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1.  The Deep-Sea Bacterium Photobacterium profundum SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions ▿ †  
Applied and Environmental Microbiology  2008;74(20):6298-6305.
Motility is a critical function needed for nutrient acquisition, biofilm formation, and the avoidance of harmful chemicals and predators. Flagellar motility is one of the most pressure-sensitive cellular processes in mesophilic bacteria; therefore, it is ecologically relevant to determine how deep-sea microbes have adapted their motility systems for functionality at depth. In this study, the motility of the deep-sea piezophilic bacterium Photobacterium profundum SS9 was investigated and compared with that of the related shallow-water piezosensitive strain Photobacterium profundum 3TCK, as well as that of the well-studied piezosensitive bacterium Escherichia coli. The SS9 genome contains two flagellar gene clusters: a polar flagellum gene cluster (PF) and a putative lateral flagellum gene cluster (LF). In-frame deletions were constructed in the two flagellin genes located within the PF cluster (flaA and flaC), the one flagellin gene located within the LF cluster (flaB), a component of a putative sodium-driven flagellar motor (motA2), and a component of a putative proton-driven flagellar motor (motA1). SS9 PF flaA, flaC, and motA2 mutants were defective in motility under all conditions tested. In contrast, the flaB and motA1 mutants were defective only under conditions of high pressure and high viscosity. flaB and motA1 gene expression was strongly induced by elevated pressure plus increased viscosity. Direct swimming velocity measurements were obtained using a high-pressure microscopic chamber, where increases in pressure resulted in a striking decrease in swimming velocity for E. coli and a gradual reduction for 3TCK which proceeded up to 120 MPa, while SS9 increased swimming velocity at 30 MPa and maintained motility up to a maximum pressure of 150 MPa. Our results indicate that P. profundum SS9 possesses two distinct flagellar systems, both of which have acquired dramatic adaptations for optimal functionality under high-pressure conditions.
doi:10.1128/AEM.01316-08
PMCID: PMC2570297  PMID: 18723648
2.  Ecotype Diversity and Conversion in Photobacterium profundum Strains 
PLoS ONE  2014;9(5):e96953.
Photobacterium profundum is a cosmopolitan marine bacterium capable of growth at low temperature and high hydrostatic pressure. Multiple strains of P. profundum have been isolated from different depths of the ocean and display remarkable differences in their physiological responses to pressure. The genome sequence of the deep-sea piezopsychrophilic strain Photobacterium profundum SS9 has provided some clues regarding the genetic features required for growth in the deep sea. The sequenced genome of Photobacterium profundum strain 3TCK, a non-piezophilic strain isolated from a shallow-water environment, is now available and its analysis expands the identification of unique genomic features that correlate to environmental differences and define the Hutchinsonian niche of each strain. These differences range from variations in gene content to specific gene sequences under positive selection. Genome plasticity between Photobacterium bathytypes was investigated when strain 3TCK-specific genes involved in photorepair were introduced to SS9, demonstrating that horizontal gene transfer can provide a mechanism for rapid colonisation of new environments.
doi:10.1371/journal.pone.0096953
PMCID: PMC4019646  PMID: 24824441
3.  CYP261 enzymes from deep sea bacteria: a clue to conformational heterogeneity in cytochromes P450 
We have explored the adaptation of the cytochromes P450 (P450) of deep-sea bacteria to high hydrostatic pressures. Strict conservation of the protein fold and functional importance of protein-bound water make P450 a unique subject for the studies of high pressure adaptation. Earlier we expressed and purified a fatty-acid binding P450 from the deep-sea bacteria Photobacterium profundum SS9 (CYP261C1). Here we report purification and initial characterization of its mesophilic ortholog from the shallow-water P. profundum 3TCK (CYP261C2), as well as another piezophilic enzyme, CYP261D1 from deep-see Moritella sp. PE36. Comparison of the three enzymes revealed a striking peculiarity of the piezophilic enzymes. Both CYP261C1 and CYP261D1 possess an apparent pressure-induced conformational toggle actuated at the pressures commensurate with the physiological pressure of habitation of the host bacteria. Furthermore, in contrast to CYP261C2, the piezophilic CYP261 enzymes may be chromatographically separated into two fractions with different properties, and different thermodynamic parameters of spin equilibrium in particular. According to our concept, the changes in the energy landscape that evolved in pressure-tolerant enzymes must stabilize the less-hydrated, closed conformers, which may be transient in the catalytic mechanisms of non-piezophilic enzymes. The studies of enzymes of piezophiles should help unravel the mechanisms that control water access during the catalytic cycle.
doi:10.1002/bab.1083
PMCID: PMC3900297  PMID: 23586990
pressure-tolerant enzymes; piezophiles; conformational heterogeneity; cytochrome; P450; monooxygenases; molecular mechanism
4.  FabF Is Required for Piezoregulation of cis-Vaccenic Acid Levels and Piezophilic Growth of the Deep-Sea Bacterium Photobacterium profundum Strain SS9 
Journal of Bacteriology  2000;182(5):1264-1271.
