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Infect Immun. Oct 2002; 70(10): 5454–5461.
PMCID: PMC128313

Role of RegM, a Homologue of the Catabolite Repressor Protein CcpA, in the Virulence of Streptococcus pneumoniae

Abstract

As part of a study of virulence gene regulation in Streptococcus pneumoniae, we have identified a gene encoding a homologue of the staphylococcal catabolite control protein CcpA in the pneumococcal genome sequence. The pneumococcal protein, designated RegM, has significant similarity to members of the LacI/GalR family of bacterial regulatory proteins. S. pneumoniae D39 derivatives with insertion-duplication or deletion mutations in regM were significantly attenuated in virulence with respect to the wild-type strain. In defined media containing either sucrose or lactose as sole carbon sources, the in vitro growth rates of D39 and the regM mutants were essentially the same. However, in the presence of galactose the regM mutants grew significantly faster than the wild-type strain, whereas growth rates were significantly lower in the presence of glucose or maltose. These data are consistent with the involvement of regM in the catabolism of carbohydrates in S. pneumoniae. RegM was a repressor of both α-glucosidase and β-galactosidase activities in S. pneumoniae, but unlike the situation in certain other bacteria, it does not mediate the repression of these enzymes by glucose. The observed attenuation in virulence was not attributable to poorer growth of the regM mutants in mouse blood ex vivo, but nevertheless, the mutants were rapidly cleared from the blood of infected mice in vivo. The regM mutation had no apparent impact on expression of several confirmed pneumococcal virulence proteins, but studies employing a lacZ transcriptional fusion construct indicated that mutation of regM resulted in a significant reduction in transcription of the capsular polysaccharide biosynthesis locus (cps). Thus, regM is the first gene outside of the cps locus to be implicated in regulation of capsular gene expression.

Streptococcus pneumoniae (the pneumococcus) is a major cause of life-threatening invasive diseases such as pneumonia, meningitis, and bacteremia, as well as other less serious, but highly prevalent infections, such as otitis media and sinusitis. Invasive pneumococcal disease is almost invariably preceded by colonization of the nasopharynx, and significant proportions of the population carry S. pneumoniae asymptomatically at any one time. Progression from carriage to invasion is associated with alteration in the levels of expression of a number of virulence factors. The capacity to sense and adapt to changing host environments is likely to play a key role in this pathogenic process. Most recent works have focused on two-component signal transduction systems. Throup et al. (31) showed that 8 of the 14 two-component systems present in the S. pneumoniae genome are involved in survival in a respiratory model, but Lange et al. (16) found no evidence for involvement in systemic virulence. Although two-component systems are an important means of sensing changes in the environment, transporters associated with cytosolic regulators also play an important role. Some of the best examples of such systems are those involved in the regulation of sugar metabolism. Since the early studies of Monod (23), we have known that in the presence of glucose, most bacteria stop using other carbohydrate sources. In low-G+C-content gram-positive bacteria, glucose or other rapidly metabolized sugars enter the glycolytic pathway and generate intermediates like fructose 1-6 bi-phosphate. These glycolytic intermediates trigger an ATP-dependent protein kinase that phosphorylates the phosphotransferase system (PTS) phosphocarrier Hpr at residue Ser46. P-Ser Hpr subsequently enhances the binding of the catabolite control protein A (CcpA) to a catabolite-responsive element (cre) present in the promoter or coding regions of catabolite-regulated genes (13, 29). Catabolite control by CcpA involves not only repression of genes involved in alternative carbon source utilization but also activation of the expression of some genes whose products are involved in excretion of excess carbon, such as the ackA gene, which encodes acetate kinase (10).

Carbon catabolite regulation of virulence gene expression has been reported for several bacteria, such as Staphylococcus aureus (14) and Listeria monocytogenes (21), but not for S. pneumoniae. However, one environmental factor which is likely to differ substantially between the nasopharynx and the blood is availability of rapidly metabolizable carbon sources, and this may have a global effect on bacterial gene expression through catabolite repression. In the present study, we examined the pneumococcal genome sequence for genes encoding homologues of CcpA and examined their role in catabolite repression and regulation of virulence-related genes.

MATERIALS AND METHODS

Bacterial strains and plasmids.

Bacterial strains and plasmids used in this study are listed in Table Table1.1. S. pneumoniae was grown without agitation in Todd-Hewitt broth with 1% (wt/vol) yeast extract (THY), in CH medium (32) with various carbohydrate supplements, or on blood agar. When required, erythromycin (0.2 μg/ml) or spectinomycin (100 μg/ml) was used for selection in S. pneumoniae, and ampicillin (50 μg/ml) or erythromycin (125 μg/ml) was used for selection in Escherichia coli.

