Identification of N-acyl-l-homoserine lactones produced by non-pigmented Chromobacterium aquaticum CC-SEYA-1T and pigmented Chromobacterium subtsugae PRAA4-1T

Many members of the genus Chromobacterium produce violacein, a characteristic purple pigment which is induced by small diffusible N-acyl homoserine lactones (AHL) quorum-sensing molecules. In this study, the production of AHL of the non-pigmented C. aquaticum CC-SEYA-1T and the pigmented C. subtsugae PRAA4-1T were determined by using a CV026 biosensor assay. The profile of AHL was identified from the extracts of stationary phase cultures using gas chromatography–mass spectroscopy (GC–MS) and thin layer chromatography (TLC). CV026 biosensor assay revealed that both the non-pigmented C. aquaticum CC-SEYA-1T and the pigmented C. subtsugae PRAA4-1T produced AHL molecules, which were identified, respectively, as N-octanoyl homoserine lactone (OHL) [also known as C-8 homoserine lactone (C8-HSL)] and N-hexanoyl homoserine lactone (HHL) [also known as C-6 homoserine lactone (C6-HSL)]. The pigment produced by C. subtsugae PRAA4-1T was similar to that of Chromobacterium violaceum ATCC12472T but no characteristic visible spectral peaks of the pigment were observed in the extracts of C. aquaticum CC-SEYA-1T. In addition, C. aquaticum CC-SEYA-1T and C. subtsugae PRAA4-1T showed hemolytic activities

Functional identification of the prnABCD operon and its regulation in Serratia plymuthica

The antibiotic pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite that plays an important role in the biocontrol of plant diseases due to its broad-spectrum of antimicrobial activities. The PRN biosynthetic gene cluster remains to be characterised in Serratia plymuthica, though it is highly conserved in PRN-producing bacteria. To better understand PRN biosynthesis and its regulation in Serratia, the prnABCD operon from S. plymuthica G3 was cloned, sequenced and expressed in Escherichia coli DH5α. Furthermore, an engineered strain prnind which is a conditional mutant of G3 prnABCD under the control of the Ptac promoter was constructed. This mutant was able to overproduce PRN with isopropylthiogalactoside (IPTG) induction by overexpressing prnABCD, whilst behaving as a conditional mutant of G3 prnABCD in the absence of IPTG. These results confirmed that prnABCD is responsible for PRN biosynthesis in strain G3. Further experiments involving lux-/dsRed-based promoter fusions, combined with site-directed mutagenesis of the putative σS extended -10 region in the prnA promoter, and liquid chromatography-mass spectrometry (LC-MS) analysis extended our previous knowledge about G3, revealing that quorum sensing (QS) regulates PRN biosynthesis through cross talk with RpoS, which may directly activated prnABCD transcription. These findings suggest that PRN in S. plymuthica G3 is produced in a tightly controlled manner, and has diverse functions, such as modulation of cell motility, in addition to antimicrobial activities. Meanwhile, the construction of inducible mutants could be a powerful tool to improve PRN production, beyond its potential use for the investigation of the biological function of PRN

Evaluation of the effects of selected phytochemicals on quorum sensing inhibition and in vitro cytotoxicity

Quorum sensing (QS) is an important regulatory mechanism in biofilm formation and differentiation. Interference with QS can affect biofilm development and antimicrobial susceptibility. This study evaluates the potential of selected phytochemical products to inhibit QS. Three isothiocyanates (allylisothiocyanate - AITC, benzylisothiocyanate - BITC and 2-phenylethylisothiocyanate - PEITC) and six phenolic products (gallic acid - GA, ferulic acid - FA, caffeic acid - CA, phloridzin - PHL, (-) epicatechin - EPI and oleuropein glucoside - OG) were tested. A disc diffusion assay based on pigment inhibition in Chromobacterium violaceum CV12472 was performed. In addition, the mechanisms of QS inhibition (QSI) based on the modulation of N-acyl homoserine lactone (AHLs) activity and synthesis by the phytochemicals were investigated. The cytotoxicity of each product was tested on a cell line of mouse lung fibroblasts. AITC, BITC and PEITC demonstrated a capacity for QSI by modulation of AHL activity and synthesis, interfering the with QS systems of C. violaceum CviI/CviR homologs of LuxI/LuxR systems. The cytotoxic assays demonstrated low effects on the metabolic viability of the fibroblast cell line only for FA, PHL and EPI.This work was supported by Operational Programme for Competitiveness Factors - COMPETE, FCT/MEC (PIDDAC) and FEDER through Projects Bioresist - PTDC/EBB-EBI/105085/2008; Phytodisinfectants - PTDC/DTP-SAP/1078/2012 (COMPETE: FCOMP-01-0124-FEDER-028765) and the PhD grants awarded to Ana Abreu (SFRH/BD/84393/2012) and Anabela Borges (SFRH/BD/63398/2009). The authors are also very grateful to Professor Robert McLean (Texas University, USA) for providing the bacterial strains