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Scientific Reports Mar 2021The genome of Pseudomonas fluorescens F113, a model rhizobacterium and a plant growth-promoting agent, encodes three putative type VI secretion systems (T6SSs); F1-, F2-...
The genome of Pseudomonas fluorescens F113, a model rhizobacterium and a plant growth-promoting agent, encodes three putative type VI secretion systems (T6SSs); F1-, F2- and F3-T6SS. Bioinformatic analysis of the F113 T6SSs has revealed that they belong to group 3, group 1.1, and group 4a, respectively, similar to those previously described in Pseudomonas aeruginosa. In addition, in silico analyses allowed us to identify genes encoding a total of five orphan VgrG proteins and eight putative effectors (Tfe), some with their cognate immunity protein (Tfi) pairs. Genes encoding Tfe and Tfi are found in the proximity of P. fluorescens F113 vgrG, hcp, eagR and tap genes. RNA-Seq analyses in liquid culture and rhizosphere have revealed that F1- and F3-T6SS are expressed under all conditions, indicating that they are active systems, while F2-T6SS did not show any relevant expression under the tested conditions. The analysis of structural mutants in the three T6SSs has shown that the active F1- and F3-T6SSs are involved in interbacterial killing while F2 is not active in these conditions and its role is still unknown.. A rhizosphere colonization analysis of the double mutant affected in the F1- and F3-T6SS clusters showed that the double mutant was severely impaired in persistence in the rhizosphere microbiome, revealing the importance of these two systems for rhizosphere adaption.
Topics: Adaptation, Physiological; Gene Expression Regulation, Bacterial; Microbial Viability; Microbiota; Multigene Family; Phylogeny; Protein Domains; Pseudomonas fluorescens; Rhizosphere; Type VI Secretion Systems
PubMed: 33707614
DOI: 10.1038/s41598-021-85218-1 -
PloS One 2016The Pseudomonas fluorescens complex includes Pseudomonas strains that have been taxonomically assigned to more than fifty different species, many of which have been...
The Pseudomonas fluorescens complex includes Pseudomonas strains that have been taxonomically assigned to more than fifty different species, many of which have been described as plant growth-promoting rhizobacteria (PGPR) with potential applications in biocontrol and biofertilization. So far the phylogeny of this complex has been analyzed according to phenotypic traits, 16S rDNA, MLSA and inferred by whole-genome analysis. However, since most of the type strains have not been fully sequenced and new species are frequently described, correlation between taxonomy and phylogenomic analysis is missing. In recent years, the genomes of a large number of strains have been sequenced, showing important genomic heterogeneity and providing information suitable for genomic studies that are important to understand the genomic and genetic diversity shown by strains of this complex. Based on MLSA and several whole-genome sequence-based analyses of 93 sequenced strains, we have divided the P. fluorescens complex into eight phylogenomic groups that agree with previous works based on type strains. Digital DDH (dDDH) identified 69 species and 75 subspecies within the 93 genomes. The eight groups corresponded to clustering with a threshold of 31.8% dDDH, in full agreement with our MLSA. The Average Nucleotide Identity (ANI) approach showed inconsistencies regarding the assignment to species and to the eight groups. The small core genome of 1,334 CDSs and the large pan-genome of 30,848 CDSs, show the large diversity and genetic heterogeneity of the P. fluorescens complex. However, a low number of strains were enough to explain most of the CDSs diversity at core and strain-specific genomic fractions. Finally, the identification and analysis of group-specific genome and the screening for distinctive characters revealed a phylogenomic distribution of traits among the groups that provided insights into biocontrol and bioremediation applications as well as their role as PGPR.
Topics: Base Sequence; DNA, Bacterial; Denitrification; Genetic Variation; Genome, Bacterial; Genomics; Phylogeny; Pseudomonas fluorescens; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Siderophores; Soil Microbiology
PubMed: 26915094
DOI: 10.1371/journal.pone.0150183 -
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 -
Journal of Evolutionary Biology Feb 2017When competing for space and resources, bacteria produce toxins known as bacteriocins to gain an advantage over competitors. Recent studies in the laboratory have...