TABLE 1.
Strains and plasmids used in this study

Bacterial transformation.

Transformation of E. coli with plasmid DNA was carried out with CaCl2-treated cells as described by Brown et al. (5). S. pneumoniae strains were transformed essentially as described previously (19) with the following modifications. After overnight growth on blood agar, cells were inoculated into THY and incubated for several hours at 37°C. The cells were subsequently diluted in competence medium (THY supplemented with 0.01% CaCl2 and 0.02% [wt/vol] bovine serum albumin) to an A600 of 0.01 and incubated at 37°C until the A600 reached 0.1. The cells were then concentrated 10 times in competence medium supplemented with 15% glycerol. For the transformation reactions, 0.1 ml of the previously prepared cells were diluted in 1 ml of competence medium supplemented with 50 ng of competence stimulating peptide 1 (12) per ml and approximately 100 ng of DNA per ml. The transformation reaction was incubated for 30 min at 32°C and 2 h at 37°C. Pneumococcal transformants were selected on blood agar containing the appropriate antibiotic.

DNA techniques.

S. pneumoniae chromosomal DNA was extracted, purified, and analyzed as described previously (25).

Plasmid constructs and inactivation of regM.

The regM knockout mutant of S. pneumoniae D39 was constructed by insertion-duplication mutagenesis (20) using the suicide vector pACH74. This vector is a derivative of pVA891 (18), from which a noncoding 0.9-kb HindIII/NruI fragment has been deleted, and an SspI/ClaI fragment containing the multiple cloning site and the lacZ region from pAlter (Promega, Madison, Wis.) inserted between the XbaI and ClaI sites adjacent to the erythromycin resistance gene. An internal fragment of regM was amplified by PCR using genomic S. pneumoniae DNA as a template and the following pair of oligonucleotide primers containing terminal restriction sites (underlined): 5′-CGGGATCCTGCTCAACATCGACAGT-3′ and 5′-CCCAAGCTTCGTGAAGCAGGTGTTT-3′. The resultant 445-bp PCR product was cloned into BamHI/HindIII-digested pACH74, and the resulting plasmid (designated pHG6) was used to transform D39.

To create pHG7 the region carrying the Eryr marker of pACH74 was amplified using the universal M13 forward sequencing primer and 5′-CTGATGCCAATCTCAAGGAATTCCGTGGGGAAGGCCATCC-3′ and cloned into BamHI/EcoRI-digested pHG2. The D39 deletion mutant carrying the ΔregM deletion, was created as follows. A 1,642-bp fragment, containing the upstream region and 196 bp of regM, was amplified from the S. pneumoniae DNA with the following pair of primers: 5′-GGTAGATGGGGCCCGAATTCGAGACCTACCACGCGCAACTGCATT-3′ and 5′-ATAGGGTACCTGCGAACCATTGTGG-3′. The product was cloned into EcoRI/KpnI-digested pHG7, and the resulting plasmid was called pHG8. The 1,324-bp downstream fragment was amplified from S. pneumoniae DNA with the following pair of primers: 5′-ATCGGAGCTCACGCTGCCAAGTATG-3′ and 5′-CCGTATTACCGAGATCTGGATCCCGGGCTTGGTGCCATTAGTATG-3′. The product was cloned into the BamHI/SacI-restricted pHG8. The resulting plasmid was called pHG9 and used to transform D39.

We constructed strain D39 lacking ebg and carrying cps::lacZ as follows. S. pneumoniae D39 was transformed with chromosomal DNA from R262, a strain in which the endogenous β-galactosidase gene (ebg) was knocked out previously (1). Chromosomal DNA from a spectinomycin-resistant transformant was used to transform D39, and the absence of endogenous β-galactosidase activity in one of the transformants was checked. We then amplified a 1,444-bp fragment corresponding to the 3′ end of the capsule locus of D39 with the following primers: 5′-CGTCTAGAACGGTCCTTATCAACAT-3′ and 5′-GGGATCCTGCCTCTTTTATTAGACTAGAAA-3′. The fragment was cloned in XbaI/BamHI-restricted pEVP3 (6). The resulting plasmid (encoding chloramphenicol resistance) was used to transform strain D39 lacking ebg. This generated strain D39 lacking ebg and carrying cps::lacZ where the promoterless lacZ reporter gene is in transcriptional fusion with the capsule operon without interruption of the locus. The specific integration of the constructs into D39 was confirmed by Southern blot hybridization. A similar procedure was used to construct strain D39 lacking ebg and carrying ΔregM cps::lacZ.

