Showing papers on "Swarming motility published in 2014"

Plate-based assay for swimming motility in Pseudomonas aeruginosa.

Abstract: Swarming motility is one of three distinct modes of motility observed in the gram-negative bacterium Pseudomonas aeruginosa. Swarming motility is defined as the movement across a semisolid surface, and in P. aeruginosa requires flagellar motility and the production of biosurfactants. Swarming motility is thought to occur on gelatinous/viscous surfaces inside a host, such as on epithelial cells. There is currently no standardized in vitro assay to visualize and study swarming motility, and the assays used can vary greatly between laboratory groups. Here, we describe a detailed, reproducible in vitro swarming motility assay for P. aeruginosa. While different protocols have previously been reported in the literature, we hope that adopting this method will improve the reproducibility of these swarming motility assays and allow comparisons of swarming motility findings between and among groups.

Cranberry proanthocyanidins have anti-biofilm properties against Pseudomonas aeruginosa

Abstract: Bacteria within a biofilm are phenotypically more resistant to antibiotics, desiccation, and the host immune system, making it an important virulence factor for many microbes Cranberry juice has long been used to prevent infections of the urinary tract, which are often related to biofilm formation Recent studies have found that the A-type proanthocyanidins from cranberries have anti-biofilm properties against Escherichia coli Using crystal violet biofilm staining, resazurin metabolism assays, and confocal imaging, we examined the ability of A-type proanthocyanidins (PACs) to disrupt the biofilm formation of Pseudomonas aeruginosa We used mass spectrometry to analyze the proteomic effects of PAC treatment We also performed synergy assays and in vitro and in vivo infections to determine whether PACs, alone and in combination with gentamicin, could contribute to the killing of P aeruginosa and the survival of cell lines and G mellonella Cranberry PACs reduced P aeruginosa swarming motility Cranberry PACs significantly disrupted the biofilm formation of P aeruginosa Proteomics analysis revealed significantly different proteins expressed following PAC treatment In addition, we found that PACs potentiated the antibiotic activity of gentamicin in an in vivo model of infection using G mellonella Results suggest that A-type proanthocyanidins may be a useful therapeutic against the biofilm-mediated infections caused by P aeruginosa and should be further tested

Anti-quorum sensing activity of Psidium guajava L. flavonoids against Chromobacterium violaceum and Pseudomonas aeruginosa PAO1.

Abstract: Psidium guajava L., which has been used traditionally as a medicinal plant, was explored for anti-quorum sensing (QS) activity. The anti-QS activity of the flavonoid (FL) fraction of P. guajava leaves was determined using a biosensor bioassay with Chromobacterium violaceum CV026. Detailed investigation of the effects of the FL-fraction on QS-regulated violacein production in C. violaceum ATCC12472 and pyocyanin production, proteolytic, elastolytic activities, swarming motility and biofilm formation in Pseudomonas aeruginosa PAO1 was performed using standard methods. Possible mechanisms of QS-inhibition were studied by assessing violacein production in response to N-acyl homoserine lactone (AHL) synthesis in the presence of the FL-fraction in C. violaceum ATCC31532 and by evaluating the induction of violacein in the mutant C. violaceum CV026 by AHL extracted from the culture supernatants of C. violaceum 31532. Active compounds in the FL-fraction were identified by liquid chromatography-mass spectrometry (LC-MS). Inhibition of violacein production by the FL-fraction in a C. violaceum CV026 biosensor bioassay indicated possible anti-QS activity. The FL-fraction showed concentration-dependent decreases in violacein production in C. violaceum 12472 and inhibited pyocyanin production, proteolytic and elastolytic activities, swarming motility and biofilm formation in P. aeruginosa PAO1. Interestingly, the FL-fraction did not inhibit AHL synthesis; AHL extracted from cultures of C. violaceum 31532 grown in the presence of the FL-fraction induced violacein in the mutant C. violaceum CV026. LC-MS analysis revealed the presence of quercetin and quercetin-3-O-arabinoside in the FL-fraction. Both quercetin and quercetin-3-O-arabinoside inhibited violacein production in C. violaceum 12472, at 50 and 100 μg/mL, respectively. Results of this study provide scope for further research to exploit these active molecules as anti-QS agents.

