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BMC Microbiology Mar 2015Pseudomonas fluorescens strain MFE01 secretes in abundance two Hcp proteins (haemolysin co-regulated proteins) Hcp1 and Hcp2, characteristic of a functional type 6...
BACKGROUND
Pseudomonas fluorescens strain MFE01 secretes in abundance two Hcp proteins (haemolysin co-regulated proteins) Hcp1 and Hcp2, characteristic of a functional type 6 secretion system. Phenotypic studies have shown that MFE01 has antibacterial activity against a wide range of competitor bacteria, including rhizobacteria and clinically relevant bacteria. Mutagenesis of the hcp2 gene abolishes or reduces, depending on the target strain, MFE01 antibacterial activity. Hcp1, encoded by hcp1, may also be involved in bacterial competition. We therefore assessed the contribution of Hcp1 to competition of P. fluorescens MFE01 with other bacteria, by studying MFE01 mutants in various competitive conditions.
RESULTS
Mutation of hcp1 had pleiotropic effects on the MFE01 phenotype. It affected mucoidy of the strain and its motility and was associated with the loss of flagella, which were restored by introduction of plasmid expressing hcp1. The hcp1 mutation had no effect on bacterial competition during incubation in solid medium. MFE01 was able to sequester another P. fluorescens strain, MFN1032, under swimming conditions. The hcp2 mutant but not the hcp1 mutant conserved this ability. In competition assays on swarming medium, MFE01 impaired MFN1032 swarming and displayed killing activity. The hcp2 mutant, but not the hcp1 mutant, was able to reduce MFN1032 swarming. The hcp1 and hcp2 mutations each abolished killing activity in these conditions.
CONCLUSION
Our findings implicate type 6 secretion of Hcp1 in mucoidy and motility of MFE01. Our study is the first to establish a link between a type 6 secretion system and flagellin and mucoidy. Hcp1 also appears to contribute to limiting the motility of prey cells to facilitate killing mediated by Hcp2. Inhibition of motility associated with an Hcp protein has never been described. With this work, we illustrate the importance and versatility of type 6 secretion systems in bacterial adaptation and fitness.
Topics: Antibiosis; Bacterial Proteins; Gene Deletion; Genetic Complementation Test; Locomotion; Polysaccharides, Bacterial; Pseudomonas fluorescens; Type VI Secretion Systems
PubMed: 25886496
DOI: 10.1186/s12866-015-0405-9 -
Applied and Environmental Microbiology Mar 1998Pseudomonas fluorescens CY091 cultures produce an extracellular protease with an estimated molecular mass of 50 kDa. Production of this enzyme (designated AprX) was...
Pseudomonas fluorescens CY091 cultures produce an extracellular protease with an estimated molecular mass of 50 kDa. Production of this enzyme (designated AprX) was observed in media containing CaCl2 or SrCl2 but not in media containing ZnCl2, MgCl2, or MnCl2. The requirement of Ca2+ (or Sr2+) for enzyme production was concentration dependent, and the optimal concentration for production was determined to be 0.35 mM. Following ammonium sulfate precipitation and ion-exchange chromatography, the AprX in the culture supernatant was purified to near electrophoretic homogeneity. Over 20% of the enzyme activity was retained in the AprX sample which had been heated in boiling water for 10 min, indicating that the enzyme is highly resistant to heat inactivation. The enzyme activity was almost completely inhibited in the presence of 1 mM 1,10-phenanthroline, but only 30% of the activity was inhibited in the presence of 1 mM EGTA. The gene encoding AprX was cloned from the genome of P. fluorescens CY091 by isolating cosmid clones capable of restoring the protease production in a nonproteolytic mutant of strain CY091. The genomic region of strain CY091 containing the aprX gene was located within a 7.3-kb DNA fragment. Analysis of the complete nucleotide sequence of this 7.3-kb fragment revealed the presence of a cluster of genes required for the production of extracellular AprX in P. fluorescens and Escherichia coli. The AprX protein showed 50 to 60% identity in amino acid sequence to the related proteases produced by Pseudomonas aeruginosa and Erwinia chrysanthemi. Two conserved sequence domains possibly associated with Ca2+ and Zn2+ binding were identified. Immediately adjacent to the aprX structural gene, a gene (inh) encoding a putative protease inhibitor and three genes (aprD, aprE, and aprF), possibly required for the transport of AprX, were also identified. The organization of the gene cluster involved in the synthesis and secretion of AprX in P. fluorescens CY091 appears to be somewhat different from that previously demonstrated in P. aeruginosa and E. chrysanthemi.
