Methionine-mediated gene expression and characterization of the CmhR regulon in Streptococcus pneumoniae

This study investigated the transcriptomic response of Streptococcus pneumoniae D39 to methionine. Transcriptome comparison of the S. pneumoniae D39 wild-type grown in chemically defined medium with 0–10 mM methionine revealed the elevated expression of various genes/operons involved in methionine synthesis and transport (fhs, folD, gshT, metA, metB-csd, metEF, metQ, tcyB, spd-0150, spd-0431 and spd-0618). Furthermore, β-galactosidase assays and quantitative RT-PCR studies demonstrated that the transcriptional regulator, CmhR (SPD-0588), acts as a transcriptional activator of the fhs, folD, metB-csd, metEF, metQ and spd-0431 genes. A putative regulatory site of CmhR was identified in the promoter region of CmhR-regulated genes and this CmhR site was further confirmed by promoter mutational experiments.


Introduction
Streptococcus pneumoniae colonizes the human nasopharynx during the first few months of life and is the causative agent of many human diseases including pneumonia, sepsis, meningitis, otitis media and conjunctivitis, resulting in over a million deaths each year worldwide (Gray et al., 1982;Ispahani et al., 2004;O'Brien et al., 2009).Proper utilization of the available nutrients is a prerequisite for the successful colonization and survival of bacteria inside the human body in addition to the virulence factors it possesses (Phillips et al., 1990;Titgemeyer & Hillen, 2002).Virulence gene screening and targeted examination of specific nutrient transporters have established the importance of nutrient acquisition for the pathogenesis of many microbial pathogens (Darwin, 2005;Lau et al., 2001;Mei et al., 1997).Amino acids are one of the most important groups of nutrients needed for proper bacterial growth.
Methionine is an amino acid that is scarcely present in physiological fluids but its importance cannot be underestimated.It is vital for protein synthesis and is an integral component of S-adenosyl methionine (SAM: the main biological methyl donor needed for the biosynthesis of phospholipids and nucleic acids) (Fontecave et al., 2004;Shelver et al., 2003).A number of genes/gene clusters are present in S. pneumoniae that can synthesize methionine from other sources in the absence of methionine.Csd and MetE are part of the methionine synthesis pathway and are involved in conversion of cystathionine to homocysteine, and homocysteine to methionine, respectively (Kanehisa et al., 2014).Cystathionine and homocysteine can also be formed from homoserine, where O-acetyl-L-homoserine is converted to cystathionine by MetB.O-Acetyl-L-homoserine can be converted to homocysteine by MetB, SPD-1073 and SPD-1074 (spd-1073 and spd-1074 encode an O-acetylhomoserine aminocarboxypropyltransferase/cysteine synthase and a hypothetical protein, respectively) (Kanehisa et al., 2014).
Methionine can be synthesized by other microbes as they may convert homoserine to homocysteine through addition of a sulphur group from either cysteine (involving MetABC), sulphide (involving MetA and CysD) or methionine using the SAM recycling pathway (MetK, Pfs and LuxS) (Kovaleva & Gelfand, 2007).MetE (methionine synthase) then methylates homocysteine in combination with MetF (methylenetetrahydrofolate reductase), and 5-methyltetrahydrofolate (FolD) provides it with the methyl group to form methionine (Kovaleva & Gelfand, 2007;Ravanel et al., 1998).It has been shown that methionine biosynthetic genes are essential for full virulence in Brucella melitensis (Lestrate et al., 2000), Haemophilus parasuis (Hill et al., 2003) and Salmonella enterica (Ejim et al., 2004).Moreover, mutation of the methionine transport regulator MtaR led to attenuated virulence in Streptococcus agalactiae (Shelver et al., 2003), which suggests that methionine synthesis is indispensable for the existence of many bacteria during invasive infection.
In the current study, we elucidated the effect of methionine on the global gene expression of S. pneumoniae and demonstrated that the transcriptional regulator CmhR (SPD-0588) acts as a transcriptional activator for fhs, folD, metB-csd, metEF, metQ and spd-0431, involved in methionine uptake and utilization.The putative regulatory site of CmhR (5 ¢ -TATAGTTTSAAACTATA-3 ¢ , where S denotes G/C/A) in the promoter regions of its regulon genes is predicted and confirmed by promoter mutational experiments.This site is also found to be highly conserved in other pneumococcal strains and streptococci.

