Comprehensive Transcriptome Profiles of Streptococcus mutans UA159 Map Core Streptococcal Competence Genes

Abstract

In Streptococcus mutans, an oral colonizer associated with dental caries, development of competence for natural genetic transformation is triggered by either of two types of peptide pheromones, competence-stimulating peptides (CSPs) (18 amino acids [aa]) or SigX-inducing peptides (XIPs) (7 aa). Competence induced by CSP is a late response to the pheromone that requires the response regulator ComE and the XIP-encoding gene comS. XIP binds to ComR to allow expression of the alternative sigma factor SigX and the effector genes it controls. While these regulatory links are established, the precise set of effectors controlled by each regulator is poorly defined. To improve the definition of all three regulons, we used a high-resolution tiling array to map global changes in gene expression in the early and late phases of the CSP response. The early phase of the CSP response was limited to increased gene expression at four loci associated with bacteriocin production and immunity. In the late phase, upregulated regions expanded to a total of 29 loci, including comS and genes required for DNA uptake and recombination. The results indicate that the entire late response to CSP depends on the expression of comS and that the immediate transcriptional response to CSP, mediated by ComE, is restricted to just four bacteriocin-related loci. Comparison of the new data with published transcriptome data permitted the identification of all of the operons in each regulon: 4 for ComE, 2 for ComR, and 21 for SigX. Finally, a core set of 27 panstreptococcal competence genes was identified within the SigX regulon by comparison of transcriptome data from diverse streptococcal species. IMPORTANCES. mutans has the hard surfaces of the oral cavity as its natural habitat, where it depends on its ability to form biofilms in order to survive. The comprehensive identification of S. mutans regulons activated in response to peptide pheromones provides an important basis for understanding how S. mutans can transition from individual to social behavior. Our study placed 27 of the 29 transcripts activated during competence within three major regulons and revealed a core set of 27 panstreptococcal competence-activated genes within the SigX regulon.

Keywords: CSP; Streptococcus; XIP; genetic competence; natural transformation systems; pheromone; quorum sensing.

FIG 1

Competence regulatory networks in S. mutans. Regulatory links are organized into two pheromone response networks acting in peptide-rich media such as THB (A) or in peptide-free CDM (B). (A) In rich media, extracellular CSP induces the expression of bacteriocin and immunity proteins (red) through the ComED TCSTS pathway. Other genes are possibly induced by direct or indirect activation by phosphorylated ComE. The bacteriocins are thought to create pores that allow internalization of the competence pheromone XIP. XIP binds to ComR to activate the expression of at least two genes (green), comS (encoding XIP) and sigX (encoding the alternative sigma factor σ^x). Induction of comS creates a positive feedback loop that increases the production of XIP and σ^x, while σ^x activates the expression of genes (blue) involved in DNA integration (e.g., comGA, comEC, and dprA). (B) In peptide-free CDM, extracellular XIP is internalized via the Opp permease and binds to ComR, activating the expression of at least two genes, comS (encoding XIP) and sigX. Induction of comS creates a positive feedback loop, while elevated σ^x activates the expression of genes involved in DNA integration (e.g., comGA, comEC, and dprA). Bacteriocin and immunity proteins are upregulated as a result of comED induction by σ^x. Uncertainties about the regulon assignment for the full range of genes that change expression in response to the pheromones are represented by question marks, and genes upregulated but not exemplified are represented by ellipses.

