Antioxidant enzyme expression in clinical isolates of Pseudomonas aeruginosa: identification of an atypical form of manganese superoxide dismutase.

Expression of superoxide dismutases (FeSOD and MnSOD) and catalases by laboratory strains of Pseudomonas aeruginosa is modulated by exogenous factors. Whether clinical isolates behave similarly and whether antioxidant enzyme expression influences P. aeruginosa virulence remain unclear. Fifty-seven P. aeruginosa blood culture isolates, plus seven pairs of blood and local-site isolates, were examined for FeSOD, MnSOD, and catalase production in vitro. Under iron-replete growth conditions FeSOD and catalase activities were maximized. MnSOD was not detected. FeSOD and catalase activity decreased under iron-limited growth conditions, whereas MnSOD activity appeared. SOD and catalase activity did not change with site of isolation or by patient. MnSOD could not be expressed by one isolate due to a missense mutation in sodA that produced a premature stop codon. Eleven percent of the isolates expressed a novel, rapidly migrating MnSOD that was associated with missense mutations in the normal stop codon of sodA. We conclude that clinical P. aeruginosa isolates vary little in FeSOD and catalase expression. Some strains produce a newly described MnSOD variant, whereas one is deficient in MnSOD production. The absence of MnSOD expression in a P. aeruginosa strain causing invasive human disease indicates that MnSOD is probably not essential for P. aeruginosa virulence.

Factors affecting catalase expression in Pseudomonas aeruginosa biofilms and planktonic cells.

Previous work with Pseudomonas aeruginosa showed that catalase activity in biofilms was significantly reduced relative to that in planktonic cells. To better understand biofilm physiology, we examined possible explanations for the differential expression of catalase in cells cultured in these two different conditions. For maximal catalase activity, biofilm cells required significantly more iron (25 microM as FeCl(3)) in the medium, whereas planktonic cultures required no addition of iron. However, iron-stimulated catalase activity in biofilms was still only about one-third that in planktonic cells. Oxygen effects on catalase activity were also investigated. Nitrate-respiring planktonic cultures produced approximately twice as much catalase activity as aerobic cultures grown in the presence of nitrate; the nitrate stimulation effect could also be demonstrated in biofilms. Cultures fermenting arginine had reduced catalase levels; however, catalase repression was also observed in aerobic cultures grown in the presence of arginine. It was concluded that iron availability, but not oxygen availability, is a major factor affecting catalase expression in biofilms.

Gene expression in Pseudomonas aeruginosa: evidence of iron override effects on quorum sensing and biofilm-specific gene regulation.

Prior studies established that the Pseudomonas aeruginosa oxidative stress response is influenced by iron availability, whereas more recent evidence demonstrated that it was also controlled by quorum sensing (QS) regulatory circuitry. In the present study, sodA (encoding manganese-cofactored superoxide dismutase [Mn-SOD]) and Mn-SOD were used as a reporter gene and endogenous reporter enzyme, respectively, to reexamine control mechanisms that govern the oxidative stress response and to better understand how QS and a nutrient stress response interact or overlap in this bacterium. In cells grown in Trypticase soy broth (TSB), Mn-SOD was found in wild-type stationary-phase planktonic cells but not in a lasI or lasR mutant. However, Mn-SOD activity was completely suppressed in the wild-type strain when TSB was supplemented with iron. Reporter gene studies indicated that sodA transcription could be variably induced in iron-starved cells of all three strains, depending on growth stage. Iron starvation induction of sodA was greatest in the wild-type strain and least in the lasR mutant and was maximal in stationary-phase cells. Reporter experiments in the wild-type strain showed increased lasI::lacZ transcription in response to iron limitation, whereas the expression level in the las mutants was minimal and iron starvation induction of lasI::lacZ did not occur. Studies comparing Mn-SOD activity in P. aeruginosa biofilms and planktonic cultures were also initiated. In wild-type biofilms, Mn-SOD was not detected until after 6 days, although in iron-limited wild-type biofilms Mn-SOD was detected within the initial 24 h of biofilm establishment and formation. Unlike planktonic bacteria, Mn-SOD was constitutive in the lasI and lasR mutant biofilms but could be suppressed if the growth medium was amended with 25 microM ferric chloride. This study demonstrated that (i) the nutritional status of the cell must be taken into account when one is evaluating QS-based gene expression; (ii) in the biofilm mode of growth, QS may also have negative regulatory functions; (iii) QS-based gene regulation models based on studies with planktonic cells must be modified in order to explain biofilm gene expression behavior; and (iv) gene expression in biofilms is dynamic.

