Douglas H. Bartlett

Pressure effects on in vivo microbial processes.

Pressures between 10 and 100 MPa can exert powerful effects on the growth and viability of organisms. Here I describe the effects of elevated pressure in this range on mesophilic (atmospheric pressure adapted) and piezophilic (high-pressure adapted) microorganisms. Examination of pressure effects on mesophiles makes use of this unique physical parameter to aid in the characterization of fundamental cellular processes, while in the case of piezophiles it provides information on the essence of the adaptation of life to high-pressure environments, which comprise the bulk of our biosphere. Research is presented on the isolation of pressure-resistant mutants, high-pressure regulation of gene expression, the role of membrane lipids and proteins in determining growth ability at high pressure, pressure effects on DNA replication and topology as well as on cell division, and the role of extrinsic factors in modulating enzyme activity at high pressure.

Prokaryotic lifestyles in deep sea habitats

Gradients of physicochemical factors influence the growth and survival of life in deep-sea environments. Insights into the characteristics of deep marine prokaryotes has greatly benefited from recent progress in whole genome and metagenome sequence analyses. Here we review the current state-of-the-art of deep-sea microbial genomics. Ongoing and future genome-enabled studies will allow for a better understanding of deep-sea evolution, physiology, biochemistry, community structure and nutrient cycling.

Structure and regulation of the omega-3 polyunsaturated fatty acid synthase genes from the deep-sea bacterium Photobacterium profundum strain SS9

Omega-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) have been shown to be of major importance in the promotion of cardiovascular health, proper human development and the prevention of some cancers. A high proportion of bacterial isolates from low-temperature and high-pressure marine environments produce EPA or DHA. This paper presents the sequence of a 33 kbp locus from the deep-sea bacterium Photobacterium profundum strain SS9 which includes four of the five genes required for EPA biosynthesis. As with other bacterial pfa (polyunsaturated fatty acid) genes, the deduced amino acid sequences encoded by the SS9 genes reveal large multidomain proteins that are likely to catalyse EPA biosynthesis by a novel polyketide synthesis mechanism. RNase protection experiments separated the SS9 pfa genes into two transcriptional units, pfaA-C and pfaD. The pfaA transcriptional start site was identified. Cultivation at elevated hydrostatic pressure or reduced temperature did not increase pfa gene expression despite the resulting increase in percentage composition of EPA under these conditions. However, a regulatory mutant was characterized which showed both increased expression of pfaA-D and elevated EPA percentage composition. This result suggests that a regulatory factor exists which coordinates pfaA-D transcription. Additional consideration regarding the activities required for PUFA synthesis is provided together with comparative analyses of bacterial pfa genes and gene products.

Identification of a regulatory protein required for pressure-responsive gene expression in the deep-sea bacterium Photobacterium species strain SS9

Here, we report the characterization of a gene necessary for hydrostatic pressure regulation of gene expression in the deep‐sea bacterium Photobacterium species strain SS9. The deduced amino acid sequence of the gene product shares extensive similarity to ToxR, a transmembrane DNA‐binding protein first discovered as a virulence determinant in the pathogenic bacterium Vibrio cholerae. Changes in hydrostatic pressure induce changes in both the abundance and the activity of the SS9 ToxR protein (or the activity of a ToxR‐regulated protein). As with other high‐pressure‐inducible phenomena observed in higher organisms, anaesthetics antagonize high‐pressure signalling mediated by ToxR. It is suggested that SS9 ToxR has evolved the ability to respond to pressure‐mediated alterations in membrane structure. V. cholerae and SS9 also share similarity in a ToxR‐regulated protein, indicating that part of the ToxR regulon is conserved in diverse members of the family Vibrionaceae. The SS9 ToxR system represents a useful model for studies of signal transduction and environmental adaptation in the largest portion of the biosphere, the deep sea.

Indole acts as an Extracellular Cue Regulating Gene Expression in Vibrio cholerae

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 sigma(54) and associated transcriptional regulators.

Environmental adaptation: genomic analysis of the piezotolerant and psychrotolerant deep-sea iron reducing bacterium Shewanella piezotolerans WP3.

Shewanella species are widespread in various environments. Here, the genome sequence of Shewanella piezotolerans WP3, a piezotolerant and psychrotolerant iron reducing bacterium from deep-sea sediment was determined with related functional analysis to study its environmental adaptation mechanisms. The genome of WP3 consists of 5,396,476 base pairs (bp) with 4,944 open reading frames (ORFs). It possesses numerous genes or gene clusters which help it to cope with extreme living conditions such as genes for two sets of flagellum systems, structural RNA modification, eicosapentaenoic acid (EPA) biosynthesis and osmolyte transport and synthesis. And WP3 contains 55 open reading frames encoding putative c-type cytochromes which are substantial to its wide environmental adaptation ability. The mtr-omc gene cluster involved in the insoluble metal reduction in the Shewanella genus was identified and compared. The two sets of flagellum systems were found to be differentially regulated under low temperature and high pressure; the lateral flagellum system was found essential for its motility and living at low temperature.

Cholix Toxin, a Novel ADP-ribosylating Factor from Vibrio cholerae

The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC50 = 4.6 ± 0.4 ng/ml) and crustaceans (Artemia nauplii LD50 = 10 ± 2 μg/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity against ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-Å resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment.

High pressure influences on gene and protein expression

Elevated hydrostatic pressure can influence gene and protein expression in both 1 atmosphere-adapted and high pressure-adapted microorganisms. Here we review experiments documenting these effects and describe their significance towards understanding the molecular bases of life in deep-sea high pressure environments.

The Unique 16S rRNA Genes of Piezophiles Reflect both Phylogeny and Adaptation

ABSTRACT In the oceans 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.

Compositional differences in particle-associated and free-living microbial assemblages from an extreme deep-ocean environment

Relatively little information is available for the composition of microbial communities present in hadal environments, the deepest marine locations. Here we present a description of the phylogenetic diversity of particle-associated (> 3 µm) and free-living (3-0.22 µm) microorganisms present in a pelagic trench environment. Small subunit ribosomal RNA gene sequences were recovered from members of the Bacteria, Archaea and Eukarya obtained from a depth of 6000 m in the Puerto Rico Trench (PRT). Species richness estimates for the bacterial particle-associated fraction were greater compared with the free-living fraction and demonstrated statistically significant compositional differences, while the archaeal fractions were not found to be significantly different. The particle-associated fraction contained more Rhodobacterales and unclassified Myxococcales along with Bacteroidetes, Planctomycetes and chloroplast sequences, whereas the free-living fraction contained more Caulobacterales, Xanthomonadales and Burkholderiales, along with Marine Group A and Gemmatimonadetes. The Eukarya contained a high abundance of Basidiomycota Fungi 18S rRNA genes, as well as representatives from the super-groups Rhizaria, Excavata and Chromalveolata. A diverse clade of diplonemid flagellates was also identified from the eukaryotic phylotypes recovered, which was distinct from previously identified deep-sea pelagic diplonemid groups. The significance of these results to considerations of deep-sea microbial life and particle colonization is discussed in comparison to the few other deep-ocean phylogenetic surveys available.