BACKGROUND
the two-component system  is one of the most ubiquitous signal transduction systems in bacteria  <cit> .
a prototypical tcs harbors a sensor histidine kinase , which is often integrated into the inner membrane, and a response regulator , which is predominantly a cytoplasmic dna-binding transcription factor.
in the presence of a specific activating signal, the sensor hk is autophosphorylated, and a phosphoryl group is subsequently transferred to a conserved aspartate residue in its cognate rr, thus changing gene expression patterns and cell physiology.
each tcs responds to specific environmental signals but elude identification even in the well-investigated organisms escherichia coli and salmonella.
due to the high levels of sequence and structure similarity among different tcss, cross-talk  may occur in at least some circumstances.
however, cross-talk is extremely rare due to the kinetic preference of a sensor hk for its cognate rr  <cit>  and their phosphatase activities  <cit> .
to date, several small proteins connecting tcss have been reported in salmonella and e. coli <cit> .
for example, the 85-amino acid pmrd protein, which is transcriptionally induced by the phop/phoq system under low mg2+ conditions, binds to the phosphorylated form of the regulator pmra and hinders its dephosphorylation by the cognate sensor pmrb  <cit> .
therefore, expression of pmra-activated genes, some of which are responsible for polymixin b resistance and iron resistance in salmonella, is induced even in the absence of an fe3+ signal  <cit> .
the small anti-adapter proteins irap and iram, which promote the stability of the stationary phase sigma s factor  of rna polymerase by hindering an rr , are also transcriptionally activated by the phop/phoq system in response to low mg2+ conditions in salmonella <cit>  and e. coli <cit> , respectively.
in contrast to these cytosolic connectors, the small inner membrane proteins safa   <cit>  and mzra  <cit>  were identified as signal transducers between two tcss by targeting downstream sensor hks.
safa elicits a response from the phoq sensor to the phop regulator even under high mg2+ conditions when the evgs <dig> mutan protein  <cit>  induces the evga-activated safa gene constitutively  <cit> .
alternatively, mzra interacts with the envz sensor to control ompr-regulated gene transcription when mzra expression is induced in a constitutively activated cpxa* mutant background  <cit>  in e. coli.
the membrane peptide mgrb  <cit> , which corresponds to a single tcs, communicates the activation status of the phop regulator to its cognate sensor phoq in e. coli and salmonella <cit> .
in contrast, the unique membrane peptide pmrr mediates the feedback control of the pmra/pmrb system indirectly in salmonella <cit> .
the cpxr/cpxa system regulates pilus assembly, adherence, and biofilm development in response to envelope stress and is required for host cell invasion in several species, including pathogenic e. coli and salmonella <cit> .
the periplasmic chaperone cpxp binds to both the cpxa periplasmic domain and to certain misfolded proteins, which are degraded by the periplasmic protease degp, therefore integrating information about their turnover status to the kinase activity of cpxa  <cit> .
the outer membrane lipoprotein nlpe activates the cpxa protein upon its overexpression  <cit>  and is required for cpxa protein activation after adhering to hydrophobic surfaces  <cit> .
additional upstream components have been proposed to integrate other stresses in a process that is independent of the cpxp and nlpe pathways  <cit> .
for example, the cpxr/cpxa system confers a copper resistance phenotype even in cpxp and nlpe mutants  <cit> .
notably, nlpe  is a pseudogene in salmonella <cit> .
here, we aimed to identify candidate connector genes that may integrate the signals of other systems.
we identified a small protein as a novel connector-like factor from screening high copy plasmid clones that could affect the cpxr/cpxa system status.
