-
Journal of Dairy Science Apr 2024Biofilm formation is usually affected by many environmental factors, including divalent cations. The purpose of the current work was to analyze how calcium (Ca) affects...
Biofilm formation is usually affected by many environmental factors, including divalent cations. The purpose of the current work was to analyze how calcium (Ca) affects the biofilm formation of dairy Pseudomonas fluorescens isolates by investigating their growth, swarming motility, biofilm-forming capacity, extracellular polymeric substance production, and biofilm structures. Moreover, the regulation mechanism of Ca involved in its biofilm formation was explored through RNA-sequencing analysis. This work revealed that supplementation of 5, 10, 15, and 20 mM Ca significantly reduced the swarming motility of P. fluorescens strains (P.F, P.F, and P.F), but the biofilm-forming ability and polysaccharide production were increased after the supplementation of 5 and 10 mM Ca. By the supplementation of Ca, complex structures with more cell clusters glued together in P. fluorescens P.F biofilms were confirmed by scanning electron microscopy, and increased biomass and coverage of P. fluorescens P.F biofilms were observed by confocal laser scanning microscopy. In addition, RNA-sequencing results showed that P. fluorescens P.F showed a transcriptional response to the supplementation of 10 mM Ca, and a total of 137 genes were significantly expressed. The differential genes were represented in 4 upregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (nonribosomal peptide structures, quorum sensing, biosynthesis of siderophore group nonribosomal peptides, and phenylalanine metabolism), and 4 downregulated KEGG pathways (flagellar assembly, amino sugar and nucleotide sugar metabolism, nitrotoluene degradation, and cationic antimicrobial peptide resistance). The results indicate that Ca might serve as an enhancer to substantially trigger the biofilm formation of dairy P. fluorescens isolates in the dairy industry.
Topics: Animals; Calcium; Pseudomonas fluorescens; Extracellular Polymeric Substance Matrix; Biofilms; RNA
PubMed: 37949404
DOI: 10.3168/jds.2023-23860 -
BMC Microbiology Jul 2020Pseudomonas fluorescens 2P24 is a rhizosphere bacterium that produces 2,4-diacetyphloroglucinol (2,4-DAPG) as the decisive secondary metabolite to suppress soilborne...
BACKGROUND
Pseudomonas fluorescens 2P24 is a rhizosphere bacterium that produces 2,4-diacetyphloroglucinol (2,4-DAPG) as the decisive secondary metabolite to suppress soilborne plant diseases. The biosynthesis of 2,4-DAPG is strictly regulated by the RsmA family proteins RsmA and RsmE. However, mutation of both of rsmA and rsmE genes results in reduced bacterial growth.
RESULTS
In this study, we showed that overproduction of 2,4-DAPG in the rsmA rsmE double mutant influenced the growth of strain 2P24. This delay of growth could be partially reversal when the phlD gene was deleted or overexpression of the phlG gene encoding the 2,4-DAPG hydrolase in the rsmA rsmE double mutant. RNA-seq analysis of the rsmA rsmE double mutant revealed that a substantial portion of the P. fluorescens genome was regulated by RsmA family proteins. These genes are involved in the regulation of 2,4-DAPG production, cell motility, carbon metabolism, and type six secretion system.
CONCLUSIONS
These results suggest that RsmA and RsmE are the important regulators of genes involved in the plant-associated strain 2P24 ecologic fitness and operate a sophisticated mechanism for fine-tuning the concentration of 2,4-DAPG in the cells.
Topics: Bacterial Proteins; Carbon; Gene Deletion; Gene Expression Regulation, Bacterial; Methyltransferases; Mutation; Phloroglucinol; Pseudomonas fluorescens; RNA-Binding Proteins; Sequence Analysis, RNA
PubMed: 32615927
DOI: 10.1186/s12866-020-01880-x -
Frontiers in Microbiology 2021is a typical spoiler of proteinaceous foods, and it is characterized by high spoilage activity. The sigma factor RpoN is a well-known regulator controlling nitrogen...