Enhanced Control of Cucumber Wilt Disease by Bacillus amyloliquefaciens SQR9 by Altering the Regulation of Its DegU Phosphorylation

Abstract: Bacillus amyloliquefaciens strain SQR9, isolated from the cucumber rhizosphere, suppresses the growth of Fusarium oxysporum in the cucumber rhizosphere and protects the host plant from pathogen invasion through efficient root colonization. In the Gram-positive bacterium Bacillus, the response regulator DegU regulates genetic competence, swarming motility, biofilm formation, complex colony architecture, and protease production. In this study, we report that stepwise phosphorylation of DegU in B. amyloliquefaciens SQR9 can influence biocontrol activity by coordinating multicellular behavior and regulating the synthesis of antibiotics. Results from in vitro and in situ experiments and quantitative PCR (qPCR) studies demonstrate the following: (i) that the lowest level of phosphorylated DegU (DegU∼P) (the degQ mutation) impairs complex colony architecture, biofilm formation, colonization activities, and biocontrol efficiency of Fusarium wilt disease but increases the production of macrolactin and bacillaene, and (ii) that increasing the level of DegU∼P by degQ and degSU overexpression significantly improves complex colony architecture, biofilm formation, colonization activities, production of the antibiotics bacillomycin D and difficidin, and efficiency of biocontrol of Fusarium wilt disease. The results offer a new strategy to enhance the biocontrol efficacy of Bacillus amyloliquefaciens SQR9.

Punicalagin inhibits Salmonella virulence factors and has anti-quorum-sensing potential

Abstract: Punicalagin, an essential component of pomegranate rind, has been demonstrated to possess antimicrobial activity against several food-borne pathogens, but its activity on the virulence of pathogens and its anti-quorum-sensing (anti-QS) potential have been rarely reported. This study investigated the efficacy of subinhibitory concentrations of punicalagin on Salmonella virulence factors and QS systems. A broth microdilution method was used to determine the MICs of punicalagin for 10 Salmonella strains. Motility assay and quantitative reverse transcription (RT)-PCR were performed to evaluate the effects of punicalagin on the virulence attributes and QS-related genes of Salmonella. The MICs of punicalagin for several Salmonella strains ranged from 250 to 1,000 μg/ml. Motility assays showed that punicalagin, at 1/16× MIC and 1/32× MIC, significantly decreased bacterial swimming and swarming motility, which corresponded to downregulation of the motility-related genes (fliA, fliY, fljB, flhC, and fimD) in RT-PCR assays. RT-PCR also revealed that punicalagin downregulated the expression of most of the selected genes involved in Salmonella virulence. Moreover, a QS inhibition assay indicated that punicalagin dose dependently inhibited the production of violacein by Chromobacterium violaceum and repressed the expression of QS-related genes (sdiA and srgE) in Salmonella. In addition, punicalagin significantly reduced Salmonella invasion of colonic cells (P<0.01) with no impact on adhesion. These findings suggest that punicalagin has the potential to be developed as an alternative or supplemental agent for prevention of Salmonella infection.

Deletion mutant library for investigation of functional outputs of cyclic diguanylate metabolism in Pseudomonas aeruginosa PA14.

Abstract: We constructed a library of in-frame deletion mutants targeting each gene in Pseudomonas aeruginosa PA14 predicted to participate in cyclic di-GMP (c-di-GMP) metabolism (biosynthesis or degradation) to provide a toolkit to assist investigators studying c-di-GMP-mediated regulation by this microbe. We present phenotypic assessments of each mutant, including biofilm formation, exopolysaccharide (EPS) production, swimming motility, swarming motility, and twitch motility, as a means to initially characterize these mutants and to demonstrate the potential utility of this library.

Coordination of swarming motility, biosurfactant synthesis, and biofilm matrix exopolysaccharide production in Pseudomonas aeruginosa.

Abstract: Biofilm formation is a complex process in which many factors are involved. Bacterial swarming motility and exopolysaccharides both contribute to biofilm formation, yet it is unclear how bacteria coordinate swarming motility and exopolysaccharide production. Psl and Pel are two key biofilm matrix exopolysaccharides in Pseudomonas aeruginosa. This opportunistic pathogen has three types of motility, swimming, twitching, and swarming. In this study, we found that elevated Psl and/or Pel production reduced the swarming motility of P. aeruginosa but had little effect on swimming and twitching. The reduction was due to decreased rhamnolipid production with no relation to the transcription of rhlAB, two key genes involved in the biosynthesis of rhamnolipids. Rhamnolipid-negative rhlR and rhlAB mutants synthesized more Psl, whereas exopolysaccharide-deficient strains exhibited a hyperswarming phenotype. These results suggest that competition for common sugar precursors catalyzed by AlgC could be a tactic for P. aeruginosa to balance the synthesis of exopolysaccharides and rhamnolipids and to control bacterial motility and biofilm formation inversely because the biosynthesis of rhamnolipids, Psl, and Pel requires AlgC to provide the sugar precursors and an additional algC gene enhances the biosynthesis of Psl and rhamnolipids. In addition, our data indicate that the increase in RhlI/RhlR expression attenuated Psl production. This implied that the quorum-sensing signals could regulate exopolysaccharide biosynthesis indirectly in bacterial communities. In summary, this study represents a mechanism that bacteria utilize to coordinate swarming motility, biosurfactant synthesis, and biofilm matrix exopolysaccharide production, which is critical for biofilm formation and bacterial survival in the environment.

