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Applied and Environmental Microbiology Dec 2012Pseudomonas fluorescens, a widespread Gram-negative bacterium, is an ideal protein manufacturing factory (PMF) because of its safety, robust growth, and high protein...
Pseudomonas fluorescens, a widespread Gram-negative bacterium, is an ideal protein manufacturing factory (PMF) because of its safety, robust growth, and high protein production. P. fluorescens possesses a type I secretion system (T1SS), which mediates secretion of a thermostable lipase (TliA) and a protease (PrtA) through its ATP-binding cassette (ABC) transporter. Recombinant proteins in P. fluorescens are attached to the C-terminal signal region of TliA for transport as fusion proteins to the extracellular medium. However, intrinsic TliA from the P. fluorescens genome interferes with detection of the recombinant protein and the secreted recombinant protein is hydrolyzed, due to intrinsic PrtA, resulting in decreased efficiency of the PMF. In this research, the lipase and protease genes of P. fluorescens SIK W1 were deleted using the targeted gene knockout method. Deletion mutant P. fluorescens ΔtliA ΔprtA secreted fusion proteins without TliA or protein degradation. Using wild-type P. fluorescens as an expression host, degradation of the recombinant protein varied depending on the type of culture media and aeration; however, degradation did not occur with the P. fluorescens ΔtliA ΔprtA double mutant irrespective of growth conditions. By homologous expression of tliA and the ABC transporter in a plasmid, TliA secreted from P. fluorescens ΔprtA and P. fluorescens ΔtliA ΔprtA cells was found to be intact, whereas that secreted from the wild-type P. fluorescens and P. fluorescens ΔtliA cells was found to be hydrolyzed. Our results demonstrate that the P. fluorescens ΔtliA ΔprtA deletion mutant is a promising T1SS-mediated PMF that enhances production and detection of recombinant proteins in extracellular media.
Topics: Culture Media; Gene Knockout Techniques; Lipase; Metabolic Engineering; Peptide Hydrolases; Protein Transport; Pseudomonas fluorescens; Recombinant Proteins; Sequence Deletion
PubMed: 23042178
DOI: 10.1128/AEM.02476-12 -
Letters in Applied Microbiology Nov 1999The effect of a commonly used biocide, 1,2-benzisothiazolin-3-one (BIT) on ppGpp accumulation in the pathogen, Pseudomonas aeruginosa PAO1, and an environmental isolate,...
The effect of a commonly used biocide, 1,2-benzisothiazolin-3-one (BIT) on ppGpp accumulation in the pathogen, Pseudomonas aeruginosa PAO1, and an environmental isolate, Ps. fluorescens, was examined. It is concluded that BIT is able to induce a stringent response in Ps. aeruginosa and Ps. fluorescens, determined by the rapid accumulation of ppGpp following addition of BIT to exponentially-growing cells. Western blot analysis of whole-cell extracts with anti-RelA antibody demonstrated that both species contain a RelA homologue. This is the first report of a RelA-like protein in pseudomonads.
Topics: Blotting, Western; Culture Media; Guanosine Tetraphosphate; Microbial Sensitivity Tests; Nitrogen; Pseudomonas aeruginosa; Pseudomonas fluorescens; Reproducibility of Results; Thiazoles
PubMed: 10664969
DOI: 10.1046/j.1365-2672.1999.00615.x -
Applied and Environmental Microbiology Sep 2013Pseudomonas species can exhibit phenotypic variation resulting from gacS or gacA mutation. P. fluorescens Pf0-1 is a gacA mutant and exhibits pleiotropic changes...
Pseudomonas species can exhibit phenotypic variation resulting from gacS or gacA mutation. P. fluorescens Pf0-1 is a gacA mutant and exhibits pleiotropic changes following the introduction of a functional allele. GacA enhances biofilm development while reducing dissemination in soil, suggesting that alternative Gac phenotypes enable Pseudomonas sp. to exploit varied environments.
Topics: Bacterial Proteins; Biofilms; Gene Deletion; Genetic Complementation Test; Pseudomonas fluorescens; Soil Microbiology
PubMed: 23811507
DOI: 10.1128/AEM.00819-13 -
Microbial Ecology Apr 2007Biofilms, and other bacterial aggregations, are of significance in both environmental microbiology and in plant and human pathogenesis. Comparative single-cell Raman...
Biofilms, and other bacterial aggregations, are of significance in both environmental microbiology and in plant and human pathogenesis. Comparative single-cell Raman spectral analysis can differentiate between planktonic bacteria and those recovered from biofilms and appears to offer a new means by which to investigate bacterial cell physiology, metabolic status, and stress under different environmental conditions.
