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Journal of Bacteriology, April 2007, p. 2976-2987, Vol. 189, No. 8
0021-9193/07/$08.00+0     doi:10.1128/JB.01919-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Physiological Effects of Crl in Salmonella Are Modulated by ^S Level and Promoter Specificity

Véronique Robbe-Saule, Miguel Dias Lopes, Annie Kolb, and Françoise Norel^*

Unité des Régulations Transcriptionnelles, URA-CNRS 2172, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France

Received 20 December 2006/ Accepted 29 January 2007

Top Abstract Introduction Materials and Methods Results Discussion References

 
The small regulatory protein Crl activates ^S (RpoS), the stationary-phase and general stress response sigma factor. Crl has been reported to bind ^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 ^S-dependent genes csgD and adrA, involved in curli and cellulose production. Here, we examined the expression of other ^S-dependent phenotypes and genes in a crl 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 ^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 ^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 ^S, a finding consistent with a role for Crl in sigma factor competition via the facilitation of ^S binding to core RNA polymerase.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacteria in their natural habitats are frequently exposed to nutrient scarcity and other stressful conditions. Consequently, they often grow and divide slowly and enter a stationary phase. Stationary-phase physiology, particularly multiple stress resistance, and even cell morphology are determined by the general stress response. The molecular mechanism controlling this response involves a sigma subunit of RNA polymerase, ^S (RpoS). In enterobacteria, RNA polymerase is composed of a core enzyme, E, with the subunit structure ₂ßß'; this core enzyme associates with one of the seven different sigma subunits to form the holoenzyme (E) (30). Sigma factors compete for association with the core enzyme, and each coordinates the transcription of a set of genes, which allows the fine control of adaptation to different physiological conditions (20). The RNA polymerase holoenzyme containing the ⁷⁰ subunit is responsible for the transcription of most genes during exponential growth. When entering a stationary phase or when under particular stress conditions during the exponential growth phase (high osmolarity, low pH, or high or low temperatures), ^S, which is encoded by the rpoS gene, accumulates in the cell; it associates with the core enzyme and directs the transcription of genes contributing to the general stress response and to stationary-phase survival (14, 16). rpoS is also involved in the virulence of the enteric pathogen Salmonella enterica serotype Typhimurium (6, 9, 18). There are numerous regulators of ^S, and they act both during transcription and posttranscriptionally (14).

Recent studies have shown the crl gene product to be a regulator of ^S activity in Escherichia coli (2, 28) and Salmonella (32). In Salmonella, Crl is required for development of the rdar morphotype, a colony morphology characterized by the production of curli and cellulose and associated with biofilm formation (32). In vitro transcription experiments with the purified Salmonella Crl protein showed that Crl stimulates ^S-dependent transcription at promoters of the adrA and csgD genes; these genes are involved in the biosynthesis of curli and cellulose (32). Crl interacts directly with the ^S protein in vitro (2), and a recent study suggested that Crl facilitates the formation of RNA polymerase holoenzyme (12). Unexpectedly, this study reports that Crl stimulates the transcriptional activity of not only ^S but also ⁷⁰ and ³² in vitro (12). It is not known, however, whether these observations are relevant in vivo.

We looked for phenotypes other than the development of the rdar morphotype that could be associated with the crl knockout mutant of Salmonella. We show that the physiological effects of Crl depend on the amount of ^S in the cell. The magnitude of the Crl-mediated activation of ^S-dependent genes and the physiological impact of Crl increased as ^S levels decreased. In addition, gene fusion analysis and in vitro transcription experiments demonstrated that the extent of Crl activation differs between promoters. Crl also contributes to negative regulation by ^S, and the crl mutant has a competitive advantage over the wild type during stationary phase. These finding are consistent with Crl in vivo being involved in sigma factor competition through the facilitation of E^S formation.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, phages, and growth conditions. The E. coli and S. enterica serovar Typhimurium strains used in this study are listed in Table 1. Transposon Tn5B21, a tetracycline-resistant derivative of Tn5, was constructed to facilitate lacZ gene fusions (37). S. enterica serovar Typhimurium C52 derivatives carrying Tn5B21 insertions in ^S-dependent genes have been described previously (15). These Tn5B21 insertions were subsequently transferred to derivatives of S. enterica serovar Typhimurium ATCC 14028 by transduction. Bacteriophage P22HT105/1int was used to transfer mutations between Salmonella strains by transduction (35). Green plates, for screening for P22-infected cells or lysogens, were prepared as described previously (38). Strains were routinely cultured at 37°C in Luria-Bertani (LB) medium (34). We used M9 minimal medium (34) containing glucose or succinate to test for the growth of Salmonella at the expense of succinate. Antibiotics were used at the following concentrations: carbenicillin (Cb), 100 µg ml^–1; chloramphenicol (Cm), 15 µg ml^–1 for the chromosomal resistance gene and 30 µg ml^–1 for the plasmid resistance gene; kanamycin (Km), 50 µg ml^–1; and tetracycline (Tet), 20 µg ml^–1.

