scholarly journals Characterization of a Type II-A CRISPR-Cas System in Streptococcus mutans

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Cas Mosterd ◽  
Sylvain Moineau
Keyword(s):  
Streptococcus Mutans ◽  
Defense Mechanisms ◽  
Bacterial Species ◽  
Oral Microbiota ◽  
Type Ii ◽  
Content Type ◽  
C Terminus ◽  
Protospacer Adjacent Motif ◽  
Cas9 Protein

ABSTRACT Streptococcus mutans and its virulent phages are important members of the human oral microbiota. S. mutans is also the primary causal agent of dental caries. To survive in this ecological niche, S. mutans must encode phage defense mechanisms, which include CRISPR-Cas systems. Here, we describe the CRISPR-Cas type II-A system of S. mutans strain P42S, which was found to display natural adaptation and interference activity in response to phage infection and plasmid transformation. Newly acquired spacers were integrated both at the 5′ end of the CRISPR locus and ectopically. In comparisons of the cas genes of P42S to those of other strains of S. mutans, cas1, cas2, and csn2 appear to be highly conserved within the species. However, more diversity was observed with cas9. While the nuclease domains of S. mutans Cas9 (SmCas9) are conserved, the C terminus of the protein, including the protospacer adjacent motif (PAM) recognition domain, is less conserved. In support of these findings, we experimentally demonstrated that the PAMs associated with SmCas9 of strain P42S are NAA and NGAA. These PAMs are different from those previously reported for the CRISPR-Cas system of the model strain S. mutans UA159. This study illustrates the diversity of CRISPR-Cas type II-A systems that can be found within the same bacterial species. IMPORTANCE CRISPR-Cas is one of the mechanisms used by bacteria to defend against viral predation. Increasing our knowledge of the biology and diversity of CRISPR-Cas systems will also improve our understanding of virus-bacterium interactions. As CRISPR-Cas systems acquiring novel immunities under laboratory conditions are rare, Streptococcus mutans strain P42S provides an alternative model to study the adaptation step, which is still the least understood step in CRISPR-Cas biology. Furthermore, the availability of a natural Cas9 protein recognizing an AT-rich PAM opens up new avenues for genome editing purposes.

scholarly journals Primed CRISPR-Cas Adaptation and Impaired Phage Adsorption in Streptococcus mutans

mSphere ◽  
2021 ◽  
Vol 6 (3) ◽  
Author(s):  
Cas Mosterd ◽  
Sylvain Moineau
Keyword(s):  
Streptococcus Mutans ◽  
Defense Mechanisms ◽  
Phage Genome ◽  
Multiplicity Of Infection ◽  
Type Ii ◽  
Content Type ◽  
Virulent Phage ◽  
Phage Adsorption ◽  
Protospacer Adjacent Motif

ABSTRACT Streptococcus mutans strain P42S possesses a type II-A CRISPR-Cas system that protects against phage infection and plasmid transformation. The analysis of 293 bacteriophage-insensitive mutants (BIMs) obtained upon exposure to the virulent phage M102AD revealed the acquisition of 399 unique spacers, including several ectopic spacer acquisitions and a few cases of native spacer deletions. The acquisition of multiple spacers was also observed and appears to be mostly due to priming, which has been rarely reported for type II-A systems. Analyses of the acquired spacers indicated that 88% of them are identical to a region of the phage M102AD genome. The remaining 12% of spacers had mismatches with the phage genome, primarily at the 5′ end of the spacer, leaving the seed sequence at the 3′ end largely intact. When a high multiplicity of infection (MOI) was used in the phage challenge assays, we also observed the emergence of CRISPR BIMs that, in addition to the acquisition of new spacers, displayed a reduced phage adsorption phenotype. While CRISPR-Cas and adsorption resistance work in tandem to protect S. mutans P42S against phage M102AD, nonidentified antiviral mechanisms are also likely at play in this strain. IMPORTANCE Bacteria are under the constant threat of viral predation and have therefore developed several defense mechanisms, including CRISPR-Cas systems. While studies on the mode of action of CRISPR-Cas systems have already provided great insights into phage-bacterium interactions, still more information is needed on the biology of these systems. The additional characterization of the type II-A CRISPR-Cas system of Streptococcus mutans P42S in this study provides novel information on the spacer acquisition step, especially regarding protospacer-adjacent motif (PAM) recognition, multiple-spacer acquisition, and priming.

scholarly journals Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems

2013 ◽  
Vol 42 (4) ◽  
pp. 2577-2590 ◽  
Author(s):  
Ines Fonfara ◽  
Anaïs Le Rhun ◽  
Krzysztof Chylinski ◽  
Kira S. Makarova ◽  
Anne-Laure Lécrivain ◽  
...  
Keyword(s):  
Genome Editing ◽  
Horizontal Transfer ◽  
Genome Engineering ◽  
Bacterial Species ◽  
Type Ii ◽  
Promising Technology ◽  
Dna Targeting ◽  
Protospacer Adjacent Motif ◽  
Cas9 Protein ◽  
Specific Manner