Topics: Benzenesulfonates; Biofilms; Discriminant Analysis; Environmental Microbiology; Fluorescent Dyes; Plankton; Principal Component Analysis; Pseudomonas fluorescens; Spectrum Analysis, Raman
PubMed: 17345138
DOI: 10.1007/s00248-006-9190-1 -
Research in Microbiology Apr 2016Pseudomonas fluorescens SF39a is a plant-growth-promoting bacterium isolated from wheat rhizosphere. In this report, we demonstrate that this native strain secretes...
Pseudomonas fluorescens SF39a is a plant-growth-promoting bacterium isolated from wheat rhizosphere. In this report, we demonstrate that this native strain secretes bacteriocins that inhibit growth of phytopathogenic strains of the genera Pseudomonas and Xanthomonas. An S-type pyocin gene was detected in the genome of strain SF39a and named pys. A non-polar pys::Km mutant was constructed. The bacteriocin production was impaired in this mutant. To identify genes involved in bacteriocin regulation, random transposon mutagenesis was carried out. A miniTn5Km1 mutant, called P. fluorescens SF39a-451, showed strongly reduced bacteriocin production. This phenotype was caused by inactivation of the ptsP gene which encodes a phosphoenolpyruvate phosphotransferase (EI(Ntr)) of the nitrogen-related phosphotransferase system (PTS(Ntr)). In addition, this mutant showed a decrease in biofilm formation and protease production, and an increase in surface motility and pyoverdine production compared with the wild-type strain. Moreover, we investigated the ability of strain SF39a-451 to colonize the wheat rhizosphere under greenhouse conditions. Interestingly, the mutant was less competitive than the wild-type strain in the rhizosphere. To our knowledge, this study provides the first evidence of both the relevance of the ptsP gene in bacteriocin production and functional characterization of a pyocin S in P. fluorescens.
Topics: DNA Transposable Elements; Gene Deletion; Mutagenesis, Insertional; Phosphotransferases; Pseudomonas fluorescens; Pyocins; Rhizosphere; Soil Microbiology; Triticum; Xanthomonas
PubMed: 26708985
DOI: 10.1016/j.resmic.2015.12.003 -
Applied and Environmental Microbiology Jan 1996The objectives of this work were (i) to use transposon mutagenesis to produce mutants of Pseudomonas fluorescens that were altered in adhesion ability and transport...
The objectives of this work were (i) to use transposon mutagenesis to produce mutants of Pseudomonas fluorescens that were altered in adhesion ability and transport through porous media and (ii) to identify the alterations in surface characteristics that were responsible for the changes in attachment. Mutants of P. fluorescens were generated with TnphoA, which enabled identification of mutants that were altered in surface proteins. Transposon mutants were screened for alterations in adhesion ability by attachment assays on hydrophobic polystyrene and water-wettable polystyrene. Four TnphoA mutants with increased adhesion to the hydrophobic surface and decreased adhesion to the water-wettable surface were obtained. Transport of the strains through porous media was evaluated by passing suspensions of each mutant and the parent through columns containing quartz sand and determining the number of cells retained in the columns. The mutants all demonstrated increased adhesion and retention in the columns. Southern analysis demonstrated two types of mutants with separate transposon insertion sites. Polyacrylamide gel electrophoresis of the strains demonstrated that the O antigen on the lipopolysaccharide was either attenuated or absent. Lack of this polysaccharide, and the consequent increased exposure of the lipid moiety of the lipopolysaccharide, is probably responsible for the increase in adhesion to the hydrophobic substrata and retention in the sand column. This work combined with previous studies of attachment of P. fluorescens demonstrates that more than one type of polymer can mediate the adhesion of this organism to nonbiological surfaces.
Topics: Bacterial Adhesion; Culture Media; Lipopolysaccharides; Mutagenesis, Insertional; Polystyrenes; Pseudomonas fluorescens
PubMed: 8572686
DOI: 10.1128/aem.62.1.100-104.1996 -
FEMS Microbiology Letters Dec 2004The influence of host growth temperature, phase and media, together with the effect of infection temperature on bacteriophage PhiS1 infection of Pseudomonas fluorescens...
The influence of host growth temperature, phase and media, together with the effect of infection temperature on bacteriophage PhiS1 infection of Pseudomonas fluorescens were examined. The rates of cell lysis and phage release were determined and showed that the efficacy of phage infection was optimal with host cells grown and infected at 26 degrees C. The host physiological state also affected these rates. Infection was dependent on the presence of cell wall proteins with molecular weights of 17.5+/-1 and 99+/-5 kDa.