When competing for space and resources, bacteria produce toxins known as bacteriocins to gain an advantage over competitors. Recent studies in the laboratory have confirmed theoretical predictions that bacteriocin production can determine coexistence, by eradicating sensitive competitors or driving the emergence of resistant genotypes. However, there is currently limited evidence that bacteriocin-mediated competition influences the coexistence and distribution of genotypes in natural environments, and what factors drive interactions towards inhibition remain unclear. Using natural soil populations of Pseudomonas fluorescens, we assessed the ability of the isolates to inhibit one another with respect to spatial proximity in the field, genetic similarity and niche overlap. The majority of isolates were found to produce bacteriocins; however, widespread resistance between coexisting isolates meant relatively few interactions resulted in inhibition. When inhibition did occur, it occurred more frequently between ecologically similar isolates. However, in contrast with results from other natural populations, we found no relationship between the frequency of inhibition and the genetic similarity of competitors. Our results suggest that bacteriocin production plays an important role in mediating competition over resources in natural settings and, by selecting for isolates resistant to local bacteriocin production, can influence the assembly of natural populations of P. fluorescens.
Topics: Bacteriocins; Genetic Variation; Population Dynamics; Pseudomonas fluorescens; Soil Microbiology
PubMed: 28000957
DOI: 10.1111/jeb.13010 -
International Journal of Molecular... Dec 2022SBW25 is a model soil- and plant-associated bacterium capable of forming a variety of air-liquid interface biofilms in experimental microcosms and on plant surfaces....
SBW25 is a model soil- and plant-associated bacterium capable of forming a variety of air-liquid interface biofilms in experimental microcosms and on plant surfaces. Previous investigations have shown that cellulose is the primary structural matrix component in the robust and well-attached Wrinkly Spreader biofilm, as well as in the fragile Viscous Mass biofilm. Here, we demonstrate that both biofilms include extracellular DNA (eDNA) which can be visualized using confocal laser scanning microscopy (CLSM), quantified by absorbance measurements, and degraded by DNase I treatment. This eDNA plays an important role in cell attachment and biofilm development. However, exogenous high-molecular-weight DNA appears to decrease the strength and attachment levels of mature Wrinkly Spreader biofilms, whereas low-molecular-weight DNA appears to have little effect. Further investigation with CLSM using an amyloid-specific fluorophore suggests that the Wrinkly Spreader biofilm might also include Fap fibers, which might be involved in attachment and contribute to biofilm strength. The robust nature of the Wrinkly Spreader biofilm also allowed us, using MALDI-TOF mass spectrometry, to identify matrix-associated proteins unable to diffuse out of the structure, as well as membrane vesicles which had a different protein profile compared to the matrix-associated proteins. CLSM and DNase I treatment suggest that some vesicles were also associated with eDNA. These findings add to our understanding of the matrix components in this model pseudomonad, and, as found in other biofilms, biofilm-specific products and material from lysed cells contribute to these structures through a range of complex interactions.
Topics: Biofilms; Deoxyribonuclease I; DNA; DNA, Bacterial; Pseudomonas fluorescens; Amyloid
PubMed: 36499433
DOI: 10.3390/ijms232315096 -
Journal of Bacteriology Sep 2018Bacteria have evolved several secretion strategies for polling and responding to environmental flux and insult. Of these, the type 1 secretion system (T1SS) is known to... (Review)
Review
Bacteria have evolved several secretion strategies for polling and responding to environmental flux and insult. Of these, the type 1 secretion system (T1SS) is known to secrete an array of biologically diverse proteins-from small, <10-kDa bacteriocins to gigantic adhesins with a mass >1 MDa. For the last several decades, T1SSs have been characterized as a one-step translocation strategy whereby the secreted substrate is transported directly into the extracellular environment from the cytoplasm with no periplasmic intermediate. Recent phylogenetic, biochemical, and genetic evidences point to a distinct subgroup of T1SS machinery linked with a bacterial transglutaminase-like cysteine proteinase (BTLCP), which uses a two-step secretion mechanism. BTLCP-linked T1SSs transport a class of repeats-in-toxin (RTX) adhesins that are critical for biofilm formation. The prototype of this RTX adhesin group, LapA of Pf0-1, uses a novel N-terminal retention module to anchor the adhesin at the cell surface as a secretion intermediate threaded through the outer membrane-localized TolC-like protein LapE. This secretion intermediate is posttranslationally cleaved by the BTLCP family LapG protein to release LapA from its cognate T1SS pore. Thus, the secretion of LapA and related RTX adhesins into the extracellular environment appears to be a T1SS-mediated two-step process that involves a periplasmic intermediate. In this review, we contrast the T1SS machinery and substrates of the BLTCP-linked two-step secretion process with those of the classical one-step T1SS to better understand the newly recognized and expanded role of this secretion machinery.
