![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Applied and Environmental Microbiology, January 2001, p. 110-117, Vol. 67, No. 1
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.1.110-117.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Characterization of a Novel Spirochete Associated with the
Hydrothermal Vent Polychaete Annelid, Alvinella
pompejana
Barbara J.
Campbell and
S. Craig
Cary*
University of Delaware Graduate College of
Marine Studies, Lewes, Delaware 19958
Received 3 July 2000/Accepted 15 September 2000
 |
ABSTRACT |
A highly integrated, morphologically diverse bacterial community is
associated with the dorsal surface of Alvinella pompejana, a polychaetous annelid that inhabits active high-temperature deep-sea hydrothermal vent sites along the East Pacific Rise (EPR). Analysis of
a previously prepared bacterial 16S ribosomal DNA (rDNA) library identified a spirochete most closely related to an endosymbiont of the
oligochete Olavius loisae. This spirochete phylotype
(spirochete A) comprised only 2.2% of the 16S rDNA clone library but
appeared to be much more dominant when the same sample was analyzed by denaturing gradient gel electrophoresis (DGGE) and the terminal restriction fragment length polymorphism procedure (12 to 18%). PCR
amplification of the community with spirochete-specific primers used in
conjunction with DGGE analysis identified two spirochete phylotypes.
The first spirochete was identical to spirochete A but was present in
only one A. pompejana specimen. The second spirochete
(spirochete B) was 84.5% similar to spirochete A and, more
interestingly, was present in the epibiont communities of all of the
A. pompejana specimens sampled throughout the geographic range of the worm (13°N to 32°S along the EPR). The sequence
variation of the spirochete B phylotype was less than 3% for the range
of A. pompejana specimens tested, suggesting that a single
spirochete species was present in the A. pompejana
epibiotic community. Additional analysis of the environments
surrounding the worm revealed that spirochetes are a ubiquitous
component of high-temperature vents and may play an important role in
this unique ecosystem.
| |
INTRODUCTION |
Symbiotic relationships between
invertebrates and bacteria are a prominent feature in
marine systems. Investigations of these marine symbiotic relationships
have been spurred on by the discovery of their dominance in
invertebrates from extreme environments, such as hydrothermal vents.
One of the first symbioses described in hydrothermal vent environments
was that of the endosymbiotic chemoautotrophic sulfur-oxidizing
bacteria associated with the deep-sea hydrothermal vent giant tubeworm,
Riftia pachyptila (8). While these bacteria
have not been cultured, their role in the symbiosis has been determined
in detail by using a variety of physiological and molecular methods
(16, 22). Other hydrothermal vent symbiotic associations
that have been described include chemoautotrophic endosymbionts
associated with other vestimentiferans and bivalves, as well as the
episymbionts associated with the shrimp Rimicaris exoculata
and the polychaetes Alvinella pompejana and Alvinella caudata (5, 7, 13, 15, 19, 28).
A. pompejana is considered to be the most thermotolerant and
eurythermal metazoan yet described (6, 9). It forms tubes and colonizes the walls of high-temperature chimneys (40 to 105°C) at
vent sites along the East Pacific Rise (EPR) from 32°S to 21°N latitude (11; C. A. Di Meo, G. Luther, and S. C. Cary,
unpublished data). A. pompejana is characterized by a dense,
specific epibiotic microflora associated with the dorsal integument of
the worm (5, 12). Although electron microscopy studies of
the A. pompejana epibionts suggest that they are highly
diverse, two bacterial morphotypes, a filamentous sheathed form and a
rod-shaped form, appear to predominate (17, 18). The
filamentous form is integrated into specialized expansions of the
intersegmentary parts, while the rod-shaped form appears to be less
abundant but evenly distributed on the dorsal surface (17,
18). The role of these epibionts in the symbiotic association
with A. pompejana is unclear. Ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO), 14C labeling, and
bicarbonate uptake assays have not implicated the dominant filamentous
morphotype in autotrophic CO2 fixation (1, 2).
However, it has been suggested that the symbionts may provide a food
source for the worm (5), as hypothesized for the
ectosymbionts of R. exoculata (28, 31),
detoxify the immediate environment surrounding the worm, or possibly
provide thermal insulation (11, 29).
In previous investigations, a combination of restriction fragment
length polymorphism (RFLP) in conjunction with sequence analysis of a
16S ribosomal DNA (rDNA) clone library and in situ hybridizations
performed with clone-specific oligonucleotides demonstrated that the
A. pompejana community is dominated by 4 families (of 32 families) that form a tight clade within the epsilon subdivision of the
Proteobacteria, at least 2 of which are made up of
filamentous organisms (5, 19). In the present study, a
novel spirochete phylotype was discovered after further screening of
the same 16S rDNA clone library. Spirochete A appeared to be much more
dominant in the community when two separate DNA fingerprinting methods
were used. However, spirochete A was not found in other A. pompejana specimens when spirochete-specific primers were used. We
also demonstrated that a second, unique spirochete (spirochete B),
while accounting for only a small percentage of the community, was a
consistent component of the A. pompejana epibiont population throughout the geographic range of the worm.
| |
MATERIALS AND METHODS |
Specimen collection and isolation of the DNAs of the A. pompejana epibionts.