Topics: Culture Media; Pseudomonas Phages; Pseudomonas fluorescens; Temperature
PubMed: 15556704
DOI: 10.1016/j.femsle.2004.06.058 -
Journal of Bacteriology Mar 2012Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) that has biocontrol activity against fungal plant pathogens and is a model for rhizosphere...
Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) that has biocontrol activity against fungal plant pathogens and is a model for rhizosphere colonization. Here, we present its complete genome sequence, which shows that besides a core genome very similar to those of other strains sequenced within this species, F113 possesses a wide array of genes encoding specialized functions for thriving in the rhizosphere and interacting with eukaryotic organisms.
Topics: DNA, Bacterial; Genome, Bacterial; Molecular Sequence Data; Plants; Pseudomonas fluorescens; Rhizosphere; Sequence Analysis, DNA
PubMed: 22328765
DOI: 10.1128/JB.06601-11 -
Brazilian Journal of Microbiology :... 2017Meat is one of the most perishable foods owing to its nutrient availability, high water activity, and pH around 5.6. These properties are highly conducive for microbial...
Meat is one of the most perishable foods owing to its nutrient availability, high water activity, and pH around 5.6. These properties are highly conducive for microbial growth. Fresh meat, when exposed to oxygen, is subjected to the action of aerobic psychrotrophic, proteolytic, and lipolytic spoilage microorganisms, such as Pseudomonas spp. The spoilage results in the appearance of slime and off-flavor in food. In order to predict the growth of Pseudomonas fluorescens in fresh meat at different pH values, stored under refrigeration, and temperature abuse, microbial mathematical modeling was applied. The primary Baranyi and Roberts and the modified Gompertz models were fitted to the experimental data to obtain the growth parameters. The Ratkowsky extended model was used to determine the effect of pH and temperature on the growth parameter μ. The program DMFit 3.0 was used for model adjustment and fitting. The experimental data showed good fit for both the models tested, and the primary and secondary models based on the Baranyi and Roberts models showed better validation. Thus, these models can be applied to predict the growth of P. fluorescens under the conditions tested.
Topics: Aerobiosis; Food Microbiology; Hydrogen-Ion Concentration; Meat; Models, Theoretical; Pseudomonas fluorescens; Temperature
PubMed: 28110805
DOI: 10.1016/j.bjm.2016.12.006 -
Microbiology (Reading, England) Dec 2015Pseudomonads produce several lipopeptide biosurfactants that have antimicrobial properties but that also facilitate surface motility and influence biofilm formation....
Pseudomonads produce several lipopeptide biosurfactants that have antimicrobial properties but that also facilitate surface motility and influence biofilm formation. Detailed studies addressing the significance of lipopeptides for biofilm formation and architecture are rare. Hence, the present study sets out to determine the specific role of the lipopeptide viscosin in Pseudomonas fluorescens SBW25 biofilm formation, architecture and dispersal, and to relate viscA gene expression to viscosin production and effect. Initially, we compared biofilm formation of SBW25 and the viscosin-deficient mutant strain SBW25ΔviscA in static microtitre assays. These experiments demonstrated that viscosin had little influence on the amount of biofilm formed by SBW25 during the early stages of biofilm development. Later, however, SBW25 formed significantly less biofilm than SBW25ΔviscA. The indication that viscosin is involved in biofilm dispersal was confirmed by chemical complementation of the mutant biofilm. Furthermore, a fluorescent bioreporter showed that viscA expression was induced in biofilms 4 h prior to dispersal. Subsequent detailed studies of biofilms formed in flow cells for up to 5 days revealed that SBW25 and SBW25ΔviscA developed comparable biofilms dominated by well-defined, mushroom-shaped structures. Carbon starvation was required to obtain biofilm dispersal in this system. Dispersal of SBW25 biofilms was significantly greater than of SBW25ΔviscA biofilms after 3 h and, importantly, carbon starvation strongly induced viscA expression, in particular for cells that were apparently leaving the biofilm. Thus, the present study points to a role for viscosin-facilitated motility in dispersal of SBW25 biofilms.
Topics: Bacterial Proteins; Biofilms; Lipopeptides; Peptide Synthases; Peptides, Cyclic; Pseudomonas fluorescens; Surface-Active Agents
PubMed: 26419730
DOI: 10.1099/mic.0.000191