The absence of Dam in Salmonella enterica serovar Enteritidis causes a defect in lipopolysaccharide (LPS) pattern associated to a reduced expression of wzz gene. Wzz is the chain length regulator of the LPS O-antigen. Here we investigated whether Dam regulates wzz gene expression through its two known regulators, PmrA and RcsB. Thus, the expression of rcsB and pmrA was monitored by quantitative real-time RT-PCR and Western blotting using fusions with 3×FLAG tag in wild type (wt) and dam strains of S. Enteritidis. Dam regulated the expression of both rcsB and pmrA genes; nevertheless, the defect in LPS pattern was only related to a diminished expression of RcsB. Interestingly, regulation of wzz in serovar Enteritidis differed from that reported earlier for serovar Typhimurium; RcsB induces wzz expression in both serovars, whereas PmrA induces wzz in S. Typhimurium but represses it in serovar Enteritidis. Moreover, we found that in S. Enteritidis there is an interaction between both wzz regulators: RcsB stimulates the expression of pmrA and PmrA represses the expression of rcsB. Our results would be an example of differential regulation of orthologous genes expression, providing differences in phenotypic traits between closely related bacterial serovars.
Citation: Sarnacki SH, Castañeda MdRA, Llana MN, Giacomodonato MN, Valvano MÁ, Cerquetti MC (2013) Dam Methylation Participates in the Regulation of PmrA/PmrB and RcsC/RcsD/RcsB Two Component Regulatory Systems in Salmonella enterica Serovar Enteritidis. PLoS ONE 8(2): e56474. https://doi.org/10.1371/journal.pone.0056474
Editor: Axel Cloeckaert, Institut National de la Recherche Agronomique, France
Received: November 23, 2012; Accepted: January 9, 2013; Published: February 13, 2013
Copyright: © 2013 Sarnacki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part from grants from Universidad de Buenos Aires, Buenos Aires, Argentina (UBACyT 20020100100541) (http://www.uba.ar/secyt/index.php) and Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina (PIP 0992) (http://www.conicet.gov.ar) (to M.C.C.) and Universidad de Buenos Aires, Buenos Aires, Argentina (UBACyT 20020100300040) (http://www.uba.ar/secyt/index.php) (to S.H.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The lipopolysaccharide (LPS) is the most abundant component of outer membrane of Gram negative bacteria which structure is divided in three regions: O-antigen polysaccharide, core oligosaccharide, and lipid A . LPS synthesis is a complex process involving various steps. In particular, O-antigen production and assembly in Salmonella occurs by mechanisms that require Wzy (polymerase of the repeating subunits), Wzx (flippase that translocated subunit across the membrane) and Wzz (a chain length determinant) (previously Cld or Rol) , , , , , , , . Even though there is a significant amount of information on biochemistry and genetics of the LPS synthesis, the regulatory mechanisms that modulate its production are complex and poorly understood. However, it is known that LPS structure is dynamic, showing changes in response to local microenvironment signal. Many of these signals are detected as stimuli by signal transduction cascades. Usually, these systems are composed by a histidine kinase (HK) (sensor protein) that transmits the signal, through a phosphorylation cascade, to a second component, named response regulator , , , , , , . Often, the response regulator is a transcription factor, thereby the result of its phosphorylation is the activation or repression of gene transcription which product is involved in the adaptation to that given microenvironment. The most important two-component regulatory systems involved in LPS modification are PhoP/PhoQ, PmrA/PmrB and RcsC/RcsD/RcsB. PmrA/PmrB and RcsC/RcsD/RcsB two-component regulatory systems of Salmonella enterica serovar Typhimurium (S. Typhimurium), each activated by different stimuli, independently promote transcription of the wzz gene . The expression of wzz is also regulated by PhoP/PhoQ via PhoP-mediated upregulation of PmrD, which binds to the phosphorylated form of PmrA protecting it from dephosphorylation by PmrB , .
In Salmonella, regulation of the long chain distribution of the O-antigen contributes not only to an effective barrier  but also affect serum resistance and entry into eukaryotic cells , , , , . Furthermore, O-antigen length can also modulate acquired immunity. Indeed, Phalipon and coworkers demonstrated that in Shigella flexneri induction of an O-antigen-specific antibody response depends on the length of the polysaccharide chain . Also, Helicobacter pylori alters its O-antigen structure expressing O-antigen of high molecular weight in response to acidic pH; an important adaptation that would facilitate colonization of the acidic gastric environment .
In gammaproteobacteria the DNA adenine methyltransferase (Dam) introduces a methyl group at the N6 position of the adenine of GATC sequence in the newly synthesized DNA strand after DNA replication, generating methylated DNA , , , . DNA methylation status can affect interactions between DNA and proteins such as RNA polymerase or transcription factors  that regulate (activate or repress) gene expression generating a plethora of effects. Thus, Dam mutants of S. enterica have shown to have many defects particularly in virulence and they have been proposed as candidate vaccines , , , , , , , . We have previously shown that a dam null mutant of S. Enteritidis presents a reduced expression of wzz gene and a defective O-antigen polysaccharide chain length distribution .
In this work we study the regulation of pmrA and rcsB expression by Dam methylation in S. Enteritidis. In addition, we found that both wzz regulators have a regulatory influence on each other.
