BACKGROUND
salmonella enterica are enteric pathogens that acquired a type iii secretion system  through horizontal gene transfer of a genomic island termed salmonella pathogenicity island  <dig>   <cit> .
the spi-2-encoded t3ss and its translocated effectors modify the intracellular host niche for salmonella replication  <cit> .
spi- <dig> also has genes, ssra and ssrb, which code for ssrab, a two-component regulatory system needed for expression of the t3ss  <cit> .
ssrb regulates the expression of spi- <dig> encoded substrate effectors including ssab, as well as several integrated virulence effectors such as ssel  <cit>  and srfn  <cit>  that are encoded elsewhere on the chromosome but that have integrated into the ssrb regulon.
mutants lacking ssrab are unable to survive within macrophages and are avirulent in mice  <cit> .
alternative sigma factors coordinate gene expression in response to environmental cues sensed by the bacterium.
sigma factors have a specific recognition motif at the - <dig> and - <dig> positions and function to concentrate rna polymerase at a subset of promoters  <cit> .
one alternative sigma factor, rpoe  responds to envelope stress at the cell surface.
release of σe from its inner membrane anchored anti-sigma factor, rsea, leads to induction of genes required to maintain cell envelope integrity  <cit> .
ssrb-regulated translocated effectors protect s. typhimurium against host cell defences such as oxidative stress and antimicrobial peptides that perturb bacterial membrane integrity and provide a stimulus for σe release  <cit> .
although proficient at cellular invasion, rpoe or ssrb mutants are highly attenuated for intracellular survival in both cultured cells and animal hosts  <cit> .
in addition, the expression of rpoe and ssrb is up-regulated within macrophages  <cit> .
links between rpoe and virulence gene expression is evident in other bacterial systems as well.
deletion of rsea in yersinia pseudotuberculosis causes elevated virulence effector synthesis and secretion  <cit> , establishing links between alternative sigma factors and virulence-specific regulators.
taken together, a connection between σe and ssrb is suggested from the available literature, however the role of σe in activating ssrb-regulated genes has not been studied.
we tested the hypothesis that rpoe is involved in expression of genes that use the ssrb response regulator for activation.
by testing six promoters representing four classes of ssrb-regulated promoters  two type iii secretion structural operons in spi- <dig>   the effector operon in spi- <dig>   two effector genes unlinked with spi- <dig>  and  an integrated virulence gene unlinked with spi-2) we demonstrate that rpoe elicits an effect on a subset of ssrb-regulated genes.
this effect was bidirectional depending on the promoter and was downstream of ssrb expression itself, since deletion of rpoe had no effect on ssrb levels in the mutant cells.
these data help unite the virulence phenotypes of strains lacking ssrb and rpoe, and highlight new transcriptional regulation that might be essential for appropriate temporal and spatial control of the virulence-associated type iii secretion system during host infection.
RESULTS
deletion of rpoe affects a subset of ssrb-regulated virulence genes
salmonella virulence gene expression is coordinated in vivo and may be regulated, in part, by alternative sigma factor in order to quickly respond to the host environment.
to date, no sigma factor has been identified as regulating ssrb-dependent virulence genes.
to start, we first screened four alternative sigma factor mutants of s. typhimurium  for their ability to express a key virulence gene, sseb, that requires ssrb for expression and whose gene product is essential for intracellular pathogenesis.
for an rpoh deletion, this strain was only viable at temperatures below 30°c.
since spi- <dig> gene activation is integrated into a thermosensing circuit  <cit>  we were unable to test the role of σh in this study .
in this screen, rpos deletion resulted in a slight increase in sseb levels  indicating a role for rpos in the repression of spi- <dig>  both rpoe and rpon deletions resulted in decreased sseb levels with a more pronounced effect in the rpoe deletion.
since we were predominantly interested in sigma factors that activate spi- <dig> and which could be linked to the previous observation that rpoe mutants are highly attenuated in vivo we choose to focus on rpoe in the current study, which had the most influence on sseb levels in the cell.
an unmarked in-frame deletion of rpoe was then generated in s. typhimurium strain sl <dig> and we verified that this in-frame deletion had the same effect on sseb as the rpoe::cat mutant used previously .
