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Traci L. Kinkel,1,2,
Stephanie J. Day,1 and
Kevin S. McIver1,2*
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9048,1 Department of Cell Biology and Molecular Genetics and Maryland Pathogen Research Institute, University of Maryland, College Park, Maryland 20742-44512
Received 30 June 2007/ Accepted 18 September 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In gram-positive organisms, regulation of carbon catabolism centers on a component of the phosphoenolpyruvate phosphotransferase system (PTS) (4). The primary purpose of the PTS is to regulate sugar uptake through phosphorylation, which is achieved by shuttling a phosphate from phosphoenolpyruvate to a cytosolic enzyme called EI, to HPr (heat-stable protein), and then to a sugar-specific membrane-bound enzyme, EII, before being attached to an incoming sugar (18). Unlike gram-negative organisms, which use cyclic AMP levels to detect the energy level of the cell, gram-positive bacteria use HPr (6). Therefore, the PTS is multifunctional in gram-positive bacteria, being involved in sugar transport as well as signal transduction in response to sugar availability.
During sugar transport, HPr is phosphorylated on a conserved histidine residue (H15). However, in the presence of glucose, an enzyme called HPr kinase phosphorylates HPr on a serine residue (S46) in response to products of glycolysis (7). This phosphorylation event allows HPr-Ser to bind to the catabolite control protein (CcpA), which belongs to the LacI/GalR family of transcription factors (5). CcpA then is capable of binding with specificity to a catabolite-responsive element (cre) located in promoters or coding sequences of genes with products that are involved in the metabolism of alternative sugars. Although CcpA primarily acts to repress expression of these operons, it also is known to activate transcription of genes important for growth in glucose (11, 14).
There is accumulating evidence that CCR is important for virulence in several low-G+C gram-positive pathogens. For instance, CCR has been linked to virulence in Clostridium perfringens (42), Staphylococcus aureus (29), and Listeria monocytogenes (26). In Streptococcus pneumoniae, the inactivation of CcpA, also called RegM, significantly attenuated virulence in various mouse models of pneumococcal colonization and infection (13, 16). CcpA (RegM) may be affecting systemic infection through the control of capsular gene expression (13), whereas its influence on mucosal colonization and pneumoniae could reflect alterations in basic cellular metabolism (16).
The group A streptococcus (GAS; Streptococcus pyogenes) is an important gram-positive pathogen capable of eliciting a wide array of diseases in humans. Surface M protein is a major virulence determinant that is capable of such functions as inhibiting phagocytosis, binding fibrinogen, and facilitating host cell invasion. Previous studies on the GAS have shown that M protein production is affected by the sugar source (33), suggesting that CCR plays a role in its expression. Transcription of the gene encoding M protein (emm) is activated by the stand-alone response regulator Mga, which also is responsible for transcriptional activation of other virulence genes involved in adhesion, invasion, and immune evasion. Expression of mga is autoregulated, and its promoter (Pmga) has two Mga-binding sites (MBSs) as well as two transcriptional start sites, named P1 (distal) and P2 (proximal) based on their proximity to the translational start site of mga (25, 30). Mga is known to be responsive to environmental signals such as growth phase, and the entire promoter, including 84 bp of sequence upstream of P1, has been reported to be necessary for full activity (3, 21, 23, 30, 34). Given this information, it was hypothesized that CCR could be involved in the control of M protein expression through the regulation of mga. In the present study, the GAS genome was scanned for cre based on a published Bacillus subtilis consensus. A functional cre involved in activation at the P1 start of transcription was identified within Pmga, suggesting a direct link between carbon metabolism and Mga regulation in S. pyogenes.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Construction of pKSM711 and purification of GAS His-CcpA. An amino-terminal fusion of 6x His to CcpA from M6 GAS was constructed as follows. A 1,019-bp region containing the entire ccpA gene was PCR amplified from serotype M6 GA19681 (Table 1) genomic DNA (gDNA) using the primer pair M6ccpA_NcoI-L and M6ccpA_XhoI-R (Table 2). The resulting product was digested with NcoI/XhoI and ligated into NcoI/XhoI-digested pProEX-HTb to produce pKSM711 (Table 1). Following verification by PCR and DNA sequence analysis, pKSM711 was transformed into BL21[DE3] Gold (Stratagene) for protein expression.
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Electrophoretic mobility shift assay (EMSA).