Pseudomonas : methods and protocols

Abstract: Gene Transfer: Transduction -- Gene Transfer: Transformation/Electroporation -- Gene transfer: Conjugation -- Pseudomonas Bacteriophage Isolation and Production -- Genotyping Methods -- Drug Susceptibility Testing by Dilution Methods -- Plate-based Assay for Swimming Motility in Pseudomonas aeruginosa -- Plate-based Assay for Swarming Motility in Pseudomonas aeruginosa -- Motility Assay: Twitching Motility -- Qualitative and Quantitative Assays for Flagellum-mediated Chemotaxis -- Microscopic Analysis: Morphotypes and Cellular Appendages -- Determination of Lipolytic Activities -- Elastinolytic and proteolytic enzymes -- In vitro Assays to Monitor the Activity of Pseudomonas aeruginosa Type III Secreted Proteins -- Cell Fractionation -- Characterization of Molecular Interactions using Isothermal Titration Calorimetry -- Proteomic Analysis -- Membrane Proteomics of Pseudomonas aeruginosa -- Construction of Pseudomonas Two-hybrid Libraries for High-throughput Assays -- Biosensors for Qualitative and Semi-quantitative Analysis of Quorum Sensing Signal Molecules -- LC-MS/MS Quantitative Analysis of Quorum Sensing Signal Molecules -- LC/MS/MS-Based Quantitative Assay for the Secondary Messenger Molecule, c-di-GMP -- Metabolic Footprinting: Extracellular Metabolomic Analysis -- Pyoverdine and Pyochelin Measurements -- Measurement of Phenazines in Bacterial Cultures -- Extraction and Measurement of NAD(P)+ and NAD(P)H -- Cyanide Measurements in Bacterial Culture and Sputum -- Monitoring Iron Uptake by Siderophores -- Exopolysaccharide Quantification -- Liquid Chromatography/Mass Spectrometry for the Identification and Quantification of Rhamnolipids -- LPS quantitation Procedures -- Monitoring Lectin Interaction with Carbohydrates -- Mining the Pseudomonas Genome -- Identification of Bacterial Small RNAs by RNA Sequencing -- Gene Amplification and qRT-PCR -- The Standard European Vector architecture (SEVA) plasmid toolkit -- Chromosomal integration of transcriptional fusions -- A Method to Capture Large DNA Fragments from Genomic DNA -- Transposon Mutagenesis -- Site-directed Mutagenesis and Gene Deletion using Reverse Genetics -- Signature Tagged Mutagenesis -- Construction of a Pseudomonas aeruginosa Genomic Bank -- Strategy for Genome Sequencing Analysis and Assembly for Comparative Genomics of Pseudomonas Genomes -- Promoter Fusions with Optical Outputs in Individual Cells and in Populations -- Chromatin Immunoprecipitation for ChIP-chip and ChIP-Seq -- Transcriptional Analysis of Pseudomonas aeruginosa Infected Caenorhabditis elegans -- Methods for Studying Biofilm Formation: Flow Cells and Confocal Laser Scanning Microscop -- Biofilm Formation in the 96-well Microtiter Plate -- Methods for Studying Biofilm Dispersal in Pseudomonas aeruginosa -- Pseudomonas aeruginosa PA14 Pathogenesis in Caenorhabditis Elegans -- Assessing Pseudomonas Virulence using a Non-mammalian Host Model: Dictyostelium Discoideum -- Assessing Pseudomonas virulence with non-mammalian host: Galleria mellonella -- Assessing Pseudomonas Virulence with the Non-mammalian Host: Arabidopsis Thaliana -- Assessing Pseudomonas aeruginosa Persister/Antibiotic Tolerant Cells -- Assessing Pseudomonas Virulence with Non-mammalian Host: Zebrafish -- Assessing Pseudomonas Virulence with a Non-mammalian Host: Drosophila Melanogaster -- Assessing Pseudomonas Virulence using Host Cells -- Assessing Pseudomonas aeruginosa Virulence and the Host Response using Murine Models of Acute and Chronic Lung Infection -- Assessing Pseudomonas Virulence using Mammalian Models: Acute Infection Model -- Burn Mouse Models.

SinR controls enterotoxin expression in Bacillus thuringiensis biofilms.

