Adaptations to high pressure of Nautilia sp. strain PV-1, a piezophilic Campylobacterium (aka Epsilonproteobacterium) isolated from a deep-sea hydrothermal vent

Abstract

Physiological and gene expression studies of deep-sea bacteria under pressure conditions similar to those experienced in their natural habitat are critical for understanding growth kinetics and metabolic adaptations to in situ conditions. The Campylobacterium (aka Epsilonproteobacterium) Nautilia sp. strain PV-1 was isolated from hydrothermal fluids released from an active deep-sea hydrothermal vent at 9° N on the East Pacific Rise. Strain PV-1 is a piezophilic, moderately thermophilic, chemolithoautotrophic anaerobe that conserves energy by coupling the oxidation of hydrogen to the reduction of nitrate or elemental sulfur. Using a high-pressure-high temperature continuous culture system, we established that strain PV-1 has the shortest generation time of all known piezophilic bacteria and we investigated its protein expression pattern in response to different hydrostatic pressure regimes. Proteogenomic analyses of strain PV-1 grown at 20 and 5 MPa showed that pressure adaptation is not restricted to stress response or homeoviscous adaptation but extends to enzymes involved in central metabolic pathways. Protein synthesis, motility, transport, and energy metabolism are all affected by pressure, although to different extents. In strain PV-1, low-pressure conditions induce the synthesis of phage-related proteins and an overexpression of enzymes involved in carbon fixation.

FIGURE 1

Maximum likelihood phylogenetic tree derived from 16 S rRNA gene sequences showing the position of Nautilia sp. strain PV‐1 within the Campylobacterota. Bootstrap values higher than 50% are based on 1000 replicates and are shown at each node. Bar, 0.02% substitutions per position. Sequences belonging to the Gammaproteobacteria were used as the outgroup. [Corrections added on 19 November 2022, after first online publication: italicization in Figure 1 has been corrected in this version.]

FIGURE 2

Metaproteogenomic reconstruction of the central metabolism of Nautilia sp.strain PV‐1. Enzyme names are reported along with the corresponding protein numbers as discussed in the text and reported in Table S1. Within each enzymatic complex, significantly (false discovery rate [fdr] < 0.01 for QSpec) overexpressed and underexpressed enzymes (outlined shapes) in the HPC versus LPC comparison are indicated by the upward green and downward red arrows, respectively. Abbreviations: Nitrate ammonification: AmtB, ammonia transporter; GlnA, glutamine synthetase; GltS, glutamate synthase; Hao, hydroxylamine:ubiquinone reductase; Har, hydroxylamine reductase; Hcp, putative iron‐sulfur cluster‐binding protein; NapABCFGHL, periplasmic nitrate reductase complex. Hydrogen oxidation: CooFHKLMUX, carbon monoxide‐induced hydrogenase complex; EchABCEF, Ech membrane bound hydrogenase complex, ferredoxin reduction; FdhABC, formate dehydrogenase; GltB, glutamate synthase; HupAB, cytosolic uptake/hydrogen sensing hydrogenase; HynABC, Ni‐Fe membrane‐bound hydrogenase; HypAF, hydrogenases expression/synthesis accessory proteins; HynABC, quinone‐reactive hydrogenase. Energy conservation: Aps, sulfate adenylyltransferase; NADH dehydrogenase and ATP synthetase are reported without the names of the single units; Nhe, sodium/hydrogen symporter. Sulfur reduction: PsrABC, polysulfide oxidoreductase complex; Sqr, sulfide:quinone oxidoreductase; Sud, putative rhodanese‐like domain‐containing protein. Flagellar complex: for simplicity single unit names are not reported. Reductive citric acid cycle: AclAB, ATP‐citrate lyase; AcnB, aconitate hydratase; AccABCD, acetyl‐coenzyme transferase complex; FumAB, fumarate hydratase; FrdAB, fumarate reductase; Idh2, isocitrate dehydrogenase/2‐oxoglutarate carboxylase; Mdh, malate dehydrogenase; OorABCD, 2‐oxoglutarate synthase; PorABDG, pyruvate synthase; PdhAB, pyruvate dehydrogenase; PpsA, phosphoenolpyruvate synthase; PvtK, pyruvate kinase; PepC, phosphoenolpyruvate carboxylase/kinase; SucCD, succinyl‐CoA synthetase. Gluconeogenesis: Eno, enolase; Fba, fructose‐bisphosphate aldolase; Fbp, fructose‐1,6‐bisphosphatase; Gapdh, glyceraldehyde 3‐phosphate dehydrogenase; Pgm, phosphoglycerate mutase; Pgk, phosphoglycerate kinase; Pgi, phosphoglucose isomerase; Pgm, Phosphoglucomutase; Tim, triosephosphate isomerase. Biosynthesis: FabABDFGHIZ: fatty acid biosynthesis pathway. Reactive oxygen species detoxification: Adp, rubredoxin/hydroperoxide reductase; CydBA, cytochrome d ubiquinol oxidase; CcpA, cytochrome c551 peroxidase; Sor, superoxide reductase. Stress protection: DnaJK, molecular chaperone; GrpE: heat‐shock protein; GroEL/S: heat‐shock protein 60 family. Secretion systems (SEC): GspCDEFLM, type II secretion system; FadL, long‐chain fatty acid transport protein; HlyBD, ABC transporter multidrug efflux pump/type I family secretion protein; PilABCDQT, type IV secretion system/fimbrial assembly; TolC, outer membrane efflux protein

FIGURE 3

(A) Genome structures of Nautilia sp. strain PV‐1. Features, starting with the outermost circle: 1. Comparative amino acid percent identity between Nautilia sp. strain PV‐1 and Nautilia profundicola strain Amh; 2. Gene distribution within PV‐1 genome; 3. Predicted genomic islands in the genome of strain PV‐1 by IslandPath‐DIMOB (blue), SIGI‐HMM (yellow); predicted by all tools within IslandViewer (orange); 4. GC skew; 5. Genetic information processing (blue lines); 6. Membrane transport (orange lines); 7. Carbon metabolism: glycolysis/gluconeogenesis reductive citric cycle (green lines); 8. Energy metabolism: hydrogenase (grey lines) nitrogen metabolism (purple lines) sulfur metabolism (yellow lines). (B) Complete genome of the prophage of Nautilia sp. strain PV‐1

FIGURE 4

Variation of growth parameters of Nautilia sp. strain PV‐1 over the course of the 1633‐hour experiment in the chemostat at different pressures. (A) Cell concentration over time. (B) Dilution rate/growth rate (D or μ) over time. The different pressure regimes are indicated at the top of the graphs.

FIGURE 5

Variation of grow rate of Nautilia sp. strain PV‐1 in relation to the concentration of dissolved hydrogen at 0.5 and 20 MPa

FIGURE 6

Relative abundance (%) of differentially expressed proteins by cellular localization as predicted by the DEseq2 and QSpec algorithms in the HPC versus LPC and LPC versus LPB treatments

FIGURE 7

Rates of hydrogen utilization (A) and carbon fixation (B) in Nautilia sp. strain PV‐1 relative to the dilution rate at 0.5 and 20 MPa