Virulence studies.

S. pneumoniae strains were grown overnight on blood agar (supplemented with erythromycin where appropriate), inoculated into serum broth (meat extract broth plus 10% horse serum), and incubated at 37°C. Cultures were diluted to a density of 5 × 103 CFU/ml or 5 × 105 CFU/ml, and 0.1-ml volumes were injected intraperitoneally into groups of 10 BALB/c mice. Survival time was recorded. Differences in median survival time were analyzed using the Mann-Whitney U test; differences in overall survival rate were analyzed using the Fisher exact probability test.

Growth rate studies.

After growth overnight on blood agar plates (with erythromycin [0.2 μg/ml] for the mutants), the cells were harvested and washed in CH medium without sugars. Cells were then diluted to an A600 between 0.02 and 0.05 in CH medium with 10 mM carbohydrate and incubated at 37°C. The A600 was monitored, and the doubling time was calculated. Comparative growth rates were measured in three experiments, and differences in doubling time were analyzed using Student's t test (two tailed).

Determination of enzyme activities.

After overnight growth on blood agar (supplemented with erythromycin when required), S. pneumoniae cells were harvested from the plates and washed in CH medium without sugars. Cells were then grown in CH medium plus a 10 mM concentration of the indicated carbohydrate(s) to an A600 of ~0.2. The cells were harvested by centrifugation and lysed in 0.1 M sodium phosphate buffer-0.1% Triton for 10 min at 37°C. The pH of the buffer was 6.6 for the lysates used for determination of α-glucosidase activity and 7.5 for lysates used for determination of β-galactosidase activity. Protein concentrations were determined using the method of Bradford (4). For β-galactosidase assays, 10 μl (2 to 10 μg of protein) of lysates was added to 1 ml of 0.1 M sodium phosphate (pH 7.5)-1 mM MgCl2-50 mM β-mercaptoethanol-10 mM O-nitrophenol-β-d-galactopyranoside (Sigma). For α-glucosidase assays, the pH of the buffer was 6.6 and the substrate was replaced by p-nitrophenyl α-d-glucopyranoside (Sigma). The assays were performed at 37°C. The release of nitrophenol was monitored at 410 nm. Activities were expressed in nanomoles of nitrophenol released per minute per microgram of protein. Data were analyzed using Student's t test (two tailed).

RESULTS

Genomic structure of a novel lacI/galR regulator in S. pneumoniae.

In an attempt to identify putative regulators involved in control of pneumococcal virulence factors, we used the partial sequence of S. aureus CcpA to search the S. pneumoniae genome database of The Institute for Genomic Research (TIGR). The products of two pneumococcal open reading frames (ORFs) were found to have similarity to this protein. One of these was MalR, a previously characterized transcriptional repressor of maltosaccharide uptake and utilization (26). The second ORF had not been described previously, but BLASTP analysis (2) indicated significant similarity with members of the LacI/GalR family of bacterial regulatory proteins produced by several other gram-positive species (Table (Table2).2). We have designated this protein RegM because of its high degree of similarity with RegM of Streptococcus mutans (30). Analysis of the region of the S. pneumoniae chromosome surrounding the regM gene revealed the presence of the two-component system hk11-rr11 (16) immediately upstream, while an ORF with unknown function and a gene encoding a putative l-asparaginase were located downstream (Fig. (Fig.1A).1A). Reverse transcription-PCR analysis of S. pneumoniae RNA extracts using primers within rr11 and regM did not result in a product, suggesting that the two-component system and regM are not cotranscribed (result not presented). An analysis of the promoter region of regM revealed the presence of a putative catabolite responsive element (cre) 150 nucleotides upstream of the probable initiation codon (Fig. (Fig.1B).1B). The cre-like sequence matches the consensus cre sequence proposed by Weickert and Chambliss (34) at 13 of 14 positions (Fig. (Fig.1C1C).

FIG. 1.
(A) Genetic organization of the regM region in S. pneumoniae. The relative positions of the oligonucleotides used to create the knockout mutant (a and b) and the deletion mutant (c, d, e, and f) are indicated by the arrows. (B) Sequence of the promoter ...
TABLE 2.
Comparison of S. pneumoniae RegM with similar gene products

Effect of inactivation of regM on the virulence of S. pneumoniae.