Methods
Bacterial strains, growth conditions and DNA isolation and modification.Bacterial strains and plasmids used in this study are listed in Table 1. S. pneumoniae D39 wild-type was grown as described previously (Afzal et al., 2014).For b-galactosidase assays, derivatives of S. pneumoniae D39 were grown in chemically defined medium (CDM) (Neves et al., 2002), supplemented with different concentrations of methionine as indicated in the Results.For selection on antibiotics, the medium was supplemented with the following concentrations of antibiotics: spectinomycin at 150 mg ml À1 and tetracycline at 2.5 mg ml À1 for S. pneumoniae; and ampicillin at 100 mg ml À1 for Escherichia coli.All bacterial strains used in this study were stored in 10 % (v/v) glycerol at À80 C. For PCR amplification, chromosomal DNA of S. pneumoniae D39 (Lanie et al., 2007) was used as a template.
Primers used in this study are based on the sequence of the D39 genome and are listed in Table S1, available in the online Supplementary Material.
Construction of a cmhR mutant.A cmhR (spd-0588) deletion mutant (MA1100) was made by allelic replacement with a spectinomycin-resistance cassette.Briefly, primers cmhR-1/cmhR-2 and cmhR-3/cmhR-4 were used to generate PCR fragments of the left and right flanking regions of cmhR.PCR products of left and right flanking regions of cmhR contain AscI and NotI restriction enzyme sites, respectively.The spectinomycin-resistance marker, which is amplified by primers SpecR/SpecF from pORI38, also contains AscI and NotI restriction enzyme sites on its ends.Then, by restriction and ligation, the left and right flanking regions of cmhR were fused to the spectinomycin-resistance gene.The resulting ligation products were transformed to S. pneumoniae D39 wild-type and selection of the mutant strains was done with the appropriate concentration of spectinomycin.
For transformation, cells were grown at 37 C until an OD 600 of ~0.1.Then, 0.2 % BSA and 1 mM CaCl 2 were added to the cells.A 1 ml aliquot of the grown culture was transferred to a 1.5 ml tube and 100 ng ml À1 of CSP1 (Competence Stimulating Peptide 1) was added to the culture.Cells were incubated at 37 C for 10-12 min.Then, the ligation mixture was added to the incubated cells and the cells were allowed to grow for 90-120 min at 37 C.After growth, the culture was spun for 1 min at 7000 r.p.m.

