Life and death of deep-sea vents: bacterial diversity and ecosystem succession on inactive hydrothermal sulfides

Abstract

Hydrothermal chimneys are a globally dispersed habitat on the seafloor associated with mid-ocean ridge (MOR) spreading centers. Active, hot, venting sulfide structures from MORs have been examined for microbial diversity and ecology since their discovery in the mid-1970s, and recent work has also begun to explore the microbiology of inactive sulfides--structures that persist for decades to millennia and form moderate to massive deposits at and below the seafloor. Here we used tag pyrosequencing of the V6 region of the 16S rRNA and full-length 16S rRNA sequencing on inactive hydrothermal sulfide chimney samples from 9°N on the East Pacific Rise to learn their bacterial composition, metabolic potential, and succession from venting to nonventing (inactive) regimes. Alpha-, beta-, delta-, and gammaproteobacteria and members of the phylum Bacteroidetes dominate all inactive sulfides. Greater than 26% of the V6 tags obtained are closely related to lineages involved in sulfur, nitrogen, iron, and methane cycling. Epsilonproteobacteria represent <4% of the V6 tags recovered from inactive sulfides and 15% of the full-length clones, despite their high abundance in active chimneys. Members of the phylum Aquificae, which are common in active vents, were absent from both the V6 tags and full-length 16S rRNA data sets. In both analyses, the proportions of alphaproteobacteria, betaproteobacteria, and members of the phylum Bacteroidetes were greater than those found on active hydrothermal sulfides. These shifts in bacterial population structure on inactive chimneys reveal ecological succession following cessation of venting and also imply a potential shift in microbial activity and metabolic guilds on hydrothermal sulfides, the dominant biome that results from seafloor venting.

FIG 1

Rarefaction curves for inactive sulfide samples. All samples were randomly resampled down to the smallest sample size, 9,149 tags (9M4O). The preclustering option was used in mothur for rarefaction calculation, and OTUs were calculated with a cutoff of 3%. Vertical bars represent 95% confidence intervals.

FIG 2

Bacterial distribution among the different samples. Only groups that represent >1% of the tags in at least one of the samples are included. The category “other” refers to all groups that represent <1% of the tags in all of the samples. These are shown in detail in Fig. S3 in the supplemental material. The total number of tags obtained for each sample is given in parentheses. The rightmost column (Shared) represents tags shared by at least two samples in this study.

FIG 3

Bacterial distribution among the different samples as determined via full-length Sanger sequencing. The total number of clones is shown in parentheses.

FIG 4

Phylogenetic tree of OTUs represented by more than three clones from a single sample and/or clones from multiple samples. OTUs are defined by a 97% similarity cutoff. OTUs were aligned using MAFFT (48) with the G-INS-I algorithm and 200PAM scoring matrix in Geneious 5.4 (49), and then the tree was constructed using the neighbor-joining algorithm with 1,000 bootstraps. Symbols next to the OTUs from this study represent the samples from which they were recovered, as indicated by the sample key at the top left. The number of clones belonging to each OTU is in parentheses. Bootstrap values of >50% are shown at nodes. Aquifex pyrophilus and Hydrogenobacter subterraneus were used as outgroups.

FIG 5

Bacterial distribution in composite inactive chimneys and a composite active black smoker chimney. “Inactive Chimneys Pyrotags” (n = 206,647) is the sum of all of the tags in this study. “Inactive Chimneys Full-Length” (n = 452) is the sum of all of the full-length clones in this study. The composite active chimney (“Active Chimneys Full-Length,” n = 834) was generated by compiling data from previously published studies of full-length 16S rRNA clones on active black smoker chimneys and represents the sum of all of the clones in these studies (2–9, 36, 37). Only studies where clone frequency was reported were used. The color code is same as that in Fig. 1, and the category “other” is shown in detail in Fig. S4 in the supplemental material.