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Molecular Microbiology Feb 2022Many bacteria form mats at the air-liquid interface of static microcosms. These structures typically involve the secretion of exopolysaccharides, the production of which...
Many bacteria form mats at the air-liquid interface of static microcosms. These structures typically involve the secretion of exopolysaccharides, the production of which is often controlled by the secondary messenger c-di-GMP. Mechanisms of mat formation have been particularly well characterized in Pseudomonas fluorescens SBW25; stimuli or mutations that increase c-di-GMP production by diguanylate cyclases (WspR, AwsR, and MwsR) result in the secretion of cellulose and mat formation. Here, we characterize and compare mat formation in two close relatives of SBW25: Pseudomonas simiae PICF7 and P. fluorescens A506. We find that PICF7-the strain more closely related to SBW25-can form mats through mutations affecting the activity of the same three diguanylate cyclases as SBW25. However, instead of cellulose, these mutations activate production of the exopolysaccharide Pel. We also provide evidence for at least two further-as yet uncharacterized-routes to mat formation by PICF7. P. fluorescens A506, while retaining the same mutational routes to mat formation as SBW25 and PICF7, preferentially forms mats by a semi-heritable mechanism that culminates in Psl and Pga over-production. Our results demonstrate a high level of evolutionary flexibility in the molecular and structural routes to mat formation, even among close relatives.
Topics: Biofilms; Biological Evolution; Cyclic GMP; Mutation; Pseudomonas; Pseudomonas aeruginosa; Pseudomonas fluorescens
PubMed: 34856020
DOI: 10.1111/mmi.14855 -
Microbiology (Reading, England) Aug 2023Evolutionary innovation of transcription factors frequently drives phenotypic diversification and adaptation to environmental change. Transcription factors can gain or...
Evolutionary innovation of transcription factors frequently drives phenotypic diversification and adaptation to environmental change. Transcription factors can gain or lose connections to target genes, resulting in novel regulatory responses and phenotypes. However the frequency of functional adaptation varies between different regulators, even when they are closely related. To identify factors influencing propensity for innovation, we utilise a SBW25 strain rendered incapable of flagellar mediated motility in soft-agar plates via deletion of the flagellar master regulator (). This bacterium can evolve to rescue flagellar motility via gene regulatory network rewiring of an alternative transcription factor to rescue activity of FleQ. Previously, we have identified two members (out of 22) of the RpoN-dependent enhancer binding protein (RpoN-EBP) family of transcription factors (NtrC and PFLU1132) that are capable of innovating in this way. These two transcription factors rescue motility repeatably and reliably in a strict hierarchy – with NtrC the only route in a ∆ background, and PFLU1132 the only route in a ∆∆ background. However, why other members in the same transcription factor family have not been observed to rescue flagellar activity is unclear. Previous work shows that protein homology cannot explain this pattern within the protein family (RpoN-EBPs), and mutations in strains that rescued motility suggested high levels of transcription factor expression and activation drive innovation. We predict that mutations that increase expression of the transcription factor are vital to unlock evolutionary potential for innovation. Here, we construct titratable expression mutant lines for 11 of the RpoN-EBPs in . We show that in five additional RpoN-EBPs (FleR, HbcR, GcsR, DctD, AauR and PFLU2209), high expression levels result in different mutations conferring motility rescue, suggesting alternative rewiring pathways. Our results indicate that expression levels (and not protein homology) of RpoN-EBPs are a key constraining factor in determining evolutionary potential for innovation. This suggests that transcription factors that can achieve high expression through few mutational changes, or transcription factors that are active in the selective environment, are more likely to innovate and contribute to adaptive gene regulatory network evolution.
Topics: Transcription Factors; Transcription, Genetic; Gene Expression Regulation; Pseudomonas fluorescens; Gene Expression Regulation, Bacterial; Bacterial Proteins
PubMed: 37584667
DOI: 10.1099/mic.0.001378 -
Applied and Environmental Microbiology Feb 2016Pseudomonas fluorescens is able to produce the medically and industrially important exopolysaccharide alginate. The proteins involved in alginate biosynthesis and...