a low-copy plasmid containing full-length rpoe and the three endogenous promoters that can drive its expression  <cit>  was able to restore wild type levels of sseb to Δrpoe cells  demonstrating that the results were specific to the rpoe deletion.
in these complementation experiments, attempts were made to examine the levels of sseb secreted into the culture supernatant  <cit> , however consistent with previous observations  <cit>  perturbations to the rpoe pathway increased cell lysis resulting in contamination of secreted fractions with cytosolic proteins which precluded accurate interpretation .
in order to examine the effect of σe  on the expression of a broad range of ssrb-regulated virulence genes, we tested whether or not the effect of rpoe deletion was specific to sseb or if it extended to other ssrb-regulated genes.
to do this we examined the levels of ssel-2ha, sifa-2ha and srfn-2ha expressed from their endogenous promoters under spi- <dig> inducing conditions .
consistent with the results for sseb, there was a decrease in sifa-2ha levels in Δrpoe compared to wild type, although deletion of rpoe did not have an effect on ssel-2ha.
relative to its expression in wild type cells, the level of srfn-2ha was reproducibly increased in the Δrpoe cells, suggesting a role for σe in the repression of srfn, although it is unlikely that this is through a direct mechanism.
rpoe is involved in transcriptional activity of a subset of virulence genes
in order to confirm the effect of σe on the expression of a broad range of ssrb-regulated virulence genes, we used wild type and Δrpoe cells and integrated into the chromosome individually six single-copy transcriptional fusions representing promoters for four classes of ssrb-dependent genes or operons  type iii secretion effector operon ;  structural operon i ;  structural operon ii ;  effectors encoded outside of spi- <dig> ; and  integrated virulence genes unlinked to spi- <dig>   <cit> .
transcriptional fusions to lacz of the ssea, ssab, ssag, ssel, sifa and srfn promoters , whereas promoter activity from the ssag and srfn reporters was upregulated in the rpoe mutant .
β-galactosidase activity observed from the ssel reporter was unaltered in the rpoe deletion compared to that in wild type cells .
these data are consistent with the protein levels detected for these gene products.
together, these data indicate that σe can have a variable and bidirectional effect on ssrb-regulated virulence genes.
the effect of rpoe on virulence genes is downstream of ssrb expression
the variable effects of rpoe deletion on ssrb-regulated effectors suggested that rpoe might direct transcription downstream of ssrb expression.
to test this, we replaced the ssrb gene in Δrpoe and wild type cells with an ssrb::flag allele  <cit>  and examined the levels of ssrb protein in the strains by western blot.
there was no change in the levels of ssrb-flag between wild type and Δrpoe cells , indicating that the effects of rpoe on the four classes of virulence gene promoters examined here was not mediated through changes to ssrb protein levels.
together these data establish a role for rpoe in the fine-tuning of virulence gene expression in s. typhimurium.
discussion
in this work we found that the alternative sigma factor, σe, is involved in fine tuning the expressing of a subset of ssrb-regulated virulence genes required for salmonella pathogenesis.
although the effect of rpoe deletion on promoter activity in some cases was mild, we have previously shown that gene regulators providing only modest transcriptional input have a profound influence on bacterial fitness in a host animal  <cit> .
in cases where the regulator is deleted, the loss of genetic fine-tuning causes incongruous changes in the timing and magnitude of virulence gene expression, leading to fitness loss and strong attenuation.
we predict that rpoe confers a similar fine-tuning effect on salmonella virulence gene expression that is required for optimal within-host fitness during infection.
when we examined the - <dig> and - <dig> positions of the promoters studied here relative to the transcriptional start sites identified previously  <cit> , these promoters did not appear to contain σe consensus sequences.
instead they appeared to have consensus sites for σ <dig>  although a bioinformatics screen identified σe consensus sequences upstream of the spi- <dig> genes ssau, ssaj, ssca and ssac  <cit> , these genes were not tested for σe-dependence in the present study because the identified consensus sites are in coding sequence within operons, and as a result may not be directly relevant.
due to the high degree of conservation in σ factor binding sequences, σe may not be directly regulating ssrb-dependent promoters.
the lack of a canonical σe sequence at these promoters suggests that another regulatory gene may be epistatic to σe or that these promoters encode functional, but non-canonical σe-binding sites due to their horizontal acquisition and gradual integration into the σe regulatory network.
