Download Isolation and characterization of a Nocardiopsis sp. from honeybee

RESEARCH LETTER
Isolation and characterization of a Nocardiopsis sp. from
honeybee guts
Preeti B. Patil, Yu Zeng, Tami Coursey, Preston Houston, Iain Miller & Shawn Chen
Molecular and Cellular Biology Program, Department of Biological Sciences, Ohio University, Athens, OH, USA
Correspondence: Shawn Chen, Molecular
and Cellular Biology Program, Department of
Biological Sciences, Ohio University, Athens,
OH 45701, USA. Tel.: 11 740 597 3112; fax:
11 740 593 0300; e-mail: [email protected]
Received 21 April 2010; revised 24 June 2010;
accepted 23 August 2010.
Final version published online 16 September
2010.
DOI:10.1111/j.1574-6968.2010.02104.x
Editor: Michael Bidochka
MICROBIOLOGY LETTERS
Keywords
actinomycetes; honeybee gut microbiota;
Nocardiopsis; antagonistic activities;
phenazines.
Abstract
Although actinomycetes are the plant-associated environmental bacteria best
known for producing thousands of antibiotics, their presence in the guts of
flower-feeding honeybees has rarely been reported. Here, we report on the selective
isolation of actinomycetes from the gut microbiota of healthy honeybees, and their
inhibitory activity against honeybee indigenous bacteria. More than 70% of the
sampled honeybees (N 4 40) in a season carried at least one CFU of actinomycete.
The isolates from bees of one location produced inhibitory bioactivities that were
almost exclusively against several bee indigenous Bacillus strains and Grampositive human pathogens but not Escherichia coli. An antibiotic-producing
actinomycete closely related to Nocardiopsis alba was isolated from the guts in
every season of the year. A DNA fragment encoding a homologous gene (phzD)
involved in phenazine biosynthesis was identified in the isolate. Expression of the
phzD detected by reverse transcription-PCR can explain the survival of this
organism in anaerobic environments as some redox-active extracellular phenazines
are commonly regarded as respiratory electron acceptors. The results raise
important questions concerning the roles of the antibiotic-producing actinomycetes and the phenazine-like molecules in honeybee guts and honey.
Introduction
Insect digestive tracts support communities of symbiotic
and transient microorganisms that are increasingly the
subjects of studies of microbial diversity and novel bioactive
microbial products (Breznak, 2004; Evans & Armstrong,
2006). In general, insect gut microbiota make significant
contributions to the nutrition of the insect host, as demonstrated in well-studied examples such as termites, cockroaches, wood-feeding beetles and aphids (Douglas, 1998;
Dillon & Dillon, 2004). With the advancement of new
sequencing methods, gut microbial communities have been
analyzed in an even wider range of insects (Broderick et al.,
2004; Xiang et al., 2006; Sen et al., 2009). Honeybees, Apis
mellifera, are an interesting model for studies of gut microorganisms because they have a complex digestive tract.
Workers collect nectar (carbohydrate source) and pollen
(source of protein, fatty acids, sterols, vitamins and minerals) and bring them back to hives to feed larvae and house
bees by oral regurgitation. The nectar and pollen mixed with
water are temporarily stored in the crop (honey stomach),
an enlargement of the esophagus. The ventriculus (midgut)
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c
is the functional stomach followed by an anterior intestine
and rectum. Recent metagenomic surveys have shown
diverse bacteria in this insect host (Jeyaprakash et al., 2003;
Mohr & Tebbe, 2006; Cox-Foster et al., 2007). Understanding their specific contributions to the physiology of
honeybees requires isolation of the microorganisms and
subsequent biochemical and genetic characterizations.
The sporulating actinomycetes are ubiquitous in terrestrial habitats and include common genera such as Streptomyces, Frankia, Nocardia and Micromonospora (Ventura
et al., 2007). They are well known for their metabolic
capabilities, with Streptomyces being the major producers of
thousands of antibiotics (Berdy, 2005). Actinomycetes, as
one of the rhizosphere bacteria, also produce a wide range of
hydrolytic exoenzymes (e.g. chitinases, cellulase, etc.), and
are therefore primary contributors to the cycling of carbon
in organic matter derived from fungi and plants. Because of
the importance and potential growth advantages of these
bacteria, several studies have focused on the isolation and
visualization of actively growing actinomycetes in the guts of
beetles, termites and millipedes (Bignell et al., 1979; Gozev &
Byzov, 2006; Scott et al., 2008). Previously, nonpathogenic
FEMS Microbiol Lett 312 (2010) 110–118
111
Actinomycetes in honeybee guts
microbiota associated with honeybees have mostly been
examined using classical culture-based techniques, and
chemotaxonomic characterization of the isolates, which
have described a group of Gram-variable pleomorphic
bacteria in honeybee guts but not in adequate detail
(Gilliam, 1997). Although data from the latest pyrosequencing technology applied to honeybee gut microbiota are yet
to be published, few metagenomic studies have revealed the
presence of actinomycetes in this environment (Cox-Foster
et al., 2007). Also, it is known that PCR amplification of
bacterial 16S rRNA genes with universal primers could have
dramatically underestimated the population of high-GC Actinobacteria in a complex community (Stach et al., 2003).