Pseudomonas fluorescens is able to produce the medically and industrially important exopolysaccharide alginate. The proteins involved in alginate biosynthesis and secretion form a multiprotein complex spanning the inner and outer membranes. In the present study, we developed a method by which the porin AlgE was detected by immunogold labeling and transmission electron microscopy. Localization of the AlgE protein was found to depend on the presence of other proteins in the multiprotein complex. No correlation was found between the number of alginate factories and the alginate production level, nor were the numbers of these factories affected in an algC mutant that is unable to produce the precursor needed for alginate biosynthesis. Precursor availability and growth phase thus seem to be the main determinants for the alginate production rate in our strain. Clustering analysis demonstrated that the alginate multiprotein complexes were not distributed randomly over the entire outer cell membrane surface.
Topics: Alginates; Glucuronic Acid; Hexuronic Acids; Membrane Transport Proteins; Microscopy, Immunoelectron; Multienzyme Complexes; Porins; Pseudomonas fluorescens
PubMed: 26655760
DOI: 10.1128/AEM.03114-15 -
Microbial Pathogenesis Jun 2024Olive knot disease, caused by Pseudomonas savastanoi, poses a significant threat to olive cultivation, necessitating sustainable alternatives to conventional chemical...
Olive knot disease, caused by Pseudomonas savastanoi, poses a significant threat to olive cultivation, necessitating sustainable alternatives to conventional chemical control. This study investigates the biocontrol effectiveness of Bacillus sp. (Og2) and Pseudomonas fluorescens (Oq5), alone and combined, against olive knot disease. Olive plants were sprayed with 5 ml of the bacteria until uniformly wet, with additional application to the soil surface. Pathogen injection occurred 24 h later. The results revealed that treating plants with a combination of both bacteria provided the highest reduction in disease severity (89.58 %), followed by P. fluorescens alone (69.38 %). Significant improvements were observed in shoot height, particularly with the combination of Bacillus sp. and P. fluorescens. The root length of olive seedlings treated with P. fluorescens and Bacillus sp., either alone or in combination, was significantly longer compared to the control and pathogen-treated seedlings. In terms of root dry weight, the most effective treatments were treated with P. fluorescens was the highest (82.94 g) among all treatments followed by the combination of both isolates with seedlings inoculated with P. savastanoi. These findings underscore the potential of Bacillus sp. and Pseudomonas fluorescens as effective biocontrol agents against olive knot disease and promoting olive seedlings growth, providing a sustainable and environmentally friendly approach to disease management.
Topics: Olea; Pseudomonas fluorescens; Bacillus; Plant Diseases; Biological Control Agents; Seedlings; Plant Roots; Antibiosis
PubMed: 38631412
DOI: 10.1016/j.micpath.2024.106645 -
Biochimica Et Biophysica Acta. Proteins... 2019Kynureninase from Pseudomonas fluorescens (Pfkynase) catalyzes the pyridoxal-5'-phosphate (PLP) dependent hydrolytic cleavage of L-kynurenine to give anthranilate and...
Kynureninase from Pseudomonas fluorescens (Pfkynase) catalyzes the pyridoxal-5'-phosphate (PLP) dependent hydrolytic cleavage of L-kynurenine to give anthranilate and L-alanine. Asp-132 and Asp-201 are located in the structure near the pyridine NH of the PLP, with Asp-201 forming a hydrogen bond. Mutation of Asp-132 to alanine and glutamate and Asp-201 to glutamate results in reduced catalytic activity with L-kynurenine and β-benzoyl-L-alanine, but not O-benzoyl-l-serine. D132A, D132E D201E and S36A mutant Pfkynases all can form quinonoid and vinylogous amide intermediates with β-benzoyl-L-alanine, similar to wild-type enzyme. D132A, D132E, and D201E Pfkynase react more slowly with β-benzoyl-L-alanine and benzaldehyde to form an aldol product absorbing at 490 nm than wild-type, with D132E reacting the slowest. The H NMR spectra of wild-type and D201E Pfkynase are very similar in the low field region from 10 to 18 ppm, but that of D132A Pfkynase is missing a resonance at 13.1 ppm. These results show that these residues modulate the reactivity of the PLP at different stages during the reaction cycle. Ser-36 is located near the expected location of the carbonyl oxygen of the substrate. Mutation of Ser-36 to alanine results in a 230-fold reduction of k and 30-fold reduction in k/K with L-kynurenine, but very little effect on the reaction of O-benzoyl-l-serine. Thus, the rate-determining step in the reaction of S36A Pfkynase is the C-C bond cleavage. These results support the hypothesis that Ser-36 together with Tyr-226 is part of an oxyanion hole that polarizes the carbonyl of the substrate in the catalytic mechanism of Pfkynase.
