A novel, dual cytokine-secretion assay for the purification of human Th22 cells that do not co-produce IL-17A.

BACKGROUND: Interleukin-22 is produced by certain T helper cells subsets (Th17, Th22) and at lower levels by γ-δ T cells, NKT and innate lymphoid cells. Th22 cells are unique immune cells that regulate tissue responses by IL-22 production. The exact discrimination between Th17 cells that co-produce IL-22 and single IL-22-producing Th22 cells has not been possible until the present study. Isolation of pure Th22 cells without co-expression of cytokines of other T-cell subsets is essential to better understand their function in humans. The aim of this study is the isolation and characterization of viable, human IL-22-producing CD4+ T cells that do not produce IL-17A. METHODS: Isolation of viable Th22 cells was performed with the combination of two cytokine secretion assays detecting IL-17A- and IL-22-producing cells in a single purification step. RESULTS: The newly developed cytokine secretion assay consists of anti-IL-22 and anti-IL-17A catch antibodies, which via biotin-streptavidin interaction are bound to the biotinylated surface of the target cell, and anti-IL-22 and IL-17A detection antibody labelled with a fluorescent dye, which detects cytokines bound to these catch antibodies. A unique population of human Th22 cells, which do not produce IL-17A, was sorted, and cytokine expression pattern was confirmed by quantitative PCR analysis and ELISA. The presented technique allows the detection and isolation of pure human Th22 cells. CONCLUSIONS: This technique may allow the purification of any single cytokine-producing cell subset, and the combination of several different cytokine secretion assays can be used to purify and characterize novel and unique cell subsets.

Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components.

Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd 's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and impact of the study: Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining.

Cloning and functional characterization of the Pseudomonas aeruginosa rhlC gene that encodes rhamnosyltransferase 2, an enzyme responsible for di-rhamnolipid biosynthesis.

Pseudomonas aeruginosa is an opportunistic pathogen capable of producing a wide variety of virulence factors, including extracellular rhamnolipids and lipopolysaccharide. Rhamnolipids are tenso-active glycolipids containing one (mono-rhamnolipid) or two (di-rhamnolipid) L-rhamnose molecules. Rhamnosyltransferase 1 (RhlAB) catalyses the synthesis of mono-rhamnolipid from dTDP-L-rhamnose and beta-hydroxydecanoyl-beta-hydroxydecanoate, whereas di-rhamnolipid is produced from mono-rhamnolipid and dTDP-L-rhamnose. We report here the molecular characterization of rhlC, a gene encoding the rhamnosyltransferase involved in di-rhamnolipid (L-rhamnose-L-rhamnose-beta-hydroxydecanoyl-beta-hydroxydecanoate) production in P. aeruginosa. RhlC is a protein consisting of 325 amino acids with a molecular mass of 35.9 kDa. It contains consensus motifs that are found in other glycosyltransferases involved in the transfer of L-rhamnose to nascent polymer chains. To verify the biological function of RhlC, a chromosomal mutant, RTII-2, was generated by insertional mutagenesis and allelic replacement. This mutant was unable to produce di-rhamnolipid, whereas mono-rhamnolipid was unaffected. In contrast, a null rhlA mutant (PAO1-rhlA) was incapable of producing both mono- and di-rhamnolipid. Complementation of mutant RTII-2 with plasmid pRTII-26 containing rhlC restored the level of di-rhamnolipid production in the recombinant to a level similar to that of the wild-type strain PAO1. The rhlC gene was located in an operon with an upstream gene (PA1131) of unknown function. A sigma54-type promoter for the PA1131-rhlC operon was identified, and a single transcriptional start site was mapped. Expression of the PA1131-rhlC operon was dependent on the P. aeruginosa rhl quorum-sensing system, and a well-conserved lux box was identified in the promoter region. The genetic regulation of rhlC by RpoN and RhlR was in agreement with the observed increasing RhlC rhamnosyltransferase activity during the stationary phase of growth. This is the first report of a rhamnosyltransferase gene responsible for the biosynthesis of di-rhamnolipid.

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.

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.

Characterization of the 2-ketogluconate utilization operon in Pseudomonas aeruginosa PAO1.

