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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 -
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 -
Italian Journal of Food Safety Jun 2022Bacteria belonging to the genus are ubiquitous and characterized by a high adaptation capability to different environmental conditions and wide range of temperatures....
Bacteria belonging to the genus are ubiquitous and characterized by a high adaptation capability to different environmental conditions and wide range of temperatures. They may colonize food, sometimes causing alteration. Quite recently, a blue pigmentation due to has been widely reported in mozzarella cheese. In this report, we describe a blue coloration occurred on rabbit meat stored in the refrigeration cell of a slaughterhouse. The alteration was observed after about 72 hours of storage at 4-6°C. Bacteriological analyses were performed, and a microorganism included in the group was identified. The experimental contamination was planned, using a bacterial suspension with 1×10 UFC/ml load to spread on rabbit carcasses. The blue pigmentation appeared after 24 hours of storage in a cell with the same conditions of temperature. The bacterium was reisolated and identified as responsible for the alteration on meat. These findings highlight the importance of considering the members of the genus and, more specifically, of the group when the microbiological quality of food is to be ascertained. In fact, even if these bacteria are not considered a public health problem, their presence should be monitored by food industry operators in self-control plans because they may cause alteration in food. In fact, any altered product should be withdrawn from the market in agreement with Regulation (EC) No 178/2002 of the European Parliament and of the Council.
PubMed: 35795461
DOI: 10.4081/ijfs.2022.9998 -
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 -
Journal of Dairy Science Sep 2020The objective of this study was to investigate the effect of adding different levels of a thermoresistant protease produced by a Pseudomonas fluorescens strain to milk...
The objective of this study was to investigate the effect of adding different levels of a thermoresistant protease produced by a Pseudomonas fluorescens strain to milk on the manufacture and quality of Cheddar cheese. Fresh raw milk was collected, standardized, and pasteurized at 72°C for 15 s, and the enzyme was added to give a protease activity of 0.15 or 0.60 U/L (treatments P1 and P4, respectively), while one sample had no enzyme added (control). Milk was stored at 4°C for 48 h and Cheddar cheese was manufactured after 0 and 48 h of storage. Results indicated that the protease was active in milk during 48 h of storage; however, its effect on milk composition was minimal. The protein that was preferentially hydrolyzed by the protease over storage was β-casein, followed by κ-casein. The mean cheese yield and recovery of fat and protein obtained for all cheeses were not affected by protease activity. The protease showed low activity during cheese manufacture, possibly because of unfavorable conditions, including low pH. One of the factors that might have influenced protease activity was the pH of the curd (approximately 6.55 after acidification and 5.35 at milling), which was lower than that at which the enzyme would have optimum activity (pH 7 to 9). Consequently, the composition, pH, patterns of proteolysis, and hardness of all cheeses produced were similar and in accordance with values expected for that type of cheese, independently of the protease activity level. However, slight increases in proteolysis were observed in P4 cheeses and produced using milk stored for 48 h. Both the P1 and P4 cheeses had higher concentrations of free amino acids (FAA) compared with the control, whereas urea-PAGE electrophoretograms indicated a greater breakdown of caseins in the P4 cheese samples, which may be related to possible increases in numbers of proteolytic bacteria in milk during storage. Therefore, the thermoresistant psychrotrophic bacterial protease(s) tested in this study may affect the manufacture or quality of Cheddar cheese during ripening to a relatively limited extent. However, controlling initial levels of proteolytic bacteria in raw milk remains essential, because proteolysis affects the development of flavor and texture in cheese.
