Sodium houttuyfonate in vitro inhibits biofilm dispersion and expression of bdlA in Pseudomonas aeruginosa

Molecular Biology Reports - Biofilm dispersion is the last step in the development of biofilms, and allows bacteria to spawn novel biofilms in new locales. In the previous studies, we found that...     

Identification of a new gene PA5017 involved in flagella-mediated motility, chemotaxis and biofilm formation in Pseudomonas aeruginosa

Flagella-mediated motility is recognized as one of the major factors contributing to virulence in Pseudomonas aeruginosa. During a screening of a mini-Mu transposon mutant library of P. aeruginosa PA68, a mutant partially deficient in swimming and swarming motility was identified in a new locus that encodes a predicted protein of unknown function annotated PA5017 in the P. aeruginosa PAO1 genome sequence. Chemotaxis plate assay indicated that inactivation of the PA5017 gene led to a decreased chemotactic response. Complementation of the PA5017 mutant with the wild-type PA5017 gene restored normal motility and chemotaxis phenotype. A promoter-lacZ reporter activity assay of the cheYZAB operon from chemotaxis gene cluster 1 showed that there was almost a twofold difference in expression levels of the wild-type PA68 and the PA5017 mutant. This suggested that the PA5017 affected expression of the cheYZAB operon negatively. Further study showed that inactivation of the PA5017 gene in PA68 led to increased biofilm formation in a static system and to the formation of a heterogeneous biofilm in a flow-chamber system. These results suggested that PA5017 possibly affected flagellum-dependent motility and in turn biofilm formation via the chemotaxis signal transduction pathway.     

Identification and functional characterization of pfm, a novel gene involved in swimming motility of Pseudomonas aeruginosa

Pseudomonas aeruginosa, an important opportunistic pathogen, has a single polar flagellum which is an important virulence and colonization factor by providing swimming motility. This paper describes the functional characterization of a novel gene pfm (PA2950) of P. aeruginosa. The pfm encodes a protein that is similar to a number of short-chain alcohol dehydrogenases of other bacterial species. Mutation of this gene results in a defect in swimming motility which can be completed back to that of wild type by a plasmid containing the pfm. Interestingly, the pfm mutant possesses an intact flagellum which does not rotate, thus giving rise to a non-motile phenotype. The pfm gene is encoded on an operon together with two upstream genes which code for electron transfer flavoprotein (ETF). Yeast two-hybrid tests indicated that the PFM interacts with the ETF, suggesting that the putative dehydrogenase (PFM) is involved in energy metabolism that is critical for the rotation of flagellum in P. aeruginosa.     

铜绿假单胞菌PA1550基因对泳动及蹭动的影响

[目的]:研究与铜绿假单胞菌运动能力相关的基因.[方法]:以一株临床分离的铜绿假单胞菌PA68做受体菌,应用人工Mu转座技术建立了库容为2000的突变子文库,从中筛选出泳动能力和蹭动能力丧失或减弱的突变子,通过基因克隆、测序,GenBankBLAST比对测序结果,互补基因表达确定与铜绿假单胞菌运动能力相关的基因.[结果]:突变子Y46在丧失了泳动运动能力的同时,蹭动能力也发生了减弱.在Y46突变子中,Mu转座子插入到功能完全未知的基因PA1550中.对极性效应及PA1550所在操纵子的分析表明,Mu转座子对插入点下游的基因的转录并不造成影响.[结论]:PA1550与铜绿假单胞菌的泳动及蹭动能力有关.     