However, one culture-based report indicated that Streptomyces
sometimes could become dominant in bee guts (Mohr &
Tebbe, 2007). To our knowledge, no antibiotic-producing
actinomycetes from the guts of honeybees have ever been
characterized, though Streptomyces are among the microorganisms found in honey (Snowdon & Cliver, 1996) and honey
products have well-known antimicrobial properties (Kwakman et al., 2008). Honey has been a popular folk medicine for
healing wound and soothing sore throat since ancient times.
In this report, selective media were used to isolate
actinomycetes from the digestive tract of adult honeybees.
The antibiotic activities produced under laboratory conditions were evaluated against bee indigenous Bacillus strains,
Escherichia coli and two drug-resistant human pathogens.
One frequently encountered isolate identified as a species of
Nocardiopsis was further characterized and the expression of
an antibiotic biosynthetic gene was analyzed.
Materials and methods
Isolation of actinomycetes from honeybee guts
and the growth media
Adult worker honeybees were collected from six locations,
most of which have o 10 isolated hives. Within 12 h of
capture, bees were externally sterilized with 70–100% alcohol and dissected under sterile conditions. The digestive
tracts, from crop to rectum, were pooled, lightly homogenized and suspended in saline and plated on selective
agar plates. The gut contents from each bee were spread
on one plate. To better investigate the actinomycete diversity
in the complex microbial milieu of the insect gut, different
selective media were used for the colony isolation. Four
out of 10 selective media tested yielded better results in
that the growth of actinomycetes was favored over other
bacteria, fungi and molds (Bredholdt et al., 2007; Babalola
et al., 2009; Maldonado et al., 2009; Qin et al., 2009). They
were as follows: actinomycete isolation agar (AIA) supplemented with cycloheximide (50 mg mL1) and rifamycin
(5 mg mL1) (sodium caseinate 2 g; asparagine 0.1 g; sodium
propionate 4 g; K2HPO4 0.5 g; MgSO4 0.1 g; FeSO4 0.001 g;
glycerol 10 g and agar 15 g L1 distilled water), MSM agar
(microcrystalline cellulose 10 g; casein 0.3 g; KNO3 0.2 g;
K2HPO4 0.5 g; CaCO3 0.02 g; FeSO4 0.01 g; NaCl 5 g;
MgCl2 6H2O 30 g; KCl 20 g; agar 15 g L1 distilled water),
IM5 agar (humic acid 1.0 g; K2HPO4 0.5 g, FeSO4 7H2O
1 mg, vitamin B solution 1 mL, agar 20 g L1 distilled water,
adjusted to pH 8.2) and IM7 agar (similar to IM5 but the
humic acid is replaced with chitin 2.0 g L1). After incubation at 30 1C for 3–7 days, filamentous bacterial colonies
that appeared powdery, fuzzy or leathery were selected
and purified (Fig. 1a). Gram stain followed by examination
under light microscope confirmed that isolates had
the morphology of actinomycetes. Spores of actinomycete
isolates were scraped off the agar and mixed with
20% glycerol to be stored in 80 1C. To make duplicates
for long-term storage, the spores of each strain were
also suspended in 5% nonfat dry milk and lyophilized. The
solid growth media for BE74 were AIA and mannitol
soya flour (MS) agar (Kieser et al., 2000). The liquid
growth media for BE74 were AIB (broth with the ingredients
same as AIA without agar) and ISP1 (Shirling & Gottlieb,
1966).
(a)
(b)
Fig. 1. (a) An example of an actinomycete
isolation agar plate that selectively allowed the
growth of actinomycetes from the guts of one
honeybee. The white colonies have identical
characteristic colonial morphology of
actinomycete. (b) An example of a MH plate used
in the agar diffusion bioassay. The test organism
was a bee indigenous Bacillus marisflavi strain.