To more fully explore the role of unsaturated fatty acids in high-pressure, low-temperature growth, the fabF gene from the psychrotolerant, piezophilic deep-sea bacterium Photobacterium profundum strain SS9 was characterized and its role and regulation were examined. An SS9 strain harboring a disruption in the fabF gene (strain EA40) displayed growth impairment at elevated hydrostatic pressure concomitant with diminished cis-vaccenic acid (18:1) production. However, growth ability at elevated pressure could be restored to wild-type levels by the addition of exogenous 18:1 to the growth medium. Transcript analysis did not indicate that the SS9 fabF gene is transcriptionally regulated, suggesting that the elevated 18:1 levels produced in response to pressure increase result from posttranscriptional changes. Unlike many pressure-adapted bacterial species such as SS9, the mesophile Escherichia coli did not regulate its fatty acid composition in an adaptive manner in response to changes in hydrostatic pressure. Moreover, an E. coli fabF strain was as susceptible to elevated pressure as wild-type cells. It is proposed that the SS9 fabF product, β-ketoacyl–acyl carrier protein synthase II has evolved novel pressure-responsive characteristics which facilitate SS9 growth at high pressure.
PMCID: PMC94411  PMID: 10671446
5.  The transcriptional landscape of the deep-sea bacterium Photobacterium profundum in both a toxR mutant and its parental strain 
BMC Genomics  2012;13:567.
Background
The deep-sea bacterium Photobacterium profundum is an established model for studying high pressure adaptation. In this paper we analyse the parental strain DB110 and the toxR mutant TW30 by massively parallel cDNA sequencing (RNA-seq). ToxR is a transmembrane DNA-binding protein first discovered in Vibrio cholerae, where it regulates a considerable number of genes involved in environmental adaptation and virulence. In P. profundum the abundance and activity of this protein is influenced by hydrostatic pressure and its role is related to the regulation of genes in a pressure-dependent manner.
Results
To better characterize the ToxR regulon, we compared the expression profiles of wt and toxR strains in response to pressure changes. Our results revealed a complex expression pattern with a group of 22 genes having expression profiles similar to OmpH that is an outer membrane protein transcribed in response to high hydrostatic pressure. Moreover, RNA-seq allowed a deep characterization of the transcriptional landscape that led to the identification of 460 putative small RNA genes and the detection of 298 protein-coding genes previously unknown. We were also able to perform a genome-wide prediction of operon structure, transcription start and termination sites, revealing an unexpected high number of genes (992) with large 5′-UTRs, long enough to harbour cis-regulatory RNA structures, suggesting a correlation between intergenic region size and UTR length.
Conclusion
This work led to a better understanding of high-pressure response in P. profundum. Furthermore, the high-resolution RNA-seq analysis revealed several unexpected features about transcriptional landscape and general mechanisms of controlling bacterial gene expression.
doi:10.1186/1471-2164-13-567
PMCID: PMC3505737  PMID: 23107454
High-pressure adaptation; Deep sea; Extremophile; Transcription; Operon; RNA-seq; UTR; Vibrionaceae; Photobacterium profundum; ToxR
6.  Monounsaturated but Not Polyunsaturated Fatty Acids Are Required for Growth of the Deep-Sea Bacterium Photobacterium profundum SS9 at High Pressure and Low Temperature 
There is considerable evidence correlating the production of increased proportions of membrane unsaturated fatty acids (UFAs) with bacterial growth at low temperatures or high pressures. In order to assess the importance of UFAs to microbial growth under these conditions, the effects of conditions altering UFA levels in the psychrotolerant piezophilic deep-sea bacterium Photobacterium profundum SS9 were investigated. The fatty acids produced by P. profundum SS9 grown at various temperatures and pressures were characterized, and differences in fatty acid composition as a function of phase growth, and between inner and outer membranes, were noted. P. profundum SS9 was found to exhibit enhanced proportions of both monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids when grown at a decreased temperature or elevated pressure. Treatment of cells with cerulenin inhibited MUFA but not PUFA synthesis and led to a decreased growth rate and yield at low temperature and high pressure. In addition, oleic acid-auxotrophic mutants were isolated. One of these mutants, strain EA3, was deficient in the production of MUFAs and was both low-temperature sensitive and high-pressure sensitive in the absence of exogenous 18:1 fatty acid. Another mutant, strain EA2, produced little MUFA but elevated levels of the PUFA species eicosapentaenoic acid (EPA; 20:5n-3). This mutant grew slowly but was not low-temperature sensitive or high-pressure sensitive. Finally, reverse genetics was employed to construct a mutant unable to produce EPA. This mutant, strain EA10, was also not low-temperature sensitive or high-pressure sensitive. The significance of these results to the understanding of the role of UFAs in growth under low-temperature or high-pressure conditions is discussed.