RESULTS
identification of a plasmid clone that activates cpxp transcription
to conduct a genetic screen for novel connector proteins acting on the cpxr/cpxa system, we constructed a strain harboring a cpxplac transcriptional fusion in salmonella.
the cpxp gene was chosen as a readout of the activation status of the cpxr/cpxa system because it is likely directly regulated exclusively by this system, unlike other cpxr-activated genes that are also controlled by envelope stress-responsive systems  <cit> .
the laczy genes were inserted after the cpxp stop codon to ensure that the cpxp protein retained the ability to repress the cpxr/cpxa system.
then, salmonella chromosomal dna was partially digested with sau3ai and ligated with the high-copy-number plasmid puc <dig>  to generate a dna fragment library.
of approximately  <dig>  cpxp-lac salmonella transformants, a plasmid clone termed pwn <dig> yielded stable blue colonies on lb plates containing 5-bromo-4-chloro-3-indolyl-β-d-galactoside  and ampicillin and was isolated four times.
the blue color of the pwn <dig> strain was due to elevated cpxp-lac fusion expression.
we demonstrated that this strain exhibit ~8-fold higher β-galactosidase activity than the same strain harboring the vector control or the plasmid clone puc19-r <dig> that was randomly selected during the screening as a negative control .
sequence analysis revealed that pwn <dig> harbors only the intact stm <dig> open reading frame , which appeared to encode a 63-amino acid protein with no homology to any protein of known function, as well as the 3’ region of stm <dig> and the 5’ region of ppha .
expression of stm <dig> from tetracycline-  or l-arabinose-  inducible promoters recapitulated the increase in the β-galactosidase activity observed in the cpxplac strain, supporting the hypothesis that stm <dig> was affecting cpxp transcription in the pwn <dig> plasmid.
here, we named stm <dig> “cpx activating connector-like factor a”, or caca.
caca-mediated cpxp activation is dependent on the cpxr/cpxa system
the results described above demonstrated that cpxp transcription was induced when caca was expressed from a high-copy-number plasmid or from a heterologous promoter in an inducer-dependent manner.
next, we compared the β-galactosidase activities of the cpxp-lac fusion from cpxr and cpxa mutant strains harboring pbad18-caca to an isogenic cpxr+a+ strain containing the same plasmid .
we determined that caca acts upstream of the cpxr/cpxa system because the activities of the cpxr and cpxa mutant strains expressing caca were comparable to that of the isogenic cpxr+a+ strain with vector  alone .
this was further supported by the observation that another cpxr-activated gene, spy, was induced by caca protein overexpression .
moreover, caca likely acts on the cpxr/cpxa system specifically because expression of caca did not affect genes under the direct control of other tcss .
caca transcription is activated by rpos but repressed by rssb
next, we asked whether the caca gene might be regulated by an undefined upstream tcs.
to examine candidate tcss that could potentially affect caca transcription, we constructed a strain with a caca promoter-lac fusion  <dig>  at the pgtp locus on the salmonella chromosome.
then,  <dig> rr mutant stocks were independently transduced into the pcaca-lac  <dig> strain by phage p <dig>  whereas most rr mutants exerted minor or no effects on transcription from the caca promoter , the rssb mutant exhibited a ~ <dig> -fold increase in caca promoter activity .
because rssb is the adaptor protein that recruits rpos to the clpxp protease, we examined the effect of a Δrpos mutant on transcription from the caca promoter.
as expected, the rpos gene was required for caca expression .
consistent with these observations, an alignment of the caca promoter regions from salmonella and its related enteric species revealed a conserved sequence that is present in an rpos-dependent consensus - <dig> region sequence   <cit>  .
moreover, although the location of the predicted - <dig> region correlates well with a transcription start site  determined by a genome-scale precise mapping of tsss that covered 78% of the salmonella orfs  <cit> , no obvious typical - <dig> region sequence exists upstream of the - <dig> nucleotides .
we mutated this - <dig> sequence from tcctacact to tcgtacact , acgtacact , or tccttcaca  and analyzed their effects on caca transcription .
in the Δrpos mutant, the β-galactosidase activity of the caca promoter was approximately 1/ <dig> of wild-type levels .
however, the β-galactosidase activities from the caca promoter containing -14c/g or -16t/a -14c/g substitutions were not affected by the Δrpos mutation after  <dig> h of growth in lb, indicating that these substitution mutations rendered the caca promoter rpos-independent .
conversely, when the essential nucleotides -12a and -8t of the canonical - <dig> region sequence, which permits recognition by both rpod and rpos, were mutated, caca promoter activity was abolished independent of rpos presence .