A. pompejana specimens were
collected from five geographically distinct hydrothermal vent sites
along the EPR. A single A. pompejana specimen (APG1B) was
collected from the Elsa vent site, (latitude, 13°N) during the joint
French U.S. Hydrothermal Environment Research Observatory expeditions
in 1991. Other A. pompejana samples were collected at
latitudes of 32°S and 18°S (dives 3340 and 3333, respectively;
Southern EPR cruise, December 1998 to January 1999, voyage 3, leg 30, R/V Atlantis), 9°N (dives 3317 and 3308, November 1998, voyage 3, leg 29, R/V Atlantis), and 13°N (dives 2874 and 2875, November 1994, cruise 131, leg 25, R/V Atlantis; dives
AM-01 and AM-08, June 1999, AMISTAD cruise, L'Atalante)
(Table 1). After the worms were washed
three times in sterile seawater, bacterial filaments were carefully cut
from the dorsal surface of each worm, homogenized in a lysis buffer (5 M guanidinium isothiocyanate, 50 mM Tris [pH 7.4], 25 mM EDTA, 0.8%
2-mercaptoethanol), frozen at 
80°C on the ship, and stored until
extractions were performed in the laboratory. Total genomic DNAs were
isolated from the frozen lysed samples by using an IsoQuick extraction
method according to the instructions of the manufacturer (ORCA
Research, Bothwell, Wash.). The extraction efficiency of the method was
comparable to those of other DNA extraction methods (5; data not
shown).
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Spirochete sequence similarities for various A. pompejana epibiont communities and environmental samples
|
|
Sequencing and phylogenetic analysis of epibionts.
A 16S
rDNA clone (APG10), representing an RFLP family (3 of 139 clones
screened) in a previously prepared 16S rDNA library (19),
was bidirectionally sequenced at the 5' end by using primers 27F and
519R (25) and a Perkin-Elmer Big Dye terminator sequencing kit (Applied Biosystems Inc., Foster City, Calif.). The reaction mixtures were ethanol precipitated and sequenced with an ABI 310 automated sequencer (ABI, Foster City, Calif.). The resulting sequences
were edited and aligned with their complementary strands by using
AutoAssembler DNA sequence assembly software (ABI).
Community structure analysis.
Denaturing gradient gel
electrophoresis (DGGE) was performed as described by Muyzer et al.
(25), with some modifications. Briefly, 25 ng of isolated
genomic DNA or 2.5 ng of plasmid DNA from the clone described above
(unless otherwise noted) was used for PCR amplification of the V3
region of 16S rDNA with the following primers: 338F/GC clamp and 519R
(25) or 804R (5'-CTACCAGGGTATCTAATCC-3'; universal bacterial primer designed from a Ribosomal Database Project II (RDP II) alignment of the 16S rDNA of all prokaryotes in the
database). Triplicate PCR were performed in 50-µl mixtures which
contained each primer at a concentration of 0.1 µM, 1.25 U of
Taq polymerase (Promega, Madison, Wis.), 2.5 U of
Pfu polymerase (Stratagene, La Jolla, Calif.), 1× Promega
Taq PCR buffer, 2 mM MgCl2, and each
deoxyribonucleoside triphosphate at a concentration of 200 µM.
Samples were pooled, and the DNA was precipitated and quantified by
densitometry by using an AlphaImager 2000 document and analysis system,
version 3.3b (Alpha Innotech Corporation, Calif.).
Amplification products (approximately 250 ng for the plasmid clones and
approximately 1,500 ng for the genomic DNA products) were
electrophoresed through a 25 to 55% denaturing gradient, 6 or 8%
acrylamide gel (100% denaturant was 7 M urea plus 40% deionized
formamide) for 5 h at a constant voltage of 130 V and a temperature of
60°C by using a Dcode universal mutation detection system (Bio-Rad,
Hercules, Calif.). DNA bands were visualized with a UV transilluminator
and were photographed by using the AlphaImager system (Alpha Innotech).
Individual separated DGGE fragments were stabbed with an aerosol-free
pipette tip, and each resulting gel piece was resuspended in 20 µl of
sterile water. One-half of the sample was used in a 50-µl PCR mixture
as described above. Amplification products were checked for purity by
DGGE of a portion of the sample. If necessary, a second round of
stabbing and amplification was performed. Excess primers were removed
from PCR products by passage through a Qiagen PCR purification column
(Qiagen, Valencia, Calif.) according to the manufacturer's
instructions and were quantified by UV spectrophotometry. Approximately
40-ng portions of purified products were used in 10-µl Big Dye
terminator sequencing reaction mixtures (ABI) along with either forward
primer 338F (3) or reverse primer 519R. Sequence analysis
of the resulting fragments was performed as described above.