PMCID: PMC91242  PMID: 10103272
7.  Photobacterium profundum under Pressure: A MS-Based Label-Free Quantitative Proteomics Study 
PLoS ONE  2013;8(5):e60897.
Photobacterium profundum SS9 is a Gram-negative bacterium, originally collected from the Sulu Sea. Its genome consists of two chromosomes and a 80 kb plasmid. Although it can grow under a wide range of pressures, P. profundum grows optimally at 28 MPa and 15°C. Its ability to grow at atmospheric pressure allows for both easy genetic manipulation and culture, making it a model organism to study piezophily. Here, we report a shotgun proteomic analysis of P. profundum grown at atmospheric compared to high pressure using label-free quantitation and mass spectrometry analysis. We have identified differentially expressed proteins involved in high pressure adaptation, which have been previously reported using other methods. Proteins involved in key metabolic pathways were also identified as being differentially expressed. Proteins involved in the glycolysis/gluconeogenesis pathway were up-regulated at high pressure. Conversely, several proteins involved in the oxidative phosphorylation pathway were up-regulated at atmospheric pressure. Some of the proteins that were differentially identified are regulated directly in response to the physical impact of pressure. The expression of some proteins involved in nutrient transport or assimilation, are likely to be directly regulated by pressure. In a natural environment, different hydrostatic pressures represent distinct ecosystems with their own particular nutrient limitations and abundances. However, the only variable considered in this study was atmospheric pressure.
doi:10.1371/journal.pone.0060897
PMCID: PMC3669370  PMID: 23741291
8.  Large-Scale Transposon Mutagenesis of Photobacterium profundum SS9 Reveals New Genetic Loci Important for Growth at Low Temperature and High Pressure▿  
Journal of Bacteriology  2007;190(5):1699-1709.
Microorganisms adapted to piezopsychrophilic growth dominate the majority of the biosphere that is at relatively constant low temperatures and high pressures, but the genetic bases for the adaptations are largely unknown. Here we report the use of transposon mutagenesis with the deep-sea bacterium Photobacterium profundum strain SS9 to isolate dozens of mutant strains whose growth is impaired at low temperature and/or whose growth is altered as a function of hydrostatic pressure. In many cases the gene mutation-growth phenotype relationship was verified by complementation analysis. The largest fraction of loci associated with temperature sensitivity were involved in the biosynthesis of the cell envelope, in particular the biosynthesis of extracellular polysaccharide. The largest fraction of loci associated with pressure sensitivity were involved in chromosomal structure and function. Genes for ribosome assembly and function were found to be important for both low-temperature and high-pressure growth. Likewise, both adaptation to temperature and adaptation to pressure were affected by mutations in a number of sensory and regulatory loci, suggesting the importance of signal transduction mechanisms in adaptation to either physical parameter. These analyses were the first global analyses of genes conditionally required for low-temperature or high-pressure growth in a deep-sea microorganism.
doi:10.1128/JB.01176-07
PMCID: PMC2258685  PMID: 18156275
9.  Effects of Hydrostatic Pressure on Growth and Luminescence of a Moderately-Piezophilic Luminous Bacteria Photobacterium phosphoreum ANT-2200 
PLoS ONE  2013;8(6):e66580.
Bacterial bioluminescence is commonly found in the deep sea and depends on environmental conditions. Photobacterium phosphoreum ANT-2200 has been isolated from the NW Mediterranean Sea at 2200-m depth (in situ temperature of 13°C) close to the ANTARES neutrino telescope. The effects of hydrostatic pressure on its growth and luminescence have been investigated under controlled laboratory conditions, using a specifically developed high-pressure bioluminescence system. The growth rate and the maximum population density of the strain were determined at different temperatures (from 4 to 37°C) and pressures (from 0.1 to 40 MPa), using the logistic model to define these two growth parameters. Indeed, using the growth rate only, no optimal temperature and pressure could be determined. However, when both growth rate and maximum population density were jointly taken into account, a cross coefficient was calculated. By this way, the optimum growth conditions for P. phosphoreum ANT-2200 were found to be 30°C and, 10 MPa defining this strain as mesophile and moderately piezophile. Moreover, the ratio of unsaturated vs. saturated cellular fatty acids was found higher at 22 MPa, in agreement with previously described piezophile strains. P. phosphoreum ANT-2200 also appeared to respond to high pressure by forming cell aggregates. Its maximum population density was 1.2 times higher, with a similar growth rate, than at 0.1 MPa. Strain ANT-2200 grown at 22 MPa produced 3 times more bioluminescence. The proposed approach, mimicking, as close as possible, the in situ conditions, could help studying deep-sea bacterial bioluminescence and validating hypotheses concerning its role into the carbon cycle in the deep ocean.
