Cysteine-Mediated Gene Expression and Characterization of the CmbR Regulon in Streptococcus pneumoniae

In this study, we investigated the transcriptomic response of Streptococcus pneumoniae D39 to cysteine. Transcriptome comparison of the D39 wild-type grown at a restricted concentration of cysteine (0.03 mM) to one grown at a high concentration of cysteine (50 mM) in chemically-defined medium (CDM) revealed elevated expression of various genes/operons, i.e., spd-0150, metQ, spd-0431, metEF, gshT, spd-0618, fhs, tcyB, metB-csd, metA, spd-1898, yvdE, and cysK, likely to be involved in the transport and utilization of cysteine and/or methionine. Microarray-based data were further confirmed by quantitative RT-PCR. Promoter lacZ-fusion studies and quantitative RT-PCR data showed that the transcriptional regulator CmbR acts as a transcriptional repressor of spd-0150, metEF, gshT, spd-0618, tcyB, metA, and yvdE, putatively involved in cysteine uptake and utilization. The operator site of CmbR in the promoter regions of CmbR-regulated genes is predicted and confirmed by mutating or deleting CmbR operator sites from the promoter regions of these genes.


INTRODUCTION
The major human pathogen Streptococcus pneumoniae colonizes the human nasopharynx and is the causal agent of many diseases, including pneumonia, sepsis, meningitis, and others. Pneumococcal nitrogen metabolism and regulation have been studied extensively as the appropriate acquisition and metabolism of nutrients are important for its lifestyle (Hendriksen et al., 2008). Sulfur is an integral part of many essential components of the cell, such as cysteine, methionine, thiamine, biotin, lipoic acid, coenzyme A, etc. Among these compounds, cysteine plays a key role, as it is the most important sulfur-containing compound-forming metabolite and its de novo synthesis signifies the central pathway of sulfur acquisition in microorganisms and plants (Sperandio et al., 2005). Many important proteins (such as cytochromes and aconitase) also have cysteine as an essential amino acid in their catalytic domains. Moreover, cysteine (and the dimer cysteine) helps in protein folding, assembly and stability, being involved in the formation of disulfide bounds. Cysteine-derived proteins (such as thioredoxin and glutathione) help in countering oxidative stress (Sperandio et al., 2005). Methionine is another sulfur-containing amino acid, regulating the initiation of translation and is vital to several methyl-transferase reactions (Sperandio et al., 2005). Microorganisms can synthesize methionine by converting homoserine to homocysteine through addition of a sulfur group from either cysteine (requiring MetABC), sulfide (requiring MetA and CysD) or by using the SAM (S-adenosylmethionine) recycling pathway (MetK, Pfs, and LuxS) (Kovaleva and Gelfand, 2007). Homocysteine is then methylated by methionine synthase (MetE) in conjunction with a methylenetetrahydrofolate reductase (MetF), with the methyl group supplied by 5methyltetrahydrofolate, to form methionine (Kovaleva and Gelfand, 2007).
Cysteine and methionine concentrations might be regulating bacterial growth in different conditions, such as pathogenic events or fermentation processes, as these amino acids have essential roles in metabolism (Schell, 1993). In Brucella melitensis (Lestrate et al., 2000), Haemophilus parasuis (Hill et al., 2003) and, Salmonella enterica (Ejim et al., 2004), sulfur-containing amino acid biosynthesis genes have been characterized as virulence factors. The cysDNC operon involved in the sulfate activation pathway forms a stress-induced operon in Mycobacterium tuberculosis (Pinto et al., 2004), whereas several thiol-and cysteine metabolism genes comprise the sigH regulon necessary for optimal existence of the bacterium in macrophages (Manganelli et al., 2002). Furthermore, cysteine metabolism also controls the regulation of toxin formation in Bordetella pertussis (Bogdan et al., 2001). Similarly, cysteine regulates a signaling molecule derivative of sulfur metabolism, autoinducer 2, which is conserved in both Gram-positive and -negative bacteria and is involved in interspecies communication and regulation of virulence factors (Sperandio et al., 1999;Marouni and Sela, 2003).
Our current study elucidates the effect of cysteine on the global gene expression of S. pneumoniae and characterizes the role of the transcriptional regulator CmbR in regulation of spd-0150, metEF, gshT, spd-0618, tcyB, metA, and yvdE. The transcriptional regulator CmbR acts as a transcriptional repressor for a number of genes/operons involved in cysteine uptake and utilization. The putative operator site (5 -GYGATAAAAAWWAYTTATMAC-3 where Y = T/C, W = A/T and M = A/C) of CmbR in the promoter regions of spd-0150, metEF, gshT, spd-0618, tcyB, metA, and yvdE is predicted and confirmed by promoter mutational/deletion experiments. Moreover, this site is found highly conserved in other pneumococcal strains and streptococci.

