The genome-wide transcription profile of Escherichia coli cells treated with hydrogen peroxide was examined with a DNA microarray composed of 4,169 E. coli open reading frames. By measuring gene expression in isogenic wild-type and oxyR deletion strains, we confirmed that the peroxide response regulator OxyR activates most of the highly hydrogen peroxide-inducible genes. The DNA microarray measurements allowed the identification of several new OxyR-activated genes, including the hemH heme biosynthetic gene; the six-gene suf operon, which may participate in Fe-S cluster assembly or repair; and four genes of unknown function. We also identified several genes, including uxuA, encoding mannonate hydrolase, whose expression might be repressed by OxyR, since their expression was elevated in the ⌬oxyR mutant strain. In addition, the induction of some genes was found to be OxyR independent, indicating the existence of other peroxide sensors and regulators in E. coli. For example, the isc operon, which specifies Fe-S cluster formation and repair activities, was induced by hydrogen peroxide in strains lacking either OxyR or the superoxide response regulators SoxRS. These results expand our understanding of the oxidative stress response and raise interesting questions regarding the nature of other regulators that modulate gene expression in response to hydrogen peroxide.
In Escherichia coli the amplification of sdiA, a positive activator of ftsQAZ, genes that are essential for septation, results in mitomycin C resistance. To help us understand this resistance phenotype, genes whose expression was altered by increased sdiA dosage were identified using a DNA microarray-based, comprehensive transcript profiling method. The expression of 62 genes was reduced by more than threefold; of these, 41 are involved in motility and chemotaxis. Moreover, the expression of 75 genes, 36 of which had been previously characterized, was elevated at least threefold. As expected, increased sdiA dosage led to significantly elevated sdiA and ddlB-ftsQAZ-lpxC operon expression. Transcription of two genes, uvrY and uvrC, located downstream of sdiA and oriented in the same direction, was elevated about 10-fold, although the intervening gene, yecF, of opposite polarity was unaffected by increased sdiA dosage. Three genes (mioC and gidAB) flanking the replication origin, oriC, were transcribed more often when sdiA dosage was high, as were 12 genes within 1 min of a terminus of replication, terB. Transcription of the acrABDEF genes, mapping in three widely spaced loci, was elevated significantly, while several genes involved in DNA repair and replication (e.g., nei, recN, mioC, and mcrC) were moderately elevated in expression. Such global analysis provides a link between septation and the response to DNA-damaging agents.
Biochemical and genetic analyses of the bacterium Salmonella typhimurium suggest that accumulation of a-ketobutyrate partially mediates the herbicidal activity of acetolactate synthase inhibitors. Growth inhibition of wild-type bacteria by the herbicide sulfometuron methyl was prevented by supplementing the medium with isoleucine, an allosteric inhibitor of threonine deaminase-catalyzed synthesis of oa-ketobutyrate. In contrast, isoleucine did not rescue the growth of a mutant containing a threonine deaminase unresponsive to isoleucine. Moreover, the hypersensitivity of seven Tn1O insertion mutants to growth inhibition by sulfometuron methyl and a-ketobutyrate correlated with their inability to convert a-ketobutyrate to less noxious metabolites. We propose that oa-ketobutyrate accumulation is an important component of sulfonylurea and imidazolinone herbicide action.
Plasmids were constructed in which DNA damage-inducible promoters recA, uvrA, and alkA from Escherichia coli were fused to the Vibrio fischeri luxCDABE operon. Introduction of these plasmids into E. coli allowed the detection of a dose-dependent response to DNA-damaging agents, such as mitomycin and UV irradiation. Bioluminescence was measured in real time over extended periods. The fusion of the recA promoter to luxCDABE showed the most dramatic and sensitive responses. lexA dependence of the bioluminescent SOS response was demonstrated, confirming that this biosensor's reports were transmitted by the expected regulatory circuitry. Comparisons were made between luxCDABE and lacZ fusions to each promoter. It is suggested that the lux biosensors may have use in monitoring chemical, physical, and genotoxic agents as well as in further characterizing the mechanisms of DNA repair. Bacterial repair of DNA damage is mediated by at least two inducible systems, the recA-independent, ada-controlled adaptive response and the recA-dependent, lexA-controlled SOS response. The former responds specifically to the presence of methylated phosphotriesters generated by DNA alkylation (39). This signal activates the ada gene product, which in turn triggers the transcription of genes such as ada, alkA, alkB, and aid (7, 20, 28, 39). In contrast, the nature of the specific inducing signal of the SOS response is not yet fully defined (21, 23, 53). Upon SOS induction, the recA gene product is converted into an active and specific protease (21). Activated RecA protein cleaves the LexA repressor and other repressors such as the phage cI product (22, 35), resulting in the transcriptional derepression of several genes, among them uvrA, recA, those needed in the lytic pathway of phage (17), and others, such as sulA, that couple DNA damage to cell division (14, 15). Recent reviews have focused upon one or both of these repair systems (39, 53). Activation of such repair systems is a measure of the mutagenic and genotoxic effects of various chemical and physical treatments. Many of the gene products, however, are difficult to assay because of the nature of their enzymatic activities and the particular substrates upon which they act. Thus, investigators have used relatively inexpensive and rapid alternative approaches. Measuring the reversion of specific auxotrophic bacterial mutations is the strategy used in the Ames tests (1, 24), and detecting restoration of bioluminescence is used in the Mutatox assay (47). The use of various transcriptional fusions also allows detection of agents that interact with DNA. The umu test (31), the rec-lac test (30), the SOS chromotest (34), and the Pro-Tox assay (32) exploit the ease with which -galactosidase specific activities can be determined (26), while the biochemical prophage induction assay (11, 37) measures additional effects of the SOS response. The use of these assays by several pharmaceutical companies in Europe, Japan, and the
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