Materials and Methods
Bacterial strains, plasmids, strain construction, and growth conditions
Bacterial strains and plasmids used are listed in Table 1. S. Enteritidis #5694 was kindly given by Dr. Anne Morris Hooke, Miami University; originally from Dr. F. Collins'collection, Trudeau Institute, Saranac Lake, New York. Strains #SS218, #SS219 and #SS220 are S. Enteritidis isolates from poultry collected from argentine farms. Wild type strains were used to construct mutant strains listed in Table 1. Gene deletions were performed as described by Datsenko and Wanner . Addition of a DNA fragment encoding 3×FLAG epitope tag at the 3′ end of protein-coding DNA sequences was carried out as previously described using plasmid pSUB11 as a template  and oligonucleotides pmrA-3×FLAG-5′ and pmrA-3×FLAG-3′ for PmrA, and rcsB-3×FLAG-5′ and rcsB-3×FLAG-3′ for RcsB. The mutagenic primers used are listed in Table 2. S. Enteritidis was transformed by electroporation as previously described . Gene deletion and the correct fusion of the ORF with 3×FLAG coding sequence were confirmed by sequencing (Macrogen Inc.), and analyzed with Sequencher (Gene Codes Corporation) and Vector NTI software. Bacteria were grown in Luria-Bertani (LB) broth  supplemented, as required, with antibiotics at the following final concentrations: ampicilin, 100 µg/ml; chloramphenicol, 30 µg/ml; kanamycin, 40 µg/ml; and tetracycline, 20 µg/ml. For PmrA and RcsB overproduction experiments bacteria were grown at 37°C in N-minimal medium , supplemented with 0.2% (w/v) glucose, 0.1 mg/ml casaminoacids, 2 µg/ml Vitamin B1 and 10 mM MgCl2 (high Mg2+ concentration) or 10 µM MgCl2 100 µM FeSO4 (low Mg2+ concentration plus Fe3+) . Dam mutants were evaluated, phenotypically, determining the absence of methylated GATC sequences . To confirm pmrA deletion and ppmrA functionality, the resistance to the antimicrobial peptide Polymyxin B assay was carried out as previously described .
Molecular cloning of Salmonella pmrA and rcsB genes
DNA extracted from the parental strains of S. Enteritidis was used as template for PCR reaction to amplify pmrA and rcsB genes. PCR amplification was performed with either Pwo polymerase (Roche) (for amplification cloning fragments) or Taq polymerase (Qiagen). PCR fragments products were separated in agarose gels, purified using a Gel Extraction kit (Qiagen), and then digested using EcoRI restriction enzyme (Roche Diagnostics). Ligation with T4 DNA ligase (Rapid Ligation kit, Roche Diagnostics) into pUC18, also digested with EcoRI, and dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics) was performed. Competent E. coli DH5α cells were transformed with the ligation mixture by the calcium chloride protocol . Colonies with a white color phenotype from plates with ampicillin and 0.2% (w/v) X-Gal were pooled and screened by PCR using the primers downlacz18 combined with rcsB-F and rcsB-R for rcsB, and pmrA-F or pmrA-R for pmrA. Also, pooled colonies were screened by restriction digestion to preliminary identify the orientation of the inserts (with respect the plasmid promoter on sense or antisense). The integrity of the inserts were confirmed by DNA sequencing (Macrogen Inc.), using the sequencing primer M13 forward and M13 reverse, and the inserts were analyzed with Sequencher (Gene Codes Corporation) and Vector NTI software.
LPS was extracted as described by Marolda et al . Briefly, from overnight plate culture, samples were adjusted to OD600 of 2.0 in a final volume of 100 µl. Then, samples were suspended in lysis buffer containing proteinase K as described by Hitchcock and Brown , followed by hot phenol extraction and a subsequent extraction of the aqueous phase with ether. LPS was resolved by electrophoresis in 14% polyacrylamide gels using a tricine-sodium dodecyl sulfate (SDS) system ,  and visualized by silver staining. Each well was loaded with the same LPS concentration determined by the keto-deoxyoctulosonic (KDO) assay . A densitometry analysis was performed using ImageJ software. The ratio of the relative intensity of the lipid A-core band to the average intensity of the bands corresponding to total O-antigen and core+n was calculated by quantifying the pixels in a narrow window across the center of each lane. The densitometric analysis was calibrated by determining the ratio of the relative intensity of the lipid A-core region to the average intensity of the O-antigen bands.
Reverse transcription-PCR and quantitative real-time PCR
Bacteria were grown at 37°C with agitation to an OD600 of 0.6. Cells were lysed, and total RNA was isolated using Trizol reagent (Invitrogen) according to the method described by the manufacturer. Contaminating DNA was digested with RNase-free DNase I (Epicentre Biotechnologies), and the purity of all RNA preparations was confirmed by subjecting them to PCR analysis using primers specific for the gene encoding the 16S rRNA (Table 2). After inactivation of DNase, RNA was used as a template for reverse transcription-PCR. Complementary cDNA was synthesized using random hexamer primers (Invitrogen), deoxynucleoside triphosphates, and Moloney murine leukemia virus M-MLV reverse transcriptase (Invitrogen). Relative quantitative real-time PCR was performed with an appropriate primer set, cDNAs, and Mezcla Real (Biodynamics) that contained nucleotides, polymerase, reaction buffer, and Green dye, using a Rotor-Gene 6000 real-time PCR machine (Corbett Research). The amplification program consisted of an initial incubation for 3 min at 95°C, followed by 40 cycles of 95°C for 20 s, 60°C for 30 s, and 72°C 20 s. The primers used are depicted in Table 2. A no-template control was included for each primer set. Melting curve analysis verified that each reaction contained a single PCR product. For the relative gene expression analysis, a comparative cycle threshold method (ΔΔCT) was used . The number of copies of each sample transcript was determined with the aid of the software. Briefly, the amplification efficiencies of the genes of interest and the 16S rRNA gene used for normalization were tested. Then each sample was first normalized for the amount of template added by comparison to the 16S rRNA gene (endogenous control). The normalized values were further normalized using the wild-type sample (calibrator treatment). Hence, the results were expressed relative to the value for the calibrator sample, which was 1. Student's t test was used to determine if the differences in retrotranscribed mRNA content observed in different backgrounds were statistically significant.