this integration may help salmonella coordinate expression of the virulence-associated t3ss in response to host factors that compromise bacterial membrane integrity .
this mechanism would activate a restorative σe pathway, which is consistent with the enhanced susceptibility of rpoe mutants to oxidative stress and antimicrobial peptides  <cit> , both of which perturb membrane integrity in vivo.
although there is no evidence that σe can directly repress transcription, the negative effect on two promoters observed here might be due to an intermediate rpoe-regulated repressor or compensatory effect where loss of rpoe increases the relative abundance of another sigma factor that can directly activate the ssag and srfn promoters.
future work will be required to resolve these possibilities.
our results indicate that rpoe deletion has no effect on ssrb levels under spi- <dig> inducing conditions suggesting that the σe pathway regulates effector expression downstream of ssrab transcription.
unlinking ssrab transcription from the σe regulon would be advantageous to the cell to prevent commitment to a virulence gene expression program in response to envelope stress not associated with infection.
the results from this study demonstrate that σe has the ability to affect expression of ssrb-regulated virulence genes and offers potential insight into the virulence attenuation of rpoe mutants.
although when considered individually, each promoter was modestly affected by deletion of rpoe, the cumulative effects of mild rewired inputs on multiple virulence promoters has been shown to severely compromise in-host fitness and virulence ability  <cit> .
CONCLUSIONS
based on these and other data  <cit> , the genetic interaction between σe and a subset of ssrb-regulated genes may serve to coordinate the spatial and temporal activation of virulence genes in a host setting, likely in response to membrane damage resulting from oxidative anti-microbial systems and membrane-targeted host defence peptides.

 salmonella enterica are enteric pathogens that acquired a type iii secretion system through horizontal gene transfer of a genomic island termed salmonella pathogenicity island. the spi-2-encoded t3ss and its translocated effectors modify the intracellular host niche for salmonella replication. spi- also has genes, ssra and ssrb, which code for ssrab, a two-component regulatory system needed for expression of the t3ss. ssrb regulates the expression of spi- encoded substrate effectors including ssab, as well as several integrated virulence effectors such as ssel and srfn that are encoded elsewhere on the chromosome but that have integrated into the ssrb regulon. mutants lacking ssrab are unable to survive within macrophages and are avirulent in mice. alternative sigma factors coordinate gene expression in response to environmental cues sensed by the bacterium. sigma factors have a specific recognition motif at the - and - positions and function to concentrate rna polymerase at a subset of promoters. one alternative sigma factor, rpoe responds to envelope stress at the cell surface. release of σe from its inner membrane anchored anti-sigma factor, rsea, leads to induction of genes required to maintain cell envelope integrity. ssrb-regulated translocated effectors protect s. typhimurium against host cell defences such as oxidative stress and antimicrobial peptides that perturb bacterial membrane integrity and provide a stimulus for σe release. although proficient at cellular invasion, rpoe or ssrb mutants are highly attenuated for intracellular survival in both cultured cells and animal hosts. in addition, the expression of rpoe and ssrb is up-regulated within macrophages. links between rpoe and virulence gene expression is evident in other bacterial systems as well. deletion of rsea in yersinia pseudotuberculosis causes elevated virulence effector synthesis and secretion, establishing links between alternative sigma factors and virulence-specific regulators. taken together, a connection between σe and ssrb is suggested from the available literature, however the role of σe in activating ssrb-regulated genes has not been studied. we tested the hypothesis that rpoe is involved in expression of genes that use the ssrb response regulator for activation. by testing six promoters representing four classes of ssrb-regulated promoters two type iii secretion structural operons in spi- the effector operon in spi- two effector genes unlinked with spi- and an integrated virulence gene unlinked with spi-2) we demonstrate that rpoe elicits an effect on a subset of ssrb-regulated genes. this effect was bidirectional depending on the promoter and was downstream of ssrb expression itself, since deletion of rpoe had no effect on ssrb levels in the mutant cells. these data help unite the virulence phenotypes of strains lacking ssrb and rpoe, and highlight new transcriptional regulation that might be essential for appropriate temporal and spatial control of the virulence-associated type iii secretion system during host infection.  