Project description:Here we report the massively parallel cDNA sequencing (RNA-seq) analysis performed using high throughput sequencing of wild type (DB110) and toxR (TW30) mutant strains of the deep-sea bacterium Photobacterium profundum. ToxR is a transmembrane DNA-binding protein first discovered in Vibrio cholerae and able to regulate numerous genes involved in virulence. In P. profundum the abundance and activity of the same protein is influenced by hydrostatic pressure and is able to regulate genes in a pressure-dependent manner. To better characterize the ToxR regulon, we have compared the genes differentially expressed in response to pressure changes with those whose expression is altered between wild type and toxR mutant strains.
Project description:Here we report the massively parallel cDNA sequencing (RNA-seq) analysis performed using high throughput sequencing of wild type (DB110) and toxR (TW30) mutant strains of the deep-sea bacterium Photobacterium profundum. ToxR is a transmembrane DNA-binding protein first discovered in Vibrio cholerae and able to regulate numerous genes involved in virulence. In P. profundum the abundance and activity of the same protein is influenced by hydrostatic pressure and is able to regulate genes in a pressure-dependent manner. To better characterize the ToxR regulon, we have compared the genes differentially expressed in response to pressure changes with those whose expression is altered between wild type and toxR mutant strains. Four samples were analyzed: DB110 strain grown at 0.1 MPa, DB110 strain grown at 28 MPa, TW30 strain grown at 0.1 MPa, TW30 strain grown at 28 MPa. Two independent coltures (replicates) were grown for each sample, RNA was extracted from each replicate and RNAs from the two replicates were pooled together to reduce biological variability. No replicates were included in experimental design.
Project description: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.
Project description:Genomic DNA extracted from two different Photobacterium profundum strains: SS9 strain (completely sequenced and used to made the microarray) and DSJ4 strain were labeled with Cy3 and Cy5 fluorophores and competitively hybridized on the microarray built on the basis of the SS9 strain genomic sequence. Aim: the identification of the genomic regions absent in the DSJ4 strain with respect to the SS9 strain. The SS9 strain was isolated from the Sulu Trench and display an optimum growth at 28 MPa (2800 metres of depth). The DSJ4 strain was recovered from a sediment sample obtained from the Ryukyu Trench (Japan) at a depth of 5110 m and displays an optimum growth at 10 MPa (but shows no significant change in growth at pressure up to 50 MPa).
Project description:BackgroundOceans 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 degrees C; 16 degrees C) conditions was analyzed taking into consideration the differentially expressed genes belonging to the flexible gene pool.ResultsThese 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.ConclusionThese 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.
Project description:Thioesterase activity is typically required for the release of products from polyketide synthase enzymes, but no such enzyme has been characterized in deep-sea bacteria associated with the production of polyunsaturated fatty acids. In this work, we have expressed and purified the Orf6 thioesterase from Photobacterium profundum. Enzyme assays revealed that Orf6 has a higher specific activity toward long-chain fatty acyl-CoA substrates (palmitoyl-CoA and eicosapentaenoyl-CoA) than toward short-chain or aromatic acyl-CoA substrates. We determined a high resolution (1.05 ?) structure of Orf6 that reveals a hotdog hydrolase fold arranged as a dimer of dimers. The putative active site of this structure is occupied by additional electron density not accounted for by the protein sequence, consistent with the presence of an elongated compound. A second crystal structure (1.40 ?) was obtained from a crystal that was grown in the presence of Mg(2+), which reveals the presence of a binding site for divalent cations at a crystal contact. The Mg(2+)-bound structure shows localized conformational changes (root mean square deviation of 1.63 ?), and its active site is unoccupied, suggesting a mechanism to open the active site for substrate entry or product release. These findings reveal a new thioesterase enzyme with a preference for long-chain CoA substrates in a deep-sea bacterium whose potential range of applications includes bioremediation and the production of biofuels.
Project description: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.