LGC
100%
90%
80%
HGC
34
70%
53
60%
50%
40%
25
30%
20%
25
30
10%
14
0%
Soddy-Podzolic Soil
L. terrestris cast
Planctomycetes,
Verrucomicrobia,
Deltaproteobactria
CFB
Gamma
Beta
Alpha
Figure 11. Composition of different phylogenetic groups of microorganisms in the soddy-podzolic
soil and cast L. terrestris.
3. Results
44
________________________________________________________________________________
Numbers of Deltaproteobacteria, Verrucomicrobia and Planctomycetes were very low (less than
1%) and did not change notably upon passage through the earthworm gut (Fig. 11).
Ratio of EUB338/DAPI in the soddy-podzolic soil was similar (0,21) to that determined during the
previous studies, this value was higher in the cast samples of L. terrestris and A. caliginosa (0,27
and 0,26 respectively).
Bacterial population changes upon passage through the gut of A. caliginosa
Bacterial population alterations upon passage through the gut of A. caliginosa were similar to those
L. terrestris (Fig. 12). Bacteria of CFB group also became dominant in the cast, Alpha-,
Gammaproteobacteria, and Firmicutes (LGC) diminished their numbers. Actinobacteria (HGC)
and Planctomycetes retained same relative abundances and Betaproteobacteria slightly increased
their density (Fig. 12). Similarly to the cast of L. terrestris, Verrucomicrobia, Planctomycetes and
Deltaproteobacteria were not abundant in the cast of A. caliginosa.
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
LGC
HGC
34
46
Planctomycetes,
Verrucomicrobia,
Deltaproteobacteria
CFB
25
30
Gamma
30
20
Soddy-Podzolic A. caliginosa cast
Soil
Beta
Alpha
Figure 12. Composition of different phylogenetic groups of microorganisms in the soddy-podzolic
soil and cast A. caliginosa.
Bacteria of the phyla Verrucomicrobia, Planctomycetes and Deltaproteobacteria were the minor
members of bacterial communities in the soil and earthworm casts, since their proportion
hybridized with specific probes was about 1%.
3. Results
45
________________________________________________________________________________
3.1.2.2 SSCP analysis
SSCP analysis with the nonspecific primer set
SSCP profiles of the samples generated from RNA and DNA pool were similar for all samples (Fig.
13A). Many bands (3-9, 12, 13) were common for all samples. Phylogenetic analysis of the
sequences from separate bands revealed microorganisms to belong to the following taxonomic
groups: α-, β-, γ-, δ-Proteobacteria, CFB, Actinobacteria, Acidobacteria, and Gemmatimonadetes.
Some of the bands running at the same position on the gel consisted of DNA from different
resources. The bacteria of CFB phyla (particularly from genus Flavobacteria) were the most
numerous and diverse on the species- and strain-taxonomy level (Fig. 13B). Band S-RL-9a from the
casts of L. terrestris did not exhibit a strong affiliation to any bacterial taxonomic group and was
but related to some uncultured bacterium (AY274120). Among the bacterial amplicons we detected
12S rRNA genes from two different eucaryiotic mitochondrions (S-RL-1a, b) related to the plantpathogenic fungus Phytophthora infestans in the casts samples of L. terrestris (Fig. 13B).
A
RS
RL
RA
DS
DL DA
1
1
2
2
2
2
2/1
2/1
2/1
3
4
4/1
5
6
7
8
9/1
9
10
11
13
8/1
9/1
10/1
8/1
8/1
9/1
8/2
9/1
12
12
12
12
14
13
13
13
13
3. Results
46
________________________________________________________________________________
B
0.1
Methanobacterium aarhusense AY386124 Archaea
S-9a
uncultures bacterium AY274120
mitochondrion Saprolegnia ferax AY534144
mitochondrion Phytophthora infestans U17009
Eucaryota
S-RS/RL-1a
S-RS/RL-1b
S-8b
Flexibacter sancti AB078067
S-RL-4/1b
Adhaeribacter aquaticus AJ626894
S-DA-8/2a
Flexibacter flexilis AB078053
S-RL-4b
S-2
Flavobacterium limicola AB075232 CFB
Flavobacterium hibernum L39067
S-RL/DL/DA-2/1
S-3
S-9b
S-4a, 4/1, 5, 9/1, 10, DA-8/2b
Flavobacterium granuli AB180738
Bacteriovorax litoralis AF084859
S-8a
δ-proteobacteria
uncultures bacterium AY499971
S-R/D-12a
Coriobacterium glomerans X79048 Actinobacteria
Rhodococcus opacus X80631
Acidimicrobium ferrooxidans U75647
Gemmatimonas aurantiaca AB073527
S-11b
Gemmatimonadetes
S-DS-10/1a
uncultured Gemmatimonadetes bacterium AY921858
S-RL-4/1a
uncultured Acidobacteria bacterium AY921697
Geothrix fermentans U41563
Holophaga foetida X77215
Acidobacteria
Acidobacterium capsulatum D26171
Bacteria
uncultured Acidobacteria bacterium AY281358
S-DS-10/1b
uncultured Acidobacteria bacterium AY281355
S-11a
Azospirillum brasilense AY324110
Azospirillum oryzae AB185396
S-RS-14
Methylocystis sp. 5FB1 AJ86842
S-13a
Terasakiella pusilla AB000482
α-proteobacteria
S-D-8/1b
Mesorhizobium loti X67229
S-R/D-12b
Paracoccus denitrificans Y16929
S-13b
Sulfitobacter pontiacus AY1598
S-RS-15
S-D-8/1a
Leptothrix discophora L33974
β-proteobacteria
Nitrosovibrio tenuis AY123803
Limnobacter thiooxidans AJ289885
Cellvibrio fulvus AF448514
S-6
Pseudomonas aeruginosa Z76672
Pseudomonas tolaasii AF255336
γ-proteobacteria
S-R-7
S-D-7
Pseudomonas putida Z76667
Pseudomonas entomophila AY907566
Figure 13. A: SSCP analysis of microbial community with universal SSCP primers. The PCR
products were generated from the total RNA (R) and DNA (D). Lanes: soil (S); earthworm
L. terrestris (L); A. caliginosa (A); B: Phylogenetic analysis of the bands performed with the
unspecific SSCP analysis. Several ribotypes detected in one band represented on phylogenetic tree
with number and small letters (a and b). Sequences of the bands running at the same level with
identity >98,5% were recognized as the single OTU and are shown on the phylogenetic tree as a
single ID.
3. Results
47
________________________________________________________________________________
Alphaproteobacteria-specific assay
SSCP analysis has demonstrated a high number of the bands in the samples and the most numerous
were the soil patterns generated from both DNA and RNA pools (Fig. 14A). The phylogenetic
analysis of the bands generated from the RNA revealed the bacteria belonged solely to the
unclassified Sphingomonadaceae, which were very diverse on the species-, and subspeciestaxonomy level. Although some bands were differently running on the gel, they exhibited more
similarity between each other, than those running at the same positions even in the neighboring
lanes (Fig. 14B). Bands amplified from the DNA belonged to the microorganisms from orders
(unclassified
Sphingomonadaceae,
Sphingomonadaceae,
Caulobacteraceae,
and
Methylobacteraceae); the sequence A-DA-2 detected in the A. caliginosa cast sample was
uncertainly affiliated with the orders Bradyrhizobiaceae or Rhizobiaceae. Among the bands
generated from DNA pool a single band A-DL-6 extracted from L. terrestris casts war derived from
someorganism from the order Myxococcales (class Deltaproteobacteria).
A
RS
1
RL
1
RA
1
2
2
2
3
3
3
4
4
4
DS
1
2
3
4
5
6
7
8
9
10
1
11
2
3
3
13
4
5
6
7
8
9
5
5
5
6
6
6
7
7
7
17
18
19
20
21
2
23
2
24
25
26
27
28
29
8
8
9
9
9
10
10
10
11
12
11
12
11
12
13
14
DA
1
2
12
14
15
16
8
DL
10
11
12
13
14
4
5
6
7
8
9
15
16
10
11
12
3. Results
48
________________________________________________________________________________
B
Alphaproteobacteria
A-RS-9
A-RL/RA-9
A-RS-4
A-RA-4
A-RL-4
A-RS/RL/RA-2
A-RS/RA-11
A-RL-11
A-RS/RL/RA-8
A-RS-14
A-RA/RL-7
A-RS-7
unclassified
A-RS/RL/RA-6
A-RA-5
Sphingomonadaceae
A-RS/RL-5
A-RS/RA-10
A-DA-5
A-RS/RL/RA-3
A-RS/RL/RA-12
A-RS/RL/RA-1
A-RL-10
A-DA-6
A-RS-13
A-DA-7
Asticcacaulis excentricus AJ007800
Caulobacter leidyi AJ227812
Caulobacter endosymbiont of Tetranychus urticae AY753176
Asticcacaulis biprosthecium AJ007799
Caulobacter subvibrioides CB81 M83797
Sphingomonas echinoides AJ0124461
Sphingomonas suberifaciens D13737
A-DL-15
Sphingomonadaceae
A-DL-16
uncultured alpha proteobacteri
Sphingomonas wittichi AB021492
Caulobacter vibrioides AJ22775
A-DA-9
A-DA-8
Caulobacter henricii AJ007805
Caulobacteraceae
Caulobacter fusiformis AJ22775
Brevundimonas mediterranea AJ244706
Brevundimonas nasdae AB071954
Brevundimonas vesicularis DQ111026
A-DA-3
A-DA-11
A-DA-12
Methylobacteraceae
Methylobacterium fujisawaense AB175634
Methylobacterium organophilum D32226
Rhizobium tropici AY166841
Rhizobium rhizogenes AY626390
A-DA-2
Rhodoblastus sphagnicola AM040096
Plesiocystis pacifica AB083432
Haliangium ochraceum AB016470
Deltaproteobacteria
Chondromyces apiculatus AJ233938
order Myxococcales
A-DL-6
Myxococcus fulvus AJ233917
0.1
Figure 14. A: SSCP analysis of Alphaproteobacteria-specific PCR products generated from total
RNA (using reverse transcription) (R) and total DNA (D). Lanes: soil (S); earthworm L. terrestris
(L); A. caliginosa (A); B: Phylogenetic analysis of the sequences from the Alphaproteobacteriaspecific SSCP analysis. Several ribotypes detected in one band represented on phylogenetic tree
with the number and small letters (a and b). Sequences with identity >98,5% were recognized as a
single OTU and are shown as one ID. .
3. Results
49
________________________________________________________________________________
Betaproteobacteria-specific assay
This class of Proteobacteria was represented by a single band in the soil as in the earthworm cast
samples generated from RNA (Fig. 15A). The sequences of those bands were similar each to other
and could be attributed to the same OTU closely related to Alcaligenes faecalis AF155157 (99% of
sequence identity). Samples generated from DNA showed numerous bands (13 in the soil and 9 in
each cast sample) and similar sequence composition. The sequences were related to bacteria of
families
Comamonadaceae,
Nitrosomonadaceae,
Oxalobacteraceae,
Burkholderiaceae,
Alcaligenaceae, Rhodocyclaceae, and unclassified Burkholderiales. The sequences in the samples
generated from the DNA and related to A. fecalis had >99% of sequence similarity to those
generated from RNA (data not shown). Sequences from the top part of the gel (numbers 1-4, 4/1,
5/1) formed a cluster not strongly affiliated to any known bacterial name but apparently belonged to
the class Betaproteobacteria (Fig. 15B).
3. Results
50
________________________________________________________________________________
A
RS RL RA DS DL DA
1
2
3/1
3/2
3
4
5
10
12
5/1
5
6
7
8
9
10
11
12/1
13
12/1
4/1
5/1
B
B-DS-7b
Variovorax paradoxus AJ420329
Variovorax sp. K6 AF532867
B-DS-6a
endosymbiont of Acanthamoeba sp. AY549551 Commamonadaceae
B-DS-8b
Hylemonella gracilis AJ565423
B-DS-1b
B-DS-5b
Acidovorax avenae subsp. citrulli AF137506
Acidovorax delafieldii AF07876
B-DS-5a
B-DS-6b
uncultured beta proteobacterium AY212711
Aquabacterium citratiphilum AF035050
uncultured Aquabacterium sp. AF523022
B-DS-9b
unclassified
Ideonella dechloratans X72724
Schlegelella thermodepolymerans AY538709 Burkholderiales
B-DS-8a
uncultured beta proteobacterium AY921703
B-DL-10a
B-DL-10b
B-DS-10a
BDS-10b
B-RS-1
B-RL-1
B-RA-1
Alcaligenaceae
Alcaligenes faecalis AF155147
Achromobacter xylosoxidans AF411021
Duganella violaceinigra AY376163
B-DS-11
Duganella zoogloeoides D14256 Oxalobacteraceae
B-DS-13
B-DS-9a
Oxalobacter sp. p8E AJ496038
B-DS-7a
uncultured beta proteobacterium AJ575695 Nitrosomonadaceae
Nitrosospira briensis L35505
Nitrosolobus multiformis L35509
Burkholderia cepacia X87275
Derxia gummosa AB089481
Azonexus fungiphilus AF011350
B-DS-4
B-DA-14
B-DS-4/1
B-D-3, 3/1, 3/2
unclassified Betaproteobacteria
BDS-2
B-DA-15b
B-DS-1a
0.1
Figure 15. A: SSCP analysis of Betaproteobacteria -specific PCR products generated from total
RNA (using reverse transcription) (R) and total DNA (D). Bands represented in all profiles marked
with arrows. Lanes: soil (S); earthworm L. terrestris (L); A. caliginosa (A); B: Phylogenetic
analysis of the phylotypes from the Betaproteobacteria-specific SSCP analysis. Several ribotypes
detected in one band represented on phylogenetic tree with the number and small letters (a and b).
Sequences with identity >98,5% were recognized as a single OTU and are shown as one ID. .
3. Results
51
________________________________________________________________________________
Gammaproteobacteria-specific SSCP assay
PCR product from total DNA with primers specific for Gammaproteobacteria was obtained only
from soil sample and the fingerprint looked similar to that from generated from RNA, but the latter
yielded a higher number of bands. Gammaproteobacteria appeared more diverse in the soil in
comparison with the samples from the cast of both earthworm species, which were very similar one
to another (Fig. 16A).
Sequences of Gammaproteobacteria were distributed between 6 clusters related to families
Pseudomonadaceae (genera Pseudomonas and Cellvibrio), Legionellaceae, Chromatiaceae (linked
to genus Nitrosococcus), Xanthomonadaceae, the unclassified bacteria, which were not strongly
affiliated
to
any
described
bacterial
genus
but
clearly
clustered
within
the
class
Gammaproteobacteria, and unclassified bacteria that are not strongly affiliated neither to classes
Gamma-, nor to Betaproteobacteria (detected in the A. caliginosa cast samples band G-RA-34).
Three groups of bacteria (genus Cellvibrio, family Chromatiaceae, and unclassified
Gammaproteobacteria) were detected only in the soil sample but never in the casts. Bacteria linked
to genus Legionella were more diverse in the soil samples; the only ribotype detected in the cast of
A. caliginosa appeared to have a low sequence similarity (<85%) to both described species and
sequences found in the soil sample. Composition of Pseudomonas-related bacteria was different in
the soil samples amplified directly from total DNA (bands 5, 6/1, 6/2 – three OTUs) in comparison
to those generated from total RNA by using reverse transcription (bands G-RS-18, 21 (the same
OTU) and G-RS-20/1) (Fig. 16B). Most of the recognized ribotypes in the cast samples were linked
to P. putida and P. entomophila. Among them two bands (G-RL-30 and G-RA-37) in the casts of
each earthworm species belonged to the same OTU, while the rest of the bands (4 in each cast
sample) were associated with another single OTU (Fig. 16B). Passage through the earthworm gut
caused decreasing diversity of Gammaproteobacteria and increasing detectable number of speciesand subspecies-level variants of bacteria closely related to P. putida and P. entomophila (Fig. 16B).