doi:10.1371/journal.pone.0066580
PMCID: PMC3688590  PMID: 23818946
10.  The Unique 16S rRNA Genes of Piezophiles Reflect both Phylogeny and Adaptation▿ †  
In the ocean's most extreme depths, pressures of 70 to 110 megapascals prevent the growth of all but the most hyperpiezophilic (pressure-loving) organisms. The physiological adaptations required for growth under these conditions are considered to be substantial. Efforts to determine specific adaptations permitting growth at extreme pressures have thus far focused on relatively few γ-proteobacteria, in part due to the technical difficulties of obtaining piezophilic bacteria in pure culture. Here, we present the molecular phylogenies of several new piezophiles of widely differing geographic origins. Included are results from an analysis of the first deep-trench bacterial isolates recovered from the southern hemisphere (9.9-km depth) and of the first gram-positive piezophilic strains. These new data allowed both phylogenetic and structural 16S rRNA comparisons among deep-ocean trench piezophiles and closely related strains not adapted to high pressure. Our results suggest that (i) the Circumpolar Deep Water acts as repository for hyperpiezophiles and drives their dissemination to deep trenches in the Pacific Ocean and (ii) the occurrence of elongated helices in the 16S rRNA genes increases with the extent of adaptation to growth at elevated pressure. These helix changes are believed to improve ribosome function under deep-sea conditions.
doi:10.1128/AEM.01726-06
PMCID: PMC1800765  PMID: 17158629
11.  The First Genomic and Proteomic Characterization of a Deep-Sea Sulfate Reducer: Insights into the Piezophilic Lifestyle of Desulfovibrio piezophilus 
PLoS ONE  2013;8(1):e55130.
Desulfovibrio piezophilus strain C1TLV30T is a piezophilic anaerobe that was isolated from wood falls in the Mediterranean deep-sea. D. piezophilus represents a unique model for studying the adaptation of sulfate-reducing bacteria to hydrostatic pressure. Here, we report the 3.6 Mbp genome sequence of this piezophilic bacterium. An analysis of the genome revealed the presence of seven genomic islands as well as gene clusters that are most likely linked to life at a high hydrostatic pressure. Comparative genomics and differential proteomics identified the transport of solutes and amino acids as well as amino acid metabolism as major cellular processes for the adaptation of this bacterium to hydrostatic pressure. In addition, the proteome profiles showed that the abundance of key enzymes that are involved in sulfate reduction was dependent on hydrostatic pressure. A comparative analysis of orthologs from the non-piezophilic marine bacterium D. salexigens and D. piezophilus identified aspartic acid, glutamic acid, lysine, asparagine, serine and tyrosine as the amino acids preferentially replaced by arginine, histidine, alanine and threonine in the piezophilic strain. This work reveals the adaptation strategies developed by a sulfate reducer to a deep-sea lifestyle.
doi:10.1371/journal.pone.0055130
PMCID: PMC3559428  PMID: 23383081
12.  Constrained water access to the active site of cytochrome P450 from the piezophilic bacterium Photobacterium profundum 
High pressure research  2010;30(4):466-474.
Living species inhabiting ocean deeps must adapt to high hydrostatic pressure. This adaptation, which must enable functioning under conditions of promoted protein hydration, is especially important for proteins such as cytochromes P450 that exhibit functionally important hydration-dehydration dynamics. Here we study the interactions of substrates with cytochrome P450-SS9, a putative fatty acid hydroxylase from the piezophilic bacterium Photobacterium profundum SS9, and characterize the protein's barotropic properties. Comparison of P450-SS9 with cytochrome P450BM-3, a mesophilic fatty acid hydroxylase, suggests that P450-SS9 is characterized by severely confined accessibility and low water occupancy of the active site. This feature may reveal a mechanism of structural adaptation of the piezophilic enzyme. We also demonstrate that saturated and unsaturated fatty acids exert opposite effects on solvent accessibility and hydration of the active site. Modulation of the protein conformation by fatty acids is hypothesized to have an important physiological function in the piezophile.
doi:10.1080/08957959.2010.535208
PMCID: PMC3070315  PMID: 21475616
pressure-perturbation spectroscopy; cytochrome P450; piezophilic organism; protein hydration; substrate binding
13.  Protein evolution in deep sea bacteria: an analysis of amino acids substitution rates 
Background
Abyssal microorganisms have evolved particular features that enable them to grow in their extreme habitat. Genes belonging to specific functional categories are known to be particularly susceptible to high-pressure; therefore, they should show some evidence of positive selection. To verify this hypothesis we computed the amino acid substitution rates between two deep-sea microorganisms, Photobacterium profundum SS9 and Shewanella benthica KT99, and their respective shallow water relatives.
