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PubMed1992

Siderophore-specific induction of iron uptake in Pseudomonas aeruginosa.

Pseudomonas aeruginosa has two siderophore-based high-affinity iron-uptake systems utilizing pyoverdin and pyochelin. Using strain IA1, a mutant deficient in production of both siderophores, we have shown that addition of purified siderophore to the growth medium induces expression of specific iron-regulated outer-membrane proteins and increases 55Fe-siderophore transport. Addition of pyoverdin from the parent strain PAO1 or from a clinical strain 0:12 induced expression of an 85 kDa IROMP and increased the rate of 55Fe-pyoverdin transport. Transport rates for 55Fe-PAO1 pyoverdin increased from 1.27 to 3.57 pmol Fe min-1 per 10(9) cells. Addition of purified pyochelin induced expression of a 75 kDa IROMP accompanied with increased 55Fe-pyochelin uptake without affecting 55Fe-pyoverdin transport. 55Fe-pyochelin transport increased from 0.3 to 10.6 pmol min-1 per 10(9) cells. Addition of pyoverdin from the parent strain or a chromatographically distinct pyoverdin caused increased reactivity with an anti-85 kDa mAb in Western blotting, indicating that the same receptor is being induced. These results suggest that P. aeruginosa can respond specifically to the presence of siderophore and moreover that not only can the pyoverdin receptor transport its cognate ferri-pyoverdin but also different ferri-pyoverdins, albeit at a reduced rate.

Bacterial Outer Membrane Proteins↗

PubMed1999

Emergence of multidrug-resistant mutants is increased under antibiotic selective pressure in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most important opportunistic pathogens involved in nosocomial infections, cystic fibrosis patients included. Hospital isolates frequently present multidrug-resistance (MDR) phenotypes as the consequence of constant antibiotic selective pressure. The kinetics of emergence of P. aeruginosa MDR mutants under antibiotic selective pressure indicated that long-term incubation in the presence of the bacteriostatic antibiotic tetracycline increases the mutation rate per cell per day of P. aeruginosa PAO1 by several orders of magnitude. The tetracycline-resistant mutants obtained were stable, showed decreased susceptibility to antibiotics belonging to different structural families, and contained an outer-membrane protein not present in the wild-type P. aeruginosa strain PAO1. These data are consistent with the hypothesis that incubation in the presence of tetracycline favours the emergence of MDR mutants in P. aeruginosa. The results are relevant for understanding the rapid emergence of antibiotic-resistant mutants among bacterial populations during infections. Their relationship to other models of increased mutagenesis under stress is discussed with respect to the adaptive mutation phenomenon.

Anti-Bacterial Agents↗

PubMed1977

Hydrolysis of lithocholate sulfate by Pseudomonas aeruginosa.

Pseudomonas aeruginosa was found to be able to hydrolyze bile sulfate. This property was observed when lithocholate sulfate was substituted for the sulfur source in the culture medium. The addition of MgSO4 to the medium inhibited the hydrolysis of the bile sulfate.

Cholic Acids↗

PubMed1994

Acute otitis media increases middle ear susceptibility to nasal injection of Pseudomonas aeruginosa.

Pseudomonas aeruginosa was injected intranasally into four groups of chinchillas to determine if these bacteria can invade the middle ear by way of the eustachian tube. One group completed penicillin treatment of bilateral penumococcal otitis media (POM), and the second group started penicillin treatment of bilateral POM at the time of P. aeruginosa injection. A third group had no POM, but completed a course of penicillin treatment before P. aeruginosa injection, and a fourth group had no POM and received no penicillin. Middle ear susceptibility to nasally injected P. aeruginosa was significantly higher in animals with POM (61%) than in animals without POM (32%, p = 0.001). Forced eustachian tube opening pressures did not correlate with P. aeruginosa susceptibility. Thus, P. aeruginosa, the principle pathogen of chronic suppurative otitis media, can invade the middle ear by way of the eustachian tube, and acute otitis media predisposes to middle ear infection by P. aeurginosa.

