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The impact of arsenite [As(III)] on several levels of cellular metabolism and gene regulation was examined in Pseudomonas aeruginosa. P. aeruginosa isogenic mutants devoid of antioxidant enzymes or defective in various metabolic pathways, DNA repair systems, metal storage proteins, global regulators, or quorum sensing circuitry were examined for their sensitivity to As(III). Mutants lacking the As(III) translocator (ArsB), superoxide dismutase (SOD), catabolite repression control protein (Crc), or glutathione reductase (Gor) were more sensitive to As(III) than wild-type bacteria. The MICs of As(III) under aerobic conditions were 0.2, 0.3, 0.8, and 1.9 mM for arsB, sodA sodB, ctc, and gor mutants, respectively, and were 1.5- to 13-fold less than the MIC for the wild-type strain. A two-dimensional gel/matrix-assisted laser desorption ionization--time of flight analysis of As(III)-treated wild-type bacteria showed significantly (>40-fold) increased levels of a heat shock protein (IbpA) and a putative allo-threonine aldolase (GlyI). Smaller increases (up to 3.1-fold) in expression were observed for acetyl-coenzyme A acetyltransferase (AtoB), a probable aldehyde dehydrogenase (KauB), ribosomal protein L25 (RpIY), and the probable DNA-binding stress protein (PA0962). In contrast, decreased levels of a heme oxygenase (HemO/PigA) were found upon As(III) treatment. Isogenic mutants were successfully constructed for six of the eight genes encoding the aforementioned proteins. When treated with sublethal concentrations of As(III), each mutant revealed a marginal to significant lag period prior to resumption of apparent normal growth compared to that observed in the wild-type strain. Our results suggest that As(III) exposure results in an oxidative stress-like response in P. aeruginosa, although activities of classic oxidative stress enzymes are not increased. Instead, relief from As(III)-based oxidative stress is accomplished from the collective activities of ArsB, glutathione reductase, and the global regulator Crc. SOD appears to be involved, but its function may be in the protection of superoxide-sensitive sulfhydryl groups.

Under conditions of iron limitation, Pseudomonas aeruginosa secretes a high-affinity siderophore pyoverdine to scavenge Fe(III) in the extracellular environment and shuttle it into the cell. Uptake of the pyoverdine--Fe(III) complex is mediated by a specific outer-membrane receptor protein, FpvA (ferripyoverdine receptor). Three P. aeruginosa siderovars can be distinguished, each producing a different pyoverdine (type I-III) and a cognate FpvA receptor. Growth of an fpvA mutant of P. aeruginosa PAO1 (type I) under iron-limiting conditions can still be stimulated by its cognate pyoverdine, suggesting the presence of an alternative uptake route for type I ferripyoverdine. In silico analysis of the PAO1 genome revealed that the product of gene PA4168 has a high similarity with FpvA. Inactivation of PA4168 (termed fpvB) in an fpvA mutant totally abolished the capacity to utilize type I pyoverdine. The expression of fpvB is induced by iron limitation in Casamino acids (CAA) and in Mq-glucose medium, but, unlike fpvA, not in a complex deferrated medium containing glycerol as carbon source. The fpvB gene was also detected in other P. aeruginosa isolates, including strains producing type II and type III pyoverdines. Inactivation of the fpvB homologues in these strains impaired their capacity to utilize type I ferripyoverdine as a source of iron. Accordingly, introduction of fpvB in trans restored the capacity to utilize type I ferripyoverdine.

Results demonstrate that organic hydroperoxide resistance (ohr) gene encoded product is involved in the decomposition (resistance) of organic hydroperoxides. Data reveal that upon exposure to organic hydroperoxides, a 15-fold increase in the transcriptional activity of ohr is observed.

Results demonstrate that Pseudomonas aeruginosa mutants carrying xylE fusion plasmids loose greater than 95% catechol-2,3-dioxygenase on exposure to hydrogen peroxide. Data indicate that enzyme activity loss is due to the oxidation of the active-site ferrous-ion.

Results show that oxyR mutants of Pseudomonas aeruginosa are sensitive to hydrogen peroxide, can not survive aerobically, and tolerate aminoglycosides. However, the mutants survive in presence of either catalase KatA or under anaerobic conditions.

Results show the periplasmic ankyrin-like protein AnkB exhibit four 33-amino-acid ankyrin repeats in a 183 amino acids stretch of the protein. Data further indicate that AnkB together with a hydrogen peroxide-inducible KatB protein forms a protective lattice work for hydrogen peroxide detoxification.