Protein extracts and Western blotting analysis
Total protein extracts were prepared from bacterial cultures grown at 37°C in LB medium and harvested at an OD600 of 0.6. Cells were pelleted by centrifugation and resuspended with Laemmli buffer . Three independent extractions for each sample were added together to minimize differences in protein recovery from sample to sample. For Western blot assays total proteins were boiled for 5–10 min in Laemmli sample buffer, and each lane was loaded with material from approximately 106 CFU before resolved by 12% SDS-polyacrylamide gel electrophoresis (PAGE) gel. Prestained SDS-PAGE standards (Bio-Rad) were used as molecular weight markers (not shown). The gels were blotted onto a Hybond-P membrane (GE Health-care, Madrid, Spain). Ponceau S red staining was used as loading control before blocking in 5% (w/v) dried skimmed milk in PBS. Finally, 3×FLAG fusion proteins were immunodetected using mouse-monoclonal anti-FLAG M2-peroxidase (HRP) antibodies (1∶5,000; Sigma, St Louis, MO). The reacting bands were detected by enhanced chemiluminescence (ECL) (Luminol, Santa Cruz Biotechnology, Santa Cruz, CA) in an Image Quant 300 cabinet (GE Healthcare) following the manufacturer instructions. Blots were photographed, and the intensity of the signals expressed in arbitrary units was determined by densitometry analysis using the public domain NIH Image J software (http://rsb.info.nih.gov/nihimage/). We randomly selected three different bands from the Ponceau S stained membrane to normalize the intensity of the band of interest. Data were analyzed for statistical significance using a nonparametric Mann-Whitney test.
Dam methylation participates in the regulation of pmrA and rcsB genes
PmrA and RcsB two-component regulatory system are the only two known wzz regulators described in S. Typhimurium. To determine whether the LPS phenotype of the dam mutant of S. Enteritidis (SEΔdam) is related to a diminished expression of these two regulators we analyzed the effect of overproduction of either RcsB or PmrA on the LPS pattern in the dam background. Recombinant plasmids containing the rcsB and pmrA genes cloned into pUC18 were transferred by electroporation in SEΔdam and wild type strains. As we previously described, the LPS pattern of the dam mutant showed many more visible bands in the intermediate region of the gel (Fig. 1, lane 2) compared with the banding pattern of the wild-type LPS (Fig. 1, lane 1). The LPS O-antigen profiles of the transformed strains were analyzed in bacteria cultured in LB and under growing conditions known to activate the PmrA/PmrB two-component regulatory system. Results are depicted in Fig. 1. Regardless the culture media used, high Mg2+ or low Mg2+ + Fe3+, we found that RcsB overexpression in SEΔdam mutant (Fig. 1A, lanes 4 and 7) generates an LPS banding pattern comparable to that of the wild type (Fig. 1A, lanes 1 and 5). Similar results were observed when bacteria were cultured in LB medium (not shown). It seems that the presence of high amounts of RcsB in a dam background reduces the intermediate region bands observed for SEΔdam mutant (Fig. 1A, lanes 2 and 6). On the contrary, no changes were evident in the LPS pattern of SEΔdam mutant overexpressing PmrA regardless the growth environmental condition, high Mg2+ or low Mg2++Fe3+ (Fig. 1B, lanes 4 and 7). ppmrA plasmid functionality was confirmed by Polymyxin B resistance assay as described in materials and methods (data not shown). Again, similar results for LPS pattern were obtained when bacteria were cultured in LB medium (data not shown). Transformation with plasmids bearing the genes cloned in antisense orientation to the Plac promoter; (ppmrAas, prcsBas), or with empty plasmid vector (pUC18) produced no changes in the O-antigen LPS pattern of any strain studied (data not shown). These data would indicate that the dam mutant produces a reduced amount of RcsB protein, suggesting that rcsB gene expression is up-regulated by Dam.
Equal amount of LPS was loaded in each lane and analyzed by Tricine/SDS-PAGE on a 14% (w/v) acrylamide gel followed by silver staining. The concentration of LPS was determined by measuring KDO using the purpald assay. A. Lanes 1–4: bacteria grown in N-minimal medium containing 10 mM MgCl2; lanes 5–8: bacteria grown in N-minimal medium containing 10 µM MgCl2 100 µM FeSO4. B. Lanes 1–4: bacteria grown in N-minimal medium containing 10 mM MgCl2; lanes 5–7: bacteria grown in N-minimal medium containing 10 µM MgCl2 100 µM FeSO4. Plasmids pIZ833, prcsB and ppmrA bears the dam, rcsB and pmrA genes respectively.
Next, we analyzed LPS pattern in the absence of RcsB and PmrA. For this purpose we constructed rcsB and pmrA deletion mutants of S. Enteritidis (SEΔrcsB and SEΔpmrA strains, respectively) using the lambda Red recombination system. As shown in Fig. 2A, the LPS phenotype of SEΔrcsB is similar to that observed in SEΔdam mutant (lanes 2 and 3, respectively). Complementation with the plasmid bearing the rcsB gene restored LPS pattern to that found in the wild type strain of S. Enteritidis (Fig. 2A). The lack of pmrA did not modify LPS pattern in S. Enteritidis. As shown in Fig. 2B, deletion mutant SEΔpmrA (lane 2) presents an LPS pattern similar to that of the wild type strain (lane 1). Collectively, these experiments indicate that the reduced wzz gene expression observed in SEΔdam mutant correlates with a diminished expression of rcsB rather than pmrA.
Equal amount of LPS was loaded in each lane and analyzed by Tricine/SDS-PAGE on a 14% (w/v) acrylamide gel followed by silver staining. The concentration of LPS was determined by measuring KDO using the purpald assay. Plasmids prcsB and ppmrA bears the rcsB and pmrA genes respectively.
In silico analysis has shown the presence of GATC motifs in the coding sequence and/or surrounding nucleotides of pmrA and rcsB genes . Then we investigated whether Dam methylation regulates the expression of pmrA, rcsB or both by analyzing the transcription of these genes in the dam mutant and the parental strain of S. Enteritidis grown to exponential phase in LB medium. By real-time quantitative PCR, the relative expression of both pmrA and rcsB genes in SEΔdam is reduced (56% and 59%, respectively) compared with the parental strain (Fig. 3). Complementation of dam mutation with plasmid pIZ833 restored the expression of pmrA, rcsB and wzz genes to wild type levels (Fig. 3). Thus, a functional Dam results in upregulation of the expression of pmrA and rcsB genes in S. Enteritidis. To analyze whether the reduction in the amount of pmrA and rcsB mRNA observed in the absence of Dam correlated with the amount of proteins, we quantified PmrA and RcsB in SEΔdam mutant. Because murine anti PmrA or anti RcsB antibodies are not commercially available, we constructed SEΔdam mutants harboring either pmrA::3×Flag or rcsB::3×Flag transcriptional fusions in the chromosome. Total bacterial proteins were extracted and the relative amount of PmrA and RcsB was determined by Western blot developed with anti-FLAG antibodies (Fig. 4). Densitometry analysis showed that the amount of PmrA produced by the dam mutant (as well as the complemented strains) was similar to that produced by the wild type strain (Fig. 4A). On the other hand the relative amount of the RcsB produced by the dam mutant was significantly reduced to 63% compared with that of the parental strain (Fig. 4 B).