Flagella, motility and invasive virulence of Pseudomonas aeruginosa

The role of motility as a virulence factor in Pseudomonas aeruginosa burn wound sepsis was examined using mutants deficient in the Fla or Mot phenotype. Physiological profiles of parental strains and Fla- and Mot- mutants were similar with respect to antibiograms, O antigen types, growth rates, and proteolytic, exotoxin A and phospholipase activities, providing evidence for isogenicity. Lethality studies using a subcutaneous mouse burn model showed that three Fla- mutants and one Mot- mutant were much less virulent (10(2) to 10(5) times) than the parent wild-type. Topical challenges in the flame burn model showed that a Fla- mutant of strain M-2 was approximately tenfold less virulent. A reduction in virulence, although somewhat less than tenfold, was also observed in the scald burn model for M-2 Fla-, and Mot- strains. Tissue colonization experiments revealed a characteristic, rapidly systemic infection in burned mice challenged with wild-type organisms. Nonmotile mutants similarly proliferated in the burn wound, but the characteristic bacteraemia and systemic invasion were markedly absent. The infection remained localized in the skin wound and the mice survived. The pattern of infection by nonmotile mutants in the colonization studies was very similar to that obtained with Fla+ cells in burned animals passively treated with antiflagellar antibody. These results add substantial support to the concept of motility as a P. aeruginosa virulence factor in invasive infections.     

Characterization of a new Pseudomonas aeruginosa strain NJ-15 as a potential biocontrol agent

Phylogenetic characterization of soil isolate NJ-15, based on sequence homology of a partial 746-bp fragment of 16SrDNA amplicon, with the ribosomal database sequences (http://www.msu.edu/RDP/cgis/phylip.cgi), validated the strain as Pseudomonas aeruginosa. The strain NJ-15 produced a substantial amount of indole acetic acid (IAA) in tryptophan-supplemented medium. Besides, the strain also exhibited significant production of both the siderophore and hydrogen cyanide (HCN) on chrome azurol S and King's B media, respectively. The data revealed lower HCN production under iron-limiting conditions vis-à-vis higher HCN release with iron stimulation. Significant growth inhibition of phytopathogenic fungi occurred in the order as Fusarium oxysporum > Trichoderma herizum > Alternaria alternata > Macrophomina phasiolina upon incubation with strain NJ-15 cells. Thus, the secondary metabolites producing new Pseudomonas aeruginosa strain NJ-15 exhibited innate potential of plant growth promotion and biocontrol activities in vitro.     

Assessment of Pseudomonas aeruginosa N5,N10-methylenetetrahydrofolate dehydrogenase-cyclohydrolase as a potential antibacterial drug target

The bifunctional enzyme methylenetetrahydrofolate dehydrogenase – cyclohydrolase (FolD) is identified as a potential drug target in Gram-negative bacteria, in particular the troublesome Pseudomonas aeruginosa. In order to provide a comprehensive and realistic assessment of the potential of this target for drug discovery we generated a highly efficient recombinant protein production system and purification protocol, characterized the enzyme, carried out screening of two commercial compound libraries by differential scanning fluorimetry, developed a high-throughput enzyme assay and prosecuted a screening campaign against almost 80,000 compounds. The crystal structure of P. aeruginosa FolD was determined at 2.2 Å resolution and provided a template for an assessment of druggability and for modelling of ligand complexes as well as for comparisons with the human enzyme. New FolD inhibitors were identified and characterized but the weak levels of enzyme inhibition suggest that these compounds are not optimal starting points for future development. Furthermore, the close similarity of the bacterial and human enzyme structures suggest that selective inhibition might be difficult to attain. In conclusion, although the preliminary biological data indicates that FolD represents a valuable target for the development of new antibacterial drugs, indeed spurred us to investigate it, our screening results and structural data suggest that this would be a difficult enzyme to target with respect to developing the appropriate lead molecules required to underpin a serious drug discovery effort.     

