Download Conference22 - Zodletone Spring

Phylogenetic and metabolic diversity of the phylum Planctomycetes from an anaerobic, sulfide and sulfur-rich
spring (Zodletone Spring, OK, USA)
Mostafa S. Elshahed1, Noha H. Youssef1, Qingwei Luo2, Fares Z. Najar2, Bruce A. Roe2, Tracy M. Sisk2, Solveig M. Buehring3, Kai-Uwe Hinrichs3, and Lee R. Krumholz2
1Oklahoma State University, Stillwater, OK, 2University of Oklahoma, Norman, OK, and 3University of Bremen, Germany
Abstract
Culture-independent analysis of the Planctomycetes community at Zodletone spring source sediments
• We investigated the phylogenetic diversity and metabolic capabilities of the phylum Planctomycetes in
the anaerobic sediments of a mesophilic spring (Zodletone spring) in southwestern Oklahoma.
1. Anaerobic reduction of elemental sulfur
• Culture-independent analysis of 16S rRNA gene sequences suggested that an extremely diverse
community of Planctomycetes is present at the spring. Although sequences that are phylogenetically
affiliated with cultured heterotrophic Planctomycetes were identified, the majority of the sequences
belonged to several globally distributed, yet-uncultured Planctomycetes lineages.
• 16S rRNA gene sequences generated using
Planctomycetes-biased
primer
pairs
(Pln46F/U1390R,
and
Eub338F-0III/Pln930R suggested that an extremely
diverse community of Planctomycetes is
present at the spring.
• Using complex organic media, we isolated two novel strains that belonged to the PirellulaRhodopirellula-Blastopirellula clade within the Planctomycetes. Both strains had an identical 16S rRNA
gene sequence, and their closest relatives were isolates from Kiel Fjord (Germany), Keauhou beach
(Hawaii), a marine aquarium, and tissues of marine organisms (Aplysina sponge and post larvae of the
giant tiger prawn Penaeus monodon). The closest validly described cultured relative of these strains was
Blastopirellula marina (93.9% sequence similarity).
• Only a small fraction of the clones in this
study (3 OTUs) were monophyletic with
heterotrophic cultured representatives of this
phylum (class Planctomyceteacea, genera
Planctomyces, Pirellula, Blastopirellula,
Rhodopirellula, Isosphaera, and Gemmata).
• Detailed characterization of one of the isolates (strain Zi62) revealed its ability to reduce elemental
sulfur to sulfide under anaerobic conditions, as well as its ability to produce acids from sugars, both
characteristics could potentially allow strain Zi62 to survive and grow in the anaerobic, sulfide, and
sulfur-rich environment at the spring source.
• The majority of sequences described here
using the Planctomycetes-biased primers were
associated with candidate class WPS-1 (27
OTUs). These OTUs belonged to three
different monophyletic lineages within this
class (group a, b, and c in figure 1).
Interestingly, many of the most similar clones
to Zodletone OTUs have been reported to
occur in anaerobic environments.
• Based on the geochemical characteristics of this site, we have concluded that the capability of
anaerobic metabolism is widely distributed among all major Planctomycetes lineages. Carbohydrate
fermentation and sulfur reduction are possible mechanisms employed by heterotrophic Planctomycetes
for growth and survival under anaerobic conditions.
• In addition, several non-Planctomycetes
type rRNA clones were identified, and most
of these phylotypes belonged to yetuncultured bacterial candidate divisions that
have previously been shown to be prevalent in
anaerobic environments, such as candidate
divisions OP11, OD1, WW1, WW2, WS3,
OP3, in addition to Chlorobia, and
Verrucomicrobia.
Introduction
Phylum Planctomycetes
• First described based on microscopic observation in 1924, first pure culture representative obtained in pure
culture in 1973 in dilute organic media (1).
• Planctomycetes strains had been isolated from a variety of marine, and freshwater environments, such as
marine brackish sediments, freshwater sediments, tissues of marine organisms, hot springs, and soil (2-6)
Quic kTime™ and a
TIFF (Unc ompres sed) dec ompres sor
are needed to see t his pic ture.
National Science Foundation
Microbial Observatories Program
Potential adaptations of strain Zi62 to anaerobic environments
2500
• Strain Zi62 reduced elemental sulfur to sulfide under
anaerobic conditions, and the production of sulfide was
coupled to the disappearance of the sulfur precipitate in
active incubations.
•Anaerobic sulfur respiration is mediated by
polysulfide reductase, which catalyzes the reduction of
polysulfide to sulfide. Analysis of the R. Baltica and B.
marina genomes indicates the presence of putative
genes encoding the three different enzyme subunits
(PsrA, B, C) in both microorganisms.
Sulfide concentration (M)
N 147
Funding Provided By
Oklahoma State University
Department of Microbiology and Molecular
Genetics
315 LSE building
Stillwater, OK 74078
[email protected]
2000
1500
1000
500
0
0
25
50
Time (days)
75
Plot of sulfide
production over time
in anaerobic medium
(--) inoculated, with
elemental sulfur and
substrate (sucrose
0.1%) added, (--)
inoculated, with
elemental sulfur but
no substrate, (--)
uninoculated, with
elemental sulfur and
substrate (--)
Uninoculated,with no
elemental sulfur and
no substrate. All
100 values shown are
average of triplicate
tubes.
putative psrC gene
identified in Zi62 using
PCR. The tree was
constructed using
partial (920bp)
sequence of putative
psrC amplified using
primer pair 262F and
1162R (R. baltica) PsrC
• Primers designed to target conserved sequences in the
psrC gene of R. baltica and B. marina (Psr262F,
Psr1162R) was used to test for the presence of a
polysulfide reductase gene homolog in strain Zi62.