Acute Disease↗

PubMed1999

RpoS-dependent stress tolerance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is able to persist during feast and famine in many different environments including soil, water, plants, animals and humans. The alternative sigma factor encoded by the rpoS gene is known to be important for survival under stressful conditions in several other bacterial species. To determine if the P. aeruginosa RpoS protein plays a similar role in stationary-phase-mediated resistance, an rpoS mutant was constructed and survival during exposure to hydrogen peroxide, high temperature, hyperosmolarity, low pH and ethanol was investigated. Disruption of the rpoS gene resulted in a two- to threefold increase in the rate of kill of stationary-phase cells. The rpoS mutant also survived less well than the parental strain during the initial phase of carbon or phosphate-carbon starvation. However, after 25 d starvation the remaining population of culturable cells was not significantly different. Stationary-phase cells of the RpoS-negative strain were much more stress resistant than exponentially growing RpoS-positive cells, suggesting that factors other than the RpoS protein must be associated with stationary-phase stress tolerance in P. aeruginosa. Comparison of two-dimensional PAGE of the rpoS mutant and the parental strain showed four major modifications of protein patterns associated with the rpoS mutation.

Bacterial Proteins↗

PubMed2000

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

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 waaPPa or waaPEc 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 WaaPPa could add one phosphate to O4 of HepI in a Salmonella typhimurium waaP mutant. The expression of WaaPPa 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.

Carbohydrate Sequence↗

PubMed1998

Synthesis of the A-band polysaccharide sugar D-rhamnose requires Rmd and WbpW: identification of multiple AlgA homologues, WbpW and ORF488, in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is capable of producing various cell-surface polysaccharides including alginate, A-band and B-band lipopolysaccharides (LPS). The D-mannuronic acid residues of alginate and the D-rhamnose (D-Rha) residues of A-band polysaccharide are both derived from the common sugar nucleotide precursor GDP-D-mannose (D-Man). Three genes, rmd, gmd and wbpW, which encode proteins involved in the synthesis of GDP-D-Rha, have been localized to the 5' end of the A-band gene cluster. In this study, WbpW was found to be homologous to phosphomannose isomerases (PMIs) and GDP-mannose pyrophosphorylases (GMPs) involved in GDP-D-Man biosynthesis. To confirm the enzymatic activity of WbpW, Escherichia coli PMI and GMP mutants deficient in the K30 capsule were complemented with wbpW, and restoration of K30 capsule production was observed. This indicates that WbpW, like AlgA, is a bifunctional enzyme that possesses both PMI and GMP activities for the synthesis of GDP-D-Man. No gene encoding a phosphomannose mutase (PMM) enzyme could be identified within the A-band gene cluster. This suggests that the PMM activity of AlgC may be essential for synthesis of the precursor pool of GDP-D-Man, which is converted to GDP-D-Rha for A-band synthesis. Gmd, a previously reported A-band enzyme, and Rmd are predicted to perform the two-step conversion of GDP-D-Man to GDP-D-Rha. Chromosomal mutants were generated in both rmd and wbpW. The Rmd mutants do not produce A-band LPS, while the WbpW mutants synthesize very low amounts of A band after 18 h of growth. The latter observation was thought to result from the presence of the functional homologue AlgA, which may compensate for the WbpW deficiency in these mutants. Thus, WbpW AlgA double mutants were constructed. These mutants also produced low levels of A-band LPS. A search of the PAO1 genome sequence identified a second AlgA homologue, designated ORF488, which may be responsible for the synthesis of GDP-D-Man in the absence of WbpW and AlgA. Polymerase chain reaction (PCR) amplification and sequence analysis of this region reveals three open reading frames (ORFs), orf477, orf488 and orf303, arranged as an operon. ORF477 is homologous to initiating enzymes that transfer glucose 1-phosphate onto undecaprenol phosphate (Und-P), while ORF303 is homologous to L-rhamnosyltransferases involved in polysaccharide assembly. Chromosomal mapping using pulsed field gel electrophoresis (PFGE) and Southern hybridization places orf477, orf488 and orf303 between 0.3 and 0.9 min on the 75 min map of PAO1, giving it a map location distinct from that of previously described polysaccharide genes. This region may represent a unique locus within P. aeruginosa responsible for the synthesis of another polysaccharide molecule.