Double-stranded DNA probes were generated by annealing 30-bp sense and antisense oligonucleotide pairs representing PmgaCRE, PccpACRE, a mutated PmgaCRE, and randomly rearranged PmgaCRE or PccpACRE, termed scrambled (see Fig. 2A). Briefly, gel-purified oligonucleotide pairs were annealed by being heated to 85°C for 5 min in 12.5 µg of each pair in 10 mM Tris-HCl, pH 8.0, 5 mM MgCl2 and slowly being cooled to room temperature for 30 min. Annealed oligonucleotides were end labeled with [
-32P]ATP using T4 polynucleotide kinase (New England Biolabs), and the resulting radiolabeled probes were separated on a 5% polyacrylamide gel and extracted by crush-and-soak elution.
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Construction of the Mga– M6 GAS strain RTG229.150Lg. A chloramphenicol-resistant GAS suicide plasmid for inactivation of mga was constructed by ligation of a 1.5-kb cat194 fragment of HincII-digested pLZ12 (31) into XmnI/BsaI-digested pBluescript II KS– to form pBlue-cat194 (Table 1). A 454-bp internal fragment of mga was PCR amplified from M6 JRS4 gDNA using OYR-4/OYL-13 (Table 2) and was ligated into EcoRV-digested pBlue-cat194 to form pKSM150Lg (Table 1). This plasmid was introduced into RTG229 by electroporation, and chloramphenicol-resistant insertion mutants (RTG229.150Lg) were selected and verified by PCR.
Chromosomal GusA-based transcriptional reporters at the VIT locus. A 706-bp fragment of M6 Pmga containing sequence downstream of the putative cre (see Fig. 3) was PCR amplified from pPmga-blue using the 1201/1211 primer pair (Table 2), digested with SspI/EcoRI, and ligated into the HpaI/EcoRI-digested pPmga-gusA to form pKSM435 (Table 1). A 520-bp fragment of M6 Pmga (see Fig. 3) containing sequence downstream of P1 was amplified from pPmga-blue (41) using the 1201/OYR-25 primer pair (Table 2), digested with EcoRI, and ligated into the HpaI/EcoRI-digested pPmga-gusA (41) to form pKSM427 (Table 1). A 371-bp fragment of M6 Pmga containing sequences downstream of MBS I (see Fig. 3) was PCR amplified from pPmga-blue using the 1201/OYR-1 primer pair (Table 2), digested with EcoRI, and ligated into the HpaI/EcoRI-digested pPmga-gusA to form pKSM428 (Table 1). A 265-bp fragment of M6 Pmga containing sequence downstream of MBS II (see Fig. 3) was PCR amplified from pPmga-blue using the 1201/MgaL3_Bam primer pair (Table 2), digested with EcoRI, and ligated into the HpaI/EcoRI-digested pPmga-gusA to form pKSM429 (Table 1). A PstI fragment of pPmga-gusA containing the gusA gene was ligated into the PstI-digested pVIT164 to form pKSM540 (Table 1).
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cre-R_Bam/OYL-14 primer pair (Table 2) and was ligated into the HpaI-digested pKSM540 to form pKSM445 (Table 1). A promoterless GusA transcriptional reporter plasmid (see Fig. 5) was constructed by ligation of the 1.9-kb BamHI gusA fragment from pKSM148 (35) into BamHI-digested pVIT164 (12) to form pVIT-gusA (Table 1).
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GusA assays were performed as previously described (10). Briefly, cells were grown to late logarithmic phase, lysed using a FastPrep cell disruptor (Bio101, Inc.), and assayed for GusA activity. Results are reported in GusA units, which are equivalent to the A420 of the lysate divided by the concentration of total lysate protein (in micrograms/microliter).
Construction of Pmga deletions at the native locus.
