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Journal of Food Science Oct 2017Under the cold storage and processing conditions of raw milk, the psychrotrophic Pseudomonas fluorescens is usually found as predominant bacteria causing its spoilage....
UNLABELLED
Under the cold storage and processing conditions of raw milk, the psychrotrophic Pseudomonas fluorescens is usually found as predominant bacteria causing its spoilage. In this study, a multiplex PCR assay was developed for rapid and selective detection of P. fluorescens with biofilm formation ability. The target sequences were 2 genes (adnA and fliC) related to biofilm formation and flagella biosynthesis of P. fluorescens. The specificity of the mPCR assay was evaluated with 7 reference strains, isolated from raw milk, belonging to P. fluorescens, Pseudomonas fragi, Pseudomonas lundensis, Pseudomonas putida, Pseudomonas monteilii, and 2 unclassified Pseudomonas species (Pseudomonas sp1 and Pseudomonas sp8). The detection limit for the target strain was 10 CFU/mL. Seventy-three strains were evaluated by the mPCR assay. The adnA gene was detected in 23 strains while fliC gene was detected in only 3 strains. However, both target genes (adnA and fliC) were detected by amplification in 12 strains belonging to P. fluorescens species. The biofilm formation ability of P. fluorescens following cultivation in 10% UHT milk at 30 °C or 4 °C were evaluated by the microtiter plate assay. The result showed that all the P. fluorescens strains with the target gene (adnA or fliC, or both 2 genes) had the biofilm-forming ability. The phylogenetic analysis showed that adnA gene tree had a higher resolution than rpoB tree, and the strains in adnA phylogenetic dendrogram could be divided into 4 different groups according with the matrix of their biofilm-forming ability. The results indicated a promising use of adnA gene as a taxonomic marker for subdividing P. fluorescens.
PRACTICAL APPLICATION
A mPCR assay targeting adnA and fliC genes showed rapid and reliable detection of P. fluorescens with biofilm formation ability, which could be useful to detect this contamination in milk samples.
Topics: Animals; Biofilms; Cattle; Food Contamination; Milk; Multiplex Polymerase Chain Reaction; Phylogeny; Pseudomonas fluorescens
PubMed: 28950041
DOI: 10.1111/1750-3841.13845 -
International Journal of Molecular... May 2023The search for and characterization of new lipases with excellent properties has always been urgent and is of great importance to meet industrial needs. In this study, a...
The search for and characterization of new lipases with excellent properties has always been urgent and is of great importance to meet industrial needs. In this study, a new lipase, from SBW25, belonging to the lipase subfamily I.3, was cloned and expressed in WB800N. Enzymatic properties studies of recombinant LipB found that it exhibited the highest activity towards -nitrophenyl caprylate at 40 °C and pH 8.0, retaining 73% of its original activity after incubation at 70 °C for 6 h. In addition, Ca, Mg, and Ba strongly enhanced the activity of LipB, while Cu, Zn, Mn, and CTAB showed an inhibiting effect. The LipB also displayed noticeable tolerance to organic solvents, especially acetonitrile, isopropanol, acetone, and DMSO. Moreover, LipB was applied to the enrichment of polyunsaturated fatty acids from fish oil. After hydrolyzing for 24 h, it could increase the contents of polyunsaturated fatty acids from 43.16% to 72.18%, consisting of 5.75% eicosapentaenoic acid, 19.57% docosapentaenoic acid, and 46.86% docosahexaenoic acid, respectively. The properties of LipB render it great potential in industrial applications, especially in health food production.
Topics: Lipase; Pseudomonas fluorescens; Fatty Acids, Unsaturated; Docosahexaenoic Acids; Eicosapentaenoic Acid; Enzyme Stability
PubMed: 37240270
DOI: 10.3390/ijms24108924 -
Biotechnology and Applied Biochemistry Jan 2020With the versatile metabolic diversity, Pseudomonas fluorescens is a potential candidate in petroleum aromatic hydrocarbon (PAH) bioremediation. Genome-scale metabolic...
With the versatile metabolic diversity, Pseudomonas fluorescens is a potential candidate in petroleum aromatic hydrocarbon (PAH) bioremediation. Genome-scale metabolic model (GSMM) can provide systematic information to guide the development of metabolic engineering strategy to improve microbial activity. In this study, a GSMM for P. fluorescens SBW25 was reconstructed, which is termed as lCW1057. The reconstruction was based on automatic reannotation and manual curation. The periplasmic compartment was constructed to better represent the proton gradient profile. The reconstructed proton transport chain has a P/O (ATP generated per atom oxygen consumed by the respiratory chain) ratio of 11/8. Flux balance analysis (FBA) was performed to explore the whole-cell metabolic flow. The model suggested that instead of Embden-Meyerhof-Parnas pathway, Entner-Doudoroff pathway was used in glycolytic metabolism of P. fluorescens, indicating that the growth of P. fluorescens is more energy dependent. Furthermore, P. fluorescens can use nitrate as the terminal electron acceptor for the glucose metabolism. The β-ketoadipate pathway was involved in catechol metabolism. The uptake of oxygen is mandatory for the aromatic ring cleavage. The in silico and in vitro maximum specific growth rate was compared, resulting in 10% difference when catechol was used as the sole carbon source.