FIG 2

Effect of CSP18 on induction of competence and expression of cipB, sigX, comGA, and comEC during growth in TSB. (A) During growth of S. mutans UA159 in the presence (●) or absence (○) of CSP, transformation efficiency (solid line) and OD₆₀₀ (broken line) were determined at the times indicated. Exposure to pVA838 was for 20 min and was followed by further incubation with DNase I for 40 min before plating. Arrows show the times of sample harvesting for microarray analysis. (B) Luciferase reporters were used in parallel cultures to measure the effect of CSP on the expression of cipB (nlmC, SMU.1914) (triangles) and sigX (squares). The strains were grown in the presence of 50 nM CSP (filled symbols) or without CSP (empty symbols). CSP was added at t = 0. Relative light units (RLU) and OD₆₀₀ were measured in a 96-well plate with a multidetection microplate reader (SynergyHT; BioTek). Expression levels are relative to the highest value of each reporter (100%). Error bars indicate standard deviations of triplicate assays. (C to E) Relative expression of selected early responsive gene cipB (C) and late responsive genes comGA and comEC (D and E). Real-time PCR data were normalized to the expression values of the respective genes in the WT strain at 5 min without CSP. Mean values and standard errors for three replicates at 5, 10, and 100 min without CSP (red bars) and with CSP (blue bars) are shown.

FIG 3

Genetic organization of TARs assigned to ComE, ComR, and SigX regulons. Operons (gray boxes) and terminators (black pins) are indicated as predicted by DOOR (38). The arrow borders of the genes in the ComE, ComR, and SigX regulons are red, green, and blue, respectively. The four TARs induced at 10 min in the WT and at 100 min in the comS mutant belong to the ComE regulon and comprise genes encoding bacteriocins (red), bacteriocin immunity proteins (yellow), and hypothetical proteins (gray). The 25 TARs induced only in the WT at 100 min (late response) belong to the ComR regulon (2 TARs), the SigX regulon (21 TARs), or unassigned regulons (others; 2 TARs). These include genes with products acting in autolysis (red), DNA transport (sky blue), recombination (green), and DNA methylation (orange) and genes that encode hypothetical proteins (gray). Three late genes encode key regulatory elements in the CSP response pathway (dark blue), SigX, ComS (ComR activator), and ComED. Binding site consensus elements are indicated by pennants for ComE (E), ComR (R), and SigX (X). Genes upregulated in the antisense direction are black. The mean fold changes in the expression of ORFs (black numbers) and intergenic regions (red umbers) for the WT at 10 min (ComE regulon) and 100 min (ComR, SigX, and other regulons) are shown above the black arrows indicating the directions of the transcripts. The images shown were drawn with Easyfig (70).

FIG 4

Correlations between gene expression changes induced by short (10 min) or long (100 min) exposure to CSP in S. mutans UA159 or by long exposure to CSP in the comS mutant. Fold changes are shown as log₂ values for all induced ORF sequences in the S. mutans UA159 genome (accession no. AE014133) and represent mean values for comparisons of CSP-treated and untreated samples from two independent biological experiments. (A) Correlation of ratios of short CSP exposure of S. mutans UA159 to long CSP exposure of the comS mutant. The same genes were upregulated under the two conditions, with two exceptions (inner dashed circle). These are grouped as early genes. (B) Same correlation as for panel A but for changes in antisense transcripts. (C) Correlation of ratios of long CSP exposure of the comS mutant to long CSP exposure of the sigX mutant in reference 15 reveals candidate genes for the ComR regulon (dashed circle). (D) Correlation of induction ratios of long CSP exposure of S. mutans UA159 to the comS mutant formed two groups. Early genes, as in panel A, were induced in both transcriptomes, and late genes were those induced only in UA159 upon long CSP exposure. (E) Correlations as in panel D but for antisense transcripts. Early genes are defined as those induced by short CSP exposure in WT UA159 and by long CSP exposure in the comS mutant (red borders), and late genes are defined as those that showed induction in the WT only upon long CSP exposure (green borders for putative genes of the ComR regulon and blue borders for the remaining genes). Circles corresponding to genes encoding upregulated bacteriocins are filled in red, circles corresponding to bacteriocin immunity proteins are filled in yellow, circles corresponding to proteins involved in DNA uptake are filled in light blue, and circles corresponding to proteins involved in DNA recombination are filled in green. The points corresponding to comE, comD, comS, and sigX are indicated by name (D; dark blue). Light-gray-filled circles correspond to other genes. Multiplot v.2 was used to draw the scatterplots (http://www.broadinstitute.org/cancer/software/genepattern/modules/docs/Multiplot/2).

