Abstract
Chromobacterium violaceum abounds in soil and water ecosystems in tropical and subtropical regions and occasionally causes severe and often fatal human and animal infections. The quorum sensing (QS) system and biofilm formation are essential for C. violaceum's adaptability and pathogenicity, however, their interrelation is still unknown. C. violaceum's cell and biofilm morphology were examined by atomic force microscopy (AFM) in comparison with growth rates, QS-dependent violacein biosynthesis and biofilm biomass quantification. To evaluate QS regulation of these processes, the wild-type strain C. violaceum ATCC 31532 and its mini-Tn5 mutant C. violaceum NCTC 13274, cultivated with and without the QS autoinducer N-hexanoyl-L-homoserine lactone (C6-HSL), were used. We report for the first time the unusual morphological differentiation of C. violaceum cells, associated with biofilm development and directed by the QS autoinducer. AFM revealed numerous invaginations of the external cytoplasmic membrane of wild-type cells, which were repressed in the mutant strain and restored by exogenous C6-HSL. With increasing bacterial growth, polymer matrix extrusions formed in place of invaginations, whereas mutant cells were covered with a diffusely distributed extracellular substance. Thus, quorum sensing in C. violaceum involves a morphological differentiation that organises biofilm formation and leads to a highly differentiated matrix structure.
Highlights
Chromobacterium violaceum is a Gram-negative, facultative anaerobic, non-sporing beta-proteobacterium that is abundant in soil and water ecosystems in tropical and subtropical regions [1,2,3]
Using atomic force microscopy (AFM), we have revealed for the first time a morphological differentiation of C. violaceum cells associated with biofilm formation and directed by C6-HSL
In order to evaluate if violacein production and biofilm formation were dependent on cell density, C. violaceum ATCC 31532 was grown in LB broth for 24, 48 and 72 h (Fig. 1)
Summary
Chromobacterium violaceum is a Gram-negative, facultative anaerobic, non-sporing beta-proteobacterium that is abundant in soil and water ecosystems in tropical and subtropical regions [1,2,3]. The remarkable versatility and adaptability of C. violaceum have been explained by complete genome sequencing of this bacterium [9]; this revealed 4,431 open reading frames (ORFs) associated with energy generation, transport, signal transduction, cell motility, secretion, and secondary metabolism [10,11], such as is important for mammalian pathogenicity proteins [12]. A large number of ORFs related to the regulation of gene expression were found, and widespread utilisation of inducible quorum sensing systems to detect and respond to changes in cell population density have been demonstrated. C. violaceum uses a LuxIR-type quorum sensing system [13,14,15] in which CviI is an N-hexanoyl-L-homoserine lactone (C6-HSL) synthase, and CviR is a cytoplasmic receptor protein Expressed at high cell density, VioA – E proteins carry out the enzymatic oxidation and coupling of two molecules of tryptophan to give a rearranged pyrrolidone-containing scaffold in the final purple pigment violacein [18]
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