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Douglas H Bartlett - One of the best experts on this subject based on the ideXlab platform.
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Ecotype Diversity and Conversion in Photobacterium profundum Strains
PloS one, 2014Co-Authors: Federico M. Lauro, Emiley A. Eloe-fadrosh, Taylor K. S. Richter, Nicola Vitulo, Steven Ferriera, Justin Johnson, Douglas H BartlettAbstract: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.
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Adaptations of the Psychrotolerant Piezophile Photobacterium profundum Strain SS9
High-Pressure Microbiology, 2014Co-Authors: Douglas H Bartlett, Gail P. Ferguson, Giorgio ValleAbstract:Strains of Photobacterium profundum and Shewanella violacea have become the work-horses for examining the adaptations of cold-adapted deep-sea microbes to high pressure. The chapter focuses on P. profundum, with an emphasis on strain SS9. Transposon mutagenesis, insertional mutagenesis, lacZ-based transcriptional reporter studies, allelic exchange, and complementation are all possible. SS9 is both the most psychrotolerant and the most piezophilic microorganism for which such studies have been reported. More recently its entire genome sequence has been determined, and transcriptome and comparative genomic analyses have been undertaken. The sections describe the roles of the membrane, membrane-associated signaling systems, and DNA recombination in SS9 piezoadaptation, and provide recent insights obtained from the SS9 genome sequence and functional and comparative genomics. Advances in genomic approaches have dramatically altered the understanding of the evolution, diversity, biochemistry, and physiology of life. These technologies have also been applied to two piezophiles: P. profundum strain SS9 and Shewanella violaceae strain DSS12, and additional sequencing projects are in the works. Genetic manipulations are needed to establish which genes actually influence growth ability at high pressure. Additional important issues are which piezophile-specific genes were acquired by horizontal gene transfer (HGT) and which were lost by the mesophilic strain 3TCK, which genes 3TCK might have needed to acquire for adaptation to shallow-water and atmospheric pressure, and whether the last common ancestor to the P. profundum strains was piezophilic or mesophilic.
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Microbial Adaptations to the Psychrosphere/Piezosphere 93 Microbial Adaptations to the Psychrosphere/Piezosphere
2013Co-Authors: Douglas H BartlettAbstract:Low temperature and high pressure deep-sea environments occupy the largest fraction of the biosphere. Nevertheless, the molecular adaptations that enable life to exist under these conditions remain poorly understood. This article will provide an overview of the current picture on high pressure adaptation in cold oceanic environments, with an emphasis on genetic experiments performed on Photobacterium profundum. Thus far genes which have been found or implicated as important for pressure-sensing or pressure-adaptation include genes required for fatty acid unsaturation, the membrane protein genes toxR and rseC and the DNA recombination gene recD. Many deep-sea bacteria possess genes for the production of omega-3 polyunsaturated fatty acids. These could be of biotechnological significance since these fatty acids reduce the risk of cardiovascular disease and certain cancers and are useful as dietary supplements
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CYP261 enzymes from deep sea bacteria: a clue to conformational heterogeneity in cytochromes P450.
Biotechnology and applied biochemistry, 2013Co-Authors: Dmitri R. Davydov, Douglas H Bartlett, Elena V. Sineva, Nadezhda Y. Davydova, James R. HalpertAbstract: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-sea 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 nonpiezophilic enzymes. The studies of enzymes of piezophiles should help unravel the mechanisms that control water access during the catalytic cycle.
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The transcriptional landscape of the deep-sea bacterium Photobacterium profundum in both a toxR mutant and its parental strain
BMC genomics, 2012Co-Authors: Stefano Campanaro, Douglas H Bartlett, Fabio De Pascale, Andrea Telatin, Riccardo Schiavon, Giorgio ValleAbstract: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.
Giorgio Valle - One of the best experts on this subject based on the ideXlab platform.