The overlaying agar plugs were taken from
separate agar plates pre-grown with individual
actinomycete isolates.
FEMS Microbiol Lett 312 (2010) 110–118
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112
Agar diffusion assay of antagonistic activity
Actinomycete isolates were individually cultured on Petri
dishes that have four sections or 24-well tissue culture plates
for 3–6 days. Two agar media, Müller–Hinton (MH) agar
(Difco) and diagnostic sensitivity test (DST) agar (Oxoid),
were used to grow the test organisms. Most test organisms
here could grow to a full lawn on MH agar plate within 12 h
but the Enterococcus grew better on DST agar. In the assay, a
fresh culture of the test organisms (at OD600 nm 0.04–0.08)
was swiped across an MH agar plate with a cotton Q-tip. A
sterile 200 mL pipette tip was used with its wide-opening end
to bore through the agar plate (0.5 cm thickness) grown
with an actinomycete lawn. The agar plug (estimated
0.11 cm3) lifted out was overlaid on the seeded MH agar
plate. Two plugs were separated about 1.5 cm in distance.
About 15–18 plugs could be arrayed on the surface area of a
plate of 100 mm diameter and about 30–40 plugs on a
150 mm plate (Fig. 1b). After incubation at 30 1C overnight,
a clearing zone (Z2 mm) surrounding the agar plug
indicated that the actinomycete produced a level of diffusible
substance that inhibited the growth of the test organism.
Genetic identification of microorganisms
and phylogenetic analysis
Genomic DNA isolation followed a salting-out procedure
(Kieser et al., 2000), but started with 2–3 mL liquid culture
and the volume of the solution used was one-tenth of that used
in the standard procedure. Mycelia were lysed by bead beating
(Biospec) with 0.1-mm-glass beads. PCR amplification (with
SuperTaq, Ambion) of the partial 16S rRNA gene of the interested colony was attempted with universal bacterial primers
(27F, 50 -AGAGTTTGATCMTGGCTCAG; 63F, 50 -CAGGCCTA
ACACATGCAAGTC; 907R, 50 -CCGTCAATTCMTTTRAGT
TT; 1378R, 5 0 -ACGGGCGGTGTGTACAAG and 1492R, 5 0 AAGGAGGTGATCCAGCC) as well as Actinobacteria classspecific primers (Stach et al., 2003). Sequences of the PCR
products were analyzed by BLAST search, and the most closely
related species were determined. DNA or protein sequences
were aligned with CLUSTALW algorithm implemented in
BIOEDIT software using the default parameters. The aligned
and trimmed sequence regions were used as the input files to
infer phylogenetic trees based on neighbor joining of genetic
distance with bootstrapping in MOLECULAR EVOLUTIONARY
GENETICS ANALYSIS (MEGA) software version 4.0.2. The accession numbers for the partial sequences of BE74 16S rRNA
gene and phzD genes are HM588007 and HM588008.
Amplification of partial phzD from BE74, the
RNA isolation and reverse transcription (RT)-PCR
The primers used for amplifying the 340-bp phzD fragment were as follows: PhzD254-282F, AAC AGC GCG GYC
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P.B. Patil et al.
TSC TCA AGG ACT TCT GG and PhzD571-592R, SSG CRC
AGC GCT CGG CGG CGT A. Mycelia of BE74 were
collected from one agar plate or 1 mL liquid culture for
RNA isolation using an RNA isolation kit (RibopureBacteria, Ambion). Total RNAs were treated with DNase
for half an hour and extracted using the standard phenol–
chloroform method. Reverse transcription (RT) was
performed with 200 ng RNA, SuperScript II reverse
transcriptase (Invitrogen) and random hexamers. Two microliters of the RT reaction were subjected to a PCR reaction
with the primers designed for an 162-nt fragment within
the phzD gene of BE74: NocPhzD_F1, AAC AGC GCG GCC
TCC TCA AGG ACT TCT GG and NocPhzD_R2, TTG GTG
AGC AGG AGG TCC TCA CCG TCG. The annealing
temperature was 64 1C and there were 30 PCR cycles.