Topics: Carbohydrate Metabolism; Carbon; Metabolic Engineering; Models, Biological; Pseudomonas fluorescens
PubMed: 31721286
DOI: 10.1002/bab.1852 -
Molecular Microbiology Sep 2001In vivo expression technology (IVET) analysis of rhizosphere-induced genes in the plant growth-promoting rhizobacterium (PGPR) Pseudomonas fluorescens SBW25 identified a...
In vivo expression technology (IVET) analysis of rhizosphere-induced genes in the plant growth-promoting rhizobacterium (PGPR) Pseudomonas fluorescens SBW25 identified a homologue of the type III secretion system (TTSS) gene hrcC. The hrcC homologue resides within a 20-kb gene cluster that resembles the type III (Hrp) gene cluster of Pseudomonas syringae. The type III (Rsp) gene cluster in P. fluorescens SBW25 is flanked by a homologue of the P. syringae TTSS-secreted protein AvrE. P. fluorescens SBW25 is non-pathogenic and does not elicit the hypersensitive response (HR) in any host plant tested. However, strains constitutively expressing the rsp-specific sigma factor RspL elicit an AvrB-dependent HR in Arabidopsis thaliana ecotype Col-0, and a host-specific HR in Nicotiana clevelandii. The inability of wild-type P. fluorescens SBW25 to elicit a visible HR is therefore partly attributable to low expression of rsp genes in the leaf apoplast. DNA hybridization analysis indicates that rsp genes are present in many plant-colonizing Pseudomonas and PGPR, suggesting that TTSSs may have a significant role in the biology of PGPR. However, rsp and rsc mutants retain the ability to reach high population levels in the rhizosphere. While functionality of the TTSS has been demonstrated, the ecological significance of the rhizosphere-expressed TTSS of P. fluorescens SBW25 remains unclear.
Topics: Bacterial Proteins; DNA Transposable Elements; DNA, Bacterial; DNA, Ribosomal; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Mutagenesis, Insertional; Plant Development; Plant Leaves; Plant Roots; Plants; Plasmids; Pseudomonas fluorescens; RNA, Ribosomal, 16S; Sequence Analysis, DNA
PubMed: 11555282
DOI: 10.1046/j.1365-2958.2001.02560.x -
Microbiology (Reading, England) Jul 2019In natural habitats, bacterial species often coexist in biofilms. They interact in synergetic or antagonistic ways and their interactions can influence the biofilm...
In natural habitats, bacterial species often coexist in biofilms. They interact in synergetic or antagonistic ways and their interactions can influence the biofilm development and properties. Still, very little is known about how the coexistence of multiple organisms impact the multispecies biofilm properties. In this study, we examined the behaviour of a dual-species biofilm at the air-liquid interface composed by two environmental bacteria: Bacillus licheniformis and a phenazine mutant of Pseudomonas fluorescens. Study of the planktonic and biofilm growths for each species revealed that P. fluorescens grew faster than B. licheniformis and no bactericidal effect from P. fluorescens was detected, suggesting that the growth kinetics could be the main factor in the dual-species biofilm composition. To validate this hypothesis, the single- and dual-species biofilm were characterized by biomass quantification, microscopy and rheology. Bacterial counts and microscale architecture analysis showed that both bacterial populations coexist in the mature pellicle, with a dominance of P. fluorescens. Real-time measurement of the dual-species biofilms' viscoelastic (i.e. mechanical) properties using interfacial rheology confirmed that P. fluorescens was the main contributor of the biofilm properties. Evaluation of the dual-species pellicle viscoelasticity at longer time revealed that the biofilm, after reaching a first equilibrium, created a stronger and more cohesive network. Interfacial rheology proves to be a unique quantitative technique, which combined with microscale imaging, contributes to the understanding of the time-dependent properties within a polymicrobial community at various stages of biofilm development. This work demonstrates the importance of growth kinetics in the bacteria competition for the interface in a model dual-species biofilm.
Topics: Bacillus licheniformis; Biofilms; Kinetics; Pseudomonas fluorescens; Staining and Labeling
PubMed: 31145677
DOI: 10.1099/mic.0.000819 -
Biotechnology Journal May 2011
Topics: Crops, Agricultural; Plant Development; Plants; Pseudomonas fluorescens; Rhizosphere
PubMed: 21538893
DOI: 10.1002/biot.201000478 -
Environmental Microbiology Jan 2008Growth in a biofilm modulates microbial metal susceptibility, sometimes increasing the ability of microorganisms to withstand toxic metal species by several orders of...