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TABLE 1. Bacterial strains and plasmids used in this study

Stress resistance assays. For stress resistance assays, cells were grown to stationary phase (optical density at 600 nm [OD₆₀₀] of 4) in LB medium at 37°C. For the oxidative shock survival assay, cells were washed and resuspended in 0.9% NaCl to an OD₆₀₀ of 1.0, and H₂O₂ was added to a final concentration of 15 mM. For the heat shock survival assay, cells were washed and diluted in 0.9% NaCl to about 3,000 cells ml^–1, and aliquots were placed into glass tubes that had been prewarmed at 55°C. For the acid shock survival assay, cells were diluted 1:1,000 in LB medium (pH 3). In all experiments, aliquots of bacteria were removed at timed intervals, and numbers of viable cells on LB plates were determined.

Survival and competition in stationary phase. For survival assays, cultures grown overnight in LB medium were washed, resuspended in 0.9% NaCl to an OD₆₀₀ of 1.0, diluted in fresh LB medium to about 3,000 cells ml^–1, 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. For competition assays, cultures grown overnight in LB medium 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, and the mixture was incubated at 37°C with shaking. Aliquots of bacteria were removed at timed intervals, and numbers of viable cells of each strain were determined on LB plates containing the appropriate antibiotics.

DNA manipulations. We used standard molecular biology techniques for DNA manipulations (33, 34). DNA was sequenced by Genomexpress (Paris, France). Oligonucleotides were obtained from Distribio (France). Strains were constructed either by generalized transduction using the phage P22HT105/ 1 int (35) or by linear DNA transformation as specified below.

Construction of plasmids and DNA templates for in vitro transcription. The plasmids used in this study are listed in Table 1. pUCK3 contains a 3-kb ScaI-HindIII fragment carrying rpoS and the downstream open reading frames (ORFs) (STM2923 and STM2922) from S. enterica serovar Typhimurium (18). pUCK3 DNA was digested with SmaI and HpaI and religated, yielding pUCK3Sma-Hpa, from which the 5' end of rpoS is deleted. This plasmid carries a single BamHI restriction site within STM2922. A 1.3-kb BamHI fragment carrying the Km resistance cartridge from pUC4K was then ligated into this BamHI restriction site in pUCK3Sma-Hpa, resulting in pUCC52-2922K, which thus contains an STM2922::Km mutation. The DNA templates for in vitro transcription were prepared as follows. First, katE and katN promoter fragments were amplified by PCR from strain ATCC 14028 using primers 5'-CCGGAATTCGCCCCCGACGTCCTG-3' and 5'-GGCGGATCCATCACTGAAATGGGCGTGG-3' for katE and primers 5'-GGCGAATTCAGGGCCGCAGATAGTG-3' and 5'-CCGGGATCCTGTCTCGTTGCTTGCTG-3' for katN. The katN promoter is upstream from the yciGEF katN operon (31) such that the katN primers hybridize to the sequence of the yciG gene. The fragments were digested with BamHI and EcoRI (underlined) and inserted into pJCD01, which was also digested with BamHI and EcoRI (21). The resulting plasmids were named pJCDkatE and pJCDkatN, and the inserts were verified by sequencing. Second, transcription templates including the rrnBT1 terminator were generated by PCR using primers 5'-CTGGCAGATGCGTCTTCCG-3' and 5'-GGATTTGTCCTACTCAGGAG-3' for katE and primers 5'-CCCGAATTCGGTAAATCACAACTATTTCCG-3' and 5'-GGATTTGTCCTACTCAGGAG-3' for katN. The katE and katN promoter fragments were 296 and 256 bp long, respectively.

