JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M309433200 on September 26, 2003

J. Biol. Chem., Vol. 278, Issue 50, 50588-50595, December 12, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/50/50588    most recent
M309433200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mouslim, C.
Right arrow Articles by Groisman, E. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mouslim, C.
Right arrow Articles by Groisman, E. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Signal-dependent Requirement for the Co-activator Protein RcsA in Transcription of the RcsB-regulated ugd Gene*

Chakib Mouslim, Tammy Latifi, and Eduardo A. Groisman{ddagger}

From the Department of Molecular Microbiology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, August 26, 2003 , and in revised form, September 22, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The RcsC/YojN/RcsB phosphorelay system controls gene expression in response to a variety of signals, including changes in temperature, osmolarity, and overproduction of membrane proteins. Transcription of certain RcsB-activated genes, such as the capsule synthesis cps operon, requires the co-activator protein RcsA, whereas expression of other RcsB-activated genes is RcsA-independent. We have established previously that a tolB mutation induces transcription of the Salmonella UDP-glucose dehydrogenase ugd gene in an RcsA- and RcsB-dependent manner. This induction is independent of the two-component systems PhoP/PhoQ and PmrA/PmrB, which are required for ugd expression in response to low Mg2+. We now report that the RcsC/YojN/RcsB system is activated in a pmrA mutant experiencing Fe3+ and low Mg2+, resulting in expression of both cps and ugd genes. However, whereas cps transcription remained RcsA-dependent, ugd transcription became RcsA-independent but dependent on the PhoP protein. S1 mapping experiments demonstrated that RcsA-dependent and -independent transcription of the ugd gene use the same promoter. DNase footprinting analysis identified a PhoP-binding site in the ugd promoter. Yet, PhoP-mediated ugd transcription required either the RcsC/YojN/RcsB or the PmrA/PmrB systems.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The RcsC/YojN/RcsB phosphorelay system controls a variety of cellular functions including the synthesis of colanic acid capsule (1-4), the decoration of the lipopolysaccharide with 4-aminoarabinose (5) as well as motility and chemotaxis (6, 7) in several bacterial species. RcsC is a sensor protein, RcsB is its cognate response regulator, and YojN is a histidine-containing phosphotransfer protein that is apparently used as an intermediary in the phosphoryl transfer from RcsC to RcsB (7). Genes regulated by RcsB can be divided into two classes based on their requirement for the co-activator protein RcsA: RcsA-dependent (e.g. cps and rcsA; Refs. 2, 8-10) and RcsA-independent (e.g. ftsA, osmC, rprA and tviA; Refs. 11-14). The RcsB and RcsA proteins bind to a sequence motif, termed RcsAB box, present in the cps genes of several species (15, 16), whereas the RcsB protein binds to a different sequence motif, termed RcsB box, found in the promoter of the RcsA-independent osmC gene (17). The RcsB protein fails to bind stably to DNA by itself (18), suggesting that RcsB may function with other co-factors at RcsA-independent promoters. For example, RcsB activation of the tviA gene requires the TviA protein (11), and full activation of the osmC and ftsA genes needs an unidentified factor(s) (12, 14).

The Salmonella ugd gene encodes UDP-glucose dehydrogenase, an enzyme required for the production of both colanic acid (19) and 4-aminoarabinose (5). Transcription of the ugd gene is induced by three different signals by means of three different two-component regulatory systems (Fig. 1). Low Mg2+ promotes ugd transcription in a process that requires the Mg2+-responsive PhoP/PhoQ system, the PhoP-regulated protein PmrD, and the PmrA/PmrB system (21). Fe3+ promotes ugd transcription in a process that is dependent on the Fe3+-responsive PmrA/PmrB system but independent of the PhoP/PhoQ system and the PmrD protein (22). A tolB mutation induces ugd transcription via the RcsC/YojN/RcsB system and its co-activator RcsA protein but independently of the PhoP/PhoQ and PmrA/PmrB systems (20).



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 1.
Signals and pathways promoting transcription of the Salmonella ugd gene. Low Mg2+ is sensed by the PhoQ protein, which activates its cognate regulator PhoP. Activation of PhoP results in expression of the PmrD protein, which activates the PmrA/PmrB system at a posttranscriptional level, so the PmrA protein can bind to the ugd promoter and activate ugd transcription. Fe3+ is recognized by the sensor protein PmrB, which activates the cognate regulatory protein PmrA, so the PmrA protein can bind to the ugd promoter and activate ugd transcription. In a tolB mutant, the RcsC/YojN/RcsB system is activated, promoting transcription of the ugd gene in an RcsB- and RcsA-dependent manner. Boxed region indicates what was previously known about the regulation of ugd transcription by the PhoP/PhoQ, PmrA/PmrB, and RcsC/YojN/RcsB systems and RcsA protein before the studies described in this paper. We have now determined that Fe3+ and low Mg2+ promote ugd transcription in a pmrA mutant by means of a pathway that requires the RcsC/YojN/RcsB and PhoP/PhoQ systems but independently of the RcsA protein.