Results
A statistical analysis of all the orthologs, led to the identification of positive selected (PS) genes, which were then used to evaluate adaptation strategies. We were able to establish "Motility" and "Transport" as two classes significantly enriched with PS genes. The prevalence of transporters led us to analyze variable amino acids (PS sites) by mapping them according to their membrane topology, the results showed a higher frequency of substitutions in the extra-cellular compartment. A similar analysis was performed on soluble proteins, mapping the PS sites on the 3D structure, revealing a prevalence of substitutions on the protein surface. Finally, the presence of some flagellar proteins in the Vibrionaceae PS list confirms the importance of bacterial motility as a SS9 specific adaptation strategy.
Conclusion
The approach presented in this paper is suitable for identifying molecular adaptations to particular environmental conditions. The statistical method takes into account differences in the ratio between non-synonymous to synonymous substitutions, thus allowing the detection of the genes that underwent positive selection. We found that positive selection in deep-sea adapted bacteria targets a wide range of functions, for example solute transport, protein translocation, DNA synthesis and motility. From these data clearly emerges an involvement of the transport and metabolism processes in the deep-sea adaptation strategy of both bathytypes considered, whereas the adaptation of other biological processes seems to be specific to either one or the other. An important role is hypothesized for five PS genes belonging to the transport category that had been previously identified as differentially expressed in microarray experiments. Strikingly, structural mapping of PS sites performed independently on membrane and soluble proteins revealed that residues under positive selection tend to occur in specific protein regions.
doi:10.1186/1471-2148-8-313
PMCID: PMC2600651  PMID: 19014525
14.  Importance of Proteins Controlling Initiation of DNA Replication in the Growth of the High-Pressure-Loving Bacterium Photobacterium profundum SS9▿  
Journal of Bacteriology  2009;191(20):6383-6393.
The molecular mechanism(s) by which deep-sea bacteria grow optimally under high hydrostatic pressure at low temperatures is poorly understood. To gain further insight into the mechanism(s), a previous study screened transposon mutant libraries of the deep-sea bacterium Photobacterium profundum SS9 and identified mutants which exhibited alterations in growth at high pressure relative to that of the parent strain. Two of these mutants, FL23 (PBPRA3229::mini-Tn10) and FL28 (PBPRA1039::mini-Tn10), were found to have high-pressure sensitivity and enhanced-growth phenotypes, respectively. The PBPRA3229 and PBPRA1039 genes encode proteins which are highly similar to Escherichia coli DiaA, a positive regulator, and SeqA, a negative regulator, respectively, of the initiation of DNA replication. In this study, we investigated the hypothesis that PBPRA3229 and PBPRA1039 encode DiaA and SeqA homologs, respectively. Consistent with this, we determined that the plasmid-carried PBPRA3229 and PBPRA1039 genes restored synchrony to the initiation of DNA replication in E. coli mutants lacking DiaA and SeqA, respectively. Additionally, PBPRA3229 restored the cold sensitivity phenotype of an E. coli dnaA(Cs) diaA double mutant whereas PBPRA1039 suppressed the cold sensitivity phenotype of an E. coli dnaA(Cs) single mutant. Taken together, these findings show that the genes disrupted in FL23 and FL28 encode DiaA and SeqA homologs, respectively. Consequently, our findings add support to a model whereby high pressure affects the initiation of DNA replication in P. profundum SS9 and either the presence of a positive regulator (DiaA) or the removal of a negative regulator (SeqA) promotes growth under these conditions.
doi:10.1128/JB.00576-09
PMCID: PMC2753030  PMID: 19700526
15.  Cytochrome P450 from Photobacterium profundum SS9, a piezophilic bacterium, exhibits a tightened control of water access to the active site † 
Biochemistry  2010;49(50):10636-10646.
We report cloning, expression in E. coli, and purification of cytochrome P450 from a deep-sea bacterium Photobacterium profundum strain SS9 (P450-SS9). The enzyme, which is predominately high-spin (86%) in the absence of any added ligand, binds fatty acids and their derivatives and exhibits the highest affinity for myristic acid. Binding of the majority of saturated fatty acids displaces the spin equilibrium further towards the high-spin state, whereas the interactions with unsaturated fatty acids and their derivatives (arachidonoyl glycine) have the opposite effect. Pressure perturbation studies showed that increasing pressure fails to displace the spin equilibrium completely to the low-spin state in the ligand-free P450-SS9 or in the complexes with either myristic acid or arachidonoyl glycine. Stabilization of high-spin P450-SS9 signifies a pressure-induced transition to a state with reduced accessibility of the active site. This transition, which is apparently associated with substantial hydration of the protein, is characterized by the reaction volume change (ΔV) around −100 – −200 mL/mol and P½ of 300-800 bar, which is close to the pressure of habitation of P. profundum. The transition to a state with confined water accessibility is hypothesized to represent a common feature of cytochromes P450 that serves to coordinate heme pocket hydration with ligand binding and the redox state. Displacement of the conformational equilibrium towards the “closed” state in P450-SS9 (even ligand-free) may have evolved to allow the protein to adapt to enhanced protein hydration at high hydrostatic pressures.
doi:10.1021/bi101466y
PMCID: PMC3027307  PMID: 21082780
16.  RNA Arbitrarily Primed PCR Survey of Genes Regulated by ToxR in the Deep-Sea Bacterium Photobacterium profundum Strain SS9 
Journal of Bacteriology  2001;183(5):1688-1693.