Construction of 2922KrpoS_LT2. We determined the nucleotide sequence of a PCR-amplified rpoS gene from S. enterica serovar Typhimurium ATCC 14028; it is identical to that in Salmonella strain LT2 (rpoS_LT2) except for the start codon, which is a rare UUG codon in rpoS_LT2 (22). According to the genomic map of S. enterica serovar Typhimurium LT2 (22), rpoS is followed by two ORFs, STM2923 and STM2922. STM2922 encodes a putative decarboxylase and is not regulated by rpoS (1). pUCC52-2922K contains the 3' end of rpoS, STM2923, and STM2922 into which a Km cartridge has been inserted. pUCC52-2922K was introduced into S. enterica serovar Typhimurium ATCC 14028 by electroporation and appeared to be unstable. Recombination of the Km cartridge into the host genome with the simultaneous loss of pUCC52-2922K resulted in the isolation of recombinants that were resistant to Km and sensitive to Cb. A Km^r Cb^s recombinant was selected, checked by PCR for the presence of the STM2922::Km mutation, and designated 2922K. The STM2922::Km mutation was then moved by transduction into S. enterica serovar Typhimurium strain LT2 (leading to strain LT2-2922K) and subsequently transduced from LT2-2922K to strain ATCCrpoS that contains the rpoS::Cm mutation. Transductants that were Km^r but Cm^s were selected, and transduction of the STM2922::Km mutation and simultaneous replacement of the rpoS::Cm mutation by the rpoS_LT2 allele were confirmed by PCR. One Km^r Cm^s strain, designated 2922KrpoS_LT2, was also checked by DNA sequencing for the presence of the rpoS_LT2 allele.

One-step inactivation of chromosomal genes and construction of chromosomal lac fusions. We created chromosomal mutations in the sdhA gene and replaced the Km^r cartridge of the sdhA-lacZY-Km fusion with a Cm^r cartridge using PCR-generated linear DNA fragments and the Red recombination method as described previously by Datsenko and Wanner (7). We used the primer pairs SdhA-P1 and SdhA-P2 for the disruption of the sdhA gene and Km-P1 and Km-P2 for the Km^r cartridge (Table 2). We characterized the mutant strains by PCR using both locus-specific primers and common test primers (7). Finally, isogenic strains were constructed by P22 HT int-mediated transduction of the mutations into the appropriate strains. When required, the Cm^r cassette was eliminated using a temperature-sensitive helper plasmid, pCP20, which encodes the FLP recombinase (7). We constructed single-copy sdhA-lacZY-Km transcriptional gene fusions from mutant ATCCsdhA::Cm using conditional plasmids containing promoterless lacZY genes and the FLP recognition target site as described previously (8). Integration of the plasmids in the correct location and the presence of multiple plasmid integrants were tested by PCR (using common test primers, such as those described in reference 8). We also used flanking locus-specific primers to amplify junction fragments that were then analyzed by DNA sequencing. Isogenic strains were constructed by the P22 HT int-mediated transduction of the mutations into the appropriate strains.

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TABLE 2. Primers used for gene mutagenesis

Electrophoresis and immunoblot analysis of proteins. Whole-cell extract preparations and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were carried out as described previously by Silhavy et al. (36). We determined the amount of protein in whole-cell lysates using the DC protein assay kit (Bio-Rad). Equal amounts of protein were loaded into each slot. The molecular weights of the proteins were estimated using molecular weight standards (Euromedex). Denaturing SDS-polyacrylamide gels were stained with Bio-Safe Coomassie (Bio-Rad). Antibodies against the Crl protein of Salmonella were described previously by Robbe-Saule et al. (32). Rabbit antibodies against the ^S protein of S. enterica serovar Typhimurium were obtained from Coynault et al. (6). Proteins were transferred onto Hybond P membranes (Amersham Life Sciences) and incubated with the polyclonal rabbit antibody serum as previously described (6). Bound antibodies were detected using a secondary anti-rabbit antibody linked to peroxidase and the ECL Plus Western blotting detection system kit (Amersham Life Sciences).

Enzymatic assays. ß-Galactosidase activity was measured as described previously by Miller (23) and is expressed in Miller units.

Mouse infection. Female BALB/c mice, which are innately susceptible to S. enterica serovar Typhimurium, were obtained from the Centre d'Elevage IFFA CREDO (Domaine des Oncins, L'Arbresle, France) and were used when approximately 7 to 8 weeks old. For inoculation of mice, bacteria were freshly streaked onto LB agar plates, and the antigenic formulae of S. enterica serovar Typhimurium strains were confirmed by slide agglutination using rabbit antisera specific for O- and H-antigen factors (Bio-Rad). Single colonies were used to inoculate LB broth, and the cultures were incubated overnight at 37°C with gentle shaking. The cultures were then diluted into fresh medium, incubated at 37°C until an OD₆₀₀ of approximately 0.5 was reached, and centrifuged. The cells were resuspended in phosphate-buffered saline (pH 7.2), and dilutions of this suspension in phosphate-buffered saline were used to inoculate mice. The numbers of CFU per ml in suitable dilutions were determined by plate countings. For oral inoculation, 0.2-ml aliquots were administered to mice, lightly anesthetized with ether, with 1-ml disposable syringes to which polyethylene catheters (Biotrol) were attached. Animal care and handling were in accordance with institutional guidelines.