To investigate the role of regM in the virulence of S. pneumoniae D39, we constructed a regM knockout mutant by insertion-duplication mutagenesis, as described in Materials and Methods. Interruption of regM was confirmed by Southern hybridization analysis (result not shown). For virulence studies, groups of six mice were challenged intraperitoneally with wild-type D39 or the regM knockout mutant, at doses of approximately 4 × 103 or 1 × 105 CFU (Fig. (Fig.2).2). At both doses, all mice challenged with D39 died, with median survival times of 2 to 3 days. In contrast, five of the six mice challenged with the lower dose of the D39 regM knockout mutant were alive and symptom-free at the end of the experiment (Fig. (Fig.2A),2A), but all mice succumbed to the higher dose of the D39 regM knockout mutant (Fig. (Fig.2B).2B). However, pneumococci isolated from the heart blood of all of the mice that died as a result of the challenge with either dose of the D39 regM knockout mutant were all erythromycin sensitive. This suggested that the vector sequences encoding erythromycin resistance had been eliminated in vivo, thereby potentially reconstituting an intact regM gene. Instability of such insertion-duplication mutants is highly unusual and suggests that regM confers a powerful selective advantage on S. pneumoniae in vivo.

FIG. 2.
Comparative virulence of D39 and the D39 regM knockout mutant (D39regM). Groups of six mice were challenged intraperitoneally with either 4 × 103 CFU (A) or 1 × 105 CFU (B). Survival times of individual mice are indicated.

To avoid the emergence of such revertants in vivo, we constructed a D39 derivative with a deletion in regM (D39 carrying ΔregM) (see Materials and Methods) and repeated the intraperitoneal virulence studies. Groups of 10 mice were then challenged intraperitoneally with approximately 500 or 4 × 104 CFU of D39 and D39 carrying ΔregM (Fig. (Fig.3).3). At the lower dose, all but one of the mice challenged with D39 died, and the median survival time was 3 days; all of the mice challenged with D39 carrying ΔregM survived. At the higher dose, all of the D39-challenged mice died, with a median survival time of 1 day, whereas only one of the mice challenged with D39 carrying ΔregM died. At both doses, differences in median survival time and overall survival rate were highly significant (P < 0.001 in all cases). The heart blood of the single mouse challenged with D39 carrying ΔregM that died 6 days after challenge with the higher dose contained only erythromycin-resistant pneumococci, confirming that reversion had not occurred. These results indicate that regM is extremely important for survival of S. pneumoniae in the mouse and de facto for the virulence of this organism.

FIG. 3.
Comparative virulence of D39 and D39 carrying ΔregM. Groups of 10 mice were challenged intraperitoneally with approximately 500 CFU (A) or 4 × 104 CFU (B). Survival times of individual mice are indicated.

Analysis of growth characteristics.

Given the putative role of RegM as a catabolite repressor, we investigated the growth of D39, the D39 regM knockout mutant, and D39 carrying ΔregM in the presence of different carbohydrate sources (Table (Table3).3). In the presence of sucrose or lactose, the growth rates of D39, the D39 regM knockout mutant, and D39 carrying ΔregM were essentially the same, as judged by doubling time. When the cells were grown in the presence of galactose, the regM mutants grew significantly faster than the wild-type strain (P < 0.01), whereas growth rates were significantly lower than those of the wild type in the presence of glucose (P < 0.001) or maltose (P < 0.02). The differential effect of mutation of regM on growth rate in the presence of different sugars is consistent with the involvement of regM in the catabolism of carbohydrates in S. pneumoniae.

TABLE 3.
Growth rates in the presence of various sugarsa

Catabolic enzyme activities.

The influence of the regM deletion on catabolite regulation was determined for two enzyme activities that were previously found to be subject to catabolite regulation in Staphylococcus xylosus (8). For investigation of β-galactosidase and α-glucosidase activities, cells were grown in CH medium in the presence of inducing sugar (10 mM lactose or maltose, respectively). To test for catabolite repression, 10 mM glucose was added. Samples were taken during mid-exponential growth and assayed for enzyme activity as described in Materials and Methods. In wild-type D39 the addition of 10 mM glucose to the medium decreased endogenous β-galactosidase activity by a factor of 3.6, demonstrating significant (P < 0.01) catabolite repression (Fig. (Fig.4).4). In D39 carrying ΔregM, catabolite repression still occurred in the presence of glucose; endogenous β-galactosidase activity was 9.4-fold lower than that in the absence of glucose (P < 0.05). Thus, catabolite repression of endogenous β-galactosidase by glucose is not relayed via RegM. However, in the presence of either lactose or lactose plus glucose, the β-galactosidase activity of the mutant was significantly higher than that of D39 (P < 0.05 and P < 0.01, respectively). This suggests that RegM is indeed a repressor of the endogenous β-galactosidase but that it does not mediate the catabolite repression due to glucose.

