Paradoxically, nitric oxide (NO) has been found to exhibit cytotoxic, antiproliferative, or cytoprotective activity under different conditions. We have utilized Salmonella mutants deficient in antioxidant defenses or peptide transport to gain insights into NO actions. Comparison of three NO donor compounds reveals distinct and independent cellular responses associated with specific redox forms of NO.The peroxynitrite (OONO-) generator 3-morpholinosydnonimine hydrochloride mediates oxygen-dependent Salmonella killing, whereas S-nitrosoglutathione (GSNO) causes oxygenindependent cytostasis, and the NO-donor diethylenetriamine-nitric oxide adduct has no antibacterial activity. GSNO has the greatest activity for stationary cells, a characteristic relevant to latent or intracellular pathogens. Moreover, the cytostatic activity of GSNO may best correlate with antiproliferative or antimicrobial effects of NO, which are unassociated with overt cell injury. dpp mutants defective in active dipeptide transport are resistant to GSNO, implicating heterolytic NO+ transfer rather than homolytic NO-release in the mechanism of cytostasis. This transport system may provide a specific pathway for GSNO-mediated signaling in biological systems. The redox state and associated carrier molecules are critical determinants of NO activity.Nitric oxide (NO) cytotoxicity has been demonstrated for a rapidly expanding list of helminths, protozoa, yeasts, bacteria, and viruses (reviewed in ref. 1), as well as for tumor cells (2). This property of NO has important implications for understanding mechanisms of antimicrobial defense, antitumor defense, cell injury in inflammatory diseases, and food preservation by nitrites. Potential molecular targets of NO include transition metals, thiols, lipids, and DNA (3). Interaction with reactive oxygen intermediates is generally believed to be required for NO cytotoxicity (4), but the precise mechanisms are incompletely understood. Moreover, it is not presently understood how NO can possess cytotoxic, antiproliferative, or cytoprotective activity under different conditions (5).Under physiologic conditions, NO may react with thiolcontaining molecules such as glutathione (GSH) to form S-nitrosothiols (6-8). S-Nitrosothiols have been detected in human bronchoalveolar lavage fluid, plasma, platelets, and polymorphonuclear neutrophils (7), with higher concentrations measured in inflammatory states (8). Although conventionally viewed as NO donor compounds that undergo spontaneous homolytic release of NO-, S-nitrosothiols are also capable of heterolytic transfer of nitrosonium (NO') to other sulfhydryl centers (7). S-Nitrosothiols have been recognized to possess antimicrobial activity, including against Salmonella (9). These compounds have also been proposed to mediate NO' transfer to outer membrane thiols in Bacillus, which inhibits spore outgrowth (10). Differences in stability and target responses relative to NO-lend credence to the suggestion that S-nitrosothiols are important physiologic redox forms...

A 5.9-kb DNA fragment was cloned from Pseudomonas aeruginosa PA103 by its ability to functionally complement a fur mutation in Escherichia coli. A fur null mutant E. coli strain that contains multiple copies of the 5.9-kb DNA fragment produces a 15-kDa protein which cross-reacts with a polyclonal anti-E. coli Fur serum. Sequencing of a subclone of the 5.9-kb DNA fragment identified an open reading frame predicted to encode a protein 53% identical to E. coli Fur and 49% identical to Vibrio cholerae Fur and Yersinia pestis Fur. While there is extensive homology among these Fur proteins, Fur from P. aeruginosa differs markedly at its carboxy terminus from all of the other Fur proteins. It has been proposed that this region is a metal-binding domain in E. coli Fur. A positive selection procedure involving the isolation of manganese-resistant mutants was used to isolate mutants of strain PA103 that produce altered Fur proteins. These manganese-resistant Fur mutants constitutively produce siderophores and exotoxin A when grown in concentrations of iron that normally repress their production. A multicopy plasmid carrying the P. aeruginosa fur gene restores manganese susceptibility and wild-type regulation of exotoxin A and siderophore production in these Fur mutants.