Impact Statement
This study demonstrates methionine-mediated gene regulation in Streptococcus pneumoniae and identifies methionine transport and biosynthesis genes.S. pneumoniae is a human nasopharyngeal pathogen that is responsible for millions of deaths each year.Methionine is one of the important amino acids for pneumococci and some of the methionine-regulated genes have been shown to have a role in virulence in different bacteria including S. pneumoniae.In other bacteria, two to three transcriptional regulators have been shown to be involved in the regulation of sulphur-containing amino acids.The current study highlights the transcriptomic response of S. pneumoniae to methionine and identifies an important transcriptional regulator, CmhR, which acts as an activator for its regulon genes.The regulatory site of CmhR in the promoter regions of its regulon genes is predicted and confirmed through mutagenesis studies.CmhR (also called MtaR in other bacteria) has been demonstrated to have a role in virulence in many bacteria.Therefore, investigation of the involvement of CmhR in virulence in S. pneumoniae might be of interest.and most of the supernatant was discarded.The cell pellet was dissolved in the remaining medium (50-100 ml) and plated on blood agar plates with 1 % sheep blood.The cmbR mutant was further confirmed by PCR and DNA sequencing.
The b-galactosidase assays were performed as described before (Halfmann et al., 2007;Israelsen et al., 1995) using cells that were harvested in the mid-exponential growth phase and grown in CDM.
Microarray analysis.Microarray analysis was performed as described before (Afzal et al., 2015a;Shafeeq et al., 2015).For DNA microarray analysis of S. pneumoniae in the presence of methionine, the transcriptome of S. pneumoniae D39 wildtype, grown in replicates in CDM with 10 mM methionine, was compared to that grown in CDM with 0 mM methionine, and harvested at respective mid-exponential growth phases.For the identification of differentially expressed genes a Bayesian P-value <0.001 and a fold-change cut-off >1.5 was applied.
For RNA isolation the following procedure was performed: the pellet of the harvested cells was resuspended in 400 ml TE buffer (diethylpyrocarbonate) and the resuspended cells were added into RNA-free screw-cap tubes containing 0.5 g glass beads, 50 ml 10 % SDS, 500 ml phenol/chloroform: isoamylalcohol, macaloid layer (150-175 ml, not exact as it is highly viscous).To break the cells the screw-cap tubes were placed in a bead beater and two 1 min pulses were applied with 1 min interval on ice.The samples were centrifuged for 10 min at 10 000 r.p.m. (4 C).Then, 500 ml chloroform/isoamylalcohol (24 : 1) was added to the tubes containing the upper phase of the centrifuged tubes and the samples were again centrifuged for 5 min at 10 000 r.p.m. (4 C).A 500 ml aliquot of the upper phase was transferred to fresh tubes and total RNA was isolated using the High pure RNA isolation kit (Roche life science) according to the manufacturer's instructions.RNA quality was assessed on a chip using an Agilent 2100 Bioanalyzer according to the manufacturer's instructions and an RNA integrative number (RIN) value above 8 was considered good.All other procedures regarding the DNA microarray experiment and data analysis were performed as previously described (Afzal et al., 2015b;Shafeeq et al., 2011a, b).Microarray data have been submitted to GEO under accession number GSE88766.
Reverse transcription (RT)-PCR and purification for quantitative RT-PCR.For quantitative RT-PCR, S. pneumoniae D39 wild-type and D39 DcmhR were grown in CDM.RNA isolation was done as described above.First, cDNA synthesis was performed on RNA (Shafeeq et al., 2011b;Yesilkaya et al., 2008).cDNA (2 ml) was amplified in a 20 ml reaction volume that contained 3 pmol of each primer (Table S1) and the reactions were performed in triplicate (Shafeeq et al., 2011b).The transcription level of specific genes was normalized to gyrA transcription, amplified in parallel with primers gyrA-F and gyrA-R.The results were interpreted using the comparative CT method (Schmittgen & Livak, 2008).Differences in expression of twofold or greater relative to the control were considered significant.