deletion of rpoe affects a subset of ssrb-regulated virulence genes salmonella virulence gene expression is coordinated in vivo and may be regulated, in part, by alternative sigma factor in order to quickly respond to the host environment. to date, no sigma factor has been identified as regulating ssrb-dependent virulence genes. to start, we first screened four alternative sigma factor mutants of s. typhimurium for their ability to express a key virulence gene, sseb, that requires ssrb for expression and whose gene product is essential for intracellular pathogenesis. for an rpoh deletion, this strain was only viable at temperatures below 30°c. since spi- gene activation is integrated into a thermosensing circuit we were unable to test the role of σh in this study. in this screen, rpos deletion resulted in a slight increase in sseb levels indicating a role for rpos in the repression of spi- both rpoe and rpon deletions resulted in decreased sseb levels with a more pronounced effect in the rpoe deletion. since we were predominantly interested in sigma factors that activate spi- and which could be linked to the previous observation that rpoe mutants are highly attenuated in vivo we choose to focus on rpoe in the current study, which had the most influence on sseb levels in the cell. an unmarked in-frame deletion of rpoe was then generated in s. typhimurium strain sl and we verified that this in-frame deletion had the same effect on sseb as the rpoe::cat mutant used previously. a low-copy plasmid containing full-length rpoe and the three endogenous promoters that can drive its expression was able to restore wild type levels of sseb to Δrpoe cells demonstrating that the results were specific to the rpoe deletion. in these complementation experiments, attempts were made to examine the levels of sseb secreted into the culture supernatant, however consistent with previous observations perturbations to the rpoe pathway increased cell lysis resulting in contamination of secreted fractions with cytosolic proteins which precluded accurate interpretation. in order to examine the effect of σe on the expression of a broad range of ssrb-regulated virulence genes, we tested whether or not the effect of rpoe deletion was specific to sseb or if it extended to other ssrb-regulated genes. to do this we examined the levels of ssel-2ha, sifa-2ha and srfn-2ha expressed from their endogenous promoters under spi- inducing conditions. consistent with the results for sseb, there was a decrease in sifa-2ha levels in Δrpoe compared to wild type, although deletion of rpoe did not have an effect on ssel-2ha. relative to its expression in wild type cells, the level of srfn-2ha was reproducibly increased in the Δrpoe cells, suggesting a role for σe in the repression of srfn, although it is unlikely that this is through a direct mechanism. rpoe is involved in transcriptional activity of a subset of virulence genes in order to confirm the effect of σe on the expression of a broad range of ssrb-regulated virulence genes, we used wild type and Δrpoe cells and integrated into the chromosome individually six single-copy transcriptional fusions representing promoters for four classes of ssrb-dependent genes or operons type iii secretion effector operon; structural operon i; structural operon ii; effectors encoded outside of spi-; and integrated virulence genes unlinked to spi-. transcriptional fusions to lacz of the ssea, ssab, ssag, ssel, sifa and srfn promoters, whereas promoter activity from the ssag and srfn reporters was upregulated in the rpoe mutant. β-galactosidase activity observed from the ssel reporter was unaltered in the rpoe deletion compared to that in wild type cells. these data are consistent with the protein levels detected for these gene products. together, these data indicate that σe can have a variable and bidirectional effect on ssrb-regulated virulence genes. the effect of rpoe on virulence genes is downstream of ssrb expression the variable effects of rpoe deletion on ssrb-regulated effectors suggested that rpoe might direct transcription downstream of ssrb expression. to test this, we replaced the ssrb gene in Δrpoe and wild type cells with an ssrb::flag allele and examined the levels of ssrb protein in the strains by western blot. there was no change in the levels of ssrb-flag between wild type and Δrpoe cells, indicating that the effects of rpoe on the four classes of virulence gene promoters examined here was not mediated through changes to ssrb protein levels. together these data establish a role for rpoe in the fine-tuning of virulence gene expression in s. typhimurium. discussion in this work we found that the alternative sigma factor, σe, is involved in fine tuning the expressing of a subset of ssrb-regulated virulence genes required for salmonella pathogenesis. although the effect of rpoe deletion on promoter activity in some cases was mild, we have previously shown that gene regulators providing only modest transcriptional input have a profound influence on bacterial fitness in a host animal. in cases where the regulator is deleted, the loss of genetic fine-tuning causes incongruous changes in the timing and magnitude of virulence gene expression, leading to fitness loss and strong attenuation. we predict that rpoe confers a similar fine-tuning effect on salmonella virulence gene expression that