 
The ugd promoter has putative binding sites for the PmrA, RcsB, and PhoP proteins (20). The PmrA protein binds to a region of the ugd promoter (23) that harbors a motif found in the promoter of other PmrA-activated genes, such as pbgP and pmrC (24). Although binding of the RcsB protein to the ugd promoter has not been reported, mutation of the putative RcsB-binding site abolished ugd transcription promoted by the tolB mutation (20). On the other hand, the presence of a putative PhoP-binding site in the ugd promoter is intriguing because (i) the low Mg2+ induction of the ugd gene, like that of other PmrA-activated genes, is dependent on the PmrD protein and the PmrA/PmrB system (21), and (ii) there is no clear -10 region near the putative PhoP-binding site, which is in contrast to genes that are directly regulated by PhoP, such as phoP, mgtA, and mgrB (25).

In this paper, we report a new condition that activates the RcsC/YojN/RcsB system and establish that the requirement for the RcsA protein in RcsB-mediated transcription of the ugd gene is dependent on the inducing condition, but that cps transcription is RcsA-dependent, regardless of the inducing condition. Our results uncover a potential new function for the PhoP protein in activating gene expression. The participation of several combinations of regulatory systems in the control of ugd expression may be due to the participation of UDP-glucuronic acid (i.e. the product of the reaction catalyzed by UDP-glucose dehydrogenase) in the biosynthesis of different cellular structures produced under different conditions.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains, Plasmids, Recombinant Molecular Techniques, and Growth Conditions—Bacterial strains and plasmids used in this study are listed in Table I. Mutants were constructed by phage P22-mediated transductions as described (26). Recombinant DNA techniques were performed according to standard protocols (27). The allele flhDC5213::MudJ was a gift from Kelly T. Hughes (University of Washington). Bacteria were grown at 37 °C in LB medium or in modified N minimal medium (50 mM Trizma base, 50 mM bis-Tris,1 50 mM KCl, 75 mM (NH4)2 SO4, 5 mM K2SO4, and 10 mM KH2PO4) containing 0.1% casamino acids and 38 mM glycerol in which 100 mM Tris-HCl was replaced by a mixture of 50 mM bis-Tris and 50 mM Tris adjusted to pH 7.7 or 5.8 with HCl. MgCl2 was added to a concentration of 10 µM FeSO4 and was used at a final concentration of 0.1 mM from a freshly prepared 0.1 M stock solution. Kanamycin was used at a final concentration of 50 µg/ml, chloramphenicol at 25 µM/ml, ampicillin at 50 µg/ml, and tetracycline at 10 µM/ml.


View this table:
[in this window]
[in a new window]
  		TABLE I
Bacterial strains and plasmids

 

Construction of Chromosomal Deletions and Chromosomal Mutants—A chromosomal deletion mutant of the PhoP-binding site was constructed as described (28), using primers 1744 (5'-GAAGGCATTTTCCATACCGAATGGTTGGAATAATTTCTGCAAAAATGTAGGCTGGAGCTGCTTCG-3') and 2502 (5'-GCGATAAATCAATAAGAATAGAGATAAAAAAAAGCCCGGTATCATATGAATATCCTCCTTAG-3'). The construction of base substitutions at the RcsB site was accomplished through a combination of multiple PCRs based on a described method (28). To retain an intact PhoP-binding site and to conserve the distance between the transcription factor-binding sites described in this study, we constructed strain EG13866, in which a chloramphenicol cassette from plasmid pKD3 was placed upstream of the PhoP-binding site using primers 2475 (5'-AAAAAAGCCCGGTATTAAACCGGGCTTAAACATTTTTGCAGCATATGAATATCCTCCTTAAG-3') and 2476 (5'-TGATAAAGAAGGCATTTTCCATACCGAATGGTTGGAATAAGTGTAGGCTGGAGCTGCTTCG-3'). To introduce the GAA -> CTT substitution at the RcsB-binding site, we first generated two PCR products with strain EG13866 DNA as template: one using primers 1502 (5'-CGGGATCCGTCCCGGATATCGTGATTTTC-3') and 2100 (5'-CAGATTATCGGTATTAC-3'), and another using primers 1501 (5'-GGAATTCAGATCACCGATGCTTACGC-3') and 2009 (5'-GTAATCCGATAATCTG-3'). Then, the two PCR products were used as template for a PCR with primers 1501 and 1502. An analogous protocol was used to generate CTG -> GGC substitutions using the mutagenic primers 2101 (5'-GAAGATAATGGCAATTG-3') and 2102 (5'-CAATTGCCATTATCTTC-3').

Plasmids—Plasmids harboring transcriptional fusion to the ugd promoter have been described (20).

S1 Nuclease Assay—The S1 nuclease protection assay was performed as described (29), with RNA harvested form mid-exponential phase cultures (A600 of 0.4-0.6) grown in 50 ml of N minimal medium, pH 7.7, containing either 10 µM MgCl2 or 10 µM MgCl2 and 0.1 mM FeSO4. Total RNA was isolated with TRIzol (Invitrogen) according to the manufacturer's specifications. A PCR product generated with primers 1501 and 1007 (5'-GCAATAAGCAAACCATTAGA-3') and Salmonella chromosomal DNA as template was used as probe. This probe was labeled at the 5' end by phosphorylation with [{gamma}-32P]ATP using T4 polynucleotide kinase (Invitrogen) as described (20).