is a typical spoiler of proteinaceous foods, and it is characterized by high spoilage activity. The sigma factor RpoN is a well-known regulator controlling nitrogen assimilation and virulence in many pathogens. However, its exact role in regulating the spoilage caused by . is unknown. Here, an in-frame deletion mutation of was constructed to investigate its global regulatory function through phenotypic and RNA-seq analysis. The results of phenotypic assays showed that the mutant was deficient in swimming motility, biofilm formation, and resistance to heat and nine antibiotics, while the mutant increased the resistance to HO. Moreover, the mutant markedly reduced extracellular protease and total volatile basic nitrogen (TVB-N) production in sterilized fish juice at 4°C; meanwhile, the juice with the mutant showed significantly higher sensory scores than that with the wild-type strain. To identify RpoN-controlled genes, RNA-seq-dependent transcriptomics analysis of the wild-type strain and the mutant was performed. A total of 1224 genes were significantly downregulated, and 474 genes were significantly upregulated by at least two folds at the RNA level in the mutant compared with the wild-type strain, revealing the involvement of RpoN in several cellular processes, mainly flagellar mobility, adhesion, polysaccharide metabolism, resistance, and amino acid transport and metabolism; this may contribute to the swimming motility, biofilm formation, stress and antibiotic resistance, and spoilage activities of . . Our results provide insights into the regulatory role of RpoN of . in food spoilage, which can be valuable to ensure food quality and safety.
PubMed: 34135871
DOI: 10.3389/fmicb.2021.641844 -
Food Microbiology Feb 2022In this study, P. fluorescens-infecting phages were isolated, characterized, and evaluated to their potential to control the bacterial counts and, consequently, the...
In this study, P. fluorescens-infecting phages were isolated, characterized, and evaluated to their potential to control the bacterial counts and, consequently, the proteolytic spoilage of raw milk during cold storage. The UFJF_PfDIW6 and UFJF_PfSW6 phages showed titers of 9.7 and 7.6 log PFU/ml; latent period of 115 and 25 min, and burst size of 145 and 25 PFU/infected cell, respectively. They also were highly specific to the host bacterium, morphologically classified as the Podoviridae family, stable at pH 5 to 11 and were not inactivated at 63 °C or 72 °C for 30 min. These phages found to be effective against P. fluorescens, reducing bacterial count throughout the entire exponential growth phase in broth formulated with milk at both 4 °C and 10 °C. This effect on bacteria growth led to inhibition by at least 2 days in proteases production, delaying the degradation of milk proteins. When applied together in raw milk stored at 4 °C, they reduced the total bacteria, psychrotrophic, and Pseudomonas by 3 log CFU/ml. This study's findings indicate that these phages have a great potential to prevent the growth of Pseudomonas and, consequently, to retard proteolytic spoilage of raw milk during chilled storage.
Topics: Animals; Bacteriophages; Cold Temperature; Food Contamination; Food Microbiology; Food Storage; Milk; Peptide Hydrolases; Pseudomonas fluorescens
PubMed: 34579852
DOI: 10.1016/j.fm.2021.103892 -
ELife Oct 2019Plant leaves constitute a huge microbial habitat of global importance. How microorganisms survive the dry daytime on leaves and avoid desiccation is not well understood....
Plant leaves constitute a huge microbial habitat of global importance. How microorganisms survive the dry daytime on leaves and avoid desiccation is not well understood. There is evidence that microscopic surface wetness in the form of thin films and micrometer-sized droplets, invisible to the naked eye, persists on leaves during daytime due to deliquescence - the absorption of water until dissolution - of hygroscopic aerosols. Here, we study how such microscopic wetness affects cell survival. We show that, on surfaces drying under moderate humidity, stable microdroplets form around bacterial aggregates due to capillary pinning and deliquescence. Notably, droplet-size increases with aggregate-size, and cell survival is higher the larger the droplet. This phenomenon was observed for 13 bacterial species, two of which - and - were studied in depth. Microdroplet formation around aggregates is likely key to bacterial survival in a variety of unsaturated microbial habitats, including leaf surfaces.
Topics: Microbial Viability; Plant Leaves; Water Microbiology
PubMed: 31610846
DOI: 10.7554/eLife.48508 -
International Journal of Molecular... Nov 2020Biofilms consist of a complex microbial community adhering to biotic or abiotic surfaces and enclosed within a protein/polysaccharide self-produced matrix. The formation...