Results
Methionine-dependent gene regulation in S. pneumoniae D39 The importance of methionine acquisition and synthesis for S. pneumoniae growth and virulence has been reported before (Basavanna et al., 2013).In this study, we explored the impact of methionine on the transcriptome of S. pneumoniae D39.To do so, we performed transcriptome comparison of S. pneumoniae D39 wild-type grown in CDM with 0-10 mM methionine.The concentration of methionine in CDM is around 0.67 mM.A number of genes/gene clusters were differentially regulated under our tested conditions (Table 2).Putative methionine pathway genes were significantly upregulated in the absence of added methionine in CDM.These genes include spd-0150 [coding for a predicted glutathione ATP binding cassette (ABC) transporter, substrate-binding protein], metQ-hmrB-metNP (a gene cluster putatively involved in methionine transport, whereas hmrB codes for a putative N-acyl-L-amino acid amidohydrolase), spd-0431 (hypothetical protein), metEF (coding for 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase and 5,10-methylenetetrahydrofolate reductase, respectively), gshT (coding for a predicted glutathione ABC transporter, substrate-binding protein), spd-0616-18 (coding for a predicted polar amino acid ABC transporter), folD [coding for a methylenetetrahydrofolate dehydrogenase (NADP+)], fhs (coding for a formate-tetrahydrofolate ligase), metA (coding for a homoserine O-succinyltransferase), metB-csd (coding for a cystathionine gamma-synthase and cysteine desulfurase, respectively) and tcyBC (coding for a cysteine ABC transporter).The genetic organization of these putative methionine genes is shown in Fig. 1.There were some other genes (spd-0360-63, spd-0372-74, spd-0385-91, spd-0447-49, spd-0616-18, spd-1073-75, spd-1098-99 and spd-2037) whose expression was also altered under our tested conditions and whose role in methionine biosynthesis and transport may be of interest.Some of the genes differentially regulated in our transcriptome comparison are putatively involved in carbohydrate transport and utilization (spd-0424-28 and spd-0559-62), and these genes have been studied in our previous studies (Afzal et al., 2014;Shafeeq et al., 2012).The spd-0424-28 genes putatively encode a cellobiose/lactose-specific phosphotransferase system, and transcriptional regulator RokA acts as a transcriptional repressor of this operon (Shafeeq  , 2012).The spd-0559-62 genes putatively code for a lactose/galactose-specific phosphotransferase system and the regulatory mechanism of this gene cluster is not yet known.A gene, spd-0588, coding for a transcriptional regulator is also upregulated in our methionine microarray.
Our bioinformatics analysis shows that this gene shares high homology with mtaR present in other bacteria and because of its putative involvement in cysteine and methionine metabolism we call it cmhR.The upregulation of cmhR under our tested conditions might be an indication of its involvement in the regulation of the methionine genes.

Prediction of the CmhR regulatory site in CmhRregulated genes
A number of genes/gene clusters were differentially regulated under our tested conditions.One important gene that was significantly upregulated in our microarray results was cmhR (spd-0588).This gene codes for a transcriptional regulator CmhR, which putatively belongs to the LysR family of proteins (Shelver et al., 2003).CmhR is a homologue of MtaR of other bacteria and our bioinformatics analysis shows that the MtaR binding site is also present in the genes of the CmhR regulon in S. pneumoniae.Using Genome2D software (Baerends et al., 2004) and a MEME †D39 annotation/TIGR4 annotation (Lanie et al., 2007).‡Ratio represents the fold increase/decrease in the expression of genes in CDM with 0-10 mM methionine.Errors in the ratios never exceeded 10 % of the given values.
motif sampler search (Bailey & Elkan, 1994), a 17 bp palindromic sequence was found in the promoter regions of several genes in S. pneumoniae D39.These genes were identified in our methionine microarray suggesting their role in methionine transport and metabolism.The CmhR regulatory site present in the promoter regions of methionine genes is shown in Fig. 3(a).A weight matrix of these putative CmhR regulatory sites (5 ¢ -TATAGTTTSAAAC-TATA-3 ¢ ) was constructed using these DNA regions (Fig. 3b).This DNA sequence may serve as the CmhR regulatory site in S. pneumoniae.From this study, we conclude that the CmhR regulatory sequence is highly conserved in these streptococci as well (Fig. S1).
To investigate the role of CmhR in the regulation of the proposed CmhR regulon, we made a cmhR deletion mutant and transformed the lacZ-fusions of the promoter regions of these genes in to D39 DcmhR and performed bgalactosidase assays in CDM (Fig. 4).The results of the bgalactosidase assays showed that the activity of all these promoters decreased significantly in D39 DcmhR compared to the D39 wild-type, suggesting a role for CmhR as a transcriptional activator of these genes.
To further confirm our b-galactosidase assay results mentioned above, we performed quantitative RT-PCR on CmhR-regulated genes (metQ, spd-0431, metEF, folD, fhs and metB).Our quantitative RT-PCR results also demonstrated that the expression of these genes increased significantly in the D39 wild-type compared to D39 DcmhR (Fig. 5).These results further confirm that CmhR acts as a transcriptional activator of metQ, spd-0431, metEF, folD, fhs and metB-csd.