DNase I Footprinting—DNase I protection assays were carried out using the appropriately labeled primer. DNA fragments used for DNase I footprinting were amplified by the PCR using Salmonella enterica serovar Typhimurium chromosomal DNA as a template. Prior to the PCR, primers 2001 (5'-GACTACTTTGGTGCGCAC-3') and 1827 (5'-CGGGATCCTCATTACGATAAACACA-3'), which anneal to the coding and non-coding strand of ugd respectively, were labeled with T4 polynucleotide kinase and [{gamma}-32P]ATP. The ugd promoter region was amplified with the labeled primers 2001 and 2021 (5'-CGGGATCCAACTTAATTATGAATCATGA-3') for the coding strand or with the labeled primers 1827 and 2000 (5'-CGGGATCCGGCATTTTCCATACCG-3') for the non-coding strand. Approximately 25 pmol of labeled DNA and 0, 10, 12.5, or 50 pmol of PhoP-H6 protein in a 100-µl volume were incubated at room temperature for 15 min. The buffer used for protein-DNA incubations was 20 mM Tris, pH 7.4, 5 mM MgCl2, 50 mM KCl, 50 µg/ml bovine serum albumin, 2.5 µg/ml salmon sperm DNA, and 10% glycerol. DNase I (Invitrogen) (0.05 units) was added and incubated for 3 min at room temperature. The reaction was stopped by adding 10 µl of 25 mM EDTA. DNA fragments were purified with Wizard DNA cleanup system (Promega, Madison, WI) and dissolved in 30 µl of H2O. Samples (6 µl) were analyzed by denaturing 6% polyacrylamide gel electrophoresis by comparison with a DNA sequence ladder generated with the appropriate primer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Fe3+ Promotes ugd Transcription Independently of the PmrA/PmrB System—Transcription of PmrA-activated genes is coordinately induced by Fe3+ in a process that requires the Fe3+ sensor PmrB and the DNA-binding transcriptional regulator PmrA (22). Although this is true for the PmrA-activated pbgP and pmrC genes, we determined that Fe3+ could still promote ugd transcription in pmrA (Fig. 2A) and pmrB mutants (data not shown). This is in contrast to the induction of PmrA-activated genes that takes place in low Mg2+, where inactivation of the pmrA gene abolished transcription of both the ugd (Fig. 2B) and pbgP (Fig. 2D) genes. As expected, expression of the PhoP-activated PmrA-independent mgtA gene was not affected by Fe3+ or by inactivation of the pmrA gene (Fig. 2, E and F). These data demonstrate that Fe3+ can specifically activate ugd transcription independently of the PmrA/PmrB system.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 2.
Fe3+ promotes transcription of the ugd gene in a PmrA-independent manner. {beta}-galactosidase activity (Miller units) expressed by strains grown in N minimal medium, pH 7.7, 10 µM MgCl2 with (A, C, E) or without (B, D, F) 100 µM FeSO4 was determined for mutants harboring lac-transcriptional fusions to the PmrA-activated genes ugd (A, B) and pbgP (C, D) and the PhoP-activated mgtA (E, F) gene. The transcriptional activity was investigated in three genetic backgrounds: wild type, pmrA, and phoP. Data correspond to mean values of four independent experiments performed in duplicate. Error bars correspond to the standard deviation (shown only if greater than the resolution of the figure).

 
Fe3+ Activates the RcsC/YojN/RcsB System, which Mediates the PmrA-independent Transcription of the ugd Gene—We hypothesized that the RcsC/YojN/RcsB system might be responsible for the Fe3+-promoted PmrA-independent activation of the ugd gene because, like a tolB mutation (30, 31), Fe3+ treatment of a pmrA mutant perturbs the outer membrane (32) and because a tolB mutation promotes ugd transcription in an RcsC/YojN/RcsB-dependent manner (20). Consistent with this notion, Fe3+ could not promote ugd transcription in rcsB pmrA (Fig. 3A), rcsC pmrA, and yojN pmrA double mutants (data not shown). On the other hand, the rcsB mutant displayed wild-type levels of ugd expression in response to the Fe3+ and low Mg2+ signals (Fig. 3A), which are mediated by the PmrA/PmrB system and the PhoP/PhoQ-PmrD-PmrA/PmrB pathway, respectively (Fig. 1). Fe3+ treatment of the pmrA mutant appears to activate the RcsC/YojN/RcsB system (as opposed to acting solely on the ugd promoter) because it also promoted transcription of the cps operon (Fig. 3B) and repressed expression of the flhDC genes (Fig. 3C). These results establish that Fe3+ treatment of a pmrA mutant activates the RcsC/YojN/RcsB system, which is necessary for the pmrA-independent transcription of the ugd gene.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 3.
Fe3+ and low Mg2+ activate the RcsC/YojN/RcsB system in a pmrA mutant, promoting ugd transcription in an RcsA-independent manner. {beta}-galactosidase activity (Miller units) expressed by strains grown in N minimal medium, pH 7.7, 10 µM MgCl2, and 100 µM FeSO4 (A, B, C) were determined for strains harboring a lac-transcriptional fusion to the ugd gene (A), cps operon (B), and flhDC operon (C). The transcriptional activity was investigated in six genetic backgrounds: wild type, pmrA, rcsB, pmrA rcsB, pmrA rcsA, and rcsA. Data correspond to mean values of four independent experiments performed in duplicate. Error bars correspond to the standard deviation (shown only if greater than the resolution of the figure).

