Figure 1.
Transcript levels for acid resistance genes.
Gene transcript levels as determined by qRT-PCR are plotted for genes of the GDAR system (panel A) and genes of the ADAR system (panel B). Mean transcript levels are normalized to the 16S rRNA gene rrsH. Transcript levels are plotted against WT TW14359 (filled), TW14359ΔrpoN (empty), TW14359ΔrpoN ΔrpoS (hatched), and TW14359ΔrpoS (stippled, gadX and gadE only) for panel A. Asterisks denote significant differences by Tukey’s HSD following a significant F-test (n≥3, p<0.05 [*]; p<0.01 [**]). Error bars indicate standard error of the mean.
Table 1.
Acid resistance by the GDAR and ADAR mechanisms.
Figure 2.
Transcript levels for LEE genes.
(Panel A): gene transcript levels as determined by qRT-PCR are plotted for representative LEE genes in WT TW14359 (filled) and TW14359ΔrpoN (empty). (Panel B): ler transcript levels by qRT-PCR are plotted against TW14359 and various mutant derivative strains of TW14359. Mean transcript levels are normalized to the 16S rRNA gene rrsH. For panel A, an asterisk denotes a significant difference between TW14359 and TW14359ΔrpoN for each gene by Welch’s t-test (n≥3, p<0.05). For panel B, the asterisk denotes a significant difference between TW14359ΔrpoN and the remaining strains by Tukey’s HSD following a significant F-test (n≥3, p<0.05). Error bars indicate standard error of the mean.
Figure 3.
Stability of rpoS mRNA and σS.
(Panel A): Mean rpoS transcript levels (1st ordinate) and ratio of rpoS transcript (2nd ordinate) plotted against time following addition of rifampin at t = 0 min for WT TW14359 (filled) and TW14359ΔrpoN (empty); ratio is indicated by the dotted line. Error bars denote standard error of the mean (n≥3). (Panel B): Representative western immunoblot for σS as a function to time following addition of tetracycline at t = 0 min for TW14359 (WT) and TW14359ΔrpoN (ΔrpoN); blots are in increments of 4 min. Stationary phase (Stat.) protein extracts were used as a positive control for σS, and TW14359ΔrpoS (ΔrpoS) as a negative control. Equal loading was controlled for by westerns for GroEL (top row is ΔrpoN, bottom row is WT).
Figure 4.
Effect of rpoNR456A expression in TW14359ΔrpoN on σS stability, gadE and ler transcription.
(Panel A): Representative western immunoblots for σS in TW14359 (WT), TW14359ΔrpoN complemented with rpoN+ (TW14359ΔrpoNpRAM-1), TW14359ΔrpoN (ΔrpoN), TW14359ΔrpoN complemented with rpoNR456A (TW14359ΔrpoNpRAM-2) before (t = 0 min) and 4 min after addition of tetracycline (Tet.). Stationary phase (Stat.) protein extracts were used as a positive control for σS, and TW14359ΔrpoS (ΔrpoS) as a negative control. Equal gel loading was controlled for by westerns for GroEL. (Panel B): Mean gadE and ler transcript levels by qRT-PCR are plotted against TW14359 (WT) and derivative strains from Panel A. Transcript levels are normalized to the 16S rRNA gene rrsH. Asterisks denote significant differences between WT and TW14359ΔrpoNpRAM-1 when compared to TW14359ΔrpoN and TW14359ΔrpoNpRAM-2 by Tukey’s HSD following a significant F-test (n≥3, p<0.05). Error bars indicate standard error of the mean.
Figure 5.
Effect of the serine protease inhibitor 3,4-DCI on σS stability and ler expression.
(Panel A): Representative western immunoblot for σS stability in TW14359 (WT) and TW14359ΔrpoN (ΔrpoN) during exponential phase (Expo.) 4 min after the addition of tetracycline, and with or without 3,4-DCI, as well as in WT and TW14359ΔrpoS (ΔrpoS) during stationary phase (Stat.) with 3,4-DCI. Equal gel loading was controlled for by westerns for GroEL. (Panel B): Expression from lerP430-lacZ as measured by mean percent β-galactosidase activity following addition of 3,4-DCI and relative to untreated controls during exponential growth for TW14359 (circles) and TW14359ΔrpoN (squares). Asterisks denote significant differences between TW14359 and TW14359ΔrpoN at each OD600 by Welch’s t-test (n≥3, p<0.05 [*]; p<0.01 [**]).
Figure 6.
Expression from lerP430-lacZ in σN enhancer binding protein mutants.
Mean expression from lerP430-lacZ represented as β-galactosidase activity during exponential growth for TW14359 (triangles), TW14359ΔrpoN (circles), TW14359ΔfhlA (squares), TW14359ΔntrC (diamonds) and empty vector pRS551 (hatched line). The asterisk denotes a significant difference for TW14359ΔrpoN and TW14359ΔntrC when compared to the remaining strains by Tukey’s HSD following a significant F-test (n≥3, p<0.05).
Figure 7.
Stability of σS in σN enhancer binding protein mutants.
Representative western immunoblots for σS in TW14359 (WT), TW14359ΔrpoN (ΔrpoN), TW14359ΔfhlA (ΔfhlA), and TW14359ΔntrC (ΔntrC) before (t = 0 min) and 4 min after addition of tetracycline (Tet.). Stationary phase (Stat.) protein extracts were used as a positive control for σS, and TW14359ΔrpoS (ΔrpoS) as a negative control. Equal loading was controlled for by westerns for GroEL.
Table 2.
Strains and plasmids used in this study.