The band G-RA-33 was derived from family Xanthomonadaceae, similarly to G-RS-12 related to
genus Stenotrophomonas generated from RNA of the soil sample.
3. Results
52
________________________________________________________________________________
A
A
DS RS
RS
DS
22
33
44
55
6/1
6/1
66
6/2
6/2
77
88
99
RL RA
RA
RL
10
10
11
11
12
12
13
13
14
14
B
B
B
33
33
34
34
15
15
15/1
15/1
16
16
17
17
18
18
19
19
20
20
20/1
20/1
21
21
22
22
23
23
24
24
25
25
26
26
27
27
28
28
35
35
29
29
30
30
36
36
37
37
31
31
32
32
32/1
32/1
39
39
40
40
41
41
0.1
0.1
G-RA-38
G-RA-38
G-RA-38
G-RL-29
G-RL-29
G-RL-29
G-RL-32-1
G-RL-32-1
G-RL-32-1
G-RS-21
G-RS-21
G-RS-21
G-RS-18
G-RS-18
G-RS-18
G-RL-32
G-RL-32
G-RL-32
G-RL-31
G-RL-31
G-RL-31
G-RL-28b, -RA35a
-RA35a
G-RL-28b,
G-RL-28b,
-RA35a
G-RL-28a,
-RA-35b
G-RL-28a,
-RA-35b
G-RL-28a,
-RA-35b
G-RL-30
G-RL-30
G-RL-30
Pseudomonas
putida Z76667
Z76667
Pseudomonas
putida
Pseudomonas
putida
Z76667
genus
Pseudomonas
entomophila
AY9075
genus
Pseudomonas
entomophila
AY9075
genus
Pseudomonas
entomophila
AY9075
Pseudomonas
syringae
AY574914
Pseudomonas syringae
syringae AY574914
AY574914
Pseuomonas
Pseudomonas
Pseuomonas
Pseudomonas sp.
sp. U81870
U81870
Pseuomonas
Pseudomonas
Pseudomonas
G-RS-13b sp. U81870
Pseudomonadaceae
G-RS-13b
Pseudomonadaceae
G-RS-13b
Pseudomonadaceae
G-RS-17a
G-RS-17a
G-RS-17a
Pseudomonas
alcaligenes Z76653
Z76653
Pseudomonas
alcaligenes
Pseudomonas
alcaligenes
Z76653
G-DS-5
G-DS-5
G-DS-5
G-DS-6-2
G-DS-6-2
G-DS-6-2
G-DS-6
G-DS-6
G-DS-6
G-DS-6-1
G-DS-6-1
G-DS-6-1
Cellvibrio
vulgaris AF448513
AF448513
Cellvibrio vulgaris
vulgaris
Cellvibrio
AF448513
Cellvibrio
fibrivorans
AJ289164
Cellvibrio
fibrivorans
AJ289164
Cellvibrio
fibrivorans
AJ289164
G-DS-7
G-DS-7
G-DS-7
G-RS-19-1
G-RS-19-1
genus
G-RS-19-1
genus
G-RS-19a
genus
G-RS-19a
G-RS-19a
Cellvibrio
gandavensisAJ289162
AJ289162 Cellvibrio
Cellvibrio
Cellvibrio
gandavensis
AJ289162
Cellvibrio
gandavensis
Cellvibrio
Cellvibriojaponicus
japonicusAF452103
AF452103
Cellvibrio
japonicus
AF452103
Cellvibrio
G-RS-22
G-RS-22
G-RS-22
G-RS-20
G-RS-20
G-RS-20
G-RS-15b
G-RS-15b
G-RS-15b
G-RS-26
G-RS-26
G-RS-26
G-RS-27
G-RS-27
G-RS-27
G-RS-25
G-RS-25
G-RS-25
G-DS-8
G-DS-8
G-DS-8
G-DS-9
G-DS-9
unclassified
G-DS-9
unclassified
unclassified
G-RS-23
G-RS-23
G-RS-23
G-RS-24
G-RS-24
Gammaproteobacteria
G-RS-24
Gammaproteobacteria
Gammaproteobacteria
unculturedgamma
gammaproteobacterium
proteobacteriumAY212658
AY212658
uncultured
gamma
proteobacterium
AY212658
uncultured
uncultured
gammaproteobacterium
proteobacteriumAJ318139
AJ318139
uncultured
gamma
proteobacterium
AJ318139
uncultured
gamma
unculturedgamma
gammaproteobacterium
proteobacteriumAY212747
AY212747
uncultured
gamma
proteobacterium
AY212747
uncultured
G-RS-17b
G-RS-17b
G-RS-17b
G-RS-18/2a
G-RS-18/2a
G-RS-18/2a
G-RS-18/2b
G-RS-18/2b
G-RS-18/2b
G-RS-19b
G-RS-19b
G-RS-19b
Legionellaceae
Legionellapneumophila
pneumophilasubsp.
subsp. pascullei
pascullei AF122885
AF122885 Legionellaceae
Legionellaceae
Legionella
pneumophila
subsp.
Legionella
Legionelladrancourtii
drancourtiiX97366
X97366 pascullei AF122885
Legionella
drancourtii
X97366
Legionella
Legionella
busanensis
AF424887
Legionella busanensis
busanensis AF424887
AF424887
Legionella
Legionellalongbeachae
longbeachaeM36029
M36029
Legionella
longbeachae
M36029
Legionella
G-RA-33b
G-RA-33b
G-RA-33b
G-RS-13a
G-RS-13a
G-RS-13a
G-RS-15a
G-RS-15a
G-RS-15a
uncultured
gammaproteobacterium
proteobacteriumAY921670
AY921670
uncultured
gamma
proteobacterium
AY921670
uncultured
gamma
G-RS-11
G-RS-11
G-RS-11
Chromatiaceae
Chromatiaceae
G-RS-10
Chromatiaceae
G-RS-10
G-RS-10
G-DS-7/1b
G-DS-7/1b
G-DS-7/1b
Nitrosococcusoceanus
oceanusM96398
M96398
Nitrosococcus
oceanus
M96398
Nitrosococcus
G-DS-7/1a
G-DS-7/1a
G-DS-7/1a
Azonexusfungiphilus
fungiphilusAF011350
AF011350
Azonexus
fungiphilus
AF011350
Azonexus
Massiliatimonae
timonaeAY157761
AY157761
Massilia
timonae
AY157761
Massilia
Burkholderia
cepacia
X87275
Burkholderia
cepacia
X87275
Burkholderia cepacia X87275
Achromobacterxylosoxidans
xylosoxidansAF411021
AF411021
Achromobacter
xylosoxidans
AF411021
Achromobacter
Bordetellapertussis
pertussisBX640420
BX640420
Bordetella
pertussis
BX640420
Bordetella
Betaproteobacteria
Betaproteobacteria
Betaproteobacteria
Alcaligenes
faecalis
AF155147
Alcaligenes faecalis
faecalis AF155147
AF155147
Alcaligenes
Comamonastestosteroni
testosteroniAB064318
AB064318
Comamonas
testosteroni
AB064318
Comamonas
Delftiaacidovorans
acidovoransAF538930
AF538930
Delftia
acidovorans
AF538930
Delftia
Azoarcuscommunis
communisAF011343
AF011343
Azoarcus
communis
AF011343
Azoarcus
Azovibriorestrictus
restrictusAF011348
AF011348
Azovibrio
restrictus
AF011348
Azovibrio
G-RA-34a
G-RA-34a
G-RA-34a
G-RA-34b
G-RA-34b
G-RA-34b
G-DS-3
G-DS-3
G-DS-3
uncultured
gammaproteobacterium
proteobacteriumAY922016
AY922016
uncultured gamma
gamma
proteobacterium
AY922016
uncultured
Hydrocarboniphagaeffusa
effusaAY363244
AY363244
Hydrocarboniphaga
effusa
AY363244
Hydrocarboniphaga
Nevskiaramosa
ramosaAJ001343
AJ001343
Nevskia
ramosa
AJ001343
Nevskia
G-RS-12
G-RS-12
G-RS-12
Xanthomonadaceae
Xanthomonadaceae
Xanthomonadaceae
G-RA-33a
G-RA-33a
G-RA-33a
Stenotrophomonasmaltophilia
maltophiliaX95924
X95924
Stenotrophomonas
maltophilia
X95924
Stenotrophomonas
Stenotrophomonasrhizophila
rhizophilaAJ293463
AJ293463
Stenotrophomonas
rhizophila
AJ293463
Stenotrophomonas
Stenotrophomonasacidaminiphila
acidaminiphilaAF273080
AF273080
Stenotrophomonas
acidaminiphila
AF273080
Stenotrophomonas
Stenotrophomonas
nitritireducens
AJ012229
Stenotrophomonas
nitritireducens
AJ012229
Stenotrophomonas nitritireducens AJ012229
Figure 16. A: SSCP analysis of Gammaproteobacteria-specific PCR products generated from total
RNA (using reverse transcription) (R) and total DNA (D). Lanes: soil (S); earthworm L. terrestris
(L); A. caliginosa (A); B: Phylogenetic analysis of the sequences from the Alphaproteobacteriaspecific SSCP analysis. Several ribotypes detected in one band represented on phylogenetic tree
with the number and small letters (a and b). Sequences with identity >98,5% were recognized as a
single OTU and are shown as one ID.
3. Results
53
________________________________________________________________________________
CFB-specific SSCP assay
Amplifications with primers specific for Cytophaga-Flavobacteria group of Bacteroidetes were
successful from both total dsDNA and ssDNA (generated from total RNA). SSCP profiles did not
appear to be significantly different among the soil and earthworm cast patterns generated from
DNA and RNA pools (Fig. 17A). Phylogenetic analysis showed, that sequences related to bacteria
of class Flavobacteria run on the above half of the gel, while Sphingobacteria run in the bottomhalf. Bands were more numerous in the soil in comparison with the cast samples amplified after RT.
Bands 1, 1/1, 2, 2/1, 3-5, 8-16 were found to be common for all investigated samples. Among them,
the bands 1-5 belonged to the class Flavobacteria, and bands 8-18 linked to the class
Sphingobacteria. Bands 6 and 7 appeared in the earthworm casts samples amplified after RT and
presented also in all samples amplified from DNA, and related to genus Flavobacterium (Fig. 17B).
A
RS
RL RA DS
DL DA
1
1/1
2
3
4
1/1
2/2
2/1
2/1
3/1
3/1
5/1
5
6
7
8
9
10
11
12 12/1
13
1/1
6
3/1
5/1
5/1
6
6
7
6
10/1 10/1
13/1
14
15
16
17
17
18
18
14/1
17
B
C-RS-1
saliperosum DQ021903
DQ021903
Flavobacterium saliperosum
C-(RS/DS/DL)-1/1
C-RS-2/1
johnsoniae AB078043
AB078043
Flavobacterium johnsoniae
limicola AB075232
AB075232
Flavobacterium limicola
psychrophilum AB078060
AB078060
Flavobacterium psychrophilum
frigidimaris AB183888
AB183888
Flavobacterium frigidimaris
C-RS-3
Flavobacterium hibernum
hibernum L39067
L39067
class Flavobacteria
Flavobacterium columnare M58781
C-RS-5
C-5/1
C-(RL/DL)-7
C-RS-6
AB18073
Flavobacterium granuli AB18073
C-RS-4
C-RL-2/2
C-RS-2
AB07807
Flexibacter tractuosus AB07807
AB078053
Flexibacter flexilis AB078053
C-RS-9
C-RL-12/1
Cyclobacterium marinum AY533665
C-(DL/DA)-10/1
AB078046
Flexibacter canadensis AB078046
spiritivorum M58778
M58778
Sphingobacterium spiritivorum
Sphingobacterium sp.
sp. AY599659
AY599659
uncultured Sphingobacterium
C-(RL/DS/DA)-3/1
C-RS-8a
Flavobacterium of
of Tetraponera
Tetraponera binghami
binghami AF459795
AF459795
symbiont cf. Flavobacterium
C-RS-8b
bacterium DQ201672
DQ201672
uncultured Bacteroidetes bacterium
C-RS-10
C-RS-8c
Flexibacter sancti AB078067
AB078049
Flexibacter filiformis AB078049
C-RS-12
C-RS-11
class Sphingobacteria
C-RS-13
C-(RS/RL/DL)-17
C-RS-14
C-RS16
C-RS-15
C-RS/RL-18
0.1
Figure 17. A: SSCP analysis of CFB -specific PCR products generated from total RNA (using
reverse transcription) (R) and total DNA (D). Bands represented in all profiles marked with arrows.
Lanes: soil (S); earthworm L. terrestris (L); A. caliginosa (A); B: Phylogenetic analysis of the
sequences from the CFB-specific SSCP analysis. Several ribotypes detected in one band
represented on phylogenetic tree with the number and small letters (a and b). Sequences with
identity >98,5% were recognized as a single OTU and are shown as one ID.
3. Results
54
________________________________________________________________________________
Bacilli (Firmicutes) – specific SSCP assay
PCR product with Bacilli-specific primers was obtained from all RNA samples and only from one
soil DNA sample. SSCP analysis showed similarity in band distribution between soil samples
generated from RNA and DNA (Fig. 18A). Sequence analysis revealed non-specifically amplified
Clostridia (Firmicutes) and Myxococcales (Deltaproteobacteria) (Fig. 18B). Uncultured Firmicutes
detected with this approach strongly related to genera Bacillus, Paenibacillus, Sporosarcina (class
Bacilli) and genera Thermoactinomyces and Desulfosporosinus (class Clostridia) (Fig. 18B).
A
RS
DS
1
2
RL
RA
B
L-RA-3/1a
L-DS-11
L-DS/RS-10b
Sporosarcina aquamarinus AF202056
Sporosarcina ureae AF202057
Sporosarcina globispora X68415
Sporosarcina psychrophila D16277
L-RA-3/1b
Sporosarcina macmurdoensis AJ514408
L-DS/RS-2b
Bacillus sp. IDA3504 AJ544784
Bacillus megaterium AY030338
Bacillus licheniformis AF387514
class
L-DS/RS-3a
Bacilli
Bacillus subtilis AY030331
Bacillus clausii X76440
L-RL-8/1
Firmicutes
L-DS-9b
Paenibacillus kobensis D78471
Paenibacillus curdlanolyticus D78466
L-DS/RS-5b
L-DS-9a
Paenibacillus mendelii AF537343
Paenibacillus polymyxa AJ320493
Paenibacillus amylolyticus D85396
L-DS-8a
Desulfosporosinus auripigmenti AJ493051
Desulfosporosinus orientis Y11570
class
Desulfosporosinus meridiei AF076247
Clostridia
L-DS/RS-3b
L-DS/RS-10a
Thermoactinomyces vulgaris AF138732
L-DS-5a
L-DS/RS-6
uncultured delta proteobacterium AY922176
L-DS/RS-2a
L-DS-7
Chondromyces lanuginosus AJ233939
Deltaproteobacteria
Chondromyces apiculatus AJ233938
order Myxococcales.
Chondromyces pediculatus AJ233940
Chondromyces crocatus M94275
L-DS/RS-1
Haliangium ochraceum AB016470
Polyangium vitellinum AJ233944
Nannocystis aggregans AJ233945
1
2
3/1
3
3
5
5
6
6
7
8
9
10
11
8/1
10
0.1
Figure 18. A: SSCP analysis of Bacilli-specific PCR products generated from total RNA (using
reverse transcription) (R) and total DNA (D). Lanes: soil (S); earthworm L. terrestris (L); A.
caliginosa (A); B: Phylogenetic analysis of the sequences from the Bacilli-specific SSCP analysis.
Several ribotypes detected in one band represented on phylogenetic tree with the number and small
letters (a and b). Sequences with identity >98,5% were recognized as a single OTU and are shown
as one ID.
