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1.  Single Cells within the Puerto Rico Trench Suggest Hadal Adaptation of Microbial Lineages 
Applied and Environmental Microbiology  2015;81(24):8265-8276.
Hadal ecosystems are found at a depth of 6,000 m below sea level and below, occupying less than 1% of the total area of the ocean. The microbial communities and metabolic potential in these ecosystems are largely uncharacterized. Here, we present four single amplified genomes (SAGs) obtained from 8,219 m below the sea surface within the hadal ecosystem of the Puerto Rico Trench (PRT). These SAGs are derived from members of deep-sea clades, including the Thaumarchaeota and SAR11 clade, and two are related to previously isolated piezophilic (high-pressure-adapted) microorganisms. In order to identify genes that might play a role in adaptation to deep-sea environments, comparative analyses were performed with genomes from closely related shallow-water microbes. The archaeal SAG possesses genes associated with mixotrophy, including lipoylation and the glycine cleavage pathway. The SAR11 SAG encodes glycolytic enzymes previously reported to be missing from this abundant and cosmopolitan group. The other SAGs, which are related to piezophilic isolates, possess genes that may supplement energy demands through the oxidation of hydrogen or the reduction of nitrous oxide. We found evidence for potential trench-specific gene distributions, as several SAG genes were observed only in a PRT metagenome and not in shallower deep-sea metagenomes. These results illustrate new ecotype features that might perform important roles in the adaptation of microorganisms to life in hadal environments.
PMCID: PMC4644660  PMID: 26386059
2.  Identification of Free-Living and Particle-Associated Microbial Communities Present in Hadal Regions of the Mariana Trench 
Relatively few studies have described the microbial populations present in ultra-deep hadal environments, largely as a result of difficulties associated with sampling. Here we report Illumina-tag V6 16S rRNA sequence-based analyses of the free-living and particle-associated microbial communities recovered from locations within two of the deepest hadal sites on Earth, the Challenger Deep (10,918 meters below surface-mbs) and the Sirena Deep (10,667 mbs) within the Mariana Trench, as well as one control site (Ulithi Atoll, 761 mbs). Seawater samples were collected using an autonomous lander positioned ~1 m above the seafloor. The bacterial populations within the Mariana Trench bottom water samples were dissimilar to other deep-sea microbial communities, though with overlap with those of diffuse flow hydrothermal vents and deep-subsurface locations. Distinct particle-associated and free-living bacterial communities were found to exist. The hadal bacterial populations were also markedly different from one another, indicating the likelihood of different chemical conditions at the two sites. In contrast to the bacteria, the hadal archaeal communities were more similar to other less deep datasets and to each other due to an abundance of cosmopolitan deep-sea taxa. The hadal communities were enriched in 34 bacterial and 4 archaeal operational taxonomic units (OTUs) including members of the Gammaproteobacteria, Epsilonproteobacteria, Marinimicrobia, Cyanobacteria, Deltaproteobacteria, Gemmatimonadetes, Atribacteria, Spirochaetes, and Euryarchaeota. Sequences matching cultivated piezophiles were notably enriched in the Challenger Deep, especially within the particle-associated fraction, and were found in higher abundances than in other hadal studies, where they were either far less prevalent or missing. Our results indicate the importance of heterotrophy, sulfur-cycling, and methane and hydrogen utilization within the bottom waters of the deeper regions of the Mariana Trench, and highlight novel community features of these extreme habitats.
PMCID: PMC4860528  PMID: 27242695
dark biosphere; deep-sea microbiology; hadal; marine microbial diversity; piezophile
3.  Effects of High Hydrostatic Pressure on Coastal Bacterial Community Abundance and Diversity 
Applied and Environmental Microbiology  2014;80(19):5992-6003.
Hydrostatic pressure is an important parameter influencing the distribution of microbial life in the ocean. In this study, the response of marine bacterial populations from surface waters to pressures representative of those under deep-sea conditions was examined. Southern California coastal seawater collected 5 m below the sea surface was incubated in microcosms, using a range of temperatures (16 to 3°C) and hydrostatic pressure conditions (0.1 to 80 MPa). Cell abundance decreased in response to pressure, while diversity increased. The morphology of the community also changed with pressurization to a predominant morphotype of small cocci. The pressure-induced community changes included an increase in the relative abundance of Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, and Flavobacteria largely at the expense of Epsilonproteobacteria. Culturable high-pressure-surviving bacteria were obtained and found to be phylogenetically similar to isolates from cold and/or deep-sea environments. These results provide novel insights into the response of surface water bacteria to changes in hydrostatic pressure.