Abstract: The entomopathogen Bacillus thuringiensis produces dense biofilms under various conditions. Here, we report that the transition phase regulators Spo0A, AbrB and SinR control biofilm formation and swimming motility in B. thuringiensis, just as they control biofilm formation and swarming motility in the closely related saprophyte species B. subtilis. However, microarray analysis indicated that in B. thuringiensis, in contrast to B. subtilis, SinR does not control an eps operon involved in exopolysaccharides production, but regulates genes involved in the biosynthesis of the lipopeptide kurstakin. This lipopeptide is required for biofilm formation and was previously shown to be important for survival in the host cadaver (necrotrophism). Microarray analysis also revealed that the SinR regulon contains genes coding for the Hbl enterotoxin. Transcriptional fusion assays, Western blots and hemolysis assays confirmed that SinR controls Hbl expression, together with PlcR, the main virulence regulator in B. thuringiensis. We show that Hbl is expressed in a sustained way in a small subpopulation of the biofilm, whereas almost all the planktonic population transiently expresses Hbl. The gene coding for SinI, an antagonist of SinR, is expressed in the same biofilm subpopulation as hbl, suggesting that hbl transcription heterogeneity is SinI-dependent. B. thuringiensis and B. cereus are enteric bacteria which possibly form biofilms lining the host intestinal epithelium. Toxins produced in biofilms could therefore be delivered directly to the target tissue.

Variations in motility and biofilm formation of Salmonella enterica serovar Typhi.

Abstract: Background: Salmonella enterica serovar Typhi (S. Typhi) exhibits unique characteristics as an intracellular human pathogen. It causes both acute and chronic infection with various disease manifestations in the human host only. The principal factors underlying the unique lifestyle of motility and biofilm forming ability of S. Typhi remain largely unknown. The main objective of this study was to explore and investigate the motility and biofilm forming behaviour among S. Typhi strains of diverse background. Results: Swim and swarm motility tests were performed with 0.25% and 0.5% agar concentration, respectively; while biofilm formation was determined by growing the bacterial cultures for 48 hrs in 96-well microtitre plate. While all S. Typhi strains demonstrated swarming motility with smooth featureless morphology, 58 out of 60 strains demonstrated swimming motility with featureless or bull’s eye morphology. Interestingly, S. Typhi strains of blood-borne origin exhibited significantly higher swimming motility (P<0.05) than stool-borne strains suggesting that swimming motility may play a role in the systemic invasion of S. Typhi in the human host. Also, stool-borne S. Typhi displayed a negative relationship between motility and biofilm forming behaviour, which was not observed in the blood-borne strains. Conclusion: In summary, both swimming and swarming motility are conserved among S. Typhi strains but there was variation for biofilm forming ability. There was no difference observed in this phenotype for S. Typhi strains from diverse background. These findings serve as caveats for future studies to understand the lifestyle and transmission of this pathogen.

To settle or to move? : the interplay between two classes of cyclic lipopeptides in the biocontrol strain Pseudomonas CMR12a

Abstract: Pseudomonas CMR12a is a biocontrol strain that produces phenazine antibiotics and as yet uncharacterized cyclic lipopeptides (CLPs). The CLPs of CMR12a were studied by chemical structure analysis and in silico analysis of the gene clusters encoding the nonribosomal peptide synthetases responsible for CLP biosynthesis. CMR12a produces two different classes of CLPs: orfamides B, D and E, whereby the latter two represent new derivatives of the orfamide family, and sessilins A-C. The orfamides are made up of a 10 amino acid peptide coupled to a β-hydroxydodecanoyl or β-hydroxytetradecanoyl fatty acid moiety, and are related to orfamides produced by biocontrol strain P. protegens Pf-5. The sessilins consist of an 18 amino acid peptide linked to a β-hydroxyoctanoyl fatty acid, and differ in one amino acid from tolaasins, toxins produced by the mushroom pathogen P. tolaasii. CLP biosynthesis mutants were constructed and tested for biofilm formation and swarming motility. Orfamides appeared indispensable for swarming, while sessilin mutants showed reduced biofilm formation, but enhanced swarming motility. The interplay between the two classes of CLPs fine-tunes these processes. The presence of sessilins in wild type CMR12a interferes with swarming by hampering the release of orfamides and by co-precipitating orfamides to form a white-line-in agar.