Topics: Amino Acid Sequence; Bacterial Proteins; Calcium Chloride; Cloning, Molecular; Endopeptidases; Molecular Sequence Data; Pseudomonas fluorescens
PubMed: 9501431
DOI: 10.1128/AEM.64.3.914-921.1998 -
Microbiology Spectrum Jun 2024Swarming motility in pseudomonads typically requires both a functional flagellum and the production/secretion of a biosurfactant. Published work has shown that the...
Swarming motility in pseudomonads typically requires both a functional flagellum and the production/secretion of a biosurfactant. Published work has shown that the wild-type Pf0-1 is swarming deficient due to a point mutation in the gene, which until recently was thought to inactivate rather than attenuate the Gac/Rsm pathway. As a result, little is known about the underlying mechanisms that regulate swarming motility by Pf0-1. Here, we demonstrate that a Δ Δ Δ mutant, which phenotypically mimics Gac/Rsm pathway overstimulation, is proficient at swarming motility. RsmA and RsmE appear to play a key role in this regulation. Transposon mutagenesis of the Δ Δ Δ mutant identified multiple factors that impact swarming motility, including pathways involved in flagellar synthesis and biosurfactant production/secretion. We find that loss of genes linked to biosurfactant Gacamide A biosynthesis or secretion impacts swarming motility, as does loss of the alternative sigma factor FliA, which results in a defect in flagellar function. Collectively, these findings provide evidence that Pf0-1 can swarm if the Gac/Rsm pathway is activated, highlight the regulatory complexity of swarming motility in this strain, and demonstrate that the cyclic lipopeptide Gacamide A is utilized as a biosurfactant for swarming motility.IMPORTANCESwarming motility is a coordinated process that allows communities of bacteria to collectively move across a surface. For Pf0-1, this phenotype is notably absent in the parental strain, and to date, little is known about the regulation of swarming in this strain. Here, we identify RsmA and RsmE as key repressors of swarming motility modulating the levels of biosurfactant production/secretion. Using transposon mutagenesis and subsequent genetic analyses, we further identify potential regulatory mechanisms of swarming motility and link Gacamide A biosynthesis and transport machinery to swarming motility.
Topics: Pseudomonas fluorescens; Movement; Bacterial Proteins; Methyltransferases; Surface-Active Agents; Mutagenesis; Sigma Factor
PubMed: 38687073
DOI: 10.1128/spectrum.00166-24 -
Biofouling Jul 2010Despite the recent enthusiasm for using bacteriophages as bacterial control agents, there are only limited studies concerning phage interaction with their respective...
Despite the recent enthusiasm for using bacteriophages as bacterial control agents, there are only limited studies concerning phage interaction with their respective hosts residing in mixed biofilm consortia and especially in biofilms where the host species is a minor constituent. In the present work, a study was made of mono and dual species biofilms formed by Pseudomonas fluorescens (Gram-negative) and/or Staphylococcus lentus (Gram-positive) and their fate after infection with phages. The dual species biofilms consisted predominantly of S. lentus. The exposure of these biofilms to a cocktail containing both P. fluorescens and S. lentus phages effectively killed and removed the hosts from the substratum. Additionally, this cocktail approach also controlled the hosts released from the biofilms to the planktonic phase. The ability of phages to control a host population present in minority in the mixed species biofilm was also assessed. For this objective, the biofilms were challenged only with phage phiIBB-PF7A, specific for P. fluorescens and the results obtained were to some extent unpredicted. First, phiIBB-PF7A readily reached the target host and caused a significant population decrease. Secondly, and surprisingly, this phage was also capable of causing partial damage to the biofilms leading to the release of the non-susceptible host (S. lentus) from the dual species biofilms to the planktonic phase. The efficiency of phage treatment of biofilms was to some extent dependent on the number of cells present and also conditioned by the infection strategy (dynamic or static) utilized in the infection of the biofilms. Nevertheless, in most circumstances phages were well capable of controlling their target hosts.
