Nitric oxide is a free radical (NO) formed biologically through the oxidation of L-arginine by nitric oxide synthases. NO is produced transiently in mammalian cells for intercellular signaling and in copious quantities to cause cytostasis and cytotoxicity. In the latter situation, NO is a deliberate cytotoxic product of activated macrophages, along with other reactive oxygen species such as hydrogen peroxide (H202) and superoxide (2--). Escherichia coli has a complex set of responses to H202 and 02--that involves =80 inducible proteins; we wondered whether these bacteria might induce analogous defenses against nitric oxide. We show here that a multigene system controlled by the redox-sensitive transcriptional regulator SoxR is activated by NO in vivo. This induction confers bacterial resistance to activated murine macrophages with kinetics that parallel the production of NO by these cells.

Escherichia coli responds to the redox stress imposed by superoxide-generating agents such as paraquat by activating the synthesis of as many as 80 polypeptides. Expression of a key group of these inducible proteins is controlled at the transcriptional level by the soxRS locus (the soxRS regulon). A two-stage control system was hypothesized for soxRS, in which an intracellular redox signal would trigger the SoxR protein as a transcriptional activator of the soxS gene and the resulting increased levels of SoxS protein would activate transcription of the various soxRS regulon genes (B. Demple and C.F. Amábile Cuevas, Cell 67:837-839, 1990). We have constructed operon fusions of the E. coli lac genes to the soxS promoter to monitor soxS transcription. Expression from the soxS promoter is strongly inducible by paraquat in a manner strictly dependent on a functional soxR gene. Several other superoxide-generating agents also trigger soxR(+)-dependent soxS expression, and the inductions by paraquat and phenazine methosulfate were dependent on the presence of oxygen. Numerous other oxidative stress agents (H2O2, gamma rays, heat shock, etc.) failed to induce soxS, while aerobic growth of superoxide dismutase-deficient bacteria triggered soxR-dependent soxS expression. These results indicate a specific redox signal for soxS induction. A direct role for SoxR protein in the activation of the soxS gene is indicated by band-shift and DNase I footprinting experiments that demonstrate specific binding of the SoxR protein in cell extracts to the soxS promoter. The mode of SoxR binding to DNA appears to be similar to that of its homolog MerR in that the SoxR footprint spans the -10 to -35 region of the soxS promoter.

The complete genomic sequence of the archaeon Thermoplasma volcanium, possessing optimum growth temperature (OGT) of 60°C, is reported. By systematically comparing this genomic sequence with the other known genomic sequences of archaea, all possessing higher OGT, a number of strong correlations have been identified between characteristics of genomic organization and the OGT. With increasing OGT, in the genomic DNA, frequency of clustering purines and pyrimidines into separate dinucleotides rises (e.g., by often forming AA and TT, whereas avoiding TA and AT). Proteins coded in a genome are divided into two distinct subpopulations possessing isoelectric points in different ranges (i.e., acidic and basic), and with increasing OGT the size of the basic subpopulation becomes larger. At the metabolic level, genes coding for enzymes mediating pathways for synthesizing some coenzymes, such as heme, start missing. These findings provide insights into the design of individual genomic components, as well as principles for coordinating changes in these designs for the adaptation to new environments.

We measured the generation of hydroxyl radical (OH ⅐ ) and oxidative DNA lesions in aerobically grown Escherichia coli cells lacking in both superoxide dismutases (SodA SodB) and repressor of iron uptake (Fur) using electroparamagnetic resonance and gas chromatography-mass spectrometry with a selected-ion monitoring method. A specific signal corresponding to OH ⅐ generation and an increase in oxidative DNA lesions such as 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine were detected in the strain deficient in sodA sodB fur. We showed that iron metabolism deregulation in fur mutant produced a 2.5-fold iron overload. The sodA sodB fur strain was about 100-fold higher mutability than the wild-type strain. The mutation spectrum in the strain was found to induce GC 3 TA and AT 3 CG transversions predominantly. The hypermutability of the strain was suppressed by the tonB mutation which reduces iron transport. Thus, excess iron and excess superoxide were responsible for OH ⅐ generation, oxidative DNA lesion formation, and hypermutability in E. coli.Reactive oxygen species such as superoxide anion radical (O 2 . ), 1 hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (OH ⅐ ), can react with various biological molecules due to their high reactivities (1, 2). The genetic materials such as DNA, RNA, and their precursors in nucleotide pools are important targets of reactive oxygen species (3-5). Reactive oxygen species produces a large number of DNA lesions including 7,8-dihydro-8-oxoguanine (8-oxo-G), 1,2-dihydro-2-oxoadenine (2-oxo-A), thymine glycol and strand breaks, and also oxidizes nucleotides to form 8-oxo-7,8-dihydro-2Ј-deoxyguanosine 5Ј-triphosphate (8-oxo-dGTP) (4) and 2-oxo-1,2-dihydro-2Ј-deoxyadenosine 5Ј-triphosphate (2-oxo-dATP) (6), which cause mutation and replication block (5).To reduce such mutagenic and cytotoxic effects caused by oxidative DNA lesions, Escherichia coli have evolved DNA repair enzymes and a clean-up enzyme. The former includes DNA glycosylases for 8-oxo-G (encoded by mutM), for adenine at 8-oxo-G:A mispairing site (encoded by mutY) and for thymine glycol (encoded by nth and nei), and the latter contains a hydrolase for 8-oxo-dGTP in the nucleotide pool, 8-oxo-dGTPase (encoded by mutT) (7-10). Mutations in mutM and mutY, and in mutT lead E. coli cells to be spontaneously hypermutagenic (10 -100-fold higher than wild-type), predominantly inducing GC 3 TA and AT 3 CG transversions, respectively (8). These results suggest the importance of these enzymes in minimizing oxidative DNA lesions and in keeping spontaneous mutation rate as low as 10 Ϫ8 -10 Ϫ9 .A long-standing proposal for the mechanism of the first step of oxidative mutagenesis is the reaction of bases in DNA or nucleotides in the pool with OH ⅐ , a highly reactive reactive oxygen species, which can be generated during the HarberWeiss/Fenton reaction that consists of an iron reduction step by O 2. and an OH ⅐ generation step via the Fenton reaction,It has been suggested that these reactions may enable endogenous oxidants to co...