Variations in motility and biofilm formation of salmonella enterica serovar typhi gut pathogens

Abstract: Background: Salmonella enterica serovar Typhi (S. Typhi) exhibits unique characteristics as an intracellular human pathogen. It causes both acute and chronic infection with various disease manifestations in the human host only. The principal factors underlying the unique lifestyle of motility and biofilm forming ability of S. Typhi remain largely unknown. The main objective of this study was to explore and investigate the motility and biofilm forming behaviour among S. Typhi strains of diverse background. Results: Swim and swarm motility tests were performed with 0.25% and 0.5% agar concentration, respectively; while biofilm formation was determined by growing the bacterial cultures for 48 hrs in 96-well microtitre plate. While all S. Typhi strains demonstrated swarming motility with smooth featureless morphology, 58 out of 60 strains demonstrated swimming motility with featureless or bull’s eye morphology. Interestingly, S. Typhi strains of blood-borne origin exhibited significantly higher swimming motility (P < 0.05) than stool-borne strains suggesting that swimming motility may play a role in the systemic invasion of S. Typhi in the human host. Also, stool-borne S. Typhi displayed a negative relationship between motility and biofilm forming behaviour, which was not observed in the blood-borne strains. Conclusion: In summary, both swimming and swarming motility are conserved among S. Typhi strains but there was variation for biofilm forming ability. There was no difference observed in this phenotype for S. Typhi strains from diverse background. These findings serve as caveats for future studies to understand the lifestyle and transmission of this pathogen. Keywords: Salmonella Typhi, Biofilm, Motility swarming, Swimming

NrsZ: a novel, processed, nitrogen-dependent, small non-coding RNA that regulates Pseudomonas aeruginosa PAO1 virulence.

Abstract: The opportunistic pathogen Pseudomonas aeruginosa PAO1 has a remarkable capacity to adapt to various environments and to survive with limited nutrients. Here, we report the discovery and characterization of a novel small non-coding RNA: NrsZ (nitrogen-regulated sRNA). We show that under nitrogen limitation, NrsZ is induced by the NtrB/C two component system, an important regulator of nitrogen assimilation and P. aeruginosa's swarming motility, in concert with the alternative sigma factor RpoN. Furthermore, we demonstrate that NrsZ modulates P. aeruginosa motility by controlling the production of rhamnolipid surfactants, virulence factors notably needed for swarming motility. This regulation takes place through the post-transcriptional control of rhlA, a gene essential for rhamnolipids synthesis. Interestingly, we also observed that NrsZ is processed in three similar short modules, and that the first short module encompassing the first 60 nucleotides is sufficient for NrsZ regulatory functions.

A nanoscale characterization of the interaction of a novel alginate oligomer with the cell surface and motility of pseudomonas aeruginosa

Abstract: Pseudomonas aeruginosa (PA) biofilm-associated infections are a common cause of morbidity in chronic respiratory disease and represent a therapeutic challenge. Recently, the ability of a novel alginate oligomer (OligoG) to potentiate the effect of antibiotics against gram-negative, multi–drug-resistant bacteria and inhibit biofilm formation in vitro has been described. Interaction of OligoG with the cell surface of PA was characterized at the nanoscale using atomic force microscopy (AFM), zeta potential measurement (surface charge), and sizing measurements (dynamic light scattering). The ability of OligoG to modify motility was studied in motility assays. AFM demonstrated binding of OligoG to the bacterial cell surface, which was irreversible after exposure to hydrodynamic shear (5,500 × g). Zeta potential analysis (pH 5–9; 0.1–0.001 M NaCl) demonstrated that binding was associated with marked changes in the bacterial surface charge (−30.9 ± 0.8 to −47.0 ± 2.3 mV; 0.01 M NaCl [pH 5]; P < 0.001). Sizing analysis demonstrated that alteration of surface charge was associated with cell aggregation with a 2- to 3-fold increase in mean particle size at OligoG concentrations greater than 2% (914 ± 284 to 2599 ± 472 nm; 0.01 M NaCl [pH 5]; P < 0.001). These changes were associated with marked dose-dependent inhibition in bacterial swarming motility in PA and Burkholderia spp. The ability of OligoG to bind to a bacterial surface, modulate surface charge, induce microbial aggregation, and inhibit motility represents important direct mechanisms by which antibiotic potentiation and biofilm disruption is affected. These results highlight the value of combining multiple nanoscale technologies to further our understanding of the mechanisms of action of novel antibacterial therapies.

The prevalence and origin of exoprotease-producing cells in the Bacillus subtilis biofilm

Abstract: Biofilm formation by the Gram-positive bacterium Bacillus subtilis is tightly controlled at the level of transcription. The biofilm contains specialized cell types that arise from controlled differentiation of the resident isogenic bacteria. DegU is a response regulator that controls several social behaviours exhibited by B. subtilis including swarming motility, biofilm formation and extracellular protease (exoprotease) production. Here, for the first time, we examine the prevalence and origin of exoprotease-producing cells within the biofilm. This was accomplished using single-cell analysis techniques including flow cytometry and fluorescence microscopy. We established that the number of exoprotease-producing cells increases as the biofilm matures. This is reflected by both an increase at the level of transcription and an increase in exoprotease activity over time. We go on to demonstrate that exoprotease-producing cells arise from more than one cell type, namely matrix-producing and non-matrix-producing cells. In toto these findings allow us to add exoprotease-producing cells to the list of specialized cell types that are derived during B. subtilis biofilm formation and furthermore the data highlight the plasticity in the origin of differentiated cells.