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Adaptations of the Psychrotolerant Piezophile Photobacterium profundum Strain SS9
High-Pressure Microbiology, 2014Co-Authors: Douglas H Bartlett, Gail P. Ferguson, Giorgio ValleAbstract:Strains of Photobacterium profundum and Shewanella violacea have become the work-horses for examining the adaptations of cold-adapted deep-sea microbes to high pressure. The chapter focuses on P. profundum, with an emphasis on strain SS9. Transposon mutagenesis, insertional mutagenesis, lacZ-based transcriptional reporter studies, allelic exchange, and complementation are all possible. SS9 is both the most psychrotolerant and the most piezophilic microorganism for which such studies have been reported. More recently its entire genome sequence has been determined, and transcriptome and comparative genomic analyses have been undertaken. The sections describe the roles of the membrane, membrane-associated signaling systems, and DNA recombination in SS9 piezoadaptation, and provide recent insights obtained from the SS9 genome sequence and functional and comparative genomics. Advances in genomic approaches have dramatically altered the understanding of the evolution, diversity, biochemistry, and physiology of life. These technologies have also been applied to two piezophiles: P. profundum strain SS9 and Shewanella violaceae strain DSS12, and additional sequencing projects are in the works. Genetic manipulations are needed to establish which genes actually influence growth ability at high pressure. Additional important issues are which piezophile-specific genes were acquired by horizontal gene transfer (HGT) and which were lost by the mesophilic strain 3TCK, which genes 3TCK might have needed to acquire for adaptation to shallow-water and atmospheric pressure, and whether the last common ancestor to the P. profundum strains was piezophilic or mesophilic.
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The transcriptional landscape of the deep-sea bacterium Photobacterium profundum in both a toxR mutant and its parental strain
BMC genomics, 2012Co-Authors: Stefano Campanaro, Douglas H Bartlett, Fabio De Pascale, Andrea Telatin, Riccardo Schiavon, Giorgio ValleAbstract: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.
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Protein evolution in deep sea bacteria: an analysis of amino acids substitution rates
BMC evolutionary biology, 2008Co-Authors: Stefano Campanaro, Laura Treu, Giorgio ValleAbstract: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.
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Large-Scale Transposon Mutagenesis of Photobacterium profundum SS9 Reveals New Genetic Loci Important for Growth at Low Temperature and High Pressure
Journal of bacteriology, 2007Co-Authors: Federico M. Lauro, Nicola Vitulo, Alessandro Vezzi, Giorgio Valle, Khiem Tran, Douglas H BartlettAbstract: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.
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Large-Scale Transposon Mutagenesis of Photobacterium profundum SS9 Reveals New Genetic Loci Important for Growth at Low
2007Co-Authors: High Pressure, Giorgio Valle, Douglas H BartlettAbstract: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-temper-ature or high-pressure growth in a deep-sea microorganism. The bulk of deep-sea environments select for microorgan-isms which grow preferentially at low temperatures (psychro-philes) and high hydrostatic pressures (piezophiles) and which are capable of tolerating sporadic inputs of organic nutrient
Abel Baerga-ortiz - One of the best experts on this subject based on the ideXlab platform.
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Artificial covalent linkage of bacterial acyl carrier proteins for fatty acid production.
Scientific reports, 2019Co-Authors: Carlos Rullán-lind, Melissa Ortiz-rosario, Andrea García-gonzález, Vivian Stojanoff, Nataliya E. Chorna, Ruth Pietri, Abel Baerga-ortizAbstract:Acyl carrier proteins (ACPs) are essential to the production of fatty acids. In some species of marine bacteria, ACPs are arranged into tandem repeats joined by peptide linkers, an arrangement that results in high fatty acid yields. By contrast, Escherichia coli, a relatively low producer of fatty acids, uses a single-domain ACP. In this work, we have engineered the native E. coli ACP into tandem di- and tri-domain constructs joined by a naturally occurring peptide linker from the PUFA synthase of Photobacterium profundum. The size of these tandem fused ACPs was determined by size exclusion chromatography to be higher (21 kDa, 36 kDa and 141 kDa) than expected based on the amino acid sequence (12 kDa, 24 kDa and 37 kDa, respectively) suggesting the formation of a flexible extended conformation. Structural studies using small-angle X-ray scattering (SAXS), confirmed this conformational flexibility. The thermal stability for the di- and tri-domain constructs was similar to that of the unfused ACP, indicating a lack of interaction between domains. Lastly, E. coli cultures harboring tandem ACPs produced up to 1.6 times more fatty acids than wild-type ACP, demonstrating the viability of ACP fusion as a method to enhance fatty acid yield in bacteria.