Total mRNA was harvested from cultures of SEΔdam, S. Enteritidis #5694 (wild type) and complemented strains. The relative mRNA amount was determined by reverse transcription real-time quantitative PCR and related to mRNA levels in wild type strain, set as 1. Values are means ± SD of five independent mRNA extractions performed in triplicates. Plasmid pIZ833 bears the dam gen. * significant difference p<0.01 with respect to wild type strain.
Western blot analysis of total proteins from S. Enteritidis #5694 wild type strain and dam mutant strains harboring an rcsB::3×FLAG (A) or pmrA::3×FLAG (B) transcriptional fusion in the chromosome grown in LB medium and harvested at an OD600 of 0.6. Protein loading was normalized to 106 CFU. Blots were probed with anti-FLAG antibodies. Band intensity was determined by densitometry; relative intensities are presented in arbitrary units (a.u.). Panel A. wt: wild type strain #5694 SErcsB::3×FLAG; dam: dam mutant strain SErcsB::3×FLAG. Panel B. wt: wild type strain #5694 SEpmrA::3×FLAG; dam: dam mutant strain SEpmrA::3×FLAG. Plasmid pIZ833 bears the dam gene. * Significant difference p<0.05. Data are expressed as means ± SD of percent change in band intensity relative to wild type of five independent experiments performed in duplicates.
RcsB induces the expression of wzz and pmrA, whereas PmrA represses the expression of wzz and rcsB
Next we analyzed to what extent the expression of wzz was reduced in the absence of its two regulators in S. Enteritidis. To do that, real-time quantitative PCR was performed using mRNA obtained from knockout rcsB and pmrA mutants and from wild type strains grown in LB medium. As shown in Fig. 5A, the expression of wzz was reduced to 29% in SEΔrcsB mutant compared with the wild type (strain #5694). In contrast, we observed 50% increased expression of wzz in SEΔpmrA with respect to the wild type (strain #5694) (Fig. 5A). These features would not be exclusive to wild type strain #5694, since similar results were found using pmrA and rcsB mutants constructed from clinical isolates of S. Enteritidis (data not shown).
Total mRNA was harvested from cultures of SEΔrcsB, SEΔpmrA and S. Enteritidis wild type #5694 (wild type) grown in LB medium (A,B,C) or grown in low Mg2+, low Mg2++Fe3+ and high Mg2+ (D). The relative amount of wzz mRNA was determined by reverse transcription real-time quantitative PCR and related to mRNA levels in wild type strain #5694 (A,B,C) or in wild type strain #5694 grown in low Mg2+ (D), set as 1. Values are means ± SD of five independent mRNA extractions performed in triplicates. * significant difference p<0.01 with respect to wild type strain #5694 grown in the same media; § significant difference p<0.01 with respect to the same strain grown in pmrA-inducing conditions (low Mg2+ and low Mg2++Fe3+ ); ¥ significant difference p<0.05 with respect to the same strain grown in pmrA-inducing conditions (low Mg2+ and low Mg2++Fe3+).
These findings prompted us to investigate whether an interaction exists between both wzz regulators. Therefore, we determined the expression of rcsB in the absence of pmrA (SEΔpmrA) and the expression of pmrA in the absence of rcsB (SEΔrcsB). As shown in Fig. 5B, the expression of rcsB in the mutant lacking pmrA was increased by 24% with respect to the parental strain cultured in LB medium. In contrast, deficiency in rcsB diminished the expression of pmrA to 30% compared with the wild type strain grown in the same medium (Fig. 5C). The expression of rcsB and pmrA was restored in complemented strains (data not shown). To further investigate these interactions, we analyzed wzz expression in the wild type, rcsB and pmrA mutants grown in conditions that stimulate or repress pmrA. As shown in Fig. 5D, similar patterns in the expression of wzz were found between bacteria cultured under conditions known to activate (low Mg2+; low Mg2++Fe3+) or repress (high Mg2+) pmrA. We found that regardless the culture media utilized, wzz expression was reduced in SEΔrcsB mutant and increased in SEΔpmrA mutant compared with the parental strain. Interestingly, when the wild type strain was cultured under conditions that repress pmrA (high Mg+2), the expression of wzz was 3 or 2 fold higher compared with the wild type grown in low Mg+2 or low Mg+2+Fe3+, respectively. This increase was even higher in the absence of the pmrA gene for any culture medium tested (Fig. 5D). Additional experiments revealed that concurring with the augmented expression of wzz (Fig. 5D), the wild type strain increased the expression of rcsB and reduced the expression of pmrA in high Mg+2 compared with low Mg+2 (data not shown). These results confirm that wzz expression is induced by RcsB and repressed by PmrA. In all cases, the expression of wzz was restored in complemented strains (data not shown).
Is there a third regulator of wzz in S. Enteritidis?