 
The Fe3+-promoted PmrA-independent Activation of the ugd Gene Does Not Require the RcsA Protein—Genes under transcriptional control of the RcsC/YojN/RcsB system can be divided into two classes based on their requirement for the RcsA protein: RcsA-dependent (e.g. cps and rcsA) and RcsA-independent (e.g. osmC, ftsZ and tviA). Although the tolB-promoted transcription of the ugd gene requires RcsA (20), the Fe3+-promoted PmrA-independent transcription of the ugd gene seems to be RcsA independent because ugd expression was similar in pmrA and pmrA rcsA strains (i.e. only 35% lower in the latter; Fig. 3A). This is in contrast to cps transcription, which was reduced nearly 8-fold by the rcsA mutation. These results indicate that the requirement for the RcsA protein in the RcsB-mediated transcription of the ugd gene is signal dependent.

The Regulatory Protein PhoP Is Necessary for the Fe3+-promoted PmrA-independent Transcription of the ugd Gene—We investigated the possibility that the PhoP protein might be involved in the Fe3+-promoted PmrA-independent transcription of the ugd gene because (i) these experiments were carried out in low Mg2+, which is the signal that activates the PhoP protein (29), and (ii) the ugd promoter harbors a putative PhoP-binding site (20). We established that Fe3+ promoted ugd transcription in phoP, pmrA, and rcsB single mutants and in a phoP rcsB double mutant but not in phoP pmrA or rcsB pmrA double mutants (Fig. 4). This finding indicates that bacteria experiencing Fe3+ and low Mg2+ promote ugd transcription via two pathways: the PmrA/PmrB pathway (operating when the RcsB protein is not active) and a pathway requiring both the PhoP/PhoQ and RcsC/YojN/RcsB systems (operating when the PmrA protein is not active; Fig. 1).



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 4.
The PhoP/PhoQ and RcsC/YojN/RcsB systems are required for the Fe3+-promoted PmrA-independent transcription of ugd gene. {beta}-galactosidase activity (Miller units) expressed by strains grown in N minimal medium, pH 7.7, 10 µM MgCl2, and 100 µM FeSO4 were determined in mutants harboring a lac-transcriptional fusion to the ugd gene. The transcriptional activity was investigated in seven genetic backgrounds: wild type, pmrA, phoP, rcsB, pmrA phoP, phoP rcsB, and pmrA rcsB. Data correspond to mean values of four independent experiments performed in duplicate. Error bars correspond to the standard deviation (shown only if greater than the resolution of the figure).

 
RcsA-independent and -dependent ugd Transcription Is Mediated by the Same Promoter—To define the ugd promoters used by the various regulatory proteins, we performed S1 mapping experiments using RNA harvested from bacteria grown under different conditions. When grown in low Mg2+, the wild-type strain produced an S1 product that originated from the PmrA-dependent promoter because it was absent from RNA prepared from the pmrA mutant (Fig. 5A). This S1 product was more intense in wild-type cells induced by low Mg2+ and Fe3+ (Fig. 5A) and of the same size as that detected in organisms grown in LB broth (Ref. 20; Fig. 5B), which is an environment of relatively low Mg2+. On the other hand, a pmrA mutant exposed to Fe3+ produced a smaller S1 product (Fig. 5A), which is of the same size as that induced by the tolB mutation in an RcsB-dependent fashion (Ref. 20; Fig. 5B). These experiments indicate that two promoters mediate ugd transcription: a distal promoter that is PmrA-dependent but RcsB-independent, and a proximal promoter that is RcsB-dependent but PmrA-independent (Fig. 5C). Moreover, they indicate that both RcsA-dependent and -independent transcription of the ugd gene use the same promoter.