Topics: Amino Acid Substitution; Aspartic Acid; Bacterial Proteins; Hydrolases; Mutation, Missense; Pseudomonas fluorescens; Serine
PubMed: 31100408
DOI: 10.1016/j.bbapap.2019.05.005 -
Journal of Agricultural and Food... Aug 2023Carbon metabolism is critical for microbial physiology and remarkably affects the outcome of secondary metabolite production. The production of...
Carbon metabolism is critical for microbial physiology and remarkably affects the outcome of secondary metabolite production. The production of 2,4-diacetylphloroglucinol (2,4-DAPG), a bacterial secondary metabolite with a broad spectrum of antibiotic activity, is a major mechanism used by the soil bacterium 2P24 to inhibit the growth of plant pathogens and control disease occurrence. Strain 2P24 has evolved a complex signaling cascade to regulate the production of 2,4-DAPG. However, the role of the central carbon metabolism in modulating 2,4-DAPG production has not been fully determined. In this study, we report that the gene, which encodes citrate synthase, affects the expression of the 2,4-DAPG biosynthesis gene and is essential for the biocontrol capacity of strain 2P24. Our data showed that the mutation of remarkably decreased the biosynthesis of 2,4-DAPG. Consistent with this result, the addition of citrate in strain 2P24 resulted in increased 2,4-DAPG production and decreased levels of RsmA and RsmE. In comparison with the wild-type strain, the mutant was severely impaired in terms of biocontrol activity against the bacterial wilt disease of tomato plants caused by . Moreover, the mutant exhibited increased antioxidant activity, and the expression of oxidative, stress-associated genes, including , , and , was significantly upregulated in the mutant compared to the wild-type strain. Overall, our data indicate that the citrate synthase GltA plays an important role in the production of 2,4-DAPG and oxidative stress and is required for biocontrol capacity.
Topics: Pseudomonas fluorescens; Citrate (si)-Synthase; Bacterial Proteins; Phloroglucinol
PubMed: 37523467
DOI: 10.1021/acs.jafc.3c03051 -
ELife Dec 2021Agricultural soil harbors a diverse microbiome that can form beneficial relationships with plants, including the inhibition of plant pathogens. spp. are one of the most...
Agricultural soil harbors a diverse microbiome that can form beneficial relationships with plants, including the inhibition of plant pathogens. spp. are one of the most abundant bacterial genera in the soil and rhizosphere and play important roles in promoting plant health. However, the genetic determinants of this beneficial activity are only partially understood. Here, we genetically and phenotypically characterize the population in a commercial potato field, where we identify strong correlations between specialized metabolite biosynthesis and antagonism of the potato pathogens and . Genetic and chemical analyses identified hydrogen cyanide and cyclic lipopeptides as key specialized metabolites associated with inhibition, which was supported by in planta biocontrol experiments. We show that a single potato field contains a hugely diverse and dynamic population of bacteria, whose capacity to produce specialized metabolites is shaped both by plant colonization and defined environmental inputs.