Terminal RFLP (T-RFLP) analysis was performed as described by Liu et
al. (23), with several modifications. Briefly, triplicate 50-µl PCR mixtures were amplified by using fluorescently labeled primers 27f-HEX and 926r-TET and the cycling conditions described previously except that 50 ng of genomic DNA was added to each reaction
mixture, the annealing temperature was changed to 54°C, and the
number of cycles was reduced from 35 to 30. Fifty nanograms of each
pooled amplification product was digested with 5 U of CfoI,
HaeIII, or AluI (Promega) in a final volume of 10 µl for 6 h at 37°C. These enzymes were chosen because of their
frequent cutting potential (23). Two microliters of each
digest was resuspended in 20 µl of formamide, and 0.5 µl of a 0-500 TAMRA size standard (ABI) was added to each tube. Denatured samples
were electrophoresed with a model 310 automated sequencer (ABI).
Individual T-RFLP fragments were visualized, and the sizes and
integrated areas under the peaks were automatically determined with the
ABI 310 GeneScan software (GeneScan, version 2.1).
Molecular analyses of spirochete members.
To amplify the
DNAs of the majority of spirochetes in a mixed microbial community, a
spirochete-specific forward primer, Spi33F (5'GGCGGCGCGTWTTAAG-3'), was designed based on a survey of
the GenBank database and a previously published phylogenetic analysis of the spirochetes (26). This forward primer was used in
PCR amplifications with reverse primer 519R or 926R under the following conditions: 94°C for 1 min, 68°C for 1 min (56°C with the 926R primer), and 72°C for 1 min for 35 cycles. Three sets of DNA
extraction mixtures were amplified: (i) mixtures from the A. pompejana specimens mentioned above, (ii) mixtures from a
bacterial film present on a sample of chimney (dive 2864, Pillar site,
9°N, voyage 131, leg 25, November 1994), and (iii) mixtures from the
outsides of A. pompejana tubes (sample 137, dive 2857, 13°N, Elsa; sample 150, dive 2859, 9°N, X5; both from the cruise
mentioned above). The negative 16S rDNA controls used were from
representatives of the alpha, beta, gamma, delta, and epsilon
subdivisions of the Proteobacteria, as well as the cytophaga
group (data not shown). RFLPs were determined by digestion of each
amplification product as described previously (19).
A 0.5-µl portion of each spirochete-specific amplified product (from
the preparations described above) was used in a second PCR performed
with primers 338F/GC clamp and 519R and was subjected to DGGE analysis
as described above. The bands were stabbed, amplified, sequenced, and
phylogenetically grouped as described above. DGGE analysis with primers
338F/GC clamp and 804R was also performed directly with the A. pompejana and environmental DNAs described above.
Nucleotide sequence accession numbers.
The consensus
nucleotide sequence of 16S rDNA clone APG10 has been deposited in the
GenBank database (4) under accession number AF180309. The
nucleotide sequences of the amplification products from DGGE fragments
have been deposited in the GenBank database under accession numbers
AF180311 to AF180319. The nucleotide sequences of spirochete-specific
amplified products have been deposited in the GenBank database under
accession numbers AY007429 to AY007433 and AF300986.
| |
RESULTS |
Community analysis of A. pompejana epibionts.
Previous molecular characterization of the epibiont community
associated with A. pompejana demonstrated a dominance of
phylotypes belonging to the epsilon subdivision of the
Proteobacteria (5, 19). Molecular
characterization of the minor members of a community in many cases may
reveal important information regarding a symbiotic association,
especially when diverse bacteria are found in the association, as in
A. pompejana (19). Therefore, in this study, we
focused on a distinct microbe present in the A. pompejana
epibiont community. A clone from a previously prepared 16S rDNA library (19) (APG10 [= spirochete A]; frequency in 16S rDNA
library, 2.2%) was characterized, and this clone phylogenetically
grouped with the Spirochaeta species (sequence similarity
with Olavius loisae endosymbiont, 86.5%). Two techniques
(DGGE and T-RFLP analysis) were used to confirm the clone dominance
predicted from the 16S rDNA library analysis. More than 11 fragments
were identified from a short, amplified region of the 16S
rDNA from the alvinellid community (APG1B) DNA by using DGGE (Fig.
1). DGGE analysis confirmed that the
majority of the epibionts were members of the epsilon subdivision of
the Proteobacteria; 10 of the 11 bands in Fig. 1 were from
members of this group, compared to 9 of 11 clones from the 16S rDNA
library. The total frequency of members of the epsilon subdivision in
the library was, therefore, at least 80% (not all of the clone
families have been characterized). DGGE analysis also indicated that
one of the bands (Fig. 1, band 5: corresponding to spirochete A) was
present at a significantly higher level (15.5%) than in the 16S rDNA
library (2.2%). Similar relative frequencies were obtained in T-RFLP
analyses with another set of primers, which confirmed the DGGE data.
The relative frequencies of the spirochete A 5'-terminal restriction
fragment (T-RF) obtained with AluI, CfoI, and
HaeIII, were 18, 12, and 15%, respectively. Frequencies
were determined by comparing the relative peak areas of T-RF with the
sum of all T-RF areas by using the Gene Scan software.
View larger version (69K):
[in this window]
[in a new window]
|
FIG. 1.