Analysis of FimX, a phosphodiesterase that governs twitching motility in Pseudomonas aeruginosa

Type IV pili (Tfp) are polar surface structures of Pseudomonas aeruginosa required for twitching motility, biofilm formation and adherence. One protein required for the assembly of tfp is FimX, which possesses both GGDEF and EAL domains characteristic of diguanylate cyclases and phosphodiesterases respectively. In this work we demonstrate that FimX has phosphodiesterase activity towards bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), but does not show diguanylate cyclase activity. Instead, the imperfect GGDEF domain of FimX likely serves to activate phosphodiesterase activity when bound to GTP, as has recently been described for the Caulobacter crescentus composite GGDEF-EAL protein, CC3396. Bacteria expressing FimX in which either the GGDEF or EAL domain is deleted or mutated have phenotypes indistinguishable from a DeltafimX strain, demonstrating the importance of both domains to function. Previous work has shown that FimX localizes to the bacterial pole. In this work we show that restriction of FimX to a single pole requires intact GGDEF and EAL domains. Deletion of the amino-terminal REC domain of FimX, which contains a putative polar localization signal, results in a protein that still supports intermediate levels of pilus assembly and function. RFP-FimXDeltaREC, unlike RFP-FimX, is no longer localized to the bacterial pole, while transmission electron microscopy shows that surface pili can originate from non-polar sites in this mutant. Although DeltafimX mutants show limited in vitro cytotoxicity, they are as virulent as the wild-type strain in a murine model of acute pneumonia.     

BifA, a cyclic-Di-GMP phosphodiesterase, inversely regulates biofilm formation and swarming motility by Pseudomonas aeruginosa PA14

The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner membrane and, in biochemical studies, that purified BifA protein exhibits phosphodiesterase activity in vitro but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the ΔbifA mutant exhibits increased cellular pools of c-di-GMP relative to the wild type and increased synthesis of a polysaccharide produced by the pel locus. This increased polysaccharide production is required for the enhanced biofilm formed by the ΔbifA mutant but does not contribute to the observed swarming defect. The ΔbifA mutation also results in decreased flagellar reversals. Based on epistasis studies with the previously described sadB gene, we propose that BifA functions upstream of SadB in the control of biofilm formation and swarming.     

An antibacterial pyrazole derivative from Burkholderia glumae, a bacterial pathogen of rice

Burkholderia glumae, a bacterial pathogen on rice, produced compounds in liquid culture that, in agar diffusion assays, gave strong inhibitory action against Erwinia amylovora, the bacterium responsible for fire blight disease of apple and pear trees. Products were isolated from culture medium by cation exchange and then purified by bioassay-guided chromatographic methods. Two major products were obtained, one of which was not active when fully purified. Each product showed a single ninhydrin-staining spot on TLC and a single HPLC peak. The non-active product was deduced from NMR, MS, and chemical data, to be the tripeptide l-alanyl-l-homoserinyl-l-aspartate. The NMR data for the active product demonstrated that it contained the same tripeptide, but functionalised at the β-carboxyl of the C-terminal aspartate, by a moiety that provided an additional 98 mass units to the parent tripeptide. Various data led to the interpretation that this moiety was a highly unusual oxygenated pyrazole structure, and thus the bioactive product was deduced to be 3-[l-alanyl-l-homoserinyl-l-aspartyl-β-carboxy]-4-hydroxy-5-oxopyrazole. This compound was found to inhibit the growth of a number of different bacterial species.     

Inverse regulation of biofilm formation and swarming motility by Pseudomonas aeruginosa PA14

We previously reported that SadB, a protein of unknown function, is required for an early step in biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa. Here we report that a mutation in sadB also results in increased swarming compared to the wild-type strain. Our data are consistent with a model in which SadB inversely regulates biofilm formation and swarming motility via its ability both to modulate flagellar reversals in a viscosity-dependent fashion and to influence the production of the Pel exopolysaccharide. We also show that SadB is required to properly modulate flagellar reversal rates via chemotaxis cluster IV (CheIV cluster). Mutational analyses of two components of the CheIV cluster, the methyl-accepting chemotaxis protein PilJ and the PilJ demethylase ChpB, support a model wherein this chemotaxis cluster participates in the inverse regulation of biofilm formation and swarming motility. Epistasis analysis indicates that SadB functions upstream of the CheIV cluster. We propose that P. aeruginosa utilizes a SadB-dependent, chemotaxis-like regulatory pathway to inversely regulate two key surface behaviors, biofilm formation and swarming motility.     

