Characterization of a novel spirochete associated with the hydrothermal vent polychaete annelid, Alvinella pompejana

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 degrees N to 32 degrees 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.

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).

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.

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.

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.