Small RNAs and small proteins involved in resistance to cell envelope stress and acid shock in Escherichia coli: analysis of a bar-coded mutant collection

Abstract

More than 80 small regulatory RNAs (sRNAs) and 60 proteins of 16 to 50 amino acids (small proteins) are encoded in the Escherichia coli genome. The vast majority of the corresponding genes have no known function. We screened 125 DNA bar-coded mutants to identify novel cell envelope stress and acute acid shock phenotypes associated with deletions of genes coding for sRNAs and small proteins. Nine deletion mutants (ssrA, micA, ybaM, ryeF, yqcG, sroH, ybhT, yobF, and glmY) were sensitive to cell envelope stress and two were resistant (rybB and blr). Deletion mutants of genes coding for four small proteins (yqgB, mgrB, yobF, and yceO) were sensitive to acute acid stress. We confirmed each of these phenotypes in one-on-one competition assays against otherwise-wild-type lacZ mutant cells. A more detailed investigation of the SsrA phenotype suggests that ribosome release is critical for resistance to cell envelope stress. The bar-coded deletion collection we generated can be screened for sensitivity or resistance to virtually any stress condition.

FIG. 1.

Diagram of bar-coded antibiotic resistance cassettes. Kanamycin resistance cassettes flanked by two unique 20-mer DNA bar code sequences (UP and DN) were generated by a two-step PCR process for each deleted gene. The bar-coded kanamycin resistance cassettes were incorporated at loci coding for sRNAs and small proteins by homologous recombination. For the analysis of the large-scale competition experiments, bar codes upstream and downstream of every kanamycin resistance cassette were amplified by means of common primer sequences (indicated by small black arrows) encoded within the regions bordering the UP and DN bar codes. The amplified bar codes were then hybridized to a DNA microarray to score each bar-coded deletion mutant within the population.

FIG. 2.

Most strains have no membrane stress phenotype under conditions of cell envelope stress. A representative histogram of RA values obtained from measurements of the fluorescence intensities of the DN tags in one experiment shows that the majority of strains have an RA value close to 1 (denoted by a solid black line). This indicates that they exhibit no phenotype under conditions of cell envelope stress. Sensitive strains have the lowest RA values, while resistant strains have the highest RA values.

FIG. 3.

Small-scale competition assays illustrate a range of phenotypes. Otherwise-wild-type LacZ⁻ cells (NM601) were evaluated against one of four LacZ⁺ competitor strains, Δblr (GSO280), wild-type E. coli K-12 MG1655, ΔsroH (GSO278), or ΔssrA (GSO279), as described in Materials and Methods. The total numbers of blue and white colonies varied in the mock-treated samples, but the ratio of blue to white colonies was roughly 1:1 in all instances. For wild-type cells, this ratio was unchanged in the stress-treated sample. However, blr mutants were more resistant to cell envelope stress than lacZ mutants, as evidenced by the preponderance of blue colonies in the corresponding stress-treated samples. In contrast, sroH and ssrA mutant cells were sensitive to cell envelope stress, as shown by the reduced number of blue colonies relative to white colonies. The calculated CIs for these individual experiments are provided beneath each strain name.

FIG. 4.

sRNA and small protein deletion mutants that were sensitive or resistant to cell envelope stress. (A and B) Competitor strains were grown in competition with LacZ⁻ cells (NM601) under mock treatment conditions or conditions of cell envelope stress as described in Materials and Methods. A CI was calculated for each experiment; the CI values reported for all strains are the means of three trials, except for MG1655 (n = 4 trials). The error bars represent 1 standard deviation from the mean. Wild-type MG1655 cells did not exhibit a cell envelope stress phenotype and were employed as controls in both panels. (A) Cells mutant for ssrA (GSO279), ybaM (GSO283), micA (GSO271), ryeF (GSO277), or yqcG (GSO288), were sensitive to cell envelope stress. Cells mutant for sroH (GSO278), ybhT (GSO284), yobF (GSO287) or glmY (GSO269) exhibited more modest sensitivity to cell envelope stress, while yqgB (GSO289) deletion mutants were effectively wild type. (B) Cells mutant for blr (GSO280) or rybB (GSO276) were resistant to cell envelope stress. (C) Complemented LacZ⁻ deletion mutants of ssrA (GSO298), ybaM (GSO299), micA (GSO297), and ybhT (GSO300) and uncomplemented LacZ⁻ deletion mutants of ssrA (GSO294), ybaM (GSO295), micA (GSO293), and ybhT (GSO296) were evaluated against LacZ⁺ wild-type MG1655 cells as described in Materials and Methods.

FIG. 5.

Strains deficient in SsrA-mediated proteolysis are only moderately sensitive to cell envelope stress. Uncomplemented LacZ⁻ deletion mutants of ssrA (GSO294) (n = 4) as well as deletion mutants of ssrA-deficient mutants complemented with either a wild-type allele of ssrA [ssrA⁺ (GSO301)] (n = 6) or an ochre codon mutant [ssrA^O (GSO302)] (n = 5) were evaluated against LacZ⁺ wild-type MG1655 cells. A deletion mutant of clpP (GSO303) was also evaluated against LacZ⁻ (NM601) cells (n = 3). A CI was calculated for each experiment. The error bars represent 1 standard deviation from the mean.

FIG. 6.

Deletion mutants of most OMP-regulating sRNAs exhibit wild-type cell envelope stress phenotypes. Otherwise wild-type LacZ⁻ mutant cells (NM601) were evaluated against one of nine LacZ⁺ competitor strains, ΔmicA (GSO271), ΔmicA ΔrybB (GSO290), ΔomrAB (GSO274), ΔmicC (GSO272), ΔmicF (GSO273), ΔrseX (GSO275), ΔipeX (GSO270), ΔcyaR (GSO268), and ΔrybB (GSO276), as described in Materials and Methods. Wild-type MG1655 cells did not exhibit a cell envelope stress phenotype and were employed as a control. A CI was calculated for each experiment; the CI values reported for each strain are the means of three trials, except for MG1655 (n = 4). The error bars represent 1 standard deviation from the mean.

FIG. 7.

Four small protein deletion mutants were sensitive to acid stress. Otherwise-wild-type LacZ⁻ mutant cells (NM601) were evaluated against one of seven LacZ⁺ competitor strains, ΔyqgB (GSO289), ΔmgrB (GSO282), ΔyobF (GSO287), ΔyceO (GSO285), ΔylcG (GSO286), ΔhokE (GSO281), and wild-type MG1655, as described in Materials and Methods. A CI was calculated for each experiment; the CI values reported for each strain are the means of three trials, except for ΔylcG (GSO286) and ΔhokE (GSO281) (n = 5 trials). The error bars represent 1 standard deviation from the mean.