The Pseudomonas aeruginosa protein PtxS negatively regulates its own synthesis by binding to the upstream region of its gene. We have recently identified a 14 bp palindromic sequence within the ptxS upstream region as the PtxS operator site (OP1). In this study, we searched the P. aeruginosa genomic sequence to determine whether this 14 bp sequence exists in other regions of the P. aeruginosa chromosome. Another PtxS operator site (OP2) was located 47 bp downstream of ptxS. DNA gel shift experiments confirmed that PtxS specifically binds to a 520 bp fragment that carries OP2. The DNA segment 3' of OP2 contains four open reading frames (ORF1-ORF4), which code for 29, 32, 48 and 35 kDa proteins respectively. The molecular weight of the products of ORFs 2 and 3 were confirmed by T7 expression experiments. Computer analyses suggest that ORF2 encodes an ATP-dependent kinase; ORF3, a transporter; and ORF4, a dehydrogenase. The predicted product of ORF1 showed no homology to previously identified proteins and contains all the conserved amino acids within the aldose 1-epimerase protein motif. Examination of the ptxs-ORF1 intergenic region (using promoter fusion experiments) showed that no potential promoter exists. An isogenic mutant defective in ORF1 was constructed in the P. aeruginosa strain PAO1. In contrast to its parent strain, the mutant failed to grow on a minimal medium in which 2-ketogluconate was the sole carbon source. Similarly, a previously constructed ptxS isogenic mutant of PAO1 did not grow in a minimal medium containing 2-ketogluconate as the sole carbon source. Furthermore, a plasmid carrying a fragment that contains ptxS and ORFs 1-4 complemented the defect of the previously described P. aeruginosa 2-ketogluconate-negative mutant. In the presence of 10 mM 2-ketogluconate, the in vitro binding of PtxS to a DNA fragment that carries either OP1 or OP2 was inhibited. These results suggest that: (i) ptxS together with the other four ORFs constitute the 2-ketogluconate utilization operon (kgu) in P. aeruginosa. Therefore, ORFs 1-4 were designated kguE, kguK, kguT and kguD respectively. (ii) PtxS regulates the expression of the kgu operon by binding to two operators (OP1 and OP2) within the operon; and (iii) 2-ketogluconate is the molecular inducer of the kgu operon or the molecular effector of PtxS.

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.

The oxygen- and iron-dependent sigma factor pvdS of Pseudomonas aeruginosa is an important virulence factor in experimental infective endocarditis.

In Pseudomonas aeruginosa, pvdS, a key oxygen (O2)-dependent locus, regulates expression of a number of virulence genes, including toxA (which encodes exotoxin A production). To define the in vivo role of differing O2 tensions on pseudomonal virulence, 2 knockout mutants, DeltapvdS and DeltatoxA, were compared with their parental strain, PA01, in rabbit aortic and tricuspid endocarditis models (representing aerobic vs. microaerobic conditions in vivo, respectively). In aortic endocarditis, DeltapvdS densities were significantly less than those of PA01 in vegetations, kidneys, and spleen (P<.01). In contrast, in tricuspid endocarditis, there were no significant differences between DeltapvdS and PA01 tissue densities in these same target tissues. The DeltatoxA mutant proliferated within target tissues to the same extent as the parental strain. Thus, pvdS (but not toxA) appears to be required for optimal virulence of P. aeruginosa, particularly in tissues preferentially exposed to high O2 tensions (e.g., aortic vegetations).

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.

The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence.

During the past decade significant progress has been made towards identifying some of the schemes that Pseudomonas aeruginosa uses to obtain iron and towards cataloguing and characterizing many of the genes and gene products that are likely to play a role in these processes. This review will largely recount what we have learned in the past few years about how P. aeruginosa regulates its acquisition, intake and, to some extent, trafficking of iron, and the role of iron acquisition systems in the virulence of this remarkable opportunistic pathogen. More specifically, the genetics, biochemistry and biology of an essential regulator (Ferric uptake regulator - Fur) and a Fur-regulated alternative sigma factor (PvdS), which are central to these processes, will be discussed. These regulatory proteins directly or indirectly regulate a substantial number of other genes encoding proteins with remarkably diverse functions. These genes include: (i) other regulatory genes, (ii) genes involved in basic metabolic processes (e.g. Krebs cycle), (iii) genes required to survive oxidative stress (e.g. superoxide dismutase), (iv) genes necessary for scavenging iron (e.g. siderophores and their cognate receptors) or genes that contribute to the virulence (e.g. exotoxin A) of this opportunistic pathogen. Despite this recent expansion of knowledge about the response of P. aeruginosa to iron, many significant biological issues surrounding iron acquisition still need to be addressed. Virtually nothing is known about which of the distinct iron acquisition mechanisms P. aeruginosa brings to bear on these questions outside the laboratory, whether it be in soil, in a pipeline, on plants or in the lungs of cystic fibrosis patients.

