RpoS-Regulated Genes ofEscherichia coliIdentified by RandomlacZFusion Mutagenesis

Abstract

RpoS is a conserved alternative sigma factor that regulates the expression of many stress response genes in Escherichia coli. The RpoS regulon is large but has not yet been completely characterized. In this study, we report the identification of over 100 RpoS-dependent fusions in a genetic screen based on the differential expression of an operon-lacZ fusion bank in rpoS mutant and wild-type backgrounds. Forty-eight independent gene fusions were identified, including several in well-characterized RpoS-regulated genes, such as osmY, katE, and otsA. Many of the other fusions mapped to genes of unknown function or to genes that were not previously known to be under RpoS control. Based on the homology to other known bacterial genes, some of the RpoS-regulated genes of unknown functions are likely important in nutrient scavenging.RpoS, an alternative sigma factor, controls a large regulon that is specifically expressed when the cell is nutrient deprived or is subjected to external stress, such as osmotic shock (32). Genes that are dependent on RpoS have been identified in different contexts from many studies of regulation in various gram-negative bacteria (38). From these studies it is clear that the regulon encodes many proteins that help bacteria adapt to adverse conditions, including those that are involved in nutrient scavenging, DNA repair, protein turnover, and protection from external environmental insult (31).Characterization of the RpoS regulon has relied on several experimental approaches, including two-dimensional gel electrophoresis (50), identification of genes that are induced by RpoS-related stimuli such as carbon starvation (85), and by identifying gene fusions that depend on RpoS for expression (66). Use of bacterial cDNA microarrays or macroarrays, while potentially useful in identifying additional members of the RpoS regulon, have been employed in only a few studies of Escherichia coli and have not yet been used to directly assess RpoS dependence of genes in isogenic wild-type and rpoS mutant strains. In a previous study (66), our lab introduced an RpoS-null allele into a bank of random operon-lacZ fusions to facilitate the identification of RpoS-dependent genes based solely on RpoS requirement. This resulted in the identification of several new highly RpoS-dependent genes in E. coli (66), and similar approaches have been employed with Salmonella spp. where other RpoS-dependent genes have been identified (37). Lac gene fusions are useful measures of gene expression, because the product, ␤-galactosidase, is stable and easily assayed (16). This makes lacZ fusions particularly suitable for the study of genes that are expressed in stationary phase, where the stability of the RNA and protein products of the affected gene is not known.In this study, we report the completion of the identification of reporter fusions identified in a previous genetic screen of lacZ-expressing fusion mutants. In addition, we examined the expression of the RpoS-dependent operon fusions in rich and minimal media and classified ...

The Stationary-Phase Sigma Factor σ S Is Responsible for the Resistance of Escherichia coli Stationary-Phase Cells to mazEF -Mediated Cell Death

“…ROS have been previously implicated in programmed cell death in eukaryotes (21,29), including in yeast (12,17), in the life span of several organisms (25,33), in the senescence of bacteria (6), and in the mode of action of some antibiotics (13)(14)(15). It was previously reported that the stationary-phase sigma factor S , encoded by rpoS (16,19), positively regulates the formation of catalase and is responsible for the elevated levels of this enzyme during stationary growth phase (24,34,35). Since catalase detoxifies ROS, we asked whether resistance of stationary-phase cells to mazEF-mediated cell death was caused by the elevated levels of catalase produced at that time.…”

A DNA damage response in Escherichia coli involving the alternative sigma factor, RpoS

“…The oxidative stress response controlled by RpoS in stationary phase is well studied, and a number of proteins that metabolize reactive oxygen species, such as catalase and superoxide dismutase, are under regulation of S (25,26). Our demonstration of sensitivity of rpoS mutants to the replication inhibitor AZT during exponential growth suggests that S may control other functions important for DNA damage repair or tolerance.…”

The SMC-Like Protein Complex SbcCD Enhances DNA Polymerase IV-Dependent Spontaneous Mutation inEscherichia coli

“…RpoS regulates over 100 genes during stationary phase and up to 500 genes in response to various other stresses (54,68,71). In addition, RpoS has been found by transcription microarray analysis to regulate directly or indirectly almost 300 genes in exponential-phase cells (14a).…”