snr-1 gene is required for nitrate reduction in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is able to use nitrate for both assimilation and anaerobic respiration. One set of genes, designated snr (for "shared nitrate reduction"), have been recently cloned and partially characterized. In this study, we demonstrate that the snr-1 gene encodes a predicted 52.5-kDa protein that is 82% similar to a unique cytochrome c of Desulfomonile tiedjei DCB-1. Importantly, the Snr-1 protein sequence of P. aeruginosa differed from that of the cytochrome c of D. tiedjei primarily in the first 25 amino acids, which are required for membrane attachment in D. tiedjei. In P. aeruginosa, the Snr-1 protein hydropathy profile indicates that it is a soluble protein. An isogenic snr-1::Gm insertional mutant was unable to grow aerobically with nitrate as a sole nitrogen source or anaerobically with nitrate as an electron acceptor. Complementation of the snr-1::Gm mutant with the snr-1 gene restored the wild-type phenotypes. Interestingly, anaerobic growth rates were significantly higher in the snr-1 mutant harboring a multicopy plasmid containing snr-1. In contrast, aerobic growth rates of the restored mutant using nitrate as the sole nitrogen source were similar to those of the wild type. Transcriptional lacZ fusions demonstrated that snr-1 was not regulated by molybdate, oxygen, or nitrate.

Genetic and physiological characterization of ohr, encoding a protein involved in organic hydroperoxide resistance in Pseudomonas aeruginosa.

The ohr (organic hydroperoxide resistance) gene product of Pseudomonas aeruginosa was essential for optimal resistance to organic hydroperoxides (OHPs) but not to hydrogen peroxide or paraquat. A Deltaohr mutant was hypersusceptible to OHPs in disk inhibition assays and showed enhanced killing by OHPs in liquid culture. The ohr gene product was demonstrated to contribute to the decomposition of OHPs. Transcription of ohr was induced up to 15-fold upon exposure to OHPs, and this induction was independent of OxyR. Somewhat enhanced ohr-lacZ activity was detected in mutant strains affected in ohr, ahpC, and oxyR, and this phenotype correlated with hypersusceptibility to OHPs, suggesting overlapping or compensatory functions of the ohr and ahpC gene products. A single transcriptional start site for ohr was determined, and ohr transcripts were abundant in cells treated with a sublethal dose of OHPs but not in cells treated with paraquat. An 84-bp portion upstream of the ohr mRNA start site was sufficient for ohr induction by OHPs. Thus, the ohr gene appears to encode an antioxidant enzyme that is not part of the OxyR regulon yet is specifically induced by OHPs.

Role of cystic fibrosis transmembrane conductance regulator in pulmonary clearance of Pseudomonas aeruginosa in vivo.