Guava leaf extract inhibits quorum-sensing and Chromobacterium violaceum induced lysis of human hepatoma cells: whole transcriptome analysis reveals differential gene expression.

Abstract: Quorum sensing (QS) is a process mediated via small molecules termed autoinducers (AI) that allow bacteria to respond and adjust according to the cell population density by altering the expression of multitudinous genes. Since QS governs numerous bioprocesses in bacteria, including virulence, its inhibition promises to be an ideal target for the development of novel therapeutics. We found that the aqueous leaf extract of Psidium guajava (GLE) exhibited anti-QS properties as evidenced by inhibition of violacein production in Chromobacterium violaceum and swarming motility of Pseudomonas aeruginosa. The gram-negative bacterium, C. violaceum is a rare pathogen with high mortality rate. In this study, perhaps for the first time, we identified the target genes of GLE in C. violaceum MTCC 2656 by whole transcriptome analysis on Ion Torrent. Our data revealed that GLE significantly down-regulated 816 genes at least three fold, with p value≤0.01, which comprises 19% of the C. violaceum MTCC 2656 genome. These genes were distributed throughout the genome and were associated with virulence, motility and other cellular processes, many of which have been described as quorum regulated in C. violaceum and other gram negative bacteria. Interestingly, GLE did not affect the growth of the bacteria. However, consistent with the gene expression pattern, GLE treated C. violaceum cells were restrained from causing lysis of human hepatoma cell line, HepG2, indicating a positive relationship between the QS-regulated genes and pathogenicity. Overall, our study proposes GLE as a QS inhibitor (QSI) with the ability to attenuate virulence without affecting growth. To the best of our knowledge, this is the first report which provides with a plausible set of candidate genes regulated by the QS system in the neglected pathogen C. violaceum.

Identification of Novel Factors Involved in Modulating Motility of Salmonella enterica Serotype Typhimurium

Abstract: Salmonella enterica serotype Typhimurium can move through liquid using swimming motility, and across a surface by swarming motility. We generated a library of targeted deletion mutants in Salmonella Typhimurium strain ATCC14028, primarily in genes specific to Salmonella, that we have previously described. In the work presented here, we screened each individual mutant from this library for the ability to move away from the site of inoculation on swimming and swarming motility agar. Mutants in genes previously described as important for motility, such as flgF, motA, cheY are do not move away from the site of inoculation on plates in our screens, validating our approach. Mutants in 130 genes, not previously known to be involved in motility, had altered movement of at least one type, 9 mutants were severely impaired for both types of motility, while 33 mutants appeared defective on swimming motility plates but not swarming motility plates, and 49 mutants had reduced ability to move on swarming agar but not swimming agar. Finally, 39 mutants were determined to be hypermotile in at least one of the types of motility tested. Both mutants that appeared non-motile and hypermotile on plates were assayed for expression levels of FliC and FljB on the bacterial surface and many of them had altered levels of these proteins. The phenotypes we report are the first phenotypes ever assigned to 74 of these open reading frames, as they are annotated as ‘hypothetical genes’ in the Typhimurium genome.

Emergence of the P2 Phenotype in Pseudomonas aeruginosa PAO1 Strains Involves Various Mutations in mexT or mexF

Abstract: We recently demonstrated that Pseudomonas aeruginosa PAO1 undergoes a pronounced phenotypic change when introduced into the intestines of rats during surgical injury. Recovered strains displayed a specific phenotype (termed the P2 phenotype) characterized by altered pyocyanin production, high collagenase activity, high swarming motility, low resistance to chloramphenicol, and increased killing of Caenorhabditis elegans compared to the inoculating strain (termed the P1 phenotype). The aims of this study were to characterize the differences between the P. aeruginosa P1 and P2 phenotypes in quorum sensing and competitiveness. We then determined the presence of the P2 phenotype among PAO1 strains from various laboratories. Results demonstrated that P2 cells display accelerated growth during early exponential phase and early activation of quorum-sensing systems and overcome the growth of P1 cells in a mixed population. Among eight PAO1 strains obtained from different laboratories, four exhibited the P2 phenotype. Of 27 mutants analyzed from the P. aeruginosa MPAO1 transposon library, 25 displayed P2 phenotypes. The P2 phenotype in both cases correlated with a lack of expression of mexE or mexF due to mutations in mexT and mexF genes. In summary, strains possessing the P2 phenotype are distributed among PAO1 strains commonly used across a variety of research laboratories. Genetically, they are characterized by various mutations in mexT or mexF.