Results presented in Fig. 5D also show that the expression of wzz is induced in the absence of rcsB by high Mg+2 (pmrA repressive condition). This finding is interesting since it suggests the existence of another wzz regulator; therefore, we decided to investigate the expression of wzz in a double mutant of S. Enteritidis lacking pmrA and rcsB genes (SEΔrcsBΔpmrA strain). As shown in Fig. 6A and B, this double mutant was able to express wzz mRNA. We found that regardless the culture condition used the expression of wzz was decreased significantly in the double mutant compared with the parental strain. Nevertheless, it is worth noting that for the double mutant the expression of wzz was 2.5 fold higher in high Mg2+ than in low Mg2+ (Fig. 6B). Moreover, LPS analysis showed that -concomitantly with the expression of wzz- the double mutant was capable to synthesize O-antigen (Fig. 6C, lane 3). Note that in the absence of Wzz, S. Enteritidis (SEΔwzz mutant) is unable to generate O-antigen (Fig. 6B, lane 2). Altogether, our results indicate that, in addition to PmrA and RcsB, another regulator(s) of wzz exists in S. Enteritidis.
A,B. Total mRNA was harvested from cultures of SEΔrcsBΔpmrA double mutant and S. Enteritidis wild type #5694 grown in LB media (A) or grown in low Mg2+, low Mg2++Fe3+ and high Mg2+ (B). The relative mRNA amount was determined by reverse transcription real-time quantitative PCR and related to mRNA levels in wild type strain (A) or in wild type strain grown in low Mg2+ (B), set as 1. Values are means ± SD of five independent mRNA extractions performed in triplicates. * significant difference p<0.01 with respect to wild type strain grown in the same media; † significant difference p<0.05 with respect to same strain grown in pmrA-inducing conditions (low Mg2+ and low Mg2++Fe3+). C. Equal amount of LPS was loaded in each lane and analyzed by Tricine/SDS-PAGE on a 14% (w/v) acrylamide gel followed by silver staining. The concentration of LPS was determined by measuring KDO using the purpald assay.
We have reported earlier that the absence of Dam in S. Enteritidis causes a defect in the O polysaccharide chain length distribution associated to reduced wzz gene expression. Here we investigated whether Dam regulates wzz gene expression through its two known regulators, PmrA and RcsB. We found that Dam regulates the expression of both rcsB and pmrA genes; nevertheless, the dam LPS phenotype of S. Enteritidis is only associated with RcsB. The fact that SEΔdam mutant exhibits reduced levels of rcsB mRNA and a diminished amount of RcsB indicates that the expression of rcsB gene is controlled (directly or indirectly) by Dam methylation. The lack (SEΔrcsB mutant) or even a diminished amount (SEΔdam strain) of RcsB resulted in an increased amount of shorter polysaccharide chains similar to the dam LPS phenotype. Furthermore, we found that overproduction of RcsB in SEΔdam mutant restores the O-antigen LPS pattern back to that of S. Enteritidis wild type. The involvement of RcsB in the regulation of polymerization was reported earlier in S. Typhimurium . It was shown that the lack of RcsB affects the mobility in those bands containing 6–10 and 16–22 O-antigen subunits. Unlike serovar Enteritidis, no increase in the amount of shorter polysaccharides was reported for the rcsB mutant of S. Typhimurium. These subtle differences in the regulation of the O-antigen chain length between two serovars of Salmonella enterica would allow them to colonize specific ecological and immunological niches .
In S. Typhimurium, PmrA not only stimulates wzz expression, regulating the O-antigen chain length, but also participates in core and lipid A modifications , , , , , , . Therefore, it would be reasonable to expect a direct participation of PmrA in the O polysaccharide chain length phenotype of S. Enteritidis; we found, however, that the absence of PmrA does not cause alterations in the LPS pattern. This is in agreement with the fact that overproduction of PmrA in the dam mutant does not restore the defective LPS pattern. On the other hand, our data indicate that Dam methylation (directly or indirectly) does modulate pmrA expression. Indeed, pmrA mRNA was reduced in the dam mutant. This finding is in agreement with microarray analysis data reported by Balbontin et al. in S. Typhimurium dam mutants . Interestingly, despite the diminished amount of pmrA mRNA found in the dam mutant, PmrA levels remained unchanged. Discrepancies between mRNA transcription and protein translation have been reported earlier , , . In this regard, different mechanisms related to mRNA stability have been proposed to play a critical role in this phenomenon. Therefore, we conclude that, in S. Enteritidis, a functional Dam is required for adequate levels of pmrA and rcsB gene expression. Also, the diminished amount of RcsB in SEΔdam strain could explain the reduced wzz gene expression found earlier in this mutant . We also analyzed the individual participation of PmrA and RcsB in the expression of wzz gene in S. Enteritidis. As expected, we found that the relative amount of wzz is reduced in rcsB mutant, indicating that RcsB induces wzz gene expression. Surprisingly, in pmrA deletion mutant the amount of wzz mRNA was higher than in the wild type, indicating that, unlike RcsB, PmrA represses wzz gene expression. This finding could explain the normal LPS phenotype of SEΔpmrA (this mutant would not lack Wzz protein).
In order to investigate a putative regulatory effect between both wzz regulators, we determined the expression of rcsB in a pmrA mutant, and pmrA expression in an rcsB mutant. We found that both regulators affect each other expression. The relative expression of pmrA mRNA decreases in the absence of rcsB, whereas in the absence of pmrA, the relative amount of rcsB mRNA increases. These results would indicate that, under the growth conditions used, RcsB stimulates pmrA whereas PmrA represses rcsB. Also, these findings could explain the elevated expression of wzz found in the pmrA mutant; in the absence of PmrA, RcsB is derepressed and therefore wzz is induced. Regulatory interactions between two-component regulatory systems, coordinating responses to diverse stimuli, have been described. The mechanisms involved in these regulations include phosphatases interrupting phosphoryl transfer in phosphorelays and transcriptional and post-transcriptional modifications , , , , . Then, it is possible that an interaction between both PmrA/PmrB and RcsC/RcsD/RcsB two-component regulatory systems would exist in S. Enteritidis. In favor of a direct RcsB-mediated regulation of pmrA, alignment analysis revealed a potential RcsB protein binding site in pmrA gene of S. Enteritidis (see Fig. S1 for the bioinformatics analysis performed). Similar results were obtained when the alignment analysis was performed between the conserved regulatory sequences of PmrA binding sites and a putative PmrA binding motif found in rcsB gene (supplemental data). Altogether these results would indicate a direct regulation of PmrA protein on rcsB gene and RcsB protein on pmrA gene. The balance between the expression and repression of pmrA and rcsB in response to environmental signals suggests a fine tuning of selective genes required for the adaptation to a specific niche.