Topics: Bacterial Adhesion; Biofilms; Colony Count, Microbial; Dairying; Food Industry; Pest Control, Biological; Plankton; Pseudomonas Phages; Pseudomonas fluorescens; Staphylococcus; Staphylococcus Phages
PubMed: 20544433
DOI: 10.1080/08927014.2010.494251 -
Journal of Bacteriology Oct 2022Cells in microbial communities on surfaces live and divide in close proximity, which greatly enhances the potential for social interactions. Spatiogenetic structures are...
Cells in microbial communities on surfaces live and divide in close proximity, which greatly enhances the potential for social interactions. Spatiogenetic structures are manifested through competitive and cooperative interactions among the same and different genotypes within a shared space, and extracellular secretions appear to function dynamically at the forefront. A previous experimental evolution study utilizing Pseudomonas fluorescens Pf0-1 colonies demonstrated that diverse mutations in the gene were repeatedly and exclusively selected through the formation of a dominant spatial structure. RsmE's primary molecular function is translation repression, and its homologs regulate various social and virulence phenotypes. Pseudomonas spp. possess multiple paralogs of Rsm proteins, and RsmA, RsmE, and RsmI are the most prevalent. Here, we demonstrate that the production of a mucoid polymer and a biosurfactant are exclusively regulated through RsmE, contradicting the generalized notion of functional redundancy among the Rsm paralogs. Furthermore, we identified the biosurfactant as the cyclic lipopeptide gacamide A. Competition and microscopy analyses showed that the mucoid polymer is solely responsible for creating a space of low cellular density, which is shared exclusively by the same genotype. Gacamide A and other RsmE-regulated products appear to establish a physical boundary that prevents the encroachment of the competing genotype into the newly created space. Although cyclic lipopeptides and other biosurfactants are best known for their antimicrobial properties and reducing surface tension to promote the spreading of cells on various surfaces, they also appear to help define spatial structure formation within a dense community. In densely populated colonies of the bacterium Pseudomonas fluorescens Pf0-1, diverse mutations in the gene are naturally selected by solving the problem of overcrowding. Here, we show that RsmE-regulated secretions function together to create and protect space of low cell density. A biosurfactant generally promotes the spreading of bacterial cells on abiotic surfaces; however, it appears to function atypically within a crowded population by physically defining genotypic boundaries. Another significant finding is that these secretions are not regulated by RsmE's paralogs that share high sequence similarity. The experimental pipeline described in this study is highly tractable and should facilitate future studies to explore additional RsmE-regulated products and address why RsmE is functionally unique from its paralogs.
Topics: Pseudomonas fluorescens; Gene Expression Regulation, Bacterial; Bacterial Proteins; Pseudomonas; Peptides, Cyclic; Lipopeptides; Polymers
PubMed: 36165622
DOI: 10.1128/jb.00285-22 -
Microbiological Research Mar 2019Banana is the second largest export crop in Colombia. To meet the demand of international markets, high amounts of chemical fertilizers are required, which represent...
Screening, plant growth promotion and root colonization pattern of two rhizobacteria (Pseudomonas fluorescens Ps006 and Bacillus amyloliquefaciens Bs006) on banana cv. Williams (Musa acuminata Colla).
