Characterization of the RpoS status of clinical isolates of Salmonella enterica

The stationary-phase-inducible sigma factor, sigma(S) (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella: We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the sigma(S) protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of sigma(S), showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease.     

Growth dynamics of Salmonella enterica strains on alfalfa sprouts and in waste seed irrigation water

Alfalfa sprouts and other seed sprouts have been implicated in numerous outbreaks of salmonellosis. The source of these epidemics appears to have been low-level contamination of seeds by Salmonella bacteria that developed into clinically significant populations during the seed germination process. To test the possibility that Salmonella enterica strains carry host range determinants that allow them to grow on alfalfa, strains isolated from alfalfa or other sources were surveyed for their ability to grow on germinating alfalfa seeds. An S. enterica serovar Cubana strain originally isolated from contaminated alfalfa sprouts multiplied most rapidly during the initial 24 h of the seed germination process. Germinating alfalfa seeds supported the multiplication of S. enterica cells prior to the emergence of the root radicle at 72 h. Thereafter, much lower rates of multiplication were apparent. The ability of S. enterica to grow on germinating alfalfa seeds was independent of the serovar, isolation source, or virulence of the strain. Isolates obtained from alfalfa attained population levels similar to those observed for strains isolated from contaminated meat products or stools. Each of the strains could be detected in the waste irrigation water, with populations being strongly correlated with those detected on the germinating alfalfa seeds. The S. enterica strains were capable of utilizing the waste irrigation water as a sole carbon and nitrogen source. S. enterica strains thus appear to grow saprophytically on soluble organics released from seeds during early phases of germination. The ability to detect S. enterica in the waste irrigation water early in the germination process indicates that this method may be used as a simple way to monitor the contamination of sprouts during commercial operations.     

Identification of RpoS (sigma(S))-regulated genes in Salmonella enterica serovar typhimurium

The rpoS gene encodes the alternative sigma factor sigma(S) (RpoS) and is required for survival of bacteria under starvation and stress conditions. It is also essential for Salmonella virulence in mice. Most work on the RpoS regulon has been in the closely related enterobacterial species Escherichia coli. To characterize the RpoS regulon in Salmonella, we isolated 38 unique RpoS-activated lacZ gene fusions from a bank of Salmonella enterica serovar Typhimurium mutants harboring random Tn5B21 mutations. Dependence on RpoS varied from 3-fold to over 95-fold, and all gene fusions isolated were regulated by growth phase. The identities of 21 RpoS-dependent fusions were determined by DNA sequence analysis. Seven of the fusions mapped to DNA regions in Salmonella serovar Typhimurium that do not match any known E. coli sequence, suggesting that the composition of the RpoS regulon differs markedly in the two species. The other 14 fusions mapped to 13 DNA regions very similar to E. coli sequences. None of the insertion mutations in DNA regions common to both species appeared to affect Salmonella virulence in BALB/c mice. Of these, only three (otsA, katE, and poxB) are located in known members of the RpoS regulon. Ten insertions mapped in nine open reading frames of unknown function (yciF, yehY, yhjY, yncC, yjgB, yahO, ygaU, ycgB, and yeaG) appear to be novel members of the RpoS regulon. One insertion, that in mutant C52::H87, was in the noncoding region upstream from ogt, encoding a O(6)-methylguanine DNA methyltransferase involved in repairing alkylation damage in DNA. The ogt coding sequence is very similar to the E. coli homolog, but the ogt 5' flanking regions were found to be markedly different in the two species, suggesting genetic rearrangements. Using primer extension assays, a specific ogt mRNA start site was detected in RNAs of the Salmonella serovar Typhimurium wild-type strains C52 and SL1344 but not in RNAs of the mutant strains C52K (rpoS), SL1344K (rpoS), and C52::H87. In mutant C52::H87, Tn5B21 is inserted at the ogt mRNA start site, with lacZ presumably transcribed from the identified RpoS-regulated promoter. These results indicate that ogt gene expression in Salmonella is regulated by RpoS in stationary phase of growth in rich medium, a finding that suggests a novel role for RpoS in DNA repair functions.     

