Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones.
Quorum sensing relies upon the interaction of a diffusible signal molecule with a transcriptional activator protein to couple gene expression with cell population density. In Gram-negative bacteria, such signal molecules are usually N-acylhomoserine lactones (AHLs) which differ in the structure of their N-acyl side chains. Chromobacterium violaceum, a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein. Previously the authors described a violacein-negative, mini-Tn5 mutant of C. violaceum (CV026) in which pigment production can be restored by incubation with supernatants from the wild-type strain. To develop this mutant as a general biosensor for AHLs, the natural C. violaceum AHL molecule was first chemically characterized. By using solvent extraction, HPLC and mass spectrometry, a single AHL, N-hexanoyl-L-homoserine lactone (HHL), was identified in wild-type C. violaceum culture supernatants which was absent from CV026. Since the production of violacein constitutes a simple assay for the detection of AHLs, we explored the ability of CV026 to respond to a series of synthetic AHL and N-acylhomocysteine thiolactone (AHT) analogues. In CV026, violacein is inducible by all the AHL and AHT compounds evaluated with N-acyl side chains from C4 to C8 in length, with varying degrees of sensitivity. Although AHL compounds with N-acyl side chains from C10 to C14 are unable to induce violacein production, if an activating AHL (e.g. HHL) is incorporated into the agar, these long-chain AHLs can be detected by their ability to inhibit violacein production. The versatility of CV026 in facilitating detection of AHL mixtures extracted from culture supernatants and separated by thin-layer chromatography is also demonstrated. These simple bioassays employing CV026 thus greatly extend the ability to detect a wide spectrum of AHL signal molecules.
McClean
Kay H
KH
Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.|Department of Applied Biochemistry and Food Science, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
Winson
Michael K
MK
Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.|Department of Applied Biochemistry and Food Science, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
Fish
Leigh
L
Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.|Department of Applied Biochemistry and Food Science, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
Taylor
Adrian
A
Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.|Department of Applied Biochemistry and Food Science, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
Chhabra
Siri Ram
SR
Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
Camara
Miguel
M
Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
Daykin
Mavis
M
Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.|Department of Applied Biochemistry and Food Science, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
Lamb
John H
JH
MRC Toxicology Unit, University of Leicester, Leicester LE1 9HN, UK.
Swift
Simon
S
Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.|Department of Applied Biochemistry and Food Science, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
Bycroft
Barrie W
BW
Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
Stewart
Gordon S A B
GSAB
Department of Applied Biochemistry and Food Science, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
Williams
Paul
P
Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
D002861:Chromobacterium / Q000235:genetics / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial; D006714:Homoserine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D007211:Indoles; D007783:Lactones / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D014344:Trypanocidal Agents
D007211:Indoles; D007783:Lactones; D014344:Trypanocidal Agents; D006714:Homoserine; C063155:violacein