Membrane-association determinants of the ω-amino acid monooxygenase PvdA, a pyoverdine biosynthetic enzyme from Pseudomonas aeruginosa
Francesco ImperiLorenza PutignaniFederica TiburziCecilia AmbrosiRita CipollonePaolo AscenziPaolo Visca
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Abstract:
The l-ornithine N δ -oxygenase PvdA catalyses the N δ -hydroxylation of l-ornithine in many Pseudomonas spp., and thus provides an essential enzymic function in the biogenesis of the pyoverdine siderophore. Here, we report a detailed analysis of the membrane topology of the PvdA enzyme from the bacterial pathogen Pseudomonas aeruginosa. Membrane topogenic determinants of PvdA were identified by computational analysis, and verified in Escherichia coli by constructing a series of translational fusions between PvdA and the PhoA (alkaline phosphatase) reporter enzyme. The inferred topological model resembled a eukaryotic reverse signal-anchor (type III) protein, with a single N-terminal domain anchored to the inner membrane, and the bulk of the protein spanning the cytosol. According to this model, the predicted transmembrane region should overlap the putative FAD-binding site. Cell fractionation and proteinase K accessibility experiments in P. aeruginosa confirmed the membrane-bound nature of PvdA, but excluded the transmembrane topology of its N-terminal hydrophobic region. Mutational analysis of PvdA, and complementation assays in a P. aeruginosa ΔpvdA mutant, demonstrated the dual (structural and functional) role of the PvdA N-terminal domain.Keywords:
Pyoverdine
Structural gene
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ABSTRACT Escherichia coli atp mutants, which lack a functional H + -ATPase complex, are capable of growth on glucose but not on succinate or other C 4 -dicarboxylates (Suc − phenotype). Suc + revertants of an atp deletion strain were isolated which were capable of growth on succinate even though they lack the entire H + -ATPase complex. Complementation in trans with the yhiF gene suppressed the growth of the Suc + mutants on succinate, which implicates the yhiF gene product in the regulation of C 4 -dicarboxylate metabolism. Indeed, when the E. coli C 4 -dicarboxylate transporter (encoded by the dctA gene) was expressed in trans , the Suc − phenotype of the atp deletion strain reverted to Suc + , which shows that the reason why the E. coli atp mutant is unable to grow aerobically on C 4 -dicarboxylates is insufficient transport capacity for these substrates.
Adenosine triphosphate
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Lotus japonicus
Symbiotic bacteria
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Plasmids carrying cloned segments of the unc operon of Escherichia coli have been used in genetic complementation analyses to identify three independent mutants defective in the uncH gene, which codes for the delta subunit of the ATP synthetase. Mutations in other unc genes have also been mapped by this technique. ATPase activity was present in extracts of the uncH mutants, but the enzyme was not as tightly bound to the membrane as it was in the parental strain. ATP-dependent membrane energization was absent in membranes isolated from the uncH mutants and could not be restored by adding normal F1 ATPase from the wild-type strain. F1 ATPase prepared from uncH mutants could not restore ATP-dependent membrane energization when added to wild-type membranes depleted of F1. Membranes of the uncH mutants were not rendered proton permeable as a result of washing with low-ionic-strength buffer.
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Lambda phage
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Mutants affecting lactose metabolism in Escherichia coli K12 have been shown by functional and genetic analysis to belong to a linked set of adjacent genes: the Lac operon. The Z gene of this group is the structural gene for β-galactosidase (Gz) as shown by the fact that all Z− mutants lack the ability to form active wild type enzyme and that many of them form altered proteins (CRM) called Cz which cross-react immunologically with β-galactosidase.
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Flavodoxin (Fld) is a bacterial electron-transfer protein that possesses flavin mononucleotide as a prosthetic group. In the genomes of the Pseudomonas species, the mioC gene is the sole gene, annotated Fld, but its function remains unclear. In this study, phenotype microarray analysis was performed using the wild-type and mioC mutant of pathogenic Pseudomonas aeruginosa PAO1. Our results showed that the mioC mutant is very resistant to oxidative stress. Different antibiotics and metals worked differently on the sensitivity of the mutant. Other pleiotropic effects of mutation in the mioC gene, such as biofilm formation, aggregation ability, motility and colony morphology, were observed under iron stress conditions. Most of the phenotypic and physiological changes could be recovered in the wild type by complementation. Mutation of the mioC gene also influenced the production of pigments. The mioC mutant and mioC over-expressed complementation cells, over-produced pyocyanin and pyoverdine, respectively. Various secreted chemicals were also changed in the mutant, which was confirmed by (1) H NMR analysis. Interestingly, physiological alterations of the mutant strain were restored by the cell-free supernatant of the wild type. The present study demonstrates that the mioC gene plays an important role in the physiology of P. aeruginosa and might be considered as a suitable drug target candidate in pathogenic P. aeruginosa.
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Seventy three temperature-sensitive mutants of Pseudomonas aeruginosa SM phage have been obtained using different mutagens and assigned to thirteen complementation groups. Representative mutants of each group have been studied with the aim of characterizing tentatively the time of genes expression in infected bacteria. Two genes appear to function during the first minutes after infection, whereas the remaining genes are needed for late functions. Most of the temperature-sensitive functions in the different mutants are reversible, i.e. they become active when the infected cells are shifted-down to the permissive temperature.
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Escherichia coli UB1005 (DCO), an envelope mutant (DC2), Pseudomonas aeruginosa 799 and an envelope mutant (799/61) were exposed to sodium deoxycholate (DOC), sarkosyl and sodium lauryl sulphate (SLS). DOC was the least effective lytic agent, but the two Ps. aeruginosa strains, especially 799/61, were more susceptible to DOC and sarkosyl than the E. coli ones. SLS was an efficient lysing agent, although Ps. aeruginosa 799 was the least susceptible of the four strains. DC2 was lysed more rapidly and to a greater extent than UB1005 by all three agents. The mutant strains, especially DC2, were more sensitive to selective media than the wild‐type ones.
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