taken together, these results demonstrated that the activation of the caca promoter is dependent on the - <dig> region sequence, which harbors an rpos recognition site.
transcription of the cpxr-activated genes cpxp and spy is attenuated in a caca mutant
because rpos activates caca expression, we assessed whether a caca deletion mutation would affect transcription of the cpxa/cpxr-dependent cpxp and spy genes in low mg2+, the conditions under which the phoq/phop-activated irap prevents the rssb/clpxp-mediated degradation of rpos, even at log phase  <cit> .
we determined that caca participates in cpxa/cpxr system activation because cpxp and spy expression levels were reduced by approximately 30% and 50%, respectively, in the caca deletion mutant compared with wild-type .
thioredoxin  <dig> is required for the caca-mediated activation of the cpxr/cpxa system
pull-down experiment of the glutathione s transferase -caca fusion protein recovered the groel and thioredoxin  <dig>  proteins, suggesting that they interact directly with caca .
because groel has been shown to associate with proteins that are overexpressed, we did not investigate its role further.
instead, we focused on the effect of trxa on the caca-mediated activation of the cpxr/cpxa system because caca orthologs contain four conserved cysteine residues  and because trxa catalyzes thiol disulfide redox reactions in a variety of substrate proteins  <cit> .
we investigated trxc, another thioredoxin, and trxb, which participates in the regeneration of reduced trxa and trxc  <cit> , as controls.
whereas mutations in trxa, trxb, and trxc did not affect cpxp transcription in strains harboring vector alone, the trxa mutant expressing caca significantly decreased the levels of cpxp transcription compared to wild-type expressing caca .
these results indicate that trxa is required for the caca-mediated activation of the cpxr/cpxa system.
this suggests that cysteine thiol-disulfide exchanges participate in caca-dependent cpx activation.
discussion
we identified caca, encoded on a plasmid clone, as a novel connector-like factor that activated the cpxr/cpxa system from screening a library of high-copy-number plasmids containing various salmonella chromosomal dna fragments.
caca appears to exclusively act on the cpxr/cpxa system because a similar induction was not observed in other tcs reporter strains with the same clone.
this observation was not just an artifact of caca overexpression or from its expression driven by a heterologous promoter because deleting this gene revealed a moderate decrease in transcription of the cpxp and spy genes, which are directly regulated by the cpxr/cpxa system.
moreover, the activation of the caca gene promoter is, at least in part, dependent on rpos, the stability of which is subject to rssb/clpxp-mediated processability and the - <dig> region sequence.
taken together, we hypothesize that caca may integrate information about the regulatory status of rssb/rpos into the cpxr/cpxa system .
however, future investigations are necessary to fully elucidate the mechanism of caca-mediated cpxr/cpxa activation.
several assessments of how the caca protein activates cpxr-regulated genes were attempted.
however, we did not detect a physical association between caca and the cpxr/cpxa system.
for example, no significant interaction was observed between the caca protein and the cpxr/cpxa system in our bacterial two-hybrid system analyses , although we cannot completely dismiss that these proteins do not interact directly.
instead, thioredoxin  <dig> amino acid sequences were recovered by our pull-down assay.
trxa inactivation impacted the activation of the cpxr/cpxa system by caca, which possesses the conserved cysteine residues.
this is in contrast to a report that demonstrated that a dsbd mutation activated the cpxr/cpxa system in vibrio cholerae <cit> , where the dsbc-dsbd pathway promotes proper folding of substrate proteins with disulfide bond at the periplasm using the cytoplasmic reducing ability of thioredoxin  <cit> .
moreover, the cysteine residues of nlpe are critical for activating the cpxr/cpxa system in e. coli <cit> , and a periplasmic lola derivative with an artificial disulfide bond activates the cpxr/cpxa system  <cit> .
notably, perturbing the oxidizing environment of the periplasm in dsba and dsbb mutants or treating wild-type cells with dithiothreitol  activated the phop/phoq system in e. coli <cit> .
disruption of disulfide bond formation affects this system largely via an additional small protein component, mgrb, and its conserved cysteine residues.