A 1,196-bp fragment upstream of the putative Pmga cre was amplified from M6 JRS4 gDNA using the primer pair OYR-17/cre-L (Table 2) and was cloned blunt into EcoRV-digested pJRS233 (32) to form pKSM436 (Table 1). A fragment of Pmga downstream of the cre was PCR amplified from JRS4 using the primer pair cre-R_Bam/Pmga_Bam (Table 2), digested with BamHI, and cloned into BamHI-digested pKSM436 to form pKSM437 (Table 1). Similarly, the primer pair OYR-22/Pmga_Bam (Table 2) was used to amplify wild-type Pmga from JRS4, digested with BamHI, and cloned into SmaI/BamHI-digested pKSM436 to form pKSM439 (Table 1). Finally, Pmga P1 was amplified from JRS4 using the primer pair OYR-25/Pmga_Bam (Table 2), digested with BamHI, and cloned into SmaI/BamHI-digested pKSM436 to form pKSM441 (Table 1). The 2.1-kb SmaI-digested 
Km2 from pUC4Km2 (31) was cloned into either SmaI-digested pKSM437 or PstI-digested and -blunted plasmids pKSM439 and pKSM441 to form pKSM438 (cre), pKSM440 (Full), and pKSM442 (P1), respectively (Table 1). Plasmids were introduced into JRS4 by electroporation at 30°C, and plasmid integrants were selected by passage of cells at 37°C with screening for kanamycin resistance and erythromycin sensitivity as described previously (35). Strains constructed from plasmids pKSM438, pKSM440, and pKSM442 were named KSM438 (cre), KSM440 (Full), and KSM442 (P1), respectively (Table 1; also see Fig. 4).
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Real-time RT-PCR.
gDNA was removed from total RNA by use of the MessageClean kit (GenHunter Corp.). Real-time reverse transcription-PCR (RT-PCR) was performed on 25 ng of RNA mixed with 5 pmol of each primer, 6.25 U of MultiScribe RT (Applied Biosystems), and 1x SYBR green PCR master mix (Applied Biosystems). Reaction mixtures were transferred in triplicate into a 96-well optical reaction plate (Applied Biosystems), and the plate was covered with optical adhesive covers (Applied Biosystems). An Applied Biosystems 7500 real-time PCR system was used to detect transcript levels in the absolute quantification mode with reaction conditions of 48°C for 30 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s followed by 60°C for 1 min. Analysis of data was performed using Sequence Detection Software, version 1.3 (Applied Biosystems). The mga RT and mga P1 RT primer pairs (Table 2) were used to detect levels of total mga and mga P1 transcripts, respectively, in relation to gyrA transcript levels (detected with the gyrA RT primer pair; Table 2) in RNA isolated from JRS4 (wild type), KSM440 (Full), KSM438 (cre), and KSM442 (P1). A standard curve using total RNA was used to quantify the levels of each transcript.
Inactivation of ccpA in the GAS. To produce the ccpA mutant strain KSM310.700, a 511-bp internal region of ccpA was amplified from JRS4 gDNA using the primer pair ccpAL/ccpAR (Table 2). The resulting fragment was ligated into EcoRV-digested pJRS233 to form pKSM700 (Table 1) and was verified by PCR using the primer pair ccpAL/ccpAR (Table 2). pKSM700 was transformed into KSM310 at 30°C, and erythromycin-resistant integrants were isolated at 37°C by following the protocol described previously (35). Mutants were verified by PCR using the primer pairs 1201/ccpAR1 and 1211/PccpA-L1 (Table 2). GusA assays were performed as described above.
GAS protein extracts and Western blot analyses.
Whole-cell GAS protein extractions, separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis, were performed as previously described (22). Blots were incubated with a 1:1,000 dilution of 
-Mga-pep2 antiserum (22), incubated with a 1:25,000 dilution of -rat (Santa Cruz Biotechnologies) horseradish peroxidase-conjugated secondary antibody, and visualized using the Western Lightning chemiluminescence system (Perkin Elmer).
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RESULTS |
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The catabolite control protein, CcpA, specifically binds to PccpA and Pmga in vitro. To determine if CcpA interacts with the cre identified in our bioinformatic screen of the GAS M1 genome, EMSAs were performed on PccpA and Pmga. Double-stranded oligonucleotide probes (30 bp) were generated that contained either the PccpA (positive control) or the Pmga cre (14 bp each) centered within the sequence (Fig. 2A). To address the specificity of CcpA binding, probes consisting of a random rearrangement of the respective nucleotides (scrambled PccpA and Pmga) or a probe containing four specific mutations in the Pmga cre (mutated Pmga) were generated (Fig. 2A). Since CcpA is capable of binding DNA in the absence of phosphorylated HPr-Ser in vitro (2), assays were performed using purified GAS His-CcpA alone (see Materials and Methods).
Studies with other gram-positive bacteria predict that CcpA will bind to a cre located within its own promoter (19, 44); therefore, we first tested the ability of purified His-CcpA to bind to the identified GAS PccpA cre probe (Fig. 2A). Increasing amounts of His-CcpA (5.0 to 7.5 µM) resulted in a mobility shift of labeled PccpA, indicating DNA binding (Fig. 2B, lanes 1 to 3). The addition of 700 ng of cold PccpA to the reaction was able to compete for His-CcpA interaction, whereas the addition of the same amount of a cold scrambled PccpA probe had no effect (Fig. 2B, lanes 4 and 5). Thus, GAS His-CcpA is able to bind specifically to the PccpA cre in vitro.