The experiments performed using double mutant rcsB pmrA of S. Enteritidis indicate that wzz gene is expressed even in the absence of both regulators. Early studies on serovar Typhimurium showed that in the absence of rcsB and pmrA genes (both wzz inducers), the activity of the wzz promoter is barely detected and consequently the O-antigen is not synthesized. In fact, the LPS phenotype of rcsB pmrA double mutant of S. Typhimurium closely resembles that of a wzz mutant . On the contrary, our experiments demonstrate that the LPS pattern of S. Enteritidis lacking both rcsB and pmrA genes (wzz inducer and repressor, respectively) does conserve O-antigen. These results indicate that, in S. Enteritidis, full expression of wzz would not depend exclusively on PmrA and RcsB. Although the wzz mRNA amount found in rcsB pmrA double mutant could be related to a basal expression of wzz (but still enough to allow the synthesis of O-antigen), the induction of wzz by high Mg2+ observed in rcsB mutant as well as in rcsB pmrA double mutant strongly support the possibility of a third gene regulating wzz expression in S. Enteritidis.
In summary, we showed that in S. Enteritidis Dam methylation regulates wzz expression through rcsB and pmrA genes; whereas RcsB induces wzz gene expression PmrA represses it. We also present evidence that rcsB and pmrA genes regulate each other; RcsB stimulates the expression of pmrA and PmrA represses rcsB gene expression. Finally, our results support the existence of a third gene regulating wzz expression in S. Enteritidis, that can be induced when bacteria is grown in high Mg2+. The regulatory network of wzz gene expression proposed, including the involvement of the hypothetical third wzz regulator, is shown in Fig. 7. Thereby, results presented here would be an example of differential regulation of orthologous genes expression providing differences in phenotypic traits between closely related bacterial serovars.
The regulatory cascade for wzz gene expression involves Dam methylation, PmrB/PmrA and RcsC/RcsD/RcsB two-component regulatory system and a putative third regulator (X). Proteins are indicated by ovals, whereas genes are symbolized by block arrows. Black dots indicate methylation sites (5′-GATC-3′ sequences). Dashed lines indicate direct interactions demonstrated in S. Typhimurium. Positive regulation (induction) is labeled with ↑ and (+), whereas negative regulation (repression) is labeled with ⊥ and (−). The question mark indicates a putative regulation.
Bioinformatics analysis. A. Conserved sequence of PmrA-binding motif. The conserved nucleotides of the sequences corresponding to PmrA binding motif are boxed. B. Molecular analysis of rcsB gene region. Diagram of the DNA sequence corresponding to rcsB region based on Refseq NC_011294 sequence of S. enterica serovar Enteritidis. Alignment analysis performed between the conserved regulatory sequences of PmrA motif and the potential PmrA protein binding site sequences found in rcsB gen region are depicted in the correspondent localization. The two know rcsB promoters PrcsB (located within rcsD coding region) and PrcsDB (located at −32 pb upstream of the rcsD ORF) are marked with arrows. C. Alignment analysis of one of the potential RscB-binding motifs found in pmrA gene region with the reported RcsB-dependent regulatory sequences of different enterobacteria. Homologous sequences of the potential RcsB-binding site found in comparison with the reported RcsB motif are in bold. D. Molecular analysis of pmrA gene region. Diagram of the DNA sequence corresponding to pmrA region based on Refseq NC_011294 sequence of S. enterica serovar Enteritidis. Potential RcsB protein binding site sequences found in pmrA gen are depicted in the correspondent localization. Next to each potential sequence is indicated the orientation (direct, + or complementary, −), the position relative to the ATG sequence of the gene and the amount of mismatches found in the alignment (mm).
Conceived and designed the experiments: SHS MRAC MAV MCC. Performed the experiments: SHS MRAC. Analyzed the data: SHS MRAC MNLL MNG MAV MCC. Contributed reagents/materials/analysis tools: SHS MRAC MNLL MNG MCC. Wrote the paper: SHS MAV MCC.
- 1. Raetz CR, Whitfield C (2002) Lipopolysaccharide endotoxins. Annu Rev Biochem 71: 635–700.
- 2. Batchelor RA, Haraguchi GE, Hull RA, Hull SI (1991) Regulation by a novel protein of the bimodal distribution of lipopolysaccharide in the outer membrane of Escherichia coli. J Bacteriol 173: 5699–704.
- 3. Liu D, Cole RA, Reeves PR (1996) An O-antigen processing function for Wzx (RfbX): a promising candidate for O-unit flippase. J Bacteriol 178: 2102–2107.
- 4. Marino PA, McGrath BC, Osborn MJ (1991) Energy dependence of O-antigen synthesis in Salmonella typhimurium. J Bacteriol 173: 3128–3133.
- 5. McGrath BC, Osborn MJ (1991) Localization of the terminal steps of O-antigen synthesis in Salmonella typhimurium. J Bacteriol 173: 649–654.
- 6. Mulford CA, Osborn MJ (1983) An intermediate step in translocation of lipopolysaccharide to the outer membrane of Salmonella typhimurium. Proc Natl Acad Sci USA 80: 1159–1163.
- 7. Rick PD, Barr K, Sankaran K, Kajimura J, Rush JS, et al. (2003) Evidence that the wzxE gene of Escherichia coli K-12 encodes a protein involved in the transbilayer movement of a trisaccharidelipid intermediate in the assembly of enterobacterial common antigen. J Biol Chem 278: 16534–16542.
- 8. Valvano MA (2003) Export of O-specific lipopolysaccharide. Front Biosci 8: s452–471.
- 9. Nixon BT, Ronson CW, Ausbel FM (1986) Two-component regulatory systems responsive to environmental stimuli share strong conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC. Proc Natl Acad Sci USA 83: 7850–7854.