PqsR-dependent and PqsR-independent regulation of motility and biofilm formation by PQS in Pseudomonas aeruginosa PAO1.

Abstract: Pseudomonas aeruginosa is an opportunistic pathogen capable of group behaviors including swarming motility and biofilm formation. Swarming motility plays an important role in the bacterium's spread to new environments, attachment to surfaces, and biofilm formation. Bacterial biofilm is associated with many persistent infections and increased resistance to antibiotics. In this study, we tested the effect of a 2-alkyl-4(1H)-quinolone (AHQ) signal, the Pseudomonas quinolone signal (PQS) on P. aeruginosa swarming and biofilm formation. Our results show that PQS repressed the swarming motility of P. aeruginosa PAO1. Such repression was independent of its cognate receptor PqsR and was not related to changes in the flagellae, type IV pili or the production of the surface-wetting agent rhamnolipid surfactant. While PQS did not affect twitching motility in PAO1, a pqsR deletion abolished twitching motility, indicating that pqsR is required for twitching motility. Our results also indicate that the enhancement of biofilm formation by PQS is at least partially dependent on the GacAS-Rsm regulatory pathway but does not involve the las or rhl QS systems.

Transposon Mutagenesis of the Plant-Associated Bacillus amyloliquefaciens ssp. plantarum FZB42 Revealed That the nfrA and RBAM17410 Genes Are Involved in Plant-Microbe-Interactions

Abstract: Bacillus amyloliquefaciens ssp. plantarum FZB42 represents the prototype of Gram-positive plant growth promoting and biocontrol bacteria. In this study, we applied transposon mutagenesis to generate a transposon library, which was screened for genes involved in multicellular behavior and biofilm formation on roots as a prerequisite of plant growth promoting activity. Transposon insertion sites were determined by rescue-cloning followed by DNA sequencing. As in B. subtilis, the global transcriptional regulator DegU was identified as an activator of genes necessary for swarming and biofilm formation, and the DegU-mutant of FZB42 was found impaired in efficient root colonization. Direct screening of 3,000 transposon insertion mutants for plant-growth-promotion revealed the gene products of nfrA and RBAM_017140 to be essential for beneficial effects exerted by FZB42 on plants. We analyzed the performance of GFP-labeled wild-type and transposon mutants in the colonization of lettuce roots using confocal laser scanning microscopy. While the wild-type strain heavily colonized root surfaces, the nfrA mutant did not colonize lettuce roots, although it was not impaired in growth in laboratory cultures, biofilm formation and swarming motility on agar plates. The RBAM17410 gene, occurring in only a few members of the B. subtilis species complex, was directly involved in plant growth promotion. None of the mutant strains were affected in producing the plant growth hormone auxin. We hypothesize that the nfrA gene product is essential for overcoming the stress caused by plant response towards bacterial root colonization.

Synthetic brominated furanone F202 prevents biofilm formation by potentially human pathogenic Escherichia coli O103:H2 and Salmonella ser. Agona on abiotic surfaces

Abstract: Aims Investigate the use of a synthetic brominated furanone (F202) against the establishment of biofilm by Salmonella ser. Agona and E. coli O103:H2 under temperature conditions relevant for the food and feed industry as well as under temperature conditions optimum for growth. Methods and Results Effect of F202 on biofilm formation by Salmonella ser. Agona and E. coli O103:H2 was evaluated using a microtiter plate assay and confocal microscopy. Effect of F202 on bacterial motility was investigated using swimming and swarming assays. Influence on flagellar synthesis by F202 was examined by flagellar staining. Results showed that F202 inhibited biofilm formation without being bactericidal. F202 was found to affect both swimming and swarming motility without, however, affecting the expression of flagella. Conclusions F202 showed its potential as a biofilm inhibitor of Salmonella ser. Agona and E. coli O103:H2 under temperature conditions relevant for the feed and food industry as well as temperatures optimum for growth. One potential mode of action of F202 was found to be by targeting flagellar function. Significance and Impact of the Study The present study gives valuable new knowledge to the potential use of furanones as a tool in biofilm management in the food and feed industry.