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Solution Structure of the Tandem Acyl Carrier Protein Domains from a Polyunsaturated Fatty Acid Synthase Reveals Beads-on-a-String Configuration
2016Co-Authors: Uldaeliz Trujillo, Delise Oyola-robles, Loren J. Stagg, David A. Vassallo, Irving E. Vega, Stefan T. Arold, Abel Baerga-ortizAbstract:The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed tha
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Expression of dehydratase domains from a polyunsaturated fatty acid synthase increases the production of fatty acids in Escherichia coli
Enzyme and microbial technology, 2013Co-Authors: Delise Oyola-robles, Carlos Rullán-lind, Néstor M. Carballeira, Abel Baerga-ortizAbstract:Increasing the production of fatty acids by microbial fermentation remains an important step toward the generation of biodiesel and other portable liquid fuels. In this work, we report an Escherichia coli strain engineered to overexpress a fragment consisting of four dehydratase domains from the polyunsaturated fatty acid (PUFA) synthase enzyme complex from the deep-sea bacterium, Photobacterium profundum. The DH1-DH2-UMA enzyme fragment was excised from its natural context within a multi-enzyme PKS and expressed as a stand-alone protein. Fatty acids were extracted from the cell pellet, esterified with methanol and quantified by GC-MS analysis. Results show that the E. coli strain expressing the DH tetradomain fragment was capable of producing up to a 5-fold increase (80.31 mg total FA/L culture) in total fatty acids over the negative control strain lacking the recombinant enzyme. The enhancement in production was observed across the board for all the fatty acids that are typically made by E. coli. The overexpression of the DH tetradomain did not affect E. coli cell growth, thus showing that the observed enhancement in fatty acid production was not a result of effects associated with cell density. The observed enhancement was more pronounced at lower temperatures (3.8-fold at 16 °C, 3.5-fold at 22 °C and 1.5-fold at 30 °C) and supplementation of the media with 0.4% glycerol did not result in an increase in fatty acid production. All these results taken together suggest that either the dehydration of fatty acid intermediates are a limiting step in the E. coli fatty acid biosynthesis machinery, or that the recombinant dehydratase domains used in this study are also capable of catalyzing thioester hydrolysis of the final products. The enzyme in this report is a new tool which could be incorporated into other existing strategies aimed at improving fatty acid production in bacterial fermentations toward accessible biodiesel precursors.
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Solution Structure of the Tandem Acyl Carrier Protein Domains from a Polyunsaturated Fatty Acid Synthase Reveals Beads-on-a-String Configuration
PloS one, 2013Co-Authors: Uldaeliz Trujillo, Delise Oyola-robles, Edwin Vázquez-rosa, Loren J. Stagg, David A. Vassallo, Irving E. Vega, Stefan T. Arold, Abel Baerga-ortizAbstract:The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP domains for increasing the yield of fatty acids in bacterial cultures.
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Structure, Activity, and Substrate Selectivity of the Orf6 Thioesterase from Photobacterium profundum
The Journal of biological chemistry, 2013Co-Authors: Maria M Rodriguez-guilbe, Delise Oyola-robles, Eric R. Schreiter, Abel Baerga-ortizAbstract: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 Å) 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 Å) was obtained from a crystal that was grown in the presence of Mg2+, which reveals the presence of a binding site for divalent cations at a crystal contact. The Mg2+-bound structure shows localized conformational changes (root mean square deviation of 1.63 Å), 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.
Federico M. Lauro - One of the best experts on this subject based on the ideXlab platform.
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Ecotype Diversity and Conversion in Photobacterium profundum Strains
PloS one, 2014Co-Authors: Federico M. Lauro, Emiley A. Eloe-fadrosh, Taylor K. S. Richter, Nicola Vitulo, Steven Ferriera, Justin Johnson, Douglas H BartlettAbstract: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.