Discussion
Bacterial pathogens need to acquire the essential nutrients from their surroundings inside the human host in order to survive and cause infections (Eisenreich et al., 2010;Kilian et al., 1979).In this study, we have explored the impact of methionine on the gene expression of S. pneumoniae D39 and found that a number of genes/operons respond to methionine.The results presented in this study will significantly enhance our understanding of the role of methionine on the gene expression of S. pneumoniae.Several regulatory systems involved in the regulation of methionine biosynthesis have been reported in bacteria.In most Gram-positive bacteria, RNA structures acting on the level of premature termination of transcription control methionine biosynthesis: S-boxes present upstream of methionine biosynthesis genes in the Bacillales and Clostridia (Epshtein et al., 2003;McDaniel et al., 2003;Winkler et al., 2003), and methionine-specific T-boxes in the Lactobacillales (Grundy & Henkin, 2003;Rodionov et al., 2004).Conversely, neither S-boxes nor methionine-specific T-boxes were found in Streptococcaceae for all methionine biosynthesis genes, with metK being the only exception that is regulated by the SMK-riboswitch (Fuchs et al., 2006).Therefore, it is clear that methionine biosynthesis in the Streptococcacae is controlled by a different mechanism that evolved after the split of this group from other Firmicutes.
Three transcriptional factors have been found to play a role in regulation of methionine metabolism in streptococci: MtaR in S. agalactiae, its orthologue MetR in S. mutans and CmbR (earlier known as FhuR) in Lactococcus lactis (Fernández et al., 2002).Methionine uptake decreased fivefold in the mtaR mutant in comparison to the wild-type in S. agalactiae, suggesting its role as a transcriptional activator of the methionine transport genes (Shelver et al., 2003).By contrast, MetJ and MetR regulate the expression of methionine biosynthetic genes in E. coli and Salmonella enterica serovar Typhimurium (Weissbach & Brot, 1991).The E. coli met genes (except for metH) are negatively regulated by MetJ, a transcriptional repressor, with SAM serving as a co-repressor (Saint-Girons et al., 1988,).These genes are also positively regulated by a LysR-type transcriptional regulator MetR, with homocysteine as a co-effector (Cai et al., 1989;Cowan et al., 1993;Mares et al., 1992).CmbR in L. lactis has been demonstrated to activate most genes involved in the methionine and cysteine biosynthesis pathway in the absence of cysteine (Fernández et al., 2002;Sperandio et al., 2005).
MetR has also been shown to regulate a number of methionine biosynthesis genes (Sperandio et al., 2007) by binding to the DNA motif suggested by Rodionov et al. (2004).The regulatory proteins mentioned above belong to the LysR family of transcriptional factors, which is the most abundant family of transcriptional regulators in bacteria (Maddocks & Oyston, 2008).These transcriptional regulators control diverse biological pathways such as central metabolism, cell division, quorum sensing, virulence, motility, nitrogen fixation, oxidative stress responses, toxin production, attachment and secretion.These transcriptional regulators act as either transcriptional activators or repressors, and often are transcribed divergently with one of the regulated genes (Schell, 1993).LysR-family regulators consist of two characteristic domains, an N-terminal helix-turn-helix DNA binding domain (PF00126) and a C-terminal substrate-binding domain (PF03466).There appear to be two transcriptional regulators in S. pneumoniae that control the expression of the cysteine and methionine genes.CmhR in S. pneumoniae belongs to the LysR family of transcriptional factors and has a helixturn-helix domain and a substrate-binding domain of LysRtype transcriptional regulators.The second is CmbR and study of its regulatory mechanism might be of interest (L.lactis has also two: CmbR and CmhR).Our results showed that CmhR acts as a transcriptional activator for a number of genes involved in methionine uptake and biosynthesis and deletion of CmhR led to downregulation of the CmhR genes.
The deletion of CmhR also hampers growth of S. pneumoniae (data not shown), which might be an indication of the importance of this protein in the lifestyle of pneumococci.
In this study, we have demonstrated that the CmhR regulon consists of metQ, spd-0431, metEF, fhs, metB-csd and folD in S. pneumoniae D39.There are two bacterial methionine transport systems: the methionine ABC uptake transporter (MUT) family (Hullo et al., 2004;Merlin et al., 2002) and a secondary transporter BcaP (den Hengst et al., 2006).The MUT system is encoded by the metD locus in E. coli and consists of the MetQ substrate binding protein (SBP), MetL trans-membrane permease and the MetN cytoplasmic ATPhydrolysing protein (ATPase) (Merlin et al., 2002).In S. pneumoniae D39, the spd-0150-54 locus encodes a methionine uptake ABC transporter and deletion of the gene encoding the lipoprotein MetQ resulted in a strain that had reduced growth in methionine-restricted media and no detectable uptake of radioactive methionine (Basavanna et al., 2013).Moreover, deletion of another important locus encoding MetEF (which is also part of the CmhR regulon) increased the growth defect of the metQ deletion strain in methionine-restricted media and in blood plasma, strengthening a role for the products of these genes in methionine synthesis (Basavanna et al., 2013).Micro-organisms can synthesize methionine by converting homoserine to homocysteine through addition of a sulphur group from either cysteine (requiring MetABC), sulphide (requiring MetA and CysD) or methionine using the SAM recycling pathway (MetK, Pfs and LuxS) (Kovaleva & Gelfand, 2007).Homocysteine is then methylated by methionine synthase (MetE) in conjunction with a methylenetetrahydrofolate reductase (MetF), with the methyl group supplied by 5-methyltetrahydrofolate, to form methionine (Kovaleva & Gelfand, 2007).
Existing data show that methionine biosynthetic genes are required for the full virulence of B. melitensis (Lestrate et al., 2000), H. parasuis (Hill et al., 2003) and Salmonella enterica (Ejim et al., 2004), and that mutation of the S. agalactiae methionine regulator MtaR attenuates virulence (Shelver et al., 2003), suggesting methionine synthesis is essential for survival of many bacteria during invasive infection.These observations also suggest that CmhR might have a role in pneumococcal pathogenesis and further studies may shed more light on this.Our microarray results show that the genes discussed above are part of methionine biosynthesis and transport genes, and are differentially regulated in our methionine microarray.Therefore, further investigations of the CmhR regulon may provide valuable information regarding virulence mechanisms in S. pneumoniae, which could be very useful for devising strategies to combat pneumococcal infections.

