Bacillus pumilus of Palk Bay origin inhibits quorum-sensing-mediated virulence factors in Gram-negative bacteria.
ABSTRACT The aim of the current study was to inhibit quoring-sensing(QS)-mediated virulence factors of representative Gram-negative bacteria by marine bacterial isolates. Bacteria isolated from Palk Bay sediments were screened for anti-QS activity. Eleven strains inhibited QS signals in Chromobacterium violaceum (ATCC 12472) and C. violaceum CV026. The marine bacterial strain S8-07 reduced the accumulation of N-acyl homoserine lactone (AHLs) and showed significant inhibition of LasA protease(76%), LasB elastase(84%), caseinase(70%), pyocyanin (84%), pyoverdin and biofilm formation(87%) in Pseudomonas aeruginosa PAO1. Strain S8-07 also showed highly significant reduction (90%) in prodigiosin, secreted casienase (92%), hemolytic activity (73%) and biofilm formation (61%) in Serratia marcescens. Strain S8-07, identified as Bacillus pumilus (accession number FJ584416), showed distinct profiles of inhibition against the virulence factors of both P. aeruginosa PAO1 (las, rhl) and S. marcescens (shl). Polar extraction and proteinase K treatment of the culture supernatant confirmed that the anti-QS activity of S8-07 was indeed due to a protein molecule. Acidification assay and HPLC analysis revealed that the degradation of AHL was not due to lactonase activity, but rather, was due to acylase activity of S8-07. Thus, novel anti-QS acylase activity is reported for the first time from a B. pumilus strain of marine origin.
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ABSTRACT: Secondary metabolites, including antibiotics, are produced in nature and serve survival functions for the organisms producing them. The antibiotics are a heterogeneous group, the functions of some being related to and others being unrelated to their antimicrobial activities. Secondary metabolites serve: (i) as competitive weapons used against other bacteria, fungi, amoebae, plants, insects, and large animals; (ii) as metal transporting agents; (iii) as agents of symbiosis between microbes and plants, nematodes, insects, and higher animals; (iv) as sexual hormones; and (v) as differentiation effectors. Although antibiotics are not obligatory for sporulation, some secondary metabolites (including antibiotics) stimulate spore formation and inhibit or stimulate germination. Formation of secondary metabolites and spores are regulated by similar factors. This similarity could insure secondary metabolite production during sporulation. Thus the secondary metabolite can: (i) slow down germination of spores until a less competitive environment and more favorable conditions for growth exist; (ii) protect the dormant or initiated spore from consumption by amoebae; or (iii) cleanse the immediate environment of competing microorganisms during germination.09/2000: pages 1-39;
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ABSTRACT: Acyl-homoserine lactones (AHLs) are employed by several Proteobacteria as quorum-sensing signals. Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients. Here we describe the isolation of a soil bacterium, Pseudomonas strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth. The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of Pseudomonas aeruginosa, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42 degrees C. The quorum-sensing bacterium P. aeruginosa, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth. It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs. In contrast to the growth observed with strain PAI-A, P. aeruginosa strain PAO1 growth on AHLs commenced only after extremely long lag phases. Liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase. A P. aeruginosa gene, pvdQ (PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a Ralstonia species. Escherichia coli expressing pvdQ catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL. P. aeruginosa engineered to constitutively express pvdQ did not accumulate its 3OC12HSL quorum signal when grown in rich media. However, pvdQ knockout mutants of P. aeruginosa were still able to grow by utilizing 3OC12HSL. To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other gamma-Proteobacteria, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.Applied and Environmental Microbiology 11/2003; 69(10):5941-9. · 3.68 Impact Factor
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ABSTRACT: In gram-negative bacterial pathogens, such as Pseudomonas aeruginosa and Yersinia pseudotuberculosis, cell-to-cell communication via the N-acylhomoserine lactone (AHL) signal molecules is involved in the cell population density-dependent control of genes associated with virulence. This phenomenon, termed quorum sensing, relies upon the accumulation of AHLs to a threshold concentration at which target structural genes are activated. By using biosensors capable of detecting a range of AHLs we observed that, in cultures of Y. pseudotuberculosis and P. aeruginosa, AHLs accumulate during the exponential phase but largely disappear during the stationary phase. When added to late-stationary-phase, cell-free culture supernatants of the respective pathogen, the major P. aeruginosa [N-butanoylhomoserine lactone (C4-HSL) and N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL)] and Y. pseudotuberculosis [N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL)] AHLs were inactivated. Short-acyl-chain compounds (e.g., C4-HSL) were turned over more extensively than long-chain molecules (e.g., 3-oxo-C12-HSL). Little AHL inactivation occurred with cell extracts, and no evidence for inactivation by specific enzymes was apparent. This AHL turnover was discovered to be due to pH-dependent lactonolysis. By acidifying the growth media to pH 2.0, lactonolysis could be reversed. By using carbon-13 nuclear magnetic resonance spectroscopy, we found that the ring opening of homoserine lactone (HSL), N-propionyl HSL (C3-HSL), and C4-HSL increased as pH increased but diminished as the N-acyl chain was lengthened. At low pH levels, the lactone rings closed but not via a simple reversal of the ring opening reaction mechanism. Ring opening of C4-HSL, C6-HSL, 3-oxo-C6-HSL, and N-octanoylhomoserine lactone (C8-HSL), as determined by the reduction of pH in aqueous solutions with time, was also less rapid for AHLs with more electron-donating longer side chains. Raising the temperature from 22 to 37 degrees C increased the rate of ring opening. Taken together, these data show that (i) to be functional under physiological conditions in mammalian tissue fluids, AHLs require an N-acyl side chain of at least four carbons in length and (ii) that the longer the acyl side chain the more stable the AHL signal molecule.Infection and Immunity 11/2002; 70(10):5635-46. · 4.07 Impact Factor