DGGE gel comparing a portion of the 16S rDNA from APG1B
(uncloned) to 16S rDNA library clones. Triplicate PCR mixtures obtained
with APG1B DNA were pooled and ethanol precipitated, and approximately
1.5 µg was loaded into the first lane. Mixtures of amplified products
of other clones were loaded into the second lane (clones 72, 5A, 13B,
44B, 56B) (Clone Mix1) and third lane (clones 118, 68, 73, 10, 115, 79, 162) (Clone Mix2).
|
|
Spirochetes in the Alvinella community.
To
determine if spirochete A (= APG10) was a consistent member of the
epibiotic community associated with A. pompejana,
additional alvinellid and environmental samples (A. pompejana tubes, chimney) were screened. This was achieved in two
ways: (i) 16S rDNA RFLP analysis after amplification with
spirochete-specific primers and (ii) DGGE and sequence analysis after
amplification with the spirochete-specific primer or a universal
bacterial primer set.
Spirochete-specific amplification products from seven A. pompejana worms and two tubes, as well as a chimney sample taken from a different vent site, were screened by RFLP analysis (Fig. 2). Digestion of the PCR products
obtained with primers 33F and 926R with MboI and
HaeIII revealed that each of the samples had a dominant
phylotype, as demonstrated by the sum obtained for darkest bands
present in each lane. For instance, in Fig. 2, lane 1, the prominent
bands corresponded to approximately 190 and 700 bp, and the sum
represented the full-length product. There also was evidence of minor
members, as indicated by the fainter bands present in each of the
lanes. Interestingly, only the amplified DNAs from latitude 13°N had
minor RFLP bands identical to the bands obtained for spirochete A,
which was also from latitude 13°N (Fig. 2, lanes 4 through 8).
View larger version (109K):
[in this window]
[in a new window]
|
FIG. 2.
RFLP analysis of the 16S rDNA gene of various microbial
communities after amplification with spirochete-specific primers and
digestion with MboI and HaeIII. Lanes 1 and 11, molecular weight marker; lane 2, A. pompejana community from
dive 3317; lane 3, community from dive 3308; lane 4, community from
dive 2874; lane 5, community from dive 2875; lane 6, APG1B; lane 7, community from dive AM-08; lane 8, community from dive AM-01; lane 9, bacterial film on chimney; lane 10, community from outside A. pompejana tube in dive 2859. Geographic locations are indicated at
the bottom.
|
|
To determine if the spirochete association was a ubiquitous trait found
in all A. pompejana epibiont communities, we screened A. pompejana samples from a large geographic range along the
EPR and several other environmental samples. Two sets of primers were used in PCR amplifications, and the amplicons from the second set were
subjected to DGGE analysis (Fig. 3A). The
first primer set (a spirochete primer and a universal primer) amplified
a large segment of the 16S rDNA gene. Amplicons obtained with the first set were used as templates for the second set of primers
(bacterium-specific primers used to amplify the V3 region of the 16S
rDNA gene). The majority of the geographically diverse epibiont
communities produced one major spirochete-specific band (indicated by
the arrows in Fig. 3A) and several minor bands, confirming the results
obtained in the RFLP analysis. The major bands resulting from
amplifications of the bacterial communities from different worms taken
from the same latitude (e.g., 13°N) (Fig. 3A, lanes 3 and 4) along
the EPR were identical, even though in some cases the alvinellids were
collected from different vent sites and in different years (Table 1).
The one exception to this was the APG1B sample obtained at latitude
13°N (Fig. 3A, lane 2), from which the original 16S rDNA library was
prepared. The major species in this case corresponded to spirochete A
(clone APG10) identified in the 16S rDNA library and DGGE analyses
described above. However, a minor spirochete-specific band (Fig. 3A,
lane 2, arrow) was present in the amplified sample that migrated just
like the other latitude 13°N samples (Fig. 3A, lanes 3 and 4).
View larger version (87K):
[in this window]
[in a new window]
|
FIG. 3.
DGGE gels containing the 16S rDNA gene from various
microbial communities. (A) Gel after two rounds of amplification, the
first with a spirochete-specific and bacterial primer set. Lane 1, APG10 plasmid control; lane 2, APG1B; lane 3, community from dive 2874;
lane 4, community from dive AM-08; lane 5, community from dive 3317;
lane 6, community from dive 3308; lane 7, community from dive 3333;
lane 8, community from dive 3340; lane 9, A. pompejana tube
community from dive 2857; lane 10, A. pompejana tube
community from dive 2859; lane 11, chimney sample community from dive
2864. (B) Gel after amplification of the 16S rDNA gene (E. coli positions 338 to 804). Lane 1, A. pompejana
community from dive 3317; lane 2, community from dive 3308; lane 3, community from dive 2874; lane 4, community from dive 2875; lane 5, APG1B. The arrows indicate the bands sequenced for further analysis.
