Regulatory role of the small noncoding RNA GcvB in acid resistance in Escherichia coli. (A) MG1655-K12 wild type strain (wt) and various deletion mutants were grown in Luria Broth broth (pH 7.0) for 5 hr at 37°C. Cells were acid shocked at pH 2.0 for 30 min, and then neutralized by adding alkalinized LB (pH 9.3). Cells were allowed to grow for an additional 3 hrs before measurement of optical density at 600 nm (OD600). Two-tailed paired t tests were employed to compare each mutant with the wt in terms of acid resistance. (B) Because the gcvB mutant displayed a lower growth rate than the wt (data not shown), percent acid survival was determined to confirm the data of acid recovery measured as OD600. Cell viability was determined by plate counts and percent acid survival was calculated as the number of cfu remaining after the 30 min-acid treatment divided by the initial cfu at time zero (quoted as a percentage). (C) A plasmid expressing GcvB under the control of its native promoter was transformed into a gcvB mutant of E. coli MG1655. The mutant carrying an empty vector was used as a control. Cells were incubated for 5 hrs before being acid challenged for 15 min. Error bars represent standard deviation (* P-value < 0.05).

Effects of individually deleting oppA and dppA on acid resistance in Escherichia coli. The acid-stress survival abilities of two individual deletion mutant strains lacking previously identified targets of GcvB were compared with those of the MG1655 wild type (wt) strain. The abilities of strains to survive a 30 minute acid shock were quantified by two methods: (A) acid recovery measured as optical density at 600 nm (OD600). (B) Percent survival determined by colony forming units (cfu). Error bars represent standard deviation (* P-value < 0.05).

Effects of deleting rpoS on GcvB-mediated acid resistance. (A) Acid resistance in an rpoS mutant and an rpoS gcvB double mutant. (B) Effects of GcvB overproduction on acid resistance in an rpoS mutant. Cell cultures of E. coli MG1655 mutant strains were grown in LB broth (pH 7.0) at 37°C for 5 hrs and then acid challenged (pH 2.0) for 15 min. After neutralization by adding alkalinized LB (pH 9.3), cells were cultured for an additional 3 hrs before measurement of optical density at 600 nm (OD600 nm). Error bars represent standard deviation (* P-value < 0.05).

Effects of GcvB sncRNA and OppA protein on the expression of RpoS in a chromosomal rpoS-lacZ reporter strain. (A) Effects of deleting gcvB on the expression of an rpoS-lacZ reporter gene fusion in E. coli MG1655. (B) Effect of GcvB overproduction on the expression of the rpoS-lacZ fusion. GcvB production was driven by its native promoter on a pET32a plasmid construct. (C) Effect of deleting oppA on the expression of the rpoS-lacZ reporter. Beta-galactosidase activities due to translation of chromosomal lacZ fusions were normalized to those of MG1655 carrying an rpoS-lacZ fusion with or without an empty vector (controls). Error bars represent standard deviation (* P-value < 0.05).

Effects of deleting gcvB on acid resistance in mutants lacking hns, crp or gadX. Cell cultures of E. coli MG1655 mutant strains containing single deletions of the hns, crp or gadX genes were grown in LB broth (pH 7.0) at 37°C for 5 hrs and then acid challenged (pH 2.0) for 15 min (gadX mutant) or 30 min (hns and crp mutants). After neutralization by adding alkalinized LB (pH 9.3), cells were cultured for an additional 3 hrs before measurement of optical density at 600 nm (OD600 nm). Error bars represent standard deviation (* P-value < 0.05).

Effects of GcvB overexpression on acid resistance in mutants lacking hns, crp, gadX or native promoters of rpoS. The mutant lacking the native promoters of rpoS was named d_567–1342. Cell cultures of E. coli MG1655 strains containing single deletions were grown in LB broth (pH 7.0) at 37°C for 5 hrs and then acid challenged (pH 2.0) for 15 min (gadX mutant) or 30 min (hns and crp mutants and d_567–1342). After neutralization by alkalinized LB (pH 9.3), cells were cultured for an additional 3 hrs before measurement of optical density at 600 nm (OD600 nm). Error bars represent standard deviation (* P-value < 0.05).