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Saturday, 23 September 2017
Violacein Biosynthesis PDF Print

Gram-negative bacterium Chromobacterium violaceum (ATCC 12472) has several biotechnological applications. Violacein, the characteristic pigment that gives its name, has been shown to act as an antibiotic, antitumoral, and anti-Trypanosome cruzi (the Chagas disease agent) molecule.

Our major interest in the violacein biosynthesis is related to its production by a “safe” organism (We have recently cloned the responsible genes in E. coli), so that it could be used in pre-clinical and further clinical studies.


Regulation of Violacein Biosynthesis and Quorum Sensing Signaling in Chromobacterium violaceum ATCC 12472
Quorum sensing mechanism is one of the regulatory mechanisms responsible for controlling violacein biosyntheses in Chromobacterium violaceum (strain ATCC 12472). Supernatant from stationary phase of C. violaceum cultures were used to identify acyl homoserine lactone molecules (AHL) by solvent extraction. Active molecules were purified using reverse phase HPLC. Bio-reporters were used for detection of short and long chain AHLs. We identified multiple signal molecules: N-hexanoyl homoserine-L-lactone (HHL), N-oxohexanoyl-L-homoserine lactone (OHHL) and N-oxododecanoyl-L-homoserine lactone (OdDHL). In a previous work, only one signal molecule (HHL) has been identified in another wild type strain of C. violaceum, suggesting that the quorum sensing regulatory mechanism can be different, even for two wild type strains of the same microorganism. In this work, in collaboration with The Quorum Sensing Group of the Institute of Infection, Immunity and Inflamation at The University of Nottingham, England, we constructed the first mutants of C. violaceum strain ATCC 12472 by random mini-Tn5 luxCDABE transposon mutagenesis. In order to optimize the mutant selection, resistance to ethidium bromide, and a wide variety of antibiotics were tested. Homoserine lactones activities were also performed on each mutant. Genes that regulate the violacein production were found by tracking the transposon insertion in five mutants that characterized the down-regulation of violacein. Our findings have revealed there is much greater complexity to quorum sensing in violacein regulation in C. violaceum. This may also apply to other quorum sensing systems yet to be characterized.
 
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