Proteome analysis of PA4203, a newly described regulator of fitness and

Abstract

In Pseudomonas aeruginosa the PA4203 gene, upstream of ppgL, encodes a LysR-type<br><br>regulator similar to BenM and CatM in Acinetobacter spp. We described that the<br><br>Pseudomonas aeruginosa ppgL (PA4204) gene, which encodes a protein with a signal<br><br>peptide and with a COG2706 domain corresponding to a 3-carboxy-cis, cis-muconate<br><br>lactonizing enzyme, could affect the fitness of P. aeruginosa. Cis,cis muconate is the effector<br><br>for BenM and CatM, suggesting that the product of PA4204 could be the effector for PA4203<br><br>and could be a molecule similar to cis,cis muconate. Interestingly, a deletion of PA4203<br><br>affected the level of expression of ppgL and mexGHI-opmD. Furthermorethe PA4203 gene<br><br>was highly expressed in a PA4204 mutant. This suggests that the product of PA4204 is the<br><br>effector of the LysR regulator, which negatively regulates its own transcription. The PA4203<br><br>mutant grew like the wild-type, but did not produce the blue-green pigment pyocyanin on<br><br>Pseudomonas P agar. Surprisingly, the same mutant was completely unable to produce the<br><br>siderophore pyoverdine in CAA medium, but the production of the second siderophore,<br><br>pyochelin, was not affected. The PA4203 mutant was hypomotile and also more sensitive to<br><br>H2O2 compared to that of wild-type. Proteome analysis was done using 2D-PAGE with<br><br>different staining methods (coomassie blue, silver staining, and DIGE). Cellular protein<br><br>extracts of the PA4203 showed a low expression of some outer membrane proteins (OmpA)<br><br>and extracellular chitinase. On the other hand, production of trigger factor, flagellin type B,<br><br>the two component response regulator PhoP/Q and 50S ribosomal protein L9 were more<br><br>pronounced in the PA4203 mutant. Secretome analysis showed that the production of two<br><br>different types of the flagellar capping protein FliD and FliG in the PA4203 mutant versus<br><br>WT, while the production of the chitin-binding protein CbpD precursor present in WT was<br><br>not observed in the PA4203 mutant. The above results suggest that the LysR type regulator<br><br>encoded by PA4203 is needed for the expression of ppgL (which is a modulator of quorum<br><br>sensing) and mexGHI-opmD (which are part of the PQS regulon) and for the production of<br><br>virulence factors.