Amino Acid Sequence↗

PubMed2004

Analysis of quorum sensing-deficient clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces multiple virulence factors and causes different types of infections. Previous clinical studies identified P. aeruginosa isolates that lack individual virulence factors. However, the impact of losing several virulence factors simultaneously on the in vivo virulence of P. aeruginosa is not completely understood. The P. aeruginosa cell-to-cell communication system, or quorum sensing (QS), controls the production of several virulence factors. Animal studies using constructed QS mutants indicated that loss of the QS system severely impacts the virulence of P. aeruginosa. In this study, we tried to determine if deficiency within the QS system compromises the ability of P. aeruginosa to establish infections in humans. We have identified five QS-deficient strains through screening 200 isolates from patients with urinary tract, lower respiratory tract and wound infections. These strains lacked LasB and LasA activities and produced either no or very low levels of the autoinducers N-(3-oxododecanoyl) homoserine lactone and N-butyryl homoserine lactone. PCR analysis revealed that three isolates contained all four QS genes (lasI, lasR, rhlI and rhlR) while two isolates lacked both the lasR and rhlR genes. We also examined the five isolates for other virulence factors. The isolates produced variable levels of exotoxin A and, with one exception, were deficient in pyocyanin production. One isolate produced the type III secretion system (TTSS) effector proteins ExoS and ExoT, two isolates produced ExoT only and two isolates produced no TTSS proteins. The isolates produced weak to moderate biofilms on abiotic surfaces. Analysis of the patients' data revealed that two of the isolates represented a single strain that was isolated twice from the same patient within a 1 month interval. One QS-deficient clinical isolate (CI-1) lacked all tested virulence factors and produced a weak biofilm. These results suggest that naturally occurring QS-deficient strains of P. aeruginosa do occur and are capable of causing infections; and, that besides the known virulence factors, additional factors may contribute to the ability of certain strains such as CI-1 to establish an infection.

4-Butyrolactone↗

PubMed2023

Genome-wide screen in human plasma identifies multifaceted complement evasion of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is a leading cause of bacteremia with a high mortality rate. We recently reported that P. aeruginosa forms a persister-like sub-population of evaders in human plasma. Here, using a gain-of-function transposon sequencing (Tn-seq) screen in plasma, we identified and validated previously unknown factors affecting bacterial persistence in plasma. Among them, we identified a small periplasmic protein, named SrgA, whose expression leads to up to a 100-fold increase in resistance to killing. Additionally, mutants in pur and bio genes displayed higher tolerance and persistence, respectively. Analysis of several steps of the complement cascade and exposure to an outer-membrane-impermeable drug, nisin, suggested that the mutants impede membrane attack complex (MAC) activity per se. Electron microscopy combined with energy-dispersive X-ray spectroscopy (EDX) revealed the formation of polyphosphate (polyP) granules upon incubation in plasma of different size in purD and wild-type strains, implying the bacterial response to a stress signal. Indeed, inactivation of ppk genes encoding polyP-generating enzymes lead to significant elimination of persisting bacteria from plasma. Through this study, we shed light on a complex P. aeruginosa response to the plasma conditions and discovered the multifactorial origin of bacterial resilience to MAC-induced killing.

Humans↗

PubMed1988

Purification, crystallisation and characterization of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa ATCC 17933 when grown on ethanol produces high levels of a quinoprotein ethanol dehydrogenase, which amounts to 7% of the soluble protein. The enzyme has been purified to homogeneity and it crystallizes readily in the presence of polyethylene glycol 1550 or 6000. The ethanol dehydrogenase (Km(ethanol) = 14 microM) resembles the dye-dependent quinoprotein methanol dehydrogenases of methylotrophic bacteria, but has a low affinity for methanol (Km (methanol) = 94mM). In addition the enzyme oxidizes secondary alcohols. With its catalytic properties the ethanol dehydrogenase is similar to the enzyme isolated from P. aeruginosa LMD 80.53 (Groen, B., Frank, J. Jzn. & Duine, J.A. (1984) Biochem. J. 223, 921-924). In contrast to this enzyme from P. aeruginosa LMD 80.53, which is a monomer, the ethanol dehydrogenase isolated from P. aeruginosa ATCC 17933 is a dimer of identical subunits of relative molecular mass 60,000. The N-terminal amino acid is lysine. Inactivation with cyclopropanone ethylhemiketal reveals one molecule of pyrroloquinoline quinone per subunit. As shown by active enzyme sedimentation, the dimer is the enzymatically active form.