Results
Isolation of antibiotic-producing actinomycetes
from honeybee guts
Initially, a small number of adult worker honeybees (N = 6)
were collected in September 2008 from hives at six locations
(separated by 3–20 miles) in southeastern Ohio. After the
processing and selective isolation of actinomycetes with the
AIA, the purified actinomycete colonies were analyzed using
morphology (colors of aerial and substrate mycelia, pigments and starch lysis zone, etc.) and sequences of the
amplified partial 16S rRNA gene. The results confirmed the
presence of actinomycetes, mainly a diverse group of Streptomyces, in the guts of honeybees. Three to eight different
Streptomyces species could be identified, with six bees from
each of five locations. Bees of the remaining one location did
not yield any actinomycete-like colonies on the AIA, but did
produce a large number of nonactinomycete colonies. DNA
typing showed that these nonactinomycete isolates were
related to at least five Bacillus species (identity of the 16S
rRNA gene 4 97%). They were Bacillus cereus, Bacillus
gibsonii, Bacillus pumilus, Bacillus firmus and Bacillus marisflavi. Some of them are known to be used as biocontrol
agents to inhibit the growth of pathogenic microorganisms
such as foulbrood in bees and fungi on plant roots (Alippi &
Reynaldi, 2006; Choudhary & Johri, 2009). It is unclear
whether the predominance of one or more Bacillus species
in this bee yard is related to the lower actinomycete diversity
in the guts of the bees. One location that has a few isolated
beehives was chosen to continue monitoring of actinomycetes diversity every 3 months in a year.
Antibiotic activity against the bee indigenous Bacillus
strains or E. coli was measured using an agar diffusion assay.
The details were described in the methods. Positive results
were interpreted as defensive rather than as nutritional
interactions between the microorganisms because the
FEMS Microbiol Lett 312 (2010) 110–118
113
Actinomycetes in honeybee guts
At least one actinomycete CFU
At least one actinomycete CFU producing antibiotics
90%
Fig. 2. Percent of the sampled honeybees
(N 4 40) that carried at least one actinomycete in
the gut microbial communities, the number of
actinomycetes producing detectable bioactivities
over the 1 year sampling period and the number
of total actinomycete isolates from honeybee
guts. The honeybees were from a single bee yard
in Athens, OH. Five other bee yards were also
sampled (see Results).
Percent of the sampled
honeybeesm (N > 40)
80%
70%
60%
50%
40%
30%
20%
10%
0%
Bioactive isolates
Seasonal total isolates
actinomycetes were already in late growth stage when used
in the assay and the test organisms were microorganisms
with shorter doubling times under the assay conditions.
Potential competitive growth disadvantage of the test
organisms like the Bacillus strains and E. coli can thus be
ruled out with confidence. Also, it has been argued that
actinomycetes in insects are predisposed toward engaging in
defensive antagonism (Kaltenpoth, 2009). The B. marisflavi
isolate identified in the initial experiment was used as a
Gram-positive organism for the primary screening in the
following survey because it seemed to be the most sensitive
to the antibiotic activities produced by the actinomycete
isolates.
For understanding the seasonal changes in actinomycete
diversity in honeybee guts, at least 40 bees were collected
from the chosen bee yard four times during the year. At the
times of December 5th (winter), April 21st (spring), July
16th (summer) and September 30th (fall) from 2008 to
2009, the gut microbial communities were assumed to be
most influenced by the seasonal changes. AIA with supplements was used as the main selective medium (see Materials
and methods). Over 70% of the bees in any one of the four
seasons carried at least one CFU of actinomycete in their
guts (Fig. 2). In some cases, thousands of conspicuous
actinomycete colonies were found in a single honeybee
(Fig. 1a). Between 28% and 58% of the bees at this location
produced at least one actinomycete isolate with detectable
bioactivities (Fig. 2). The highest diversity of actinomycetes
was found in honeybees collected in the summer, and the
lowest in the winter (Fig. 2). Of the 401 actinomycete
isolates obtained, 163 isolates exhibited bioactivity against
the bee indigenous B. marisflavi strain (Fig. 2). All except
four of the 163 bioactive isolates had no observable effect on
the growth of E. coli. Only one of the total 401 isolates
showed exclusive antagonism against E. coli. Therefore,
FEMS Microbiol Lett 312 (2010) 110–118
Winter
Spring
Summer
Fall
Total
27
58
45
110
60
120
31
113
163
401
there appeared to be a specificity of the bioactivities
produced by the actinomycetes from honeybee guts.