View larger version (50K):
[in this window]
[in a new window]
  		FIG. 5.
Molecular analysis of the ugd promoter. (A) S1 mapping of ugd transcripts produced by wild type, pmrA, and pmrA rcsB bacteria grown in minimal medium, pH 7.7, containing 10 µM MgCl2 or 10 µM MgCl2 and 100 µM FeSO4 and harvested during the logarithmic phase. The S1 protection assay was performed as described under "Experimental Procedures." Lanes G, A, T, and C correspond to dideoxy chain-termination reactions for this region. The transcription start sites are marked with arrows. The PmrA-dependent and RcsB-dependent transcriptional start sites are indicated by +1A and +1B, respectively. (B) S1 mapping of ugd transcripts produced by wild type, tolB, and tolB rcsB bacteria grown in LB broth and harvested during the logarithmic phase (reproduced from Ref. 20). Lanes G, A, T, and C correspond to dideoxy chain termination reactions for this region. The transcription start sites are marked with arrows. The PmrA-dependent and RcsB-dependent transcriptional start sites are indicated by +1A and +1B, respectively. Note that the RcsB-dependent transcription start site is the same in a tolB mutant grown in LB broth and in a pmrA mutant exposed to Fe3+ and low Mg2+. (C) DNA sequence corresponding to the 283-bp region upstream of the start codon of ugd containing the cis-acting elements required for transcription promoted by the PmrA/PmrB, PhoP/PhoQ, and RcsC/YojN/RcsB systems. The PhoP-binding site is indicated by a red box, the direct repeat in the PmrA-binding site by a blue box, and the putative RcsB-binding site by a green box. Underlined sequence represents DNA region footprinted by the PhoP protein. The transcription start sites are indicated by arrows. (D) Footprinting analysis of the ugd promoter region was performed with the end-labeled coding, non-coding strand, and the PhoP-H6 protein added at 0, 10, 12.5, and 50 pmol. Solid bars represent the PhoP-binding regions. The position of the binding was determined by comparison with sequence ladder, obtained by using the same labeled primers as those utilized for the probe.

 
Defining the cis-Acting Sequences Required for the Fe3+-promoted PmrA-independent Transcription of the ugd Gene—Using plasmid-borne lac fusions to the ugd promoter, we have recently established that the 283-bp region upstream of the ugd start codon (Fig. 5C) has all the necessary cis-acting sequences necessary for ugd transcription promoted by the tolB mutation (20). This region includes a PmrA-binding site (21), the putative RcsB-binding site, as well as a bona fide PhoP-binding site, because the PhoP protein footprinted the region -223 to -198 from the ugd start codon (Fig. 5D). We have now determined that this region harbors the sequence information required for the low Mg2+ PmrA-dependent, as well as for the Fe3+-promoted PmrA-dependent and -independent transcription of the ugd gene (Fig. 6). Indeed deletion of the 79-bp sequence including the PhoP-binding site abolished the Fe3+-promoted PmrA-independent transcription of the ugd gene (Fig. 6).



View larger version (10K):
[in this window]
[in a new window]
  		FIG. 6.
The cis-acting sequences required for the Fe3+-promoted PmrA-independent transcription of the ugd gene. {beta}-galactosidase activity (Miller units) expressed by strains grown in N minimal medium, pH 7.7, 10 µM MgCl2, and with or without 100 µM FeSO4 were determined for mutants harboring a lac-transcriptional fusion to the wild-type ugd promoter and to the deleted PhoP binding site ugd promoter present in the multicopy number plasmid pIC552 in two genetic backgrounds, wild-type and pmrA. For comparison purposes, we reproduced the transcriptional activity of the ugd gene in a tolB mutant grown in LB broth (20). Representative results of three independent experiments are shown. Note that deletion of the PhoP-binding site eliminates the Fe3+ and low Mg2+ activation that takes place in a pmrA mutant but had no effect on that promoted by the tolB mutation.

 
To rule out the possibility of potential phenotypes resulting from the lac fusion being in a multicopy number plasmid, we constructed strains harboring a lac fusion to the chromosomal copy of the ugd gene and mutations in the RcsB- and PhoP-binding sites. Deletion of the PhoP-binding site abolished ugd expression promoted by low Mg2+ and Fe3+ in a pmrA mutant (Fig. 7), but had no effect on ugd transcription promoted by Fe3+ or by the tolB mutation in a pmrA+ strain (Fig. 7). Mutation of the conserved GAA and CTG sequences in the RcsB-binding site abolished the Fe3+-promoted activation that takes place in the pmrA mutant (Fig. 6B) but had no effect on the low Mg2+ and Fe3+ activation that is mediated by the PmrA protein (Fig. 7). Cumulatively, these results are in agreement with the data resulting from inactivation of the regulatory genes: (i) mutation of the phoP gene or the PhoP-binding site in the ugd promoter abolished the Fe3+-promoted PmrA-independent activation of ugd but had no effect on the Fe3+-promoted activation mediated by the PmrA protein or the RcsB-mediated activation promoted by a tolB mutation; and (ii) mutation of the rcsB gene or the RcsB site in the ugd promoter abolished both the tolB- and Fe3+-promoted PmrA-independent activation of ugd transcription.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 7.
Role of the PhoP- and RcsB-binding sites on the Fe3+-promoted PmrA-independent transcription of the ugd gene. {beta}-galactosidase activity (Miller units) expressed by strains grown in N minimal medium, pH 7.7, 10 µM MgCl2, and 100 µM FeSO4 were determined for mutants harboring a lac-transcriptional fusion to the chromosomal copy of the ugd gene. The PhoP-binding site is indicated by a red box, the direct repeat in the PmrA-binding site by a blue box, and the putative RcsB-binding site by a green box. The wild-type promoter region, the chromosomal deletion of the PhoP-binding site, and base substitutions at the putative RcsB-binding site are shown. The transcriptional activity was investigated in three genetic backgrounds: wild type, pmrA, and pmrA phoP. Representative results of three experiments are shown. Note that deletion of the PhoP-binding site or site-directed mutations of the RcsB-binding site abolish Fe3+-promoted PmrA-independent transcription of the ugd gene.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have identified a new condition that activates the RcsC/YojN/RcsB system, established that the requirement for the RcsA protein in RcsB-promoted transcription depends on the particular promoter and inducing condition, and uncovered a novel interaction between two-component system proteins.