PMCID: PMC4178687  PMID: 25063663
4.  Adaptive laboratory evolution of Escherichia coli K-12 MG1655 for growth at high hydrostatic pressure 
Much of microbial life on Earth grows and reproduces under the elevated hydrostatic pressure conditions that exist in deep-ocean and deep-subsurface environments. In this study adaptive laboratory evolution (ALE) experiments were conducted to investigate the possible modification of the piezosensitive Escherichia coli for improved growth at high pressure. After approximately 500 generations of selection, a strain was isolated that acquired the ability to grow at pressure non-permissive for the parental strain. Remarkably, this strain displayed growth properties and changes in the proportion and regulation of unsaturated fatty acids that indicated the acquisition of multiple piezotolerant properties. These changes developed concomitantly with a change in the gene encoding the acyl carrier protein, which is required for fatty acid synthesis.
PMCID: PMC4285802  PMID: 25610434
adaptive laboratory evolution; Escherichia coli; high pressure; piezophile; acpP
5.  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.
PMCID: PMC3911031  PMID: 24123740
6.  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.
PMCID: PMC4019646  PMID: 24824441
7.  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.
PMCID: PMC3900297  PMID: 23586990
pressure-tolerant enzymes; piezophiles; conformational heterogeneity; cytochrome; P450; monooxygenases; molecular mechanism
8.  Complete Genome Sequence of the Thermophilic, Piezophilic, Heterotrophic Bacterium Marinitoga piezophila KA3 
Journal of Bacteriology  2012;194(21):5974-5975.
Marinitoga piezophila KA3 is a thermophilic, anaerobic, chemoorganotrophic, sulfur-reducing bacterium isolated from the Grandbonum deep-sea hydrothermal vent site at the East Pacific Rise (13°N, 2,630-m depth). The genome of M. piezophila KA3 comprises a 2,231,407-bp circular chromosome and a 13,386-bp circular plasmid. This genome was sequenced within Department of Energy Joint Genome Institute CSP 2010.
PMCID: PMC3486111  PMID: 23045491
9.  The transcriptional landscape of the deep-sea bacterium Photobacterium profundum in both a toxR mutant and its parental strain 
BMC Genomics  2012;13:567.
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.
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.
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.
PMCID: PMC3505737  PMID: 23107454
High-pressure adaptation; Deep sea; Extremophile; Transcription; Operon; RNA-seq; UTR; Vibrionaceae; Photobacterium profundum; ToxR
10.  Complete Genome Sequence of the Hyperthermophilic Archaeon Thermococcus sp. Strain AM4, Capable of Organotrophic Growth and Growth at the Expense of Hydrogenogenic or Sulfidogenic Oxidation of Carbon Monoxide 
Journal of Bacteriology  2011;193(24):7019-7020.
Analysis of the complete genome of Thermococcus sp. strain AM4, which was the first lithotrophic Thermococcales isolate described and the first archaeal isolate to exhibit a capacity for hydrogenogenic carboxydotrophy, reveals a proximity with Thermococcus gammatolerans, corresponding to close but distinct species that differ significantly in their lithotrophic capacities.
PMCID: PMC3232831  PMID: 22123768
11.  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.
PMCID: PMC3208983  PMID: 21948832
12.  Going Deeper: Metagenome of a Hadopelagic Microbial Community 
PLoS ONE  2011;6(5):e20388.
The paucity of sequence data from pelagic deep-ocean microbial assemblages has severely restricted molecular exploration of the largest biome on Earth. In this study, an analysis is presented of a large-scale 454-pyrosequencing metagenomic dataset from a hadopelagic environment from 6,000 m depth within the Puerto Rico Trench (PRT). A total of 145 Mbp of assembled sequence data was generated and compared to two pelagic deep ocean metagenomes and two representative surface seawater datasets from the Sargasso Sea. In a number of instances, all three deep metagenomes displayed similar trends, but were most magnified in the PRT, including enrichment in functions for two-component signal transduction mechanisms and transcriptional regulation. Overrepresented transporters in the PRT metagenome included outer membrane porins, diverse cation transporters, and di- and tri-carboxylate transporters that matched well with the prevailing catabolic processes such as butanoate, glyoxylate and dicarboxylate metabolism. A surprisingly high abundance of sulfatases for the degradation of sulfated polysaccharides were also present in the PRT. The most dramatic adaptational feature of the PRT microbes appears to be heavy metal resistance, as reflected in the large numbers of transporters present for their removal. As a complement to the metagenome approach, single-cell genomic techniques were utilized to generate partial whole-genome sequence data from four uncultivated cells from members of the dominant phyla within the PRT, Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes and Planctomycetes. The single-cell sequence data provided genomic context for many of the highly abundant functional attributes identified from the PRT metagenome, as well as recruiting heavily the PRT metagenomic sequence data compared to 172 available reference marine genomes. Through these multifaceted sequence approaches, new insights have been provided into the unique functional attributes present in microbes residing in a deeper layer of the ocean far removed from the more productive sun-drenched zones above.
