scholarly journals RecD Function Is Required for High-Pressure Growth of a Deep-Sea Bacterium

1999 ◽  
Vol 181 (8) ◽  
pp. 2330-2337 ◽  
Author(s):  
Kelly A. Bidle ◽  
Douglas H. Bartlett
Keyword(s):  
High Pressure ◽  
Deep Sea ◽  
Genomic Library ◽  
Plasmid Stability ◽  
Stop Codon ◽  
Dna Recombination ◽  
Repair Gene ◽  
E Coli ◽  
Pressure Sensitive ◽  
Photobacterium Profundum

ABSTRACT A genomic library derived from the deep-sea bacteriumPhotobacterium profundum SS9 was conjugally delivered into a previously isolated pressure-sensitive SS9 mutant, designated EC1002 (E. Chi and D. H. Bartlett, J. Bacteriol. 175:7533–7540, 1993), and exconjugants were screened for the ability to grow at 280-atm hydrostatic pressure. Several clones were identified that had restored high-pressure growth. The complementing DNA was localized and in all cases found to possess strong homology torecD, a DNA recombination and repair gene. EC1002 was found to be deficient in plasmid stability, a phenotype also seen inEscherichia coli recD mutants. The defect in EC1002 was localized to a point mutation that created a stop codon within the recD gene. Two additional recDmutants were constructed by gene disruption and were both found to possess a pressure-sensitive growth phenotype, although the magnitude of the defect depended on the extent of 3′ truncation of the recD coding sequence. Surprisingly, the introduction of the SS9 recD gene into an E. coli recD mutant had two dramatic effects. At high pressure, SS9recD enabled growth in the E. coli mutant strain under conditions of plasmid antibiotic resistance selection and prevented cell filamentation. Both of these effects were recessive to wild-type E. coli recD. These results suggest that the SS9recD gene plays an essential role in SS9 growth at high pressure and that it may be possible to identify additional aspects of RecD function through the characterization of this activity.

scholarly journals Importance of Proteins Controlling Initiation of DNA Replication in the Growth of the High-Pressure-Loving Bacterium Photobacterium profundum SS9

2009 ◽  
Vol 191 (20) ◽  
pp. 6383-6393 ◽  
Author(s):  
Ziad W. El-Hajj ◽  
Theodora Tryfona ◽  
David J. Allcock ◽  
Fariha Hasan ◽  
Federico M. Lauro ◽  
...  
Keyword(s):  
High Pressure ◽  
Dna Replication ◽  
Deep Sea ◽  
Negative Regulator ◽  
Cold Sensitivity ◽  
E Coli ◽  
Positive Regulator ◽  
Initiation Of Dna Replication ◽  
Photobacterium Profundum ◽  
Insight Into

ABSTRACT 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.

scholarly journals FabF Is Required for Piezoregulation ofcis-Vaccenic Acid Levels and Piezophilic Growth of the Deep-Sea Bacterium Photobacterium profundum Strain SS9

2000 ◽  
Vol 182 (5) ◽  
pp. 1264-1271 ◽  
Author(s):  
Eric E. Allen ◽  
Douglas H. Bartlett
Keyword(s):  
High Pressure ◽  
Hydrostatic Pressure ◽  
Deep Sea ◽  
Unsaturated Fatty Acids ◽  
Acyl Carrier Protein ◽  
Vaccenic Acid ◽  
Bacterial Species ◽  
Elevated Pressure ◽  
Wild Type ◽  
Photobacterium Profundum

ABSTRACT 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 bacteriumPhotobacterium 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 diminishedcis-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.

