Identification of conserved, RpoS-dependent stationary-phase genes of Escherichia coli

Abstract

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 100 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.

FIG. 1

Schematic representation of the strategy used to identify transconjugants harboring RpoS (ς^s)-dependent promoter-lacZ fusions.

FIG. 2

The 105 recipient (rpoS⁺) and transconjugant (rpoS) pairs in a GC4468 background. Strains were plated on M9 minimal media supplemented with 0.4% glucose. The ς^s-dependent and -independent control strains NC4468 (katE::lacZ) and 13C10, respectively, were placed in the top row, with rpoS derivatives placed adjacent to them. rpoS⁺ and rpoS derivative pairs are adjacent to one another in rows, starting from the top left. The rpoS status of each column is shown on the top (+, rpoS⁺; −, rpoS).

FIG. 3

Growth-phase-dependent expression of 10 highly-RpoS-dependent fusions in rich medium. Flasks containing LB broth were inoculated with exponentially growing cultures as described in Materials and Methods, sampled periodically as indicated, and assayed for growth (OD₆₀₀) and β-galactosidase activity. Each panel shows the growth of the culture and the β-galactosidase activity in strains carrying promoter-lacZ fusions to the indicated gene. The levels of growth of the wild-type strain and rpoS derivatives were equivalent, and thus only growth data for the wild-type strain are shown. ○, growth (OD₆₀₀); ■, β-galactosidase activity in the wild-type strain; □, β-galactosidase activity in the rpoS derivative.

FIG. 4

Location of points of insertion of transcriptional fusions in RpoS-regulated genes identified in this study. Arrows indicate the direction of transcription of genes. ▪, highly RpoS-dependent genes carrying the promoter-lacZ fusions identified in this study; , other RpoS-dependent genes (see text); □, genes not known to require RpoS for expression.

FIG. 5

RpoS- and growth-phase-dependent expression of rsd genes. The results of Northern analyses of total RNA isolated from exponential-phase (E) and stationary-phase (S) cultures of wild-type (GC4468) and rpoS (GC122) strains are shown. RNA was hybridized with probes specific for osmY, katE, and gabP. To confirm that equivalent amounts of RNA were extracted and loaded, control blots were probed with rrnA, an RpoS-independent gene (data not shown).

FIG. 6

Growth-phase-dependent expression of an RpoS-dependent fusion and an RpoS-independent fusion. Flasks containing LB broth were inoculated with exponentially growing cultures as described in Materials and Methods, sampled periodically as indicated, and assayed for growth (OD₆₀₀) and β-galactosidase activity. Each panel shows growth of the culture and β-galactosidase activity in strains carrying promoter-lacZ fusions to the indicated gene. The levels of growth of the wild-type strain and rpoS derivatives were equivalent, and thus only growth data for the wild-type strain are shown. (A) rsd1004 (ldcC) (RpoS dependent). (B) 13C10 (RpoS independent). ○, growth (OD₆₀₀); ■, β-galactosidase activity in the rpoS⁺ strain; □, β-galactosidase activity in the rpoS derivative.