3. Results
55
________________________________________________________________________________
Amplification
of
bacteria
from
phyla
Verrucomicrobia,
Planctomycetes
and
class
Deltaproteobacteria considered as minor members of bacterial communities soil and earthworm
casts (according to th FISH analysis) was successfully performed only from DNA pools of
corresponding samples. The diversity of those bacteria was higher in the soil, than in the cast
samples from both of the earthworm species. The phylotypes had low sequence identity (<95%) to
described bacterial species.
Deltaproteobacteria-specific SSCP assay
This class of Proteobacteria was amplified from the soil and A. caliginosa cast samples (Fig. 19A).
Phylogenetic analysis demonstrated wide-ranging taxonomic diversity in the soil sample (the lowest
sequence similarity between the ribotypes was 92%, the highest one >97%), while in the cast
sample diversity was on the strain-taxonomy level (sequence identity between the ribotypes was
99%). Most of the sequences in soil and cast samples clustered together and linked to family
Polyangiaceae. One pattern from the soil sample (D-DS-5b) was affiliate to the order Myxococcales
with uncertain placement within this taxonomic group (Fig. 19B).
B
A
D-DS-3
D-DS-4
D-DS-2
D-DS-6
D-DS-5a
D-DA2
D-DS-1
D-DA-1
DS DA
1
2
3
4
1
5a
5b
2
uncultured δ-proteobacterium AY922176
Chondromyces lanuginosus AJ233939
Chondromyces apiculatus AJ233938
Polyangium thaxteri AJ233943
D-S-5b
Cystobacter minus AJ233903
Cystobacter fuscus AJ233897
Anaeromyxobacter dehalogenans AF382398
Nannocystis exedens AJ233946
Plesiocystis pacifica AB083432
6
0.1
Figure 19. A: SSCP analysis of Deltaproteobacteria-specific PCR products generated from total
RNA (using reverse transcription) (R) and total DNA (D). Lanes: soil (S); earthworm L. terrestris
(L); A. caliginosa (A); B: Phylogenetic analysis of the sequences from the Deltaproteobacteriaspecific SSCP analysis. Several ribotypes detected in one band represented on phylogenetic tree
with the number and small letters (a and b). Sequences with identity >98,5% were recognized as a
single OTU and are shown as one ID.
3. Results
56
________________________________________________________________________________
Verrucomicrobia-specific SSCP assay
The PCR products with Verrucomicrobia-specific primers were obtained from soil and earthworm
cast but the size of amplified fragments was shorter (~300 bp), than expected. The patterns
appeared on the gel were pretty similar each to other (Fig. 10A). Phylogenetic analysis of the
sequences
revealed
unspecifically
amplified
16S
rRNA
from
bacteria
form
family
Streptomycetaceae (Actinobacteria) (Fig. 20B) and wide-ranging diversity of Verrucomicrobia
ribotypes, which were mainly linked to uncultured bacteria but some sequences clustered with O.
terrae. The pattern V-DS-3a and V-DS-2a were similar (sequence identity 95%) to the clones
detected in the L. terrestris libraries, and linked to A. mucinifila.
B
A
1
1
1
2
3
4
1/1
2
3
4
1/1
2
3
4
5
6
5
6
5
6
7
8
7
8
7
8
9
uncultured Verrucomicrobia bacterium EV101 AJ132479
uncultured Spartobacteria bacterium DQ088012
V-DS-5b
Chthoniobacter flavus AY388649
V-3b
uncultured Verrucomicrobia bacterium AJ132498
V-1b
uncultured bacterium AY212695
Verrucomicrobium spinosum X90515
candidatus Xiphinematobacter bacterium AF217462
V-2a
uncultured bacterium AY212657
V-DL/DA-1/1
Akkermansia muciniphila AY271254
Verrucomicrobia
V-7b
V-6b
V-DS/DA-9b
V-3a
uncultured Verrucomicrobia bacterium AY622244
V-1a
V-4
Opitutus terrae AJ229246
V-5a
V-6a
V-DS/DA-9a
V-2b
uncultured bacterium U81738
Streptomyces virginiae D85121
Streptomyces lateritius AF454764
V-7a
Streptomyces albus AJ621602
Actinobacteria
Streptomyces canescens Z76684
Streptomyces speibonae AF452714
V-8
Parastreptomyces abscessus DQ000673
9
0.1
Figure 20. A: SSCP analysis of Verrucomicrobia-specific PCR products generated from total RNA
(using reverse transcription) (R) and total DNA (D). Lanes: soil (S); earthworm L. terrestris (L); A.
caliginosa (A); B: Phylogenetic analysis of the sequences from the Verrucomicrobia-specific SSCP
analysis. Several ribotypes detected in one band represented on phylogenetic tree with the number
and small letters (a and b). Sequences with identity >98,5% were recognized as a single OTU and
are shown as one ID.
3. Results
57
________________________________________________________________________________
Planctomyces-specific SSCP assay
SSCP profiles of the patterns from the phylum Planctomycetes were comparable each to other in all
estimated samples (Fig. 21A) and phylogenetic analysis confirmed specificity of the primer set and
identity of the neighbor bands running in the different lines each to other. The ribotypes linked to
genera Isophaera, Gemmata, Pirellula and Planctomyces without clustering (Fig. 21B).
A
DS DL DA
B
P-DS-6a
P-DS-8a, b
Planctomyces sp. X81954
Planctomyces brasiliensis X81949
P-DS-1
P-DS-9b
uncultured planctomycete AB116422
Planctomyces maris X62910
P-DS-3a
uncultured planctomycete AB116406
P-DS-2b
uncultured sludge bacterium A40 AF234736
Pirellula staleyi X81946
Pirellula sp. X81947
Isosphaera sp. X81958
Isosphaera-like str. CJuql1 AF239699
Isosphaera pallida AJ231195
P-DS-7b, c, d
P-DS-2a
Isosphaera sp. X81959
P-DS-10
P-DS-6b
P-DS-4
P-DS-7a
P-DS-3b
Gemmata obscuriglobus AJ231191
P-DS-9a
P-DS-5a,b
1
2a
2b
3
4a
4b
5a
5b
6a
6b
7a
7b
7c
7d
8a
8b
9
10
0.1
Figure 21. A: SSCP analysis of Planctomycetes-specific PCR products generated from total RNA
(using reverse transcription) (R) and total DNA (D). Bands represented in all profiles marked with
arrows. Lanes: soil (S); earthworm L. terrestris (L); A. caliginosa (A); B: Phylogenetic analysis of
the sequences from the Planctomycetes-specific SSCP analysis. Several ribotypes detected in one
band represented on phylogenetic tree with the number and small letters (a and b). Sequences with
identity >98,5% were recognized as a single OTU and are shown as one ID.
3. Results
58
________________________________________________________________________________
3.2 Recombinant prion proteolysis assays
3.2.1 Proteolytic activity of pure isolates
Bacterial isolates (totally, 800) from the soil, compost and earthworm sources had generally a high
sequence identity (>98%) to the taxonomically recognized organisms and only a few strains had
lower sequence identity. Identification of isolates revealed a high diversity of the bacteria from
clases Gamaproteobacteria and Actinobacteria, while the isolaes from CFB group were scarce,
most likely due to the prevalently aerobic isolation approach.
Sole recPrP-digesting species were detected in the classes Alphaproteobacteria (Bosea
minatitlanensis 478-1, isolated from the cast of A. caliginosa) and Betaproteobacteria (Massilia
timonae 434-2) both of them were isolated from the cast of A. caliginosa (Fig. 22, 23).
Paracoccus kocurii D32241
Paracoccus alcaliphilus AY014177
Paracoccus sp. 365-2 (Lt/Ac-EX)
Paracoccus denitrificans Y16929
Brevundimonas sp. 337-2 (SP)
Brevundimonas intermedia AJ227786
Brevundimonas vesicularis AY169433
Brevundimonas diminuta 384-1 (SP, Ef-EX)
Methylobacterium populi 5 (Ef-GC)
Bosea minatitlanensis 478-1 (Ac-EX)
Sphingopyxis witflariensis 397 (SP)
Rhizobium sp. 539-2 (Ef-GC)
Rhizobium galegae AF025853
Rhizobium huautlense AF025852
Ochrobactrum gallinifaecis AJ519939
Ochrobactrum grignonense 629 (Ac-GC)
Ochrobactrum grignonense AJ242581
Ochrobactrum sp. 341-2 (SP)
Agrobacterium tumefaciens 19 (SP)
Defluvibacter lusatiae 598 (Ac-GC)
Ensifer adhaerens AJ420774
Rhizobiaceae bacterium 577-2 (Ac-GC)
Sinorhizobium morelense AJ420776
Aminobacter sp. 411-2 (SP)
Aminobacter aminovorans AJ011759
Aminobacter niigataensis AJ011761
0.1
Figure 22. Bacterial isolates (marked in bolt) of the class Alphaproteobacteria. Bacterium in the
gray box digested recPrP. The tree is constructed with sequences of ~500 bp length; the sequences
with identity >98,5% to each other and to corresponded described species considered as the same
OTU and presented in single example without correspondent bacteria. Sources of isolation:
L. terrestris (Lt); A. caliginosa (Ac); E. fetida (Ef); soddy-podzolic soil (SP); gut content (GC);
casts (EX).
3. Results
59
________________________________________________________________________________
Variovorax paradoxus AJ420329
Comamonas testosteroni 660kpb (Ac-GC; Ef-GC/EX)
Delftia acidovorans 623-1 (Lt/Ac/-GC/EX)
Massilia dura AY965998
Massilia timonae 434-2 (Ac-EX)
Massilia sp. 557-1 (Ac-GC)
Alcaligenes faecalis E690 (SP, Ef-EX)
Alcaligenes defragrans AB195161
Tetrathiobacter kashmirensis 1 (SP)
Alcaligenes sp. 17 (Ac-GC)
Bordetella pertussis BX640420
Achromobacter xylosoxidans 2 (Ef-GC)
Collimonas sp. b214 (commensal of fungus isolated from SP)
Bordetella bronchiseptica 337-1 (SP)
Bordetella parapertussis AJ278450
0.1
Figure 23. Bacterial isolates (marked in bolt) of the class Betaproteobacteria. Bacterium in the gray
box digested recPrP. The tree is constructed with sequences of ~500 bp length; the sequences with
identity >98,5% to each other and to corresponded described species considered as the same OTU
and presented in single example without correspondent bacteria. Sources of isolation: L. terrestris
(Lt); A. caliginosa (Ac); E. fetida (Ef); soddy-podzolic soil (SP); gut content (GC); casts (EX).
Bacterial species from the class Gammaproteobacteria able to digest recPrP were abundant between
other Proteobacteria: 9 species of total 27 isolated appeared recPrP proteolytic activity. The
bacteria from the genus Pseudomonas were the most successful (Fig. 24). The isolate
P. agglomerans 571-1 did not digest the not-structured N-terminus of the recombinant prion
(epitope of PrPc248 antibody); the isolates A. sorbia and Pseudomonas sp. 9 did not degrade the
interhelix loop at the C-terminus of the recPrP (epitope of VRQ14 antibody) (Fig. 24).
3. Results
60
________________________________________________________________________________
Pseudomonas marginalis AF364098
Pseudomonas grimontii AF268029
Pseudomonas sp. 597-2 (Ac-GC)
Pseudomonas sp. 593 (Ac-GC)
Pseudomonas sp. 596-2 (Ac-GC)
Pseudomonas rhodesiae AF064459
Pseudomonas reactans 435-1 (SPs, Ac-GC)
Pseudomonas sp. 621 (Ac-GC)
Pseudomonas sp. 329-1 (SPs)
Pseudomonas brassicacearum AJ292381
Pseudomonas sp. 614 (Ac-GC)
Pseudomonas putida 300-1 (SPs)
Pseudomonas sp. 9 ** (Ef-GC)
Pseudomonas gingeri AF320991
Pseudomonas sp. 360-2 (Lt-EX)
Pseudomonas tolaasii AF255336
Pseudomonas sp. 399-2 (SPs)
Pseudomonas syringae AY574914
Pseudomonas aeruginosa Z76672
Pseudomonas alcaligenes Z76653
Pseudomonas chloritidismutans 361-2 (Lt-EX)
Pseudomonas sp. 309-2 (SPs, Lt/Ac-EX)
Pseudomonas entomophila AY907566
Pseudomonas sp. 357-2 (Lt-EX)
Raoultella planticola 565-1 (Ac-GC/EX, Ef-GC)
Kluyvera ascorbata 303-1 (SPs, Ac/Lt-EX)
Enterobacteriaceae bacterium 552-2 (Ac-GC)
Leclercia adecarboxylata 366-1 (Lt-EX, Ac-GC)
Serratia marcescens 658 (Ef/Ac-GC)
Buttiauxella gaviniae AJ233403
Buttiauxella izardii AJ233404
Buttiauxella sp. 498-2 (Ac-EX)
Buttiauxella brennerae 624 (Ac/Lt-GC)
Pantoea agglomerans 571-2 * (Ac-GC/EX)
Morganella morganii 358-1 (Lt-EX)
Shewanella baltica 359-1 (Lt-EX)
Aeromonas sobria 368-2 ** (Lt-EX, Ef-GC)
Stenotrophomonas maltophilia 3 (SPs)
Acinetobacter lwoffii 546-2 (Ac-EX)
0.1
Figure 24. Bacterial isolates (marked in bolt) of the Gammaproteobacteria group. Bacteria in the
gray box digested recPrP. * - bacterium digested only N-terminus of recPrP (epitope PrPc248); ** bacteria digested only C-terminus of recPrP (epitope VRQ 14). The tree is constructed with
sequences of ~500 bp length; the sequences with identity >98,5% to each other and to corresponded
described species considered as the same OTU and presented in single example without
correspondent bacteria. Sources of isolation: L. terrestris (Lt); A. caliginosa (Ac); E. fetida (Ef);
soddy-podzolic soil (SP); gut content (GC); casts (EX).
None of estimated randomly choused isolates of the bacteria from CFB group did recPrP digestion
on the remarkable level (Fig. 25).
3. Results
61
________________________________________________________________________________
Flavobacterium columnare M58781
Flavobacterium limicola AB075232
Flavobacterium johnsoniae 451-1 (Ac-GC)
Flavobacterium denitrificans 375-2 (Lt-EX)
Chryseobacterium scophthalmum 597 (Lt/Ac-GC)
Chryseobacterium balustinum M58771
Sphingobacteriaceae bacterium 611-2 (Ac-GC)
Sphingobacterium spiritivorum M58778
Sphingobacterium thalpophilum M58779
Sphingobacterium comitans X91814
0.1
Figure 25. Bacterial isolates (marked in bolt) of the CFB group. Bacteria in the gray box digested
recPrP. The tree is constructed with sequences of ~500 bp length; the sequences with identity
>98,5% to each other and to corresponded described species considered as the same OTU and
presented in single example without correspondent bacteria. Sources of isolation: L. terrestris (Lt);
A. caliginosa (Ac); E. fetida (Ef); soddy-podzolic soil (SP); gut content (GC); casts (EX).
Isolates
of
the
class
Actinobacteria
capable
to
digest
recPrP
were
numerous
as
Gammaproteobacteria: 8 species among 30 isolated degraded recombinant prion (Fig. 26). The
isolate Nocardioides sp. 410-1 did not digest the not-structured N-terminus of the recombinant
prion (epitope of PrPc248 antibody) (Fig. 26).