Cystic fibrosis (CF)2 is a fatal genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) that is commonly associated with chronic pulmonary infections with mucoid Pseudomonas aeruginosa (PA). To test the hypothesis that CFTR plays a direct role in PA adhesion and clearance, we have used mouse lines expressing varying levels of human (h) or mouse (m) CFTR. A subacute intratracheal dose of 3 x 10(6) bacteria was cleared with similar kinetics in control wild-type (WT) and transgenic mice overexpressing hCFTR in the lung from the surfactant protein C (SP-C) promoter (SP-C-hCFTR+/-). In a second series of experiments, the clearance of an acute intratracheal dose of 1.5 x 10(7) PA bacteria was also similar in WT, hemizygous SP-C-hCFTR+/-, and bitransgenic gut-corrected FABP-hCFTR+/+-mCFTR-/-, the latter lacking expression of mCFTR in the lung. However, a small but significant decrease in bacterial killing was observed in lungs of homozygote SP-C-hCFTR+/+ mice. Lung pathology in both WT and SP-C-hCFTR+/+ mice was marked by neutrophilic inflammation and bacterial invasion of perivascular and subepithelial compartments. Bacteria were associated primarily with leukocytes and were not associated with alveolar type II or bronchiolar epithelial cells, the cellular sites of SP-C-hCFTR+/+ transgene expression. The results indicate that there is no direct correlation between levels of CFTR expression and bacterial clearance or association of bacteria with epithelial cells in vivo.

Hydrogen peroxide sensitivity of catechol-2,3-dioxygenase: a cautionary note on use of xylE reporter fusions under aerobic conditions.

Catechol-2,3-dioxygenase (C23O) of Pseudomonas putida, encoded by the xylE gene, was found to be sensitive to hydrogen peroxide (H(2)O(2)) when used as a reporter in gene fusion constructs. Exposure of Pseudomonas aeruginosa katA or katA katB mutants harboring katA- or katB-lacZ (encoding beta-galactosidase) or -xylE fusion plasmids to H(2)O(2) stimulated beta-galactosidase activity, while there was little or no detectable C23O activity in these strains. More than 95% of C23O activity was lost after a 5-min exposure to equimolar H(2)O(2), while a 10,000-fold excess was required for similar inhibition of beta-galactosidase. Electron paramagnetic resonance spectra of the nitrosyl complexes of C23O showed that H(2)O(2) nearly stoichiometrically oxidized the essential active-site ferrous ion, thus accounting for the loss of activity. Our results suggest using caution in interpreting data derived from xylE reporter fusions under aerobic conditions, especially where oxidative stress is present or when catalase-deficient strains are used.

Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB-ankB, ahpB, and ahpC-ahpF.

Pseudomonas aeruginosa possesses an extensive armament of genes involved in oxidative stress defense, including katB-ankB, ahpB, and ahpC-ahpF. Transcription of these genes was regulated in response to H(2)O(2), paraquat, or organic peroxides. Expression of katB-lacZ and the observed KatB catalase levels in P. aeruginosa PAO1 were induced up to 250-fold after exposure to oxidative stress-generating compounds. Also, ahpB-lacZ and ahpC-lacZ expression was 90- and 3-fold higher, respectively, upon exposure to paraquat. The dose- and time-response curves revealed that 1 microM paraquat was sufficient for half-maximal activation of each reporter fusion within 5 min of exposure. Expression of these genes was not observed in a DeltaoxyR mutant, indicating that OxyR was essential for this response. The transcriptional start sites of katB-ankB, ahpB, and ahpC-ahpF were mapped, putative OxyR-binding sites were identified upstream of the -35 promoter elements, and direct binding of purified OxyR protein to these target promoters was demonstrated. The oxyR mutant was hypersusceptible to oxidative stress-generating agents, including H(2)O(2) and paraquat, in spite of total KatA catalase activity being comparable to that of the wild type. The oxyR phenotype was fully complemented by a plasmid containing the oxyR gene, while any of the katB, ahpB, or ahpCF genes alone resulted in only marginal complementation. Increased katB-lacZ expression and higher KatB catalase levels were detected in a DeltaahpCF background compared to wild-type bacteria, suggesting a compensatory function for KatB in the absence of AhpCF. In P. aeruginosa, oxyR is located upstream of recG, encoding a putative DNA repair enzyme. oxyR-lacZ and recG-lacZ reporter activities and oxyR-recG mRNA analysis showed that oxyR and recG are organized in an operon and expressed constitutively with regard to oxidative stress from a single promoter upstream of oxyR. Mutants affected in recG but not oxyR were dramatically impaired in DNA damage repair as measured by sensitivity to UV irradiation. In conclusion, we present evidence that the oxyR-recG locus is essential for oxidative stress defense and for DNA repair.

