Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages

Ploeg, Jan R. van der

doi:10.1099/mic.0.027508-0

Cited by 102 publications

(75 citation statements)

References 39 publications

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“…SMU.63 and SMU.1402, two genes known to be differentially regulated under CSP conditions (25), were first tested. SMU.1402 encodes a CRISPR-associated Csn2 protein belonging to the CRISPR-1 region of S. mutans (49). CRISPR arrays with CRISPRassociated proteins are involved in resistance to bacteriophage, and resistance specificity is determined by the insertion of CRISPR spacer-phage sequence similarity (50).…”

Section: Resultsmentioning

confidence: 99%

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The Formation of Streptococcus mutans Persisters Induced by the Quorum-Sensing Peptide Pheromone Is Affected by the LexA Regulator

et al. 2015

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bThe presence of multidrug-tolerant persister cells within microbial populations has been implicated in the resiliency of bacterial survival against antibiotic treatments and is a major contributing factor in chronic infections. The mechanisms by which these phenotypic variants are formed have been linked to stress response pathways in various bacterial species, but many of these mechanisms remain unclear. We have previously shown that in the cariogenic organism Streptococcus mutans, the quorumsensing peptide CSP (competence-stimulating peptide) pheromone was a stress-inducible alarmone that triggered an increased formation of multidrug-tolerant persisters. In this study, we characterized SMU.2027, a CSP-inducible gene encoding a LexA ortholog. We showed that in addition to exogenous CSP exposure, stressors, including heat shock, oxidative stress, and ofloxacin antibiotic, were capable of triggering expression of lexA in an autoregulatory manner akin to that of LexA-like transcriptional regulators. We demonstrated the role of LexA and its importance in regulating tolerance toward DNA damage in a noncanonical SOS mechanism. We showed its involvement and regulatory role in the formation of persisters induced by the CSP-ComDE quorum-sensing regulatory system. We further identified key genes involved in sugar and amino acid metabolism, the clustered regularly interspaced short palindromic repeat (CRISPR) system, and autolysin from transcriptomic analyses that contribute to the formation of quorum-sensing-induced persister cells.T he classical view of bacterial survival against antibiotic killing has usually been seen as the expression of genetic resistance mechanisms that arise from mutations or gained through horizontal gene transfer. These resistance mechanisms include host target modification, degradation or modification of the antibiotic itself, and reduction in the permeability or increase in the efflux of the drug (1). A major survival mechanism of bacteria is antibiotic tolerance, whereby bacterial cells that have a slower or reduced growth rate become more tolerant toward antibiotic killing (2, 3). This reduction in bacterial growth is prominently perceived as one of the main survival mechanisms elicited in bacterial biofilms, by which the slower growth of biofilm cells contributes toward the highly recalcitrant nature of biofilm infections, even in biofilm populations that lack genetically encoded antibiotic resistance markers (4, 5).Formation of persister cells is the main factor responsible for the tolerance of pathogens to antibiotics. Persisters are nongrowing dormant cells that are produced in a clonal population of genetically identical cells. They constitute a small fraction of the bacterial population. Persisters are not mutants but phenotypic variants of the wildtype population (6). In contrast to the case with the aforementioned drug resistance mechanisms, which allow for bacterial cells to actively grow and divide unimpeded in the presence of specific antimicrobials, persisters are capable of sur...

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Section: Resultsmentioning

confidence: 99%

“…is insufficient to render sensitivity to infection by the phage M102 (49). Natural phage resistance mechanisms in S. mutans have not been previously described, mainly because phages and sensitive strains are actually quite rare in S. mutans (52).…”

Section: Figmentioning

confidence: 99%

The Formation of Streptococcus mutans Persisters Induced by the Quorum-Sensing Peptide Pheromone Is Affected by the LexA Regulator

et al. 2015

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“…Intriguingly, mutations in these active sites did not alter the affinity of the CRISPR/Cas complex for binding the protospacer (29). Importantly, protospacer-adjacent motifs (PAMs) that are short conserved nucleotide stretches next to the protospacers, such as NGG (32), NGGNG (22), NAAR (32), and NNAGAAW (33), are absolutely necessary for Cas9 binding and cleavage (29). Orthogonal Cas9 nucleases from different microorganisms require different PAM sequences (31,34).…”

Section: Type II Crispr/cas Systemmentioning

confidence: 99%

Cas9-Based Tools for Targeted Genome Editing and Transcriptional Control

Nostrand

et al. 2014

Appl Environ Microbiol

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dDevelopment of tools for targeted genome editing and regulation of gene expression has significantly expanded our ability to elucidate the mechanisms of interesting biological phenomena and to engineer desirable biological systems. Recent rapid progress in the study of a clustered, regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein system in bacteria has facilitated the development of newly facile and programmable platforms for genome editing and transcriptional control in a sequence-specific manner. The core RNA-guided Cas9 endonuclease in the type II CRISPR system has been harnessed to realize gene mutation and DNA deletion and insertion, as well as transcriptional activation and repression, with multiplex targeting ability, just by customizing 20-nucleotide RNA components. Here we describe the molecular basis of the type II CRISPR/Cas system and summarize applications and factors affecting its utilization in model organisms. We also discuss the advantages and disadvantages of Cas9-based tools in comparison with widely used customizable tools, such as Zinc finger nucleases and transcription activator-like effector nucleases.

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“…Despite this knowledge, the mechanisms responsible for resistance to M102 in S. mutans serotype c remain unknown. S. mutans strain UA159 harbors two distinct CRISPR-Cas systems: a type II-A CRISPR1-Cas system and a type I-C CRISPR2-Cas system (45)(46)(47)(48). The analysis of CRISPR cassettes in 29 S. mutans strains revealed that CRISPR spacers had high sequence similarity with M102, a virulent siphophage specific for S. mutans, suggesting that phage-derived spacers present in these strains likely resulted from M102-like phage attacks (45).…”

mentioning

confidence: 99%

“…Only five phages, designated M101, M102AD, M102, e10, and f1, have been shown to have lytic activity against S. mutans strains of serotypes c, e, and f, respectively (44,45). Except for S. mutans strain OMZ381, all S. mutans serotype c strains, including UA159, are known to be resistant to phage infection by M102; only strain OMZ381 showed sensitivity to phage infection, resulting in cell lysis (45). Despite this knowledge, the mechanisms responsible for resistance to M102 in S. mutans serotype c remain unknown.…”

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confidence: 99%

Role of the Streptococcus mutans CRISPR-Cas Systems in Immunity and Cell Physiology

et al. 2015

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b 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 (⌬C1S), CRISPR2 (⌬C2E), or both CRISPR1؉2 (⌬C1SC2E) or the removal of spacers 2 and 3 (⌬CR1SP13E) 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 ⌬C1S 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 ⌬C2E 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.

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Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages

Cited by 102 publications

References 39 publications

The Formation of Streptococcus mutans Persisters Induced by the Quorum-Sensing Peptide Pheromone Is Affected by the LexA Regulator

The Formation of Streptococcus mutans Persisters Induced by the Quorum-Sensing Peptide Pheromone Is Affected by the LexA Regulator

Cas9-Based Tools for Targeted Genome Editing and Transcriptional Control

Role of the Streptococcus mutans CRISPR-Cas Systems in Immunity and Cell Physiology

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