Virulent Salmonella typhimurium has two periplasmic Cu, Zn-superoxide dismutases.

Periplasmic Cu, Zn-cofactored superoxide dismutase (SodC) protects Gram-negative bacteria from exogenous oxidative damage. The virulent Salmonella typhimurium strain ATCC 14028s has been found to contain two discrete periplasmic Cu, Zn-SOD enzymes that are only 57% identical at the amino acid level. SodCI is carried by a cryptic bacteriophage, and SodCII is closely related to the Cu, Zn-superoxide dismutase of Escherichia coli. All Salmonella serotypes appear to carry the sodCII locus, but the phage-associated sodCI gene is found only in certain strains belonging to the most highly pathogenic serotypes. Expression of either sodC locus appears to be enhanced during stationary phase, but only sodCII is regulated by the alternative sigma factor sigmas (RpoS). Mutants lacking both sodC genes are less lethal for mice than mutants possessing either sodC locus alone, indicating that both Cu, Zn-SOD enzymes contribute to Salmonella pathogenicity. The evolutionary acquisition of an additional sodC gene has contributed to the enhanced virulence of selected Salmonella strains.

The pvc gene cluster of Pseudomonas aeruginosa: role in synthesis of the pyoverdine chromophore and regulation by PtxR and PvdS.

A putative operon of four genes implicated in the synthesis of the chromophore moiety of the Pseudomonas aeruginosa siderophore pyoverdine, dubbed pvcABCD (where pvc stands for pyoverdine chromophore), was cloned and sequenced. Mutational inactivation of the pvc genes abrogated pyoverdine biosynthesis, consistent with their involvement in the biosynthesis of this siderophore. pvcABCD expression was negatively regulated by iron and positively regulated by both PvdS, the alternate sigma factor required for pyoverdine biosynthesis, and PtxR, a LysR family activator previously implicated in exotoxin A regulation.

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 fur overlaps with a gene encoding a novel outer membrane lipoprotein, OmlA.

A novel outer membrane lipoprotein in Pseudomonas aeruginosa is encoded by the omlA gene, which was identified immediately upstream of the fur (ferric uptake regulator) gene. The omlA and fur genes were divergently transcribed and had overlapping promoter regions. The proximal fur P2 promoter and the omlA promoter shared a 5-bp DNA motif for their -10 promoter elements. The distal fur P1 promoter was located within the omlA coding sequence, and the omlA and fur T1 mRNAs overlapped by 154 nucleotides. Optimal expression of both fur and omlA required roughly 200 bp of DNA upstream of the promoter regions, suggesting the presence of cis-acting transcriptional activation elements located within the omlA and fur genes, respectively. The levels of Fur and OmlA proteins had no influence on omlA or fur expression, excluding any trans-acting cross-regulation between fur and omlA. Expression of omlA was constitutive regardless of growth phase, oxygen tension, iron concentration, pH, and temperature. OmlA contained a signal sequence typical of bacterial lipoproteins, with a cysteine as a putative cleavage and lipid attachment site. Inhibition of signal peptidase II by globomycin resulted in failure to process OmlA, thus giving strong evidence that OmlA is a lipoprotein. Cell fractionation followed by Western blot analysis indicated that all OmlA protein is localized in the outer membrane. Mature OmlA was an acidic (pI = 4. 5) protein of 17.3 kDa and had close to 40% amino acid sequence identity to SmpA (small protein A) of Escherichia coli, Vibrio cholerae, and Haemophilus influenzae, a protein of unknown function. All P. aeruginosa strains tested as well as Pseudomonas fluorescens were found to produce OmlA. A mutant strain with impaired production of OmlA but no change in the expression of the overlapping fur gene was constructed. The omlA mutant was hypersusceptible to anionic detergents such as sodium dodecyl sulfate and deoxycholate, and it showed increased susceptibility to various antibiotics, including nalidixic acid, rifampin, novobiocin, and chloramphenicol. A structural role of OmlA in maintaining the cell envelope integrity is proposed.

The fur-regulated gene encoding the alternative sigma factor PvdS is required for iron-dependent expression of the LysR-type regulator ptxR in Pseudomonas aeruginosa.

