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Journal of Food Protection Oct 1998The relationship between bacterial growth and oxymyoglobin oxidation in vitro and in meat was studied. In the in vitro study, oxymyoglobin was combined with Pseudomonas...
The relationship between bacterial growth and oxymyoglobin oxidation in vitro and in meat was studied. In the in vitro study, oxymyoglobin was combined with Pseudomonas fluorescens or sterile nutrient broth (control) in an airtight vessel. P. fluorescens samples showed greater metmyoglobin formation and oxygen consumption than controls. The P. fluorescens population in the reaction vessels was correlated with metmyoglobin formation (r = 0.85, P < 0.05) and oxygen consumption (r = 0.91, P < 0.05). When P. fluorescens and oxymyoglobin were combined in an airtight vessel, reducing the headspace from 13 ml and 9 ml to 3 ml resulted in greater metmyoglobin formation (P < 0.05). In the meat study, beef cores prepared from longissimus lumborum were inoculated with P. fluorescens (10(7) CFU/cm2) or sterile peptone water (control), packaged under 1% O2 (+99% N2), air, or 100% O2 and stored at 4 degrees C. Inoculated beef cores showed higher bacterial loads and metmyoglobin formation than their respective controls during 10 h storage in 1% O2, 3 days in air, and 7 days in 100% O2 (P < 0.05). This finding indicated that P. fluorescens could accelerate beef discoloration. Overall, studies demonstrated that oxygen consumption concomitant with P. fluorescens growth decreased partial oxygen pressure, which accelerated oxymyoglobin oxidation.
Topics: Animals; Cattle; Culture Media; Meat; Metmyoglobin; Myoglobin; Oxidation-Reduction; Oxygen Consumption; Pseudomonas fluorescens
PubMed: 9798152
DOI: 10.4315/0362-028x-61.10.1341 -
BMC Microbiology Apr 2013Pseudomonas fluorescens is a common inhabitant of soil and the rhizosphere environment. In addition to potential applications in biocontrol and bioremediation, P....
BACKGROUND
Pseudomonas fluorescens is a common inhabitant of soil and the rhizosphere environment. In addition to potential applications in biocontrol and bioremediation, P. fluorescens is of interest as a model for studying bacterial survival and fitness in soil. A previous study using in vivo expression technology (IVET) identified 22 genes in P. fluorescens Pf0-1 which are up-regulated during growth in Massachusetts loam soil, a subset of which are important for fitness in soil. Despite this and other information on adaptation to soil, downstream applications such as biocontrol or bioremediation in diverse soils remain underdeveloped. We undertook an IVET screen to identify Pf0-1 genes induced during growth in arid Nevada desert soil, to expand our understanding of growth in soil environments, and examine whether Pf0-1 uses general or soil type-specific mechanisms for success in soil environments.
RESULTS
Twenty six genes were identified. Consistent with previous studies, these genes cluster in metabolism, information storage/processing, regulation, and 'hypothetical', but there was no overlap with Pf0-1 genes induced during growth in loam soil. Mutation of both a putative glutamine synthetase gene (Pfl01_2143) and a gene predicted to specify a component of a type VI secretion system (Pfl01_5595) resulted in a decline in arid soil persistence. When examined in sterile loam soil, mutation of Pfl01_5595 had no discernible impact. In contrast, the Pfl01_2143 mutant was not impaired in persistence in sterile soil, but showed a significant reduction in competitive fitness.
CONCLUSIONS
These data support the conclusion that numerous genes are specifically important for survival and fitness in natural environments, and will only be identified using in vivo approaches. Furthermore, we suggest that a subset of soil-induced genes is generally important in different soils, while others may contribute to success in specific types of soil. The importance of glutamine synthetase highlights a critical role for nitrogen metabolism in soil fitness. The implication of Type 6 secretion underscores the importance of microbial interactions in natural environments. Understanding the general and soil-specific genes will greatly improve the persistence of designed biocontrol and bioremediation strains within the target environment.
Topics: Desert Climate; Gene Expression Profiling; Genes, Bacterial; Microbial Viability; Nevada; Pseudomonas fluorescens; Soil Microbiology; Stress, Physiological
PubMed: 23622502
DOI: 10.1186/1471-2180-13-92 -
Microbiology (Reading, England) Nov 1998Five independent fluorescent pseudomonad isolates originating from Antarctica were analysed for their pyoverdine systems. A pyoverdine-related siderotyping, which...