We are currently investigating the role of ToxR-mediated gene regulation in Photobacterium profundum strain SS9. SS9 is a moderately piezophilic (“pressure loving”) psychrotolerant marine bacterium belonging to the family Vibrionaceae. In Vibrio cholerae, ToxR is a transmembrane DNA binding protein involved in mediating virulence gene expression in response to various environmental signals. A homolog to V. cholerae ToxR that is necessary for pressure-responsive gene expression of two outer membrane protein-encoding genes was previously found in SS9. To search for additional genes regulated by ToxR in SS9, we have used RNA arbitrarily primed PCR (RAP-PCR) with wild-type and toxR mutant strains of SS9. Seven ToxR-activated transcripts and one ToxR-repressed transcript were identified in this analysis. The cDNAs corresponding to these partial transcripts were cloned and sequenced, and ToxR regulation of their genes was verified. The products of these genes are all predicted to fall into one or both of two functional categories, those whose products alter membrane structure and/or those that are part of a starvation response. The transcript levels of all eight newly identified genes were also characterized as a function of hydrostatic pressure. Various patterns of pressure regulation were observed, indicating that ToxR activation or repression cannot be used to predict the influence of pressure on gene expression in SS9. These results provide further information on the nature of the ToxR regulon in SS9 and indicate that RAP-PCR is a useful approach for the discovery of new genes under the control of global regulatory transcription factors.
doi:10.1128/JB.183.5.1688-1693.2001
PMCID: PMC95054  PMID: 11160100
17.  RecD Function Is Required for High-Pressure Growth of a Deep-Sea Bacterium 
Journal of Bacteriology  1999;181(8):2330-2337.
A genomic library derived from the deep-sea bacterium Photobacterium profundum SS9 was conjugally delivered into a previously isolated pressure-sensitive SS9 mutant, designated EC1002 (E. Chi and D. H. Bartlett, J. Bacteriol. 175:7533–7540, 1993), and exconjugants were screened for the ability to grow at 280-atm hydrostatic pressure. Several clones were identified that had restored high-pressure growth. The complementing DNA was localized and in all cases found to possess strong homology to recD, a DNA recombination and repair gene. EC1002 was found to be deficient in plasmid stability, a phenotype also seen in Escherichia coli recD mutants. The defect in EC1002 was localized to a point mutation that created a stop codon within the recD gene. Two additional recD mutants were constructed by gene disruption and were both found to possess a pressure-sensitive growth phenotype, although the magnitude of the defect depended on the extent of 3′ truncation of the recD coding sequence. Surprisingly, the introduction of the SS9 recD gene into an E. coli recD mutant had two dramatic effects. At high pressure, SS9 recD enabled growth in the E. coli mutant strain under conditions of plasmid antibiotic resistance selection and prevented cell filamentation. Both of these effects were recessive to wild-type E. coli recD. These results suggest that the SS9 recD gene plays an essential role in SS9 growth at high pressure and that it may be possible to identify additional aspects of RecD function through the characterization of this activity.
PMCID: PMC93654  PMID: 10197992
18.  Draft Genome Sequence of the Deep-Sea Bacterium Shewanella benthica Strain KT99 
Genome Announcements  2013;1(3):e00210-13.
We report the draft genome sequence of the obligately piezophilic Shewanella benthica strain KT99 isolated from the abyssal South Pacific Ocean. Strain KT99 is the first piezophilic isolate from the Tonga-Kermadec trench, and its genome provides many clues on high-pressure adaptation and the evolution of deep-sea piezophilic bacteria.
doi:10.1128/genomeA.00210-13
PMCID: PMC3668000  PMID: 23723392
19.  Isolation and Characterization of a Psychropiezophilic Alphaproteobacterium▿† 
Applied and Environmental Microbiology  2011;77(22):8145-8153.