Actinomycetes producing anti-Gram-positive
activities are enriched in the honeybee guts
To investigate whether the actinomycete isolates can stably
produce the antimicrobial activities, or whether the antagonisms are more broadly apparent against different Bacillus
species and other Gram-positive pathogens, the actinomycete spores were revived after being frozen for 4–16 months
in storage. They were grown on AIA and used in the
bioassay with the test microorganisms listed (Table 1). One
hundred and fifteen strains were able to grow well
and showed consistent inhibitory activity against the
B. marisflavi strain. In addition, nearly one-third of
them were active against the previously isolated bee indigenous B. pumilus and B. cereus strains. The growth of
Bacillus subtilis was inhibited by about the same number of
the actinomycete strains. Two human pathogens, vancomycin-resistant Enterococcus faecium and methicillin-resistant
Staphylococcus aureus, were also used as the test organisms
in screening for the antibacterial activities produced by the
actinomycete isolates. More than one quarter of the isolates
clearly produced antibacterial substances that inhibited the
growth of the two human pathogens under the assay
conditions. We also attempted to test the inhibition of
Paenibacillus larvae, the causative agent of hive disease
American foulbrood. Because of its much slower growth
rate on the MH agar in the bioassay, the agar diffusion assay
method failed to show a clear antagonism between an
actinomycete isolate and the Paenibacillus strain. Nonetheless, the important confirmation in the second-round
screening was that none of the revived strains produced
anti-E. coli activities.
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114
P.B. Patil et al.
Table 1. Microorganisms tested in the agar diffusion bioassay
Microorganisms tested
Number of antagonistic
actinomycete isolates
Bacillus marisflavi,w
Bacillus pumilus
Bacillus cereus
Bacillus subtilis ATCC6051
Enterococcus faecium ATCC51559
Staphylococcus aureus ATCC43300
Paenibacillus larvae ATCC13537
Escherichia coli ATCC25922w
163
35
38
37
30
33
–z
4
Isolated from honeybee guts and identified by partial 16S rRNA gene
sequences ( 4 97% identity).
w
Used as test organisms in the primary screening with 401 actinomycete
isolates. The rest test organisms were used with 115 isolates that were
revived and reproducibly active against Bacillus marisflavi.
z
Attempted but inconclusive with the agar diffusion assay.
The frequent occurrence of anti-Bacillus bioactivity from
insect gut actinomycetes is especially notable in comparison
to the bioactivities produced by actinomycetes from soil
samples collected in this geographic region. When the same
procedures were used to isolate soil actinomycetes, we found
more colonies producing anti-Gram-negative and broadspectrum antibiotics (together 60–70%) than anti-Bacillusspecific producers (30–40%) (S. Chen, unpublished data).
Therefore, there appeared to be an unusual enrichment of
actinomycetes producing anti-Gram-positive bacteria activities in honeybee guts. This observation is reminiscent of
early reports that the bioactivities of Streptomyces isolates
from earthworm guts were all against Gram-positive bacteria (Kristufek et al., 1993). It could also explain a number of
reports indicating that the antimicrobial activity of honey
products is mainly against Gram-positive bacteria [reviewed
in Viuda-Martos et al. (2008)].
A Nocardiopsis alba strain was one of the
actinomycetes frequently isolated from the
honeybee guts
One actinomycete isolate named BE74, which consistently
produced inhibitory activities against the B. marisflavi
strain, was noticed because of its distinctive colonial morphology on the AIA – it appeared waxy, and only started to
generate very thin aerial mycelia and poorly sporulate after
5–7 days of incubation. It produced abundant white aerial
mycelia and spores when growing on MS agar, but exhibited
much less exuberant growth on ISP1 agar. The substrate
mycelia on MS agar are brown to yellow. Diffusible brown
and light yellow pigments were observed on MS and AIA
agars. Scanning electron micrographs of BE74 grown on
AIA showed long, unbranched and not-fragmented mycelia
that can twist to form spore chains (Fig. 3). The spores have
smooth surfaces and the chains are spiral in shape.
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Fig. 3. Scanning electron micrograph of the spiral hyphae of a rare
actinomycete from the guts of the sampled honeybees. The actinomycete (BE74) identified as Nocardiopsis alba was grown on AIA for 7 days.
A BLAST search with partial 16S rRNA gene sequences
(1252 bp) of BE74 showed similarity (93–99%) to members of the genus Nocardiopsis in the Nocardiopsaceae family.