FIG 5

Putative regulatory sites at early and late CSP-induced loci. (A, top) Alignment of DNA sequences upstream of clusters of early genes. The presumptive canonical promoter −10 site is blue, bases corresponding to the DR consensus sequences are underlined, and the putative transcription start site is red (18). The previously suggested SigX box of SMU.925 is boxed (39). (A, bottom) Transcriptome map showing predicted ComE binding sites (15, 40). Vertical arrows show the ComE DR sites that appeared to be active (orange DR) and sites that did not appear as active (black DR). Conditions A and B are 10 min of CSP and 10 min of no CSP, respectively. (B, top) Alignment of DNA sequences upstream of sigX and comS. These are late CSP-induced loci upregulated by ComRS. The presumptive canonical promoter −10 site is blue, and conserved IRs known as the ComR box are bold (6). (B, bottom) Transcriptome maps at comS and sigX. Vertical arrows indicate the ComR IR sites (6). Conditions A and B are 100 min of CSP and 100 min of no CSP, respectively. Probe intensities for the comS region were above the threshold used in the visualizer program. (C, top) Alignment of DNA sequences with putative SigX boxes. The SigX boxes are in bold. Superscript letters: a, SigX box within SMU.60; b, induction in the antisense direction; c, putative SigX boxes (39) not confirmed in our transcriptome. (C, bottom) Transcriptome map showing locations of predicted SigX boxes. Vertical arrows show the sites of SigX boxes that appeared to be active. Conditions A and B are 100 min of CSP and 100 min of no CSP, respectively. Changes in gene expression are presented as log₂ ratios (condition A/condition B), with ratios of >0 in green and ratios of <0 in red. The signal intensity of each probe (50-mer) is presented as a horizontal line in green for condition A and in orange for condition B. S. mutans locus tags are as in the sequence with GenBank accession no. AE014133 (>, transcript in the forward strand showing probe intensities above the locus tag boxes; <, transcript in the reverse strand showing probe intensities below the locus tag boxes). The vertical lines are separated by a distance of 100 bp. The complete maps are available at http://bioinformatics.forsyth.org/mtd/dataset=RNAseq_smut_comS.

FIG 5

Putative regulatory sites at early and late CSP-induced loci. (A, top) Alignment of DNA sequences upstream of clusters of early genes. The presumptive canonical promoter −10 site is blue, bases corresponding to the DR consensus sequences are underlined, and the putative transcription start site is red (18). The previously suggested SigX box of SMU.925 is boxed (39). (A, bottom) Transcriptome map showing predicted ComE binding sites (15, 40). Vertical arrows show the ComE DR sites that appeared to be active (orange DR) and sites that did not appear as active (black DR). Conditions A and B are 10 min of CSP and 10 min of no CSP, respectively. (B, top) Alignment of DNA sequences upstream of sigX and comS. These are late CSP-induced loci upregulated by ComRS. The presumptive canonical promoter −10 site is blue, and conserved IRs known as the ComR box are bold (6). (B, bottom) Transcriptome maps at comS and sigX. Vertical arrows indicate the ComR IR sites (6). Conditions A and B are 100 min of CSP and 100 min of no CSP, respectively. Probe intensities for the comS region were above the threshold used in the visualizer program. (C, top) Alignment of DNA sequences with putative SigX boxes. The SigX boxes are in bold. Superscript letters: a, SigX box within SMU.60; b, induction in the antisense direction; c, putative SigX boxes (39) not confirmed in our transcriptome. (C, bottom) Transcriptome map showing locations of predicted SigX boxes. Vertical arrows show the sites of SigX boxes that appeared to be active. Conditions A and B are 100 min of CSP and 100 min of no CSP, respectively. Changes in gene expression are presented as log₂ ratios (condition A/condition B), with ratios of >0 in green and ratios of <0 in red. The signal intensity of each probe (50-mer) is presented as a horizontal line in green for condition A and in orange for condition B. S. mutans locus tags are as in the sequence with GenBank accession no. AE014133 (>, transcript in the forward strand showing probe intensities above the locus tag boxes; <, transcript in the reverse strand showing probe intensities below the locus tag boxes). The vertical lines are separated by a distance of 100 bp. The complete maps are available at http://bioinformatics.forsyth.org/mtd/dataset=RNAseq_smut_comS.