Cultivated psychropiezophilic (low-temperature- and high-pressure-adapted) bacteria are currently restricted to phylogenetically narrow groupings capable of growth under nutrient-replete conditions, limiting current knowledge of the extant functional attributes and evolutionary constraints of diverse microorganisms inhabiting the cold, deep ocean. This study documents the isolation of a deep-sea bacterium following dilution-to-extinction cultivation using a natural seawater medium at high hydrostatic pressure and low temperature. To our knowledge, this isolate, designated PRT1, is the slowest-growing (minimal doubling time, 36 h) and lowest cell density-producing (maximal densities of 5.0 × 106 cells ml−1) piezophile yet obtained. Optimal growth was at 80 MPa, correlating with the depth of capture (8,350 m), and 10°C, with average cell sizes of 1.46 μm in length and 0.59 μm in width. Through detailed growth studies, we provide further evidence for the temperature-pressure dependence of the growth rate for deep-ocean bacteria. PRT1 was phylogenetically placed within the Roseobacter clade, a bacterial lineage known for widespread geographic distribution and assorted lifestyle strategies in the marine environment. Additionally, the gene transfer agent (GTA) g5 capsid protein gene was amplified from PRT1, indicating a potential mechanism for increased genetic diversification through horizontal gene transfer within the hadopelagic environment. This study provides a phylogenetically novel isolate for future investigations of high-pressure adaptation, expands the known physiological traits of cultivated members of the Roseobacter lineage, and demonstrates the feasibility of cultivating novel microbial members from the deep ocean using natural seawater.
doi:10.1128/AEM.05204-11
PMCID: PMC3208983  PMID: 21948832
20.  Novel Psychropiezophilic Oceanospirillales Species Profundimonas piezophila gen. nov., sp. nov., Isolated from the Deep-Sea Environment of the Puerto Rico Trench 
The diversity of deep-sea high-pressure-adapted (piezophilic) microbes in isolated monoculture remains low. In this study, a novel obligately psychropiezophilic bacterium was isolated from seawater collected from the Puerto Rico Trench at a depth of ∼6,000 m. This isolate, designated YC-1, grew best in a nutrient-rich marine medium, with an optimal growth hydrostatic pressure of 50 MPa (range, 20 to 70 MPa) at 8°C. Under these conditions, the maximum growth rate was extremely slow, 0.017 h−1, and the maximum yield was 3.51 × 107 cells ml−1. Cell size and shape changed with pressure, shifting from 4.0 to 5.0 μm in length and 0.5 to 0.8 μm in width at 60 MPa to 0.8- to 1.0-μm diameter coccoid cells under 20 MPa, the minimal pressure required for growth. YC-1 is a Gram-negative, facultatively anaerobic heterotroph. Its predominant cellular fatty acids are the monounsaturated fatty acids (MUFAs) C16:1 and C18:1. Unlike many other psychropiezophiles, YC-1 does not synthesize any polyunsaturated fatty acids (PUFAs). Phylogenetic analysis placed YC-1 within the family of Oceanospirillaceae, closely related to the uncultured symbiont of the deep-sea whale bone-eating worms of the genus Osedax. In common with some other members of the Oceanospirillales, including those enriched during the Deepwater Horizon oil spill, YC-1 is capable of hydrocarbon utilization. On the basis of its characteristics, YC-1 appears to represent both a new genus and a new species, which we name Profundimonas piezophila gen. nov., sp. nov.
doi:10.1128/AEM.02288-13
PMCID: PMC3911031  PMID: 24123740
21.  Pressure Regulation of Soluble Cytochromes c in a Deep-Sea Piezophilic Bacterium, Shewanella violacea 
Journal of Bacteriology  2000;182(10):2945-2952.
Two c-type cytochromes from the soluble fraction of a deep-sea moderately piezophilic bacterium, Shewanella violacea, were purified and characterized, and the genes coding for these cytochromes were cloned and sequenced. One of the cytochromes, designated cytochrome cA, was found to have a molecular mass of approximately 8.3 kDa, and it contained one heme c per molecule. The other, designated cytochrome cB, was found to have a molecular mass of approximately 23 kDa, and it contained two heme c molecules per protein molecule. The amount of cytochrome cB expressed in cells grown at high hydrostatic pressure (50 MPa) was less than that in cells grown at atmospheric pressure, whereas cytochrome cA was constitutively expressed under all pressure conditions examined. The results of Northern blotting analysis were consistent with the above-mentioned observations and suggested that the pressure regulation of cytochrome cB gene expression occurred at the transcriptional level. These results suggest that the components of the respiratory chain of moderately piezophilic S. violacea could be exchanged according to the growth pressure conditions.
PMCID: PMC102006  PMID: 10781566
22.  Genome Sequence of Luminous Piezophile Photobacterium phosphoreum ANT-2200 
Genome Announcements  2014;2(2):e00096-14.
