Adaptations to submarine hydrothermal environments exemplified by the genome of Nautilia profundicola

Abstract

Submarine hydrothermal vents are model systems for the Archaean Earth environment, and some sites maintain conditions that may have favored the formation and evolution of cellular life. Vents are typified by rapid fluctuations in temperature and redox potential that impose a strong selective pressure on resident microbial communities. Nautilia profundicola strain Am-H is a moderately thermophilic, deeply-branching Epsilonproteobacterium found free-living at hydrothermal vents and is a member of the microbial mass on the dorsal surface of vent polychaete, Alvinella pompejana. Analysis of the 1.7-Mbp genome of N. profundicola uncovered adaptations to the vent environment--some unique and some shared with other Epsilonproteobacterial genomes. The major findings included: (1) a diverse suite of hydrogenases coupled to a relatively simple electron transport chain, (2) numerous stress response systems, (3) a novel predicted nitrate assimilation pathway with hydroxylamine as a key intermediate, and (4) a gene (rgy) encoding the hallmark protein for hyperthermophilic growth, reverse gyrase. Additional experiments indicated that expression of rgy in strain Am-H was induced over 100-fold with a 20 degrees C increase above the optimal growth temperature of this bacterium and that closely related rgy genes are present and expressed in bacterial communities residing in geographically distinct thermophilic environments. N. profundicola, therefore, is a model Epsilonproteobacterium that contains all the genes necessary for life in the extreme conditions widely believed to reflect those in the Archaean biosphere--anaerobic, sulfur, H2- and CO2-rich, with fluctuating redox potentials and temperatures. In addition, reverse gyrase appears to be an important and common adaptation for mesophiles and moderate thermophiles that inhabit ecological niches characterized by rapid and frequent temperature fluctuations and, as such, can no longer be considered a unique feature of hyperthermophiles.

Figure 1. Circular View of the N. profundicola genome.

Circles correspond to the following features, starting with the outermost circle: (1) forward strand genes (2) reverse strand genes (3) χ² deviation of local nucleotide composition from the genome average (4) GC skew (5) tRNAs (green lines) (6) rRNAs (blue lines) (7) small RNAs (red lines). Genes are colored according to their role categories.

Figure 2. Major metabolic subsystems in N. profundicola AmH as deduced from the genomic sequence.

Proteins that belong to a given subsystem are color coded as follows: energy metabolism, olive green; carbon (C) metabolism, pale red; nitrogen (N) metabolism, light blue; sulfur (S) metabolism, yellow. Numbers denote the ORFs encoding the enzymes predicted to catalyze a particular reaction. Individual subunits are not drawn to scale. Common energy and reductant carriers shared across subsystems are depicted in green type. Interactions between subsystems based on shared pools of metabolites or reductant carriers are indicated by the dashed blue double arrows. For details on each subsystem, please refer to the appropriate section of the text.

Figure 3. Deduced pathways for sulfur metabolism in N. profundicola AmH.

Enzymes are labeled with the number of the ORF that encodes the protein. The central intermediate in the pathway is periplasmic polysulfide, which is predicted to have two fates. First, polysulfide can be used as the terminal electron acceptor for energy conservation by a quinol oxidizing polysulfide reductase (right branch, 1483–1485). This process likely involves a polysulfide carrier (0471) similar to the Sud protein of Wolinella succinogenes. Second, polysulfide may be assimilated after transport after conversion to sulfide by an NAD(P)H:polysulfide oxidoreductase (center branch, 0923). This is the only route for the synthesis of cysteine and methionine as AmH lacks a reductive sulfate assimilation pathway.

Figure 4. Deduced pathways for nitrogen metabolism in N. profundicola AmH.

Enzymes are labeled with the number of the ORF that encodes the protein. The proposed alternative pathway for nitrite reduction to ammonia, predicted to be essential for the growth of AmH on nitrate, is highlighted in orange. The first step of this pathway consists of a hydroxylamine:ubiquinone redox module (HURM) composed of a novel nitrite reductase (1248; reverse hydroxylamine oxidoreductase) that produces hydroxylamine utilizing electrons donated from the (ubi)quinol pool via a cytochrome c protein (0536) in the NapC/NrfH/cM552 protein superfamily. Following transport of hydroxylamine into the cytoplasm, the second step is the reduction of hydroxylamine to ammonia by a hybrid cluster protein/hydroxylamine reductase (1044) utilizing reducing power from a flavin-containing NADH oxidase (0519).

Figure 5. Phylogenetic relationships of large subunit hydrogenase protein sequences.

Distance topologies were performed in MEGA4, based on the Neighbor-joining method after alignment with ClustalW (BLOSUM matrix). Bootstrap values (500 replicates) are indicated on branches. Indicated groupings are based on previous analyses.

Figure 6. Generation of NAD(P)H in N. profundicola via the predicted partial NUO complex from hydrogen or formate.

The ultimate reduction of NAD(P)+ from the quinone pool is endergonic and requires energy in the form of coupling to the proton motive force, which may be provided, in part, by a V-type ATPase. Numbers on complexes refer to the ORF numbers of the encoding genes.

Figure 7. Phylogenetic relationships between reverse gyrase and related topoisomerase protein sequences.

Distance topologies were performed in MEGA4, based on the Neighbor-joining method after alignment with ClustalW (BLOSUM matrix). Bootstrap values (500 replicates) are indicated on branches. Nearly identical topologies were observed with Maximum Likelihood (phyml), Minimum Evolution and Maximum Parsimony algorithms (data not shown).

Figure 8. Phylogenetic relationships between bacterial reverse gyrase sequences obtained from PCR amplification from hydrothermal vent chimneys and known sequences.

Distance topologies were performed in MEGA4, based on the Neighbor-joining method after alignment with ClustalW (BLOSUM matrix). Bootstrap values (500 replicates) are indicated on branches.

Figure 9. Abundance of rgy mRNA and 16S rRNA in N. profundicola cells incubated under the indicated temperature for 2 hours.

Values were calculated per ng of RNA. Standard errors indicated by the error bars. Y axes are on a log scale.

Figure 10. Phylogenetic associations and relative percentages of rgy clones retrieved from hydrothermal vent chimney samples.

9N-773 (Io vent); 9N-POB (P vent, bottom); 9N-243T (Alvinella tube scraping); G-Rebecca's Roost (Guaymas Basin flange); G-855 (Guaymas Basin flange). Groups were reported as clades with nearest cultured representatives because of poor bootstrap values on the distal branch points, most likely due to the length of the aligned sequence.