Lipopolysaccharide core phosphates are required for viability and intrinsic drug resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that is notorious for i ts intrinsic drug resistance. We have used chemical and genetic techniques to characterize three putative kinase genes that are involved in the additi on of phosphate to the inner core region of P. aeruginosa lipopolysaccharid e. The first gene is a waaP homologue, whereas the other two (wapP and wapQ ) are unique to P. aeruginosa. Repeated attempts using a variety of membran e-stabilizing conditions to generate waaP::Gm (Gm, gentamicin) or wapP::Gm mutants were unsuccessful. We were able to generate a chromosomal waaP muta nt that had a wild-type copy of either waaP(Pa) or waaP(Ec) in trans, but w ere unable to cure this plasmid-borne copy of the gene. These results are c onsistent with the fact that P. aeruginosa mutants lacking inner core hepto se (Hep) or phosphate have never been isolated and demonstrate the requirem ent of Hep-linked phosphate for P. aeruginosa viability. A wapQ::Gm mutant was isolated and it had an unaltered minimum inhibitory concentration (MIC) for novobiocin and only a small decrease in the MIC for sodium dodecyl sul phate (SDS), suggesting that the loss of a phosphate group transferred by W apQ may only be having a small impact on outer-membrane permeability. Nucle ar magnetic resonance and methylation linkage analysis showed that WaaP(Pa) could add one phosphate to O4 of HepI in a Salmonella typhimurium waaP mut ant. The expression of WaaP(Pa) increased the outer-membrane integrity of t hese complemented mutants, as evidenced by 35-fold and 75-fold increases in the MIC for novobiocin and SDS respectively. The S. typhimurium waaP mutan t transformed with both waaP and wapP had over 250-fold and 1000-fold incre ases, respectively, in these MICs. The inner core phosphates of P. aerugino sa appear to be playing a key role in the intrinsic drug resistance of this bacterium.