Runoff transcription assays. Reconstituted RNA polymerase (final concentration, 15 nM; sigma factor/core ratio, 2:1) was incubated in buffer A (40 mM HEPES [pH 8.0], 10 mM MgCl₂, 100 mM K-glutamate, 2 mM dithiothreitol) containing 500 µg ml^–1 bovine serum albumin with or without Crl (0.5 µM) for 20 min at 37°C. Open complex formation was started by the addition of the 296-bp katE or the 256-bp katN fragment including the rrnBT1 terminator (10 nM). After various times, a 4-µl aliquot was withdrawn and added to a 4-µl mixture containing 0.4 mM ATP, 0.4 mM GTP, 0.4 mM CTP, 40 µM UTP, 0.2 µCi [-³²P]UTP, and 240 µg ml^–1 heparin. After 10 min, the reactions were stopped by adding formamide containing 10 mM EDTA, xylene cyanol, bromophenol blue, and 1% SDS to the mixture. The samples were heated to 90°C and subjected to electrophoresis on a 7.5% polyacrylamide sequencing gel. The bands corresponding to transcripts were quantified using a PhosphorImager (Molecular Dynamics). The transcript sizes were around 151 nucleotides for katN (31) and 120 nucleotides for katE, assuming that the transcription start sites are identical in Salmonella and E. coli (39).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of a crl knockout mutation on virulence and general stress resistance of Salmonella strain ATCC 14028. Crl is involved in the development of the rdar morphotype of Salmonella, a multicellular behavior controlled by ^S (32). To investigate the role of Crl further, we examined the effect of a crl knockout mutation on the expression of other phenotypes regulated by ^S. S. enterica serovar Typhimurium infection in mice results in a systemic illness, similar to human enteric (typhoid) fever; rpoS mutants are, however, highly attenuated (6, 9, 18). Therefore, we determined the virulence of the crl strain in mice. Mice were inoculated orally with a wild-type strain of Salmonella and the crl and rpoS mutants, and mouse mortality was recorded (Fig. 1D). As expected (6), no animal died after receiving inocula of the rpoS mutant. In contrast, all mice inoculated with wild-type and crl strains died within 11 and 14 days, respectively. We also performed mixed oral inoculation experiments in which mice were inoculated with equal numbers of wild-type and crl strains simultaneously; 6 days later, the mice were killed, and the numbers of wild-type and crl bacteria in the spleens were determined. Results between individual mice differed, but there was no evidence that the crl strain was significantly more attenuated than the wild type (data not shown). In contrast, when the rpoS mutant was mixed with the wild-type strain for inoculation, no rpoS bacteria could be recovered from mice killed 6 days postinfection. Therefore, we conclude that the Crl protein does not play a major role in virulence under these conditions.

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FIG. 1. Impact of a crl knockout mutation on S-dependent phenotypes. Resistance of ATCC 14028 and its crl, rpoS, and rpoS crl 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 rpoS and crl derivatives SL1344K and SL1344crl, respectively (8 x 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 crl strains were dead within 11 and 14 days, respectively.

^S is required for bacterial resistance to various stresses during stationary phase (the so-called general stress resistance) (14, 16). Surprisingly, we found no effect of the crl mutation on the ability of Salmonella to express general stress resistance during the stationary phase (Fig. 1) and in cultures grown overnight (data not shown) in LB medium. The resistances of crl and wild-type strains to hydrogen peroxide, high temperature, and acidic pH were not significantly different, whereas rpoS and rpoS crl strains were highly susceptible to these stresses (Fig. 1A, B, and C).

^S concentration mediates Crl physiological impact. The crl mutant is unable to develop the rdar morphotype, but this phenotype is restored when ^S levels in the cell are increased (32). This suggests that the effects of Crl in vivo depend on ^S levels. To test this, we determined the effect of the crl knockout mutation on the general stress resistance level of isogenic strains containing either the natural rpoS allele of ATCC 14028 or the rpoS allele of Salmonella strain LT2 (strains 2922K and 2922KrpoS_LT2, respectively). The rpoS_LT2 allele is identical to the rpoS allele of ATCC 14028 except for the start codon, which is a rare UUG codon in rpoS_LT2 resulting in a low level of ^S production in strain LT2 (19, 22) (Fig. 2A). Expression of ^S was two- to threefold lower in 2922KrpoS_LT2 than in 2922K. Expression of Crl was similar in the two strains during both exponential and stationary growth phases (Fig. 2A).