Banana is the second largest export crop in Colombia. To meet the demand of international markets, high amounts of chemical fertilizers are required, which represent high costs and can be hazardous to the environment. Plant growth promoting rhizobacteria (PGPR) can, at least partially, replace chemical fertilizers. In this paper, we evaluated the effect of nine PGPR of the genera Bacillus and Pseudomonas on banana growth. Banana seedlings were produced through tissue culture and acclimatized in the greenhouse core. Plants were inoculated with the rhizobacteria and growth parameters (plant height, leaf number, leaf area, pseudostem thickness, root and shoot fresh weight, root and shoot dry weight) were assessed after 55 days. The two best performing PGPR, Bs006 and Ps006 previously identified as Bacillus amyloliquefaciens and Pseudomonas fluorescens, respectively, promoted banana growth similarly or even slightly superior to 100% chemical fertilization, and were selected for further characterization of root colonization by both eletron microscopy and confocal microscopy of fluorescence in situ hybridization (FISH)-stained root tissues. Both P. fluorescens Ps006 and B. amyloquifaciens Bs006 showed ability to colonize banana roots, but Bs006 appeared faster than Ps006 in the colonization dynamics. This work demonstrated that inoculation of rhizobacteria Bacillus amyloliquefaciens Bs006 and Pseudomonas fluorescens Ps006 could partially replace the chemical fertilization of tissue cultured banana plants, and therefore could be used for the formulation of a new biofertilizer.
Topics: Bacillus amyloliquefaciens; Colombia; Fertilizers; In Situ Hybridization, Fluorescence; Microscopy, Electron, Scanning; Musa; Plant Development; Plant Leaves; Plant Roots; Pseudomonas fluorescens; Seedlings; Soil; Soil Microbiology
PubMed: 30744815
DOI: 10.1016/j.micres.2018.11.006 -
MicrobiologyOpen Aug 2021Naphthenic acids (NAs) are carboxylic acids with the formula (C H O ) and are among the most toxic, persistent constituents of oil sands process-affected waters (OSPW),...
Naphthenic acids (NAs) are carboxylic acids with the formula (C H O ) and are among the most toxic, persistent constituents of oil sands process-affected waters (OSPW), produced during oil sands extraction. Currently, the proteins and mechanisms involved in NA biodegradation are unknown. Using LC-MS/MS shotgun proteomics, we identified proteins overexpressed during the growth of Pseudomonas fluorescens Pf-5 on a model NA (4'-n-butylphenyl)-4-butanoic acid (n-BPBA) and commercial NA mixture (Acros). By day 11, >95% of n-BPBA was degraded. With Acros, a 17% reduction in intensity occurred with 10-18 carbon compounds of the Z family -2 to -14 (major NA species in this mixture). A total of 554 proteins (n-BPBA) and 631 proteins (Acros) were overexpressed during growth on NAs, including several transporters (e.g., ABC transporters), suggesting a cellular protective response from NA toxicity. Several proteins associated with fatty acid, lipid, and amino acid metabolism were also overexpressed, including acyl-CoA dehydrogenase and acyl-CoA thioesterase II, which catalyze part of the fatty acid beta-oxidation pathway. Indeed, multiple enzymes involved in the fatty acid oxidation pathway were upregulated. Given the presumed structural similarity between alkyl-carboxylic acid side chains and fatty acids, we postulate that P. fluorescens Pf-5 was using existing fatty acid catabolic pathways (among others) during NA degradation.
Topics: Acyl-CoA Dehydrogenase; Biodegradation, Environmental; Carboxylic Acids; Fatty Acids; Gene Expression Regulation, Bacterial; Oxidation-Reduction; Palmitoyl-CoA Hydrolase; Pseudomonas fluorescens; Water Pollutants, Chemical
PubMed: 34459546
DOI: 10.1002/mbo3.1196 -
PloS One 2020Biofilms are microbial communities embedded in an extracellular polymeric matrix and display an enhanced tolerance to the action of antimicrobials. The emergence of...
Biofilms are microbial communities embedded in an extracellular polymeric matrix and display an enhanced tolerance to the action of antimicrobials. The emergence of novel functionalised nanoparticles is considered a promising avenue for the development of biofilm-specific antimicrobial technologies. However, there is a gap in the understanding of interactions between nanoparticles and the biofilm matrix. Particularly, questions are raised on how nanoparticle charge and surface groups play a role in aggregation when in contact with biofilm components. Herein we present the synthesis of four types of silica nanoparticles and undertake an analysis of their interactions with Pseudomonas fluorescens biofilm matrix. The effect of the biofilm matrix components on the charge and aggregation of the nanoparticles was assessed. Additionally, the study focused on the role of matrix proteins, with the in-depth characterisation of the protein corona of each nanoparticle by Liquid Chromatography with Tandem Mass Spectrometry experiments. The protein corona composition is dependent on the nanoparticle type; non-functionalised nanoparticles show less protein selectivity, whereas carboxylate-functionalised nanoparticles prefer proteins with a higher isoelectric point. These outcomes provide insights into the field of biofilm-nanoparticle interactions that can be valuable for the design of new nano-based targeting systems in future anti-biofilm applications.