In a phylogenetic tree based on the neighbor-joining algorithm, BE74 is clustered with all Nocardiopsis typing species
(Tamura et al., 2008). The closest strain to BE74 is N. alba
DSM 43377 (99% identity). The two formed a clade that was
strongly supported by a high bootstrap value (100%). The
N. alba strain BE74 was susceptible to rifamycin
(2 mg mL1) on AIA. It was isolated from bee guts in all
four seasons. However, we can only ascertain that 23% of the
sampled bees (N = 40) at this location in the winter carried
the N. alba strain. The isolate produced medium levels of
antagonism (clearing zones 3–7 mm) against the B. marisflavi strain. It showed no activities against other organisms
in Table 1 except B. cereus.
Identification of a putative phenazine
biosynthetic gene (phzD) in the Nocardiopsis
strain
Nocardiopsis species have been isolated from marine sediments (Engelhardt et al., 2010). Antibiotic biosynthetic
genes were searched in a draft of the genome of Nocardiopsis
dassonvillei DSM 43111 (Wu et al., 2009). One gene cluster
proposed for an involvement in phenazine biosynthesis has
been identified in this organism (Mentel et al., 2009).
Phenazines are a family of nitrogen-containing tricyclic
pigments produced by rhizosphere bacteria including Pseudomonas and Streptomyces (Pierson & Pierson, 2010). Interestingly, it has been shown that some secreted phenazines of
Pseudomonas aeruginosa can promote the anaerobic survival
of the producer itself via extracellular electron transfer
FEMS Microbiol Lett 312 (2010) 110–118
115
Actinomycetes in honeybee guts
(a)
Fig. 4. (a) Alignment of partial PhzD protein
sequences, each of which contains 112 continuous
amino acids. M.r., Microbispora rosea; N.d.,
Nocardiopsis dassonvillei; S.a., Streptomyces
anulatus; S.c., Streptomyces cinnamonens; S.l.,
Streptomyces lomondensis; P.a., Pseudomonas
aeruginosa; P.f., P. fluorescens 2–79. The amino
acid residues marked with asterisks are conserved
and involved in binding of the substrate (Parsons
et al., 2003). (b) Evolutionary relationships of the
eight PhzD proteins inferred using the neighborjoining method in the MEGA 4.0.2 software. The
accession numbers are in the parentheses. The
bootstrap consensus tree inferred from 1000
replicates is presented. The percentage of
replicate trees in which the associated taxa
clustered together in the bootstrap test are shown
next to the branches. (c) RT-PCR analysis of phzD
gene expression in BE74 with total RNAs isolated
from mycelia under three growth conditions: lanes
1 and 2, MS agar; lanes 3 and 4, AIA; lanes 5 and 6,
AIB broth. Samples in odd-numbered lanes are the
negative controls (no reverse transcriptase in the RT
reactions) for the samples in the following evennumbered lanes. M, 100 bp DNA marker. The
arrow indicates the amplified phzD DNA fragment.
BE74
M.r.
N d
N.d.
S.l.
S.a.
S.c.
P.a.
P.f.
BE74
M.r.
N.d.
S.l.
S.a.
S.c.
P.a.
P.f.
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
10
20
30
40
50
60
QRGLLKDFWG AGMKAVAEHT DIVPELAPDG EDLLLTKWRY SAFAQTDLAE RMAAQGRDQI
QRGLLKDFWG PGMRTDAADR EVVAELTPAE GDWVLTKWRY SAFFRSDLLE RMRAAGRDQL
QRGLLKDFWG PGMRRSPEDR LVVDELAPSP DDWMFTKLRY SAFHKSDLLE RMRAAGRDQL
RMRAAGRDQ
QRGLLKDFWG PGMRTDAADR EVVAELTPAP GDWVLTKWRY SASFRSDLLE RMRAAGRDQL
QRGLLKDFWG PGMRPEPEDR QVVDALAPAE QDWMLTKWRY SAFFKTDLLR RMRAAGRDQL
QRGLLKDFWG PGMRPAPEDR QVVDALAPTE QDWLLTKWRY SAFFKTDLLE RMRAAGRDQL
QRGLLKDFWG PGMRASPADR EVVEELAPGP DDWLLTKWRY SAFFHSDLLQ RMRAAGRDQL
QRGLLKDFWG PGMKASPTDR EVVDALAPQP GDWLLTKWRY SAFFNSDLLQ RLHASGRDQL
* *
*
*
....|....| ....|....| ....|....| ....|....| ....|....| ..