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FIG. 2. Effects of Crl on stress resistance of Salmonella wild-type and rpoSLT2 mutant strains. (A) Expression of Crl and S in strains 2922K (lane 1) and 2922KrpoSLT2 (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-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.

The crl mutation decreased the resistance of strain 2922KrpoS_LT2 to oxidative, acid, and thermal stresses during stationary phase, whereas it had no effect on the resistance of 2922K (Fig. 2B). This indicates that the physiological effects of Crl can be affected by the abundance of ^S. The rpoS_LT2 mutation decreased the resistance of 2922K to acid and thermal stresses, whereas it had no significant effect on its resistance to oxidative stress (Fig. 2B). This suggests that under these conditions, genes involved in resistance to acid and thermal stresses are more sensitive to ^S levels than are the genes responsible for oxidative stress resistance.

The extent of Crl-mediated activation differs between genes. To determine the effects of Crl on gene expression, we monitored the expression of 30 ^S-dependent transcriptional fusions in crl and wild-type strains grown to stationary phase in LB medium at 37°C (Fig. 3A). The Crl induction ratio during stationary phase was between 1.2 and 2.4 for all except two fusions, H30 in ydeJ and G57 in katN, with induction ratios of 4. These two fusions were thus more sensitive than the other fusions to Crl. We tested the expression of the fusions in cultures grown overnight. The Crl induction ratio was lower than that during stationary phase for all fusions, but ydeJ and katN were still the most responsive (induction ratio of >2) (data not shown). The kinetics of expression were assessed for genes that display high levels of sensitivity (ydeJ and katN), low levels of sensitivity (poxB), and almost no sensitivity (katE) to Crl; the high sensitivity of ydeJ and katN to Crl was confirmed (Fig. 3B). The Crl effect was greatest upon entry into stationary phase (Fig. 3B), when the Crl concentration is maximal in the cell and ^S expression is induced (32) (Fig. 2A and data not shown). During early stationary phase, the absence of activation by Crl resulted in a slightly delayed expression of genes in the crl mutant (Fig. 3B).

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FIG. 3. Effects of a crl knockout mutation on expression of S-dependent genes. (A) A collection of lacZ transcriptional fusions in S-dependent genes was constructed previously in Salmonella strain C52 (15). Each fusion was then moved to Salmonella strain ATCC 14028 and its crl 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 ß-galactosidase activity was measured according to a method described previously by Miller (23). ß-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 crl 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 ß-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).

The function of ydeJ is unknown, and the physiological role of katN that encodes an Mn-catalase is not known (31). katE is the major catalase of Salmonella responsible for resistance of the bacteria to hydrogen peroxide in stationary-phase LB cultures (3, 31). Therefore, the low responsiveness of the katE fusion to Crl activation is in agreement with the high level of resistance to H₂O₂ displayed by the crl strain (Fig. 1). Attenuation of rpoS mutants in mice is largely a consequence of the decreased expression of the virulence plasmid gene spv due to the absence of ^S (9, 18, 24). Consistent with the high virulence of the crl strain, expression of an spvRAB-lacZ fusion was only moderately lower in the crl strain than in the wild type (i.e., the crl mutation had only a small effect on entry into stationary phase) (Fig. 3B).

The ^S level does not affect the hierarchy of promoter upregulation by Crl. Interestingly, the rpoS_LT2 allele rendered the katE fusion sensitive to Crl. Expression of katE was reduced 2.5-fold and 1.7-fold by the crl mutation in cultures in stationary phase and cultures grown overnight, respectively (Fig. 4). Thus, Crl is required for katE expression in 2922KrpoS_LT2 to compensate for the reduction in ^S levels resulting from the rpoS_LT2 mutation. This is consistent with the finding described above, that Crl is required for H₂O₂ resistance of 2922KrpoS_LT2 but not for that of 2922K during stationary phase (Fig. 2B). As for katE, expression of katN was more dependent on Crl in 2922KrpoS_LT2 than in 2922K (Fig. 4). Therefore, even though decreased ^S abundance increased the magnitude of Crl activation of gene expression, it did not affect the differential responsiveness of the katE and katN genes to Crl activation.