Topics: Biofilms; Chromatography, Liquid; Humans; Metal Nanoparticles; Protein Corona; Protein Interaction Maps; Pseudomonas fluorescens; Silicon Dioxide; Tandem Mass Spectrometry
PubMed: 32701973
DOI: 10.1371/journal.pone.0236441 -
Microbes and Environments 2014Pseudomonas fluorescens Pf0-1 exhibited chemotactic responses to l-malate, succinate, and fumarate. We constructed a plasmid library of 37 methyl-accepting chemotaxis...
Pseudomonas fluorescens Pf0-1 exhibited chemotactic responses to l-malate, succinate, and fumarate. We constructed a plasmid library of 37 methyl-accepting chemotaxis protein (MCP) genes of P. fluorescens Pf0-1. To identify a MCP for l-malate, the plasmid library was screened using the PA2652 mutant of Pseudomonas aeruginosa PAO1, a mutant defective in chemotaxis to l-malate. The introduction of Pfl01_0728 and Pfl01_3768 genes restored the ability of the PA2652 mutant to respond to l-malate. The Pfl01_0728 and Pfl01_3768 double mutant of P. fluorescens Pf0-1 showed no response to l-malate or succinate, while the Pfl01_0728 single mutant did not respond to fumarate. These results indicated that Pfl01_0728 and Pfl01_3768 were the major MCPs for l-malate and succinate, and Pfl01_0728 was also a major MCP for fumarate. The Pfl01_0728 and Pfl01_3768 double mutant unexpectedly exhibited stronger responses toward the tomato root exudate and amino acids such as proline, asparagine, methionine, and phenylalanine than those of the wild-type strain. The ctaA, ctaB, ctaC (genes of the major MCPs for amino acids), Pfl01_0728, and Pfl01_3768 quintuple mutant of P. fluorescens Pf0-1 was less competitive than the ctaA ctaB ctaC triple mutant in competitive root colonization, suggesting that chemotaxis to l-malate, succinate, and/or fumarate was involved in tomato root colonization by P. fluorescens Pf0-1.
Topics: Chemotaxis; Fumarates; Gene Library; Genetic Complementation Test; Solanum lycopersicum; Malates; Mutation; Plant Roots; Plasmids; Proteins; Pseudomonas aeruginosa; Pseudomonas fluorescens; Succinic Acid
PubMed: 25491753
DOI: 10.1264/jsme2.ME14128 -
ELife Jan 2019Predicting evolutionary change poses numerous challenges. Here we take advantage of the model bacterium in which the genotype-to-phenotype map determining evolution of...
Predicting evolutionary change poses numerous challenges. Here we take advantage of the model bacterium in which the genotype-to-phenotype map determining evolution of the adaptive 'wrinkly spreader' (WS) type is known. We present mathematical descriptions of three necessary regulatory pathways and use these to predict both the rate at which each mutational route is used and the expected mutational targets. To test predictions, mutation rates and targets were determined for each pathway. Unanticipated mutational hotspots caused experimental observations to depart from predictions but additional data led to refined models. A mismatch was observed between the spectra of WS-causing mutations obtained with and without selection due to low fitness of previously undetected WS-causing mutations. Our findings contribute toward the development of mechanistic models for forecasting evolution, highlight current limitations, and draw attention to challenges in predicting locus-specific mutational biases and fitness effects.
Topics: Adaptation, Physiological; Bias; Genotype; Models, Biological; Mutation; Mutation Rate; Phenotype; Pseudomonas fluorescens
PubMed: 30616716
DOI: 10.7554/eLife.38822