Keith Poole

Crystal structure of the MexR repressor of the mexRAB-oprM multidrug efflux operon of Pseudomonas aeruginosa.

MexR is a member of the MarR family of bacterial transcriptional regulators and is the repressor for the MexAB-OprM operon, which encodes a tripartite multidrug efflux system inPseudomonas aeruginosa. Mutations in MexR result in increased resistance to multiple antibiotics due to overexpression of this efflux system. We have determined the crystal structure of MexR to 2.1-Å resolution in the absence of effector. The four copies of the MexR dimer in the asymmetric unit are observed in multiple conformations. Analysis of these conformational states in the context of a model of the MexR-DNA complex proposed in this study suggests that an effector-induced conformational change may inhibit DNA binding by reducing the spacing of the DNA binding domains. The inhibited conformation is exhibited by one of the four MexR dimers, which contains an ordered C-terminal tail from a neighboring monomer inserted between its DNA binding domains and which we propose may resemble the MexR-effector complex. Our results indicate that MexR may differ from the other described member of this family, MarR, in the nature of its effector, mode of DNA binding, and mechanism of regulation.

An anion-selective channel from the Pseudomonas aeruginosa outer membrane

Abstract Protein P from Pseudomonas aeruginosa outer membrane was reconstituted in lipid bilayer membranes from diphytanoylphosphatidylcholine. The reconstitution resulted in the formation of anion-selective channels with a conductance of 160 pS for 0.1 M chloride solution. The channels were at least 100-times more selective for anions than for cations as judged from zero-current membrane potentials. The single-channel conductance was dependent on the size of the different anions and saturated at higher salt concentrations suggesting single ion occupancy of the protein P channel.

Stress responses as determinants of antimicrobial resistance in Pseudomonas aeruginosa: multidrug efflux and more.

Pseudomonas aeruginosa is a notoriously antimicrobial-resistant organism that is increasingly refractory to antimicrobial chemotherapy. While the usual array of acquired resistance mechanisms contribute to resistance development in this organism a multitude of endogenous genes also play a role. These include a variety of multidrug efflux loci that contribute to both intrinsic and acquired antimicrobial resistance. Despite their roles in resistance, however, it is clear that these efflux systems function in more than just antimicrobial efflux. Indeed, recent data indicate that they are recruited in response to environmental stress and, therefore, function as components of the organisms stress responses. In fact, a number of endogenous resistance-promoting genes are linked to environmental stress, functioning as part of known stress responses or recruited in response to a variety of environmental stress stimuli. Stress responses are, thus, important determinants of antimicrobial resistance in P. aeruginosa. As such, they represent possible therapeutic targets in countering antimicrobial resistance in this organism.

Modification of the conductance, selectivity and concentration dependent saturation of Pseudomonas aeruginosa protein P channels by chemical acetylation

Protein P, an anion-specific channel-forming protein from the outer membrane of Pseudomonas aeruginosa was chemically modified by acetylation and syccinylation of its accessible amino groups. The chemically modified protein retained its ability to form oligomers on sodium dodecyl sulfate polyacrylamide gels, whereas only the acetylated protein formed channels in reconstitution experiments with lipid bilayers. Acetylated protein P demonstrated a substantially reduced mean single channel conductance (25 pS at 1 M KCl) compared to the native protein P channels (250 pS at 1 M KCl) when reconstituted into black lipid bilayer membranes. The homogeneous size distribution of single-channel conductances suggested that all of the protein P molecules had been acetylated. Zero-current potential measurements demonstrated that the acetylated protein P channel was only weakly selective for anions and allowed the permeation of cations, in contrast to the native protein P channels, which were more than 100-fold selective for anions over cations. The dependence of conductance on salt concentration was changed upon acetylation, in that acetylated protein P demonstrated a linear concentration-conductance relationship, whereas native protein P channels became saturated at high salt concentrations. These data strongly suggested that the basis of anion selectivity for native protein P channels is fixed amino groups. In agreement with this, we could demonstrate a 2.5-fold decrease in single-channel conductance between pH 7 and pH 9, between which pH values the epsilon-amino groups of amino acids would start to become deprotonated. Two alternative schemes for the topography of the protein P channel and localization of the fixed amino groups are presented and discussed.

Isolation of a Tn501 insertion mutant lacking porin protein P of Pseudomonas aeruginosa

SummaryIn order to demonstrate a role for anion-specific protein P channels in phosphate transport in Pseudomonas aeruginosa PAO, we wished to isolate a transposon insertion mutant deficient in protein P. A number of transposon delivery systems were tested which yielded, for the most part, whole plasmid inserts. Plasmid pMT1000 (Tsuda et al. 1984), a temperature-sensitive R68 plasmid carrying the transposon Tn501, was successfully employed in the isolation of a Tn501 insertion mutant lacking protein P under normally inducing conditions. To identify the mutant deficient in protein P, a protein P-specific polyclonal antiserum was used. This mutant, strain H576, was deficient in high-affinity phosphate transport exhibiting a Km for uptake (3.60±0.64 μM) almost ten times greater than that of the wild type strain (Km=0.39 μM). There was, however, no change in the Vmax for high-affinity phosphate transport as a result of the loss of protein P in this mutant. The protein P-deficiency of the mutant correlated with a growth defect in a phosphate-limited medium resulting in an 18%–35% decrease in growth when compared with the wild type.

Characterization and Chemical Modification of Small Anion Specific Channels formed in Lipid Bilayer Membranes by Outer Membrane Protein P or Pseudomonas aeruginosa

The movement of small molecules and ions across the outer membrane of gram-negative bacteria is mediated by a class of major proteins named porins (1). The porins form generally large water-filled pores with a diameter of 1.3-2.3 nm in the outer membrane (1) and in lipid bilayer membranes (2). These pores have a defined exclusion limit for hydrophilic solutes (3). A new outer membrane protein has been found to be induced in P. aeruginosa grown low-phosphate media. Studies with mutants have suggested that this protein, named protein P, is part of the phosphate uptake system (4). Lipid bilayer experiments suggested that the diameter of the protein-P pore is much smaller than that of the other porins(4).