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FIG. 4. Crl activation of gene expression in Salmonella wild-type and rpoSLT2 mutant strains. Expression levels of katN-lacZ and katE-lacZ fusions in LB medium at 37°C in the Salmonella strains are indicated. ß-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).

Expression of the katN-lacZ fusion was greatly affected by the rpoS_LT2 mutation; expression was reduced 35-fold during stationary phase and 3-fold in cultures grown overnight (Fig. 4). In contrast, the expression of the katE-lacZ fusion was only moderately affected by the rpoS_LT2 mutation: a reduction of 1.8-fold during stationary phase (Fig. 4). Consistent with this result, the rpoS_LT2 mutation did not modify the resistance of Salmonella to oxidative stress during stationary phase (Fig. 2B). Thus, katN expression appeared to be more sensitive than katE expression to the amount of ^S in the cell.

In vitro target specificity for Crl-mediated activation. Our findings indicate that the magnitude of the Crl activation differs from one gene to another. To determine whether this results from intrinsic properties of the promoters, transcription levels from katN and katE promoters were compared in a transcription system with purified components and E^S RNA polymerase (Fig. 5). The promoters directing katE and katN transcription were introduced upstream from the rrnBT1 terminator, and the resulting PCR fragments were assayed in single-round runoff experiments after various incubation times with E^S. This method reveals transcript formation as a function of time and reflects the formation of open complexes. At low concentrations of reconstituted E^S (15 nM core enzyme and 30 nM ^S), Crl affected both the amounts of transcripts formed and the kinetics of transcription from katN, whereas only minor effects could be detected at early incubation times for katE (Fig. 5). Increasing the amounts of the E^S holoenzyme (60 nM core and 120 nM ^S) resulted in katE becoming totally unresponsive to Crl, whereas for katN, a small activation effect persisted at early incubation times (data not shown). Decreasing the concentration of E^S (4 nM core and 8 nM ^S) led to higher levels of katE activation by Crl, which were observed even after 40 min of incubation (data not shown). These results indicate that Crl activation is dependent on the concentration of the E^S holoenzyme at both katE and katN promoters and that the katN promoter is more responsive than katE to Crl. They also suggest that the magnitude of Crl activation is the consequence of intrinsic properties of the promoter, probably the initial binding constant of RNA polymerase (K_B); promoters with lower K_B values may thus be more susceptible than others to Crl activation.

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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 ES 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 ES (E = 15 nM; 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.

Crl contributes to negative regulation by ^S. Negative regulation of ⁷⁰-dependent gene expression by ^S has been reported, and this probably results from the competition between ^S and ⁷⁰ for binding to a limiting amount of core polymerase (10, 13, 26). Consistent with this model of cellular control through sigma factor competition, RpoS protein levels influence phenotypic and nutritional properties of E. coli (5, 17, 29). For instance, rpoS mutants grow better on succinate due to an increased metabolism of tricarboxylic acid cycle intermediates (5, 29). Furthermore, the expression of the succinate dehydrogenase sdh operon, and that of many genes encoding enzymes of the tricarboxylic acid cycle, is higher in an rpoS mutant than in the wild-type strain (27, 29, 40).

To determine whether Crl contributes to negative regulation by ^S, growth of wild-type strains and that of mutant strains were compared in minimal medium with succinate as a sole carbon source (Fig. 6). The rpoS, rpoS_LT2, and crl mutants of Salmonella grew better on succinate than the wild-type strain (Fig. 6). In contrast, crl, rpoS, and wild-type strains displayed similar growth levels on glucose, which was used as the control (Fig. 6). Growth of the crl strain on M9 succinate was prevented by pACcrl-1, which expresses Crl, but not by the empty vector pACYC184 or by pACcrl-2, in which the crl gene is inserted in the promoterless orientation (Fig. 6A). This indicates that Crl negatively controls the growth of Salmonella on succinate. A similar role for Crl was observed for growth on malate and fumarate (data not shown). Growth of rpoS strains and that of rpoS crl strains on succinate and glucose were similar (Fig. 6B), indicating that the negative effect of Crl was abolished in the absence of ^S.

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FIG. 6. Effect of the crl 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 crl, rpoS, and crl rpoS derivatives in liquid M9 medium containing either succinate or glucose (0.5%) as the sole carbon source.

Consistent with these results, the expression of a lacZ gene fusion to sdhA, encoding the flavoprotein subunit of the succinate dehydrogenase, during stationary phase was 6- and 2.5-fold higher in the rpoS and rpoS_LT2 mutants, respectively, than in the wild-type strain (Fig. 7). The positive effect of the crl mutation on sdhA expression was small in strains ATCC 14028 and 2922K and slightly higher in 2922KrpoS_LT2 (Fig. 7). This suggests that the magnitude of Crl repression, like that of Crl activation, is dependent on the ^S content in the cell.