Topics: Animals; Caseins; Cheese; Food Quality; Hydrogen-Ion Concentration; Milk; Pasteurization; Peptide Hydrolases; Proteolysis; Pseudomonas fluorescens; Taste
PubMed: 32600766
DOI: 10.3168/jds.2019-18043 -
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 -
Microbial Genomics Jul 2021is a highly diverse genus that includes species that cause disease in both plants and animals. Recently, pathogenic pseudomonads from the and species complexes have...
is a highly diverse genus that includes species that cause disease in both plants and animals. Recently, pathogenic pseudomonads from the and species complexes have caused significant outbreaks in several agronomically important crops in Turkey, including tomato, citrus, artichoke and melon. We characterized 169 pathogenic strains associated with recent outbreaks in Turkey via multilocus sequence analysis and whole-genome sequencing, then used comparative and evolutionary genomics to characterize putative virulence mechanisms. Most of the isolates are closely related to other plant pathogens distributed among the primary phylogroups of , although there are significant numbers of isolates, which is a species better known as a rhizosphere-inhabiting plant-growth promoter. We found that all 39 citrus blast pathogens cluster in phylogroup 2, although strains isolated from the same host do not cluster monophyletically, with lemon, mandarin orange and sweet orange isolates all being intermixed throughout the phylogroup. In contrast, 20 tomato pith pathogens are found in two independent lineages: one in the secondary phylogroups, and the other from the species complex. These divergent pith necrosis strains lack characteristic virulence factors like the canonical tripartite type III secretion system, large effector repertoires and the ability to synthesize multiple bacterial phytotoxins, suggesting they have alternative molecular mechanisms to cause disease. These findings highlight the complex nature of host specificity among plant pathogenic pseudomonads.
Topics: Crops, Agricultural; Genome, Bacterial; Multilocus Sequence Typing; Plant Diseases; Plants; Pseudomonas fluorescens; Pseudomonas syringae; Turkey; Type III Secretion Systems; Virulence Factors; Whole Genome Sequencing
PubMed: 34227931
DOI: 10.1099/mgen.0.000585 -
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 -
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 -
Food Research International (Ottawa,... Feb 2023To better understand the microbial quality and safety of plant-based meat analogues, this study investigated the changes of native microflora present in soy- and...
To better understand the microbial quality and safety of plant-based meat analogues, this study investigated the changes of native microflora present in soy- and pea-based meat analogues (SBM and PBM) and compared them with ground beef (GB). SBM, PBM, and GB were also artificially inoculated with meat spoilage microorganisms, Pseudomonas fluorescens and Brochothrix thermosphacta, and pathogenic microorganisms, Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes; the fitness of these bacteria was evaluated during storage at refrigerated and/or abused temperatures. Results showed that the initial total aerobic plate count (APC), coliform, lactic acid bacteria (LAB), and mold/yeast (M/Y) counts for GB could be as high as 5.44, 2.90, 4.61, and 3.45 log CFU/g, while the highest initial APC, coliform, LAB, and M/Y counts found in SBM were 3.10, 2.00, 2.04, and 1.95 log CFU/g, and were 3.82, 2.51, 3.61, and 1.44 log CFU/g for PBM. The batch-to-batch differences in microbial counts were more significant in GB than in SBM and PBM. Despite the different initial concentrations, there was no difference among APC and LAB counts between the three meat types by the end of the 10-day 4 °C storage period, all approaching ca. 7.00 log CFU/g. Artificially-inoculated B. thermosphacta increased by 0.76, 1.58, and 0.96 log CFU/g in GB, PBM, and SBM respectively by the end of the refrigeration storage; P. fluorescens increased by 4.92, 3.00, and 0.40 log CFU/g in GB, PBM, and SBM respectively. Under refrigerated storage conditions, pathogenic bacteria did not change in GB and SBM. L. monocytogenes increased by 0.74 log in PBM during the 7-day storage at 4 °C. All three pathogens grew at abused storage temperatures, regardless of the meat type. Results indicated that plant-based meat could support the survival and even growth of spoilage and pathogenic microorganisms. Preventive controls are needed for ensuring the microbial quality and safety of plant-based meat analogues.
Topics: Animals; Cattle; Meat Products; Food Microbiology; Meat; Salmonella; Pseudomonas fluorescens
PubMed: 36737989
DOI: 10.1016/j.foodres.2022.112408