3. Results
62
________________________________________________________________________________
Microbacterium paraoxydans 597-1 (Ac-GC)
Microbacterium keratanolyticum Y17233
Microbacterium terregens 472-2 (Ac-EX)
Microbacterium sp. 360-1 (Ac-EX)
Leifsonia sp. 555-1 (Ac-GC)
Leifsonia poae AF116342
Agromyces sp. 423-2 (SPs)
Agromyces ramosus 462-1 (SPs; Ac-GC/EX)
Agromyces cerinus D45060
Microbacterium sp. 475-2 (SP; Ac-EX)
Microbacterium imperiale X77442
Mycetocola lacteus AB012648
Mycetocola shiratae AB012647
Micrococcaceae bacterium 521-1 (Ef-GC)
Rathayibacter tritici X77438
Rathayibacter toxicus D84127
Oerskovia paurometabola AJ314851
Oerskovia jenensis AJ314850
Oerskovia sp. 463-2 (Ac-EX)
Oerskovia enterophila 463-1 (Ac-GC)
Curtobacterium flaccumfaciens 581-1 (Ac-GC)
Kocuria palustris 560-1 (SPs; Ac-GC/EX; Ef-GC/EX)
Micrococcus luteus 536-2 536-2 (Ef-GC)
Micrococcus antarcticus AJ005932
Micrococcus sp. 532-1 (Ef-GC)
Arthrobacter globiformis 333-1 (SPs)
Micrococcus lylae AJ298940
Arthrobacter sp. 430-1 (SPs)
Arthrobacter sp. 573-1 (Ac-GC)
Arthrobacter ardleyensis AJ551163
Arthrobacter nicotianae X80739
Promicromonospora sp. 372-2 (Lt-EX)
Promicromonospora sukumoe AJ272024
Arthrobacter oxydans 304-2 (SPs)
Arthrobacter chlorophenolicus 578-2 (Ef-GC)
Arthrobacter aurescens AF467106
Nocardia globerula 456-2 (Ac-EX)
Rhodococcus erythropolis 505-2 (SPs)
Rhodococcus opacus 667 (SPs; Ef-GC)
Mycobacterium sp. b208r (commensal of fungus isolated from SPs)
Mycobacterium chitae X55603
Mycobacterium goodii AY457079
Nocardioides sp. 560-2 (SPs)
Nocardioides plantarum Z78211
Nocardioides albus AF005005
Streptomyces sp. 597-2 (Ac-GC)
Streptomyces lateritius 579-2 579-2 (SPs)
Streptomyces sp. 471-1 (SPs; Ac-EX)
Streptomyces sp. 470-1 (Ac-EX)
Streptomyces griseus 406-2 (SPs)
Streptomyces albus AJ621602
Streptomyces sp. 627 (SPs; Ac-EX)
Streptomyces somaliensis AJ007400
Streptomyces sp. 809 (SP)
Streptomyces violaceus AJ781755
Streptomyces massasporeus AJ781323
Streptomyces albidoflavus Z76685
0.1
Figure 26. Bacterial isolates (marked in bolt) of the Actinobacteria. Bacteria in the gray box
digested recPrP. The tree is constructed with sequences of ~500 bp length; the sequences with
identity >98,5% to each other and to corresponded described species considered as the same OTU
and presented in single example without correspondent bacteria. Sources of isolation: L. terrestris
(Lt); A. caliginosa (Ac); E. fetida (Ef); soddy-podzolic soil (SP); gut content (GC); casts (EX).
3. Results
63
________________________________________________________________________________
Isolates only from the genus Bacilli (5 among 20 different species) appeared recPrP proteolytic
capacity. The digestion by those bacteria was complete (Fig. 27).
Staphylococcus sp. 533-1 (Ef-GC)
Staphylococcus succinus AF004219
Staphylococcus xylosus AF515587
Staphylococcus aureus 342-2 (SPs)
Bacillus mojavensis 324-2 (SPs, Ac-GC, Ef-GC)
Bacillus subtilis 385-2 (SPs, Ac-GC)
Bacillus licheniformis 414-2 (SPs, Lt-EX)
Bacillus pumilus 403-1 (SPs, Ac-EX)
Bacillus sp. 611-1 (Ac-GC)
Bacillus bataviensis AJ542508
Bacillus soli AJ542514
Bacillus macroides 373-2 (SPs, Lt-EX)
Bacillus simplex 564-2 (SPs, Ac-GC, Lt-EX)
Bacillus megaterium 581-2 (SPs, Ac-GC/EX)
Bacillus mycoides 582-1 (Ac-GC)
Sporosarcina globispora b208 (commensal of fungus isolated from SPs)
Sporosarcina psychrophila D16277
Sporosarcina sp. 502-2 (Lt-GC)
Sporosarcina macmurdoensis AJ514408
Bacillus psychrodurans AJ277984
Bacillus psychrotolerans AJ27983
Bacillus sp. 459-1 (Ac-EX)
Bacillus sphaericus 449-1 (Ac-EX)
Pediococcus damnosus AJ318414
Pediococcus inopinatus AJ271383
Pediococcus pentosaceus AJ305321
Pediococcus sp. 691 (Ef-EX)
Paenibacillus sp. 598 (Ac-GC)
Paenibacillus polymyxa AJ320493
Paenibacillus amylolyticus 554-1 (SPs, Ac-GC/EX)
Paenibacillus sp. 412-1 (SPs)
Paenibacillus borealis AJ011321
Paenibacillus sp. 561-2 (Ac-GC/EX)
0.1
Figure 27. Bacterial isolates (marked in bolt) of the class Bacilli. Bacteria in the gray box digested
recPrP. The tree is constructed with sequences of ~500 bp length; the sequences with identity
>98,5% to each other and to corresponded described species considered as the same OTU and
presented in single example without correspondent bacteria. Sources of isolation: L. terrestris (Lt);
A. caliginosa (Ac); E. fetida (Ef); soddy-podzolic soil (SP); gut content (GC); casts (EX).
Six isolates from the most common eight fungal species were able to digest prion: Ceriporiopsis
subvermispora (Basidiomycota); Bionectria ochroleuca, Fusarium oxysporum, Tolypocladium
inflatum, Gibberella sp. (Ascomycota); and fungus of family Mucoraceae (Zygomycota). The
residual amount of recPrP in all fungal samples was below the detection level. Bjerkandera adusta
and Trichosporon dulcitum (Basidimycota) did not digest prion under given conditions.
3. Results
64
________________________________________________________________________________
3.2.2 Effect of earthworms and gut microbiota on recPrP retaining
Unspecific proteolytic activity
Recombinant PrP was not detected in the soil control samples under given extraction conditions,
hence all experiments with soil and cast samples aimed at the recPrP proteolytic potential and
specific enzymatic kinetics of the recPrP digestion were performed in vitro with aqueous extracts
from soil and casts. That could be caused by the irreversible absorption of the recPrP to the soil
particles (Vasina et al., 2005; Johnson et al., 2006).
The amount of total protein in aqueous extracts was similar each to other (2,17; 2,30; 2,23 ng/µl in
the soddy-podzolic soil, L. terrestris and A. caliginosa casts respectively).
Maximal proteolytic activity was observed in all aqueous extracts without protease inhibitors (1).
Inhibitors had affected the enzymatic activity of water-soluble fractions from the soil and cast
samples, but proteolysis has still been performed in all variants with added inhibitors; using even
two cocktail sets together (2) (Fig. 28A). Degradation of chromogenic substrate in soil extract was
mostly caused by action of proteases, which were inhibited by EDTA presented in the Cocktail Set
II, while the Inhibitor Set III did not affect so successfully (Fig. 28A). In contrast to the soil,
proteases inhibited by aprotinine and leupeptine (presented in the Cocktail Set III) played the main
proteolytic role in cast samples (Fig. 28A).
Proteinase K and trypsin did also digest commercially available chromogenic substrate differently
probably because of diverse composition of substrates available for hydrolysis; PK was more
efficient in proteolysis (Fig. 27B).
Non-specific proteolytic activity of water extract from soil sample, which contained 2,17 ng/µl of
total soluble proteins (in 40 µl of final sample volume) without additional inhibitors, was
approximately equivalent to the activity of 0,125 ng/µl proteinase K (0,15 mU). Proteolysis of
chromogenic substrate in the aqueous extracts from the casts of both earthworm species, without
inhibitors, was much stronger than in the soil sample and even exceeded the activity of the same
amount of pure proteases: 2,30 ng/µl of total proteins in the cast sample of L. terrestris and
2,23 ng/µl of total proteins in the cast sample of A. caliginosa showed the enzymatic activity
comparable to approximately 50 ng/µl (~60 mU) and 17,5 ng/µl (~20 mU) of proteinase K,
respectively (Fig. 27B).
Activities of the proteases inhibited with EDTA (Inhibitor Coctail Set II) in presence of Inhibitor
Set III (4) in all samples were very similar each to other and corresponded to the activity of
0,0625 ng/µl (0,075 mU) proteinase K. The activities of the proteases inhibited with aprotinin and
leupeptine (Inhibitor Set III) with added Inhibitor Set II (3) were higher in the water extracts from
3. Results
65
________________________________________________________________________________
the casts of both earthworm species and analogous to the action of 5 ng/µl (6 mU) proteinase K in
the contrast to that from the soil sample, which correlated to 0,0113 ng/µl (0,0135 mU) proteinase
K (Fig. 27). According to the optical density of digested chromogenic substrate, the summarize
activity of proteases in the variants with separately added Inhibitor Sets were equal to the variant
with presence of the both Inhibitor Set together in the aqueous extract derived from the same
0,18
B: Proteolysis by pure proteases
A: Proteolysis in aqueous extracts
0,18
0,16
0,119
0,12
0,102
0,1
0,096
0,02
0,035
0,004
0,035
0,017
0,045
0,04
0,032
0,08
OD of digested chromogenic substrate
0,136
0,14
0,06
2000 ng
(60 mU)
0,16
0,005
0,009
OD of digested chromagenic substrate
sample, soil or earthworm casts (Fig. 27A).
20000 ng
(600 mU)
0,14
0,12
200 ng
(6 mU)
0,1
10000 ng
20 ng
(0,6 mU)
0,08
20000 ng
0,06
1000 ng
0,04
0,02
0
0
Soil
Lt-Ex
without inhibitors
Sets II+III
Ac-Ex
Set II
-0,02
Set III
0
PK
5000
10000
15000
2000
proteins, ng
TP
Logarithmic PK
Logarithmic TP
Figure 28. A: Unspecific proteolysis of chromogenic substrate (GenoTech, USA) in the aqueous
extracts from soil and casts from L. terrestris (Lt-EX) and A. caliginosa (Ac-EX) with/without
Protease Inhibitor Cocktail Sets; B: Unspecific proteolysis of chromogenic substrate (GenoTech,
USA) by proteinase K (PK) and trypsin (TP).
Wide spectrum of proteases presented in the aqueous extracts and their broad specificity could
caused the stronger digestion of chromogenic substrates and thus, higher optical density of reaction
mixture in comparison with pure proteases. The pure proteases revealed logarithmic progression for
the digestion of chromogenic substrate, while the proteases in the aqueous extracts had linear
progression for the tested concentrations (data not shown).
Thus, proteases inhibited with EDTA played a major role in the non-specific proteolysis in soil
aqueous extracts, and the proteases inhibited with aprotinin and leupeptine augmented upon passage
through the earthworm gut caused a significantly enhanced non-specific proteolysis in watersoluble content from the earthworm casts.
3. Results
66
________________________________________________________________________________
Proteolysis of recPrP
Dynamics of recombinant prion degradation detected with both antibodies was similar each to other
(Fig. 29, 30). Visible changes of prion amounts were already observed in the soil and cast from
samples both earthworm species with bacterial inhibitor protease Cocktail Set II (3) after 12 hours
(data not shown). The reduction of recPrP quantity became remarkable after 1-day incubation in the
soil and cast extracts without protease inhibitors (1). Complete digestion of recPrP was observed in
the samples without inhibitors (1) and with protease Inhibitor Set II contained EDTA (3) using
antibody PrPc248 (Fig. 29).
S
M
1
2
Ac
Lt
3
4
1
2
3
4
1
2
3
4
C
Initial
0 days
1 day
2 days
4 days
6 days
Figure 29. Immunodetection of recPrP with the antibody PrPc248 after incubation of the recPrP
for 0, 1, 2, 3, and 6 days at 15° C with the aqueous extracts from soddy-podzolic soil (S), cast of L.
terrestris (Lt) and A. caliginosa (Ac) detected Lanes: MultiMark Multi Colored Standard (M);
control, 40 ng (C); without inhibitors (1); with protease Inhibitor Cocktail Sets (II+III) (2); with
protease Inhibitor Cocktail Set II (3); with protease Inhibitor Cocktail Set III (4).
3. Results
67
________________________________________________________________________________
Degradation of recombinant prion detected with antibody VRQ 14 was also more rapid in presence
of Inhibitor Set II (3) (Fig. 30).
M
1
2
Ac
Lt
S
3
4
1
2
3
4
1
2
3
4
C
1 day
2 days
4 days
6 days
Figure 30. Immunodetection of recPrP with the antibody VRQ14 after incubation of the recPrP for
0, 1, 2, 3, and 6 days at 15° C with the aqueous extracts from soddy-podzolic soil (S), cast of L.
terrestris (Lt) and A. caliginosa (Ac) detected Lanes: MultiMark Multi Colored Standard (M);
control, 40 ng (C); without inhibitors (1); with protease Inhibitor Cocktail Sets (II+III) (2); with
protease Inhibitor Cocktail Set II (3); with protease Inhibitor Cocktail Set III (4).
Notable prion digestion was observed neither in presence of both Inhibitor Cocktail Sets together
(2), nor with Inhibitor Cocktail Sets III (4) during the whole term of incubation with both used
antibodies (Fig 29, 30).
In general, the proteolytic property in aqueous extracts from soil and cast samples did not show
remarkable differences between for prion hydrolysis in contrast to unspecific proteolytic activity.
Pure trypsin (0,8 ng/µl) did not digest ovine recPrP during 6-days incubation time (Fig. 31), while
α-chymotrypsin and proteinase K lead to a rapid (within 15 min) hydrolysis of the full-length
recombinant prion (Rezaei et al., 2000).
3. Results
68
________________________________________________________________________________
5
M
10 15 30
1
H
2 4
6 12
1
D
2 4
C
6
A
B
Figure 31. Immunobloting of recPrP treated with pure trypsin detected by PrPc248 (A) and
VRQ 14 (B) antibodies after incubation for: 5, 10, 15, 30 minutes (M); 1, 2, 4, 6, 12 hours (H); and
1, 2, 4, 6 days at 15° C in comparison with control (C) recPrP (40 ng).
4. Discussion
69
________________________________________________________________________________
4. Discussion
4.1 Effect of the earthworm gut environment on the microbial community
The changes of bacterial community of soil or compost upon passage through the earthworm GI
tract were studied using earthworm species populating soddy-podzolic soil (L. terrestris and
A. caliginosa) and horse manure compost (E. fetida). The experimental setup exploited: (i) different
earthworm species (L. terrestris and A. caliginosa) thriving in the same substratum (soddy-podzolic
soil) and the same species of the earthworm from the same soil type but with another bacterial
community and (ii) the earthworms (E. fetida) populating quite different substratum (horse manure
compost).
On the basis of the FISH analysis we consider that bacterial communities of studied substrata (soil
and compost) were significantly modified passing through the gut of earthworms. The ratio
EUB338/DAPI (illustrated the percentage of physiologically active cells) for the soddy-podzolic
soil collected at different years was comparable each to other (0,18-0,20), while the index was
higher in the cast samples of earthworms (L. terrestris (0,27-0,29) and A. caliginosa (0,23-0,30)) .
At the same time the index EUB338/DAPI was lower in the cast of earthworm E. fetida in
comparison with the horse manure compost. This proportion of the cells hybridized with FISH
probe to those stained with DAPI had the lowest number in the guts of estimated earthworms
(excluding A. caliginosa collected at the spring 2003). In general, the ratio EUB338/DAPI was
pretty similar to those described early by Schönholzer and colleagues (2002) for soil and earthworm
L. terrestris. Augmentation with bacteria during transit was also detected elsewhere with plating
method (Daniel and Anderson, 1992) and epifluorescence microscopy (Kristufek et al., 1992).