The Pmga cre probe also demonstrated slower migration upon addition of increasing amounts of purified His-CcpA (7.5 to 12.5 µM), indicating protein-DNA interaction (Fig. 2C). The 700 ng of cold Pmga or PccpA cre probe that was added to the reaction was able to compete for binding of His-CcpA to the labeled Pmga probe to various degrees (Fig. 2C, lanes 6 and 8). In contrast, a scrambled Pmga cre probe (9/14 mismatches) was not able to compete for His-CcpA (Fig. 2B, lane 9), suggesting that the interaction with Pmga cre is specific. In support of this conclusion, mutation of only 4/14 nucleotides in the predicted Pmga cre exhibited an intermediate level of competition (Fig. 2B, lane 7). Thus, GAS His-CcpA is capable of binding directly to the predicted cre sequences located upstream of PccpA and Pmga P1 promoters in vitro.
P1 and cre are not essential for Pmga activity in the presence of existing Mga.
To address whether binding of CcpA to the Pmga cre is important for transcriptional regulation of mga, ß-glucuronidase (GusA) transcriptional reporter assays were performed. Various single-copy Pmga-gusA alleles were constructed in the JRS4-derived M6 GAS strain RTG229 (12) genome at an ectopic site (VIT) separate from the native mga locus. Promoter fragments corresponding to the full-length (wild type; KSM310) Pmga, a deletion of the cre only (cre; KSM435), a deletion of the entire P1 with cre (P1; KSM427), a deletion of P1/cre and Mga-binding site 1 (MBS I; KSM428), and a deletion of P1/cre and both Mga-binding sites (MBS I & II; KSM429) were introduced into the VIT locus of both a wild-type and Mga– M6 GAS (Fig. 3A and Table 1). The Mga– strains then were complemented by introduction of a multicopy Pspac-mga plasmid for constitutive expression of mga (22). Strains were grown to late logarithmic phase, and the levels of GusA activity were quantified (see Materials and Methods).
The wild-type Pmga, cre, and P1 strains all showed Mga-regulated GusA activity, which could be restored upon complementation (Fig. 3B). As predicted from previous studies (25), deletion of either MBS I (MBS I) or both MBSs (MBS I & II), in addition to the upstream P1 promoter and cre, eliminated all Mga-dependent regulation of Pmga (Fig. 3B). Thus, deletion of the cre alone or the entire P1/cre had little effect on wild-type activity (Fig. 3B), suggesting that the Mga produced from its native locus was sufficient to activate Pmga at the VIT locus from the P2 promoter alone. Of note, GusA levels appeared to increase slightly upon deletion of P1 (Fig. 3B), indicating that the Pmga P1 region also may contain sequences that repress mga expression.
The Pmga cre is necessary for full transcriptional activity of Pmga at its native locus.
To determine whether the cre is important for Pmga activity in the absence of exogenously produced Mga, Pmga mutations were constructed using the Km2 cassette at the native mga locus in the serotype M6 strain JRS4 chromosome. The Km2 cassette has T4 terminators that flank the antibiotic resistance marker to prevent upstream transcription through the element (31). As a wild-type control, the Km2 cassette was inserted by allelic exchange upstream of the wild-type Pmga promoter (Fig. 4A). To generate Pmga alleles lacking either cre or the entire P1 promoter, the Km2 cassette was inserted either immediately downstream of cre (cre) or downstream of the P1 transcriptional start site (P1), respectively (Fig. 4A) (see Materials and Methods).
Given that transcript levels from Pmga P1 often are quite low (25), quantitative real-time RT-PCR was utilized to assess the effects of promoter deletions on the expression of mga. Total RNA was isolated from the wild-type JRS4, full Pmga, cre, and P1 strains grown to late logarithmic phase. Transcript levels of both Pmga P1 alone (Fig. 4B) and total Pmga (Fig. 4C) were detected using the mga P1 RT and mga RT primer pairs (Table 2), respectively, and results were normalized to transcript levels of the housekeeping gene gyrA.