FIG 5

Putative regulatory sites at early and late CSP-induced loci. (A, top) Alignment of DNA sequences upstream of clusters of early genes. The presumptive canonical promoter −10 site is blue, bases corresponding to the DR consensus sequences are underlined, and the putative transcription start site is red (18). The previously suggested SigX box of SMU.925 is boxed (39). (A, bottom) Transcriptome map showing predicted ComE binding sites (15, 40). Vertical arrows show the ComE DR sites that appeared to be active (orange DR) and sites that did not appear as active (black DR). Conditions A and B are 10 min of CSP and 10 min of no CSP, respectively. (B, top) Alignment of DNA sequences upstream of sigX and comS. These are late CSP-induced loci upregulated by ComRS. The presumptive canonical promoter −10 site is blue, and conserved IRs known as the ComR box are bold (6). (B, bottom) Transcriptome maps at comS and sigX. Vertical arrows indicate the ComR IR sites (6). Conditions A and B are 100 min of CSP and 100 min of no CSP, respectively. Probe intensities for the comS region were above the threshold used in the visualizer program. (C, top) Alignment of DNA sequences with putative SigX boxes. The SigX boxes are in bold. Superscript letters: a, SigX box within SMU.60; b, induction in the antisense direction; c, putative SigX boxes (39) not confirmed in our transcriptome. (C, bottom) Transcriptome map showing locations of predicted SigX boxes. Vertical arrows show the sites of SigX boxes that appeared to be active. Conditions A and B are 100 min of CSP and 100 min of no CSP, respectively. Changes in gene expression are presented as log₂ ratios (condition A/condition B), with ratios of >0 in green and ratios of <0 in red. The signal intensity of each probe (50-mer) is presented as a horizontal line in green for condition A and in orange for condition B. S. mutans locus tags are as in the sequence with GenBank accession no. AE014133 (>, transcript in the forward strand showing probe intensities above the locus tag boxes; <, transcript in the reverse strand showing probe intensities below the locus tag boxes). The vertical lines are separated by a distance of 100 bp. The complete maps are available at http://bioinformatics.forsyth.org/mtd/dataset=RNAseq_smut_comS.

FIG 6

Comparison of gene expression maps from available S. mutans competence transcriptome studies. The directions of the activated TARs (arrows) from S. mutans exposed for 100 min to CSP are indicated by arrows as follows: red, TARs of the ComE regulon; green, the ComR regulon; blue and black, TARs of the SigX regulon with 5′ ends starting in the sense and antisense directions, respectively. The genes proximal to the ComE DR are red, those proximal to the ComR indirect repeat are green, and those proximal to the SigX box are blue. Gene numbers are as in the sequence with GenBank accession no. AE014133. Gene expression data for corresponding regions are indicated for five transcriptome studies, as indicated at the left. The top row is UA159 (100 min). The transcriptomes in studies 1, 3, and 5 used traditional microarrays with probes limited to annotated ORFs (15, 32, 39). The transcriptome in study 2 used a tiling microarray covering most of the S. mutans genome (28). Study 4 used RNA sequencing in the analysis of the response to CSP and XIP in CDM (27). The CSP response in this medium does not support activation of competence but is included for comparative analysis of early responses. The transcriptome for XIP in CDM used a 30-min exposure to XIP, which was probably too short to detect the full range of core genes regulated by SigX. The comS gene was not represented in the arrays for the transcriptomes in studies 1, 2, 3, and 5 (empty space).

