Expanding the Vocabulary of Peptide Signals in Streptococcus mutans

Abstract

Streptococci, including the dental pathogen Streptococcus mutans, undergo cell-to-cell signaling that is mediated by small peptides to control critical physiological functions such as adaptation to the environment, control of subpopulation behaviors and regulation of virulence factors. One such model pathway is the regulation of genetic competence, controlled by the ComRS signaling system and the peptide XIP. However, recent research in the characterization of this pathway has uncovered novel operons and peptides that are intertwined into its regulation. These discoveries, such as cell lysis playing a critical role in XIP release and importance of bacterial self-sensing during the signaling process, have caused us to reevaluate previous paradigms and shift our views on the true purpose of these signaling systems. The finding of new peptides such as the ComRS inhibitor XrpA and the peptides of the RcrRPQ operon also suggests there may be more peptides hidden in the genomes of streptococci that could play critical roles in the physiology of these organisms. In this review, we summarize the recent findings in S. mutans regarding the integration of other circuits into the ComRS signaling pathway, the true mode of XIP export, and how the RcrRPQ operon controls competence activation. We also look at how new technologies can be used to re-annotate the genome to find new open reading frames that encode peptide signals. Together, this summary of research will allow us to reconsider how we perceive these systems to behave and lead us to expand our vocabulary of peptide signals within the genus Streptococcus.

Keywords: LC-MS/MS; RNA-Seq; Ribo-Seq; bacterial communication; cell-to-cell signaling; genetic competence; peptides; transformation.

Figure 1

Current view of the genetic competence pathway in S. mutans. Competence activation is centrally mediated by the ComRS system, consisting of the Rgg-like transcriptional regulator ComR, and ComS, a 17-aa precursor of the 7-aa XIP peptide. ComS is processed into XIP and exported by unknown mechanism(s), potentially facilitated by dedicated transport systems such as PptAB or through cell lysis. Externalized XIP can activate nearby cells in an intercellular signaling mechanism or is re-imported by oligopeptide permeases (Opp) in an intracellular signaling manner. Cytosolic ComS or XIP interacts with the transcriptional regulator ComR, forming the ComR-XIP complex. The complex recognizes a select palindromic sequence termed the ComR-box, located within the promoter of both comX and comS, the latter creating a positive feedback loop for the system. Several negative regulators can also impact ComRS signaling, including decreased environmental pH and XrpA, located intragenically within the comX coding sequence on a separate reading frame that inhibits ComRS signaling through direct interaction with ComR. After competence activation, the alternative sigma factor ComX directs the RNA polymerase to a regulon consisting of late competence genes whose products make up the machinery needed for DNA uptake and potential homologous recombination of the single stranded DNA into the genome. However, the RcrRPQ operon, that is negatively regulated by RcrR, can shut down ComX production through overexpression of the ABC transporters RcrP and RcrQ that results in the processing/degradation of comX mRNA. Additionally, two peptides located at the 3′ end of the rcrQ coding region termed Pep1 and Pep2 are believed to modulate competence signaling through interactions with the (p)ppGpp enzymes RelA and RelP. Several distal regulators, including two component systems ComDE, ScnKR, and HdrMR, membrane protein DivIB and (p)ppGpp concentration mediated by the dual synthetase/hydrolase enzyme RelA can impact ComRS signaling.

Figure 2

(A) Genomic organization surrounding the rcrRPQ operon of S. mutans. Genes are divided into their respective reading frames. rcrR encodes for a MarR-like transcriptional regulator that negatively regulates the transcription of the operon. rcrP and rcrQ are ABC-type multidrug/protein/lipid transporters. At the end of rcrQ are two small peptides encoded in separate reading frames. The start codon of Pep 1 overlaps with the stop codon of rcrQ. Pep 2's reading frame begins inside the rcrQ coding sequence and overlaps with the beginning of Pep 1. Downstream of the rcrRPQ operon is tpx, a thiol peroxidase, and cipI, a bacteriocin immunity protein. Finally relP, a small alarmone (p)ppGpp synthetase, along with the two component system relRS are further downstream. Mutations within rcrRPQ effect the expression of the relPRS operon and vice versa. Red lines denote operon structures within the region. (B) Two different mutants of rcrR are mainly used to investigate the operon that differ only by the presence of a transcriptional terminator (red box) within the antibiotic resistance cassette used to replace the gene. The ΔrcrR-P strain expresses the rcrP and rcrQ transporters at wild-type levels due to the presence of the strong transcriptional terminator and is hyper-transformable in terms of genetic competence, while the ΔrcrR-NP strain produces the transporters at 100x higher than wild-type background, leading to a non-transformable state. Differences in transformability between ΔrcrR-P and ΔrcrR-NP is the result of an altered comX transcript between the two strains. RNA-Seq read counts (white boxes) between the 5′ and 3′ ends of comX are even in the ΔrcrR-P background and a full transcript is produced. However, in the ΔrcrR-NP background, reads only map to the 3′ end and a smaller transcript is produced that corresponds to the intragenic coding region of xrpA that led to its discovery.

Figure 3

Description of potential coding open reading frames (ORFs) within the S. mutans UA159 genome. The S. mutans UA159 genome was downloaded from NCBI (Biosample SAMN02604090, Accession PRJNA333) with all annotations and loaded into Geneious (v 11.1.4). All ORFs (>100 nucleotides) were predicted with Geneious. (A) Chord plot showing the relationship between the type of predicted ORF and the direction (forward or reverse strand). From the total of 18,276 predicted ORFs, around 69% (12,599 ORFs) are completely intragenic or within another coding sequence. The count of ORFs that overlap with another on its 5′ end is 23% (4231 ORFs) and < 0.001% (1 ORF of length 123 bp) that overlap on its 3′ end. Finally, 8% (1445 ORFs) are located entirely within intergenic regions. Regarding the orientation of the ORFs, from the plot it can be concluded that roughly half of the total ORF count are oriented in opposite directions. Chord plot was generated with the function “chordDiagram” from the R package “circlize” (Gu et al., 2014). (B) Histogram of the nucleotide length distribution for intergenic, left, and right overlapping ORFs. Histogram was generated with the R package “ggplot2” (Wickham, 2016).