PMCID: PMC3101246  PMID: 21629664
13.  Defining the Mode of Action of Tetramic Acid Antibacterials Derived from Pseudomonas aeruginosa Quorum Sensing Signals 
Journal of the American Chemical Society  2009;131(40):14473-14479.
In Nature, bacteria rarely exist as single, isolated entities, but rather as communities comprised of many other species including higher host organisms. To survive in these competitive environments, microorganisms have developed elaborate tactics such as the formation of biofilms and the production of antimicrobial toxins. Recently, it was discovered that the Gram-negative bacterium Pseudomonas aeruginosa, an opportunistic human pathogen, produces an antibiotic, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione (C12-TA), derived from one of its quorum sensing molecules. Here, we present a comprehensive study of the expanded spectrum of C12-TA antibacterial activity against microbial competitors encountered by P. aeruginosa in Nature as well as significant human pathogens. The mechanism of action of C12-TA was also elucidated and C12-TA was found to dissipate both the membrane potential and pH gradient of Gram-positive bacteria, correlating well with cell death. Notably, in stark contrast to its parent molecule 3-oxo-dodecanoyl homoserine lactone (3-oxo-C12-HSL), neither activation of cellular stress pathways nor cytotoxicity was observed in human cells treated with C12-TA. Our results suggest that the QS machinery of P. aeruginosa has evolved for a dual-function, both to signal others of the same species, and also to defend against both host immunity and competing bacteria. Because of the broad-spectrum antibacterial activity, established mode of action, lack of rapid resistance development, and tolerance by human cells, the C12-TA scaffold may also serve as a new lead compound for the development of antimicrobial therapeutics.
PMCID: PMC2760024  PMID: 19807189
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.
PMCID: PMC2753030  PMID: 19700526
15.  Indole Acts as an Extracellular Cue Regulating Gene Expression in Vibrio cholerae▿ †  
Journal of Bacteriology  2009;191(11):3504-3516.
Indole has been proposed to act as an extracellular signal molecule influencing biofilm formation in a range of bacteria. For this study, the role of indole in Vibrio cholerae biofilm formation was examined. It was shown that indole activates genes involved in vibrio polysaccharide (VPS) production, which is essential for V. cholerae biofilm formation. In addition to activating these genes, it was determined using microarrays that indole influences the expression of many other genes, including those involved in motility, protozoan grazing resistance, iron utilization, and ion transport. A transposon mutagenesis screen revealed additional components of the indole-VPS regulatory circuitry. The indole signaling cascade includes the DksA protein along with known regulators of VPS production, VpsR and CdgA. A working model is presented in which global control of gene expression by indole is coordinated through σ54 and associated transcriptional regulators.
PMCID: PMC2681914  PMID: 19329638
16.  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.
PMCID: PMC2570297  PMID: 18723648
17.  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.
PMCID: PMC2258685  PMID: 18156275
18.  Vibrio cholerae Strains Possess Multiple Strategies for Abiotic and Biotic Surface Colonization▿ † 
Journal of Bacteriology  2007;189(14):5348-5360.
Despite its notoriety as a human pathogen, Vibrio cholerae is an aquatic microbe suited to live in freshwater, estuarine, and marine environments where biofilm formation may provide a selective advantage. Here we report characterization of biofilms formed on abiotic and biotic surfaces by two non-O1/O139 V. cholerae strains, TP and SIO, and by the O1 V. cholerae strain N16961 in addition to the isolation of 44 transposon mutants of SIO and TP impaired in biofilm formation. During the course of characterizing the mutants, 30 loci which have not previously been associated with V. cholerae biofilms were identified. These loci code for proteins which perform a wide variety of functions, including amino acid metabolism, ion transport, and gene regulation. Also, when the plankton colonization abilities of strains N16961, SIO, and TP were examined, each strain showed increased colonization of dead plankton compared with colonization of live plankton (the dinoflagellate Lingulodinium polyedrum and the copepod Tigriopus californicus). Surprisingly, most of the biofilm mutants were not impaired in plankton colonization. Only mutants impaired in motility or chemotaxis showed reduced colonization. These results indicate the presence of both conserved and variable genes which influence the surface colonization properties of different V. cholerae subspecies.
PMCID: PMC1951843  PMID: 17496082
19.  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.
PMCID: PMC1800765  PMID: 17158629
20.  Antagonistic Interactions among Marine Bacteria Impede the Proliferation of Vibrio cholerae 
Applied and Environmental Microbiology  2005;71(12):8531-8536.