Five independent fluorescent pseudomonad isolates originating from Antarctica were analysed for their pyoverdine systems. A pyoverdine-related siderotyping, which involved pyoverdine-induced growth stimulation, pyoverdine-mediated iron uptake, pyoverdine analysis by electrophoresis and isoelectric focusing, revealed three different pyoverdine-related siderotypes among the five isolates. One siderotype, including Pseudomonas fluorescens 1W and P. fluorescens 10CW, was identical to that of P. fluorescens ATCC 13525. Two other strains, P. fluorescens 9AW and Pseudomonas putida 9BW, showed identical pyoverdine-related behaviour to each other, whereas the fifth strain, P. fluorescens 51W, had unique features compared to the other strains or to a set of 12 fluorescent Pseudomonas strains used as comparison material. Elucidation of the structure of the pyoverdines produced by the Antarctic strains supported the accuracy of the siderotyping methodology by confirming that pyoverdines from strains 1W and 10CW had the same structures as the P. fluorescens ATCC 13525 pyoverdine, whereas the 9AW and 9BW pyoverdines are probably identical with the pyoverdine of P. fluorescens strain 244. Pyoverdine from strain 51W appeared to be a novel pyoverdine since its structure was different from all previously established pyoverdine structures. Together with the conclusion that the Antarctic Pseudomonas strains have no special features at the level of their pyoverdines and pyoverdine-mediated iron metabolism compared to worldwide strains, the present work demonstrates that siderotyping provides a rapid means of screening for novel pyoverdines.
Topics: Antarctic Regions; Bacterial Typing Techniques; Biological Transport; Fluorescence; Iron; Oligopeptides; Pigments, Biological; Pseudomonas fluorescens; Pseudomonas putida; Siderophores
PubMed: 9846748
DOI: 10.1099/00221287-144-11-3119 -
FEBS Letters Jun 2021The histidine brace (His-brace) is a copper-binding motif that is associated with both oxidative enzymes and proteinaceous copper chaperones. Here, we used biochemical...
The histidine brace (His-brace) is a copper-binding motif that is associated with both oxidative enzymes and proteinaceous copper chaperones. Here, we used biochemical and structural methods to characterize mutants of a His-brace-containing copper chaperone from Pseudomonas fluorescens (PfCopC). A total of 15 amino acid variants in primary and second-sphere residues were produced and characterized in terms of their copper binding and redox properties. PfCopC has a very high affinity for Cu(II) and also binds Cu(I). A high reorganization barrier likely prevents redox cycling and, thus, catalysis. In contrast, mutations in the conserved second-sphere Glu27 enable slow oxidation of ascorbate. The crystal structure of the variant E27A confirmed copper binding at the His-brace. Unexpectedly, Asp83 at the equatorial position was shown to be indispensable for Cu(II) binding in the His-brace of PfCopC. A PfCopC mutant that was designed to mimic the His-brace from lytic polysaccharide monooxygenase-like family X325 did not bind Cu(II), but was still able to bind Cu(I). These results highlight the importance of the proteinaceous environment around the copper His-brace for reactivity and, thus, the difference between enzyme and chaperone.
Topics: Amino Acid Motifs; Amino Acid Substitution; Bacterial Proteins; Copper; Histidine; Molecular Chaperones; Mutation, Missense; Pseudomonas fluorescens
PubMed: 33896006
DOI: 10.1002/1873-3468.14092 -
PloS One 2012One of the most important micronutrients for bacterial growth is iron, whose bioavailability in soil is limited. Consequently, rhizospheric bacteria such as Pseudomonas...
One of the most important micronutrients for bacterial growth is iron, whose bioavailability in soil is limited. Consequently, rhizospheric bacteria such as Pseudomonas fluorescens employ a range of mechanisms to acquire or compete for iron. We investigated the transcriptomic and proteomic effects of iron limitation on P. fluorescens Pf-5 by employing microarray and iTRAQ techniques, respectively. Analysis of this data revealed that genes encoding functions related to iron homeostasis, including pyoverdine and enantio-pyochelin biosynthesis, a number of TonB-dependent receptor systems, as well as some inner-membrane transporters, were significantly up-regulated in response to iron limitation. Transcription of a ribosomal protein L36-encoding gene was also highly up-regulated during iron limitation. Certain genes or proteins involved in biosynthesis of secondary metabolites such as 2,4-diacetylphloroglucinol (DAPG), orfamide A and pyrrolnitrin, as well as a chitinase, were over-expressed under iron-limited conditions. In contrast, we observed that expression of genes involved in hydrogen cyanide production and flagellar biosynthesis were down-regulated in an iron-depleted culture medium. Phenotypic tests revealed that Pf-5 had reduced swarming motility on semi-solid agar in response to iron limitation. Comparison of the transcriptomic data with the proteomic data suggested that iron acquisition is regulated at both the transcriptional and post-transcriptional levels.