Geographic locations are indicated at the bottom.
|
|
DNA distance analysis of sequences obtained from all the major bands
(and the minor band in the APG1B sample) indicated that closely related
spirochetes were present in all the A. pompejana epibiont
communities tested (Table 1). The levels of DNA similarity between
spirochetes obtained from A. pompejana were greater than 97.8%, except for the value for clone APG10 from APG1B, which was
84.5% similar to AP32. Since the phylotypes were more than 97%
identical, they were considered members of a single species (spirochete
B). Phylogenetic analysis of the 16S rDNA gene from several spirochetes
demonstrated that all of the hydrothermal vent spirochetes
characterized in this study cluster within the genus
Spirochaeta (Fig. 4). Three
distinct phylogenetic clusters were observed, and one of these groups
contained all but one of the spirochetes associated with A. pompejana. A second cluster included spirochete A (= APG10) as
identified by 16S rDNA library screening and DGGE analysis and the
endosymbiont from O. loisae (14). The third
group contained the spirochete from an A. pompejana tube and
Spirochaeta litoralis. Values from phylogenetic bootstrap analyses of the sequences obtained directly from the DGGE bands (amplified region from position 338 to position 519 of the
Escherichia coli 16S rDNA gene) were low, most likely due to
the small fragment available for sequencing. Since the bootstrap values
were low, the tree topologies were confirmed by obtaining nearly
identical trees with the maximum-likelihood and neighbor-joining
algorithms (data not shown).
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 4.
Consensus bootstrapped cladogram (100 replicates)
depicting the ancestral relationships indicated. The cladogram was
calculated by using the DNA parsimony algorithm (PHYLIP, version 3.5c).
The analysis was based on the region corresponding to bases 338 to 519 of the E. coli 16S rDNA gene, excluding insertions,
deletions, and ambiguous bases. Various members of the genus
Spirochaeta were included (GenBank accession numbers in
parentheses). Treponema socranskii was used as the outgroup
species.
|
|
In order to compare the relative levels of the two unique spirochetes
obtained from A. pompejana, the relative frequencies of both
were estimated by DGGE analysis. Several different bands were observed
on a DGGE gel prepared from bacterial 16S rDNA products (region
corresponding to 16S rDNA of E. coli at positions 338 to
804) amplified from alvinellid epibiont samples obtained from the 9°N
and 13°N sites (Fig. 3B). The calculated relative frequency of
spirochete A (arrow indicating APG10 in Fig. 3B, lane 5) as determined
by densitometry was 15.5%. Sequence analysis of a similarly migrating
band (arrow in lane 1) demonstrated that it did not correspond to a
spirochete; however, sequence analysis of 20 additional bands revealed
a band corresponding to spirochete B (arrow S in lane 3). The relative
frequency of this band was approximately 1%, as determined by
densitometry. All of the other bands were identical or closely related
to APG5A, APG13B, APG44B, or APG56B, the four clones identified in a
previous investigation (19; data not shown).
| |
DISCUSSION |
A. pompejana is characterized by a dense coating of
epibionts whose role in the symbiotic association remains unknown. A
previous study of the complexity of this association revealed the
prevalence of two filamentous epsilon-proteobacterial phylotypes as
determined by both 16S rDNA library screening and in situ
hybridization, confirming previous morphological evidence of dominant
filamentous symbionts (5, 19). Although these two closely
related bacterial phylotypes dominate the community, there is
morphological and molecular evidence of a more diverse assemblage of
bacteria on the dorsal surface of A. pompejana (5, 10,
17-19, 29). In this investigation, besides confirming the
diversity of members of the epsilon subdivision of the
Proteobacteria by DGGE analysis, we determined the presence
of spirochetes in the A. pomepjana community and found that
one of these spirochetes is a minor, but consistent member.
Spirochete B was detected in all A. pompejana epibiont
communities surveyed (at latitudes from 13°N to 32°S along the
EPR), although it was a minor member of the community as determined by
DGGE analysis, accounting for (~1%) of the population. More importantly, its close phylogenetic relationship throughout almost the
entire geographic range of A. pompejana (more than 97%
similarity) and the fact that identical spirochetes were found at the
same vent sites in different years suggest that this spirochete may be
involved in an integrated symbiosis with A. pompejana. The fidelity of this relationship has yet to be determined. None of the
other spirochetes detected were consistently present in the A. pompejana epibiont community, and most were found in the
hydrothermal vent environment separate from the worm.
The genus Spirochaeta is comprised of obligately and
facultatively chemoheterotrophic anaerobes that are generally
free-living freshwater and marine species (26). However,
Dubilier et al. demonstrated the presence of three symbionts associated
with cuticular invaginations of O. loisae, an annelid
inhabiting marine sediments, one of which is a member of the
Spirochaeta family (14). Until our study, only
one spirochete had been discovered in a hydrothermal vent environment
(21). Although this spirochete was not phylogenetically characterized, its growth characteristics were consistent with those of
other anaerobic chemoheterotrophic Spirochaeta strains, the
majority of which ferment available carbohydrates to produce acetate,
CO2, and H2 (21). Therefore, the
metabolism of the A. pompejana spirochete epibiont most
likely is fermentative, and the spirochete probably uses available
carbohydrates in the mucus surrounding the worm and potentially
supplies carbon sources and electron donors to the other epibionts
(members of the epsilon subdivision of the Proteobacteria).