currently, we cannot exclude the possibility that the interaction between caca and trxa is an artifact caca protein overexpression because trxa interacts with many proteins, including the rr rcsb  <cit> .
because we were unable to detect the 63-amino acid caca protein at native levels, we employed a larger tag or carrier protein in several biochemical experiments, including the pull-down assay.
protein instability likely precludes thorough analysis of small proteins of less than  <dig> amino acids or so  <cit> .
notably, deletion of trxa did not impact cpxp transcription levels in normal growth conditions .
more strict conditions need to be tested, as some small proteins accumulated within bacterial cells upon exposure to sodium dodecyl sulfate  and ethylenediaminetetraacetic acid   <cit> .
the specificity that tcs connectors exhibit for their targets is likely a key contributing factor in the fidelity of the integration of tcs signals at a post-translational level.
in fact, the pmrd connector protein can inhibit the dephosphorylation of phospho-pmra but not of its closest homolog, the response regulator ygix  <cit> .
although recognizing novel connectors in genomic sequences based on their uniqueness is far from trivial, genetic approaches will continue to help elucidate links amongst tcss.
CONCLUSIONS
in this study, we identified the caca protein as an activator of the cpxr/cpxa system.
this factor may be another example of an emerging class of small proteins  <cit>  that function as nodes in the tcs network and function to integrate their signaling pathways in salmonella.

 the two-component system is one of the most ubiquitous signal transduction systems in bacteria. a prototypical tcs harbors a sensor histidine kinase, which is often integrated into the inner membrane, and a response regulator, which is predominantly a cytoplasmic dna-binding transcription factor. in the presence of a specific activating signal, the sensor hk is autophosphorylated, and a phosphoryl group is subsequently transferred to a conserved aspartate residue in its cognate rr, thus changing gene expression patterns and cell physiology. each tcs responds to specific environmental signals but elude identification even in the well-investigated organisms escherichia coli and salmonella. due to the high levels of sequence and structure similarity among different tcss, cross-talk may occur in at least some circumstances. however, cross-talk is extremely rare due to the kinetic preference of a sensor hk for its cognate rr and their phosphatase activities. to date, several small proteins connecting tcss have been reported in salmonella and e. coli. for example, the 85-amino acid pmrd protein, which is transcriptionally induced by the phop/phoq system under low mg2+ conditions, binds to the phosphorylated form of the regulator pmra and hinders its dephosphorylation by the cognate sensor pmrb. therefore, expression of pmra-activated genes, some of which are responsible for polymixin b resistance and iron resistance in salmonella, is induced even in the absence of an fe3+ signal. the small anti-adapter proteins irap and iram, which promote the stability of the stationary phase sigma s factor of rna polymerase by hindering an rr, are also transcriptionally activated by the phop/phoq system in response to low mg2+ conditions in salmonella and e. coli, respectively. in contrast to these cytosolic connectors, the small inner membrane proteins safa and mzra were identified as signal transducers between two tcss by targeting downstream sensor hks. safa elicits a response from the phoq sensor to the phop regulator even under high mg2+ conditions when the evgs mutan protein induces the evga-activated safa gene constitutively. alternatively, mzra interacts with the envz sensor to control ompr-regulated gene transcription when mzra expression is induced in a constitutively activated cpxa* mutant background in e. coli. the membrane peptide mgrb, which corresponds to a single tcs, communicates the activation status of the phop regulator to its cognate sensor phoq in e. coli and salmonella. in contrast, the unique membrane peptide pmrr mediates the feedback control of the pmra/pmrb system indirectly in salmonella. the cpxr/cpxa system regulates pilus assembly, adherence, and biofilm development in response to envelope stress and is required for host cell invasion in several species, including pathogenic e. coli and salmonella. the periplasmic chaperone cpxp binds to both the cpxa periplasmic domain and to certain misfolded proteins, which are degraded by the periplasmic protease degp, therefore integrating information about their turnover status to the kinase activity of cpxa. the outer membrane lipoprotein nlpe activates the cpxa protein upon its overexpression and is required for cpxa protein activation after adhering to hydrophobic surfaces. additional upstream components have been proposed to integrate other stresses in a process that is independent of the cpxp and nlpe pathways. for example, the cpxr/cpxa system confers a copper resistance phenotype even in cpxp and nlpe mutants. notably, nlpe is a pseudogene in salmonella. here, we aimed to identify candidate connector genes that may integrate the signals of other systems. we identified a small protein as a novel connector-like factor from screening high copy plasmid clones that could affect the cpxr/cpxa system status.  