Previous studies have shown that several genes take part in the regulation of AlgU activation and alginate overproduction. MucA is a trans-membrane protein that negatively regulates mucoidy by acting as an anti-sigma factor

via sequestering AlgU to the cytoplasmic membrane [7]; MucB and intra-membrane proteases AlgW, MucP and ClpXP were reported to affect alginate production by affecting the stability of MucA [8]. A small envelope protein called MucE was found to be a positive regulator for mucoid conversion in P. aeruginosa strains with a wild type MucA [9]. The mechanism for mucE induced mucoidy is due to its C-terminal –WVF signal, which can activate the protease AlgW possibly by interaction with the PDZ domain [9]. Upon activation, AlgW initiates the proteolytic degradation of the periplasmic portion of MucA, causing the release of AlgU to drive expression of the alginate biosynthetic operon [9]. While INCB024360 datasheet the function of MucE as an alginate inducer was identified, its physiological role, and its role in the regulation of mucoidy in clinical isolates, remains unknown. Comparative analysis through Basic Local Alignment Search Tool (BLAST) using the

genomes of Pseudomonas species from the public databases reveals that MucE orthologues are found only in the strains of P. aeruginosa[9]. In order to study the role selleck kinase inhibitor and regulation of MucE in P. aeruginosa, we first mapped the mucE transcriptional start site. We then examined the effect of five different sigma factors on the expression of mucE in vivo. Different cell wall stress agents were tested for the induction of mucE transcription. Expression of MucE was also analyzed in non-mucoid CF isolates to determine its ability to induce alginate overproduction. Methods Bacteria strains, plasmids, and growth conditions Bacterial strains and plasmids used in this eltoprazine study are shown in Additional file 1: Table S1. E. coli strains were grown at 37°C in Luria broth (LB, Tryptone 10 g/L, Yeast extract 5 g/L and sodium chloride

5 g/L) or LB agar. P. aeruginosa strains were grown at 37°C in LB or on Pseudomonas isolation agar (PIA) plates (Difco). When required, carbenicillin, tetracycline or gentamicin were added to the growth media. The concentration of carbenicillin, tetracycline or gentamycin was added at the following concentrations: for LB broth or plates 100 μg ml-1, 20 μg ml-1 or 15 μg ml-1, respectively. The concentration of carbenicillin, tetracycline or gentamycin to the PIA plates was 300 μg ml-1, 200 μg ml-1 or 200 μg ml-1, respectively. The mucE primer extension assay Total RNA was isolated from P. aeruginosa PAO1 grown to an OD600 of 0.6 in 100 ml LB at 37°C as previously described [10]. The total RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA) per the manufacturer’s instructions.