Bacterial Strains and Growth Conditions
Bacterial strains and plasmids used in this study are listed in Table 1. S. pneumoniae D39 was grown as described previously (Kloosterman et al., 2006a;Afzal et al., 2014). For β-galactosidase assays, derivatives of S. pneumoniae D39 were grown in a chemically defined medium (CDM) (Kloosterman and Kuipers, 2011) supplemented either with 0.03 or 50 mM cysteine. CDM was prepared without cysteine. For selection on antibiotics, the medium was supplemented with the following concentrations of antibiotics: tetracycline: 2.5 µg/ml for S. pneumoniae; ampicillin: 100 µg/ml for Escherichia coli and erythromycin: 0.25 µg/ml for S. pneumoniae and 120 µg/ml for E. coli. All bacterial strains used in this study were stored in 10% (v/v) glycerol at −80 • 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 S. pneumoniae D39 genome and are listed in Table 2.

Construction of a cmbR Mutant
A markerless cmbR mutant (MA1000) was constructed in S. pneumoniae D39 using pORI280, as described before (Kloosterman et al., 2006a). Primer pairs cmbR-1/cmbR-2 and cmbR-3/cmbR-4 were used to generate PCR fragments of the left and right flanking regions of cmbR. The integrity of the cmbR mutant (MA1000) was further confirmed by PCR and DNA sequencing.
β-galactosidase assays were performed as described before (Israelsen et al., 1995;Halfmann et al., 2007) using cells that were harvested in the mid-exponential growth phase, and grown in CDM supplemented either with 0.03 or 50 mM cysteine.

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 cysteine, the transcriptomes of S. pneumoniae D39 wild-type, grown in replicates in CDM with 0.03 mM cysteine, was compared to that grown in CDM with 50 mM cysteine and harvested at respective mid-exponential growth phases. For the identification of differentially expressed genes, a Bayesian p-value of <0.001 and a fold-change cut-off >1.5 was applied. RNA isolation was performed as described before (Afzal et al., 2015a). All other procedures regarding the DNA microarray experiments and data analysis were performed as previously described (Shafeeq et al., 2011a,b;Afzal et al., 2015b). Microarray data have been submitted to GEO under the accession number GSE89458.

Reverse Transcription (RT)-PCR and Purification for Quantitative RT-PCR
For quantitative RT-PCR, S. pneumoniae D39 wild-type and D39 cmbR were grown in replicates in CDM supplemented with either 0.03 mM or 50 mM cysteine. RNA isolation was done as described before (Afzal et al., 2015a). First, strand cDNA synthesis was performed on RNA (Shafeeq et al., 2011b). cDNA (2 µl) was amplified in a 20 µl reaction volume that contained 3 pmol of each primer ( Table 2) and the reactions were performed in three technical replicates on two biological replicates of RNA (Shafeeq et al., 2011b). The transcription level of specific genes was normalized to gyrA transcription, amplified in parallel with gyrA-F and gyrA-R primers. The results were interpreted using the comparative CT method (Schmittgen and Livak, 2008). Differences in expression of twofold or greater relative to control were considered as significant.

Cysteine-Dependent Gene Regulation in S. pneumoniae D39
Cysteine is one of the most important amino acids for bacteria. It is also present in human blood plasma at concentration of 0.03 mM (Lopez, 2013). To study the impact of cysteine on the transcriptome of S. pneumoniae D39 wild-type, we performed microarray comparison of S. pneumoniae D39 grown in CDM with 0.03-50 mM cysteine. 0.03 mM concentration was chosen, as this is the concentration of cysteine in human blood plasma (Lopez, 2013). 50 mM concentration of cysteine is normally used to prepare CDM. A number of genes/gene clusters were differentially regulated under our tested conditions ( Table 3). The expression of spd-0447-49 and spd-1098-99 was altered under our tested conditions. These genes belong to the glutamine regulon and their expression has been reported to be downregulated in the presence of a nitrogen source (Kloosterman et al., 2006b). The expression of important metal-related genes (prtA, psaBC and spd-1402) was downregulated under our tested conditions. These genes belong to the PsaR regulon and repressed by transcriptional regulator PsaR in the presence of manganese (Johnston et al., 2006). These genes have been shown to have role in virulence of pneumococcus (Kloosterman et al., 2008). Therefore, it might be interesting to further explore the role of cysteine in the regulation of these genes.