identification of a plasmid clone that activates cpxp transcription to conduct a genetic screen for novel connector proteins acting on the cpxr/cpxa system, we constructed a strain harboring a cpxplac transcriptional fusion in salmonella. the cpxp gene was chosen as a readout of the activation status of the cpxr/cpxa system because it is likely directly regulated exclusively by this system, unlike other cpxr-activated genes that are also controlled by envelope stress-responsive systems. the laczy genes were inserted after the cpxp stop codon to ensure that the cpxp protein retained the ability to repress the cpxr/cpxa system. then, salmonella chromosomal dna was partially digested with sau3ai and ligated with the high-copy-number plasmid puc to generate a dna fragment library. of approximately cpxp-lac salmonella transformants, a plasmid clone termed pwn yielded stable blue colonies on lb plates containing 5-bromo-4-chloro-3-indolyl-β-d-galactoside and ampicillin and was isolated four times. the blue color of the pwn strain was due to elevated cpxp-lac fusion expression. we demonstrated that this strain exhibit ~8-fold higher β-galactosidase activity than the same strain harboring the vector control or the plasmid clone puc19-r that was randomly selected during the screening as a negative control. sequence analysis revealed that pwn harbors only the intact stm open reading frame, which appeared to encode a 63-amino acid protein with no homology to any protein of known function, as well as the 3’ region of stm and the 5’ region of ppha. expression of stm from tetracycline- or l-arabinose- inducible promoters recapitulated the increase in the β-galactosidase activity observed in the cpxplac strain, supporting the hypothesis that stm was affecting cpxp transcription in the pwn plasmid. here, we named stm “cpx activating connector-like factor a”, or caca. caca-mediated cpxp activation is dependent on the cpxr/cpxa system the results described above demonstrated that cpxp transcription was induced when caca was expressed from a high-copy-number plasmid or from a heterologous promoter in an inducer-dependent manner. next, we compared the β-galactosidase activities of the cpxp-lac fusion from cpxr and cpxa mutant strains harboring pbad18-caca to an isogenic cpxr+a+ strain containing the same plasmid. we determined that caca acts upstream of the cpxr/cpxa system because the activities of the cpxr and cpxa mutant strains expressing caca were comparable to that of the isogenic cpxr+a+ strain with vector alone. this was further supported by the observation that another cpxr-activated gene, spy, was induced by caca protein overexpression. moreover, caca likely acts on the cpxr/cpxa system specifically because expression of caca did not affect genes under the direct control of other tcss. caca transcription is activated by rpos but repressed by rssb next, we asked whether the caca gene might be regulated by an undefined upstream tcs. to examine candidate tcss that could potentially affect caca transcription, we constructed a strain with a caca promoter-lac fusion at the pgtp locus on the salmonella chromosome. then, rr mutant stocks were independently transduced into the pcaca-lac strain by phage p whereas most rr mutants exerted minor or no effects on transcription from the caca promoter, the rssb mutant exhibited a ~ -fold increase in caca promoter activity. because rssb is the adaptor protein that recruits rpos to the clpxp protease, we examined the effect of a Δrpos mutant on transcription from the caca promoter. as expected, the rpos gene was required for caca expression. consistent with these observations, an alignment of the caca promoter regions from salmonella and its related enteric species revealed a conserved sequence that is present in an rpos-dependent consensus - region sequence. moreover, although the location of the predicted - region correlates well with a transcription start site determined by a genome-scale precise mapping of tsss that covered 78% of the salmonella orfs, no obvious typical - region sequence exists upstream of the - nucleotides. we mutated this - sequence from tcctacact to tcgtacact, acgtacact, or tccttcaca and analyzed their effects on caca transcription. in the