Changes in global climate have raised concerns about the emergence and resurgence of infectious diseases. Vibrio cholerae is a reemerging pathogen that proliferates and is transported on marine particles. Patterns of cholera outbreaks correlate with sea surface temperature increases, but the underlying mechanisms for rapid proliferation of V. cholerae during ocean warming events have yet to be fully elucidated. In this study, we tested the hypothesis that autochthonous marine bacteria impede the spread of V. cholerae in the marine environment. It was found that some marine bacteria are capable of inhibiting the growth of V. cholerae on surfaces and that bacterial isolates derived from pelagic particles show a greater frequency of V. cholerae inhibition than free-living bacteria. Vibrio cholerae was less susceptible to antagonism at higher temperatures, such as those measured during El Niño-Southern Oscilliation and monsoonal events. Using a model system employing green fluorescent protein-labeled bacteria, we found that marine bacteria can directly inhibit V. cholerae colonization of particles. The mechanism of inhibition in our model system was linked to the biosynthesis of andrimid, an antibacterial agent. Antibiotic production by the model antagonistic strain decreased at higher temperatures, thereby explaining the increased competitiveness of V. cholerae under warmer conditions. These findings suggest that bacterium-bacterium antagonism is a contributing mechanism in regulating the proliferation of V. cholerae on marine particles.
PMCID: PMC1317384  PMID: 16332844
21.  Laterally transferred elements and high pressure adaptation in Photobacterium profundum strains 
BMC Genomics  2005;6:122.
Oceans cover approximately 70% of the Earth's surface with an average depth of 3800 m and a pressure of 38 MPa, thus a large part of the biosphere is occupied by high pressure environments. Piezophilic (pressure-loving) organisms are adapted to deep-sea life and grow optimally at pressures higher than 0.1 MPa. To better understand high pressure adaptation from a genomic point of view three different Photobacterium profundum strains were compared. Using the sequenced piezophile P. profundum strain SS9 as a reference, microarray technology was used to identify the genomic regions missing in two other strains: a pressure adapted strain (named DSJ4) and a pressure-sensitive strain (named 3TCK). Finally, the transcriptome of SS9 grown under different pressure (28 MPa; 45 MPa) and temperature (4°C; 16°C) conditions was analyzed taking into consideration the differentially expressed genes belonging to the flexible gene pool.
These studies indicated the presence of a large flexible gene pool in SS9 characterized by various horizontally acquired elements. This was verified by extensive analysis of GC content, codon usage and genomic signature of the SS9 genome. 171 open reading frames (ORFs) were found to be specifically absent or highly divergent in the piezosensitive strain, but present in the two piezophilic strains. Among these genes, six were found to also be up-regulated by high pressure.
These data provide information on horizontal gene flow in the deep sea, provide additional details of P. profundum genome expression patterns and suggest genes which could perform critical functions for abyssal survival, including perhaps high pressure growth.
PMCID: PMC1239915  PMID: 16162277
22.  A Glimpse into the Expanded Genome Content of Vibrio cholerae through Identification of Genes Present in Environmental Strains†  
Journal of Bacteriology  2005;187(9):2992-3001.
Vibrio cholerae has multiple survival strategies which are reflected both in its broad distribution in many aquatic environments and its high genotypic diversity. To obtain additional information regarding the content of the V. cholerae genome, suppression subtractive hybridization (SSH) was used to prepare libraries of DNA sequences from two southern California coastal isolates which are divergent or absent in the clinical strain V. cholerae O1 El Tor N16961. More than 1,400 subtracted clones were sequenced. This revealed the presence of novel sequences encoding functions related to cell surface structures, transport, metabolism, signal transduction, luminescence, mobile elements, stress resistance, and virulence. Flanking sequence information was determined for loci of interest, and the distribution of these sequences was assessed for a collection of V. cholerae strains obtained from southern California and Mexican environments. This led to the surprising observation that sequences related to the toxin genes toxA, cnf1, and exoY are widespread and more common in these strains than those of the cholera toxin genes which are a hallmark of the pandemic strains of V. cholerae. Gene transfer among these strains could be facilitated by a 4.9-kbp plasmid discovered in one isolate, which possesses similarity to plasmids from other environmental vibrios. By investigating some of the nucleotide sequence basis for V. cholerae genotypic diversity, DNA fragments have been uncovered which could promote survival in coastal environments. Furthermore, a set of genes has been described which could be involved in as yet undiscovered interactions between V. cholerae and eukaryotic organisms.
PMCID: PMC1082809  PMID: 15838025
23.  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.
PMCID: PMC95054  PMID: 11160100
24.  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
25.  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

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