Topics: Adhesins, Bacterial; Bacterial Proteins; Biofilms; Cell Membrane; Computational Biology; Cysteine Proteases; Periplasm; Phylogeny; Pseudomonas fluorescens; Transglutaminases; Type I Secretion Systems
PubMed: 29866808
DOI: 10.1128/JB.00168-18 -
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 -
Journal of Food Protection Mar 2022Thermostable alkaline protease (TAP) harbored by Pseudomonas fluorescens decomposes protein in milk and dairy products, leading to milk and dairy product spoilage during...
ABSTRACT
Thermostable alkaline protease (TAP) harbored by Pseudomonas fluorescens decomposes protein in milk and dairy products, leading to milk and dairy product spoilage during storage. Thus, a specific, sensitive, rapid, and simple method is required to detect TAP-harboring P. fluorescens. Two sets of primers targeting the aprX and gyrB genes of P. fluorescens were designed. The detection system and conditions were optimized, and a real-time loop-mediated isothermal amplification (real-time LAMP) method was developed for the simultaneous detection of TAP-harboring P. fluorescens in two separate reaction tubes. The phylogenetic tree targeting aprX showed that P. fluorescens and Pseudomonas lurida clustered on the same branch. The phylogenetic tree targeting gyrB showed that P. fluorescens clustered on the same branch with 95% confidence value, whereas P. lurida clustered on different branches. DNA of 16 strains of P. fluorescens and 34 strains of non-P. fluorescens was detected by real-time LAMP. TAP-harboring P. fluorescens can only be identified when the real-time LAMP detection results of both aprX and gyrB are positive. The dissociation temperatures of aprX and gyrB in the real-time LAMP-amplified products were approximately 90.0 and 88.0°C, respectively. The detection limits of the real-time LAMP targeting aprX and gyrB were 4.9 CFU per reaction in pure culture and 2.2 CFU per reaction in skimmed milk. The coefficient of variation of the repeatability test was less than 2%, indicating that the established real-time LAMP of P. fluorescens targeting gyrB and aprX has good stability and repeatability. Real-time LAMP was used to test 200 raw milk samples for the presence of TAP-harboring P. fluorescens in 3 h, and the coincidence rate of the results with those obtained using the traditional method, which takes at least 5 to 7 days, was 100%. Real-time LAMP will be a practical and effective method for accurate and rapid identification of TAP-harboring P. fluorescens in raw milk.
Topics: Bacterial Proteins; Endopeptidases; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Phylogeny; Pseudomonas fluorescens; Sensitivity and Specificity
PubMed: 34855939
DOI: 10.4315/JFP-21-272 -
Microbiological Research Sep 2021Pseudomonas fluorescens ATCC13525 is an important growth-promoting rhizobacteria (PGPR) and plant disease biocontrol bacterium. However, due to poor stress resistance,...