- 10. Ronson CW, Nixon BT, Ausbel FM (1987) Conserved domains in bacterial regulatory proteins that respond to environmental stimuli. Cell 49: 579–581.
- 11. Kofoid EC, Parkinson JS (1988) Transmitter and receiver modules in bacterial signalling proteins. Proc Natl Acad Sci USA 85: 4981–4985.
- 12. Msadek T, Kunst F, Rapoport G (1993) Two-component regulatory systems. In: Bacillus subtilis and other gram-positive bacteria; biochemistry, physiology, and molecular genetics. Hoch, J.A., Sonenshein, A.L., and Losick, R. (eds). American Society for Microbiology Washington, D.C., pp. 729–745
- 13. Alex LA, Simon MI (1994) Protein histidine kinases and signal transduction in prokaryotes and eukaryotes. Trends Genet 10: 133–138.
- 14. Swanson RV, Alex LA, Simon MI (1994) Histidine and Aspartate phosphorylation: two-component systems and the limits of homology. Trends Biochem Sci 19: 485–490.
- 15. Parkinson JS (1995) Genetic approaches for signaling pathways and proteins. In: Hoch JA, Sihavy TS, editors. Two-component transduction. Washington, DC: ASM Press. pp. 9–23.
- 16. Delgado MA, Mouslim C, Groisman EA (2006) The PmrA/PmrB and RcsC/YojN/RcsB systems control expression of the Salmonella O-antigen chain length determinant. Mol Microbiol 60: 39–50.
- 17. Kato A, Groisman EA (2004) Connecting two-component regulatory systems by a protein that protects a response regulator from dephosphorylation by its cognate sensor. Gene Dev 18: 2302–2313.
- 18. Kox LF, Wösten MM, Groisman EA (2000) A small protein that mediates the activation of a two-component system by another two-component system. EMBO J 19: 1861–1872.
- 19. Murata T, Tseng W, Guina T, Miller SI, Nikaido H (2007) PhoPQmediated regulation produces a more robust permeability barrier in the outer membrane of Salmonella enterica serovar Typhimurium. J Bacteriol 189: 7213–7222.
- 20. Grossman N, Schmetz MA, Foulds J, Klima EN, Jiminez V, et al. (1987) Lipopolysaccharide size and distribution determine serum resistance in Salmonella montevideo. J Bacteriol 169: 856–863.
- 21. Hoare A, Bittner M, Carter J, Alvarez S, Zaldivar M, et al. (2006) The outer core lipopolysaccharide of Salmonella enterica serovar Typhi is required for bacterial entry into epithelial cells. Infect Immun 74: 1555–1564.
- 22. Murray GL, Attridge SR, Morona R (2003) Regulation of Salmonella Typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz. Mol Microbiol 47: 1395–1406.
- 23. Murray GL, Attridge SR, Morona R (2005) Inducible serum resistance in Salmonella Typhimurium is dependent on wzz(fepE)-regulated very long O antigen chains. Microbes Infect 7: 1296–1304.
- 24. Murray GL, Attridge SR, Morona R (2006) Altering the length of the lipopolysaccharide O antigen has an impact on the interaction of Salmonella enterica serovar Typhimurium with macrophages and complement. J Bacteriol 188: 2735–2739.
- 25. Phalipon A, Costachel C, Grandjean C, Thuizat A, Guerreiro C, et al. (2006) Characterization of functional oligosaccharide mimics of the Shigella flexneri serotype 2a O-antigen: implications for the development of a chemically defined glycoconjugate vaccine. J Immunol 176: 1686–1694.
- 26. McGowan CC, Necheva A, Thompson SA, Cover TL, Blaser MJ (1998) Acid-induced expression of an LPS-associated gene in Helicobacter pylori. Mol Microbiol 30: 19–31.
- 27. Løbner-Olesen A, Skovgaard O, Marinus MG (2005) Dam methylation: coordinating cellular processes. Curr Opin Microbiol 8: 154–160.
- 28. Low DA, Weyand NJ, Mahan MJ (2001) Roles of DNA adenine methylation in regulating gene expression and virulence. Infect Immun 69: 7197–7204.
- 29. Marinus MG (1996) Methylation of DNA. In: Neidhardt FC, Curtiss III R, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger ME, editors. Escherichia coli and Salmonella: cellular and molecular biology. Washington, DC: ASM Press. pp. 782–791.
- 30. Wion D, Casadesús J (2006) N6-methyl-adenine: an epigenetic signal for DNA-protein interactions. Nat Rev Microbiol 4: 183–192.
- 31. Balbontín R, Rowley G, Pucciarelli MG, López-Garrido J, Wormstone Y, et al. (2006) DNA adenine methylation regulates virulence gene expression in Salmonella enterica serovar Typhimurium. J Bacteriol 188: 8160–8168.
- 32. Campellone KG, Roe AJ, Løbner-Olesen A, Murphy KC, Magoun L, et al. (2007) Increased adherence and actin pedestal formation by dam-deficient enterohaemorrhagic Escherichia coli O157:H7. Mol Microbiol 63: 1468–1481.
- 33. Chessa D, Winter MG, Nuccio SP, Tükel C, Bäumler AJ (2008) RosE represses Std fimbrial expression in Salmonella enterica serotype Typhimurium. Mol Microbiol 68: 573–587.
- 34. Fälker S, Schilling J, Schmidt MA, Heusipp G (2007) Overproduction of DNA adenine methyltransferase alters motility, invasion, and the lipopolysaccharide O-antigen composition of Yersinia enterocolitica. Infect Immun 75: 4990–4997.
- 35. Heithoff DM, Sinsheimer RL, Low DA, Mahan MJ (1999) An essential role for DNA adenine methylation in bacterial virulence. Science 284: 967–970.
- 36. Jakomin M, Chessa D, Bäumler AJ, Casadesús J (2008) Regulation of the Salmonella enterica std fimbrial operon by DNA adenine methylation, SeqA, and HdfR. J Bacteriol 190: 7406–7413.
- 37. Dueger EL, House JK, Heithoff DM, Mahan MJ (2003) Salmonella DNA adenine methylase mutants elicit early and late onset protective immune responses in calves. Vaccine 21: 3249–3258.