Alteration of the rugose phenotype in waaG and ddhC mutants of Salmonella enterica serovar Typhimurium DT104 is associated with inverse production of curli and cellulose

The rugose (also known as wrinkled or rdar) phenotype in Salmonella enterica serovar Typhimurium DT104 Rv has been associated with cell aggregation and the ability, at low temperature under low-osmolarity conditions, to form pellicles and biofilms. Two Tn5 insertion mutations in genes that are involved in lipopolysaccharide (LPS) synthesis, ddhC (A1-8) and waaG (A1-9), of Rv resulted in diminished expression of colony rugosity. Scanning electron micrographs revealed that the ddhC mutant showed reduced amounts of extracellular matrix, while there was relatively more, profuse matrix production in the waaG mutant, compared to Rv. Both mutants appeared to produce decreased levels of curli, as judged by Western blot assays probed with anti-AgfA (curli) antibodies but, surprisingly, were observed to have increased amounts of cellulose relative to Rv. Comparison with a non-curli-producing mutant suggested that the alteration in curli production may have engendered the increased presence of cellulose. While both mutants had impaired biofilm formation when grown in rich medium with low osmolarity, they constitutively formed larger amounts of biofilms when the growth medium was supplemented with either glucose or a combination of glucose and NaCl. These observations indicated that LPS alterations may have opposing effects on biofilm formation in these mutants, depending upon either the presence or the absence of these osmolytes. The phenotypes of the waaG mutant were further confirmed in a constructed, nonpolar deletion mutant of S. enterica serovar Typhimurium LT2, where restoration to the wild-type phenotypes was accomplished by complementation. These results highlight the importance of an integral LPS, at both the O-antigen and core polysaccharide levels, in the modulation of curli protein and cellulose production, as well as in biofilm formation, thereby adding another potential component to the complex regulatory system which governs multicellular behaviors in S. enterica serovar Typhimurium.     

Differences in attachment of Salmonella enterica serovars and Escherichia coli O157:H7 to alfalfa sprouts

Numerous Salmonella enterica and Escherichia coli O157:H7 outbreaks have been associated with contaminated sprouts. We examined how S. enterica serovars, E. coli serotypes, and nonpathogenic bacteria isolated from alfalfa sprouts grow on and adhere to alfalfa sprouts. Growth on and adherence to sprouts were not significantly different among different serovars of S. enterica, but all S. enterica serovars grew on and adhered to alfalfa sprouts significantly better than E. coli O157:H7. E. coli O157:H7 was essentially rinsed from alfalfa sprouts with repeated washing steps, while 1 to 2 log CFU of S. enterica remained attached per sprout. S. enterica Newport adhered to 3-day-old sprouts as well as Pantoea agglomerans and 10-fold more than Pseudomonas putida and Rahnella aquatilis, whereas the growth rates of all four strains throughout seed sprouting were similar. S. enterica Newport and plant-associated bacteria adhered 10- to 1,000-fold more than E. coli O157:H7; however, three of four other E. coli serotypes, isolated from cabbage roots exposed to sewage water following a spill, adhered to sprouts better than E. coli O157:H7 and as well as the Pseudomonas and Rahnella strains. Therefore, attachment to alfalfa sprouts among E. coli serotypes is variable, and nonpathogenic strains of E. coli to be used as surrogates for the study of pathogenic E. coli may be difficult to identify and should be selected carefully, with knowledge of the biology being examined.     

Evaluation of phoP and rpoS mutants of Salmonella enterica serovar Typhi as attenuated typhoid vaccine candidates: virulence and protective immune responses in intranasally immunized mice

The attenuation and immunoenhancing effects of rpoS and phoP Salmonella enterica serovar strain Typhi (Salmonella typhi) mutants have not been compared. Here, three S. typhi deletion mutants (phoP, rpoS, and rpoS-phoP double mutant) are constructed and these mutants are characterized with respect to invasiveness, virulence, and protective immune response compared with wild-type Ty2. It was found that phoP and phoP-rpoS deletion mutants are less invasive to HT-29 cells than the wild-type Ty2 and the rpoS single-deleted strain. The LD(50) of immunized mice was higher for phoP than for rpoS mutants, and the highest for the phoP-rpoS double mutant. In addition, all S. typhi mutants showed an increase in the specific serum IgG levels and T-cell-mediated immunity, and showed equal protection abilities against a wild-type Ty2 challenge after two rounds of immunization in BALB/c mice. It is concluded that phoP genes appear to play a more important role than rpoS genes in both cellular invasion and virulence of S. typhi, but not in immunogenicity in mice. Furthermore, the data indicate that the phoP-rpoS double mutant may show promise as a candidate for an attenuated typhoid vaccine.     

