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Lipopolysaccharide core phosphates are required for viability and intrinsic drug resistance in Pseudomonas aeruginosa
Author(s) -
Walsh Andrew G.,
Matewish Mauricia J.,
Burrows Lori L.,
Monteiro Mario A.,
Perry Malcolm B.,
Lam Joseph S.
Publication year - 2000
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2000.01741.x
Subject(s) - biology , pseudomonas aeruginosa , mutant , novobiocin , bacterial outer membrane , microbiology and biotechnology , regulon , heptose , lipid a , gene , biochemistry , bacteria , escherichia coli , genetics , antibiotics
Pseudomonas aeruginosa is an opportunistic pathogen that is notorious for its intrinsic drug resistance. We have used chemical and genetic techniques to characterize three putative kinase genes that are involved in the addition of phosphate to the inner core region of P. aeruginosa lipopolysaccharide. 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 membrane‐stabilizing conditions to generate waaP::Gm (Gm, gentamicin) or wapP::Gm mutants were unsuccessful. We were able to generate a chromosomal waaP mutant that had a wild‐type copy of either waaP Pa or waaP Ec in trans , but were unable to cure this plasmid‐borne copy of the gene. These results are consistent with the fact that P. aeruginosa mutants lacking inner core heptose (Hep) or phosphate have never been isolated and demonstrate the requirement 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 sulphate (SDS), suggesting that the loss of a phosphate group transferred by WapQ may only be having a small impact on outer‐membrane permeability. Nuclear magnetic resonance and methylation linkage analysis showed that WaaP Pa could add one phosphate to O4 of HepI in a Salmonella typhimurium waaP mutant. The expression of WaaP Pa increased the outer‐membrane integrity of these complemented mutants, as evidenced by 35‐fold and 75‐fold increases in the MIC for novobiocin and SDS respectively. The S. typhimurium waaP mutant transformed with both waaP and wapP had over 250‐fold and 1000‐fold increases, respectively, in these MICs. The inner core phosphates of P. aeruginosa appear to be playing a key role in the intrinsic drug resistance of this bacterium.