Biofilms consist of a complex microbial community adhering to biotic or abiotic surfaces and enclosed within a protein/polysaccharide self-produced matrix. The formation of this structure represents the most important adaptive mechanism that leads to antibacterial resistance, and therefore, closely connected to pathogenicity. Antimicrobial peptides (AMPs) could represent attractive candidates for the design of new antibiotics because of their specific characteristics. AMPs show a broad activity spectrum, a relative selectivity towards their targets (microbial membranes), the ability to act on both proliferative and quiescent cells, a rapid mechanism of action, and above all, a low propensity for developing resistance. This article investigates the effect at subMIC concentrations of Temporin-L (TL) on biofilm formation in () both in static and dynamic conditions, showing that TL displays antibiofilm properties. Biofilm formation in static conditions was analyzed by the Crystal Violet assay. Investigation of biofilms in dynamic conditions was performed in a commercial microfluidic device consisting of a microflow chamber to simulate real flow conditions in the human body. Biofilm morphology was examined using Confocal Laser Scanning Microscopy and quantified via image analysis. The investigation of TL effects on showed that when subMIC concentrations of this peptide were added during bacterial growth, TL exerted antibiofilm activity, impairing biofilm formation both in static and dynamic conditions. Moreover, TL also affects mature biofilm as confocal microscopy analyses showed that a large portion of preformed biofilm architecture was clearly perturbed by the peptide addition with a significative decrease of all the biofilm surface properties and the overall biomass. Finally, in these conditions, TL did not affect bacterial cells as the live/dead cell ratio remained unchanged without any increase in damaged cells, confirming an actual antibiofilm activity of the peptide.
Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacterial Adhesion; Biofilms; Biomass; Drug Resistance, Bacterial; Microbial Sensitivity Tests; Microfluidics; Microscopy, Confocal; Polymers; Polysaccharides, Bacterial; Pseudomonas fluorescens; Shear Strength; Stress, Mechanical; Surface Properties
PubMed: 33198325
DOI: 10.3390/ijms21228526 -
Anais Da Academia Brasileira de Ciencias 2023Pseudomonas fluorescens is known to have the ability to adhere and produce biofilm. The formation of biofilms is enhanced by cellular motility, particularly when...
Pseudomonas fluorescens is known to have the ability to adhere and produce biofilm. The formation of biofilms is enhanced by cellular motility, particularly when mediated by flagella. Biofilm formed on surfaces such as those used for food production act as points of contamination, releasing pathogenic or deteriorating microorganisms and compromising the quality of products. We assessed two strains of Pseudomonas fluorescens PL5.4 and PL7.1, sampled from raw, chilled, buffalo milk, which was obtained from a dairy farm. Twitching and swarming motility assays were performed, in addition to the biofilm production evaluations at a temperature of 7 °C. Regarding the motility assays, only the PL5.4 strain scored positive for the swarming assay. On microplates, both strains presented themselves as strong biofilm producers at 7 °C. The PL5.4 strain was also able to form biofilm on a stainless steel structure and maintain this structure for up to 72 hours at refrigeration. The Pseudomonas fluorescens PL5.4 isolate was identified on the basis of a 99% sequence identity with Pseudomonas fluorescens A506, a strain used as a biocontrol in agriculture. Biofilm-forming bacteria, when adapted to low temperatures, become a constant source of contamination, damaging the production, quality, safety and shelf-life of products.
Topics: Animals; Pseudomonas fluorescens; Milk; Biofilms; Temperature
PubMed: 37466543
DOI: 10.1590/0001-3765202320220982 -
Journal of Bacteriology Sep 2023Biofilm formation by the Gram-negative, Gammaproteobacteria relies on the repeats-in-toxin adhesins LapA and MapA in the cytoplasm, secretion of these adhesins through...