Confirmation of Cysteine-Dependent
Expression of spd-0150, metQ, spd-0431, metEF, gshT, spd-0618, fhs, tcyB, metB-csd, metA, spd-1898, yvdE, and cysK To confirm our microarray results and to study the expression of spd-0150, metQ, spd-0431, metEF, gshT, spd-0618, fhs, tcyB, metB-csd, metA, spd-1898, yvdE, and cysK under limiting cysteine concentration in CDM, we performed quantitative RT-PCR on these genes. Our quantitative RT-PCR results demonstrated that the expression of these genes was increased significantly in CDM with 0.03 mM cysteine, when compared to 50 mM (Figure 1). These data not only confirms our microarray results mentioned above, but also suggests a direct role of these genes in cysteine transport and biosynthesis.
There are three LysR-type transcriptional regulators in different bacteria, which have been shown to be involved in the regulation of sulfur amino acids (Sperandio et al., 2005(Sperandio et al., , 2010. S. pneumoniae also has two LysR-type transcriptional regulators (CmhR and CmbR), which are proposed to be involved in the regulation of the sulfur amino acids (Novichkov et al., 2010). Our recent study has revealed the regulatory mechanism of CmhR in S. pneumoniae and demonstrates that CmhR acts as a transcriptional activator of the fhs, folD, metB-csd, metEF, metQ, and spd-0431 in the presence of methionine (Afzal et al., 2016). The presence of CmbR (putative Cysteine Methionine Biosynthesis Regulator) in S. pneumoniae suggests its involvement in the regulation of cysteine-responsive genes. Therefore, we decided to further study the role of transcriptional regulator CmbR in the regulation of cysteine transport and biosynthesis genes.
Prediction of the CmbR Regulatory Site and the Role of CmbR as a Transcriptional Repressor of spd-0150, metEF, gshT, spd-0618, tcyB, metA, and yvdE The presence of cmbR in the S. pneumoniae genome suggests its involvement in the regulation of cysteine-responsive genes. cmbR codes for the putative transcriptional regulator CmbR, which belongs to the LysR family of proteins. CmbR is a homolog of a LysR-type regulator (also called FhuR) of Lactococcus lactis and Streptococcus mutans (Fernández et al., 2002;Sperandio et al., 2005Sperandio et al., , 2010. To study the role of CmbR in S. pneumoniae D39, we analysed the promoter regions of cysteine-regulated genes and predicted a 21-bp palindromic-like sequence (that has high homology with the FhuR binding site of L. lactis and S. mutans) in the promoter regions of spd-0150, metEF, gshT, spd-0618, tcyB, metA, and yvdE indicating that CmbR regulon in S. pneumoniae D39 is comprised of these genes. This DNA sequence might serve as the CmbR operator site in S. pneumoniae. The CmbR site present in the promoter regions of spd-0150, metEF, gshT, spd-0618, tcyB, metA, and yvdE is shown in Figure 2A. A weight matrix of these CmbR sites (5 -GYGATAAAAAWWAYTTATMAC-3 ) was constructed ( Figure 2B). Promoter regions of these genes were also examined in other streptococcal species (Streptococcus mitis, Streptococcus agalactiae, Streptococcus gallolyticus, Streptococcus gordonii, S. mutans, Streptococcus sanguinis, Streptococcus suis, and Streptococcus thermophilus) to check whether the CmbR site is also conserved in those streptococci. The CmbR site is highly conserved in these streptococci as well. Moreover, we constructed a phylogenetic tree of CmbR present in different streptococci, which shows that it is highly conserved in these streptococci (Figure 3).
bases in the cmbR sites were mutated in Pspd-0150 (5 -CCCATAAAAAAAACTTATCAC-3 to 5 -CCCGCGAAAAA AACTTATCAC-3 ), PmetE (5 -CTTATAAGAATTACTA ATAAC-3 to 5 -CTTGCAAGAATTACTAGCAAC-3 ), Pspd-0618 (R1: 5 -ATGGTAAAAAATCCTTATAAC-3 to 5 -ATGG CCAAAAATCCTGCGAAC-3 and R2: 5 -GCAATAAAAAA TAGATATTAT-3 to 5 -GCAGCGAAAAATAGAGCGTAT-3 ), and terminated in PmetA (CmbR site is deleted), and β-galactosidase assays were performed on cells grown in CDM with 50 mM cysteine. β-galactosidase assays data revealed that mutating the CmbR operator site in Pspd-0150 and PmetE, and deletion of the CmbR operator site in PmetA led to significantly increased expression of these promoter lacZ-fusions in the presence of 50 mM cysteine (Figure 6). These data confirm that the predicted CmbR sites present in the promoter regions of these genes are functional and intact in S. pneumoniae D39. Two putative operator sites of CmbR are present in Pspd-0618 (R1 and R2). We mutated both sites individually. We could only observe derepression (caused by CmbR) of Pspd-0618 when operator site 1 (R1) was mutated and did not witness any change in the activity of Pspd-0618 due to mutations in operator site 2 (R2). This suggests that only operator site 1 (R1) is a functional operator site for CmbR in Pspd-0618.