Crl binds to domain 2 of σ(S) and confers a competitive advantage on a natural rpoS mutant of Salmonella enterica serovar Typhi

The RpoS sigma factor (σ(S)) is the master regulator of the bacterial response to a variety of stresses. Mutants in rpoS arise in bacterial populations in the absence of stress, probably as a consequence of a subtle balance between self-preservation and nutritional competence. We characterized here one natural rpoS mutant of Salmonella enterica serovar Typhi (Ty19). We show that the rpoS allele of Ty19 (rpoS(Ty19)) led to the synthesis of a σ(S)(Ty19) protein carrying a single glycine-to-valine substitution at position 282 in σ(S) domain 4, which was much more dependent than the wild-type σ(S) protein on activation by Crl, a chaperone-like protein that increases the affinity of σ(S) for the RNA polymerase core enzyme (E). We used the bacterial adenylate cyclase two-hybrid system to demonstrate that Crl bound to residues 72 to 167 of σ(S) domain 2 and that G282V substitution did not directly affect Crl binding. However, this substitution drastically reduced the ability of σ(S)(Ty19) to bind E in a surface plasmon resonance assay, a defect partially rescued by Crl. The modeled structure of the Eσ(S) holoenzyme suggested that substitution G282V could directly disrupt a favorable interaction between σ(S) and E. The rpoS(Ty19) allele conferred a competitive fitness when the bacterial population was wild type for crl but was outcompeted in Δcrl populations. Thus, these results indicate that the competitive advantage of the rpoS(Ty19) mutant is dependent on Crl and suggest that crl plays a role in the appearance of rpoS mutants in bacterial populations.     

Positive selection for loss of RpoS function in Escherichia coli

Though RpoS, an alternative sigma factor, is required for survival and adaptation of Escherichia coli under stress conditions, many strains have acquired independent mutations in the rpoS gene. The reasons for this apparent selective loss and the nature of the selective agent are not well understood. In this study, we found that some wild type strains grow poorly in succinate minimal media compared with isogenic strains carrying defined RpoS null mutations. Using an rpoS+ strain harboring an operon lacZ fusion to the highly-RpoS dependent osmY promoter as an indicator strain, we tested if this differential growth characteristic could be used to selectively isolate mutants that have lost RpoS function. All isolated (Suc+) mutants exhibited attenuated beta-galactosidase expression on indicator media suggesting a loss in either RpoS or osmY promoter function. Because all Suc+ mutants were also defective in catalase activity, an OsmY-independent, RpoS-regulated function, it was likely that RpoS activity was affected. To confirm this, we sequenced PCR-amplified products containing the rpoS gene from 20 independent mutants using chromosomal DNA as a template. Sequencing and alignment analyses confirmed that all isolated mutants possessed mutated alleles of the rpoS gene. Types of mutations detected included single or multiple base deletions, insertions, and transversions. No transition mutations were identified. All identified point mutations could, under selection for restoration of beta-galactosidase, revert to rpoS+. Revertible mutation of the rpoS gene can thus function as a genetic switch that controls expression of the regulon at the population level. These results may also help to explain why independent laboratory strains have acquired mutations in this important regulatory gene.     

Low-Shear modeled microgravity alters the Salmonella enterica serovar typhimurium stress response in an RpoS-independent manner

We have previously demonstrated that low-shear modeled microgravity (low-shear MMG) serves to enhance the virulence of a bacterial pathogen, Salmonella enterica serovar Typhimurium. The Salmonella response to low-shear MMG involves a signaling pathway that we have termed the low-shear MMG stimulon, though the identities of the low-shear MMG stimulon genes and regulatory factors are not known. RpoS is the primary sigma factor required for the expression of genes that are induced upon exposure to different environmental-stress signals and is essential for virulence in mice. Since low-shear MMG induces a Salmonella acid stress response and enhances Salmonella virulence, we reasoned that RpoS would be a likely regulator of the Salmonella low-shear MMG response. Our results demonstrate that low-shear MMG provides cross-resistance to several environmental stresses in both wild-type and isogenic rpoS mutant strains. Growth under low-shear MMG decreased the generation time of both strains in minimal medium and increased the ability of both strains to survive in J774 macrophages. Using DNA microarray analysis, we found no evidence of induction of the RpoS regulon by low-shear MMG but did find that other genes were altered in expression under these conditions in both the wild-type and rpoS mutant strains. Our results indicate that, under the conditions of these studies, RpoS is not required for transmission of the signal that induces the low-shear MMG stimulon. Moreover, our studies also indicate that low-shear MMG can be added to a short list of growth conditions that can serve to preadapt an rpoS mutant for resistance to multiple environmental stresses.