AnkB, a periplasmic ankyrin-like protein in Pseudomonas aeruginosa, is required for optimal catalase B (KatB) activity and resistance to hydrogen peroxide.

In this study, we have cloned the ankB gene, encoding an ankyrin-like protein in Pseudomonas aeruginosa. The ankB gene is composed of 549 bp encoding a protein of 183 amino acids that possesses four 33-amino-acid ankyrin repeats that are a hallmark of erythrocyte and brain ankyrins. The location of ankB is 57 bp downstream of katB, encoding a hydrogen peroxide-inducible catalase, KatB. Monomeric AnkB is a 19.4-kDa protein with a pI of 5.5 that possesses 22 primarily hydrophobic amino acids at residues 3 to 25, predicting an inner-membrane-spanning motif with the N terminus in the cytoplasm and the C terminus in the periplasm. Such an orientation in the cytoplasmic membrane and, ultimately, periplasmic space was confirmed using AnkB-BlaM and AnkB-PhoA protein fusions. Circular dichroism analysis of recombinant AnkB minus its signal peptide revealed a secondary structure that is approximately 65% alpha-helical. RNase protection and KatB- and AnkB-LacZ translational fusion analyses indicated that katB and ankB are part of a small operon whose transcription is induced dramatically by H(2)O(2), and controlled by the global transactivator OxyR. Interestingly, unlike the spherical nature of ankyrin-deficient erythrocytes, the cellular morphology of an ankB mutant was identical to that of wild-type bacteria, yet the mutant produced more membrane vesicles. The mutant also exhibited a fourfold reduction in KatB activity and increased sensitivity to H(2)O(2), phenotypes that could be complemented in trans by a plasmid constitutively expressing ankB. Our results suggest that AnkB may form an antioxidant scaffolding with KatB in the periplasm at the cytoplasmic membrane, thus providing a protective lattice work for optimal H(2)O(2) detoxification.

A protease-resistant catalase, KatA, released upon cell lysis during stationary phase is essential for aerobic survival of a Pseudomonas aeruginosa oxyR mutant at low cell densities.

A Pseudomonas aeruginosa oxyR mutant was dramatically sensitive to H(2)O(2), despite possessing wild-type catalase activity. Oxygen-dependent oxyR phenotypes also included an inability to survive aerobic serial dilution in Luria broth and to resist aminoglycosides. Plating the oxyR mutant after serial dilution in its own spent culture supernatant, which contained the major catalase KatA, or under anaerobic conditions allowed for survival. KatA was resistant to sodium dodecyl sulfate, proteinase K, pepsin, trypsin, chymotrypsin and the neutrophil protease cathepsin G. When provided in trans and expressed constitutively, the OxyR-regulated genes katB, ahpB, and ahpCF could not restore both the serial dilution defect and H(2)O(2) resistance; only oxyR itself could do so. The aerobic dilution defect could be complemented, in part, by only ahpB and ahpCF, suggesting that the latter gene products could possess a catalase-like activity. Aerobic Luria broth was found to generate approximately 1.2 microM H(2)O(2) min(-1) via autoxidation, a level sufficient to kill serially diluted oxyR and oxyR katA bacteria and explain the molecular mechanism behind the aerobic serial dilution defect. Taken together, our results indicate that inactivation of OxyR renders P. aeruginosa exquisitely sensitive to both H(2)O(2) and aminoglycosides, which are clinically and environmentally important antimicrobials.

Effect of catalase on hydrogen peroxide penetration into Pseudomonas aeruginosa biofilms.

The penetration of hydrogen peroxide into biofilms formed by wild-type and catalase-deficient Pseudomonas aeruginosa strains was measured using microelectrodes. A flowing stream of hydrogen peroxide (50 mM, 1 h) was unable to penetrate or kill wild-type biofilms but did penetrate and partially kill biofilms formed by an isogenic strain in which the katA gene was knocked out. Catalase protects aggregated bacteria by preventing full penetration of hydrogen peroxide into the biofilm.

Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide.

Quorum sensing (QS) governs the production of virulence factors and the architecture and sodium dodecyl sulphate (SDS) resistance of biofilm-grown Pseudomonas aeruginosa. P. aeruginosa QS requires two transcriptional activator proteins known as LasR and RhlR and their cognate autoinducers PAI-1 (N-(3-oxododecanoyl)-L-homoserine lactone) and PAI-2 (N-butyryl-L-homoserine lactone) respectively. This study provides evidence of QS control of genes essential for relieving oxidative stress. Mutants devoid of one or both autoinducers were more sensitive to hydrogen peroxide and phenazine methosulphate, and some PAI mutant strains also demonstrated decreased expression of two superoxide dismutases (SODs), Mn-SOD and Fe-SOD, and the major catalase, KatA. The expression of sodA (encoding Mn-SOD) was particularly dependent on PAI-1, whereas the influence of autoinducers on Fe-SOD and KatA levels was also apparent but not to the degree observed with Mn-SOD. beta-Galactosidase reporter fusion results were in agreement with these findings. Also, the addition of both PAIs to suspensions of the PAI-1/2-deficient double mutant partially restored KatA activity, while the addition of PAI-1 only was sufficient for full restoration of Mn-SOD activity. In biofilm studies, catalase activity in wild-type bacteria was significantly reduced relative to planktonic bacteria; catalase activity in the PAI mutants was reduced even further and consistent with relative differences observed between each strain grown planktonically. While wild-type and mutant biofilms contained less catalase activity, they were more resistant to hydrogen peroxide treatment than their respective planktonic counterparts. Also, while catalase was implicated as an important factor in biofilm resistance to hydrogen peroxide insult, other unknown factors seemed potentially important, as PAI mutant biofilm sensitivity appeared not to be incrementally correlated to catalase levels.

Pseudomonas aeruginosa biofilm sensitivity to biocides: use of hydrogen peroxide as model antimicrobial agent for examining resistance mechanisms.

The biofilm mode of bacterial growth may be the preferred form of existence in nature. Because of the global impact of problematic biofilms, study of the mechanisms affording resistance to various biocides is of dire importance. Furthermore, understanding the physiological differences between biofilm and planktonic organisms ranks particularly high on the list of important and necessary research. Such contributions will only serve to broaden our knowledge base, especially regarding the development of better antimicrobials while also fine-tuning the use of current highly effective antimicrobials. Using H2O2 as a model oxidizing biocide, we demonstrate the marked resistance of biofilm bacteria relative to planktonic cells. Because many biocides are good oxidizing agents (e.g., H2O2, HOCl), understanding the mechanisms by which genes involved in combating oxidative stress are activated is important in determining the overall efficacy of such biocides. Future studies will focus on determining mechanisms of oxidative stress gene regulation in bacterial biofilms.

Protective role of catalase in Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide.

The role of the two known catalases in Pseudomonas aeruginosa in protecting planktonic and biofilm cells against hydrogen peroxide (H(2)O(2)) was investigated. Planktonic cultures and biofilms formed by the wild-type strain PAO1 and the katA and katB catalase mutants were compared for their susceptibility to H(2)O(2). Over the course of 1 h, wild-type cell viability decreased steadily in planktonic cells exposed to a single dose of 50 mM H(2)O(2), whereas biofilm cell viability remained at approximately 90% when cells were exposed to a flowing stream of 50 mM H(2)O(2). The katB mutant, lacking the H(2)O(2)-inducible catalase KatB, was similar to the wild-type strain with respect to H(2)O(2) resistance. The katA mutant possessed undetectable catalase activity. Planktonic katA mutant cultures were hypersusceptible to a single dose of 50 mM H(2)O(2), while biofilms displayed a 10-fold reduction in the number of culturable cells after a 1-h exposure to 50 mM H(2)O(2). Catalase activity assays, activity stains in nondenaturing polyacrylamide gels, and lacZ reporter genes were used to characterize the oxidative stress responses of planktonic cultures and biofilms. Enzyme assays and catalase activity bands in nondenaturing polyacrylamide gels showed significant KatB catalase induction occurred in biofilms after a 20-min exposure to H(2)O(2), suggesting that biofilms were capable of a rapid adaptive response to the oxidant. Reporter gene data obtained with a katB::lacZ transcriptional reporter strain confirmed katB induction and that the increase in total cellular catalase activity was attributable to KatB. Biofilms upregulated the reporter in the constant presence of 50 mM H(2)O(2), while planktonic cells were overwhelmed by a single 50 mM dose and were unable to make detectable levels of beta-galactosidase. The results of this study demonstrated the following: the constitutively expressed KatA catalase is important for resistance of planktonic and biofilm P. aeruginosa to H(2)O(2), particularly at high H(2)O(2) concentrations; KatB is induced in both planktonic and biofilm cells in response to H(2)O(2) insult, but plays a relatively small role in biofilm resistance; and KatB is important to either planktonic cells or biofilm cells for acquired antioxidant resistance when initial levels of H(2)O(2) are sublethal.

Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa.

The sigma factor RpoS (sigmaS) has been described as a general stress response regulator that controls the expression of genes which confer increased resistance to various stresses in some gram-negative bacteria. To elucidate the role of RpoS in Pseudomonas aeruginosa physiology and pathogenesis, we constructed rpoS mutants in several strains of P. aeruginosa, including PAO1. The PAO1 rpoS mutant was subjected to various environmental stresses, and we compared the resistance phenotype of the mutant to that of the parent. The PAO1 rpoS mutant was slightly more sensitive to carbon starvation than the wild-type strain, but this phenotype was obvious only when the cells were grown in a medium supplemented with glucose as the sole carbon source. In addition, the PAO1 rpoS mutant was hypersensitive to heat shock at 50 degrees C, increased osmolarity, and prolonged exposure to high concentrations of H2O2. In accordance with the hypersensitivity to H2O2, catalase production was 60% lower in the rpoS mutant than in the parent strain. We also assessed the role of RpoS in the production of several exoproducts known to be important for virulence of P. aeruginosa. The rpoS mutant produced 50% less exotoxin A, but it produced only slightly smaller amounts of elastase and LasA protease than the parent strain. The levels of phospholipase C and casein-degrading proteases were unaffected by a mutation in rpoS in PAO1. The rpoS mutation resulted in the increased production of the phenazine antibiotic pyocyanin and the siderophore pyoverdine. This increased pyocyanin production may be responsible for the enhanced virulence of the PAO1 rpoS mutant that was observed in a rat chronic-lung-infection model. In addition, the rpoS mutant displayed an altered twitching-motility phenotype, suggesting that the colonization factors, type IV fimbriae, were affected. Finally, in an alginate-overproducing cystic fibrosis (CF) isolate, FRD1, the rpoS101::aacCI mutation almost completely abolished the production of alginate when the bacterium was grown in a liquid medium. On a solid medium, the FRD1 rpoS mutant produced approximately 70% less alginate than did the wild-type strain. Thus, our data indicate that although some of the functions of RpoS in P. aeruginosa physiology are similar to RpoS functions in other gram-negative bacteria, it also has some functions unique to this bacterium.

Bacterioferritin A modulates catalase A (KatA) activity and resistance to hydrogen peroxide in Pseudomonas aeruginosa.

