Investigating the function and regulation of two CRISPR systems of Streptococcus mutans.

摘要

CRISPR-Cas (clustered, regularly interspaced short palindromic repeats--CRISPR-associated proteins) provide adaptive microbial immunity systems against invading viruses and natural transformation via plasmids. Naturally competent Streptococcus mutans UA159 harbors two CRISPR-Cas systems: type II-A system (CRISPR1) and type I-C system (CRISPR2) and several spacers matching sequences of phage M102 or genomic sequences of other S. mutans strains. In addition, previous transcriptome studies in S. mutans linked CRISPR/Cas systems to stress response and virulence. The goal of this work was to determine the role of CRISPR/Cas systems in phage defense and natural transformation in S. mutans, and also to investigate if they play additional functions in the cell physiology. Deletion of CRISPR1 and/or CRISPR2 cas genes or removal of the spacers in S. mutans UA159 did not affect its M102 phage resistant phenotype, suggesting that CRISPR-independent mechanisms contribute to the phage resistance. Using a plasmid-based interference assay, we identified DNA interference activity in S. mutans UA159, which is mediated by its type II-A CRISPR/Cas system (CRISPR1). Spacers 2 and 3 from type II-A, both matching sequences from phage M102, were found to be essential for CRISPR interference against engineered plasmids containing matching proto-spacer sequences. Functional analysis of the cas deletion mutants revealed that CRISPR1-Cas system modulates stress tolerance induced by low pH, high temperature, oxidative and cell membrane stress, as well as DNA damaging conditions, whereas the CRISPR2-Cas participates in the tolerance associated with heat shock. Transcriptional analysis identified that VicR/K two-component signal transduction system differentially regulates the expression of cas genes for both systems in S. mutans. Further, structural, biochemical and functional studies found that the Cas5d protein SMU.1763c, a putative endoribonuclease associated with the CRISPR system I-C, acts on structured RNA substrates that is likely to be involved in CRISPR RNA processing but not in sensing cell envelope stress or preserving cell integrity in S. mutans. Together our data provide in vivo evidence that the CRISPR-Cas systems of S. mutans play novel roles in resistance against incoming plasmids that carry matching protospacer sequences and stress response.