DISCUSSION
The sulfur-containing amino acids cysteine and methionine play essential role in various metabolic processes in the cell, especially because of their sulfur group, which plays a vital role in the catalytic sites of many enzymes and participates in ion-and redox metabolism (Ayala-Castro et al., 2008). Involvement of   multiple mechanisms in the regulation of these pathways in various groups of bacteria makes the regulatory phenomenon even more interesting, as it seems to evolve faster than that of many other regulatory pathways (Fernández et al., 2002;Sperandio et al., 2005Sperandio et al., , 2010. CmbR regulates most of the cysteine and methionine genes in L. lactis, where it binds to a 13-bp box centered 46-53 bp upstream of transcriptional start sites, with a second box with a same consensus sequence is located upstream of the first binding box (separated by 8-10 bp) (Fernández et al., 2002;Golic et al., 2005;Sperandio et al., 2005). In other members of the closely related Streptococcaceae family, the existence of FIGURE 5 | The relative increase in the expression of spd-0150, metEF, gshT, spd-0618, tcyB, metA, and yvdE in S. pneumoniae D39 cmbR compared to D39 wild-type grown in CDM with 50 mM cysteine. The expression of these genes was normalized with that of housekeeping gene gyrA. a different motif upstream of several potential cysteine genes (Kovaleva and Gelfand, 2007) suggests that the regulation of sulfur amino acid metabolism may be diverse. In this study, we studied the cysteine-dependent gene expression and the role of CmbR in S. pneumoniae D39, and demonstrated that CmbR acts as a transcriptional repressor of spd-0150, metEF, gshT, spd-0618, tcyB, metA, and yvdE.
The number of transcriptional factors regulating cysteine/methionine genes varies among different bacteria. This number is three in S. mutans (MetR, CysR and HomR), where these transcriptional regulators are phylogenetically related (Sperandio et al., 2010). CysR activates the transcription of cysK (codes for the cysteine biosynthesis enzyme), tcyABC, gshT (code for the cysteine and glutathione transporter systems), and homR. HomR is needed for the activation of metBC (code for the methionine biosynthesis enzymes), tcyDEFGH (involved in cysteine transport) and thiosulfate metabolism genes. Control of HomR by CysR emulates a cascade regulation for sulfur amino acid metabolism in S. mutans. Similarly, MtaR has been found to have a role in the regulation of the cysteine/methionine metabolism in S. agalactiae (Shelver et al., 2003). MetJ and MetR, regulate the expression of methionine biosynthetic genes in E. coli and S. enterica serovar Typhimurium (Weissbach and Brot, 1991). The E. coli met genes (except for metH) are negatively regulated by MetJ, a transcriptional repressor, with SAM (S-adenosylmethionine) serving as a co-repressor (Saint-Girons et al., 1988). These genes are also under the positive influence of a LysR-type transcriptional regulator MetR, with homocysteine as a co-effector (Cai et al., 1989;Mares et al., 1992;Cowan et al., 1993). CmbR in L. lactis also activates most genes involved in the methionine and cysteine biosynthesis pathway in the absence of cysteine (Sperandio et al., 2005). The regulatory proteins mentioned above belong to LysR family of transcription factors, which is the most abundant family of regulators in bacteria (Maddocks and Oyston, 2008). These 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 activators or repressors, and often are transcribed divergently with one of the regulated genes (Schell, 1993). LysR-family transcriptional regulators consist of two characteristic domains, an N-terminal HTH DNA binding domain (PF00126) and a C-terminal substrate binding domain (PF03466). There are two transcriptional regulators in S. pneumoniae that control the expression of the cysteine and methionine genes (CmhR and CmbR) (Novichkov et al., 2010). CmhR in S. pneumoniae belongs to the LysR family of transcriptional factors and has an HTH (helix-turn-helix) domain and a substrate binding domain of LysR-type transcriptional regulators (LTTRs). CmhR acts as a