We have cloned a 3.6-kb genomic DNA fragment from Pseudomonas aeruginosa harboring the rpoA, rplQ, katA, and bfrA genes. These loci are predicted to encode, respectively, (i) the alpha subunit of RNA polymerase; (ii) the L17 ribosomal protein; (iii) the major catalase, KatA; and (iv) one of two iron storage proteins called bacterioferritin A (BfrA; cytochrome b1 or b557). Our goal was to determine the contributions of KatA and BfrA to the resistance of P. aeruginosa to hydrogen peroxide (H2O2). When provided on a multicopy plasmid, the P. aeruginosa katA gene complemented a catalase-deficient strain of Escherichia coli. The katA gene was found to contain two translational start codons encoding a heteromultimer of approximately 160 to 170 kDa and having an apparent Km for H2O2 of 44.7 mM. Isogenic katA and bfrA mutants were hypersusceptible to H2O2, while a katA bfrA double mutant demonstrated the greatest sensitivity. The katA and katA bfrA mutants possessed no detectable catalase activity. Interestingly, a bfrA mutant expressed only approximately 47% the KatA activity of wild-type organisms, despite possessing wild-type katA transcription and translation. Plasmids harboring bfrA genes encoding BfrA altered at critical amino acids essential for ferroxidase activity could not restore wild-type catalase activity in the bfrA mutant. RNase protection assays revealed that katA and bfrA are on different transcripts, the levels of which are increased by both iron and H2O2. Mass spectrometry analysis of whole cells revealed no significant difference in total cellular iron levels in the bfrA, katA, and katA bfrA mutants relative to wild-type bacteria. Our results suggest that P. aeruginosa BfrA may be required as one source of iron for the heme prosthetic group of KatA and thus for protection against H2O2.

Pseudomonas aeruginosa alginate is a potent secretagogue in the isolated ferret trachea.

Airway mucus hypersecretion is in part a response to infection and inflammation. Pseudomonas aeruginosa infection is nearly universal in advanced cystic fibrosis (CF) lung disease. Mucoid strains of P. aeruginosa produce an exopolysaccharide product called alginate. The purpose of this study was to determine whether P. aeruginosa alginate stimulates secretion from mucous or serous cells in the ferret trachea exposed to alginate at concentrations reported to be present in the CF airway. We used a sandwich enzyme-linked lectin assay (ELLA) to measure mucin secretion and spectrophotometry to measure lysozyme secretion from isolated ferret tracheal segments. Purified Pseudomonas aeruginosa alginate stimulated mucin and lysozyme secretion in a dose-dependent fashion (mucin = +111%: P = 0.003; lysozyme = +20%: P = 0.024 at 200 microg/mL). This stimulated secretion was not due to proteolytic activity, and alginate exposure did not produce ultrastructural damage to the trachea. We conclude that alginate may contribute to mucus hypersecretion and respiratory morbidity associated with P. aeruginosa infection in patients with CF.

Cloning and characterization of the Pseudomonas aeruginosa zwf gene encoding glucose-6-phosphate dehydrogenase, an enzyme important in resistance to methyl viologen (paraquat).

In this study, we cloned the Pseudomonas aeruginosa zwf gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), an enzyme that catalyzes the NAD+- or NADP+-dependent conversion of glucose-6-phosphate to 6-phosphogluconate. The predicted zwf gene product is 490 residues, which could form a tetramer with a molecular mass of approximately 220 kDa. G6PDH activity and zwf transcription were maximal in early logarithmic phase when inducing substrates such as glycerol, glucose, or gluconate were abundant. In contrast, both G6PDH activity and zwf transcription plummeted dramatically when bacteria approached stationary phase, when inducing substrate was limiting, or when the organisms were grown in a citrate-, succinate-, or acetate-containing basal salts medium. G6PDH was purified to homogeneity, and its molecular mass was estimated to be approximately 220 kDa by size exclusion chromatography. Estimated Km values of purified G6PDH acting on glucose-6-phosphate, NADP+, and NAD+ were 530, 57, and 333 microM, respectively. The specific activities with NAD+ and NADP+ were calculated to be 176 and 69 micromol/min/mg. An isogenic zwf mutant was unable to grow on minimal medium supplemented with mannitol. The mutant also demonstrated increased sensitivity to the redox-active superoxide-generating agent methyl viologen (paraquat). Since one by-product of G6PDH activity is NADPH, the latter data suggest that this cofactor is essential for the activity of enzymes critical in defense against paraquat toxicity.

Phosphorylation-independent activity of the response regulators AlgB and AlgR in promoting alginate biosynthesis in mucoid Pseudomonas aeruginosa.