Topics: Hydrogen Cyanide; Lipopeptides; Peptides, Cyclic; Phytophthora infestans; Plant Diseases; Pseudomonas fluorescens; Solanum tuberosum; Streptomyces
PubMed: 34792466
DOI: 10.7554/eLife.71900 -
Microbial Ecology Nov 2016Bacterial species exhibit biogeographical patterns like those observed in larger organisms. The distribution of bacterial species is driven by environmental selection...
Bacterial species exhibit biogeographical patterns like those observed in larger organisms. The distribution of bacterial species is driven by environmental selection through abiotic and biotic factors as well dispersal limitations. We asked whether interference competition, a biotic factor, could explain variability in habitat use by Pseudomonas species in the human home. To answer this question, we screened almost 8000 directional, pairwise interactions between 89 Pseudomonas strains including members of the Pseudomonas aeruginosa (n = 29), Pseudomonas fluorescens (n = 21), and Pseudomonas putida (n = 39) species groups for the presence of killing. This diverse set of Pseudomonas strains includes those isolated from several different habitats within the home environment and includes combinations of strains that were isolated from different spatial scales. The use of this strain set not only allowed us to analyze the commonality and phylogenetic scale of interference competition within the genus Pseudomonas but also allowed us to investigate the influence of spatial scale on this trait. Overall, the probability of killing was found to decrease with increasing phylogenetic distance, making it unlikely that interference competition accounts for previously observed differential habitat use among Pseudomonas species and species groups. Strikingly, conspecific P. aeruginosa killing accounted for the vast majority of the observed killing, and this killing was found to differ across the habitat type and spatial scale of the strains' isolation. These data suggest that interference competition likely plays a large role in the within-species dynamics of P. aeruginosa but not other household Pseudomonas species.
Topics: Bacteriocins; Ecosystem; Humans; Microbial Interactions; Phylogeny; Pseudomonas aeruginosa; Pseudomonas fluorescens; Pseudomonas putida; Pyocins; Residence Characteristics
PubMed: 26276409
DOI: 10.1007/s00248-015-0652-1 -
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 -
Food Microbiology Jun 2024Bacterial volatile compounds (BVCs) facilitate interspecies communication in socio-microbiology across physical barriers, thereby influencing interactions between...
Bacterial volatile compounds (BVCs) facilitate interspecies communication in socio-microbiology across physical barriers, thereby influencing interactions between diverse species. The impact of BVCs emitted from Pseudomonas on the biofilm formation characteristics of Listeria monocytogenes within the same ecological niche has been scarcely investigated under practical conditions of food processing. The objective of this study was to explore the motility and biofilm formation characteristics of L. monocytogenes under the impact of Pseudomonas BVCs. It was revealed that BVCs of P. fluorescens, P. lundensis, and P. fragi significantly promoted swimming motility of L. monocytogenes (P < 0.05). As evidenced by crystal violet staining, the L. monocytogenes biofilms reached a maximum OD value of approximately 3.78 at 4 d, which was 0.65 units markedly higher than that of the control group (P < 0.05). Despite a decrease in adherent cells of L. monocytogenes biofilms among the BVCs groups, there was a remarkable increase in the abundance of extracellular polysaccharides and proteins with 3.58 and 4.90 μg/cm, respectively (P < 0.05), contributing to more compact matrix architectures, which suggested that the BVCs of P. fluorescens enhanced L. monocytogenes biofilm formation through promoting the secretion of extracellular polymers. Moreover, the prominent up-regulated expression of virulence genes further revealed the positive regulation of L. monocytogenes under the influence of BVCs. Additionally, the presence of BVCs significantly elevated the pH and TVB-N levels in both the swimming medium and biofilm broth, thereby exhibiting a strong positive correlation with increased motility and biofilm formation of L. monocytogenes. It highlighted the crucial signaling regulatory role of BVCs in bacterial interactions, while also emphasizing the potential food safety risk associated with the hitchhiking behavior of L. monocytogenes, thereby shedding light on advancements in control strategies for food processing.
Topics: Pseudomonas fluorescens; Listeria monocytogenes; Coculture Techniques; Swimming; Biofilms; Pseudomonas
PubMed: 38431335
DOI: 10.1016/j.fm.2024.104494