FIG 7

Core genes of the SigX regulon. Conservation of synteny to S. mutans SigX-induced TARs (top row) in S. pneumoniae, S. gordonii, S. sanguinis, S. pyogenes, and S. thermophilus. Red pentagons correspond to genes immediately downstream of a SigX box. Competence-induced genes, black border; upregulated genes oriented antisense to the gene proximal to the SigX box, dashed black border; no change in gene expression, borderless faded colors; orthologs in each of the 15 groups, similar colors; no orthologs in the regions analyzed, gray. Core genes in the DpnII group are shown within dashed squares. The induced lytF gene in S. mutans is also induced in S. gordonii and S. sanguinis and encodes a CHAP domain found in CbpD, an induced nonortholog in S. pneumoniae, S. thermophilus, and S. pyogenes. LytF and CbpD have conserved lytic functions in the different species. Three other transcripts induced in S. mutans are not shown, as they have no matches in the other species (SMU.1400, comE) or are internal to an ORF (3′ end of SMU.60). Orthology is as annotated at KEGG (http://www.genome.jp/kegg/). Note that for all of the transcriptomes compared with S. mutans, there is no information on TAR polarity or length. SP¹, S. pneumoniae Rx (13); gene locus tag as in GenBank (S. pneumoniae TIGR4, accession no. AE005672). SP², S. pneumoniae R6 (44); gene locus tag as in GenBank (S. pneumoniae TIGR4, accession no. AE005672). SP³, S. pneumoniae G54 (56) DpnII restriction-methylase group (competence transcriptome not available); gene locus tag as in GenBank (S. pneumoniae G54, accession no. CP001015). SGO⁴, S. gordonii Challis (14); gene locus tag as in GenBank (accession no. CP000725). SSA⁵, S. sanguinis SK36 (45); gene locus tag as in GenBank (accession no. CP000387.1). SPy⁶, S. pyogenes MGAS315 (9) and derivative strain D471 (46); gene locus tag as in GenBank (S. pyogenes M1 GAS SF370, accession no. AE004092.2). Shown are S. pyogenes induced genes in one or both studies. STER⁷, S. thermophilus LMD-9 (43); gene locus tag as in GenBank (accession no. GCA_000014485.1). ^aSMU.354 is homologous to ccs50, but the SigX box is distal to SMU.352. ^b,c,dUpregulation of comFC, cilC, and coiA was not detected in the S. sanguinis transcriptome but is essential for competence (45). ^eSSA.2233 is distal to the SigX box but not homologous to SMU.2076.

FIG 8

Model of the transcriptional organization of the S. mutans response to CSP in peptide-rich medium. In step 1, extracellular CSP activates the two-component system ComED, which then induces the expression of four operons comprising several bacteriocin and bacteriocin immunity genes. The bacteriocins are suggested to create pores in the membrane that allow the entrance of the XIP pheromone into the cells. In step 2, XIP binds to ComR, which then assumes a conformation that activates the expression of two operons, one initiated at comS, creating a positive feedback loop, and the other initiated at sigX, activating the competence response. In step 3, SigX activates the expression of 22 operons, including 4 sense transcripts that are not seen in other streptococcal transcriptomes (noncore), 3 transcripts in the antisense direction (not yet investigated in other streptococci), and 15 operons encoding genes that are in the core of the streptococcal SigX response. Three groups of core genes were identified: (i) core orthologs found in at least four of the available transcriptomes, (ii) core orthologs found only in strains that belong to the DpnII group of restriction-modification systems, and (iii) core nonorthologs that have in common a conserved domain associated with similar functions (in this class are CbpD of S. pneumoniae and S. thermophilus and LytF of S. mutans, S. gordonii, and S. sanguinis, both of which have the lytic CHAP domain in common).