During entry into stationary phase, many free-living, gram-negative bacteria express genes that impart cellular resistance to environmental stresses, such as oxidative stress and osmotic stress. Many genes that are required for stationary-phase adaptation are controlled by RpoS, a conserved alternative sigma factor, whose expression is, in turn, controlled by many factors. To better understand the numbers and types of genes dependent upon RpoS, we employed a genetic screen to isolate more than

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... 00 independent RpoS-dependent gene fusions from a bank of several thousand mutants harboring random, independent promoter-lacZ operon fusion mutations. Dependence on RpoS varied from 2-fold to over 100-fold. The expression of all fusion mutations was normal in an rpoS/rpoS ؉ merodiploid (rpoS background transformed with an rpoS-containing plasmid). Surprisingly, the expression of many RpoS-dependent genes was growth phase dependent, albeit at lower levels, even in an rpoS background, suggesting that other growth-phase-dependent regulatory mechanisms, in addition to RpoS, may control postexponential gene expression. These results are consistent with the idea that many growth-phase-regulated functions in Escherichia coli do not require RpoS for expression. The identities of the 10 most highly RpoS-dependent fusions identified in this study were determined by DNA sequence analysis. Three of the mutations mapped to otsA, katE, ecnB, and osmY-genes that have been previously shown by others to be highly RpoS dependent. The six remaining highly-RpoS-dependent fusion mutations were located in other genes, namely, gabP, yhiUV, o371, o381, f186, and o215. Like many other free-living bacteria, Escherichia coli lives in environments that may change rapidly with respect to both nutrients and physical conditions. To survive stresses associated with starvation, E. coli expresses many stationary-phasespecific genes whose expression depends largely on an alternative sigma factor, s , encoded by rpoS (27, 30). Inactivation of this gene renders the cell sensitive to heat shock (25, 29), oxidative stress (25, 29), osmotic challenge (29), and near-UV light (40). Proteins that depend on RpoS include catalase HPII (33, 39, 42) and catalase HPI (32), exonuclease III (39), penicillin-binding proteins (15), and osmoprotective proteins (21, 22, 53) . RpoS is required for virulence (17) and acid tolerance (6) in Salmonella typhimurium. Although the signal(s) giving rise to increased expression of RpoS itself is not completely understood, homoserine lactone (23), UDP-6-glucose (10), and weak acids, such as acetate (42) , have been shown to be inducers of RpoS. Several approaches have been used to enumerate and identify RpoS-regulated functions. Many of these genes, however, are probably still unidentified. Two-dimensional gel electrophoresis studies of proteins expressed in wild-type and rpoS strains have revealed that the RpoS regulon is quite large (30). Mutagenesis with random lacZ (16, 51) or lux insertions (46) , coupled with screening for RpoS-related characteristic phenotypes, has also been successfully employed to identify new RpoS-regulated genes (51). However, unlike other regulons, the RpoS regulon does not have a single unifying characteristic or differentiating phenotype that all members share. These factors, in addition to its suspected large size, have delayed complete characterization of the regulon. To circumvent the problems associated with the characteristics described above, we have employed a mutant identification scheme in which an rpoS null allele is introduced into strains containing random promoter-lacZ fusions to directly identify RpoS dependency. Since this procedure does not rely on a phenotype specific for the regulon (e.g., carbon starvation), this method should be of general use in the identification of members of any regulon for which a null allele of a positive-acting regulator is available.