70
80
90
100
110
VVTGVYAHIG CQLTAADAFM RDIRPFLIAD ALADFNADYH RMAVRYAAER CA
VLCGVYAHVG VLATALEAFT NDIQTFLAAD ALGSFSEAHH RLALDYAAER CA
VVCGVYAHVG VLMTAVEAYT NDIQTFLVAD AVADFNADYH RMAVRYAAER CA
VLCGVYAHVG VLATALEAFT NDIQTFLAAD ALGDFSEAHH RLALDYAAER CA
VLCGVYAHVG VLATAVEAFT HDIQPFFVAD ATADFSEHYH RSALTYAAER CA
ILCGVYAHVG VLATAVDAFT HDIQPFFVAD ATADFSQDYH RSALTYAAER CA
VLCGVYAHVG VLISTVDAYS NDIQPFLVAD AIADFSEAHH RMALEYAASR CA
ILCGVYAHVG VLISSVDAYS NDIQPFLVAD AIADFSKEHH WMAMEYAASR CA
** *
(b)
M
(c)
2
3
4
5
6
phzD
100 bp
(Wang et al., 2010). Therefore, we were interested in whether
N. alba from the honeybee gut has the phenazine biosynthetic genes and whether they are expressed.
The phenazine biosynthetic pathway is branched from the
shikimate pathway in bacteria (Mentel et al., 2009). Five
genes, phzB, phzD, phzE, phzF and phzG, are required for
biosynthesis of the core structure, and they are highly
conserved in all known phenazine biosynthetic gene clusters. phzF has been used as a genetic marker for analyzing
the diversity and evolution of phenazine biosynthetic pathways in many Gram-negative bacteria, most of which are
pseudomonads (Mavrodi et al., 2010). The PCR primers for
phzF were tested with BE74 genomic DNA but the reactions
did not yield products under the suggested conditions.
Instead, PCR primers based on the alignments of phzD
genes encoding an isochorismatase from Streptomcyes cinnamonensis DSM 1042, Streptomyces anulatus LU9663 and
N. dassonvillei DSM 43111 yielded an 340-bp fragment
with the BE74 DNA. Putative protein sequences encoded by
this DNA fragment showed the highest homology to a
part of PhzD from N. dassonvillei DSM 43111 and other
FEMS Microbiol Lett 312 (2010) 110–118
1
homologs (similarity 70–90%) involved in isochorismate
metabolism. The protein is unlikely a member of the
hydrolase family of primary metabolism that substantially
differ from the PhzDs. The major amino acid residues
of PhzD involved in binding an isochorismate substrate
were found to be encoded in the sequences (Fig. 4a)
(Parsons et al., 2003). The two primers were also used to
amplify the same region of PhzD homologs from the
genomes of two other actinomycetes, Streptomyces lomondensis ATCC25299 and Microbispora rosea ATCC15738,
previously known to produce phenazines. Alignments of
the partial sequences (112 out of total 207 amino acids) of
six actinomycete PhzD proteins allowed the construction of
phylogenetic trees (Fig. 4a). The trees constructed with
several algorithms have the same topology. Streptomyces
lomondensis and M. rosea PhzDs are more closely associated
with each other compared with the PhzDs of other two
Streptomcyes. Nocardiopsis PhzDs also form their own
group, although the sequence of BE74 PhzD is somewhat
divergent from that of N. dassonvillei (Fig. 4b). This
observation is in contrast to the higher homology (98%)
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116
of the 16S rRNA genes between the two species, which
suggests that the two biosynthetic genes in Nocardiopsis
species may have evolved differently.
To preliminarily investigate the expression of the putative
phzD gene, RT-PCR was used to detect the phzD transcript.
Total RNAs were isolated from mycelia harvested from MS
and AIA agar plates and actinomycete isolation broth (AIA
without agar). Cells grown with these media should be in
significantly different physiological states. Nonetheless, the
phzD gene was always expressed under the three conditions
(Fig. 4c). Although regulation of phz gene expression in
actinomycetes is unknown, the result herein suggests that
the phz mRNAs might be expressed in the Nocardiopsis BE74
cells in various environments.