A multicopy plasmid containing the Escherichia coli fur gene was introduced into Pseudomonas aeruginosa strain PA103C. This strain contains a toxA-lacZ fusion integrated into its chromosome at the toxA locus. Beta-galactosidase synthesis in this strain is regulated by iron, as is seen for exotoxin A production. Beta-galactosidase synthesis and exotoxin A production in PA103C containing multiple copies of E. coli fur was still repressed in low iron conditions. The transcription of regA, a positive regulator of toxA, was also found to be inhibited by multiple copies of the E. coli fur gene. In addition, the ability of PA103C containing multiple copies of E. coli fur to produce protease was greatly reduced relative to PA103C containing a vector control. A polyclonal rabbit serum containing antibodies that recognize E. coli Fur was used to screen whole-cell extracts from Vibrio cholerae, Shigella flexneri, Salmonella typhimurium and Pseudomonas aeruginosa. All strains tested expressed a protein that was specifically recognized by the anti-Fur serum. These results and those described above suggest that Fur structure and function are conserved in a variety of distinct bacterial genera and that at least some of these different genera use this regulatory protein to control genes encoding virulence factors.

The Escherichia coli aidB gene is regulated by two different mechanisms, an ada-dependent pathway triggered by methyl damage to DNA and an ada-independent pathway triggered when cells are grown without aeration. In this report we describe our search for mutations afecting the ada-independent aidB induction pathway. The mutant strain identified carries two mutations affecting aidB expression. These mutations are named abrB (aidB regulator) and abrD. The abrB mutation is presently poorly characterized because of instability of the phenotype it imparts. The second mutation, abrDl, reduces the expression of aidB observed when aeration is ceased and oxygen becomes limiting. Genetic and phenotypic analysis of the abrDI mutation demonstrates that it is an allele of rpoS. Thus, aidB is a member of the family of genes that are transcribed by a crs-directed RNA polymerase holoenzyme. Examination ofaidB expression in an rpoS insertion mutant strain indicates that both rpoSl3::TnlO and abrDl mutations reduce aidB expression under oxygen-limiting conditions that prevail in unaerated cultures, reduce aidB induction by acetate at a low pH, but have little or no elect on the adadependent alkylation induction of aidB.The Escherichia coli aidB gene is one of several genes induced in response to alkylation damage caused by treatments with agents that methylate DNA. The aidB gene encodes a protein that has a high degree of homology to several mammalian coenzyme A dehydrogenases (12). In agreement with this homology, it has been shown that the aidB gene product has isovaleryl coenzyme A dehydrogenase activity (12), an activity known to be required for leucine metabolism in mammalian cells (8,9). Overexpression of aidB protein has also been shown to reduce the mutagenic effects of the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine, although the mechanism responsible for this resistance is unclear (12). Thus, aidB has roles in basic metabolism as well as defenses against methylation damage.In E. coli, the Ada protein mediates the response to alkylation damage, which has been called the adaptive response. Adaptive-response induction occurs when the Ada protein transfers a methyl group from methylphosphotriesters formed in DNA by methylating agents to its own Cys-69 residue (for reviews, see references 12, 15, and 36). Methylation of Ada converts this protein to a positive regulatory factor that binds sequences upstream of the ada, alkA, and aidB promoters, stimulating their transcription (12,15,36). Unlike the ada-alkB operon and the alkA gene, aidB is also subject to a second form of regulation (18,37,39). This second aidB induction pathway is activated when cells are grown in the absence of aeration and does not require a functional ada gene (37). Thus, there are two independent aidB regulatory pathways: an ada-dependent alkylation induction pathway and an ada-independent pathway induced when aeration is ceased and oxygen becomes limiting.To examine further the regulatory pathway triggered in unaerated cultures, random mini-T...

A mobilizable plasmid which carries the promoter for the exotoxin A (ETA) structural gene fused to lacZ was integrated into the chromosome of wild-type and mutant strains of Pseudomonas aeruginosa at the toxA locus by homologous recombination. beta-galactosidase synthesis in the strains (cointegrates) carrying the toxA-lacZ fusions was regulated like ETA synthesis is in P. aeruginosa. Two multicopy plasmids carrying a positive regulatory gene designated toxR were constructed which are identical except with respect to the orientation of toxR to the lacZ promoter on the plasmid. These plasmids were then introduced into P. aeruginosa cointegrate strains. When toxR was using its own promoter, synthesis of beta-galactosidase in the cointegrate strains was increased but the pattern of iron regulation was not altered. In contrast, when the lacZ promoter was directing synthesis of the toxR product in the cointegrate strains, iron regulation of beta-galactosidase and ETA synthesis were abolished.