As expected, relative levels of mga transcribed from Pmga P1 alone and total Pmga were similar between the parent strain, JRS4, and the full Pmga control strain (Fig. 4B and C), indicating that the Km2 cassette inserted upstream of Pmga had little effect on the transcription of mga (Fig. 4B and C). Importantly, deletion of the cre in Pmga caused an approximately twofold reduction in the levels of both Pmga P1 and total Pmga, suggesting that the cre is important in the activation of mga transcription. Unexpectedly, the deletion of the P1 promoter resulted in increased transcript levels from P2 (mga P1-specific transcript levels remained low) (Fig. 4B and C). As seen with the GusA reporter assays described above (Fig. 3B), this result supports previous reports of a repressor region in Pmga near P1 (25).
cre is necessary for activation of Pmga P1.
In order to further assess the effect of the cre on the activity of Pmga P1 alone, a gusA transcriptional fusion was made to Pmga P1 with and without the cre in the VIT locus of wild-type and Mga– M6 GAS to form full-length (cre+) and P1cre (cre mutant) strains, respectively (Fig. 5A). Since the level of GusA activity from the reporter strains was too low to detect, transcript levels were assessed directly using semiquantitative primer extension. Total RNA was extracted from late-logarithmic-phase cells representing the full-length Pmga P1, the P1 cre, and the no-promoter control gusA reporter strains either in the presence or absence of a functional mga. Primer extensions were performed simultaneously for both gusA and the constitutive rpsL for each of the strains.
Promoter-specific products were not observed in the absence of RNA (Fig. 5B, lane 1) or in the no-promoter control strains (Fig. 5B, lanes 6 and 7), with the exception of two light background bands (Fig. 5B). However, a product of the predicted size for Pmga P1 was detected in the cre+ strains (Fig. 5B, lanes 2 and 3), while it was reduced approximately 4.2-fold in the cre mutant strains, as determined by densitometry (Fig. 5B, lanes 4 and 5). This correlates with the cre results from the real-time RT-PCR analysis of Pmga P1 at its native locus (Fig. 4B). As expected from the absence of Mga-binding sites in P1, Mga had no detectable effect on transcription from Pmga P1 (Fig. 5B, lanes 2 to 5) when its transcript levels were normalized to rpsL. Thus, the cre is necessary for transcriptional activation from the Pmga P1 start site.
Inactivation of ccpA affects mga expression. The role of CcpA-mediated activation of Pmga P1 on mga expression in vivo was assessed using an insertion-inactivation mutant of ccpA in the M6 Pmga-gusA VIT reporter strain KSM310 (KSM310.700; Table 2). The ccpA-defective strain did not exhibit any significant growth defects compared to growth of the wild-type KSM310 when grown in rich THY medium (data not shown). Inactivation of ccpA resulted in a greater than threefold reduction in Pmga-specific GusA activity compared to the activity of the parental KSM310 samples grown to late-logarithmic phase in THY medium (Fig. 6A). The resulting GusA activity was slightly higher than background levels observed in the Mga– control strain KSM310.150 (Fig. 6A). In addition, Western analysis of whole-cell extracts found that steady-state levels of Mga also were reduced in the CcpA– mutant at the same point in growth (Fig. 6B). These data indicate that CcpA is necessary for wild-type production of Mga during logarithmic phase, a point in growth when the Mga virulence regulon shows maximal expression (23).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the case of two identified GAS cre located in Pmga and PccpA, specific binding by a purified GAS His-CcpA was demonstrated in vitro (Fig. 2), demonstrating that these are functional CcpA-binding sites. An alignment of a subset of GAS cre found that the single-nucleotide mismatches to the B. subtilis consensus were located in variable positions in the site (Fig. 1A), suggesting that a GAS-derived consensus cre might be somewhat divergent. Furthermore, this would strongly indicate that these nucleotides, although important for B. subtilis, likely fall in positions that are not essential for CcpA-cre interactions in GAS. Regardless, a more comprehensive analysis utilizing CcpA-binding studies and growth in defined sugar sources will be required to determine whether these cre are important for CCR in the GAS.