Discussion
The gut microbiota of insects is an interesting source of
microbial diversity and study of the interactions within an
ecological context. Small molecules naturally produced by
some environmental bacteria are expected to influence the
microbial community as well as the physiology of an insect
host, especially when the insects are reared in the wild. In
this report, we focused on the selective isolation of actinomycetes from honeybee guts. The majority of the bioactivities produced by the actinomycete isolates were specific
against several bee indigenous Bacillus strains and two drugresistant Gram-positive human pathogens. One rare-actinomycete isolate from the honeybee gut identified as a strain of
N. alba was preliminarily characterized. Production of
phenazine-like redox-active molecules by this isolate could
contribute to its ability to temporarily survive the anoxic or
anaerobic conditions that may occur in honeybee guts
(Andreas et al., 2000; Johnson & Barbehenn, 2000). It was
thereafter observed that one type of the modified phenazines, so-called endophenazines, was previously detected as
the metabolites of S. anulatus. Four strains of this species
producing endophenazines were isolated from the intestines
of leaf beetles, millipedes, woodlice and other arthropods
collected in various countries over Europe during a search
for symbiotic actinomycetes of the animals (Gebhardt et al.,
2002). Furthermore, the antimicrobial spectra of endophenazines were reported as having good activity against several
Gram-positive bacteria but no activity against Gram-negative
bacteria (Gebhardt et al., 2002). Preliminary analysis with the
16S rRNA genes of some isolates in our collection revealed the
presence of S. anulatus in honeybee guts, which supports our
finding here that similar redox-active molecules are produced
by the Nocardiopsis isolate from honeybee guts. Although the
relationship between the actinomycetes and insects needs to be
further characterized, production of endophenazines might be
a first step toward establishing or evolving a symbiotic
relationship. It would be interesting to investigate the fre2010 Federation of European Microbiological Societies
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c
P.B. Patil et al.
quency of occurrence of actinomycete phenazine producers in
honeybee guts. Various gene-centric pangenomic or multilocus sequence typing approaches could be used.
Naturally occurring phenazines are redox-active compounds, traditionally thought as antimicrobials that include
over 100 structures (Laursen & Nielsen, 2004). In several
Pseudomonas models, the biological roles of phenazines have
recently been expanded with implications in microbial
interaction processes such as shuttling electron, intracellular
signaling, contributing to form biofilm and enhancing
anaerobic survival (Pierson & Pierson, 2010). These roles
are also expected for phenazines produced by actinomycetes,
with possibly additional functions beyond antibiotic because the structural diversity of actinomycete phenazines is
even greater and the lifecycle of actinomycetes is generally
complex. Phenazines produced by the actinomycetes from
honeybee guts probably have structural commonalities
even though the producers can be quite different (e.g.
Nocardiopsis vs. Streptomyces). Indeed, more actinomycete
isolates in our study displayed specific antagonism against a
B. marisflavi strain than against other Bacillus strains
(Table 1). On the other hand, other microbial metabolites
that share an anthranilic acid structural moiety with phenazines, such as actinomycins and quinolones, also have
widely known electrochemical properties. In addition,
thiols, quinones and coumarins of microbial origins have
noticeable electron transfer capabilities. Voltammetric measurements of the purified compounds will shed light on the
proposed biological functions of these secondary metabolites. Lastly, some actinomycetes carry numerous stressresponsive genes for maintaining viability in anaerobiosis
(van Keulen et al., 2007). Using the extracellular redoxactive secondary metabolites as respiratory electron acceptors could be another survival strategy of actinomycetes. In
summary, studying the actinomycetes isolated from honeybee guts and the metabolites produced will yield many
insights into the fundamental biology and chemistry of
these microorganisms.
From a practical standing point, the health and well-being
of honeybees is of considerable concern as they are the
important agricultural resources. Actinomycete-produced
organic compounds have been marketed or are being
investigated as insecticides (e.g. spinosad). Given the specificity of the actinomycetes that honeybees retain in their guts
and bring back to hives, several important questions have
arisen: Are they beneficial bacteria or opportunistic pathogens to the honeybees? Are phenazines virulence factors or
contributors to a healthy gut microbial community? Are
phenazines present in raw honey and do they contribute to
its antimicrobial properties? Phenazines are often produced
in large quantities in situ and can be directly detected in the
soil or the human tissues colonized with the microorganisms (Wilson et al., 1988; Thomashow et al., 1990). Future
FEMS Microbiol Lett 312 (2010) 110–118
117
Actinomycetes in honeybee guts
investigations may open new avenues for discovering new
antibiotics in human medicine or exploring methods to
fight honeybee diseases.
Acknowledgements
We thank beekeepers John McGovern, Edward Newman and
Dr Scott Moody for providing the honeybees and for
continuous support. We are grateful to Dr Kelly Johnson
for helpful discussion. This project was supported by startup funds from Ohio University to S.C.
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