Topics: Bacterial Proteins; Biological Transport; Cell Membrane; Cluster Analysis; Electron Transport; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Iron; Membrane Proteins; Metabolome; Mutation; Oxidative Stress; Proteome; Pseudomonas fluorescens; Ribosomal Proteins; Siderophores; Sigma Factor; Transcriptome
PubMed: 22723948
DOI: 10.1371/journal.pone.0039139 -
BMC Microbiology Sep 2012Pseudomonas fluorescens biovar I MFN1032 is a clinical isolate able to grow at 37°C. This strain displays secretion-mediated hemolytic activity involving phospholipase...
BACKGROUND
Pseudomonas fluorescens biovar I MFN1032 is a clinical isolate able to grow at 37°C. This strain displays secretion-mediated hemolytic activity involving phospholipase C and cyclolipopeptides, and a cell-associated hemolytic activity distinct from the secreted hemolytic activity. Cell-associated hemolysis is independent of biosurfactant production and remains in a gacA mutant. Disruption of the hrpU-like operon (the basal part of type III secretion system from rhizospheric strains) suppresses this activity. We hypothesized that this phenotype could reflect evolution of an ancestral mechanism involved in the survival of this species in its natural niche. In this study, we evaluated the hrpU-like operon's contribution to other virulence mechanisms using a panel of Pseudomonas strains from various sources.
RESULTS
We found that MFN1032 inhibited the growth of the amoebae Dictyostelium discoideum and that this inhibition involved the hrpU-like operon and was absent in a gacA mutant. MFN1032 was capable of causing macrophage lysis, if the hrpU-like operon was intact, and this cytotoxicity remained in a gacA mutant. Cell-associated hemolytic activity and macrophage necrosis were found in other P. fluorescens clinical isolates, but not in biocontrol P. fluorescens strains harbouring hrpU-like operon. The growth of Dictyostelium discoideum was inhibited to a different extent by P. fluorescens strains without correlation between this inhibition and hrpU-like operon sequences.
CONCLUSIONS
In P. fluorescens MFN1032, the basal part of type III secretion system plays a role in D. discoideum growth inhibition and macrophage necrosis. The inhibition of D. discoideum growth is dependent on the GacS/GacA system, while cell-associated hemolytic activity and macrophage lysis are not. Virulence against eukaryotic cells based on the hrpU-like operon may be more than just a stochastic evolution of a conserved system dedicated to survival in competition with natural predators such as amoebae. It may also mean that there are some important modifications of other type III secretion system components, which remain unknown. Cell-associated hemolysis might be a good indicator of the virulence of Pseudomonas fluorescens strain.
Topics: Animals; Bacterial Secretion Systems; Cell Death; Cell Line; Dictyostelium; Macrophages; Mice; Operon; Pseudomonas fluorescens; Virulence; Virulence Factors
PubMed: 23020706
DOI: 10.1186/1471-2180-12-223 -
Applied and Environmental Microbiology Jul 2009Cyclic lipopeptides (CLPs) are produced by many Pseudomonas species and have several biological functions, including a role in surface motility, biofilm formation,...
Cyclic lipopeptides (CLPs) are produced by many Pseudomonas species and have several biological functions, including a role in surface motility, biofilm formation, virulence, and antimicrobial activity. This study focused on the diversity and role of LuxR-type transcriptional regulators in CLP biosynthesis in Pseudomonas species and, specifically, viscosin production by Pseudomonas fluorescens strain SBW25. Phylogenetic analyses showed that CLP biosynthesis genes in Pseudomonas strains are flanked by LuxR-type regulators that contain a DNA-binding helix-turn-helix domain but lack N-acylhomoserine lactone-binding or response regulator domains. For SBW25, site-directed mutagenesis of the genes coding for either of the two identified LuxR-type regulators, designated ViscAR and ViscBCR, strongly reduced transcript levels of the viscABC biosynthesis genes and resulted in a loss of viscosin production. Expression analyses further showed that a mutation in either viscAR or viscBCR did not substantially (change of <2.5-fold) affect transcription of the other regulator. Transformation of the DeltaviscAR mutant of SBW25 with a LuxR-type regulatory gene from P. fluorescens strain SS101 that produces massetolide, a CLP structurally related to viscosin, restored transcription of the viscABC genes and viscosin production. The results further showed that a functional viscAR gene was required for heterologous expression of the massetolide biosynthesis genes of strain SS101 in strain SBW25, leading to the production of both viscosin and massetolide. Collectively, these results indicate that the regulators flanking the CLP biosynthesis genes in Pseudomonas species represent a unique LuxR subfamily of proteins and that viscosin biosynthesis in P. fluorescens SBW25 is controlled by two LuxR-type transcriptional regulators.