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Insights into piezophily from genetic studies on the deep‐sea bacterium, Photobacterium profundum SS9
Annals of the New York Academy of Sciences, 2010Co-Authors: Ziad W. El-hajj, Theodora Tryfona, David J. Allcock, Federico M. Lauro, Lindsay Sawyer, Douglas H Bartlett, Gail P. FergusonAbstract:The deep-sea bacterium, Photobacterium profundum SS9, has been adopted as a model organism to understand the molecular basis of cold-adapted high-pressure-loving (piezophilic) growth. Despite growing optimally at 28 MPa (15° C), P. profundum SS9 can grow over a wide range of pressures and temperatures. The ability to grow at atmospheric pressure has enabled a limited set of genetic tools to be developed, which has provided genetic insights into the mechanism of piezophilic growth in P. profundum SS9. This review focuses on how genetic studies have uncovered the importance of processes affecting the DNA and the bacterial cell envelope in the piezophilic growth of P. profundum SS9. In addition, a method was developed to assess quantitative piezophilic colony growth of P. profundum SS9 on solid agar. Future studies, using this methodology, could provide novel insights into the molecular basis of piezophilic, surface-attached growth.
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insights into piezophily from genetic studies on the deep sea bacterium Photobacterium profundum ss9
Annals of the New York Academy of Sciences, 2010Co-Authors: Ziad W Elhajj, Theodora Tryfona, David J. Allcock, Federico M. Lauro, Lindsay Sawyer, Douglas H Bartlett, Gail P. FergusonAbstract:The deep-sea bacterium, Photobacterium profundum SS9, has been adopted as a model organism to understand the molecular basis of cold-adapted high-pressure-loving (piezophilic) growth. Despite growing optimally at 28 MPa (15° C), P. profundum SS9 can grow over a wide range of pressures and temperatures. The ability to grow at atmospheric pressure has enabled a limited set of genetic tools to be developed, which has provided genetic insights into the mechanism of piezophilic growth in P. profundum SS9. This review focuses on how genetic studies have uncovered the importance of processes affecting the DNA and the bacterial cell envelope in the piezophilic growth of P. profundum SS9. In addition, a method was developed to assess quantitative piezophilic colony growth of P. profundum SS9 on solid agar. Future studies, using this methodology, could provide novel insights into the molecular basis of piezophilic, surface-attached growth.
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Importance of Proteins Controlling Initiation of DNA Replication in the Growth of the High-Pressure-Loving Bacterium Photobacterium profundum SS9
Journal of bacteriology, 2009Co-Authors: Ziad W. El-hajj, Theodora Tryfona, David J. Allcock, Fariha Hasan, Federico M. Lauro, Lindsay Sawyer, Douglas H. Bartlett, Douglas H Bartlett, Gail P. FergusonAbstract: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 highpressure 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. Despite the fact that more than 70% of the earth’s surface is covered by oceans, which have an average temperature of 3°C and exert an average hydrostatic pressure of 38 MPa (atmospheric pressure is 0.1 MPa), little is understood about the molecular basis of cold- and high-pressure-adapted deepocean life. The discovery and isolation of the pyschrotolerant
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The Deep-Sea Bacterium Photobacterium profundum SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions
Applied and environmental microbiology, 2008Co-Authors: Emiley A. Eloe, Federico M. Lauro, Rudi F. Vogel, Douglas H BartlettAbstract: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.
Eric E Allen - One of the best experts on this subject based on the ideXlab platform.