We have demonstrated that the RcsC/YojN/RcsB system is activated in a pmrA mutant experiencing Fe3+ and low Mg2+, resulting in transcriptional activation of the ugd (Fig. 3A) and cps genes (Fig. 3B) and transcriptional repression of the flhDC operon (Fig. 3C). What this condition has in common with previously reported activating conditions of the Rcs system, such as those resulting from inactivation of the tolB gene (30, 31) and the overproduction of the DnaJ-like protein DjlA (33-37), is the perturbation of the outer membrane. Indeed, Fe3+ disrupts the outer membrane of pmrA Salmonella, rendering it susceptible to killing by vancomycin and lysis by deoxycholate (32).

We have established that the two conditions that induce RcsB-dependent transcription of the ugd gene (i.e. a tolB mutation or Fe3+ and low Mg2+ in a pmrA mutant) use the same transcription start site (Fig. 5, A and B; Ref. 20) and require the same putative RcsB-binding site at the ugd promoter (Fig. 7; Ref. 20). This is despite the fact that these activation conditions differ in their requirement for the response regulator protein PhoP and the co-activator protein RcsA (Fig. 4; Ref. 20). The PhoP protein had been implicated previously in the transcriptional induction of the ugd gene in response to low Mg2+ because of its role as transcriptional activator of the PmrD protein, which is a posttranscriptional activator of the PmrA/PmrB system (Ref. 21; Fig. 1). However, the PhoP protein plays a different role in ugd transcription in a pmrA mutant experiencing Fe3+ and low Mg2+ because (i), this activation requires the PhoP-binding site at the ugd promoter (Figs. 6 and 7), (ii) it is dependent upon the RcsB protein (Fig. 3A), (iii) it is independent of the PmrA protein (Fig. 3A), and (iv) different transcription start sites are used in the two activation conditions (Fig. 5, A and B). The PhoP protein may promote RcsB-dependent transcription of the ugd gene by interacting with the RcsB protein and/or by altering the ugd promoter in a fashion that facilitates RcsB binding.

Our results reinforce the idea that the RcsB protein uses different partners to promote transcription. RcsB-regulated genes have been divided into those that are RcsA-dependent, such as cps and rcsA (1), and those that are RcsA-independent, such as ftsZ (12, 38), tviA (11), and osmC (14). Placement of the Salmonella ugd gene into one of these groups is problematic because ugd transcription is RcsA-dependent when the inducing condition is a tolB mutation (20) but RcsA-independent when the inducing condition is Fe3+ and low Mg2+ in a pmrA mutant (Fig. 3A). This is most surprising given the fact that transcription of the Salmonella cps genes is RcsA-dependent under both inducing conditions (Fig. 3B; Ref. 20).

We suggest that the RcsB protein utilizes different co-regulatory proteins depending both on the inducing condition, which may control the levels of co-regulatory proteins, and the promoter in question, which may have binding sites for co-regulatory proteins. This is supported by the requirement for additional factors in the transcriptional activation of the RcsB-regulated RcsA-independent tviA (11), osmC, and ftsA (12, 14, 38) genes. Furthermore, it may explain the incongruent results reported for the RcsA-dependence of the RcsB-mediated repression of the master flagellar regulator flhDC locus. Repression of flhDC transcription resulting from overexpression of the RcsB protein in LB broth (39), as well as that observed in a pmrA mutant experiencing Fe3+ and low Mg2+ (Fig. 3C), or in a igaA mutant (40), was independent of RcsA. Yet, overexpression of the RcsA protein repressed flhDC transcription in an RcsB-dependent manner (39). In addition, RcsB derivatives that are predicted to mimic the phosphorylated state of the protein could bind to the osmC and flhDC promoters in the absence of the RcsA protein (17, 39), suggesting that a highly activated RcsB may promote transcription in the absence of co-regulators.

A pattern that seems to be emerging about RcsB-regulated genes is that all RcsA-dependent genes are involved in the production of capsule, whereas those that are RcsA-independent participate in a variety of different cellular functions. Thus, the dual status of the ugd gene is likely caused by its participation in both capsule and lipopolysaccharide biosynthesis.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants AI42336 and AI49561 (to E. A. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Dept. of Molecular Microbiology, Howard Hughes Medical Institute, Washington Univ. School of Medicine, Campus Box 8230, 660 S. Euclid, St. Louis, MO 63110. Tel.: 314-362-3692; Fax: 314-747-8228; E-mail: groisman{at}borcim.wustl.edu.

