Evidence of chemolithoautotrophy in the bacterial community associated with Alvinella pompejana, a hydrothermal vent polychaete

Abstract

The deep-sea polychaete Alvinella pompejana colonizes tubes on the sides of black smoker chimneys along the East Pacific Rise. A diverse, yet phylogenetically constrained episymbiotic community is obligately associated with its dorsal surface. The morphologically and phylogenetically distinct dominant episymbionts have not yet been cultured, and there are no clearly defined roles for these bacteria in this symbiosis. A large insert fosmid library was screened for the presence of the two dominant phylotypes. Two fosmids, 35.2 and 38 kb, containing phylotype-specific 16S ribosomal DNA sequences were fully sequenced. Each fosmid had a gene encoding ATP citrate lyase, a key enzyme in the reverse tricarboxylic acid (rTCA) cycle, a CO(2) fixation pathway. A selection of episymbiont communities from various geographic locations and vent sites were screened for the presence, diversity, and expression (via reverse transcription-PCR) of the ATP citrate lyase gene. Our results indicate that the ATP citrate lyase gene is not only a consistent presence in these episymbiont communities but is also expressed. Phylogenetically distinct forms of ATP citrate lyase were also found associated with and expressed by bacteria extracted from the tubes of A. pompejana. Utilizing PCR with degenerate primers based on a second key enzyme in the rTCA cycle, 2-oxoglutarate:acceptor oxidoreductase, we also demonstrated the persistent presence and expression of this gene in the episymbiont community. Our results suggest that members of both the episymbiont and the surrounding free-living communities display a chemolithoautotrophic form of growth and therefore contribute fixed carbon to other organisms in the vent community.

FIG. 1.

Phylogenetic amino acid distance trees showing the relationships between the ATP citrate lyase genes (alpha and beta) from 6C6 and 7G3 fosmids with other ATP citrate lyase genes (prokaryotic [Chlorobium limicola] and eukaryotic [Arabidopsis thaliana, Lupinus albus, Capsicum annuum, Caenorhabditis elegans, Mus musculus, Rattus norvegicus, and Homo sapiens]). The trees are based on alignment of ca. 591 and 445 translated amino acids (minus insertions and deletions) from the alpha and beta genes, respectively. Bootstrap values from 100 resamplings are indicated prior to the branch points of the tree. The scale bar represents the calculated number of changes per amino acid position.

FIG. 2.

CLUSTALW alignment of a portion of the ATP citrate lyase beta gene from the indicated translated sequence. Sequences are the same as those used in Fig. 1. Numbered amino acids are from the 6C6 and 7G3 fosmid subunits. The regions used to design primers are indicated by boldface type.

FIG. 3.

DGGE of positive PCR products after amplification with universal primers for 16S rRNA or rDNA. Lanes 1 to 11, separated amplification products obtained from genomic DNA or RNA extracted from various A. pompejana episymbiont (AP) or A. pompejana tube (APT) communities. Lane 1, APG1B (AP, 13°N EPR in 1994); lanes 2 (DNA) and 3 (RNA) from sample 3713 (AP); lanes 4 (DNA) and 5 (RNA) from sample 3713 (APT); lanes 6 (DNA) and 7 (DNA) from sample 42 (AP); lanes 8 (DNA) and 9 (RNA) from sample 3722 (APT); and lanes 10 (DNA) and 11 (RNA) from sample 64 (AP). Samples were obtained from the indicated locations.

FIG. 4.

Phylogenetic DNA distance trees showing the relationships between a section of the ATP citrate lyase β gene from genomic versus expressed amplified products. (A) Phylogenetic DNA distance tree that includes clones amplified from the indicated A. pompejana episymbiont communities. (B) Phylogenetic DNA distance tree that includes clones amplified from samples taken from the inside (sample 3713T) or entire (sample 3722) A. pompejana tubes. The “♦—♦” bracket indicates 100% amino acid identity to the 7G3 ATP citrate lyase β gene within the sequenced region. Genomic samples are indicated by a “D,” expressed samples by are indicated by an “R,” and tube samples are indicated by a “T.” The trees are based on alignment of ca. 311 bases (minus insertions, deletions, and ambiguous bases) from the β ATP citrate lyase gene. Bootstrap values from 100 resamplings are indicated prior to the branch points of the tree. The scale bar represents the calculated number of changes per nucleotide position.