Monounsaturated but Not Polyunsaturated Fatty Acids Are Required for Growth of the Deep-Sea BacteriumPhotobacterium profundum SS9 at High Pressure and Low Temperature

1999 ◽  
Vol 65 (4) ◽  
pp. 1710-1720 ◽  
Author(s):  
Eric E. Allen ◽  
Daniel Facciotti ◽  
Douglas H. Bartlett
Keyword(s):  
Fatty Acids ◽  
High Pressure ◽  
Fatty Acid ◽  
Low Temperature ◽  
Mutant Strain ◽  
Deep Sea ◽  
High Pressures ◽  
Unsaturated Fatty Acids ◽  
Temperature Sensitive ◽  
Pressure Sensitive

ABSTRACT 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 byP. 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.

scholarly journals The Deep-Sea Bacterium Photobacterium profundum SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions

2008 ◽  
Vol 74 (20) ◽  
pp. 6298-6305 ◽  
Author(s):  
Emiley A. Eloe ◽  
Federico M. Lauro ◽  
Rudi F. Vogel ◽  
Douglas H. Bartlett
Keyword(s):  
High Pressure ◽  
Gene Cluster ◽  
Deep Sea ◽  
Gene Clusters ◽  
Maximum Pressure ◽  
Swimming Velocity ◽  
Flagellar Motor ◽  
Lateral Flagellum ◽  
Photobacterium Profundum ◽  
Flagellum Gene

ABSTRACT 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.

scholarly journals Large-Scale Transposon Mutagenesis of Photobacterium profundum SS9 Reveals New Genetic Loci Important for Growth at Low Temperature and High Pressure

2007 ◽  
Vol 190 (5) ◽  
pp. 1699-1709 ◽  
Author(s):  
Federico M. Lauro ◽  
Khiem Tran ◽  
Alessandro Vezzi ◽  
Nicola Vitulo ◽  
Giorgio Valle ◽  
...  
Keyword(s):  
High Pressure ◽  
Low Temperature ◽  
Deep Sea ◽  
Large Scale ◽  
High Pressures ◽  
Cell Envelope ◽  
Transposon Mutagenesis ◽  
Chromosomal Structure ◽  
And Function ◽  
Photobacterium Profundum

ABSTRACT 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.

scholarly journals Release factor-dependent ribosome rescue by BrfA in the Gram-positive bacterium Bacillus subtilis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Naomi Shimokawa-Chiba ◽  
Claudia Müller ◽  
Keigo Fujiwara ◽  
Bertrand Beckert ◽  
Koreaki Ito ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stop Codon ◽  
Release Factor ◽  
Positive Bacterium ◽  
Gram Positive ◽  
E Coli ◽  
Cryo Electron Microscopy ◽  
Dead End ◽  
Bacterium Bacillus Subtilis ◽  
Bacterium Bacillus

AbstractRescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop-codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named BrfA. Genetic analysis shows that B. subtilis requires the function of either trans-translation or BrfA for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-electron microscopy (cryo-EM) characterization demonstrates that BrfA binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although BrfA is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.

A Study of the Sealing Performance of a New High-Pressure Cone Valve for Deep-Sea Gas-Tight Water Samplers

2010 ◽  
Vol 132 (4) ◽  
Author(s):  
Shi-Jun Wu ◽  
Can-Jun Yang ◽  
Ying Chen ◽  
Yan-Qing Xie
Keyword(s):  
Finite Element Analysis ◽  
High Pressure ◽  
Deep Sea ◽  
Hydrothermal Fluid ◽  
Contact Model ◽  
Element Analysis ◽  
Sea Trial ◽  
Sealing Performance ◽  
Pressure Cone ◽  
Gas Tight

The cone valve plays an important role in high-pressure sealing applications. In this paper, a new high-pressure cone valve, based on the titanium alloy poppet-to-polyetheretherketone seat sealing structure, is proposed for deep-sea gas-tight water samplers. In order to study the sealing performance of the new valve, both the conforming poppet-seat contact model and the nonconforming poppet-seat contact model were evaluated. Finite element analysis based on the two models was performed and validated by experiments. The results indicate that the nonconforming poppet-seat contact model has a better sealing performance than the conforming poppet-seat contact model. The new cone valve also was applied in a gas-tight hydrothermal fluid sampler and successfully tested in a sea trial during the KNOX18RR cruise from 9 July to 12 August 2008.

scholarly journals A Recombination Repair Gene of Schizosaccharomyces pombe, rhp57, Is a Functional Homolog of the Saccharomyces cerevisiae RAD57 Gene and Is Phylogenetically Related to the Human XRCC3 Gene