Results demonstrate that expression of oxidative stress defence genes katB-ankB, ahpB, and ahpC-ahpF is regulated by oxyR gene product and is located upstream of DNA repair enzyme coding gene recG. Data indicate that the oxyR-recG locus governs oxidative stress and DNA repair function in Pseudomonas aeruginosa.

Results demonstrate identification of haem iron uptake gene cluster consisting of Pseudomonas haem uptake (phu) locus and haem-acquisition system (has) locus in Pseudomonas aeruginosa. Data indicate that phu and has are controlled by Fur protein and individually encode two distinct systems for the acquisition of haem and haemoglobin.

A unique regulator of the exotoxin A gene (toxA) in Pseudomonas aeruginosa, PtxR, has been previously identified. Because toxA expression and siderophore production in this bacterium 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 studied. Findings showed that the iron-regulated expression of ptxR under microaerobic conditions from the P2 promoter of ptxR is controlled directly by Fur through the iron-regulated expression of pvdS.

The ferric uptake regulatory protein (Fur)-associated gene (fagA) of Pseudomonas aeruginosa was cloned and analyzed to determine the role of Fur in the regulation of alginate biosynthesis. Sequence analysis of the cloned Pseudomonas aeruginosa fagA gene indicated the presence of two iron boxes or Fur binding sites in the upstream region of the gene. Furthermore, the gene was directly regulated and indirectly regulated by the alternative sigma factor AlgT(U) which was mediated by iron homeostasis.

The A4 and C6 ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa are incapable of proper siderophore-mediated iron uptake and aerobic growth. This is due to a decrease in total superoxide dismutase (SOD) and catalase activity. The mutants have a higher manganese-SOD (Mn-SOD) activity, contain altered Fur proteins, and constitutively produce the siderophores pyochelin and pyoverdin. Mutants of strain PAO1, with normal pyochelin and defective pyoverdin biosynthesis, have increased Mn-SOD activity. Sensitivity to redox cycling agent paraquat and hydrogen peroxide is greater in the mutants.

A T7 expression system is used to overexpress the clone Pseudomonas aeruginosa fur gene on P. aeruginosa while ion-exchange chromatography is employed in conjunction with metal affinity chromatography to purify the gene's fur protein. Results suggest that the iron regulation of exotoxin production in P. aeruginosa involves additional factors that may be under the control of PA-fur.

Sequential analysis of a 16-kDa extracellular protein, produced by Pseudomonas aeruginosa PG201 in n-hexadecane medium, reveals the similarity of this protein with a protein-like activator for n-alkane oxidation (PA). Cloning and sequential analysis of the PA-encoding (pra) gene reveals the presence of a protein-coding region of 162 amino acids. Insertional mutagenesis leads to inactivation of pra gene and mutant strains lack the extracellular PA protein. Escherichia coli expresses the pra gene, and metal chelate affinity chromatography helps purity the recombinant protein present in the extracellular growth medium.

Rhamnolipid biosynthesis in Pseudomonas aeruginosa is activated by rhlR regulatory gene. The 2-kb long gene encodes a 28-kDa protein (RhlR) that functions as a transcriptional activator. The gene has a putative target promoter in the upstream. Rhamnolipid-deficient mutations are produced when wild-type rhlR genes are disrupted indicating the pleiotropic regulatory action of the RhlR protein.

Results indicate that catalase protects Pseudomonas aeruginosa biofilms by not allowing hydrogen peroxide penetrate full into the aggregated bacteria.

The isogenic catalase A (KatA) genes and bfrA mutants in Pseudomonas aeruginosa have been found to induce the strain's hypersusceptibility to hydrohgen peroxide (H2O2). By cloning the strain's genomic DNA fragments, including rpoA, rplQ, katA and bfrA genes, the existence of two transitional codons in KatA genes was confirmed. These codons have been found to participate in the translation of a heteromultimer of 160 to 170 kDa. The degree of gene transcription in KatA and bfrA genes has further been found to be influenced by the presence of H2O2 and iron.

Research was conducted to examine the hypothesis that Pseudomonas aeruginosa fur overlaps with a gene encoding a novel outer membrane lipoprotein, Om1A.Cell fractionation method and Western blot analysis provide strong evidence that all Om1A protein is localized in the outer membrane. The inhibition of signal peptidase I by globomycin led to failure to process Om1A which demonstrate that Om1A is a lipoprotein.