Topics: Amino Acid Substitution; Bacterial Proteins; Binding Sites; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Lipopeptides; Multigene Family; Mutagenesis, Site-Directed; Peptides, Cyclic; Phylogeny; Pseudomonas fluorescens; Repressor Proteins; Sequence Homology, Amino Acid; Trans-Activators
PubMed: 19447950
DOI: 10.1128/AEM.00575-09 -
Journal of Bacteriology Dec 2005The plant-colonizing Pseudomonas fluorescens strain SBW25 harbors a gene cluster (rsp) whose products show similarity to type III protein secretion systems found in...
The plant-colonizing Pseudomonas fluorescens strain SBW25 harbors a gene cluster (rsp) whose products show similarity to type III protein secretion systems found in plant and animal pathogens. Here we report a detailed analysis of the expression and regulation of the P. fluorescens rsp pathway, both in the phytosphere and in vitro. A combination of chromosomally integrated transcriptional reporter fusions, overexpressed regulatory genes, and specific mutants reveal that promoters controlling expression of rsp are actively transcribed in the plant rhizosphere but not (with the exception of the rspC promoter) in the phyllosphere. In synthetic medium, regulatory (rspL and rspR) and structural (rspU, plus the putative effector ropE) genes are poorly expressed; the rspC promoter is subject to an additional level of regulatory control. Ectopic expression of regulatory genes in wild-type and mutant backgrounds showed that RspR controls transcription of the alternate sigma factor, rspL, and that RspL controls expression of gene clusters encoding structural genes. Mutation of rspV did not affect RspR-mediated expression of rspU. A search for additional regulators revealed two candidates--one with a role in the conversion of alanine to pyruvate--suggesting that expression of rsp is partly dependent upon the metabolic status of the cell. Mutations in rsp regulators resulted in a significant reduction in competitive colonization of the root tips of sugar beet seedlings but also caused a marked increase in the lag phase of laboratory-grown cultures, indicating that rsp regulatory genes play a more significant general role in the function of P. fluorescens SBW25 than previously appreciated.
Topics: Arabidopsis; Artificial Gene Fusion; Bacterial Proteins; Base Sequence; Beta vulgaris; Biological Transport; Colony Count, Microbial; Culture Media; DNA-Binding Proteins; Gene Dosage; Gene Expression; Gene Expression Regulation, Bacterial; Genes, Regulator; Green Fluorescent Proteins; Molecular Sequence Data; Mutagenesis, Insertional; Mutation; Plant Leaves; Plant Roots; Plants; Promoter Regions, Genetic; Pseudomonas fluorescens; Transcription, Genetic
PubMed: 16321952
DOI: 10.1128/JB.187.24.8477-8488.2005 -
BMC Genomics Jan 2013Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) isolated from the sugar-beet rhizosphere. This bacterium has been extensively studied as a...
BACKGROUND
Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) isolated from the sugar-beet rhizosphere. This bacterium has been extensively studied as a model strain for genetic regulation of secondary metabolite production in P. fluorescens, as a candidate biocontrol agent against phytopathogens, and as a heterologous host for expression of genes with biotechnological application. The F113 genome sequence and annotation has been recently reported.
RESULTS
Comparative analysis of 50 genome sequences of strains belonging to the P. fluorescens group has revealed the existence of five distinct subgroups. F113 belongs to subgroup I, which is mostly composed of strains classified as P. brassicacearum. The core genome of these five strains is highly conserved and represents approximately 76% of the protein-coding genes in any given genome. Despite this strong conservation, F113 also contains a large number of unique protein-coding genes that encode traits potentially involved in the rhizocompetence of this strain. These features include protein coding genes required for denitrification, diterpenoids catabolism, motility and chemotaxis, protein secretion and production of antimicrobial compounds and insect toxins.
CONCLUSIONS
The genome of P. fluorescens F113 is composed of numerous protein-coding genes, not usually found together in previously sequenced genomes, which are potentially decisive during the colonisation of the rhizosphere and/or interaction with other soil organisms. This includes genes encoding proteins involved in the production of a second flagellar apparatus, the use of abietic acid as a growth substrate, the complete denitrification pathway, the possible production of a macrolide antibiotic and the assembly of multiple protein secretion systems.
Topics: Adaptation, Physiological; Animals; Bacterial Proteins; Chemotaxis; Genome, Bacterial; Genomics; Host-Pathogen Interactions; Phylogeny; Plant Development; Plants; Prophages; Pseudomonas fluorescens; Rhizosphere
PubMed: 23350846
DOI: 10.1186/1471-2164-14-54 -
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