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FIG. 7. Effect of crl, rpoS, and rpoSLT2 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 crl and rpoS 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 ß-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 rpoSLT2 and crl 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 ß-galactosidase activity was measured in cultures grown overnight as described previously by Miller (23).

The crl and rpoS_LT2 mutants show increased competitive fitness in stationary phase. One major consequence of negative regulation by ^S is the selection of rpoS mutants with a growth advantage in bacterial populations (25, 41). To assess the competitive fitness of mutant strains, we performed competition experiments in which wild-type and mutant strains of Salmonella were mixed in equal cell numbers in LB liquid medium, and the numbers of each were monitored for several days (Fig. 8B). The rpoS_LT2 mutant showed a competitive advantage during stationary phase over wild-type strain ATCC 14028 (Fig. 8B, panel e). Three days after inoculation of the medium, more than 80% of the cell population was the mutant. In similar control experiments, wild-type strain ATCC 14028 showed similar fitness as wild-type strain 2922K (Fig. 8B, panel f).

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FIG. 8. Role of Crl and 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), 2922KrpoSLT2 (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.

We reasoned that because Crl participates in the negative regulation by ^S, a crl mutant should display a competitive advantage over the wild-type strain. Alternatively, since Gaal et al. (12) recently reported that Crl increases the transcriptional activity of E⁷⁰ in vitro besides that of E^S, the absence of Crl might be disadvantageous for bacterial fitness during stationary phase. The crl mutant behaved like the rpoS_LT2 mutant, showing a competitive advantage over the wild-type strain during stationary phase (Fig. 8B, panel b). The gain of fitness afforded by the crl mutation was lost in the absence of ^S. Indeed, the rpoS crl strain was outcompeted by the wild-type strain, as were the rpoS strains (Fig. 8B, panels a, c, and d). Competition experiments were performed during exponential growth in LB medium, and no competitive advantage of the crl strain was detected (data not shown). We also compared the ability of wild-type and mutant strains to survive during stationary phase when cultivated alone (Fig. 8A). The survival of wild-type strains and that of crl and rpoS_LT2 mutant strains were similar, whereas the viability of the rpoS and rpoS crl mutants was lower (Fig. 8A). Thus, ^S is needed for survival and fitness during stationary phase, but reduced function of ^S in crl and rpoS_LT2 mutants results in a gain of fitness relative to that of wild-type strains.

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We previously reported that the Crl protein is required for the development of the rdar morphotype of Salmonella, a multicellular behavior that requires ^S activity (32). This was the only phenotype previously known to be associated with a crl mutant. Here, we further investigated the physiological effects of Crl. We show that the physiological impact of Crl is modulated by at least two factors: (i) the levels of ^S in the cell and (ii) the sensitivity of genes/promoters to Crl activation.

The first of these two factors is responsible for the differences in the physiological effects of Crl from one strain to another (Fig. 1 to 4). Clearly, the physiological effects of Crl and the magnitude of Crl activation of gene expression were greater at low levels of ^S. In vitro, Crl binds ^S (2) and enhances ^S transcriptional activity (12, 32), most likely by facilitating E^S holoenzyme formation (12). This mechanism of action of Crl is consistent with our finding that the effect of Crl in vivo is maximal at low concentrations of ^S. The affinity of ^S for the core is weaker than that of other sigma factors (20). Consequently, when ^S levels are low, as, for instance, at the end of the exponential phase, only a minute fraction of the rare ^S molecules would be able to bind the core in the absence of Crl. Hence, the role of Crl in assisting ^S binding to the core appeared to be particularly significant at the lowest concentrations of ^S. In in vitro transcription experiments also, the magnitude of the Crl effect decreased substantially as the ^S concentration increased (12).