FIG. 4.
Catabolite repression of β-galactosidase in D39 and D39 carrying ΔregM. Cells were grown at 37°C in CH medium supplemented with lactose or lactose plus glucose. Specific activity of β-galactosidase in exponentially grown ...

In contrast, we did not observe catabolite repression of α-glucosidase activity in the presence of glucose, in either D39 or D39 carrying ΔregM. However, in the presence or absence of glucose, α-glucosidase activity was significantly higher in the mutant than in the wild type (P < 0.05 in both cases) (Fig. (Fig.5).5). These results show that RegM is a repressor of both α-glucosidase and β-galactosidase activities in S. pneumoniae, but unlike the situation in certain other bacteria, it does not mediate the repression of these enzymes by glucose.

FIG. 5.
Catabolite repression of α-glucosidase in D39 and D39 carrying ΔregM. Cells were grown at 37°C in CH medium supplemented with maltose or maltose plus glucose. Specific activity of α-glucosidase in exponentially grown cells ...

Growth of D39 carrying ΔregM in blood and in mice.

We have shown above that the regM mutants have lower growth rates than the wild type in the presence of certain sugars, particularly glucose. The concentration of glucose in the blood is in the range 2 to 5 mM, and so it was possible that the extremely low virulence of the regM mutants observed in this study was a consequence of low growth rates. To examine this, five BALB/c mice were exsanguinated and fresh heparinized blood was inoculated with D39 or D39 carrying ΔregM. There was no significant difference in growth rate between the wild-type strain and the mutant (respective doubling times were 86.7 ± 4.5 and 88.1 ± 3.3 min). We also challenged BALB/c mice intraperitoneally with 4 × 103 CFU of D39 carrying ΔregM and monitored the numbers of bacteria in the blood of the mice by direct plating on blood agar. After 24 h no bacteria could be found, indicating that they had been rapidly cleared from the blood (result not shown).

Effect of RegM on virulence gene expression.

The above results suggest that RegM could be involved in regulation of the expression of genes essential for the virulence of the pneumococcus. To test this hypothesis we examined the impact of the regM mutation on the expression of several confirmed pneumococcal virulence proteins, namely, CbpA (also known as PspC and SpsA), PspA, PsaA, and Ply by Western blot, using polyclonal mouse antisera. No differences were observed in the levels of any of these proteins in D39 lysates compared with lysates of D39 carrying ΔregM (results not shown).

We also investigated the effect of the regM mutation on expression of the capsular polysaccharide biosynthesis (cps) locus. We constructed a transcriptional fusion of the β-galactosidase gene lacZ at the 5′ end of the cps locus in derivatives of D39 and D39 carrying ΔregM lacking the endogenous β-galactosidase gene (see Materials and Methods). We then monitored capsule gene expression by assaying β-galactosidase activity under various conditions (Fig. (Fig.6).6). In the presence of either glucose or sucrose, expression of the cps locus in the regM mutant was significantly lower than that in the wild type (P < 0.001 and P < 0.01, respectively).

FIG. 6.
Expression of the cps operon. D39 lacking ebg and carrying cps::lacZ and D39 lacking ebg and carrying ΔregM cps::lacZ were grown in CH supplemented with glucose, sucrose, or lactose, and expression of capsule genes was monitored as expression ...

We had noticed previously that when grown on blood agar, colonies of D39 carrying ΔregM were somewhat smaller than those of D39. We initially attributed this phenotypic difference to growth deficiency of strains with mutations in regM on media containing starch (the carbohydrate source in the blood agar plates). However, in light of the results obtained with the cps::lacZ constructs, we examined colony size after plating on CH agar containing 10 mM sucrose, a medium in which the growth rates of D39 and D39 carrying ΔregM are essentially identical (Fig. (Fig.7).7). After incubation for 16 h at 37°C, the mean diameter of colonies of D39 carrying ΔregM was less than half that of D39 colonies (P < 0.01). Thus, it is possible that differences in capsular expression are reflected in colony size.