Genetics ◽  
2000 ◽  
Vol 154 (4) ◽  
pp. 1451-1461 ◽  
Author(s):  
Yasuhiro Tsutsui ◽  
Takashi Morishita ◽  
Hiroshi Iwasaki ◽  
Hiroyuki Toh ◽  
Hideo Shinagawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Schizosaccharomyces Pombe ◽  
Genomic Library ◽  
Synthetic Lethality ◽  
Multicopy Plasmid ◽  
Repair Gene ◽  
Recombination Repair ◽  
Xrcc3 Gene ◽  
Γ Rays ◽  
Lower Temperature

Abstract To identify Schizosaccharomyces pombe genes involved in recombination repair, we identified seven mutants that were hypersensitive to both methyl methanesulfonate (MMS) and γ-rays and that contained mutations that caused synthetic lethality when combined with a rad2 mutation. One of the mutants was used to clone the corresponding gene from a genomic library by complementation of the MMS-sensitive phenotype. The gene obtained encodes a protein of 354 amino acids whose sequence is 32% identical to that of the Rad57 protein of Saccharomyces cerevisiae. An rhp57 (RAD57 homolog of S. pombe) deletion strain was more sensitive to MMS, UV, and γ-rays than the wild-type strain and showed a reduction in the frequency of mitotic homologous recombination. The MMS sensitivity was more severe at lower temperature and was suppressed by the presence of a multicopy plasmid bearing the rhp51 gene. An rhp51 rhp57 double mutant was as sensitive to UV and γ-rays as an rhp51 single mutant, indicating that rhp51 function is epistatic to that of rhp57. These characteristics of the rhp57 mutants are very similar to those of S. cerevisiae rad57 mutants. Phylogenetic analysis suggests that Rhp57 and Rad57 are evolutionarily closest to human Xrcc3 of the RecA/Rad51 family of proteins.

High Pressure Inactivation of Escherichia coli, Campylobacter jejuni, and Spoilage Microbiota on Poultry Meat

2012 ◽  
Vol 75 (3) ◽  
pp. 497-503 ◽  
Author(s):  
YANG LIU ◽  
MIRKO BETTI ◽  
MICHAEL G. GÄNZLE
Keyword(s):  
Escherichia Coli ◽  
High Pressure ◽  
Campylobacter Jejuni ◽  
Resistant Mutant ◽  
Poultry Meat ◽  
E Coli ◽  
Cell Counts ◽  
Meat Spoilage ◽  
Brochothrix Thermosphacta ◽  
Pressure Resistance

This study evaluated the high pressure inactivation of Campylobacter jejuni, Escherichia coli, and poultry meat spoilage organisms. All treatments were performed in aseptically prepared minced poultry meat. Treatment of 19 strains of C. jejuni at 300 MPa and 30°C revealed a large variation of pressure resistance. The recovery of pressure-induced sublethally injured C. jejuni depended on the availability of iron. The addition of iron content to enumeration media was required for resuscitation of sublethally injured cells. Survival of C. jejuni during storage of refrigerated poultry meat was analyzed in fresh and pressure-treated poultry meat, and in the presence or absence of spoilage microbiota. The presence of spoilage microbiota did not significantly influence the survival of C. jejuni. Pressure treatment at 400 MPa and 40°C reduced cell counts of Brochothrix thermosphacta, Carnobacterium divergens, C. jejuni, and Pseudomonas fluorescens to levels below the detection limit. Cell counts of E. coli AW1.7, however, were reduced by only 3.5 log (CFU/g) and remained stable during subsequent refrigerated storage. The resistance to treatment at 600 MPa and 40°Cof E. coli AW1.7 was compared with Salmonella enterica, Shiga toxin–producing E. coli and nonpathogenic E. coli strains, and Staphylococcus spp. Cell counts of all organisms except E. coli AW 1.7 were reduced by more than 6 log CFU/g. Cell counts of E. coli AW1.7 were reduced by 4.5 log CFU/g only. Moreover, the ability of E. coli AW1.7 to resist pressure was comparable to the pressure-resistant mutant E. coli LMM1030. Our results indicate that preservation of fresh meat requires a combination of high pressure with high temperature (40 to 60°C) or other antimicrobial hurdles.

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