Fig. 1 .Fig. 2 .
Fig. 1.Organization of the CmhR-regulated genes in S. pneumoniae D39.Black ovals represent the putative CmhR binding sites, whereas the lollipop structures represent the putative transcriptional terminators.Numbers below genes represent the number of base pairs of genes, whereas the number between putative binding sites and start of genes shows the number of bases between the translation start sites and the putative CmhR binding sites.See text for further details.

Fig. 3 .Fig. 4 .
Fig. 3. Identification of the CmhR regulatory site.(a) Weight matrix of the identified CmhR regulatory site in the promoter region of metQ, spd-0431, metE, folD, metB and fhs.(b) Position of the CmhR regulatory site in the promoter region of metQ, spd-0431, metE, folD, metB and fhs.Core promoter sequences are in bold, translational start sites are in italic and putative CmhR regulatory sites are bold-underlined.

Fig. 5 .Fig. 6 .
Fig. 5.The relative increase in the expression of metQ, spd-0431, metE, folD, fhs and metB in S. pneumoniae D39 wild-type compared to D39 DcmhR grown in CDM.Expression of metQ, spd-0431, metE, folD, fhs and metB was normalized with the housekeeping gene gyrA.Results represent the mean and standard deviation of three independent replicates.

Table 1 .
List of strains and plasmids used in this study

Table 2 .
Summary of transcriptome comparison of S. pneumoniae D39 wild-type grown in CDM with 0-10 mM methionine PTS, phosphotransferase system.
*Gene numbers refer to D39 locus tags.
Promoter regions of these genes were also examined in other streptococcal species (Streptococcus mitis, Streptococcus gordonii, Streptococcus mutans, Streptococcus thermophiles, Streptococcus uberis, Streptococcus agalactiae, Streptococcus gallolyticus, Streptococcus sanguinis and Streptococcus suis) to check if the CmhR regulatory site is also conserved in those streptococci.