Demonstration and chemical modification of a specific phosphate binding site in the phosphate-starvation-inducible outer membrane porin protein P of Pseudomonas aeruginosa (original) (raw)
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Identification of a major protein upon phosphate starvation of Pseudomonas aeruginosa PAO1
Journal of Basic Microbiology, 2003
To understand the physiology of non-differentiating bacteria exposed to nutrient deprivation and stress, various approaches have been employed in combination with detailed analysis of protein synthesis pattern. In this study, separation of proteins from clarified cell extracts of Pseudomonas aeruginosa PAO1 grown under phosphorus limiting conditions was achieved by high resolution twodimensional gel electrophoresis (2-DE). Limitation of phosphate in the growth medium revealed significant differences in the 2-DE pattern of proteins between phosphate starved cells and an unstarved control. A major protein identified as PstS, a phosphate binding protein of the pts operon was exclusively found on 2-DE gels of phosphate starved bacteria. The identity of protein was established based on the results of EDMAN degradation, amino acid analysis and mass spectrometry. PstS was also found in other pseudomonads, and therefore, it can be used as a landmark protein in proteomic studies. Additionally, we propose utilizing pstS of pseudomonads for testing bioavailable phosphate from soils and water streams.
Expression in Escherichia coli and function of Pseudomonas aeruginosa outer membrane porin protein F
Journal of bacteriology, 1986
The gene encoding porin protein F of Pseudomonas aeruginosa was cloned onto a cosmid vector into Escherichia coli. Protein F was expressed as the predominant outer membrane protein in a porin-deficient E. coli background and was clearly visible on one-dimensional sodium dodecyl sulfate-polyacrylamide gels in a porin-sufficient background. The identity of the protein F from the E. coli clone and native P. aeruginosa protein F was demonstrated by their identical mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoretograms, 2-mercaptoethanol modifiabilities, and reactivities with monoclonal antibodies specific of two separate epitopes of protein F. In the course of gene subcloning, a 2-kilobase DNA fragment was isolated, with an apparent truncation of the part of the gene encoding the carboxy terminus of protein F. This subclone produced a 24,000-molecular-weight, outer membrane-associated, truncated protein F derivative which was not 2-mercaptoethanol modifiable and whi...
Antimicrobial agents and chemotherapy, 1999
Pseudomonas aeruginosa OprD is a specific porin which facilitates the uptake of basic amino acids and imipenem, a carbapenem antibiotic. Resistance to imipenem due to the loss of OprD is an important mechanism for the loss of clinical effectiveness. To investigate the negative regulatory mechanisms influencing oprD expression, a gene upstream of the coregulated mexEF-oprN efflux operon, designated mexT, was cloned. The predicted 304-amino-acid mature MexT protein showed strong homology to LysR-type regulators. When overexpressed it induced the expression of the mexEF-oprN efflux operon while decreasing the level of expression of OprD. The use of an oprD::xylE transcriptional fusion indicated that it acted by repressing the transcription of oprD. Salicylate, a weak aromatic acid known to reduce porin expression and induce low levels of multiple antibiotic resistance in Escherichia coli, was able to induce imipenem resistance and reduce the expression of OprD but not multiple antibiot...
Structure, Dynamics, and Substrate Specificity of the OprO Porin from Pseudomonas aeruginosa
Biophysical journal, 2015
The outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier between cell and environment. For nutrient acquisition, the OM contains a number of channels that mediate uptake of small molecules by diffusion. Many of these channels are specific, i.e., they prefer certain substrates over others. In electrophysiological experiments, the OM channels OprP and OprO from Pseudomonas aeruginosa show a specificity for phosphate and diphosphate, respectively. In this study we use x-ray crystallography, free-energy molecular dynamics (MD) simulations, and electrophysiology to uncover the atomic basis for the different substrate specificity of these highly similar channels. A structural analysis of OprP and OprO revealed two crucial differences in the central constriction region. In OprP there are two tyrosine residues, Y62 and Y114, whereas the corresponding residues in OprO are phenylalanine F62 and aspartate D114. To probe the importance of these two residue...
Two different mechanisms mediate chemotaxis to inorganic phosphate in Pseudomonas aeruginosa
Scientific Reports, 2016
Inorganic phosphate (Pi) is a central signaling molecule that modulates virulence in various pathogens. In Pseudomonas aeruginosa, low Pi concentrations induce transcriptional alterations that increase virulence. Also, under low Pi levels, P. aeruginosa exhibits Pi chemotaxis—a process mediated by the two non-paralogous receptors CtpH and CtpL. Here we show that the two receptors operate via different mechanisms. We demonstrate that the ligand binding domain (LBD) of CtpH but not CtpL binds Pi directly. We identify the periplasmic ligand binding protein PstS as the protein that binds in its Pi loaded state to CtpL, resulting in receptor stimulation. PstS forms part of the Pi transporter and has thus a double function in Pi transport and chemotaxis. The affinity of Pi for CtpH was modest whereas that for PstS very high, which may explain why CtpH and CtpL mediate chemotaxis to high and low Pi concentrations, respectively. The pstS/ctpH double mutant was almost devoid of Pi taxis, ind...
Molecular Microbiology, 2000
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 membranestabilizing 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 plasmidborne 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 outermembrane 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 75fold 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.