The A. pompejana epibiont community described in this study
and many other symbiotic and nonsymbiotic communities have been characterized by using PCR. The drawbacks to PCR-based analyses of
microbial communities have been reviewed at length (33). Two of the major concerns lie with PCR bias and different resolution capabilities of the methods. A determination of whether a member is
considered dominant or minor in a community by PCR-based techniques may
be questioned if only a single parameter is used for analysis. We felt
that this study was a unique opportunity to characterize the
spirochetes in a microbial community by using three separate, PCR-based
methods. For each analysis (16S rDNA library screening, DGGE, and
T-RFLP analysis) we used different primers, cycling conditions, and
resolution techniques. DGGE and T-RFLP analyses, in which 16S rDNA
amplification products that were approximately 171 and 918 bp long,
respectively, were used, resulted in similar relative frequencies of
spirochete A. The frequency of the corresponding clone was
significantly different in the 16S rDNA library, where the entire 16S
rDNA gene had been amplified (19). Alignment of the
sequence of the 3' primer used for the 16S rDNA library with the 10 sequences most similar to spirochete A (= APG10) demonstrated that 7 of
10 members of the spirochete family had four or more mismatches in the
3' end of the primer sequence. Thus, many of the spirochetes could not
be amplified representatively with this primer and were
underrepresented in the full-length 16S rDNA community analysis. Like
the results of other investigators, our results indicate that primer
bias may be extremely important in a comparison of the members of a
community (27, 30, 34).
There have been only a few previous molecular biology-based studies
that described the microbial diversity observed at hydrothermal vents,
and in none of them were spirochetes found (20, 24, 32).
This may have been due to absence of the organisms or, as suggested by
this study, to the choice of primers used for PCR amplification. During
this investigation, we discovered several novel spirochetes not only in
the A. pompejana epibiont community but also in the
surrounding tubes and chimney samples from other hydrothermal vent
environments. Because of the difficulty of isolating and
cultivating spirochetes, detailed molecular analysis of similar environments with spirochete-specific primers would most likely reveal other, previously unknown members of the spirochete division. We
are currently screening other hydrothermal vent habitats with such
primers to identify additional novel spirochetes that are most likely
important components of the microbial communities associated with
deep-sea hydrothermal vent environments.
Unlike many other symbiotic associations in marine systems, the
bacterial community associated with A. pompejana is
morphologically and phylogenetically diverse. A group of closely
related members of the epsilon subdivision of the
Proteobacteria dominates the community (19;
this study), while a smaller percentage of the A. pompejana
epibiont community members from a wide geographic range studied in this
work consists of more diverse members of the epsilon subdivision of the
Proteobacteria and of a closely related
Spirochaeta phylogenetic cluster. The role of the
spirochetes remains unclear, but our results support the hypothesis
that some of the epibionts of A. pompejana are
chemoheterotrophic. Further research is currently being performed in
our laboratory to characterize the mixture of A. pompejana
epibionts collected from geographically, thermally, and chemically
diverse environments and to decipher their metabolic capabilities in an
effort to discover their role in this integrated episymbiosis.
| |
ACKNOWLEDGMENTS |
This research was supported by grants to S.C.C. from the National
Science Foundation (grant OCE-9314595), the LEXEN initiative (grant
OPP-9907666) and the Delaware Sea Grant Program (grant R/B37).
We thank K. Coyne, M. Cottrell, and C. Di Meo for helpful discussions
and for critically reviewing the manuscript. We thank the captain and
crew of the R/V Atlantis and especially the DSV Alvin pilots for their critical roles in the collection of
specimens. We thank D. Prieur for the invitation to participate on the
AMISTAD cruise and C. Jeanthon and the pilots and crew of
L'Atalante and DSV Nautile for their assistance.
We also thank R. Vrijenhoek for providing access to samples collected
during the Southern EPR cruise.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: College of
Marine Studies, University of Delaware, Cannon Laboratory, 700 Pilottown Road, Lewes, DE 19958. Phone: (302)645 4078. Fax: (302)645
4007. E-mail: Caryc{at}udel.edu.
| |
REFERENCES |
| 1.
|
Alayse-Danet, A. M.,
D. Desbruyères, and F. Gaill.
1987.
The possible nutritional or detoxification role of the epibiotic bacteria of alvinellid polychaetes: review of the current data.
Symbiosis
4:51-62.
|
| 2.
|
Alayse-Danet, A. M.,
F. Gaill, and D. Desbruyères.
1986.
In situ bicarbonate uptake by bacteria-Alvinella associations.
Mar. Ecol. (Pubbl. Stn. Zool. Napoli I)
7:233-240.
|
| 3.
|
Amann, R. I.,
L. Krumholz, and D. A. Stahl.
1990.
Fluorescent oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology.
J. Bacteriol.
172:762-770[Abstract/Free Full Text].
|
| 4.
|
Benson, D. A.,
I. Karsch-Mizrachi,
D. J. Lipman,
J. Ostell,
B. A. Rapp, and D. L. Wheeler.
2000.
