Cryptic Streptococcus mutans 5.6-kb plasmids encode a toxin–antitoxin system for plasmid stabilization

Abstract

BackgroundIn all Streptococcus mutans strains, 5–13% carry a 5.6-kb plasmid. Despite its frequency, little is known about its mediated functions with most of the information coming from a single study focussing on plasmid pUA140.ObjectiveHere, we describe the sequence and genetic organization of two S. mutans 5.6-kb plasmids, pDC09 and pNC101.ResultsBased on PicoGreen dsDNA quantification and Real-Time quantitative PCR (RTQ-PCR), the plasmid copy number was found to range between 10 and 74, depending on the strain tested. In contrast to literature, we identified six instead of five open reading frames (ORFs). While the putative gene products of ORF1 (as a Rep-protein) and ORF2 (as a Mob-protein) could be confirmed as being identical to those from pUA140, the functions of ORF3 (unknown) and ORF 4 (possibly AtpE homologue) could not be further revealed. However, the product of ORF5 showed a fairly high identity (38–50%) and structural similarity (58–74%) to RelE of Streptococcus pneumoniae, Streptococcus equi, and Streptococcus downei. In addition, we identified a functionally corresponding ORF6 encoding a protein with 61–68% identity (81–86% similarity) to the S. equi and S. downei antitoxin of the RelB family. RelE and RelB together form a plasmid-encoded toxin-antitoxin (TA) system, RelBEplas. Despite its rather limited sequence similarity with chromosomal TA systems in S. mutans (RelBEchro, MazEF, HicBA), we found similar tertiary structures applying I-Tasser protein prediction analysis.ConclusionType II-toxins, as the plasmid-encoded RelE, are RNA endonucleases. Depending on their mRNA cleavage activity, they might 1) kill every plasmid-free progeny, thereby stabilizing plasmid transfer at the expense of the host and/or 2) help S. mutans enter a dormant state and survive unfavourable environmental conditions. Whilst a function in plasmid stabilization has been confirmed, a function in persistence under nutritional stress, tested here by inducing amino acid starvation, could not be demonstrated so far.

Pathophysiology of Dental Caries

“…From the microbiological point of view, this approach is tolerable as electron transport within, and acid production by, the few cells is also very low in this zone. However, bacteria have several strategies to overcome harsh conditions andafter preparation, disinfection if applicable, infiltration if applicable, and restoring -might still be alive although in a dormant state [16,17] . This means the lesion and the bacteria are arrested, but only temporarily.…”

Investigating CRISPR spacer targets and their impact on genomic diversification of Streptococcus mutans

“…A spacer from smu333 mapped to pLF25067 carried by Lactobacillus fermentum [36] and a spacer from smu408 mapped to pSZ4 from Staphylococcus warneri [37]. One strain, smu441, carries a spacer that maps (88% coverage) against a small 5.6 kbp ‘cryptic’ plasmid that is carried by ∼5% of S. mutans strains [20, 21].…”

“…Plasmid sequences were downloaded from a database containing 10,892 complete and annotated plasmids [19]. In addition, known S. mutans plasmid sequences were compiled (available on the Github platform) [20][21][22]. Parameters for Blastn were set to 95% sequence coverage (qcovs) and 85% sequence identity (pident).…”

Investigating CRISPR spacer targets and their impact on genomic diversification of Streptococcus mutans

“…Plasmid sequences were downloaded from a database containing 10,892 complete and annotated plasmids (Brooks et al, 2019). In addition, known S. mutans plasmid sequences were compiled (available on the Github platform) (Zhou et al, 2001;Caufield et al, 2007;Rheinberg et al, 2013). Parameters for Blastn were set to 95% sequence coverage (qcovs) and 85% sequence identity (pident).…”