Role of the Streptococcus mutans CRISPR-Cas systems in immunity and cell physiology

Abstract

CRISPR-Cas systems provide adaptive microbial immunity against invading viruses and plasmids. The cariogenic bacterium Streptococcus mutans UA159 has two CRISPR-Cas systems: CRISPR1 (type II-A) and CRISPR2 (type I-C) with several spacers from both CRISPR cassettes matching sequences of phage M102 or genomic sequences of other S. mutans. The deletion of the cas genes of CRISPR1 (ΔC1S), CRISPR2 (ΔC2E), or both CRISPR1+2 (ΔC1SC2E) or the removal of spacers 2 and 3 (ΔCR1SP13E) in S. mutans UA159 did not affect phage sensitivity when challenged with virulent phage M102. Using plasmid transformation experiments, we demonstrated that the CRISPR1-Cas system inhibits transformation of S. mutans by the plasmids matching the spacers 2 and 3. Functional analysis of the cas deletion mutants revealed that in addition to a role in plasmid targeting, both CRISPR systems also contribute to the regulation of bacterial physiology in S. mutans. Compared to wild-type cells, the ΔC1S strain displayed diminished growth under cell membrane and oxidative stress, enhanced growth under low pH, and had reduced survival under heat shock and DNA-damaging conditions, whereas the ΔC2E strain exhibited increased sensitivity to heat shock. Transcriptional analysis revealed that the two-component signal transduction system VicR/K differentially modulates expression of cas genes within CRISPR-Cas systems, suggesting that VicR/K might coordinate the expression of two CRISPR-Cas systems. Collectively, we provide in vivo evidence that the type II-A CRISPR-Cas system of S. mutans may be targeted to manipulate its stress response and to influence the host to control the uptake and dissemination of antibiotic resistance genes.

FIG 1

Gene maps of the CRISPR-Cas systems in S. mutans UA159. (A) CRISPR1-Cas system. (B) CRISPR2-Cas system. Analysis of the promoter regions of cas genes identified the putative −10 box, −35 box, transcriptional start site (TSS), and ribosome binding site (RBS) (all underlined in boldface), as well as the putative VicR binding consensus sequence (TGTWAH-6/10 bp-TGTWAH) for cas gene regulation.

FIG 2

The CRISPR1-Cas system of S. mutans UA159 provides immunity against plasmid transformation. (A) Schematic representation of cloning vector pCG1 used for the construction of plasmids for the transformation interference assay. Plasmids for interference assays were produced by inserting a protospacer and 10 nt on both sides of the protospacers into pCG1 plasmid. (B) pCG1 constructs containing potential targets for different S. mutans UA159 spacers (spacers 2, 3, and 6 within CRISPR1 array and spacer 1 within CRISPR2 array). pCG1derivatives in the presence (C) or absence (D) of flank sequences were tested by natural transformation assays using the wild-type S. mutans UA159, ΔC1K, ΔC2E, and ΔC1SC2E strains. The transformation frequency was calculated as the transformant CFU divided by the total number of viable cells. The results shown are representative of at least two independent experiments. ***, constructs showing targeting phenotype.

FIG 3

Cleavage of the synthetic ssRNA substrates by the Cas5d protein from S. mutans UA159. 5′-³²P-labeled RNA1 or RNA2 (0.05 μm) was incubated in the absence or in the presence of 100 ng (lanes 1 and 3) or 200 ng of Cas5d (SMU.1763c) (lanes 2 and 4) or in the presence of 100 or 200 ng of purification product obtained from E. coli cells transformed with an empty p15TvL vector (lanes 5 and 6) at 37°C for 30 min in the presence of 50 mM Tris-HCl (pH 7.0), 5 mm MnCl₂, 100 mM KCl, and 1 mM DTT. Reaction products were separated on a 15% PAGE–8 M urea gel and visualized by phosphorimaging. 39-nt RNA1 and 40-nt RNA2 were prepared by using the oligonucleotides 5′-AAAUACGUUUUCUCCAUUGUCAUAUUGCGCAUAAGUUGA and 5′-UUUCAAUUCCUUUUAGGAUUAAUCUUGAAGAUAGAGUUAA, respectively.

FIG 4

Cas5d (SMU.1763c) cleavage of RNA transcripts of SMU.995 and SMU.1502c generated by in vitro transcription.

FIG 5

Effects of MMC or UV irradiation on viability of S. mutans UA159 and mutant strains. (A) S. mutans UA159, as well as ΔC1S, ΔC2E, and ΔC1SC2E strains, was exposed to 0.050 μg of MMC/ml for 1 h. The results shown are representative of at least two independent experiments. Statistical analyses were performed using the Student t test (***, P < 0.005). (B) Actively growing cells of UA159 and its mutant strains were exposed to UV irradiation for 2, 4, 6, and 8 min. The results here represent the average of two independent experiments ± the standard errors. The differences were statistically significant (P < 0.005; P < 0.05 [Student test]).

FIG 6

Growth kinetics of S. mutans UA159 and ΔC1S under various stressors: pH 7.0 or 5.5 (A), 25 mM paraquat or 0.003% H₂O₂ (B), and THYE or 0.004% SDS (C). Each point represents the average of four independent optical density values per sample. These results shown are representative of two independent experiments conducted with the mutant and UA159 parent strain.

FIG 7

Survival of S. mutans UA159 and mutant strains after exposure to 50°C temperature stress for 1 h. The results represent mean CFU counts ± the standard deviations. The differences were statistically significant (P ≤ 0.05, Student t test). These results shown are representative of two independent experiments conducted with the mutants and UA159 parent strain.

FIG 8

Expression of cas genes from the CRISPR1 (A) and CRISPR2 (B) operons. RNA analysis from mid-logarithmic-phase cultures of S. mutans UA159 and SmuvicK grown under regular or acidic conditions. The results are the averages of triplicate samples from three independent experiments ± the standard errors.