FIG. 7.
Colony size of D39 and D39 carrying ΔregM. Cells were grown on CH agar plates with 5% defibrinated blood and incubated for 16 h at 37°C in a CO2 incubator. (A) D39 carrying ΔregM; (B) D39. (C) Mean colony diameter + standard ...

DISCUSSION

In this study, we demonstrated the involvement of a CcpA homologue called RegM in the virulence of S. pneumoniae. Disruption of regM leads to pleiotropic effects, including alteration of growth rates in semisynthetic media supplemented with different sugar sources, induction of sugar metabolism enzymes, and loss of virulence. The involvement in control of endogenous β-galactosidase and α-glucosidase activities and the change in growth rates as a function of the source of carbohydrate could indicate a role for RegM in regulation of sugar metabolism. However, contrary to the situation in other bacteria (24), this CcpA homologue is not involved in the glucose-mediated repression of β-galactosidase expression. This situation is not without precedent, as in S. mutans the disruption of the homonymous gene does not relieve the glucose repression of catabolite controlled enzymes but, paradoxically, increases their repression (30). Rosenow et al. (28) have reported that in S. pneumoniae, catabolite repression of α-galactosidase is not mediated by a ccpA homologue (presumably regM) but rather is mediated directly or indirectly by a sucrose-specific PTS. We have located two potential glucose-specific PTS in the pneumococcal genome sequence (data not shown), but gene knockout experiments will be required to determine whether there is analogous control of β-galactosidase expression by glucose. We also located several cre-like sequences in promoters and coding regions of potential β-galactosidase and α-glucosidase genes. It remains to be determined whether RegM is capable of binding to these sites and blocking transcription of theses genes in the absence of an inducer.

Mutagenesis of regM demonstrated the importance of this gene for the survival of S. pneumoniae in the host. Even though reversion of the insertion-duplication mutation was not observed after prolonged growth in CH medium supplemented with glucose, in vivo growth selected strongly for excision of the inserted plasmid. Glucose is the major sugar present in the blood (at concentrations of 2 to 5 mM), and we have shown that regM mutants grow more slowly in semisynthetic medium with glucose. Thus, it was tempting to attribute the loss of virulence to inability of the mutant to grow in blood. However, when fresh heparinized mouse blood was inoculated with D39 or D39 carrying ΔregM we did not observe a difference in the growth rate of the bacteria, indicating that other factors underpinned the attenuation in virulence.

One possible explanation for this observation would be a requirement for regM for optimal expression of essential virulence proteins. We used Western blot analysis to examine levels of some of the better-characterized pneumococcal virulence proteins (namely, CbpA, PspA, PsaA, and Ply) but failed to detect any obvious differences in expression between the wild-type and mutant strains. However, we demonstrated that deletion of regM down-regulates transcription of the capsule locus as determined using a cps::lacZ fusion construct. Thus, we propose that the avirulent phenotype of regM mutants is largely due to the inability to produce sufficient capsule to survive in the host, although we do not exclude the possibility that other unknown factors regulated by RegM are also involved.

The 90 distinct S. pneumoniae capsular polysaccharides are composed of a complex range of sugar derivatives (15), and RegM is involved in the control of sugar pathways, as suggested by homology to CcpA and the change of growth rate of mutants in the presence of different sugars. We have also shown that capsule gene expression is dependent upon sugar source, since in semisynthetic medium supplemented with lactose, the expression of capsule genes is down-regulated. Thus, RegM may be a regulatory link between these sugar pathways and capsule production. Recently, two genes localized some distance from the cps locus have been found to be involved in type 3 and type 37 capsule production. The gene pgm, which encodes a phosphoglucomutase, is involved in synthesis of type 3 capsule (11), and tts, which encodes a β-glucosyltransferase, is involved in type 37 capsule synthesis (17), but neither of these genes alters cps transcription. This contrasts with our findings for regM. Interestingly, we have located several potential cre sequences in the 5′ region of the cps locus, and we are currently investigating the binding of RegM to these sites.

In summary, we have demonstrated the involvement of RegM, a LacI/GalR regulator in the virulence of S. pneumoniae. RegM directly or indirectly regulates the expression of endogenous β-galactosidase and α-glucosidase and is the first product of a gene outside of the cps locus to be directly implicated in regulation of capsule biosynthesis.

Acknowledgments

This work was supported by a grant from the National Health and Medical Research Council of Australia.

We thank David Ogunniyi for assistance with virulence studies, Angelo Guidolin for construction of pACH74, and Mathew Woodrow and David Miller for technical assistance.

Notes

Editor: E. I. Tuomanen

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