is required for optimal within-host fitness during infection. when we examined the - and - positions of the promoters studied here relative to the transcriptional start sites identified previously, these promoters did not appear to contain σe consensus sequences. instead they appeared to have consensus sites for σ although a bioinformatics screen identified σe consensus sequences upstream of the spi- genes ssau, ssaj, ssca and ssac, these genes were not tested for σe-dependence in the present study because the identified consensus sites are in coding sequence within operons, and as a result may not be directly relevant. due to the high degree of conservation in σ factor binding sequences, σe may not be directly regulating ssrb-dependent promoters. the lack of a canonical σe sequence at these promoters suggests that another regulatory gene may be epistatic to σe or that these promoters encode functional, but non-canonical σe-binding sites due to their horizontal acquisition and gradual integration into the σe regulatory network. this integration may help salmonella coordinate expression of the virulence-associated t3ss in response to host factors that compromise bacterial membrane integrity. this mechanism would activate a restorative σe pathway, which is consistent with the enhanced susceptibility of rpoe mutants to oxidative stress and antimicrobial peptides, both of which perturb membrane integrity in vivo. although there is no evidence that σe can directly repress transcription, the negative effect on two promoters observed here might be due to an intermediate rpoe-regulated repressor or compensatory effect where loss of rpoe increases the relative abundance of another sigma factor that can directly activate the ssag and srfn promoters. future work will be required to resolve these possibilities. our results indicate that rpoe deletion has no effect on ssrb levels under spi- inducing conditions suggesting that the σe pathway regulates effector expression downstream of ssrab transcription. unlinking ssrab transcription from the σe regulon would be advantageous to the cell to prevent commitment to a virulence gene expression program in response to envelope stress not associated with infection. the results from this study demonstrate that σe has the ability to affect expression of ssrb-regulated virulence genes and offers potential insight into the virulence attenuation of rpoe mutants. although when considered individually, each promoter was modestly affected by deletion of rpoe, the cumulative effects of mild rewired inputs on multiple virulence promoters has been shown to severely compromise in-host fitness and virulence ability.  based on these and other data, the genetic interaction between σe and a subset of ssrb-regulated genes may serve to coordinate the spatial and temporal activation of virulence genes in a host setting, likely in response to membrane damage resulting from oxidative anti-microbial systems and membrane-targeted host defence peptides. 
in this study we demonstrate that rpoe is involved in fine-tuning the expression of a subset of ssrb-regulated genes found in the salmonella pathogenicity island- genetic locus that encodes a horizontally acquired type iii secretion system, and unlinked genes integrated into this regulon that are required for virulence in host animals. these data point to a potential connection between the virulence phenotype of strains lacking ssrb and rpoe, and highlight new transcriptional regulation that might be essential for appropriate temporal and spatial control of the virulence-associated type iii secretion system during host infection. the survival of salmonella enterica within the intracellular host niche requires highly co-ordinated expression of virulence effectors predominantly regulated by the ssrab two-component regulatory system. mutants lacking the gene encoding the alternative sigma factor σe are also highly attenuated for intracellular survival, pointing to a potential connection with the ssrab regulatory system.
 the survival of salmonella enterica within the intracellular host niche requires highly co-ordinated expression of virulence effectors predominantly regulated by the ssrab two-component regulatory system. s. enterica serovar typhimurium mutants lacking the ssrab genes are avirulent in mice, highlighting the importance of this regulatory system in vivo. mutants lacking the gene encoding the alternative sigma factor σe are also highly attenuated for intracellular survival, pointing to a potential connection with the ssrab regulatory system.  in this study we demonstrate that rpoe is involved in fine-tuning the expression of a subset of ssrb-regulated genes found in the salmonella pathogenicity island- genetic locus that encodes a horizontally acquired type iii secretion system, and unlinked genes integrated into this regulon that are required for virulence in host animals.  these data point to a potential connection between the virulence phenotype of strains lacking ssrb and rpoe, and highlight new transcriptional regulation that might be essential for appropriate temporal and spatial control of the virulence-associated type iii secretion system during host infection. 