2-Heptyl-4-quinolone, a precursor of the Pseudomonas quinolone signal molecule, modulates swarming motility in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen capable of group behaviors, including biofilm formation and swarming motility. These group behaviors are regulated by both the intracellular signaling molecule c-di-GMP and acylhomoserine lactone quorum-sensing systems. Here, we show that the Pseudomonas quinolone signal (PQS) system also contributes to the regulation of swarming motility. Specifically, our data indicate that 2-heptyl-4-quinolone (HHQ), a precursor of PQS, likely induces the production of the phenazine-1-carboxylic acid (PCA), which in turn acts via an as-yet-unknown downstream mechanism to repress swarming motility. We show that this HHQ- and PCA-dependent swarming repression is apparently independent of changes in global levels of c-di-GMP, suggesting complex regulation of this group behavior.     

Stimulation of secretion into human and feline airways by Pseudomonas aeruginosa proteases

We have investigated the effect of elastase and alkaline protease from Pseudomonas aeruginosa on airway secretion into the trachea of anesthetized cats and from human bronchial mucosa in vitro. Secretory macromolecules were radiolabeled biosynthetically with two precursors in the cat, [3H]glucose and [35S]sulfate, and with [35S]-sulfate only in human tissue. Both enzymes (2.6 x 10(-9) to 1.3 x 10(-6)M elastase and 8 x 10(-9) to 2.4 x 10(-6)M alkaline protease) released radiolabeled macromolecules in a concentration-dependent manner from the two preparations. Purified elastase, 1.3 x 10(-6)M, released radiolabeled macromolecules (delta 3H = +397 +/- 72%, delta 35S 225 +/- 40% over control, P less than 0.001) and periodic acid-Schiff- (PAS) reactive glycoconjugates (delta PAS = +4.1 +/- 0.96 micrograms/min or +102 +/- 20%; P less than 0.01) from cat trachea, as did alkaline protease, 2.4 x 10(-6)M (delta 3H = +356 +/- 57%, delta 35S = +176 +/- 25%, delta PAS = +7.5 +/- 1.3 micrograms/min or 194 +/- 36%, P less than 0.001). Increases in 3H exceeded those of 35S, suggesting surface epithelium as the main source of secretion. Inhibition of enzyme activity abolished secretory effects. Both enzymes also stimulated secretion from human bronchus (e.g., with elastase, 1.3 x 10(-6)M: delta 35S = +331 +/- 67%, delta PAS = +4.3 +/- 0.92 micrograms/min or +131 +/- 24%, P less than 0.001; with alkaline protease, 2.4 x 10(-6)M: delta 35S = +220 +/- 67%, delta PAS = +12.7 +/- 3.2 micrograms/min or +575 +/- 245%, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

The potential biocontrol agent Pseudomonas antimicrobica inhibits germination of conidia and outgrowth of Botrytis cinerea

AIMS: Antifungal metabolites of Pseudomonas antimicrobica have previously been shown to inhibit conidial germination of the grey mould pathogen Botrytis cinerea. In this study, metabolites of the bacterium have been tested at different stages of Botrytis germination to determine their effects on germ tube production and extension. METHODS AND RESULTS: Metabolites were added to conidia that had been pre-incubated for either 120 or 255 min. Pseudomonas antimicrobica inhibited B. cinerea conidial germination and caused a significant reduction in germ tube extension, irrespective of the stage of germination. Abnormal germination and a reduction in the frequency of lateral branching of the germ tubes in the presence of the metabolites were also reported, suggesting interference with normal hyphal development. CONCLUSION: The bacterium can inhibit germination of conidia and extension of germ tubes at different stages of Botrytis development. SIGNIFICANCE AND IMPACT OF THE STUDY: The antagonistic activity of the bacterium has promising implications for its use as a biocontrol agent.