Growth in a biofilm modulates microbial metal susceptibility, sometimes increasing the ability of microorganisms to withstand toxic metal species by several orders of magnitude. In this study, a high-throughput metal toxicity screen was initiated with the aim of correlating biological toxicity data in planktonic and biofilm cells to the physiochemical properties of metal ions. To this end, Pseudomonas fluorescens ATCC 13525 was grown in the Calgary Biofilm Device (CBD) and biofilms and planktonic cells of this microorganism were exposed to gradient arrays of different metal ions. These arrays included 44 different metals with representative compounds that spanned every group of the periodic table (except for the halogens and noble gases). The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum biofilm eradication concentration (MBEC) values were obtained after exposing the biofilms to metal ions for 4 h. Using these values, metal ion toxicity was correlated to the following ion-specific physicochemical parameters: standard reduction-oxidation potential, electronegativity, the solubility product of the corresponding metal-sulfide complex, the Pearson softness index, electron density and the covalent index. When the ions were grouped according to outer shell electron structure, we found that heavy metal ions gave the strongest correlations to these parameters and were more toxic on average than the other classes of the ions. Correlations were different for biofilms than for planktonic cells, indicating that chemical mechanisms of metal ion toxicity differ between the two modes of growth. We suggest that biofilms can specifically counter the toxic effects of certain physicochemical parameters, which may contribute to the increased ability of biofilms to withstand metal toxicity.
Topics: Biofilms; Metals; Metals, Heavy; Microbial Sensitivity Tests; Pseudomonas fluorescens
PubMed: 17894814
DOI: 10.1111/j.1462-2920.2007.01448.x -
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 -
Scientific Reports Nov 2019Bacterial biofilm formation involves signaling and regulatory pathways that control the transition from motile to sessile lifestyle, production of extracellular...
Bacterial biofilm formation involves signaling and regulatory pathways that control the transition from motile to sessile lifestyle, production of extracellular polymeric matrix, and maturation of the biofilm 3D structure. Biofilms are extensively studied because of their importance in biomedical, ecological and industrial settings. Gene inactivation is a powerful approach for functional studies but it is often labor intensive, limiting systematic gene surveys to the most tractable bacterial hosts. Here, we adapted the CRISPR interference (CRISPRi) system for use in diverse strain isolates of P. fluorescens, SBW25, WH6 and Pf0-1. We found that CRISPRi is applicable to study complex phenotypes such as cell morphology, motility and biofilm formation over extended periods of time. In SBW25, CRISPRi-mediated silencing of genes encoding the GacA/S two-component system and regulatory proteins associated with the cylic di-GMP signaling messenger produced swarming and biofilm phenotypes similar to those obtained after gene inactivation. Combined with detailed confocal microscopy of biofilms, our study also revealed novel phenotypes associated with extracellular matrix biosynthesis as well as the potent inhibition of SBW25 biofilm formation mediated by the PFLU1114 operon. We conclude that CRISPRi is a reliable and scalable approach to investigate gene networks in the diverse P. fluorescens group.
Topics: Biofilms; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Cytokinesis; Gene Editing; Gene Expression Regulation, Bacterial; Gene Silencing; Pseudomonas fluorescens
PubMed: 31685917
DOI: 10.1038/s41598-019-52400-5 -
ACS Nano Feb 2014Understanding the molecular mechanisms of bacterial adhesion and biofilm formation is an important topic in current microbiology and a key in nanomedicine for developing...
Understanding the molecular mechanisms of bacterial adhesion and biofilm formation is an important topic in current microbiology and a key in nanomedicine for developing new antibacterial strategies. There is growing evidence that the production of extracellular polymeric substances at the cell-substrate interface plays a key role in strengthening bacterial adhesion. Yet, because these adhesive polymers are available in small amounts and are localized at interfaces, they are difficult to study using traditional techniques. Here, we use single-molecule atomic force microscopy (AFM) to functionally analyze the biophysical properties (distribution, adhesion, and extension) of bacterial footprints, that is, adhesive macromolecules left on substrate surfaces after removal of the attached cells. We focus on the large adhesin protein LapA from Pseudomonas fluorescens, which mediates cell attachment to a wide diversity of surfaces. Using AFM tips functionalized with specific antibodies, we demonstrate that adhesion of bacteria to hydrophobic substrates leads to the active accumulation of the LapA protein at the cell-substrate interface. We show that single LapA proteins left on the substrate after cell detachment localize into microscale domains corresponding to the bacterial size and exhibit multiple adhesion peaks reflecting the adhesion and extension of adsorbed LapA proteins. The mechanical behavior of LapA-based footprints makes them ideally suited to function as multipurpose bridging polymers, enabling P. fluorescens to attach to various surfaces. Our experiments show that single-molecule AFM offers promising prospects for characterizing the biophysics and dynamics of the cell-substrate interface in the context of bacterial adhesion, on a scale that was not accessible before.
Topics: Bacterial Adhesion; Biofilms; Microscopy, Atomic Force; Pseudomonas fluorescens
PubMed: 24456070
DOI: 10.1021/nn4060489