What is the role of Pmga P1 in transcription of mga? The Pmga region is quite complex and contains two starts of transcription, a strong gene-proximal P2 start site that is autoactivated by Mga and a weaker distal P1 start site that has been thought to be constitutive. Our studies show that CcpA was able to bind specifically to the putative cre located upstream of the P1 –35 region (Fig. 1B and 2) in a position that suggests an activating function based on recent studies on CcpA in L. lactis (44). Furthermore, deletion of the cre at the native mga locus or in the context of Pmga P1 alone leads to decreased mga transcription (Fig. 4 and 5B). Inactivation of ccpA in the serotype M6 JRS4 also shows a reduction in both Pmga activity and Mga levels in the cell (Fig. 6). Taken together, these results indicate that CcpA can activate the Pmga P1 start site through the upstream cre and that this interaction is important for normal expression of mga in the GAS during exponential growth. Previously, Pmga P1 was thought to provide low-level constitutive expression of mga early in growth that, when translated, would amplify its expression from Pmga P2 (30). Our results suggest a modification of this model, whereby CcpA may activate Pmga P1 in response to glucose levels in the cell early in growth and provide the initial trigger that leads to high production of Mga and autoactivation at Pmga P2. Experiments are currently under way to test whether Pmga P1 expression can be influenced by glucose and CCR via its cre.
Earlier Pmga deletion studies indicated that Pmga P1 was essential for the expression of mga (30). However, deletion of the entire Pmga P1 and cre did not prevent Mga-regulated expression from Pmga P2 as long as Mga was produced from its native locus (Fig. 3B). Although these data appear to contradict the published findings, the Pmga P1 deletion constructs used in this study included approximately 20 bp of extra sequence not found in the previous study and used a different reporter gene (30). Therefore, it is possible that this extra sequence is necessary for the normal expression of mga.
Rather than being essential to the activity of mga, Pmga P1 may actually play a role in repression. An increase in Pmga P2 expression is observed upon deletion of the P1 promoter at both its native locus (Fig. 4C) and at an ectopic locus (Fig. 3), suggesting that sequences in this region actually function to repress transcription initiated from Pmga P2. A repressor has been suggested to function at the MBS (MBS I) in Pmga (25); however, no such regulator has been identified to date. One possible model is that Pmga P1 repression is important for fine-tuning mga expression later in growth. However, this repression would be neutralized early on in the presence of CcpA and Pmga P1 cre (Fig. 4B). In theory, Pmga P1 functions to activate mga expression levels in conjunction with CCR and suppresses its activity later in growth in the absence of a preferred energy source. Overall, the regulation of mga requires a complex interplay between P1, P2, Mga, and other unknown regulators.
CcpA and Mga virulence regulation. In this study, a link between carbon catabolite repression and mga regulation in the GAS has been demonstrated. The significance of this regulation may be that Mga virulence gene expression is influenced by the availability of a preferred carbon source during infection. Presumably, this would enable the bacterium to sense its environment and express the genes it requires to survive during early time points in growth. The importance of CCR in the regulation of mga may be observed by the strict conservation of the cre in an activating position in the mga promoter in all sequenced serotypes of the GAS (Fig. 1A). However, this may be unique to S. pyogenes, since an examination of the promoters of mga orthologs found that this conservation is not consistent across other pathogenic streptococcal species (data not shown). In the GAS, Mga originally may have functioned to regulate genes important in carbon metabolism and later evolved to regulate virulence genes. This hypothesis is supported by a recent microarray analysis of Mga-regulated genes from different serotypes of GAS showing that inactivation of mga alters the ability of the GAS to grow in different sugars (36).
It has been known for some time that the expression of Mga is regulated by growth phase, with maximal expression occurring during exponential phase (23). The results presented here may provide a mechanism for this connection. As the GAS colonize a new tissue site with available glucose, the pathogen would enter into exponential-phase growth. CCR would be active at this time, and the expression of mga from Pmga P1 is activated. Once the mga transcripts are translated, Mga autoactivates itself from Pmga P2. Upon depletion of glucose, the bacteria enter stationary-phase growth, and CcpA would no longer activate expression of Pmga P1. However, since Mga still may be able to activate transcription from Pmga P2, an unidentified factor that suppresses Mga activity during stationary phase must come into play, either by modifying Mga to inactivate it or through competition for binding at Pmga. A complete understanding of the events that occur at Pmga may provide insights into how the GAS is able to coordinate Mga regulation in response to growth-phase-dependent cues.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the National Institutes of Health (NIH/NIAID AI47928 to K.S.M.). A.C.A. was supported in part by an NIH/NIAID Molecular Microbiology training grant (5T32 AI07520) and an NIH/NIAID research supplement for underrepresented minorities (RSUM AI-47928-S).
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FOOTNOTES |
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Published ahead of print on 28 September 2007.
Supplemental material for this article may be found at http://jb.asm.org/.
These authors contributed equally to this work.
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