Besides, almost all taxonomic groups were undergoing significant changes. The alterations in
microbial composition exhibited some commonalities in the earthworms of different species. As
such one should notice: (i) augmentation of Cytophaga-Flavobacteria group of Bacteroidetes upon
passage in all our independent experiments throughout all three years of the present study; (ii)
numbers of Firmicutes and Gammaproteobacteria were in most cases lower in gut content in
contrast to substratum; (iii) microbial composition alterations of Actinobacteria, Alpha- and
Betaproteobacteria varied and could increase or decrease upon passage; (iv) bacteria of phyla
Planctomycetes, Verrucomicrobia and Deltaproteobacteria had very low numbers (< 1%) of
physiologically active cells in population being minor members of community, furthermore the
passage through the earthworm gut did not notably effect them.
4. Discussion
70
________________________________________________________________________________
Another issue we tried to address was whether a common regularity of changes of microbial
community upon its passage through the GI tract of earthworms does exist and whether there is the
host or substratum dependence influencing these changes. Same major patterns in microbial
community composition changes were observed for two different earthworm species (L. terrestris
and A. caliginosa) populating the same soil with the same microbial inocula and containing in
contrast to those of a same species kept in the soils with different initial microbial compositions.
All these facts indicate that microbial composition of inoculum (microbial biodiversity in the
soil/compost) was the major factor affecting the further population changes during the passage
through the earthworm gut; species-specific features of the earthworm gut environment affecting
the bacterial population changes upon passage were not observed.
Among the physical-chemical factors in the earthworm gut the following items are known: (i)
anoxic conditions (Karsten, Drake, 1995; Horn et. al. 2003); (ii) higher (in comparison with soil)
content of water, organic and amino acids, sugars, nitrite, ammonium and hydrogen (Karsten,
Drake, 1995; Horn et. al. 2003); (iii) mechanical (Schönholzer et al., 2002) and biochemical
disintegration of microorganisms in the digestive tract of earthworms (Edwards and Fletcher, 1988),
killing activity of gut exudates was noticed for soil diplopods and millipedes (Byzov et al., 1996,
1998); (iv) interaction of microorganisms between each other (including the whole spectrum of
possibilities, from antagonism to mutualism) (Schönholzer et al., 2002).
Thus, earthworm gut environment has a strong selective pressure influences certain bacterial
taxonomy groups being a “bottleneck” with unfavorable conditions decreasing total number of
certain physiologically active microbial groups hybridized with FISH probes. At the same time, the
very conditions, inhibiting or even deadly to some members from bacterial population, stimulate
other bacteria flourishing in the gut or in the cast. Similar qualitative variation patterns of the same
taxonomic bacterial groups upon passage through the guts of A. caliginosa, L. terrestris and
E. fetida inhabiting various substrata with different bacterial communities were observed and were
possibly caused by similar selective pressure factors of gut environments of earthworms.
One of the most challenging questions in the earthworm microbiology is what kind of organisms we
can consider as intestine symbiotic or gut-associated. There are two possible definitions: (i) obligate
gut-associated microorganisms cannot survive out of earthworm gut or their numbers are getting
extremely low and they become in physiologically inactive; (ii) facultative gut-associated
microorganisms which can survive outside the gut where they can physiologically be active, but
their numbers are lower in comparison with gut lumen, and during the passage through the gut a
certain enrichment of these microbes occurs. According to the FISH analysis, the bacteria of CFB
4. Discussion
71
________________________________________________________________________________
satisfactory fit to the definition and could be considered as facultative gut-associated
microoragnisms.
Simultaneously, taxonomic groups reducing their numbers in the gut could contain certain bacterial
species believed to be the gut-associated bacteria but undistinguishable in the total pool of
microbes, i.e. for whom the FISH approach is too insensitive. To qualitatively evaluate bacterial
communities’ compositions and to determine gut-associated bacteria we have applied PCR-based
methods.
Phylogenetic analysis of the clone libraries showed a high diversity of microorganisms (especially
in compost), but the number of sequenced clones was relatively low and allowed just to roughly
estimate bacteria populating the substrates and earthworm sources. Application of the cloning
technique allows detection of even a single copy (although the probability of that for randomly
chosen colonies is very low). This could be an explanation for finding in the libraries from
earthworm sources (gut content and cast but not in the substrata) clones belonging to the minor part
of population.
SSCP coupled with the consequent phylogenetic analysis of separate DNA bands delivers even
though superficial, but important and visible information about bacterial community composition.
Disadvantage of this method is based on the staining the DNA bands, which, to become visible,
should harbour quite a number of DNA copies per band, and which could then be detected and
analysed by re-amplification and sequencing.
In general, taxon-specific SSCP approach allows detection of bacteria on the levels of the genus,
species and strain, bringing up a higher diversity of bacteria in comparison with universal SSCP
primers designed by Schweiger and Tebbe (1998). Primer sets designed for detection of bacteria
from classes Alphaproteobacteria, Bacilli (Firmicutes), and Verrucomicrobia appeared to possess
some minor unspecific affinity with outgrouped bacteria; Gammaproteobacteria-specific primers
annealed with unclear taxonomy-placed bacteria equally affiliated to γ- or β-proteobacteria. That
could be caused by humid acid co-extracted with nucleic acid and decreased the primer specificity
(Stach et al., 2001).
Despite of mentioned above shortcomings, the taxon-specific SSCP worked out significantly more
information than conventional SSCP. Firstly, the number of distinguishable bands obtained with
universal SSCP primers (e.g. 13 bands in the sample generated from soil RNA) was notably lower
than sum of the bands detected by using taxon-specific SSCP primers (90 distinguishable bands in
all taxon-specific bands generated from soil RNA). Gram-negative microorganisms delivered the
most of the bands on the SSCP profile. Among them, bands of the bacteria from the phyla CFB and
4. Discussion
72
________________________________________________________________________________
Proteobacteria were the most abundant. Despite of this, diversity of such bacteria estimated with
taxon-specific primers was higher. The phyla Firmicutes, Planctomycetes and Verrucomocrobia
were detected using the universal primer set neither from DNA, nor from RNA pools, while their
diversity was accessed using only taxon-specific SSCP primers. Simultaneously, the bacteria from
the phyla Acidobacteria and Gemmatinonadetes were detected only with universal SSCP primers
for 16S rDNA gene. Thus, one may consider Gram-negative (easy-lysed) bacteria with several
ribosomal operons in genome to have more chances to be detected using PCR. Perhaps this is the
reason of the presence of a relatively high number of bands from scanty Gammaproteobacteria on
the SSCP profiles generated with universal primers, which outcompete the Gram-positive bacteria
for oligonucleotides and other PCR reaction ingredients which, in turn, lead to the absence of the
bands corresponding to the latter organisms on the SSCP profiles generated with universal primers.
Another point to mention is a moderate, comparable number of the bands on the profiles generated
with universal primers (up to 15 bands per sample) and with taxon-specific primers (up to 20 –
Gammaproteobacteria generated from total soil RNA) diverged within the similar level of
magnitudes.
As far as number of physiologically inactive bacteria presented in the community was 5 times
higher than active cells (i.e. the cells hybridized with unspecific FISH probe), one could expect
higher number of bands on SSCP profiles generated from DNA. Inactive bacteria present in the
community and “waiting” for favorable conditions to initiate their growth can be accumulated in the
soil or upon the income from other sources (for instance from leaf litter) or through seasonal
changes when suddently an enormous number of species become inactive (Torsvik et al. 1990;
Trüper, 1992). Truly, composition of bacterial community estimated with taxon-specific primers
was significantly depended on the type of template (DNA or RNA). Minor members of bacterial
community (Planctomycetes, Verrucomicrobia and class Myxococcales of Deltaproteobacteria)
were amplified only from total DNA but not from RNA preps. Probably, number of the target 16S
rRNA in the total RNA pool was below the sensitivity of the method, or one of the primers was
exhausted by a non-specific binding to ribosomal RNAs from outgrouping microorganisms during
reverse transcription. The single bands linked to A. faecalis were detected in the SSCP profiles
generated from RNA pool by using Betaproteobacteria-specific primers along with many bands in
the DNA-generated profiles. But the profiles amplified from DNA pool were not always rich with
bands in comparison with those done from RNA. Gammaproteobacteria and Bacilli demonstrated
an opposite picture: PCR products were obtained from the RNA, but not from DNA (casts of
L. terrestris and A. caliginosa), or profile was more complex (Gammaproteobacteria, soil sample).
High amount of total DNA could block the amplification because of excesss of the target 16S rDNA
4. Discussion
73
________________________________________________________________________________
template in case of using total DNA as template; dilution of the template could remove or reduce
diversity of the target gene in the pool, caused low diversity of amplicons. The use of RT-PCR with
taxon-specific primers enhanced in some cases the sensitivity of the method whereas only rRNA
was amplified and thus the competition by competing molecules was omitted.
Identity of SSCP profiles in the soil and cast samples generated from DNA could be explained with
the presence of the same bacteria in both samples that remain intact upon passage, which affected
only the physiologically active part of population. This was consistent with the early results of Egert
and colleagues (2004), which could not find dominant indigenous microbes in the gut of L.
terrestris using T-RFLP analysis using DNA as template.
According to the published data, the 16S RNA is moderately stable in the dead cells (in comparison
with even more stable DNA and sensitive mRNA) and could be detected with both FISH and RTPCR techniques even some time after the cells die (Sheridan et al., 1998; McKillip et al., 1998;
Lleó et al., 2000; Keer and Birch, 2003). Nevertheless, the application of the 16S rRNA-based
methods for studying microbial communities of substrates and earthworm casts make sense and
differences between biodiversity of the samples generated from DNA and RNA were in fact very
clear. Thus, comparison the data obtained with PCR using DNA as a template with those based on
rRNA-based techniques (FISH) is believed to be pretty sophisticated.
Another task for the study microbial communities passing through the earthworm gut were the
discovering bacterial genera and species permanently or transiently associated with this
environment. Novel bands appeared on the SSCP profiles of cast samples generated from the RNA
could be considered as belonged to earthworm gut-associated bacteria. These new bands were
detected on the CFB, Alpha-, Gammaproteobacteria, and Bacilli taxon-specific SSCP profiles.
However, do these bands really belong to bacteria coupled with earthworm intestine or occur
through the possible PCR amplification errors?
The novel bands from CFB bacteria derived from the same species, F. granuli, occurring as
neighbor bands, exhibited high homology degree (sequence identity 99%) each to other and formed
a monophyletic cluster. Simultaneously, bacteria of the CFB group can thus be considered as the
main candidates to form a stable association with the gut environment. The assumption that the
novel band provided the growing number of CFB bacteria in the cast is supposed to be unrealistic.
Most probably, the members of the CFB monophyletic cluster all together became more abundant
upon the passage.
4. Discussion
74
________________________________________________________________________________
The novel bands appeared on the Gammaproteobacteria- and Bacilli-specific SSCP profiles from
cast samples belonged to bacteria being a novel taxon clustering next to genera Legionella and
Stenotrophomonas (Gammaproteobacteria) and to Bacillus and Sporosarcina (Bacilli).
In the contrary to other bacterial groups, almost all community of Alphaproteobacteria (detected on
taxon-specific SSCP profile) was composed of unique bands linked to family unclassified
Sphingomonadaceae in the soil and cast samples, although this part of total bacterial population as
well as Gammaproteobacteria and Firmicutes had a tendency to reduce their number upon the
passage.
On the basis of FISH, clone libraries and SSCP methods, one could distinguish three bacterial
groups and approximately predict the effect of earthworm gut environment upon the microbial
community of substrate (i) bacteria always increasing their numbers in the gut: classes
Flavobacteria and Sphingobacteria (Cytophaga-Flavobacteria group of Bacteroidetes) was the sole
bacterial group, which always benefited from the gut environment, being facultative earthworm gutassociated; (ii) bacteria always decreasing their numbers in the gut, bacteria from taxonomy groups
Gammaproteobacteria and Firmicutes; (iii) bacteria without clear changes in the numbers,
normally;
minor
members
of
community
(Planctomycetes,
Verrucomicrobia
and
Deltaproteobacteria), which have number of active cell less than 1% of the total population and
were abundant neither in the soil, nor in the earthworm gut environments. The rest of bacterial
taxonomy groups (Alpha-, Betaproteobacteria, and Actinobacteria) had varying response upon the
transit depending on many factors including earthworm-bacteria and bacteria-bacteria interaction.
Particularly describing the changes of microbial community estimated with FISH and SSCP
approaches one could distinguish the bacteria resistant to earthworm gut environment among the
gut-sensitive taxonomy groups. Active parts of Alphaproteobacteria and Betaproteobacteria in the
community detected with the SSCP were not very diverse. Although many unique bands were
present on the Alphaproteobacteria-specific SSCP profile, they belonged only to family
unclassified Sphingomonadaceae, while other families from the taxa (Sphingomonadaceae
Caulobacteraceae, and Methylobacteraceae) were detected on the profiles generated from the
DNA. The bacteria from the family of unclassified Spingomonas (Alphaproteobacteria), A. faecalis
(Betaproteobacteria)
and
the
bacteria
linked
to
P. enthomophila
and
P. putida
(Gammaproteobacteria) could be considered as earthworm gut-resistant bacteria. This statement is
well supported with data from FISH analysis: A. faecalis seeming to be predominant bacterium was
not influenced by the selective pressure of the gut environment and was stable or just slightly
fluctuated upon the passage which is reflected by stable numbers of Betaproteobacteria in the
4. Discussion
75
________________________________________________________________________________
community. The decreasing number of Gammaproteobacteria was caused by strong diminishing or
elimination of genera Cellvibrio, Chromatiaceae, family Legionellaceae from active part of
community, but whether the novel bands appeared on the Gammaproteobacteria-specific profile
truly belonged to original gut microflora is uncertain. Unclassified Spingomonadaceae reduced their
amount being nevertheless resistant to the earthworm gut environment among the other families of
the class Alphaproteobacteria. Firmicutes had a similar behavior as Gammaproteobacteria.
Non-diverse active part of Proteobacteria and other taxonomy groups together with high diversity
and number of hypothetical gut-associated bacteria from CFB group in the soil community could be
caused by a long-term-keeping (>3 months) the earthworm on the same soil with following
fractional substitution of bacterial population under the earthworm gut selective pressure.
The favorable growth of Cytophaga-Flavobacteria cluster in the community passing the earthworm
gut was previously reported for L. terrestris (Schönholzer et al., 2002) and termite gut (SchmittWagner et al., 2003). The bacteria from our sources were closely related to those being intracellular
symbionts of the ants Tetraponera binghami (van Borm et al., 2002b). The bacteria from CFB
group were also detected in the gut of Acromyrmex leafcutter ants (van Borm et al., 2002a), and
other insects (Jucci, 1952). The CFB division is also suggested to be predominantly associated with
mammals’ intestine, distinct subgroup of the division being ancient and coevolved with their hosts
(Bäckhed et al., 2005). Reducing numbers of Proteobacteria and Firmicutes in our study well
correlated with previously described data of plating/culturing methods and FISH analysis for the
earthworms Lumbricus spp. (Pedersen and Hendriksen, 1993; Kristufek et al., 1993; Schönholzer et
al., 2002). At the same time Actinobacteria were not detected by FISH analysis in the gut content
and excrements in that study although they were observed both in the soil and earthworm sources
during our studies.
In contrast to the previously published data, scanty bacterial clones (two OTUs) from the phylum
Acidobacteria (Fibrobacteres/Acidobacteria group) found in the cast library of autumn-collected
A. caliginosa during our investigation were reported as dominant bacterial group of L. rubellus
intestine (Singleton et al., 2003).
Bacteria from Verrucomicrobia detected on the SSCP profile from cast samples of L. terrestris and
in the libraries from gut content and cast the same earthworm species were similar each to other
(sequence identity 95%) and related to Akkermansia muciniphila mucosa-populating bacteria
isolated from the human gut (Derrien et al., 2004), hence they could be an ecological and
physiological analog of this microorganism.
4. Discussion
76
________________________________________________________________________________
The increasing number of bacteria upon passage with reduction of the population of some
Gammaproteobacteria from family Enterobacteriaceae well correlated with many previous studies
done with the plating methods.
Midgut
Soil
Cast
Earthworm‘ body
F
E
B
F
F
B
E
D
A
D
B
D
C
C
C
Earthworm gut
Figure 31. The scheme of changes of populations different microbial group in substratum upon
passage through the earthworm gut: (A) obligate host-associated microorganisms
("Lumbricoplasma"); (B, C) facultative gut-associated microorganisms (CFB group); (D, E) gutsensitive microorganisms (particularly Gammaproteobacteria, Bacilli, and Eukarya); (F) gutresistant microorganisms.
Apart from the free-living microorganisms populating the substrate we detected certain bacteria,
which could be intra- or extracellular symbionts of the earthworm. As obligate earthwormassociated bacteria one can consider bacteria from a cluster from the class Mollicutes (Firmicutes)
detected in the earthworms of family Lumbricidae (L. terrestris, A. caliginosa, and E. fetida). On
the basis of sequence analysis, source of isolation, and host specificity, the bacteria from newly
discovered taxonomic monophyletic cluster were named ‘Lumbricoplasma’ candidate division and
are thought to compise a novel family, ‘Lumbricoplasmataceae’. The ‘Lumbricoplasma’ spp. were
detected in the gut tissue, coelom intestine, muscles, and outer epidermis being located in these
tissues as small clusters. This is the first finding of this group of Mollicutes in the earthworm.
Although the Mycoplasma-like bacteria were discovered by single clone in the intestine library of
L. rubellus (Singleton et al., 2003), the sequence identity with both described Mollicutes and
‘Lumbricoplasma’ candidates was low.
Free-living amoebae harbored bacterial symbionts, which include α−, β-, γ-proteobacteria,
Flavobacteria and Actinobacteria, as postulated before (Kwaik et al., 1998; Greub and Raoult,
4. Discussion
77
________________________________________________________________________________
2004; Horn and Wagner, 2004; Molmeret et al., 2005). Even though we detected no amoebae in our
sources even accidentally, as other eukarya, many bacteria detected in our sources were related to
microbes from this group. Bacteria of the genus Legionella populate aquatic environments but two
matching clones in the L. terrestris sources (gut content and cast libraries) and several bands from
Gammaproteobacteria-specific SSCP profile of soil sample related to that genus (sequence identity
92-96%); single OTU (band G-RA-33b) from the A. caliginosa cast sample linked to the genus
Legionella not closely (sequence identity 87%). Besides, certain clones from the compost and
earthworm A. caliginosa cast libraries linked to family Anaplasmataceae, which also included
symbionts of eukarya.
Free-living amoebae could serve as vehicles for obligate intracellular bacteria and as “Troyan
horse” in pathogenesis of animals (Barker and Brown, 1994). Amoebae and fungi were considered
as the main nutrient sources for earthworms (Edwards and Fletcher, 1988; Brown, 1995). Hence,
symbionts of amoebae are hypothetically able to change the host passing through the gut of
earthworms: symbiotic bacteria released in the gut from digested protozoa could infect intestinal gut
eucarya (for example roundworms detected in the gut of the earthworms) or earthworm itself and
grow massively inside of new host. It was reported that earthworms might become a vector for
mycobacteria (Fisher et al., 2002). Consequently, the symbiotic organisms of amoebae and other
protozoa could be judged as additional subsystem of microbial community of substrate incoming
into the earthworm gut.
Throughout our study we have detected some differences in microbial composition of gut wall and
gut content. Spatial dissimilarities in the gut microenvironments could be a possible explanation of
certain augmentation with particular gut-associated microorganisms. The item whether bacterial
populations in the substrates altered by gut-associated bacteria of earthworms or the differences
were only quantitative is still unclear.
4.2 Proteolytic activity of the soil and earthworm-modified microbial communities
According to the inhibitor assay, metalloproteases were the main enzymes caused unspecific
proteolytic activity of the soil aqueous extract. Proteases inhibited with aprotinin and leupeptine
(trypsin, chymotrypsin, trypsin-like and some other proteases) were not abundant and less active.
Unspecific proteolytic activity was significantly higher in water-soluble content from the
earthworm casts than in soil extracts and was mainly comprised by proteases inhibited with
4. Discussion
78
________________________________________________________________________________
aprotinin and leupeptin. At the same time enzymatic activity of metalloproteases in the soil and
earthworm casts was on approximately same level.
Metallopeptidases are present in both prokaryotes and eukaryotes and often have much in common,
in terms of both, structure and biochemical properties (Rawlings and Barrett, 1995). Eukaryotes (as
well as bacteria) can produce proteases of trypsin and chymotrypsin family. Chymotrypsin is
mainly produced by animals, trypsin-like enzymes are found in actinomycetes of the genera
Streptomyces and Saccharopolyspora, and in the fungus Fusarium oxysporum (Rawlings and
Barrett, 1994). The enhanced unspecific proteolytic activity of water-soluble earthworm cast
content was clearly based on trypsin- and chymtrypsin-like proteases derived from earthworm gut
system (earthworm gut tissue and host gut-associated organisms).
Proteolytic activity of the earthworm lumen content was previously reported for certain tropical
earthworms (Mishra et al., 1980) and for L. terrestris (Tillinghast et al., 2001). Protease activity in
the cast of earthworm Eudrilus eugeniae was lower than in the pig slurry (Aira et al., 2005). Several
alkaline serine proteases were purified from tissues of earthworms L. rubellus and E. fetida and
later characterized. Six alkaline proteases from L. rubellus consisted of single polypeptide chain
encoded by different genes. They hydrolyzed various proteins and the caseinolytic and fibrinolytic
activities of the enzymes was much higher than plasmin. Those proteases showed similarity to
mammalian serine proteases, however, neither arginine nor lysine residues were present in the
autolysis region. The enzymes were clearly distinct from other fibrinolytic enzymes, such as
plasmin and urokinase in amino acid composition. The proteases had long-term stable activity at
room temperature and a wide range of pH, being strongly resistant to organic solvents and
detergents. They were suggested to be trypsin-like and chymotrypsin-like serine proteases (Mihara
et al., 1991; Nakajima et al., 1993, 2002, 2003; Sugimoto and Nakajima, 2001; Cho et al., 2004; Hu
et al., 2005). Most of isozymes were strongly or partially inhibited with aprotinine (Nakajima et al.,
2003) but some recombinant was sensitive to EDTA (Hu et al., 2005). Seven purified proteins from
E. fetida were characterized as a “living fossil” in the evolution track of the chymotrypsin protein
family (Wang et al., 2003, 2005). Enzymes purified from Lumbricus rubellus and E. fetida
exhibited high homology each to other (Wang et al., 2003; Zhao et al., 2003). Yang and colleagues
(1997) characterized SDS-activated fibrinolytic enzyme from Eisenia fetida to consist of two
subunits multicatalytic enzyme, which has more than one active center. Leupeptin and aprotinin did
not affect this enzyme and the protease inhibitor E-64 even activated it.
Thus, even a single species of the earthworm does produce a wide range of different proteases.
Some of them are similar to those from other species, but certain enzymes are probably unique.
Tang and colleagues (2002) suggested that this enzymatic diversity is very advantageous for
4. Discussion
79
________________________________________________________________________________
earthworms to digest proteins efficiently under specific living and nutritional conditions during
migration through the substratum or upon passage different nutritional substrate through their gut.
However, the source of the proteases we detected in our study is rather uncertain. The enzymes
could be released from destructed bacteria (Gibson et al., 1989) and could also be produced in the
tissues of earthworm gut (Tillinghast et al., 2001) and transported into blood through the intestinal
wall (Fan et al., 2001). That looks reasonable because physiologically active part of soil bacterial
community did not significantly increase its number upon passage (ratio EUB338/DAPI) and
proteolytic activity of proteases inhibited with EDTA (most probably bacterial metalloproteases) in
the earthworm cast samples was the same or almost the same as in the soil aqueous extracts.
Therefore we have concluded that proteases enriched in the earthworm gut environment and thus
caused the high unspecific proteolytic activity of water-soluble content of the cast were derived
from earthworm tissues.
Substratum
Microbiota
of substratum
Cast
Cast
Earthworm
Earthworm
gut environment
proteases produced by the
the
earthworm tissues
gut-produced proteases
proteases
Earthworm gut-produced
inactivate those derived
derived from
from
the biota of substratum
substratum ???
???
Biota of
of cast
cast
Biota
earthworm
prion
???
???
???
Substratum augemnted with
proteases derived from its biota
0,16
0,14
0,12
Days
6
0,10
4
0,08
0,06
0,04
0,02
0,00
proteases derived
from the gut-associated
Proteolytic activity
activity of
of water-soluble
water-soluble contents
contents
Proteolytic
bacteria
0,045
gut-associated bacteria
2
1
Unscpecific: recRrP proteolysis:
main inhibitor - EDTA main inhibitors – aprotinine and leupeptine
OD of chromogenic substrate
substrate
OD of chromogenic substrate
substrate
Proteolytic activity
of water-soluble content
Substratum augemnted
augemnted with
with
Substratum
proteases derived
derived from
from the
the
proteases
earthworm gut
gut environment
environment
earthworm
0,16
0,16
0,14
0,14
0,136
0,136
Days
Days
0,119
0,119
0,12
0,12
66
0,10
0,10
44
0,08
0,08
0,06
0,06
22
0,04
0,04
0,02
0,02
0,00
0,00
11
Lt-EX
Lt-EX
Ac-EX
Ac-EX
Unscpecific
Unscpecific
Lt-EX
Lt-EX
Ac-EX
Ac-EX
recRrP proteolysis
proteolysis
recRrP
main inhibitors
inhibitors –– aprotinin
aprotinin and
and leupeptine
leupeptine
main
for both
both
for
Figure 32. The scheme of protease-producing agents in variuos compartments of earthwormassociated environment. Substratum proteases (mostly inhibited with EDTA) get augmented with
enzymes derived from the earthworm gut environment (mostly inhibited with aprotinin and
leupeptine), while recPrP proteolysis kept the same rate. Lt-EX – L. terrestris and Ac-EX –
A. caliginosa casts.
4. Discussion
80
________________________________________________________________________________
In contrast to unspecific proteolytic activity of the aqueous extracts, recombinant prion proteolysis
activity in all samples was comparable among the variants. Degradation of recPrP was observed
only in the aqueous extracts without inhibitors and with added Inhibitor Cocktail Set II uninhibited
trypsin- and chymotrypsin-family proteases. DMSO used as a solvent and protease inhibitor E-64
also presented in the Inhibitor Cocktail Set II could activate enzymes (Yang et al., 1997) and caused
a tiny increase of recPrP proteolysis observed in the variant with the Set II in comparison with
variant without inhibitors. Protease Inhibitor Cocktail Set III contained aprotinin and leupeptine
blocked completely recPrP-degrading enzymatic activity. As we have concluded above, the
earthworm gut produced the proteases that greatly increased unspecific proteolytic activity of
aqueous extract from the casts. But these proteases did not remarkably affect the recPrP degradation
in water-soluble content from the casts. Thus, we think that proteases derived from earthworm gut
system (neither organisms associated with earthworm intestine nor gut tissue) do not digest the
prion, and recPrP degradation was performed primarily by the proteases derived from soil
microorganisms. Those proteases belong to trypsin- and chymotrypsin-family produced by bacteria
and eukaryotes, as it was mentioned above and showed low unspecific proteolytic activity.
Pure microbial cultures exhibited different capacities for prion proteolysis but in the most cases
recPrP-degrading microbes reduced recPrP amounts to the levels below the detection. Taxonomic
distribution of the bacterial isolates capable for recPrP proteolysis was diverse but only 19% of
isolates in total showed capacity for recombinant prion proteolysis (Fig. 33). Alpha- and
Betaproteobacteria have shown low ability for recPrP digestion (1% from each class among the
total estimated isolates). Gammaproteobacteria (mostly genus Pseudomonas), Actinobacteria, and
Bacilli (only genus Bacillus) were more capable: 7%, 6%, and 4% respectively (Fig. 33). Bacteria
of CFB group were not abundant among the isolates (even being numerous in the communities) and
none of isolates showed recPrP proteolysis activity.
4. Discussion
81
________________________________________________________________________________
4%
81%
6%
7%
1%
1%
Bacteria non-digesting recPrP, total
Betaproteobacteria, recPrP-digesting
Actinobacteria, recPrP-digesting
Alphaproteobacteria, recPrP-digesting
Gammaproteobacteria, recPrP-digesting
Firmicutes (Bacilli), recPrP-digesting
Figure 33. Correlation between digesting and not digesting recPrP bacterial isolates.
Most of bacteria able to digest recPrP have hydrolyzed whole molecule, at least both epitope sites
of it were destroyed. But some species, for instance Pantoea agglomerans 571-2 clearly degraded
only N-terminus of the recombinant prion (epitope of PrPc248), while Aeromonas sobria 368-2 and
Pseudomonas sp. 9 have digested epitope of antibody VRQ14 (α-helix and interhelix loop). Fungi
were very active in recPrP digestion. Six among eight the most common isolates appeared recPrPproteolysis activity, digesting whole recPrP molecule.
Low proteolytic activity towards recPrP of aqueous extracts from studied soil and earthworm cast
could be explained with low quantity of bacterial groups potentially able for prion proteolysis
(Gammaproteobacteria, Bacilli (Firmicutes) and Actinobacteria). The population densities of all
these groups went down upon the transit through the gut and their total number in bacterial
population did not exceed 11%. Fungi are also efficient in recPrP digestion but they also were
eliminated from community upon gut passage (Kristufek et al., 1992; Toyota and Kimura, 1994;
Schönholzer et. al., 2002).
5. Conclutions
82
________________________________________________________________________________
5. Conclusions
•
Bacteria in the substrate undergo severe changes upon the gut passage. Independently of the
earthworm species and initial substrata, the common rules are: (i) increase of relative
densities of Cytophaga-Flavobacteria group of Bacteroidetes during the transit; (ii)
densities of Firmicutes and Gammaproteobacteria decreased in the earthworm cast
compared to substrata; (iii) numbers of Alpha-, Betaproteobacteria, and Actinobacteria
varied.
•
Many bacterial isolates (depending on taxonomic affiliation, up to 33%) from earthworms
did digest recPrP in pure cultures. Gammaproteobacteria, Bacilli, Actinobacteria, and fungi
were the most active potential degraders of recPrP in vitro.
•
Recombinant recPrP was definitely degraded in vitro in aqueous extracts of the earthworm
casts and the soil. This process took under given conditions up to 2-6 days. However,
additional experiments with labeled recPrP should be performed in the future to
unambiguously confirm the same in the system with presence solid soil particles and organic
matter.
•
Non-specific proteolytic activity of soil strongly increased during the transit through the
earthworm gut. The major contribution to that were the earthworm-produced enzymes.
However, this augmentation along with modification of microbial population in the
earthworm gut environment did not enhance the recPrP digestion. Most likely, the active
enzymatic fraction for recPrP proteolysis in the soil enzyme pool was constituted by trypsinand chymotrypsin-like proteases. The contribution to this pool of the earthworm itself and
earthworm gut microflora seems to be minimal. Thus, studied microbial-earthworm gut
systems do not produce proteases notably affecting the prion proteolysis.
References
83
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Supplementary materials
93
________________________________________________________________________________
Supplementary materials
Cm-E 10
99
S o il c lo n e
S M 2F 01 _A F 445727
99
P ir e llu la
71
s t a le y i _ A J 2 3 1 1 8 3
Lt-E X-E 9
S o il c lo n e W D 2 8 7
96
_ A J292682
I s ophaera
100
T herm al
100
s p . S c h le s n e r
s p r in g c lo n e
A 12_ A F 234727
Lt-GC-C7
Lt-E X-C11
Lt-GC-H5
Lt-E X-H9
Lt-GC-G8
Lt-E X-G12
67
100
77
G e m m a t a o b s c u r ig lo b u s _ A
V o lc a n ic d e p o s it s c lo n e
X ip h in e m a t o b a c t e r
C o n t a m in a t e d a q u if e r c lo n e
P eas hrub
100
r h iz o s p h a e r e
100
V e r r u c im ic r o b iu m
A F 217462
c lo n e 2 8 _ A Y 9 4 3 0 0 2
f u s if o r m is _ U 6 0 0 1 5
G la c ia l c r y o c o n it e
A k k e r m a n s ia
72
h o le c lo n e
m u c in ip h ila _
F r e s h w a t e r a q u if e r c lo n e
88
Verrucomicrobia
s p in o s u m _ X 9 0 5 1 5
P ros thec obac ter
100
C S 9 _A Y 124349
A Y 271254
C R 98-5-68
_A F 428798
Lt-GC-D6
Lt-E X-D10
100
S o il c lo n e
b r e v ic o li_
W C H D 3 -88 _A F 050561
Ac2-GC-B8
Ac-E X-G12
E f -E X-G10
100
66
J231191
1700a-47 _ A Y 917438
C a n d id a t u s
82
A s t er_20_29. 07 _ A Y 79567 7
Ac2-E X-B3
70
S e a w a t e r c lo n e
100
S A R 307 _A U 20798
S e a w a t e r c lo n e
84
Planctomycetes
F JQ B A B 9_A M 039543
A c t iv a t e d s lu d g e c lo n e
93
Archaea
640_ X 81959
Lt-GC-H7
Lt-E X-H11
S A R 202 _ A U 20797
Ac2-E X-G2
74
T r ic h lo r o b e n z e n e - t r a n s f o r m in g
68
c o n s o r t iu m c lo n e
Y e llo w s t o n e h o t s p r in g c lo n e
P e r m a f r o s t c lo n e
65
T r ic h lo r o b e n z e n e - t r a n s f o r m in g
P a s t u r e s o il c lo n e
74
S J A -68_ A J 009475
O P B 12_ A F 027031
Chloroflexi
21 _A Y 390996
c o n s o r t iu m c lo n e
S J A -116 _A J 009487
E C 1109 _D Q 083302
Lt-GC-C8
84
T h e r m o m ic r o b iu m
99
99
100
s p. S U B T -4_ A F 361218
Ac-E X-G10
Ac-E X-A8
70
DQ 1 2 5 7 7 8
Fibrobacter / Acidobacterium
Ac-E X-G9
100
H o lo p h a g a
sp. R B 24 _ Z 95717
Alpha
98
Beta
100
86
0 .0 6 7
0 .0 2 9
100
Gamma
0 .0 4
8
0 .0 5 3
Proteobacteria
Ac2-E X-H1
100
61
98
75
H o t s p r in g c lo n e
b o le t u s _ A J 2 3 3 9 0 8
G 13_ A F 407700
Ac2-GC-H8
S u lf a t e - r e d u c in g b a c t e r iu m
75
61
C ystobacter fusc us_ A J233898
M e lit t a n g iu m
57
98
D e s u lf u r o m o n a s
A K -01 _A F 141328
p a lm it a t is _ U 2 8 1 7 2
Delta
Cm-A6
98
Ac2-E X-F2
68
P e lo b a c t e r p r o p io n ic u s _
X 70954
G e o b a c t e r b r e m e n s is _ U 9 6 9 1 7
G eobac ter s p. C dA -3_Y 19191
Actinobacteria
100
Firmicutes
0.05
Bacteroides
Figure 34. Overview of bacterial diversity in the soil, compost, gut content and casts of three
earthworm species: SPs1 (autumn-collected soddy-podzolic soil); SPs 2 (spring-collected soddypodzolic soil); Cm – compost; Lt – L. terrestris; Ac – autumn-collected A. caliginosa; Ac2 –
spring-collected A. caliginosa; Ef – E. fetida; GW – gut wall; GC – gut content; EX – excrements.
Supplementary materials
94
________________________________________________________________________________
Cm-D9
N ot affiliated
SPs-G6
100
Un cu ltu r e d so il b a cte r iu m 6 0 7 - 2 _ AY3 2 6 6 0 9
Br e vu n d imo n a sve sicu la r is_ AY1 6 9 4 3 3
100
Ac-EX-D7_A10
60
Br e vu n d imo n a sd imin u ta_ X8 7 2 7 4
50
C aulobacteraceae
SPs-E6
93
H yphomicrobiaceae
De vo sia n e p tu n ia e_ AF4 6 9 0 7 2
80
De vo sia r ib o fla vin a_ AJ5 4 9 0 8 6
Ac2-EX-F5
100
N ot affiliated
Ac2-EX-F1
86
Un cu ltu r e d so il b a cte r iu m TSS3 _ AB0 7 5 1 6 7
Ac2-EX-A3
100
Sp h in g o mo n a ssp . 1 3 _ AY9 4 2 9 8 7
54
Sp h in g o mo n a swittich i _ AB0 2 1 4 9 2
95
Sp h in g o mo n a sa g r e stis_ Y1 2 8 0 3
76
96
Sphingomonadaceae
SPs-F5
Ac2-EX-F3
Sp h in g o mo n a ssu b e r ifa cie n s_ AY5 2 1 0 0 9
Sp h in g o p yxis witfla r ie n sis_ AJ4 1 6 4 1 0
54
Erythrobacteraceae
Ac2-EX-C5
80
Po r p h yr o b a cte rn e u sto n e n sis_ AF4 6 5 8 3 8
Er yth r o micr o b iu m_ r a mo su m
_ AF4 6 5 8 3 7
Ag r o b a cte r iu mtu me fa cie n s_ AY7 0 1 9 0 0
O ch r o b a ctr u mg a llin ifa e cis_ AJ5 1 9 9 3 9
99
72
Lt-GW-G2
B rucellaceae
100
O ch r o b a ctr u m g r ig n o n e n se
_ AJ2 4 2 5 8 0
68
Me so r h izo b iu m lo ti_ X6 7 2 2 9
95
Phyllobacteriaceae
SPs2-B8_A11
87
Me so r h izo b iu m p lu r ifa r iu m_ X6 8 3 9 1
Cm-D3
100
Un cu ltu r e d Be ije r in ckia sp . 3 1 _ AY9 4 2 9 5 7
68
SPs-H4
66
N ot affiliated
Ac2-GC-H12
90
Ac-GC-C4
100
Br a d yr h izo b iu m sp . SH2 8 3 0 1 _ AY1 4 1 9 8 2
76
Ac-EX-H8
Nitr o b a cte r win o g r a d skyi_ L 1 1 6 6 1
100
Afip ia ma ssilie n sis _ AY0 2 9 5 6 2
99
Ac2-EX-B5
53
86
B radyrhizobiaceae
Ac-GC-E6
SPs2-D9
Ac2-EX-G3
Afip ia b r o o me a e
_ AY5 1 3 5 0 5
Me th ylo cystis p a r vu s_ Y1 8 9 4 5
70
97
60
51
symb io n t cf. Me th ylo b a cte r iu m_ AF4 5 9 7 9 9
Me th ylo b a cte r iu m e xto r q u e n s_ D3 4 5 3 3
100
Methylobacteriaceae
Lt-GW-F4
Cm-A2
52
Sta p p ia ste llu la ta _ D8 8 5 2 5
83
100
Pa r vib a cu lu m la va me n tivo r a n s_ AY3 8 7 3 9 8
R hodobacteraceae
Lt-GW-C4
Lt-GW-H3
Un cu ltu r e d b a cte r iu mSPO TSAUG - 0 1 _ DQ 0 0 9 2 5 7
80
An a p la sma ce n tr a le_ AF2 8 3 0 0 7
Cm-D10
60
94
Ac-EX-D11
A naplasmataceae
e n d o symb io n t o f Aca n th a mo e b a
_ AF3 6 6 5 8 3
52
Tr o ja n e llath e ssa lo n ice s_ AF0 6 9 4 9 6
Ko za kia b a lie n sis_ AB0 5 6 3 1 9
100
86
71
Acid o mo n a sme th a n o lica_ D3 0 7 7 0
SPs-H6
Ac2-GC-E9
100
56
85
Un cu ltu r e d so il b a cte r iu m C1 1 5 7 _AF5 0 7 6 6 7
Ste lla va cu o la ta_ AJ5 3 5 7 1 1
A cetobacteraceae
Ste lla h u mo sa_ AJ5 3 5 7 1 0
Lt-GW-D2_A2
Ac2-GC-C9
0.05
100
Un cu ltu r e d so il b a cte r iu m KCM- B- 2 4 _ AJ5 8 1 5 8 3
Hyp h o micr o b iu m vu lg a r e_ Y1 4 3 0 2
Figure 35. Phylogenetic affiliation of the clones from Alphaproteobacteria in the soil, compost, gut
content and casts of three earthworm species: SPs1 (autumn-collected soddy-podzolic soil); SPs 2
(spring-collected soddy-podzolic soil); Cm – compost; Lt – L. terrestris; Ac – autumn-collected A.
caliginosa; Ac2 – spring-collected A. caliginosa; Ef – E. fetida; GW – gut wall; GC – gut content;
EX – excrements.
Supplementary materials
95
________________________________________________________________________________
A c -E X -G 7
100
N i tr o s o s p ir a b r ie n s is_ L 3 5 5 0 5
Nitrosomonadaceae
100
N i tr o s o l o b u s m u l ti fo r m is _ L 3 5 5 0 7
A c -G C -E 3
100
R a l s to n i a m a n n ito l i l y ti c a_ A Y 0 4 3 3 7 9
62
R a l s to n i a p ic k e tti i _ A Y 2 6 8 1 8 0
S P s 2 -B 1 0
83
A c -E X -B 9
A c 2 -E X -F 6
Burkholderiaceae
P a n d o r a e a a p i s ta _ A F 1 3 9 1 7 2
B u r k h o l d e r i a a n d r o p o g o n i s_ A B 0 2 1 4 2 2
88
S y m b i o n t o f T e tr a p o n e r a b in g h a m i_ A F 4 5 9 7 9 6
73
B u r k h o ld e r i a c e p a c ia_ X 8 7 2 7 5
99
A c -G C -F 1
A c -G C -D 5
L im n o b a c te r th i o o x i d a n s_ A J 2 8 9 8 8 5
B r a c h y m o n a s p e tr o l e o v o r a n s_ A Y 2 7 5 4 3 2
54
H y d r o g e n o p h a g afl a v a _ A B 0 2 1 4 2 0
C m -H 5
100
54
H y l e m o n e ll a g r a c i l i s_ A J 5 6 5 4 2 3
66
E f-G C -D 6 /A 6
E f-E X -C 7
E f-E X -E 9
66
95
93
E f-E X -G 5
L t-G W -F 1 /E 3
L t-G W -C 2
Comamonadaceae
D e l fti a a c i d o v o r a n s_ A F 5 3 8 9 3 0
75
X y l o p h i lu s a m p e l in u s_ A F 0 7 8 7 5 8
C o m a m o n a s te s to s te r o ni_ A B 0 6 4 3 1 8
97
84
A c -E X -C 7
R h o d o fe r a x fe r m e n ta n s_ D 1 6 2 1 1
94
51
V a r i o v o r a x p a r a d o x u s_ A J 4 2 0 3 2 9
E n d o s y m b i o n t o f A c a n th a m o e b a_ A Y 5 4 9 5 5 1
75
L t-E X -F 9
66
L t-G C -F 5
R u b r i v i v a x g e l a ti n o s u s_ D 1 6 2 1 3
C m -H 4
P s e u d o m o n a s s a c c h a r o p h i l a_ A B 0 2 1 4 0 7
89
S P s 2 -E 7
Unclassified Burkholderiales
Id e o n e l l a d e c h l o r a ta n s_ X 7 2 7 2 4
L e p to th r i x d i s c o p h o r a_ L 3 3 9 7 5
A c h r o m o b a c te r x y l o s o x id a n s_ A F 4 1 1 0 2 1
78
B o r d e te l l a b r o n c h i s e p ti c a_ B X 6 4 0 4 4 7
A c 2 -E X -C 7
S P s 2 -B 9
A c h r o m o b a c te r i n s o l i tu s _ A Y 1 7 0 8 4 7
B o r d e te l l a a v i u m _ A F 1 7 7 6 6 6
S P s -E 5
A c 2 -E X -C 6 / G 6 /H 6 / C 3 / C 4
E f-G C -H 7
Alcaligenaceae
B o r d e te l l a h in z i i_ A F 1 7 7 6 6 7
57
S P s -D 2
82
A c h r o m o b a c te r r u h l a n d i i_ A F 2 0 5 3 7 0
B o r d e te ll a tr e m a tu m _ A J 2 7 7 7 9 8
77
B o r d e te l la p e r tu s s is _ B X 6 4 0 4 2 0
51
0 .0 2
D e r x ia g u m m o s a_ A B 0 8 9 4 8 1
S P s -B 5
Figure 36. Phylogenetic affiliation of the clones from Betaproteobacteria in the soil, compost, gut
content and casts of three earthworm species: SPs1 (autumn-collected soddy-podzolic soil); SPs 2
(spring-collected soddy-podzolic soil); Cm – compost; Lt – L. terrestris; Ac – autumn-collected A.
caliginosa; Ac2 – spring-collected A. caliginosa; Ef – E. fetida; GW – gut wall; GC – gut content;
EX – excrements.
Supplementary materials
96
________________________________________________________________________________
100
97
H y d r o c a r b o n ip h a g a
N e v s k ia
effusa_A Y 363245
ram os a _A J 001343
S P s-A 4
D e e p m a r in e s e d im e n t s
r ib o c lo n e
1 1 7 4 -1 0 9 1 -1 4 _ A B 1 2 8 8 7 9
S P s-C 6
C m -B 6
L T -E X -B 12
U N O X a c t iv a t e d s lu d g e
r ib o c lo n e
H S -36_A Y 157114
C m -F 8
C m -H 9
C m -G 4
92
S P s-D 3
P e n g u in d r o p p in g s s e d im e n t s
100
57
r ib o c lo n e
K D 8-62_A Y 218689
A c 2 -E X -G 5
S o il r ib o c lo n e
Lysobacter
56
X y le lla
Xanthomonadaceae
217_D Q 158103
brunes c ens _A B 161360
f a s t id io s a _ A F 5 3 6 7 7 0
P seudoxanthom onas
broegbernens
S tenotrophom onas
Lysobacter
m a lt o p h ilia _
_A J012231
L28166
enzym ogenes _A J29829
A c-E X -C 10
F u lv im o n a s
s o li _ A J 3 1 1 6 5 3
R hodanobacter
lin d a n o c la s t ic u s
_A F 039167
S P s-E 3
S P s-A 6
S P s-E 2
S P s-G 2
R hodanobacter
f u lv u s _ A B 1 0 0 6 0 8
S P s -B 2
68
A c2-E X -F 4
P seudom onas
p u t id a _ Z 7 6 6 6 7
S P s-D 4__F 4
A c2-G C -F 8_G 9
P seudom onas
m ig u la e _ A Y 0 4 7 2 1 8
P seudom onas
r h o d e s ia e
_A F 064459
L t-G C -H 8
P seudom onas
m a r g in a lis
_A F 36409
L t-E X -H 1 2
s y m b io n t c f . P s e u d o m o n a s
_A F 459797
A c2-G C -H 9
P seudom onas
reac tans _A F 255337
P seudom onas
t o la a s ii _ A F 2 5 5 3 3 6
P seudom onas
Pseudomonadaceae
g in g e r i _ A F 3 2 0 9 9 1
L t-G C -A 7
L t-E X -A 11
58
P seudom onas
b r a s s ic a c e a r u m
_A J292381
E f-G C -A 1 _ F 2
E f-G C -C 6
100
P seudom onas
a e r u g in o s a
P seudom onas
s p . A r c t ic - P 2 3 _ A Y 5 7 3 0 3 2
_Z 76672
S P s-E 11
A c-E X -E 11
S P s-B 7
A c -E X -B 7 _ B 8
81
S y m b io n t
LP 2
of Ly rodus
p e d ic e lla t u s _
A Y 150184
C m -C 5
N ot affiliated
C m -A 10
E f-G C -A 11
98
100
L t-G C -G 7
L t-E X -G 11
L e g io n e lla
lo n g b e a c h a e
L e g io n e lla
L e g io n e lla
_M 36029
b u s a n e n s is
_A F 424887
p n e u m o p h ila
A erom onas
Legionellaceae
s u b s p . p a s c u lle i _ A F 1 2 2 8 8 5
v e r o n ii b v . v e r o n ii _ A Y 9 2 8 4 7 3
S P s-C 11
100
A erom onas
c u lic ic o la _ A Y 3 4 7 6 8 2
A erom onas
a llo s a c c h a r o p h ila
A erom onas
h y d r o p h ila
_S 39232
_X 60404
L T -G W -A 3__G 3__H 1
A c-E X -F 7__F 10
A eromonadaceae
A c-E X -H 7__F 8
E f-G C -A 5
A c-E X -A 9
A c-E X -H 9
E f-G C -G 6/3
E f-E X -A 5
A erom onas
99
S h e w a n e lla
sp. D 6_D Q 103510
d e n it r if ic a n s _ A J 3 1 1
C m -C 8
A c-E X -G 8
L t-G W -D 1
S h e w a n e lla
b a lt ic a _ A J 0 0 0 2 1 4
S h e w a n e lla
p u t r e f a c ie n s _ A F 0 0 6 6
Shewanellaceae
C m -G 2
A c-E X -H 6
63
S P s2-D 11__B 11
B r e n n e r ia
s a lic is _ J 2 3 3 4 1 9
E f-G C -F 4_G 6/1
E s c h e r ic h ia
c o li _ N C 0 0 4 4 3 1
E s c h e r ic h ia
S h ig e lla
v u ln e r is _ X 8 0 7 3 4
f le x n e r i _ X 8 0 6 7 9
S h ig e lla
sonnei _X 80726
E s c h e r ic h ia
S h ig e lla
a lb e r t ii _ A Y 6 9 6 6 6 9
Enterobacteriaceae
b o y d ii _ A Y 6 9 6 6 6 8
A c-G C -G 2
S h ig e lla
0 .0 2
d y s e n t e r ia e _ X 8 0 6 8 0
E f-E X -C 8
S P s-B 6
A c-G C -B 2
Figure 37. Phylogenetic affiliation of the clones from Gammaproteobacteria in the soil, compost,
gut content and casts of three earthworm species: SPs1 (autumn-collected soddy-podzolic soil); SPs
2 (spring-collected soddy-podzolic soil); Cm – compost; Lt – L. terrestris; Ac – autumn-collected
A. caliginosa; Ac2 – spring-collected A. caliginosa; Ef – E. fetida; GW – gut wall; GC – gut
content; EX – excrements.
Supplementary materials
97
________________________________________________________________________________
100
SPs 2 -A 8
SPs 2 -F 9
Ef-G C -G 7
A c - G C - A 2 ,- A 3 , - D 4
S y m b i o n t c f . F l a v o b a c t e ri u m o f T e t ra p o n e ra b i n g h a m i i _ A F 4 5 9 7 9 5
SPs -G 1
92
Crenotrichaceae
S P s 2 - D 1 2 ,- G 7 , - D 7 , - E 9
SPs -A 5
SPs 2 -G 1 2
A c -G C -F 3 , -E4
A c 2 -EX-G 7 , -A 6
SPs 2 -H 7
87
F l e x i b a c t e r s a n c t i_ A B 0 7 8 0 6 7
C h i t i n o p h a g a p i n e n s i s_ A F 0 7 8 7 7 5
F l e x i b a c t e r f i l i f o rm i s_ A B 0 7 8 0 4 7
L e e u w e n h o e k i e l l a m a ri n o f l a v a_ A Y 1 6 7 3 1 5
58
80
F l e x i b a c t e r a g g re g a n s _ A B 0 7 8 0 3 9
C m -F 1
L t-G C -A 6
L t-EX-A 1 0
A c -G C -C 5
F l a v o b a c t e ri u m f ri g o ri s _ A J 5 5 7 8 8 7
L t-G C -D 8
L t-EX-D 1 2
A c - G C - H 5 ,- E 4
A c - G C - B 6 ,- F 5
93
L t-G C -H 6
L t-EX-H 1 0
L t-G C -D 5
L t-EX-D 9
100
F l a v o b a c t e ri u m x i n j i a n g e n s e_ A F 4 3 3 1 7 3
Flavobacteriaceae
A c 2 - G C - E 1 2 ,- G 8
F l a v o b a c t e ri u m l i m i c o l a_ A B 0 7 5 2 3 2
A c -EX-B 1 1
L t-G C -E7
L t-EX-E1 1
A c -G C -E5
A c 2 -G C -C 1 1 _ A 1 1 _ C 8
F l a v o b a c t e ri u m d e g e rl a c h e i_ A J 4 4 1 0 0 5
C m -F 7
52
F l a v o b a c t e ri u m c o l u m n a re_ A B 0 1 0 9 5 1
F l a v o b a c t e ri u m h y d a t i s_ M 5 8 7 6 4
F l a v o b a c t e ri u m o m n i v o ru m _ A F 4 3 3 1 7 4
F l a v o b a c t e ri u m a n t a rc t i c u m _ A Y 5 8 1 1 1 3
F l a v o b a c t e ri u m p s y c h ro p h i l u m_ A B 2 1 9 5 1 7
99
A c -G C -G 4
Ef-EX-E4
F l a v o b a c t e ri u m j o h n s o n i a e
_A J585227
A c -EX-B 1 0
B a c t e ro i d e s f ra g i l i s_ X 8 3 9 4 0
Ef-G C -B 4
100
F l e x i t h ri x d o ro t h e a e _ A F 0 3 9 2 9 6
C m -C 1 0
C m -B 1 0 , -E9
Flammeovirgaceae
Ef-G C -B 1 1
68
S p h i n g o b a c t e ri u m s p i ri t i v o ru m_ M 5 8 7 7 8
F l e x i b a c t e r c a n a d e n s i s_ A B 0 7 8 0 4 6
Ef-EX-B 7
68
C m -C 4
C m -H 2 , -B 7
Flexibacteraceae
A c 2 -G C -G 9
89
C m -H 3
F l e x i b a c t e r f l e x i l i s_ A B 0 7 8 0 5 3
0.05
C y c l o b a c t e ri u m m a ri n u m _ A Y 5 3 3 6 6 5
Figure 38. Phylogenetic affiliation of the clones from CFB in the soil, compost, gut content and
casts of three earthworm species: SPs1 (autumn-collected soddy-podzolic soil); SPs 2 (springcollected soddy-podzolic soil); Cm – compost; Lt – L. terrestris; Ac – autumn-collected A.
caliginosa; Ac2 – spring-collected A. caliginosa; Ef – E. fetida; GW – gut wall; GC – gut content;
EX – excrements.
Supplementary materials
98
________________________________________________________________________________
A c2-GC -A 12
55
R u b r o b a c t e r r a d io d u r a n s
Rubrobacteraceae
_U 65647
63 A c2-GC -A 9
P o p la r t r e e
100
r iz o s p h e r e
r ib o c lo n e
20B S U 5 _A J863183
Not affiliated
A c2-GC -B 9
70
A c2-EX-G4
C m-A 4
75
Not affiliated
A c2-GC -D 9
97
B io r e c t o r r ib o c lo n e
85
P o p la r t r e e
92
9-7 _A Y 7 55380
r iz o s p h e r e
r ib o c lo n e
20B S U 18 _A J863190
A c2-GC -E8
Not affiliated
A c2-GC -F9
96 A c2-EX-C 9
B r e v ib a c t e r iu m
p ic t u r a e
Brevibacteriaceae
B r e v ib a c t e r iu m
a u r a n t ia c u m
B r e v ib a c t e r iu m
lin e n s
B r e v ib a c t e r iu m
56
62 M ic r o b a c t e r iu m
_X 76566
_ X 77451
casei _A Y 468365
p h y llo s p h a e r a e
_A J277840
k e r a t a n o ly t ic u m
_Y 17233
d e x t r a n o ly t ic u m
_D 21341
M ic r o b a c t e r iu m
98
_A J620364
A c2-EX-C 12
58
100
Lt-GW -G1
A c2-GC -A 8
89
58
Microbacteriaceae
M ic r o b a c t e r iu m
C la v ib a c t e r
t o x ic u s _ D 8 4 1 2 7
85
R a t h a y ib a c t e r t r it ic i _ X 7 7 4 3 8
95
A c2-EX-C 2
A c2-EX-D 6
A r t h r o b a c t e r c h lo r o p h e n o lic u s
67
A r t h r o b a c t e r s u lf u r e u s
A r t h r o b a c t e r g lo b if o r m is
A r t h r o b a c t e r a g ilis
99
M ic r o c o c c u s
50
_A F 102267
A c2-EX-D 10
M ic r o c o c c u s
a n t a r c t ic u s
_X 83409
_ A B 098573
_X 80748
_A J005932
Micrococcaceae
lu t e u s _ A J 5 3 6 1 9 8
A c2-EX-A 11
A c2-EX-E12
67
A c2-EX-E10
K o c u r ia
100
p a lu s t r is
_Y 16263
A c2-EX-E8
57
N o c a r d io id e s
n it r o p h e n o lic u s
N o c a r d io id e s
p la n t a r u m
N o c a r d io id e s
87
N o c a r d io id e s
a lb u s
a q u it e r r a e
_A F 005024
_Z 78211
Nocardiodaceae
_A F 005005
_ A F 529063
Lt-GC -A 8
Lt-EX-A 12
M y c o b a c t e r iu m
g o o d ii_
M y c o b a c t e r iu m
m a g e r it e n s e
100
M y c o b a c t e r iu m
99
Lt-EX-B 11
c h it a e
A Y 547079
Mycobacteriaceae
_ A Y 457076
_X 55603
Acidimicrobiaceae
Ef-EX-F9
88
A c id im ic r o b iu m
f e r r o o x id a n s
_U 75647
SPs-B 4
100
P a e n ib a c illu s
100
62
agarex edens
A n e u r in ib a c illu s
100
_A J345020
a n e u r in ily t ic u s
B r e v ib a c illu s
_A B 210964
Paenibacillaceae
in v o c a t u s _ A F 3 7 8 2 3 2
SPs2-C 8
A c-GC -B 4
100
G e o b a c illu s
sp. T d_A J564615
A c2-EX-A 5
84
SPs2-C 7
A c-GC -H 1
Thermophilic
SPs2-H 8
A c2-EX-H 5,B 7
SPs2-A 12
93
B a c illu s
95
95
sp. H A 6_A Y 753654
C om post
r ib o c lo n e
A c-GC -H 3
A c2-EX-A 7,B 6
54
Ef-EX-C 5
75
B a c illu s
63
63
B a c illu s
B a c illu s s im p le x
P h18 _A Y 833099
C m-A 5
R u m in o c o c c u s
86
100
_A J311893
Lt-EX-F12
98
99
s p h a e r ic u s
c e reus _A Y 986507
100
Lt-GC -B 5
Lt-EX-B 9
89
0 .05
Bacillaceae
044 _A B 128824
A c-GC -C 3
a lb u s _ X 8 5 0 9 8
Lachnospiraceae
C m-H 8
C m-B 8
100
R u m in o c o c c u s
s c h in k ii _ X 9 4 9 6 4
C m-F9
Figure 39. Phylogenetic affiliation of the clones from Actinobacteria and Firmicutes in the soil,
compost, gut content and casts of three earthworm species: SPs1 (autumn-collected soddy-podzolic
soil); SPs 2 (spring-collected soddy-podzolic soil); Cm – compost; Lt – L. terrestris; Ac – autumncollected A. caliginosa; Ac2 – spring-collected A. caliginosa; Ef – E. fetida; GW – gut wall; GC –
gut content; EX – excrement.
Curriculum Vitae
99
________________________________________________________________________________
Curriculum Vitae
Personal data:
Surname:
Given names
Residence
Nechitaylo
Taras
Erlenkamp 27A
38126 Braunschweig
Date of birth 02.06.1974
Place of birth Krasnodar / Russia
Nationality
Russian
Marital status Married
School Education:
09.1981 – 06.1991 secondary school, Krasnodar / Russia; school-leaving certificate
Higher Education
07.1991 – 07.1998
Kuban State University, Krasnodar / Russia, Diploma
profession: Biologist
speciality: Zoology of Vertebrates
Positions held
02.12.1992 – 28.02.1993
Laboratory assistant, Kuban Ecologic – Biological Centre,
Krasnodar
01.03.1993 – 30.09.1994
Laboratory assistant, Biochemical Chair of Kuban State
Agricultural University, Krasnodar
22.11.1999 – 28.02.2001
Scientist, Biotechnology Centre, Kuban State University,
Krasnodar
19.03.2001 – 14.07.2002
Scientist, Research Institute of Applied Ecology, Krasnodar
01.09.2001 – 31.05.2002
Senior Teacher, Chair of Biology and Ecology, Kuban State
Agricultural University, Krasnodar
01.01.2003 – 31.12.2005
PhD Student, Helmholtz Zenrum für Infekzionsforschung
(former GBF)
Curriculum Vitae
100
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Publications
Nechitaylo, T.J., & Peskova T.J. Effect of low concentrations of heavy metals on embryonal and
larval development of amphibia. (Poster) 11. Interrepublical Scientific Conference “Live
Issues of Ecology and Natural Protection of South Regions of Russia and Frontier
Territories”, Krasnodar (1999).
Giritch, I.E., Nechitaylo, T.J. Khudokormov, A.A., & Melnicov, D.A. Selection of microbial
strains capable of utilize long-chain fractions of hydrocarbons. (Poster) 1. International
Interuniversity Seminar “Ecology – 2000”, Moscow. (2000)
Malachov, A.A., Giritch, I.E., Nechitaylo, T.J., & Karasiova, E.V. Role of hydrocarbon –
degrading microflora in the bioremediation oil-contaminated soils. (Poster) 1.
International Interuniversity Seminar “Ecology – 2000”. Moscow, (2000).
Nechitaylo, T.J. & Khudokormov, A. A. Application of different types of “Kristalon” for removal
hydrocarbons by soil’s microorganisms in model experiments. (Poster). Ecological
Problems of Kuban Region. Krasnodar, (2001).
Nechitaylo, T.J. Effect of various concentrations of crude soil on the soya and soils microflora.
(Poster) 3. Practical Conference of Young Scientists “Scientifical Maintenance of
Agriculture”, Krasnodar. (2001).
Schneiker, S., Dos Santos, V.A., Bartels, D., Bekel, T., Brecht, M., Buhrmester, J.,
Chernikova, T.N., Denaro, R., Ferrer, M., Gertler, C., Goesmann, A., Golyshina, O.V.,
Kaminski, F., Khachane, A.N., Lang, S., Linke, B., McHardy, A.C., Meyer, F.,
Nechitaylo, T., Puhler, A., Regenhardt, D., Rupp, O., Sabirova, J.S., Selbitschka, W.,
Yakimov, M.M., Timmis, K.N., Vorholter, F.J., Weidner, S., Kaiser, O., &
Golyshin, P.N. Genome sequence of the ubiquitous hydrocarbon-degrading marine
bacterium Alcanivorax borkumensis. Nat Biotechnol. Jul 30; (2006) [Epub ahead of
print].