GenBank.
Nucleic Acids Res.
28:15-18[Abstract/Free Full Text].
|
| 5.
|
Cary, S. C.,
M. T. Cottrell,
J. L. Stein,
F. Camacho, and D. Desbruyères.
1997.
Molecular identification and localization of a filamentous symbiotic bacteria associated with the hydrothermal vent annelid, Alvinella pompejana.
Appl. Environ. Microbiol.
63:1124-1130[Abstract].
|
| 6.
|
Cary, S. C.,
T. Shank, and J. Stein.
1998.
Worms bask in extreme temperatures.
Nature
391:545-546.
|
| 7.
|
Cavanaugh, C. M.
1994.
Microbiol symbiosis: patterns of diversity in the marine environment.
Am. Zool.
34:79-89.
|
| 8.
|
Cavanaugh, C. M.,
M. L. Jones,
H. W. Jannasch, and J. B. Waterbury.
1981.
Prokaryotic cells in the hydrothermal vent tube worm Riftia pachyptila Jones: possible chemoautotrophic symbionts.
Science
213:340-342[Abstract/Free Full Text].
|
| 9.
|
Chevaldonné, P.,
D. Desbruyères, and J. J. Childress.
1992.
Some like it hot, some like it hotter.
Nature
359:593-594.
|
| 10.
|
Cottrell, M. T., and S. C. Cary.
1999.
Diversity of dissimilatory bisulfite reductase genes of bacteria associated with the deep-sea hydrothermal vent polychaete annelid Alvinella pompejana.
Appl. Environ. Microbiol.
65:1127-1132[Abstract/Free Full Text].
|
| 11.
|
Desbruyères, D.,
P. Chevaldonné,
A.-M. Alayse,
D. Jollivet,
F. H. Lallier,
C. Jouin-Toulmond,
F. Zal,
P.-M. Sarradin,
R. Cosson,
J.-C. Caprais,
C. Arndt,
J. O'Brien,
J. Guezennec,
S. Hourdez,
R. Riso,
F. Gaill,
L. Laubier, and A. Toulmond.
1998.
Biology and ecology of the "Pompeii worm" (Alvinella pompejana Desbruyères and Laubier), a normal dweller of an extreme deep-sea environment: a synthesis of current knowledge and recent developments.
Deep-Sea Res. Part II
45:383-422[CrossRef].
|
| 12.
|
Desbruyères, D.,
F. Gaill,
L. Laubier, and Y. Fouquet.
1985.
Polychaetous annelids from hydrothermal vent ecosystems: an ecological overview.
Bull. Biol. Soc. Wash.
6:103-116.
|
| 13.
|
Distel, D. L.,
H. K. Lee, and C. M. Cavanaugh.
1995.
Intracellular coexistence of methano- and thioautotrophic bacteria in a hydrothermal vent mussel.
Proc. Natl. Acad. Sci. USA
92:9598-9602[Abstract/Free Full Text].
|
| 14.
|
Dubilier, N.,
R. Amann,
C. Erseus,
G. Muyzer,
S. Y. Park,
O. Giere, and C. M. Cavanaugh.
1999.
Phylogenetic diversity of bacterial endosymbionts in the gutless marine oligochete Olavius loisae (Annelida).
Mar. Ecol. Prog. Ser.
178:271-280.
|
| 15.
|
Feldman, R. A.,
M. B. Black,
C. S. Cary,
R. A. Lutz, and R. C. Vrijenhoek.
1997.
Molecular phylogenetics of bacterial endosymbionts and their vestimentiferan hosts.
Mol. Mar. Biol. Biotechnol.
6:268-277[Medline].
|
| 16.
|
Fisher, C. R.,
J. J. Childress, and E. Minnich.
1989.
Autotrophic carbon fixation by the chemoautotrophic symbionts of Riftia pachyptila.
Biol. Bull. (Woods Hole)
177:372-385[Abstract/Free Full Text].
|
| 17.
|
Gaill, F.,
D. Desbruyères, and D. Prieur.
1987.
Bacterial communities associated with "Pompeii worms" from the East Pacific Rise hydrothermal vents: SEM, TEM observations.
Microb. Ecol.
13:129-139[CrossRef].
|
| 18.
|
Gaill, F.,
D. Desbruyères,
D. Prieur, and J. P. Gourret.
1984.
Mise en évidence de communautés bactériennes épibiontes du "ver de Pompéi."
C. R. Acad. Sci. Paris Ser. III
298:553-558.
|
| 19.
|
Haddad, M. A.,
F. Camacho,
P. Durand, and S. C. Cary.
1995.
Phylogenetic characterization of the epibiotic bacteria associated with the hydrothermal vent polychaete Alvinella pompejana.
Appl. Environ. Microbiol.
61:1679-1687[Abstract].
|
| 20.
|
Harmsen, H. J. M.,
D. Prieur, and C. Jeanthon.
1997.
Distribution of microorganisms in deep-sea hydrothermal vent chimneys investigated by whole-cell hybridization and enrichment culture of thermophilic subpopulations.
Appl. Environ. Microbiol.