•Phylogenetic analysis of the translated amino acid
sequence of the PCR product obtained using Zi62
genomic DNA indicated that this peptide is most
closely related to B. marina, and R. baltica PsrC
subunits.
gene numbering).
2. Acid production from sugars.
• Strain Zi62 produced acids from carbohydrates (glucose, fructose,
sucrose, trehalose, mannose, and xylose).
• Succinate and acetate were the major products accumulating, with
minor amounts of lactate, propionate, and formate. The pattern of
product formation suggest a mixed acid fermentation pathway.
• Repeated attempts to grow strain Zi62 under anaerobic fermentative
conditions, either in complex (rumen fluid, yeast extract), or defined
medium (glucose, sucrose, N-acetylglucosamine) were not successful.
• 16S rRNA gene-based surveys suggest that the Planctomycetes are ubiquitous in aerobic and anaerobic
habitats, and that novel, yet uncultured lineages within the group exists.
• Of special interest is the phylogeny and metabolic capabilities of members of the heterotrophic Planctomycetes
in anaerobic habitats, since all described heterotrophic Planctomycetes are aerobic, chemoroganotrophic
(a)
Zodletone Spring
• An artesian sulfur-containing, mesophilic spring located in the Anadarko Basin in Southwestern Oklahoma.
Two isolates, were obtained on plates containing either source sediment aqueous extracts or 5%
rumen fluid as a sole carbon source. Strain Zi62 was successfully purified by re-streaking on
media with N-acetylglucosamine as the sole carbon and nitrogen source, and with ampicillin and
cycloheximide (50 g/ml each) to inhibit the growth of other Bacteria and Fungi.
CM
• Surface water contains abundant sulfide (8-10 mM) and zero valent sulfur (1-1.5 mM), as well as methane,
ethane, and propane.
• The source is a contained area, approximately 1
15 cm.
m2,
Pa
filled with biomass and soft sediments to a depth of at least
Isolation of heterotrophic Planctomycetes from Zodletone spring
(b)
Zi62 cells are ovoid to pear-shaped, 0.6-1.2µm x 1.6-2.2µm, and occur in singles, pairs or in
rosette formations attached at the smaller cell pole.
n
• The spring flows for approx. 20 meters before discharging into stinking creek.
• 16S rRNA gene analysis using Bacteria specific primers identified Planctomycetes as members of the bacterial
community at the spring source
P
A. Negative stained electron micrograph of strain Zi62,
showing crateriform like structures (CM) covering one
third of the cell, and a single flagellum.
B. Thin sectioned micrograph of strain Zi62 showing the
pirellulosome (Pl), the paryphoplasm (Pa), and the
nucleoid structure (n).
Negatively stained cells showed that, similar to other Planctomycetes, crateriform-like structures
are present, occurs only at one pole of the cell, and covers one-fourth to one-third of the cell
surface. Thin-sectioned cells showed the presence of an intracellular membrane (ICM), dividing
the cell into a pirellulosome and the paryphoplasm. A dense nucleoid structure was also
observed that covered 20-30% of the pirellulosome.
Strain Zi62 represents a new genus within the Planctomycetes
Strain Zi62 within the Pirellula – Rhodopirellula – Blastopirellula (PRB) clade within the family
Planctomycetaceae.
Overview of Zodletone spring
Source of the spring
Goal
Strain Zi62 formed a monophyletic lineage with strong bootstrap support with a group of 10
Planctomycetes (98.8% to 96.7% sequence similarity). All 10 strains were isolated from marine
habitats: (Postlarvae of the giant tiger prawn Penaeus monodon, tissue of the Aplysina sponge, Kiel
Fjord, a marine aquarium, and Keauhou Beach, Hawaii)
To determine the level of phylogenetic diversity as well as possible metabolic pathways
utilized by members of the Planctomycetes in this anaerobic sulfide-saturated, hydrocarbonimpacted environment.
The closest recognized species to strain Zi62 is Blastopirellula marina (93.9% 16S rRNA gene
sequence similarity). Strain Zi62 was 87.3% and 86.8% similar to the 16S rRNA genes of Pirellula
staleyi and Rhodopirellula baltica the other two recognized species within the PRB clade
Conclusions
• An extremely diverse Planctomycetes population was identified in anaerobic, sulfide-saturated sediments at the
source of Zodletone spring, suggesting that capability for anaerobic metabolism is widely distributed among members
of this phylum.
• A novel Planctomycete, strain Zi62 was isolated from the spring source using a complex soil-extract based medium.
Strain Zi62 putatively represents a novel genus within the PRB clade within the Planctomycetaceae.
• Strain Zi62 is capable of reducing elemental sulfur to sulfide, as well as acid production from sugars, suggesting
sulfur reduction and carbohydrate fermentation as two possible mechanisms employed by heterotrophic
Planctomycetes for growth and survival under anaerobic conditions.
References
1.
2.
3.
Kiel fjord strain 116 (AJ231181)
Aplysina sponge strain 16 (AF453519)
Keauhou beach strain 611 (AJ231179)
Marine aquarium strain 391 (AJ231180)
Giant tiger prawn strain AGA M12 (X86391)
Aplysina sponge strain18 (AF453518)
Strain Zi62
Blastopirellula marina (X62912)
Pirellula staleyi (AJ231183)
Rhodopirellula baltica (BX294149)
Planctomyces brasiliensis (AJ231190)
Isosphaera pallida (AJ231195)
Gemmata obscuriglobus (X56305)
Planctomyces limnophilus (X62911)
Candidatus “Kuenenia stuttgartien” (AF375995)
Candidatus “Brocadia anammoxidans” (AF375994)
Candidatus “Scalindua brodae” (AY254883)
E. Coli (J01695)
0.05 substitutions/site
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