1 The abbreviation used is: bis-Tris, bis(2-hydroxyethyl)iminotris-(hydroxymethyl)methane. Back


   ACKNOWLEDGMENTS
 
We thank F. Solomon for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gottesman, S., Trisler, P., and Torres-Cabassa, A. (1985) J. Bacteriol. 162, 1111-1119[Abstract/Free Full Text]
  2. Gottesman, S., and Stout, V. (1991) Mol. Microbiol. 5, 1599-1606[Medline] [Order article via Infotrieve]
  3. Stout, V. (1994) Res. Microbiol. 145, 389-392[Medline] [Order article via Infotrieve]
  4. Stout, V., and Gottesman, S. (1990) J. Bacteriol. 172, 659-669[Abstract/Free Full Text]
  5. Gunn, J. S., Lim, K. B., Krueger, J., Kim, K., Guo, L., Hackett, M., and Miller, S. I. (1998) Mol. Microbiol. 27, 1171-1182[CrossRef][Medline] [Order article via Infotrieve]
  6. Belas, R., Schneider, R., and Melch, M. (1998) J. Bacteriol. 180, 6126-6139[Abstract/Free Full Text]
  7. Takeda, S., Fujisawa, Y., Matsubara, M., Aiba, H., and Mizuno, T. (2001) Mol. Microbiol. 40, 440-450[CrossRef][Medline] [Order article via Infotrieve]
  8. Gottesman, S. (1995) in Two-component Signal Transduction (Silhavy, T. J., ed) pp. 253-262, American Society for Microbiology, Washington, D. C.
  9. Stout, V. (1996) J. Bacteriol. 178, 4273-4280[Abstract/Free Full Text]
  10. Ebel, W., and Trempy, J. E. (1999) J. Bacteriol. 181, 577-584[Abstract/Free Full Text]
  11. Virlogeux, I., Waxin, H., Ecobichon, C., Lee, J. O., and Popoff, M. Y. (1996) J. Bacteriol. 178, 1691-1698[Abstract/Free Full Text]
  12. Carballes, F., Bertrand, C., Bouche, J. P., and Cam, K. (1999) Mol. Microbiol. 34, 442-450[CrossRef][Medline] [Order article via Infotrieve]
  13. Majdalani, N., Hernandez, D., and Gottesman, S. (2002) Mol. Microbiol. 46, 813-826[CrossRef][Medline] [Order article via Infotrieve]
  14. Davalos-Garcia, M., Conter, A., Toesca, I., Gutierrez, C., and Cam, K. (2001) J. Bacteriol. 183, 5870-5876[Abstract/Free Full Text]
  15. Wehland, M., Kiecker, C., Coplin, D. L., Kelm, O., Saenger, W., and Bernhard, F. (1999) J. Biol. Chem. 274, 3300-3307[Abstract/Free Full Text]
  16. Wehland, M., and Bernhard, F. (2000) J. Biol. Chem. 275, 7013-7020[Abstract/Free Full Text]
  17. Sturny, R., Cam, K., Gutierrez, C., and Conter, A. (2003) J. Bacteriol. 185, 4298-4304[Abstract/Free Full Text]
  18. Pristovsek, P., Sengupta, K., Lohr, F., Schafer, B., von Trebra, M. W., Ruterjans, H., and Bernhard, F. (2003) J. Biol. Chem. 278, 17752-17759[Abstract/Free Full Text]
  19. Stevenson, G., Andrianopoulos, K., Hobbs, M., and Reeves, P. R. (1996) J. Bacteriol. 178, 4885-4893[Abstract/Free Full Text]
  20. Mouslim, C., and Groisman, E. A. (2003) Mol. Microbiol. 47, 335-344[CrossRef][Medline] [Order article via Infotrieve]
  21. Kox, L. F., Wosten, M. M., and Groisman, E. A. (2000) EMBO J. 19, 1861-1872[CrossRef][Medline] [Order article via Infotrieve]
  22. Wosten, M. M., Kox, L. F., Chamnongpol, S., Soncini, F. C., and Groisman, E. A. (2000) Cell 103, 113-125[CrossRef][Medline] [Order article via Infotrieve]
  23. Aguirre, A., Lejona, S., Vescovi, E. G., and Soncini, F. C. (2000) J. Bacteriol. 182, 3874-3876[Abstract/Free Full Text]
  24. Wosten, M. M., and Groisman, E. A. (1999) J. Biol. Chem. 274, 27185-27190[Abstract/Free Full Text]
  25. Minagawa, S., Ogasawara, H., Kato, A., Yamamoto, K., Eguchi, Y., Oshima, T., Mori, H., Ishihama, A., and Utsumi, R. (2003) J. Bacteriol. 185, 3696-3702[Abstract/Free Full Text]
  26. Davis, R. W., Bolstein, D., and Roth, J. R. (1980) Advanced Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  27. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  28. Datsenko, K. A., and Wanner, B. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6640-6645[Abstract/Free Full Text]
  29. Garcia Vescovi, E., Soncini, F. C., and Groisman, E. A. (1996) Cell 84, 165-174[CrossRef][Medline] [Order article via Infotrieve]
  30. Fognini-Lefebvre, N., Lazzaroni, J. C., and Portalier, R. (1987) Mol. Gen. Genet. 209, 391-395[CrossRef][Medline] [Order article via Infotrieve]