Alcohol Oxidoreductases↗

PubMed2002

Multidrug efflux systems play an important role in the invasiveness of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic human pathogen. Certain strains can transmigrate across epithelial cells, and their invasive phenotype is correlated with capacity to cause invasive human disease and fatal septicemia in mice. Four multidrug efflux systems have been described in P. aeruginosa, however, their contribution to virulence is unclear. To clarify the role of efflux systems in invasiveness, P. aeruginosa PAO1 wild-type (WT) and its efflux mutants were evaluated in a Madin-Darby canine kidney (MDCK) epithelial cell monolayer system and in a murine model of endogenous septicemia. All efflux mutants except a deltamexCD-oprJ deletion demonstrated significantly reduced invasiveness compared with WT. In particular, a deltamexAB-oprM deletion strain was compromised in its capacity to invade or transmigrate across MDCK cells, and could not kill mice, in contrast to WT which was highly invasive (P < 0.0006) and caused fatal infection (P < 0.0001). The other mutants, including deltamexB and deltamexXY mutants, were intermediate between WT and the deltamexAB-oprM mutant in invasiveness and murine virulence. Invasiveness was restored to the deltamexAB-oprM mutant by complementation with mexAB-oprM or by addition of culture supernatant from MDCK cells infected with WT. We conclude that the P. aeruginosa MexAB-OprM efflux system exports virulence determinants that contribute to bacterial virulence.

Animals↗

PubMed1993

Cloning and characterization of the ferric enterobactin receptor gene (pfeA) of Pseudomonas aeruginosa.

Pseudomonas aeruginosa K407, a mutant lacking a high-affinity 80,000-molecular-weight ferric enterobactin receptor protein (80K protein), exhibited poor growth (small colonies) on iron-deficient succinate minimal medium containing ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) and enterobactin. The gene encoding the ferric enterobactin receptor was cloned by complementation of this growth defect. The complementing DNA was subsequently localized to a 7.1-kilobase-pair (kb) SstI-HindIII fragment which was able to restore synthesis of the 80K protein in strain K407 and also to direct the synthesis of high levels of a protein of the same molecular weight in the outer membranes of Escherichia coli fepA strains MT912 and IR20. Moreover, the fragment complemented the fepA mutation in MT912, restoring both growth in EDDHA-containing medium and enterobactin-dependent uptake of 55Fe3+. Expression of the P. aeruginosa receptor in E. coli IR20 was shown to be regulated by both iron and enterobactin. The complementing DNA was further localized to a 5.3-kb SphI-SstI fragment which was then subjected to deletion analysis to obtain the smallest fragment capable of directing the synthesis of the 80K protein in the outer membrane of strain K407. A 3.2-kb DNA fragment that restored production of the receptor in strain K407 was subsequently isolated. The fragment also directed synthesis of the protein in E. coli MT912 but at levels much lower than those previously observed. Nucleotide sequencing of the fragment revealed an open reading frame (designated pfeA for Pseudomonas ferric enterobactin) of 2,241 bp capable of encoding a 746-amino-acid protein with a molecular weight of 80,967. The PfeA protein showed more than 60% homology to the E. coli FepA protein. Consistent with this, the two proteins showed significant immunological cross-reactivity.

Amino Acid Sequence↗

PubMed2006

Influence of high salinity on biofilm formation and benzoate assimilation by Pseudomonas aeruginosa.

Pseudomonas species were used in bioremediation technologies. In situ conditions, such as marine salinity, could limit the degradation of hydrocarbons and aromatic compounds by the bacteria. Biofilm ability to tolerate environmental stress could be used to increase biorestoration. In this report, we used scanning confocal laser microscopy and microtiter dish assay to analyse the impact of hyperosmotic stress on biofilm formation by Pseudomonas aeruginosa. We used benzoate as the sole carbon source and the effect of the stress on its degradation was also studied. Hyperosmotic shock inhibited the biofilm development and decreased the degradation of benzoate. The osmoprotectant glycine betaine partially restored both the biofilm formation and benzoate degradation, suggesting that it could be used as a complement in bioremediation processes.

Benzoates↗

PubMed1992

[Studies on the serotyping and pyocintyping of Pseudomonas aeruginosa].

Pseudomonas aeruginosa (118 strains) from Xian were typed by 12 groups O-serum and revised pyocin typing method Results indicated that VI, I, III were major serotypes, VI type were mainly from trauma infected, I type mainly from respiration system infected. Pyocintype were mainly I and UT types. Pyocintype I were mainly 1/c and 1/x subtypes. 85.7% of 1/c subtypes were serotype VI, 84.4% of 1/x subtypes were serotype I.