Biofilm formation by the Gram-negative, Gammaproteobacteria relies on the repeats-in-toxin adhesins LapA and MapA in the cytoplasm, secretion of these adhesins through their respective type 1 secretion systems, and retention at the cell surface. Published work has shown that retention of the adhesins occurs via a post-translational mechanism involving the cyclic-di-GMP receptor LapD and the protease LapG. However, little is known about the underlying mechanisms that regulate the level of these adhesins. Here, we demonstrate that the master regulator FleQ modulates biofilm formation by both transcriptionally and post-transcriptionally regulating LapA and MapA. We find that a Δ mutant has a biofilm formation defect compared to the wild-type (WT) strain, which is attributed in part to a decrease in LapA and MapA abundance in the cell, despite the Δ mutant having increased levels of and transcripts compared to the WT strain. Through transposon mutagenesis and subsequent genetic analysis, we found that overstimulation of the Gac/Rsm pathway partially rescues biofilm formation in the Δ mutant background. Collectively, these findings provide evidence that FleQ regulates biofilm formation by both transcriptionally regulating the expression of the genes and post-transcriptionally regulating the abundance of LapA and MapA, and that activation of the Gac/Rsm pathway can post-transcriptionally enhance biofilm formation by . IMPORTANCE Biofilm formation is a highly coordinated process that bacteria undergo to colonize a variety of surfaces. For , biofilm formation requires the production and localization of repeats-in-toxin adhesins to the cell surface. To date, little is known about the underlying mechanisms that regulate biofilm formation by . Here, we identify FleQ as a key regulator of biofilm formation that modulates both gene expression and abundance of LapA and MapA through both a transcriptional and post-transcriptional mechanism. We provide further evidence implicating activation of the Gac/Rsm system in FleQ-dependent regulation of biofilm formation. Together, our findings uncover evidence for a dual mechanism of transcriptional and post-transcriptional regulation of the LapA and MapA adhesins.
Topics: Pseudomonas fluorescens; Biofilms; Adhesins, Bacterial; Carrier Proteins; Bacterial Proteins; Gene Expression Regulation, Bacterial; Cyclic GMP
PubMed: 37655913
DOI: 10.1128/jb.00152-23 -
Food Research International (Ottawa,... Oct 2020The development of effective disinfection strategies, including the search for new active biocides, is a priority research for the food industry. Glycolic acid (GA) and...
The development of effective disinfection strategies, including the search for new active biocides, is a priority research for the food industry. Glycolic acid (GA) and glyoxal (GO) are two glycolysis by-products approved as biocides for surface disinfection, whose antimicrobial action remains to be understood. This study focused on the understanding of the antimicrobial activity of GA and GO against two foodborne pathogens, Bacillus cereus and Pseudomonas fluorescens. Benzalkonium chloride (BAC) and peracetic acid (PAA) were used as reference biocides for comparison. The influence of biocide concentration and exposure time on the antimicrobial activity of the selected biocides was evaluated based on the European Standard EN 1276. The mode of action of each biocide was characterized based on their effects on the cell envelope and cell replication. According to the Chick-Watson model, all biocides interacted chemically with cell targets of both bacteria, except GO that only stablished physicochemical interactions with P. fluorescens. The survival curves revealed that high concentrations of biocide induced readily effects on cell culturability. The susceptibility profile of P. fluorescens was constant over time. In general, B. cereus increased tolerance to BAC and became more susceptible to cumulative damages of GA. Overall, this study demonstrates that the biocidal activity was species-, dose- and time-dependent. GA, similarly to BAC, was a membrane-active and oxidant agent. GO had no effect on the bacterial surface as well as PAA. GO was mainly categorized as a cell replication inhibitor. For the first time, the antimicrobial activity of GA and GO were characterized revealing their potential for rational combination with other biocides commonly used in the food industry.
Topics: Bacillus cereus; Disinfectants; Glycolates; Glyoxal; Pseudomonas fluorescens
PubMed: 32846538
DOI: 10.1016/j.foodres.2020.109346 -
Microbiology (Reading, England) Oct 2021
Topics: Actinobacteria; Bacillus subtilis; Bacteriophages; Biofilms; Humans; Microbiology; Microbiota; Mycobacterium; Plasmids; Pseudomonas fluorescens
PubMed: 34672917
DOI: 10.1099/mic.0.001115