63:2876-2883[Abstract].
|
| 21.
|
Harwood, C. S.,
H. W. Jannasch, and E. Canale-Parola.
1982.
Anaerobic spirochete from a deep-sea hydrothermal vent.
Appl. Environ. Microbiol.
44:234-237[Abstract/Free Full Text].
|
| 22.
|
Laue, B. E., and D. C. Nelson.
1994.
Characterization of the gene encoding the autotrophic ATP sulfurylase from the bacterial endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila.
J. Bacteriol.
176:3723-3729[Abstract/Free Full Text].
|
| 23.
|
Liu, W.-T.,
T. L. Marsh,
H. Cheng, and L. J. Forney.
1997.
Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA.
Appl. Environ. Microbiol.
63:4516-4522[Abstract].
|
| 24.
|
Moyer, C. L.,
F. C. Dobbs, and D. M. Karl.
1995.
Phylogenetic diversity of the bacterial community from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii.
Appl. Environ. Microbiol.
61:1555-1562[Abstract].
|
| 25.
|
Muyzer, G.,
E. C. de Waal, and A. G. Uitterlinden.
1993.
Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA.
Appl. Environ. Microbiol.
59:695-700[Abstract/Free Full Text].
|
| 26.
|
Paster, B. J.,
F. E. Dewhirst,
W. G. Weisburg,
L. A. Tordoff,
G. J. Fraser,
R. B. Hespell,
T. B. Stanton,
L. Zablen,
L. Mandelco, and C. R. Woese.
1991.
Phylogenetic analysis of the spirochetes.
J. Bacteriol.
173:6101-6109[Abstract/Free Full Text].
|
| 27.
|
Polz, M. F., and C. M. Cavanaugh.
1998.
Bias in template-to-product ratios in multitemplate PCR.
Appl. Environ. Microbiol.
64:3724-3730[Abstract/Free Full Text].
|
| 28.
|
Polz, M. F., and C. M. Cavanaugh.
1995.
Dominance of one bacterial phylotype at a Mid-Atlantic Ridge hydrothermal vent site.
Proc. Natl. Acad. Sci. USA
92:7232-7236[Abstract/Free Full Text].
|
| 29.
|
Prieur, D., and C. Jeanthon.
1987.
Preliminary study of heterotrophic bacteria isolated from two deep sea hydrothermal vent invertebrates: Alvinella pompejana (polychaete) and Bathymodiolus thermophilus (bivalve).
Symbiosis
4:87-98.
|
| 30.
|
Rainey, F.,
N. Ward,
L. Sly, and E. Stackebrandt.
1994.
Dependence on the taxon composition of clone libraries for PCR amplified, naturally occurring 16S rDNA, on the primer pair and the cloning system used.
Experientia
50:796-797.
|
| 31.
|
Rieley, G.,
C. L. Van Dover,
D. B. Hedrick, and G. Eglinton.
1999.
Trophic ecology of Rimicaris exoculata: a combined lipid abundance stable isotope approach.
Mar. Biol. (New York)
133:495-499[CrossRef].
|
| 32.
|
Sievert, S. M.,
T. Brinkhoff,
G. Muyzer,
V. Ziebis, and J. Kuever.
1999.
Spatial heterogeneity of bacterial populations along an environmental gradient at a shallow submarine hydrothermal vent near Milos Island (Greece).
Appl. Environ. Microbiol.
65:3834-3842[Abstract/Free Full Text].
|
| 33.
|
Wintzingerode, F. V.,
U. B. G el, and E. Stackebrandt.
1997.
Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis.
FEMS Microbiol. Rev.
21:213-229[CrossRef][Medline].
|
| 34.
|
Zheng, D.,
E. W. Alm,
D. A. Stahl, and L. Raskin.
1996.
Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies.
Appl. Environ. Microbiol.
62:4504-4513[Abstract].
|
Applied and Environmental Microbiology, January 2001, p. 110-117, Vol. 67, No. 1
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.1.110-117.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Blazejak, A., Erseus, C., Amann, R., Dubilier, N.
(2005). Coexistence of Bacterial Sulfide Oxidizers, Sulfate Reducers, and Spirochetes in a Gutless Worm (Oligochaeta) from the Peru Margin. Appl. Environ. Microbiol.
71: 1553-1561
[Abstract]
[Full Text]
-
Campbell, B. J., Stein, J. L., Cary, S. C.
(2003). Evidence of Chemolithoautotrophy in the Bacterial Community Associated with Alvinella pompejana, a Hydrothermal Vent Polychaete. Appl. Environ. Microbiol.
69: 5070-5078
[Abstract]
[Full Text]
-
Campbell, B. J., Jeanthon, C., Kostka, J. E., Luther, G. W. III, Cary, S. C.
(2001). Growth and Phylogenetic Properties of Novel Bacteria Belonging to the Epsilon Subdivision of the Proteobacteria Enriched from Alvinella pompejana and Deep-Sea Hydrothermal Vents. Appl. Environ. Microbiol.
67: 4566-4572
[Abstract]
[Full Text]
Top
Abstract
Introduction