Overproduction of the capsular polysaccharide alginate appears to confer a selective advantage for Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. The regulators AlgB and AlgR, which are both required as positive activators in alginate overproduction, have homology with the regulator class of two-component environmental responsive proteins which coordinate gene expression through signal transduction mechanisms. Signal transduction in this class of proteins generally occurs via autophosphorylation of the sensor kinase protein and phosphotransfer from the sensor to a conserved aspartate residue, which is present in the amino terminus of the response regulator. Recently, kinB was identified downstream of algB and was shown to encode the cognate histidine protein kinase that efficiently phosphorylates AlgB. However, we show here that a null mutation in kinB in a mucoid cystic fibrosis isolate, P. aeruginosa FRD1, did not block alginate production. The role of the conserved aspartate residue in the phosphorylation of AlgB was examined. The predicted phosphorylation site of AlgB (D59) was mutated to asparagine (N), and a derivative of an AlgB lacking the entire amino-terminal phosphorylation domain (AlgB delta1-145) was constructed. A hexahistidine tag was included at the amino terminus of the wild-type (H-AlgB), H-AlgB delta1-145, and mutant (H-AlgB.59N) AlgB proteins. These derivatives were purified by Ni2+ affinity chromatography and examined for in vitro phosphorylation by the purified sensor kinase protein, KinB. The results indicated that while KinB efficiently phosphorylated H-AlgB, no phosphorylation of H-AlgB delta1-145 or H-AlgB.D59N was apparent. An allelic exchange system was developed to transfer mutant algB alleles onto the chromosome of a P. aeruginosa algB mutant to examine the effect on alginate production. Despite the defect in AlgB phosphorylation, P. aeruginosa strains expressing AlgB.D59N or H-AlgB delta1-145 remained mucoid. The roles of the conserved aspartate residues in the phosphorylation of AlgR were also examined. As seen with AlgB, mutations in the predicted phosphorylation site of AlgR (AlgR.D54N and AlgR.D85N) did not affect alginate production. These results indicate that in vivo phosphorylation of AlgB and AlgR are not required for their roles in alginate production. Thus, the mechanism by which these response regulators activate alginate genes in mucoid P. aeruginosa appears not to be mediated by conventional phosphorylation-dependent signal transduction.

Fumarase C activity is elevated in response to iron deprivation and in mucoid, alginate-producing Pseudomonas aeruginosa: cloning and characterization of fumC and purification of native fumC.

We report the discovery of fumC, encoding a fumarase, upstream of the sodA gene, encoding manganese superoxide dismutase, in Pseudomonas aeruginosa. The fumC open reading frame, which terminates 485 bp upstream of sodA, contains 1,374 bp that encode 458 amino acids. A second 444-bp open reading frame located between fumC and sodA, called orfX, showed no homology with any genes or proteins in database searches. A fumarase activity stain revealed that P. aeruginosa possesses at least two and possibly three fumarases. Total fumarase activity was at least approximately 1.6-fold greater in mucoid, alginate-producing bacteria than in nonmucoid bacteria and decreased 84 to 95% during the first 5 h of aerobic growth, followed by a rapid rise to maximum activity in stationary phase. Bacteria exposed to the iron chelator 2,2'-dipyridyl, but not ferric chloride, demonstrated an increase in fumarase activity. Mucoid bacteria produced approximately twofold-higher levels of the siderophores pyoverdin and pyochelin than nonmucoid bacteria. Northern blot analysis revealed a transcript that included fumC, orfX, and sodA, the amount of which was increased in response to iron deprivation. A P. aeruginosa fumC mutant produced only approximately 40% the alginate of wild-type bacteria. Interestingly, a sodA mutant possessed an alginate-stable phenotype, a trait that is typically unstable in vitro. These data suggest that mucoid bacteria either are in an iron-starved state relative to nonmucoid bacteria or simply require more iron for the process of alginate biosynthesis. In addition, the iron-regulated, tricarboxylic acid cycle enzyme fumarase C is essential for optimal alginate production by P. aeruginosa.