Δrpos mutant, the β-galactosidase activity of the caca promoter was approximately 1/ of wild-type levels. however, the β-galactosidase activities from the caca promoter containing -14c/g or -16t/a -14c/g substitutions were not affected by the Δrpos mutation after h of growth in lb, indicating that these substitution mutations rendered the caca promoter rpos-independent. conversely, when the essential nucleotides -12a and -8t of the canonical - region sequence, which permits recognition by both rpod and rpos, were mutated, caca promoter activity was abolished independent of rpos presence. taken together, these results demonstrated that the activation of the caca promoter is dependent on the - region sequence, which harbors an rpos recognition site. transcription of the cpxr-activated genes cpxp and spy is attenuated in a caca mutant because rpos activates caca expression, we assessed whether a caca deletion mutation would affect transcription of the cpxa/cpxr-dependent cpxp and spy genes in low mg2+, the conditions under which the phoq/phop-activated irap prevents the rssb/clpxp-mediated degradation of rpos, even at log phase. we determined that caca participates in cpxa/cpxr system activation because cpxp and spy expression levels were reduced by approximately 30% and 50%, respectively, in the caca deletion mutant compared with wild-type. thioredoxin is required for the caca-mediated activation of the cpxr/cpxa system pull-down experiment of the glutathione s transferase -caca fusion protein recovered the groel and thioredoxin proteins, suggesting that they interact directly with caca. because groel has been shown to associate with proteins that are overexpressed, we did not investigate its role further. instead, we focused on the effect of trxa on the caca-mediated activation of the cpxr/cpxa system because caca orthologs contain four conserved cysteine residues and because trxa catalyzes thiol disulfide redox reactions in a variety of substrate proteins. we investigated trxc, another thioredoxin, and trxb, which participates in the regeneration of reduced trxa and trxc, as controls. whereas mutations in trxa, trxb, and trxc did not affect cpxp transcription in strains harboring vector alone, the trxa mutant expressing caca significantly decreased the levels of cpxp transcription compared to wild-type expressing caca. these results indicate that trxa is required for the caca-mediated activation of the cpxr/cpxa system. this suggests that cysteine thiol-disulfide exchanges participate in caca-dependent cpx activation. discussion we identified caca, encoded on a plasmid clone, as a novel connector-like factor that activated the cpxr/cpxa system from screening a library of high-copy-number plasmids containing various salmonella chromosomal dna fragments. caca appears to exclusively act on the cpxr/cpxa system because a similar induction was not observed in other tcs reporter strains with the same clone. this observation was not just an artifact of caca overexpression or from its expression driven by a heterologous promoter because deleting this gene revealed a moderate decrease in transcription of the cpxp and spy genes, which are directly regulated by the cpxr/cpxa system. moreover, the activation of the caca gene promoter is, at least in part, dependent on rpos, the stability of which is subject to rssb/clpxp-mediated processability and the - region sequence. taken together, we hypothesize that caca may integrate information about the regulatory status of rssb/rpos into the cpxr/cpxa system. however, future investigations are necessary to fully elucidate the mechanism of caca-mediated cpxr/cpxa activation. several assessments of how the caca protein activates cpxr-regulated genes were attempted. however, we did not detect a physical association between caca and the cpxr/cpxa system. for example, no significant interaction was observed between the caca protein and the cpxr/cpxa system in our bacterial two-hybrid system analyses, although we cannot completely dismiss that these proteins do not interact directly. instead, thioredoxin amino acid sequences were recovered by our pull-down assay. trxa inactivation impacted the activation of the cpxr/cpxa system by caca, which possesses the conserved cysteine residues. this is in contrast to a report that demonstrated that a dsbd mutation activated the cpxr/cpxa system in vibrio cholerae, where the dsbc-dsbd pathway promotes proper folding of substrate proteins with disulfide bond at the periplasm using the cytoplasmic reducing ability of thioredoxin. moreover, the cysteine residues of nlpe are critical for activating the cpxr/cpxa system in e. coli, and a periplasmic lola derivative with an artificial disulfide bond activates the cpxr/cpxa system. notably, perturbing the oxidizing environment of the periplasm in