Bacteria of the genus Photobacterium thrive worldwide in oceans and show substantially varied lifestyles, including free-living, commensal, pathogenic, symbiotic, and piezophilic. Here, we present the genome sequence of a luminous, piezophilic Photobacterium phosphoreum strain, ANT-2200, isolated from a water column at 2,200 m depth in the Mediterranean Sea. It is the first genomic sequence of the P. phosphoreum group. An analysis of the sequence provides insight into the adaptation of bacteria to the deep-sea habitat.
doi:10.1128/genomeA.00096-14
PMCID: PMC3990738  PMID: 24744322
23.  Substrate-specific pressure-dependence of microbial sulfate reduction in deep-sea cold seep sediments of the Japan Trench 
The influence of hydrostatic pressure on microbial sulfate reduction (SR) was studied using sediments obtained at cold seep sites from 5500 to 6200 m water depth of the Japan Trench. Sediment samples were stored under anoxic conditions for 17 months in slurries at 4°C and at in situ pressure (50 MPa), at atmospheric pressure (0.1 MPa), or under methanic conditions with a methane partial pressure of 0.2 MPa. Samples without methane amendment stored at in situ pressure retained higher levels of sulfate reducing activity than samples stored at 0.1 MPa. Piezophilic SR showed distinct substrate specificity after hydrogen and acetate addition. SR activity in samples stored under methanic conditions was one order of magnitude higher than in non-amended samples. Methanic samples stored under low hydrostatic pressure exhibited no increased SR activity at high pressure even with the amendment of methane. These new insights into the effects of pressure on substrate specific sulfate reducing activity in anaerobic environmental samples indicate that hydrostatic pressure must be considered to be a relevant parameter in ecological studies of anaerobic deep-sea microbial processes and long-term storage of environmental samples.
doi:10.3389/fmicb.2012.00253
PMCID: PMC3398547  PMID: 22822404
hydrostatic pressure; deep sea; sulfate reduction; piezophile; biogeochemical processes
24.  Use of a reporter gene to follow high-pressure signal transduction in the deep-sea bacterium Photobacterium sp. strain SS9. 
Journal of Bacteriology  1993;175(23):7533-7540.
Photobacterium sp. strain SS9 is a deep-sea bacterium which modulates the abundances of several outer membrane proteins as a function of hydrostatic pressure. These proteins include the product of the previously cloned ompH gene (D. H. Bartlett, M. Wright, A. A. Yayanos, and M. Silverman. Nature (London) 342:572-574, 1989). Subsequent to conjugal plasmid delivery it was possible to cross an ompH::lacZ transcriptional fusion into the genome of SS9, replacing the wild-type ompH gene, generating strain EC10. EC10 is not impaired in growth at high pressure, indicating that under the growth conditions employed, OmpH is not required for baroadaptation. beta-Galactosidase production in EC10 is induced by high pressure to approximately the same extent that OmpH production is in the parental strain, SS9. Therefore, OmpH abundance appears to be primarily regulated at the transcriptional level. EC10 was used for the isolation of ompH regulatory mutants. Derivatives of EC10 which produce reduced levels of beta-galactosidase at both low and high pressure and which appeared to possess mutations outside the ompH::lacZ locus were obtained. All of these regulatory mutants displayed alterations in the high-pressure repression of a second outer membrane protein, designated OmpL, and two of the mutants were also deficient in the high-pressure induction of a third outer membrane protein, designated OmpI. The most dramatic phenotype was present in mutant EC1002, whose growth was extremely barosensitive. EC1002 is the first pressure-sensitive mutant ever isolated. Prolonged incubation of EC1002 at high pressure led to the accumulation of cells with wild-type growth characteristics at high pressure. These cells are suggested to possess suppressor mutations, as they remain deficient in beta-galactosidase production and maintain their high-pressure-adapted phenotype for many generations in the absence of high-pressure selection.
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PMCID: PMC206909  PMID: 8244922
25.  Iron reduction by the deep-sea bacterium Shewanella profunda LT13a under subsurface pressure and temperature conditions 
Microorganisms influence biogeochemical cycles from the surface down to the depths of the continental rocks and oceanic basaltic crust. Due to the poor recovery of microbial isolates from the deep subsurface, the influence of physical environmental parameters, such as pressure and temperature, on the physiology and metabolic potential of subsurface inhabitants is not well constrained. We evaluated Fe(III) reduction rates (FeRRs) and viability, measured as colony-forming ability, of the deep-sea piezophilic bacterium Shewanella profunda LT13a over a range of pressures (0–125 MPa) and temperatures (4–37∘C) that included the in situ habitat of the bacterium isolated from deep-sea sediments at 4500 m depth below sea level. S. profunda LT13a was active at all temperatures investigated and at pressures up to 120 MPa at 30∘C, suggesting that it is well adapted to deep-sea and deep sedimentary environments. Average initial cellular FeRRs only slightly decreased with increasing pressure until activity stopped, suggesting that the respiratory chain was not immediately affected upon the application of pressure. We hypothesize that, as pressure increases, the increased energy demand for cell maintenance is not fulfilled, thus leading to a decrease in viability. This study opens up perspectives about energy requirements of cells in the deep subsurface.
doi:10.3389/fmicb.2014.00796
PMCID: PMC4301008  PMID: 25653646
Fe reduction; pressure; Shewanella profunda; temperature; XANES

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