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genetic regulation of the bacterial omega 3 polyunsaturated fatty acid biosynthesis pathway
Journal of Bacteriology, 2020Co-Authors: Marco N Allemann, Eric E AllenAbstract:ABSTRACT A characteristic among many marine Gammaproteobacteria is the biosynthesis and incorporation of omega-3 polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) is mediated by a polyketide/fatty acid synthase mechanism encoded by a set of five genes, pfaABCDE. This unique fatty acid synthesis pathway coexists with the principal type II dissociated fatty acid synthesis pathway, which is responsible for the biosynthesis of core saturated, monounsaturated, and hydroxylated fatty acids used in phospholipid and lipid A biosynthesis. In this work, a genetic approach was undertaken to elucidate genetic regulation of the pfa genes in the model marine bacterium Photobacterium profundum SS9. Using a reporter gene fusion, we showed that expression of the pfa operon is downregulated in response to exogenous fatty acids, particularly long-chain monounsaturated fatty acids. This regulation occurs independently of the canonical fatty acid regulators, FabR and FadR, present in P. profundum SS9. Transposon mutagenesis and screening of a library of mutants identified a novel transcriptional regulator, which we have designated pfaF, to be responsible for the observed regulation of the pfa operon in P. profundum SS9. Gel mobility shift and DNase I footprinting assays confirmed that PfaF binds the pfaA promoter and identified the PfaF binding site. IMPORTANCE The production of long-chain omega-3 polyunsaturated fatty acids (PUFA) by marine Gammaproteobacteria, particularly those from deep-sea environments, has been known for decades. These unique fatty acids are produced by a polyketide-type mechanism and subsequently incorporated into the phospholipid membrane. While much research has focused on the biosynthesis genes, their products, and the phylogenetic distribution of these gene clusters, no prior studies have detailed the genetic regulation of this pathway. This study describes how this pathway is regulated under various culture conditions and has identified and characterized a fatty acid-responsive transcriptional regulator specific to PUFA biosynthesis.
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genetic regulation of the bacterial omega 3 polyunsaturated fatty acid biosynthesis pathway
bioRxiv, 2020Co-Authors: Marco N Allemann, Eric E AllenAbstract:Abstract A characteristic among many marine Gammaproteobacteria is the biosynthesis and incorporation of omega-3 polyunsaturated fatty acids into membrane phospholipids. Biosynthesis of eicosapentaenoic (EPA) and/or docosahexaenoic (DHA) acids is accomplished using a polyketide/fatty acid synthase mechanism encoded by a set of five genes pfaABCDE. This unique fatty acid synthesis (FAS) pathway co-exists with the canonical Type II dissociated fatty acid synthesis pathway, which is responsible for the biosynthesis of saturated, monounsaturated, and hydroxylated fatty acids used in phospholipid and lipid A biosynthesis. In this work, a genetic approach was undertaken to elucidate genetic regulation of the pfa genes in the model marine bacterium Photobacterium profundum SS9. Using a reporter gene fusion, we showed that expression of the pfa operon is down regulated in response to exogenous fatty acids, particularly long chain monounsaturated fatty acids. This regulation occurs independently of the canonical fatty acid regulators, FabR and FadR, present in P. profundum SS9. Transposon mutagenesis and screening of a library of mutants identified a novel transcriptional regulator, which we have designated pfaF, to be responsible for the observed regulation of the pfa operon in P. profundum SS9. Gel mobility shift and DNase I footprinting assays confirmed that PfaF binds the pfaA promoter and identified the PfaF binding site. Importance The production of polyunsaturated fatty acids (PUFA) by marine Gammaproteobacteria, particularly those from deep-sea environments, has been known for decades. These unique fatty acids are produced by a polyketide-type mechanism and subsequently incorporated into the phospholipid membrane. While much research has focused on the biosynthesis genes, their products and the phylogenetic distribution of these gene clusters, no prior studies have detailed the genetic regulation of this pathway. This study describes how this pathway is regulated under various culture conditions and has identified and characterized a fatty acid responsive transcriptional regulator specific to the PUFA biosynthesis pathway.
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structure and regulation of the omega 3 polyunsaturated fatty acid synthase genes from the deep sea bacterium Photobacterium profundum strain ss9
Microbiology, 2002Co-Authors: Eric E Allen, Douglas H BartlettAbstract: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.
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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, 2000Co-Authors: Eric E. Allen, Douglas H. Bartlett, Eric E Allen, Douglas H BartlettAbstract: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, b-ketoacyl‐acyl carrier protein synthase II has evolved novel pressure-responsive characteristics which facilitate SS9 growth at high pressure.
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Monounsaturated but not polyunsaturated fatty acids are required for growth of the deep-sea bacterium Photobacterium profundum SS9 at high pressure and low temperature.
Applied and environmental microbiology, 1999Co-Authors: Eric E Allen, Daniel Facciotti, Douglas H BartlettAbstract: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.