dsba and dsbb mutants or treating wild-type cells with dithiothreitol activated the phop/phoq system in e. coli. disruption of disulfide bond formation affects this system largely via an additional small protein component, mgrb, and its conserved cysteine residues. currently, we cannot exclude the possibility that the interaction between caca and trxa is an artifact caca protein overexpression because trxa interacts with many proteins, including the rr rcsb. because we were unable to detect the 63-amino acid caca protein at native levels, we employed a larger tag or carrier protein in several biochemical experiments, including the pull-down assay. protein instability likely precludes thorough analysis of small proteins of less than amino acids or so. notably, deletion of trxa did not impact cpxp transcription levels in normal growth conditions. more strict conditions need to be tested, as some small proteins accumulated within bacterial cells upon exposure to sodium dodecyl sulfate and ethylenediaminetetraacetic acid. the specificity that tcs connectors exhibit for their targets is likely a key contributing factor in the fidelity of the integration of tcs signals at a post-translational level. in fact, the pmrd connector protein can inhibit the dephosphorylation of phospho-pmra but not of its closest homolog, the response regulator ygix. although recognizing novel connectors in genomic sequences based on their uniqueness is far from trivial, genetic approaches will continue to help elucidate links amongst tcss.  in this study, we identified the caca protein as an activator of the cpxr/cpxa system. this factor may be another example of an emerging class of small proteins that function as nodes in the tcs network and function to integrate their signaling pathways in salmonella. 
the caca-mediated induction of cpxp transcription was affected in a trxa deletion mutant, which encodes thioredoxin suggesting a role for cysteine thiol-disulfide exchange in caca-dependent cpx activation. substitution mutations in a conserved - region harboring the rna polymerase recognition sequence, which is well conserved with a known rpos - region consensus sequence, rendered the caca promoter rpos-independent. here, we report a connector-like factor that affects the activity of the cpxr/cpxa two-component system in salmonella enterica serovar typhimurium. future investigations are necessary to thoroughly elucidate how caca activates the cpxr/cpxa system. a 63-amino acid protein, caca, was responsible for the cpxa/cpxr-dependent activation of the cpxp gene. we identified caca as an activator of the cpxr/cpxa system in the plasmid clone. we propose that caca may integrate the regulatory status of rssb/rpos into the cpxr/cpxa system. we isolated a clone that induced the expression of a cpxp-lac gene fusion from a high-copy-number plasmid pool of random salmonella genomic fragments. the cpxr-activated genes cpxp and spy exhibited approximately 30% and 50% reductions in transcription, respectively, in a clean caca deletion mutant strain in comparison to wild-type.
 bacteria integrate numerous environmental stimuli when generating cellular responses. increasing numbers of examples describe how one two-component system responds to signals detected by the sensor of another tcs. however, the molecular mechanisms underlying this phenomenon remain poorly defined.  here, we report a connector-like factor that affects the activity of the cpxr/cpxa two-component system in salmonella enterica serovar typhimurium. we isolated a clone that induced the expression of a cpxp-lac gene fusion from a high-copy-number plasmid pool of random salmonella genomic fragments. a 63-amino acid protein, caca, was responsible for the cpxa/cpxr-dependent activation of the cpxp gene. the cpxr-activated genes cpxp and spy exhibited approximately 30% and 50% reductions in transcription, respectively, in a clean caca deletion mutant strain in comparison to wild-type. from response regulator deletion mutants, we identified that the rssb regulator represses caca transcription. substitution mutations in a conserved - region harboring the rna polymerase recognition sequence, which is well conserved with a known rpos - region consensus sequence, rendered the caca promoter rpos-independent. the caca-mediated induction of cpxp transcription was affected in a trxa deletion mutant, which encodes thioredoxin suggesting a role for cysteine thiol-disulfide exchange in caca-dependent cpx activation.  we identified caca as an activator of the cpxr/cpxa system in the plasmid clone. we propose that caca may integrate the regulatory status of rssb/rpos into the cpxr/cpxa system. future investigations are necessary to thoroughly elucidate how caca activates the cpxr/cpxa system. two-component systemconnectornetworkrssbrposcacacpxr/cpxa 