transcriptional activator of the fhs, folD, metB, metEF, metQ, and spd-0431 genes and a 17-bp palindromic sequence (5 -TATAGTTTSAAACTATA-3 ) is present in the promoter regions of CmhR-regulated genes in S. pneumoniae D39 (Afzal et al., 2016). There are two methionine transport systems [the methionine ABC uptake transporter (MUT)] (Merlin et al., 2002;Hullo et al., 2004) and a secondary transporter BcaP (den Hengst et al., 2006) in bacteria. metD codes for the MUT system in E. coli and also have the MetQ substrate binding protein (SBP), MetL trans-membrane permease and the MetN cytoplasmic ATP hydrolyzing protein (ATPase) (Merlin et al., 2002). The spd-0150-54 locus codes for a methionine uptake ABC transporter in S. pneumoniae D39 and deletion of the gene encoding the lipoprotein MetQ resulted in a strain that had hampered growth in methionine-restricted medium and no measurable uptake of radioactive methionine (Basavanna et al., 2013). Furthermore, deletion of locus encoding MetEF (which is also part of the CmhR regulon) resulted in an increase in the growth defect of the metQ deletion strain in methionine-restricted medium and in blood plasma, reinforcing a role for the products of these genes in methionine synthesis (Basavanna et al., 2013). CmhR-regulated genes have important roles in the transport and biosynthesis of methionine. Csd and MetE are part of methionine synthesis pathway as Csd coverts cystathionine into homocysteine and MetE converts homocysteine into methionine (Kanehisa et al., 2014). Cystathionine and homocysteine can also be formed from homoserine, where O-acetyl-L-homoserine is converted into cystathionine by MetB. O-acetyl-L-homoserine can also be converted into homocysteine by MetB and SPD-1073-74 (spd-1073-1074 encode an O-acetylhomoserine aminocarboxypropyltransferase/cysteine synthase and a hypothetical protein, respectively) (Kanehisa et al., 2014). Methionine can also be synthesized by other microbes as they may convert homoserine to homocysteine through addition of a sulfur group from either cysteine (involving MetABC), sulfide (involving MetA and CysD) or by using the SAM recycling pathway (MetK, Pfs and LuxS) (Kovaleva and 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 (Ravanel et al., 1998;Kovaleva and Gelfand, 2007).
CmbR is the second one and acts as transcriptional repressor for spd-0150, metEF, gshT, spd-0618, tcyB, metA, and yvdE. The deletion of CmbR led to a significant increase in the activity of the CmbR-regulated promoters. Thus, it represents a different mode of regulation of the cysteine/methionine genes than in other related streptococci.
A number of metal-related genes are found to be differentially expressed in our tested conditions. These genes include psaBC and prtA, which belong to the PsaR regulon. psaBCA encode a Mn 2+ -dependent ABC transporter PsaBCA and prtA codes for a serine protease PrtA. The expression of these genes was also altered in our recent transcriptome of S. pneumoniae D39 grown in CDM with 0-10 mM methionine (Afzal et al., 2016). The DtxR-family transcriptional regulator PsaR represses the expression of the PsaR regulon in the presence of Mn 2+ (Johnston et al., 2006). Zn 2+ , Ni 2+ and Co 2+ have been demonstrated to bind with PsaR and relieve the Mn 2+ -dependent repression of the PsaR regulon (Kloosterman et al., 2008;Manzoor et al., 2015a,b). The interplay or competition of metal ions plays a significant role in the regulation of metal-responsive genes. Competition of Mn 2+ with Zn 2+ , Co 2+ , or Ni 2+ in the regulation of the PsaR regulon by transcriptional regulator PsaR has already extensively been studied in S. pneumoniae (Kloosterman et al., 2008;Manzoor et al., 2015a,b). The significant changes in the expression of some of these metalresponsive genes in our study indicate the involvement of S-containing amino acids in this interplay as well, in an as yet unknown way.

AUTHOR CONTRIBUTIONS
MA planned the experiments, performed experiments and wrote the manuscript. SS planned the experiments and wrote the manuscript. IM performed the experiments. OK planned the experiments and wrote the manuscript.