We previously identified a novel regulator of the exotoxin A gene (toxA) in Pseudomonas aeruginosa, PtxR, that belongs to the LysR family of prokaryotic regulatory proteins. Preliminary data also suggest that PtxR affects the expression of siderophores in P. aeruginosa. Because toxA expression and siderophore production in this organism are coordinately regulated by the ferric uptake regulator (Fur) and the Fur-regulated alternative sigma factor PvdS, regulation of ptxR itself in the context of these regulators was examined. RNase protection analyses of ptxR transcription revealed that there are two independent transcription initiation sites (T1 and T2). While transcription from the promoter of T1 is constitutive throughout the growth cycle of PAO1, transcription from the second promoter (P2) is negatively affected by iron. Transcription from the P2 promoter is constitutive in a fur mutant under microaerobic conditions but still iron regulated during aerobic growth. High concentrations (>100 nM) of the ferric uptake regulatory protein (Fur) failed to bind to either of the promoter regions of ptxR in either gel mobility shift assays or DNase I footprint experiments. These results indicate that Fur indirectly regulates the iron-dependent expression of ptxR. Iron-regulated transcription of ptxR from the P2 promoter, but not constitutive expression from the P1 promoter, was dependent on the Fur-regulated alternative sigma factor gene pvdS, even under aerobic conditions. Consequently, there are two levels of iron-regulated expression of ptxR. The iron-regulated expression of ptxR under microaerobic conditions from the P2 promoter of ptxR is mediated indirectly by Fur through the iron-regulated expression of pvdS. In contrast, pvdS-mediated iron regulation of ptxR under aerobic conditions is Fur independent.

An operon containing fumC and sodA encoding fumarase C and manganese superoxide dismutase is controlled by the ferric uptake regulator in Pseudomonas aeruginosa: fur mutants produce elevated alginate levels.

The activities of fumarase- and manganese-cofactored superoxide dismutase (SOD), encoded by the fumC and sodA genes in Pseudomonas aeruginosa, are elevated in mucoid, alginate-producing bacteria and in response to iron deprivation (D. J. Hassett, M. L. Howell, P. A. Sokol, M. L. Vasil, and G. E. Dean, J. Bacteriol. 179:1442-1451, 1997). In this study, a 393-bp open reading frame, fagA (Fur-associated gene), was identified immediately upstream of fumC, in an operon with orfX and sodA. Two iron boxes or Fur (ferric uptake regulatory protein) binding sites were discovered just upstream of fagA. Purified P. aeruginosa Fur caused a gel mobility shift of a PCR product containing these iron box regions. DNA footprinting analysis revealed a 37-bp region that included the Fur binding sites and was protected by Fur. Primer extension analysis and RNase protection assays revealed that the operon is composed of at least three major iron-regulated transcripts. Four mucoid fur mutants produced 1.7- to 2.6-fold-greater fumarase activity and 1.7- to 2.3-greater amounts of alginate than wild-type organisms. A strain devoid of the alternative sigma factor AlgT(U) produced elevated levels of one major transcript and fumarase C and manganase-cofactored SOD activity, suggesting that AlgT(U) may either play a role in regulating this transcript or function in some facet of iron metabolism. These data suggest that the P. aeruginosa fagA, fumC, orfX, and sodA genes reside together on a small operon that is regulated by Fur and is transcribed in response to iron limitation in mucoid, alginate-producing bacteria.

Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase.

Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide. Periplasmic localization of bacterial Cu,Zn-SOD has suggested a role of this enzyme in defense against extracellular phagocyte-derived reactive oxygen species. Sequence analysis of regions flanking the Salmonella typhimurium sodC gene encoding Cu,Zn-SOD demonstrates significant homology to lambda phage proteins, reflecting possible bacteriophage-mediated horizontal gene transfer of this determinant among pathogenic bacteria. Salmonella deficient in Cu,Zn-SOD has reduced survival in macrophages and attenuated virulence in mice, which can be restored by abrogation of either the phagocyte respiratory burst or inducible nitric oxide synthase. Moreover, a sodC mutant is extremely susceptible to the combination of superoxide and nitric oxide. These observations suggest that SOD protects periplasmic or inner membrane targets by diverting superoxide and limiting peroxynitrite formation, and they demonstrate the ability of the respiratory burst and nitric oxide synthase to synergistically kill microbial pathogens in vivo.