The second factor determining the physiological impact of Crl in the cell is the different responsivenesses of different ^S-controlled genes to Crl (Fig. 3). Different responsivenesses of genes to Crl activation were also observed in a transcription system with purified components, thereby excluding the possibility that additional regulators were responsible for the difference (Fig. 5). Our finding that Crl affects E^S-dependent promoters to different extents is consistent with the previous finding that in in vitro transcription experiments, Crl has double the effect on the cc-35 con promoter compared to that on the RNA-I promoter (12). This led to the suggestion that differences in the levels of responsiveness of different promoters to Crl activation may reflect differences in the intrinsic binding constants of these promoters for RNA polymerase. Promoters that recruit RNA polymerase inefficiently would be affected more by an increase in the RNA polymerase holoenzyme concentration caused by Crl (12). The kinetic constants of the katE and katN promoters are not known, but katN expression in vivo is more sensitive to variation in ^S levels than that of katE. When ^S levels were decreased by two- to threefold, in the rpoS_LT2 mutant, katN expression was considerably reduced, whereas katE expression was only slightly affected (Fig. 4). In addition, katE is not resistant to Crl activation per se because it responded to Crl activation when ^S levels were decreased by the rpoS_LT2 mutation (Fig. 4). The ability to recruit holoenzyme E^S is thus likely to be an important determinant of promoter responsiveness to Crl. However, even though Crl affects holoenzyme formation (12), additional, subsequent steps in the transcription initiation pathway may also be affected by Crl. It is not known whether Crl is linked to E^S and E^S-promoter complexes. The presence of Crl resulted in minor modifications of the footprints at some open complexes (2, 32), suggesting that, at least in these cases, the architecture of the complex was altered. Thus, in addition to the ability to recruit E^S, other features of a promoter may be involved in Crl responsiveness: they may include DNA flexibility or size of the spacer between the –10 and –35 regions. Moreover, additional regulatory circuits may modulate the responsiveness of a promoter to Crl in vivo.

Unexpectedly, Gaal et al. (12) recently showed that in a transcription system with purified components, Crl affects transcription not only by E^S but also by E⁷⁰ and E³². This raises an important question. If Crl activates the major sigma factor ⁷⁰ in vivo, in addition to alternative sigma factors, what is its major physiological function? One way to answer this question is to determine the role (if any) of Crl in the expression of ⁷⁰-dependent genes and phenotypes negatively regulated by RpoS. Negative control by RpoS appears to be as significant as the positive control (17, 27) and appears to result in a growth advantage of rpoS mutants and thus the selection of rpoS mutants in bacterial populations (25). As it is a sigma factor, RpoS probably does not repress genes directly. Indirect mechanisms, for example, activation of a repressor or, more likely, sigma factor competition for core binding, are presumably involved (10, 20, 25). The competition model predicts that sigma factors compete for binding to a limiting amount of core polymerase and that the superinduction of ⁷⁰-dependent genes in rpoS mutants results from an increased amount of E⁷⁰ complexes in the absence of competing ^S (10, 13, 20, 25). The finding that the crl mutant had an advantage over the wild-type strain for growth on succinate and fitness in stationary phase (Fig. 6 to 8) indicated that Crl contributes to negative regulation by ^S. According to the competition model, this would imply that Crl favors ^S at the expense of ⁷⁰. Thus, even though a role for Crl in ⁷⁰ activation under specific conditions or on particular promoters could not be excluded, it appears that under the conditions used in our study, the basic function of Crl in vivo involves ^S much more than ⁷⁰ activity. It remains to be determined whether Crl also increases the activity of other alternative sigma factors, for example, ³², in vivo.

^S is needed for survival and competitive fitness in stationary phase, but lower-than-wild-type function of ^S in the crl mutant does not affect survival (Fig. 8A) or stress resistance in stationary phase (Fig. 1 and 2). This may be because the ^S-dependent genes required for survival and stress resistance under these conditions are not highly sensitive to Crl activation, as is the case for the katE gene. Moreover, reduced ^S activity in the crl and rpoS_LT2 mutants resulted in a gain of competitive fitness (Fig. 8B). This is probably because there is a trade-off in these strains, between the reduction of expression of ^S-dependent genes required for survival and stress resistance and increased expression of ⁷⁰-dependent genes required for persistence and/or growth. Our finding that a crl mutation confers a selective advantage on Salmonella during starvation supports the speculation that the selective advantage conferred by some large-scale deletions that accumulate in long-term stab cultures of E. coli K-12 is a consequence of the loss of the crl gene (11).

 
This work was supported by research funds from the Pasteur Institut and CNRS.

 
* Corresponding author. Mailing address: Institut Pasteur, Unité des Régulations Transcriptionnelles, URA-CNRS 2172, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France. Phone: 33 140613122. Fax: 33 145688960. E-mail: francoise.norel{at}pasteur.fr

Published ahead of print on 9 February 2007.

Present address: Protein Crystallography, Instituto de Tecnologia Química e Biológica, Av. da Republica, EAN, 2781-901 Oeiras, Portugal.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, April 2007, p. 2976-2987, Vol. 189, No. 8
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