  31. Bernadac, A., Gavioli, M., Lazzaroni, J. C., Raina, S., and Lloubes, R. (1998) J. Bacteriol. 180, 4872-4878[Abstract/Free Full Text]
  32. Chamnongpol, S., Dodson, W., Cromie, M. J., Harris, Z. L., and Groisman, E. A. (2002) Mol. Microbiol. 45, 711-719[CrossRef][Medline] [Order article via Infotrieve]
  33. Chen, M. H., Takeda, S., Yamada, H., Ishii, Y., Yamashino, T., and Mizuno, T. (2001) Biosci. Biotechnol. Biochem. 65, 2364-2367[CrossRef][Medline] [Order article via Infotrieve]
  34. Genevaux, P., Wawrzynow, A., Zylicz, M., Georgopoulos, C., and Kelley, W. L. (2001) J. Biol. Chem. 276, 7906-7912[Abstract/Free Full Text]
  35. Clarke, D. J., Holland, I. B., and Jacq, A. (1997) Mol. Microbiol. 25, 933-944[CrossRef][Medline] [Order article via Infotrieve]
  36. Kelley, W. L., and Georgopoulos, C. (1997) Mol. Microbiol. 25, 913-931[CrossRef][Medline] [Order article via Infotrieve]
  37. Zuber, M., Hoover, T. A., and Court, D. L. (1995) J. Bacteriol. 177, 4238-4244[Abstract/Free Full Text]
  38. Costa, C. S., and Anton, D. N. (2001) FEMS Microbiol. Lett. 200, 201-205[CrossRef][Medline] [Order article via Infotrieve]
  39. Francez-Charlot, A., Laugel, B., Van Gemert, A., Dubarry, N., Wiorowski, F., Castanie-Cornet, M. P., Gutierrez, C., and Cam, K. (2003) Mol. Microbiol. 49, 823-832[CrossRef][Medline] [Order article via Infotrieve]
  40. Cano, D. A., Dominguez-Bernal, G., Tierrez, A., Garcia-Del Portillo, F., and Casadesus, J. (2002) Genetics 162, 1513-1523[Abstract/Free Full Text]
  41. Fields, P. I., Swanson, R. V., Haidaris, C. G., and Heffron, F. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5189-5193[Abstract/Free Full Text]
  42. Soncini, F. C., Garcia Vescovi, E., Solomon, F., and Groisman, E. A. (1996) J. Bacteriol. 178, 5092-5099[Abstract/Free Full Text]
  43. Hanahan, D. (1983) J. Mol. Biol. 166, 557-580[Medline] [Order article via Infotrieve]
  44. Macian, F., Perez-Roger, I., and Armengod, M. E. (1994) Gene 145, 17-24[CrossRef][Medline] [Order article via Infotrieve]
  45. Sanderson, K. E., Hessel, A., and Rudd, K. E. (1995) Microbiol. Rev. 59, 241-303[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J BiochemHome page
C.-T. Lin, T.-Y. Huang, W.-C. Liang, and H.-L. Peng
Homologous Response Regulators KvgA, KvhA and KvhR Regulate the Synthesis of Capsular Polysaccharide in Klebsiella pneumoniae CG43 in a Coordinated Manner
J. Biochem., September 1, 2006; 140(3): 429 - 438.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
N. Majdalani, M. Heck, V. Stout, and S. Gottesman
Role of RcsF in Signaling to the Rcs Phosphorelay Pathway in Escherichia coli
J. Bacteriol., October 1, 2005; 187(19): 6770 - 6778.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. Frirdich, C. Bouwman, E. Vinogradov, and C. Whitfield
The Role of Galacturonic Acid in Outer Membrane Stability in Klebsiella pneumoniae
J. Biol. Chem., July 29, 2005; 280(30): 27604 - 27612.
[Abstract] [Full Text] [PDF]

Home page
 Innate ImmunityHome page
E. Frirdich and C. Whitfield
Review: Lipopolysaccharide inner core oligosaccharide structure and outer membrane stability in human pathogens belonging to the Enterobacteriaceae
Innate Immunity, June 1, 2005; 11(3): 133 - 144.
[Abstract] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
I. Zwir, D. Shin, A. Kato, K. Nishino, T. Latifi, F. Solomon, J. M. Hare, H. Huang, and E. A. Groisman
Dissecting the PhoP regulatory network of Escherichia coli and Salmonella enterica
PNAS, February 22, 2005; 102(8): 2862 - 2867.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
A. Tierrez and F. Garcia-del Portillo
The Salmonella Membrane Protein IgaA Modulates the Activity of the RcsC-YojN-RcsB and PhoP-PhoQ Regulons
J. Bacteriol., November 15, 2004; 186(22): 7481 - 7489.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/50/50588    most recent
M309433200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mouslim, C.
Right arrow Articles by Groisman, E. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mouslim, C.
Right arrow Articles by Groisman, E. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit