Physiological effects of Crl in Salmonella are modulated by sigmaS level and promoter specificity

Abstract

The small regulatory protein Crl activates sigma(S) (RpoS), the stationary-phase and general stress response sigma factor. Crl has been reported to bind sigma(S) in vitro and to facilitate the formation of RNA polymerase holoenzyme. In Salmonella enterica serovar Typhimurium, Crl is required for the development of the rdar morphotype and transcription initiation of the sigma(S)-dependent genes csgD and adrA, involved in curli and cellulose production. Here, we examined the expression of other sigma(S)-dependent phenotypes and genes in a Deltacrl mutant of Salmonella. Gene fusion analyses and in vitro transcription assays indicate that the magnitude of Crl activation differs between promoters and is highly dependent on sigma(S) levels. We replaced the wild-type rpoS allele in S. enterica serovar Typhimurium strain ATCC 14028 with the rpoS(LT2) allele that shows reduced expression of sigma(S); the result was an increased Crl activation ratio and larger physiological effects of Crl on oxidative, thermal, and acid stress resistance levels during stationary phase. We also found that crl, rpoS, and crl rpoS strains grew better on succinate than did the wild type and expressed the succinate dehydrogenase sdhCDBA operon more strongly. The crl and rpoS(LT2) mutations also increased the competitive fitness of Salmonella in stationary phase. These results show that Crl contributes to negative regulation by sigma(S), a finding consistent with a role for Crl in sigma factor competition via the facilitation of sigma(S) binding to core RNA polymerase.

FIG. 1.

Impact of a crl knockout mutation on σ^S-dependent phenotypes. Resistance of ATCC 14028 and its Δcrl, ΔrpoS, and ΔrpoS Δcrl derivatives to oxidative stress (A) (15 mM H₂O₂), acid stress (B) (LB, pH 3), and thermal stress (C) (55°C) was determined in stationary-phase LB cultures (OD₆₀₀ of 4) grown at 37°C. (D) Role of Crl in oral infectivity of Salmonella in mice. BALB/c mice (five mice per group) were inoculated with Salmonella wild-type (wt) strain SL1344 and the ΔrpoS and Δcrl derivatives SL1344K and SL1344crl, respectively (8 × 10⁷ bacteria per mouse given orally), and mouse deaths were recorded. Survival was calculated as a percentage of mice remaining alive at designated times postinoculation. As expected (6), no animal died after receiving inocula of the rpoS mutant. In contrast, all mice inoculated with wild-type and Δcrl strains were dead within 11 and 14 days, respectively.

FIG. 2.

Effects of Crl on stress resistance of Salmonella wild-type and rpoS_LT2 mutant strains. (A) Expression of Crl and σ^S in strains 2922K (lane 1) and 2922KrpoS_LT2 (lane 2) grown in LB medium at 37°C. Exponential-phase cultures of Salmonella in LB medium at 37°C were diluted into LB medium prewarmed at 37°C to prolong the exponential phase. Aliquots were removed during the exponential phase (LOG) (OD₆₀₀ of 0.4) and stationary phase (STA) (OD₆₀₀ of 4) and analyzed by Western blotting with anti-Crl and anti-σ^S antibodies. Ten micrograms of total protein was loaded into each slot. (B) The Salmonella 2922K wild-type (wt) and mutant strains indicated were grown to stationary phase (OD₆₀₀ of 3.5 to 4) in LB medium at 37°C and were subjected to oxidative stress (15 mM H₂O₂), acid stress (LB, pH 3), and thermal stress (55°C). Representative experiments are shown. Similar results were obtained in repeat experiments.

FIG. 3.

Effects of a crl knockout mutation on expression of σ^S-dependent genes. (A) A collection of lacZ transcriptional fusions in σ^S-dependent genes was constructed previously in Salmonella strain C52 (15). Each fusion was then moved to Salmonella strain ATCC 14028 and its Δcrl derivative ATCCcrl by transduction. The resulting strains were grown to stationary phase in LB medium at 37°C (OD₆₀₀ of 3.5 to 4), and β-galactosidase activity was measured according to a method described previously by Miller (23). β-Galactosidase activity in the crl mutant is reported as a percentage of that in the wild-type strain. (B) Kinetics of expression of lacZ gene fusions in ydeJ, katN, poxB, katE, and spvB. Exponential-phase cultures (OD₆₀₀ of 0.4) of Salmonella wild-type strain ATCC 14028 and the Δcrl mutant carrying the fusions indicated were diluted in LB medium prewarmed to 37°C to prolong the exponential phase. Aliquots were removed at various times, and β-galactosidase activity was measured (closed symbols) according to a method described previously by Miller (23). The growth phase was determined by the measurement of culture turbidity as the OD₆₀₀ (open symbols). The measurements were repeated twice, and a representative experiment is shown. The ydeJ, katN poxB, and katE-lacZ fusions were on the chromosome, whereas the spvRAB-lacZ fusion was carried on pSTF4 (Table 1).

FIG. 4.

Crl activation of gene expression in Salmonella wild-type and rpoS_LT2 mutant strains. Expression levels of katN-lacZ and katE-lacZ fusions in LB medium at 37°C in the Salmonella strains are indicated. β-Galactosidase activity was measured in stationary-phase cultures (STA) (OD₆₀₀ of 3.5 to 4) and cultures grown overnight (ON) (OD₆₀₀ of 3.5 to 4) according to a method described previously by Miller (23).

FIG. 5.

In vitro effects of Crl on transcription initiation at the katE and katN promoters. Data for single-round runoff transcription monitored after various incubation times with Eσ^S and the promoter in the absence or presence of Crl (500 nM) are shown. (A) A typical time course experiment is shown. After preincubation of the holoenzyme Eσ^S (E = 15 nM; σ^S = 30 nM) with buffer or Crl for 20 min at 37°C, the template fragment was added at time zero. At the indicated times, an aliquot of the mixture was added to heparin and XTPs, and transcription was allowed to proceed for 10 min. The ³²P-labeled transcripts were analyzed on a 7% polyacrylamide sequencing gel, and the band intensities were quantified. (B) The graphs show the time course of transcript synthesis averaged from three independent experiments.

FIG. 6.

Effect of the Δcrl mutation on Salmonella growth on succinate. (A) Growth of the Salmonella strains indicated was evaluated on M9 minimum medium plates containing either succinate or glucose as the sole carbon source. Cultures in M9 glucose grown overnight at 37°C were resuspended in M9 medium to OD₆₀₀s of 1.0 and 0.1, and 5 μl of each dilution was spotted onto M9 plates containing either succinate or glucose (0.4%). The plates were incubated at 37°C for 24 h. The crl mutation was complemented by the crl gene on pACcrl-1 but not by either vector pACYC184 or pACcrl-2. In pACcrl-2, the crl gene is inserted in the promoterless orientation. (B) Growth of Salmonella wild type (ATCC 14028) and the Δcrl, ΔrpoS, and Δcrl ΔrpoS derivatives in liquid M9 medium containing either succinate or glucose (0.5%) as the sole carbon source.

FIG. 7.

Effect of Δcrl, ΔrpoS, and rpoS_LT2 mutations on expression of an sdhA-lacZ transcriptional fusion. (A) Kinetics of expression of an sdhA-lacZ gene fusion in Salmonella strain ATCC 14028 and the Δcrl and ΔrpoS derivatives. Exponential-phase cultures (OD₆₀₀ of 0.4) of the Salmonella strains indicated were diluted into LB medium prewarmed to 37°C to prolong the exponential phase. Aliquots were removed at various time intervals, and β-galactosidase activity was measured (plain lines) according to a method described previously by Miller (23). The growth phase was determined by measurement of culture turbidity as the OD₆₀₀ (dashed lines). The measurements were repeated twice, and a representative experiment is shown. (B) Effect of the rpoS_LT2 and Δcrl mutations on expression of an sdhA-lacZ gene fusion. The indicated strains carrying the sdhA-lacZ transcriptional fusion were grown in LB medium at 37°C, and β-galactosidase activity was measured in cultures grown overnight as described previously by Miller (23).

FIG. 8.

Role of Crl and σ^S in survival and competitive fitness during stationary phase. (A) Survival in stationary-phase cultures in LB medium at 37°C. Cells from cultures of ATCC 14028 and the indicated derivative strains grown overnight in LB medium were washed, resuspended in 0.9% NaCl to an OD₆₀₀ of 1.0, diluted into fresh LB medium, and incubated at 37°C with shaking. Aliquots of bacteria were removed at timed intervals, and numbers of viable cells on LB plates were determined. One hundred percent survival corresponds to the number of cells in cultures grown overnight (day 1). Representative experiments are shown. Similar results were obtained in repeat experiments. (B) Competition assays between wild-type strain ATCC 14028 and mutant strains ATCCrpoS (a), ATCCcrl (b), ATCCrpoS crl (c), 2922KrpoS (d), 2922KrpoS_LT2 (e), and 2922K (f). Cultures in LB medium grown overnight were washed and resuspended in 0.9% NaCl to an OD₆₀₀ of 1.0. Equal numbers of cells of wild-type strain ATCC 14028 and the mutant strain were then mixed in fresh LB medium to give a total of about 3,000 cells ml⁻¹ (time zero), and the mixtures were incubated at 37°C with shaking. Aliquots of bacteria were removed at timed intervals, and numbers of viable cells of each strain were determined. Numbers of cells of each strain are reported as a percentage of the total number of viable cells in the culture.