Burns↗

PubMed2005

Evidence for two flagellar stators and their role in the motility of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous bacterium capable of twitching, swimming, and swarming motility. In this study, we present evidence that P. aeruginosa has two flagellar stators, conserved in all pseudomonads as well as some other gram-negative bacteria. Either stator is sufficient for swimming, but both are necessary for swarming motility under most of the conditions tested, suggesting that these two stators may have different roles in these two types of motility.

Amino Acid Sequence↗

PubMed1987

Surface expression of ferripyochelin-binding protein is required for virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa mutants which do not express ferripyochelin-binding protein (FBP) on the cell surface have previously been isolated. These mutants were used to assess the role of FBP in virulence in an acute and a systemic animal infection model. In a mouse corneal infection model, the pathology of eyes infected with the mutant strains was significantly less than that of eyes infected with the parent strain. The mutants were also cleared more rapidly from the eye. In a burn infection model, the mortality rate in mice infected with mutant FBP-28 was much less than that of mice infected with the parent strain at an inoculum of 10(2) CFU. At higher inocula (10(4) CFU), the mortality rate was not significantly different but the survival time was dramatically longer with the mutant strain. Quantitative bacteriology of blood and tissue homogenates revealed that P. aeruginosa PAO could multiply in the skin and could also be cultured from the blood, livers, and spleens of infected mice. FBP-28 could only be cultured from the skin. Therefore, this mutant could colonize the skin but could not disseminate. These data indicate that functional, exposed FBP is required for virulence of PAO.

Animals↗

PubMed2005

Methylcrotonyl-CoA and geranyl-CoA carboxylases are involved in leucine/isovalerate utilization (Liu) and acyclic terpene utilization (Atu), and are encoded by liuB/liuD and atuC/atuF, in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is able to grow on acyclic monoterpenes (citronellol, citronellate, geraniol and geranylate), and on other methyl-branched compounds such as leucine or isovalerate. The catabolic pathway of citronellol (Atu, acyclic terpene utilization) enters that of leucine/isovalerate (Liu, leucine and isovalerate utilization) at the level of methylcrotonyl-CoA. Key enzymes of the combined pathways are geranyl-CoA carboxylase (GCase) and methylcrotonyl-CoA carboxylase (MCase). In this study, isovalerate-grown cells specifically expressed MCase (apparent molecular mass of the biotin-containing subunit, 74 kDa) only, and the GCase biotin-containing subunit (71 kDa) was not detected. Citronellol- or citronellate-grown cells produced both carboxylases. Biotin-dependent proteins were purified from crude extracts by avidin-affinity chromatography, and assigned to the corresponding coding genes by trypsin fingerprint analysis. The two subunits of MCase corresponded to liuB/liuD (PA2014/PA2012) of the P. aeruginosa genome database, and atuC/atuF (PA2888/PA2891) encoded GCase subunits. This finding is contrary to that reported by others. The identified genes are part of two separate gene clusters [liuRABCDE (PA2011-PA2016) and atuABCDEFGH (PA2886-PA2893)] that are thought to encode most of the genes of the Atu and Liu pathways.

Acyclic Monoterpenes↗

PubMed1990

Production and properties of crude enterotoxin of Pseudomonas aeruginosa.

Pseudomonas aeruginosa CTM-3 was found to be the most potentially enterotoxigenic strain out of the 12 isolates recovered from milk, as a high fluid length ratio, i.e. F/L (1.1) in rabbit gut and a strong permeability response in rabbit skin (38.5 mm2 necrotic zone) was obtained with this culture. No clear-cut relationship between the two tests was observed. Six of the ethidium bromide (300 micrograms/ml) cured variants of this culture completely lost their ability to produce enterotoxin indicating the possible involvement of a plasmid in enterotoxin synthesis. The crude enterotoxin from P. aeruginosa CTM-3 was completely inactivated in 15 s at 72 degrees C. However, it was fairly stable at pH values in the range 4.5-7.5. Both pepsin and trypsin inactivated the enterotoxin activity at a concentration of 40 micrograms/ml. Organic acids, formalin and hydrogen peroxide had no significant effect on the enterotoxin activity. The need for further investigations with purified preparations is emphasized.

Animals↗