Pseudomonas fluorescens ATCC13525 is an important growth-promoting rhizobacteria (PGPR) and plant disease biocontrol bacterium. However, due to poor stress resistance, it is prone to be inactivated by preparation, drying and storage. In this study, we investigated the effects of different stress preadaptation methods (2.0∼3.0 wt% NaCl, 0.01∼0.20 wt% HO, and 35∼44 °C) and two stress adaptation genes (rpoS, and hfq) on the stress resistance of P. fluorescens ATCC13525 (PF-WT). After stress preadaptation with low stress of 3.0 wt% NaCl, 0.05 wt% HO, and 41 °C for 30 min, the tolerance of PF-WT toward high lethal stress environments (20.0 wt% NaCl, 1.00 wt% HO, and 47 °C) were significantly improved. Moreover, knockout of rpoS and hfq genes resulted in slower culture growth than PF-WT under the sublethal stress culture conditions (5.0 wt% NaCl, 0.08 wt% HO, and 35 °C), whereas rpoS and hfq overexpressed strains (PF-pBBR2-rpoS and PF-pBBR2-hfq) obviously grew better than the control strain PF-pBBR2. Further, we prepared biocontrol agents (BACs) of different strains after different stress preadaptation treatments. Compared to PF-WT without stress preadaptation, preadaptation by 0.05 wt% HO for 30 min resulted in 5.65 times higher survival rate, while treatment with 3.0 wt% NaCl for 30 min of PF-pBBR2-rpoS led to 5.60 times higher survival rate. This finding provides the simple and effective protection methods for P. fluorescens ATCC13525 BACs preparation by stress preadaptation and overexpression of stress adaptation genes.
Topics: Adaptation, Physiological; Bacterial Proteins; Biological Control Agents; Host Factor 1 Protein; Hydrogen Peroxide; Pseudomonas fluorescens; Sigma Factor; Stress, Physiological
PubMed: 34144508
DOI: 10.1016/j.micres.2021.126804 -
Applied and Environmental Microbiology Jul 2022Pseudomonas fluorescens 2P24 is a beneficial plant root-associated microorganism capable of suppressing several soilborne plant diseases. The capacity of P. fluorescens...
Pseudomonas fluorescens 2P24 is a beneficial plant root-associated microorganism capable of suppressing several soilborne plant diseases. The capacity of P. fluorescens to aggressively colonize the rhizosphere is an important requirement for its biocontrol trait. We previously found that the PcoI/PcoR quorum-sensing system (QS) is involved in regulating the rhizosphere colonization of P. fluorescens. Here, we revealed a sophisticated regulatory network that connects PcoR, RsaL, and MvaT proteins to fine-tune the PcoI/PcoR QS system. Our data showed that PcoR could directly bind to the promoter region of thereby inducing the PcoI/PcoR QS system, whereas RsaL binds simultaneously with PcoR to the promoter region of and represses the PcoR-dependent activation of gene. In addition, RsaL indirectly downregulates the expression of . Furthermore, we showed that disruption of enhanced the expression of , , and , whereas MvaT controls the PcoI/PcoR QS in a RsaL-independent manner. Overall, this study elucidates that PcoR, RsaL, and MvaT regulate the PcoI/PcoR QS through a multi-tiered regulatory mechanism and that PcoR is necessary in the RsaL- and MvaT-mediated repression on the expression of . The PcoI/PcoR quorum-sensing system of Pseudomonas fluorescens 2P24 is important for its effective colonization in the plant rhizosphere. Many regulatory elements appear to directly or indirectly influence the QS system. Here, we found a complex regulatory network employing transcriptional factors PcoR, RsaL, and MvaT to influence the expression of the PcoI/PcoR QS in P. fluorescens 2P24. Our results indicate that PcoR and RsaL directly bind to the promoter region of and then positively and negatively regulate the expression of , respectively. Furthermore, the H-NS family protein MvaT negatively controls the PcoI/PcoR QS in a RsaL-independent manner. Taken together, our data provide new insights into the interplays between different regulatory elements that fine-tune the QS system of P. fluorescens.
Topics: Bacterial Proteins; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa; Pseudomonas fluorescens; Quorum Sensing; Transcription Factors
PubMed: 35695573
DOI: 10.1128/aem.00625-22