Transcriptional analysis of pha genes in Pseudomonas mediterranea CFBP 5447 grown on glycerol

We analysed the draft genome sequence of Pseudomonas mediterranea CFBP 5447 in order to identify firstly the central metabolic pathways that convert fatty acids or carbohydrate intermediates into mcl-PHA and secondly the genes involved in glycerol metabolism (glpF, glpK, glpD, glpR). Absence of the glpF gene, which codifies for the “glycerol uptake facilitator protein”, was highlighted. In order to understand the expression of the pha gene cluster, we investigated the promoter activity of phaC1, phaC2, phaZ, phaD and phaI genes. When glycerol was present as the carbon source, PI was found to be the most active promoter. Expression analysis of the knock-out mutant of the phaD gene, which is a transcriptional regulator belonging to the TetR family, showed that PhaD acts as an activator of the phaI promoter which, in turn, triggers the transcription of the phaIF operon. The activation of PC1, which controls the phaC1ZC2D, by PhaD, was less efficient than PI

Proof of principle of a novel impedance microbiology method based on bacteriophages functionalized paramagnetic nanobeads

A novel and unconventional approach to impedance microbiology for the detection of Escherichia coli is under investigation. The detection principle of solution conductivity variation is based on intracellular content escape resulting from bacteriophage generated cell lysis. Bare electrodes on chip included in a PDMS chamber were applied to the impedance spectra measurements. Proofs of principle experiments were performed. In parallel, paramagnetic nano-beads were functionalised with selective phages for sample magnetic concentration and future methods integration. The system potential detection limit is about 10 CFU/chamber and provides the means for selective detection of viable cells only. The methods integration could provide cost–effective results in less than 1 hour