Engineering of Bacillus subtilis Strains To Allow Rapid Characterization of Heterologous Diguanylate Cyclases and Phosphodiesterases

Abstract: Microbial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often encode dozens of redundant enzymes that synthesize and hydrolyze c-di-GMP, diguanylate cyclases (DGCs), and c-di-GMP phosphodiesterases (PDEs); thus, determining the contribution of any one particular enzyme is challenging. In an effort to develop a facile system to study c-di-GMP metabolic enzymes, we have engineered a suite of Bacillus subtilis strains to assess the effect of individual heterologously expressed proteins on c-di-GMP levels. As a proof of principle, we characterized all 37 known genes encoding predicted DGCs and PDEs in Clostridium difficile using parallel readouts of swarming motility and fluorescence from green fluorescent protein (GFP) expressed under the control of a c-di-GMP-controlled riboswitch. We found that 27 of the 37 putative C. difficile 630 c-di-GMP metabolic enzymes had either active cyclase or phosphodiesterase activity, with agreement between our motility phenotypes and fluorescence-based c-di-GMP reporter. Finally, we show that there appears to be a threshold level of c-di-GMP needed to inhibit motility in Bacillus subtilis.

Influence of lysogeny of Tectiviruses GIL01 and GIL16 on Bacillus thuringiensis growth, biofilm formation, and swarming motility.

Abstract: Bacillus thuringiensis is an entomopathogenic bacterium that has been used as an efficient biopesticide worldwide. Despite the fact that this bacterium is usually described as an insect pathogen, its life cycle in the environment is still largely unknown. B. thuringiensis belongs to the Bacillus cereus group of bacteria, which has been associated with many mobile genetic elements, such as species-specific temperate or virulent bacteriophages (phages). Temperate (lysogenic) phages are able to establish a long-term relationship with their host, providing, in some cases, novel ecological traits to the bacterial lysogens. Therefore, this work focuses on evaluating the potential influence of temperate tectiviruses GIL01 and GIL16 on the development of different life traits of B. thuringiensis. For this purpose, a B. thuringiensis serovar israelensis plasmid-cured (nonlysogenic) strain was used to establish bacterial lysogens for phages GIL01 and GIL16, and, subsequently, the following life traits were compared among the strains: kinetics of growth, metabolic profiles, antibiotics susceptibility, biofilm formation, swarming motility, and sporulation. The results revealed that GIL01 and GIL16 lysogeny has a significant influence on the bacterial growth, sporulation rate, biofilm formation, and swarming motility of B. thuringiensis. No changes in metabolic profiles or antibiotic susceptibilities were detected. These findings provide evidence that tectiviruses have a putative role in the B. thuringiensis life cycle as adapters of life traits with ecological advantages.

RecA Protein Plays a Role in the Chemotactic Response and Chemoreceptor Clustering of Salmonella enterica

Abstract: The RecA protein is the main bacterial recombinase and the activator of the SOS system. In Escherichia coli and Salmonella enterica sv. Typhimurium, RecA is also essential for swarming, a flagellar-driven surface translocation mechanism widespread among bacteria. In this work, the direct interaction between RecA and the CheW coupling protein was confirmed, and the motility and chemotactic phenotype of a S. Typhimurium ΔrecA mutant was characterized through microfluidics, optical trapping, and quantitative capillary assays. The results demonstrate the tight association of RecA with the chemotaxis pathway and also its involvement in polar chemoreceptor cluster formation. RecA is therefore necessary for standard flagellar rotation switching, implying its essential role not only in swarming motility but also in the normal chemotactic response of S. Typhimurium.

Legume seed exudates and Physcomitrella patens extracts influence swarming behavior in Rhizobium leguminosarum

Abstract: Plants are known to secrete chemical compounds that can change the behavior of rhizosphere-inhabiting bacteria We investigated the effects of extracts from legume host plants on the swarming behavior of Rhizobium leguminosarum bv viciae We also investigated the effects on swarming when Rhizobium is exposed to extracts from an ancestor to vascular plants, the model bryophyte Physcomitrella patens Lentil and faba bean seed exudates enhanced and inhibited swarming motility, respectively, whereas pea seed exudates had no observable effect on swarming Swarming was also enhanced by the moss extracts Exposure to lentil seed exudates and the moss extract increased flaA expression 2-fold, while faba bean seed exudates exposure decreased expression 3-fold, suggesting that the swarming effect could, in part, be due to regulation of flagellin gene expression However, the exudates and extracts did not significantly affect flaA gene expression in planktonic motile cells, indicating that the response to flagellar regulation is specific to a physiology unique to the swarming cell Transmission electron microscopy demonstrated that addition of the lentil seed exudate and the moss extract results in earlier differentiation into swarmer cells, which could contribute to the development of a larger swarming surface area To gain further mechanistic insight into the effect of the moss extract on swarming, a moss strigolactone-deficient mutant (Ppccd8Δ) was tested A reduction in the promotive effect was observed, suggesting that the plant hormone strigolactone may be a signalling molecule activating swarming motility in R leguminosarum