- 38. Dueger EL, House JK, Heithoff DM, Mahan MJ (2003) Salmonella DNA adenine methylase mutants prevent colonization of newly hatched chickens by homologous and heterologous serovars. Int J Food Microbiol 80: 153–159.
- 39. Sarnacki SH, Marolda CL, Noto Llana M, Giacomodonato MN, Valvano MA, et al. (2009) Dam methylation controls O-antigen chain length in Salmonella enterica serovar enteritidis by regulating the expression of Wzz protein. J Bacteriol 191: 6694–700.
- 40. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97: 6640–6645.
- 41. Uzzau S, Figueroa-Bossi N, Rubino S, Bossi L (2001) Epitope tagging of chromosomal genes in Salmonella. Proc Natl Acad Sci U S A 98: 15264–15269.
- 42. Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16: 6127–6145.
- 43. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
- 44. Snavely MD, Gravina SA, Cheung TT, Miller CG, Maguire ME (1991) Magnesium transport in Salmonella typhimurium. Regulation of mgtA and mgtB expression. J Biol Chem 266: 824–829.
- 45. Kawasaki K, China K, Nishijima M (2007) Release of the Lipopolysaccharide Deacylase PagL from Latency Compensates for a Lack of Lipopolysaccharide Aminoarabinose Modification-Dependent Resistance to the Antimicrobial Peptide Polymyxin B in Salmonella enterica. J bacteriol 189: 4911–4919.
- 46. Cohen SN, Chang AC, Hsu L (1972) Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci USA 69: 2110–2114.
- 47. Marolda CL, Welsh J, Dafoe L, Valvano MA (1990) Genetic analysis of the O7-polysaccharide biosynthesis region from the Escherichia coli O7:K1 strain VW187. J Bacteriol 172: 3590–3599.
- 48. Hitchcock PJ, Brown TM (1983) Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol 154: 269–277.
- 49. Lesse AJ, Campagnari AA, Bittner WE, Apicella MA (1990) Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Immunol Methods 126: 109–117.
- 50. Schagger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166: 368–379.
- 51. Osborn MJ (1963) Studies in the Gram-negative cell wall. I. Evidence for the role of 2-keto-3-deoxyoctonoate in the lipopolysaccharide of Salmonella typhimurium. Proc. Natl Acad Sci USA 50: 499–506.
- 52. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25: 402–408.
- 53. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
- 54. Wales AD, Davies RH (2011) A critical review of Salmonella Typhimurium infection in laying hens. Avian Pathol 40: 429–436.
- 55. Gunn JS, Lim KB, Krueger J, Kim K, Guo L, et al. (1998) PmrA-PmrB-regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance. Mol Microbiol 27: 1171–1182.
- 56. Trent MS, Ribeiro AA, Lin SH, Cotter RJ, Raetz CRH (2001) An inner membrane enzyme in Salmonella and Escherichia coli that transfers 4-amino-4-deoxy-Larabinose to lipid A – induction in polymyxin-resistant mutants and role of a novel lipid-linked donor. J Biol Chem 276: 43122–43131.
- 57. Zhou ZM, Ribeiro AA, Lin SH, Cotter RJ, Miller SI, et al. (2001) Lipid A modifications in polymyxin-resistant Salmonella typhimurium: PmrAdependent 4-amino-4-deoxy-L-arabinose, and phosphoethanolamine incorporation. J Biol Chem 276: 43111–43121.
- 58. Breazeale SD, Ribeiro AA, Raetz CRH (2003) Origin of lipid A species modified with 4-amino-4-deoxy-Larabinose in polymyxin-resistant mutants of Escherichia coli: an aminotransferase (ArnB) that generates UDP-4-amino-4-deoxy-L-arabinose. J Biol Chem 278: 24731–24739.
- 59. Lee H, Hsu FF, Turk J, Groisman EA (2004) The PmrA-regulated pmrC gene mediates phosphoethanolamine modification of lipid A and polymyxin resistance in Salmonella enterica. J Bacteriol 186: 4124–4133.
- 60. Nishino K, Hsu FF, Turk J, Cromie MJ, Wosten MM, et al. (2006) Identification of the lipopolysaccharide modifications controlled by the Salmonella PmrA/PmrB system mediating resistance to Fe(III) and Al(III). Mol Microbiol 61: 645–654.
- 61. Lin HH, Lin CH, Hwang SM, Tseng CP (2012) High growth rate downregulates fumA mRNA transcription but is dramatically compensated by its mRNA stability in Escherichia coli. Curr Microbiol 64: 412–417.
- 62. Brockmann R, Beyer A, Heinisch JJ, Wilhelm T (2007) Posttranscriptional expression regulation: what determines translation rates? PLoS Comput Biol 3: e57.
- 63. Mittal N, Roy N, Babu MM, Janga SC (2009) Dissecting the expression dynamics of RNA-binding proteins in posttranscriptional regulatory networks. Proc Natl Acad Sci USA 106: 20300–20305.
- 64. Bijlsma JJ, Groisman EA (2003) Making informed decisions: regulatory interactions between two-component systems. Trends Microbiol 11: 359–366.
- 65. Eguchi Y, Utsumi R (2005) A novel mechanism for connecting bacterial two-component signal-transduction systems. Trends Biochem Sci 30: 70–72.
- 66. Kato A, Groisman EA (2008) Howard Hughes Medical Institute. The PhoQ/PhoP regulatory network of Salmonella enterica. Adv Exp Med Biol 631: 7–21.
- 67. Gunn JS (2008) The Salmonella PmrAB regulon: lipopolysaccharide modifications, antimicrobial peptide resistance and more. Trends Microbiol 16: 284–290.
- 68. Goulian M (2010) Two-component signaling circuit structure and properties. Curr Opin Microbiol 13: 184–189.
- 69. Torreblanca J, Marques S, Casadesus J (1999) Synthesis of FinP RNA by plasmids F and pSLT is regulated by DNA adenine methylation. Genetics 152: 31–45.