Abstract The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool.

scholarly journals Transition of Bacterial Diversity and Composition in Tongue Microbiota during the First Two Years of Life

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Shinya Kageyama ◽  
Mikari Asakawa ◽  
Toru Takeshita ◽  
Yukari Ihara ◽  
Shunsuke Kanno ◽  
...  
Keyword(s):  
Oral Cavity ◽  
Bacterial Diversity ◽  
Bacterial Species ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Oral Microbiota ◽  
Bacterial Composition ◽  
Content Type ◽  
Operational Taxonomic Units ◽  
Rrna Gene Sequencing

ABSTRACTNewborns are constantly exposed to various microbes from birth; hence, diverse commensal bacteria colonize the oral cavity. However, how or when these bacteria construct a complex and stable ecosystem remains unclear. This prospective cohort study examined the temporal changes in bacterial diversity and composition in tongue microbiota during infancy. We longitudinally collected a total of 464 tongue swab samples from 8 infants (age of <6 months at baseline) for approximately 2 years. We also collected samples from 32 children (aged 0 to 2 years) and 73 adults (aged 20 to 29 years) cross-sectionally as control groups. Bacterial diversities and compositions were determined by 16S rRNA gene sequencing. The tongue bacterial diversity in infancy, measured as the number of observed operational taxonomic units (OTUs), rapidly increased and nearly reached the same level as that in adults by around 80 weeks. The overall tongue bacterial composition in the transitional phase, 80 to 120 weeks, was more similar to that of adults than to that of the early exponential phase (EEP), 10 to 29 weeks, according to analysis of similarities. Dominant OTUs in the EEP corresponding toStreptococcus perorisandStreptococcus lactariusexponentially decreased immediately after EEP, around 30 to 49 weeks, whereas several OTUs corresponding toGranulicatella adiacens,Actinomyces odontolyticus, andFusobacterium periodonticumreciprocally increased during the same period. These results suggest that a drastic compositional shift of tongue microbiota occurs before the age of 1 year, and then bacterial diversity and overall bacterial composition reach levels comparable to those in adults by the age of 2 years.IMPORTANCEEvaluating the development of oral microbiota during infancy is important for understanding the subsequent colonization of bacterial species and the process of formation of mature microbiota in the oral cavity. We examined tongue microbiota longitudinally collected from 8 infants and found that drastic compositional shifts in tongue microbiota occur before the age of 1 year, and then bacterial diversity and overall bacterial composition reach levels comparable to those in adults by the age of 2 years. These results may be helpful for preventing the development of various diseases associated with oral microbiota throughout life.

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

2015 ◽  
Vol 197 (6) ◽  
pp. 1083-1094 ◽  
Author(s):  
Vincent Leung ◽  
Dragana Ajdic ◽  
Stephanie Koyanagi ◽  
Céline M. Lévesque
Keyword(s):  
Quorum Sensing ◽  
Streptococcus Mutans ◽  
Bacterial Species ◽  
Regulatory System ◽  
Bacterial Survival ◽  
Chronic Infections ◽  
Persister Cells ◽  
Gene Encoding ◽  
Content Type ◽  
Peptide Pheromone

The 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 organismStreptococcus mutans, the quorum-sensing 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 oflexAin 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.

scholarly journals Characterization of a Feruloyl Esterase from Lactobacillus plantarum

2013 ◽  
Vol 79 (17) ◽  
pp. 5130-5136 ◽  
Author(s):  
María Esteban-Torres ◽  
Inés Reverón ◽  
José Miguel Mancheño ◽  
Blanca de las Rivas ◽  
Rosario Muñoz
Keyword(s):  
Lactobacillus Plantarum ◽  
Bacterial Species ◽  
Hydrolytic Activity ◽  
Feruloyl Esterase ◽  
Gene Encoding ◽  
Content Type ◽  
Cell Extracts ◽  
Methyl Ferulate ◽  
Methyl Caffeate

ABSTRACTLactobacillus plantarumis frequently found in the fermentation of plant-derived food products, where hydroxycinnamoyl esters are abundant.L. plantarumWCFS1 cultures were unable to hydrolyze hydroxycinnamoyl esters; however, cell extracts from the strain partially hydrolyze methyl ferulate and methylp-coumarate. In order to discover whether the protein Lp_0796 is the enzyme responsible for this hydrolytic activity, it was recombinantly overproduced and enzymatically characterized. Lp_0796 is an esterase that, among other substrates, is able to efficiently hydrolyze the four model substrates for feruloyl esterases (methyl ferulate, methyl caffeate, methylp-coumarate, and methyl sinapinate). A screening test for the detection of the gene encoding feruloyl esterase Lp_0796 revealed that it is generally present amongL. plantarumstrains. The present study constitutes the description of feruloyl esterase activity inL. plantarumand provides new insights into the metabolism of hydroxycinnamic compounds in this bacterial species.

scholarly journals Genomic Characterization of VIM Metallo-β-Lactamase-Producing Alcaligenes faecalis from Gaza, Palestine

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Nahed Al Laham ◽  
Kalyan D. Chavda ◽  
Astrid V. Cienfuegos-Gallet ◽  
Barry N. Kreiswirth ◽  
Liang Chen
Keyword(s):  
Bacterial Species ◽  
Alcaligenes Faecalis ◽  
Gram Negative Bacteria ◽  
Sequencing Analysis ◽  
Large Hospital ◽  
Content Type ◽  
Next Generation Sequencing Analysis ◽  
Genomic Characterization ◽  
Generation Sequencing

ABSTRACT Carbapenemase-producing Gram-negative bacteria (CP-GNB) have increasingly spread worldwide, and different families of carbapenemases have been identified in various bacterial species. Here, we report the identification of five VIM metallo-β-lactamase-producing Alcaligenes faecalis isolates associated with a small outbreak in a large hospital in Gaza, Palestine. Next-generation sequencing analysis showed bla VIM-2 is harbored by a chromosomal genomic island among three strains, while bla VIM-4 is carried by a novel plasmid in two strains.

scholarly journals ClpP is required for proteolytic regulation of type II toxin–antitoxin systems and persister cell formation in Streptococcus mutans

2019 ◽  
Vol 1 (8) ◽  
Author(s):  
Xiao-Lin Tian ◽  
Miao Li ◽  
Zachariah Scinocca ◽  
Heather Rutherford ◽  
Yung-Hua Li
Keyword(s):  
Streptococcus Mutans ◽  
Type Species ◽  
Critical Role ◽  
Cell Formation ◽  
Type Ii ◽  
Wild Type ◽  
Content Type ◽  
Link Type ◽  
Viability Assay ◽  
Persister Cell

The type II toxin–antitoxin (TA) modules, mazEF and relBE, in Streptococcus mutans have been implicated in stress response, antibiotic tolerance and persister cell formation. However, how S. mutans regulates these systems to prevent unwanted toxin activation and persister cell formation is unclear. In this study, we provide evidence that ClpP is required for the proteolytic regulation of these TA systems and persister cell formation in S. mutans following antibiotic challenge. A persister viability assay showed that S. mutans UA159 (WT) formed a larger quantity of persister cells than its isogenic mutant ΔclpP following antibiotic challenge. However, the lux reporter assay revealed that clpP deletion did not affect the transcriptional levels of mazEF and relBE, since no significant differences (P>0.05) in the reporter activities were detected between the wild-type and ΔclpP background. Instead, all antibiotics tested at a sub-minimum inhibitory concentration (sub-MIC) induced transcriptional levels of mazEF and relBE operons. We then examined the protein profiles of His-tagged MazE and RelB proteins in the UA159 and ΔclpP backgrounds by Western blotting analysis. The results showed that S. mutans strains grown under non-stress conditions expressed very low but detectable levels of MazE and RelB antitoxin proteins. Antibiotics at sub-MICs induced the levels of the MazE and RelB proteins, but the protein levels decreased rapidly in the wild-type background. In contrast, a stable level of MazE and RelB proteins could be detected in the ΔclpP mutant background, suggesting that both proteins accumulated in the ΔclpP mutant. We conclude that ClpP is required for the proteolytic regulation of cellular levels of the MazE and RelB antitoxins in S. mutans , which may play a critical role in modulating the TA activities and persister cell formation of this organism following antibiotic challenge.

scholarly journals Distinct Requirements for Tail-Anchored Membrane Protein Biogenesis in Escherichia coli

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Markus Peschke ◽  
Mélanie Le Goff ◽  
Gregory M. Koningstein ◽  
Norbert O. Vischer ◽  
Abbi Abdel-Rehim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Transmembrane Domain ◽  
Signal Recognition Particle ◽  
Membrane Insertion ◽  
Type Ii ◽  
Content Type ◽  
E Coli ◽  
C Terminus

ABSTRACT Tail-anchored membrane proteins (TAMPs) are a distinct subset of inner membrane proteins (IMPs) characterized by a single C-terminal transmembrane domain (TMD) that is responsible for both targeting and anchoring. Little is known about the routing of TAMPs in bacteria. Here, we have investigated the role of TMD hydrophobicity in tail-anchor function in Escherichia coli and its influence on the choice of targeting/insertion pathway. We created a set of synthetic, fluorescent TAMPs that vary in the hydrophobicity of their TMDs and corresponding control polypeptides that are extended at their C terminus to create regular type II IMPs. Surprisingly, we observed that TAMPs have a much lower TMD hydrophobicity threshold for efficient targeting and membrane insertion than their type II counterparts. Using strains conditional for the expression of known membrane-targeting and insertion factors, we show that TAMPs with strongly hydrophobic TMDs require the signal recognition particle (SRP) for targeting. Neither the SecYEG translocon nor YidC appears to be essential for the membrane insertion of any of the TAMPs studied. In contrast, corresponding type II IMPs with a TMD of sufficient hydrophobicity to promote membrane insertion followed an SRP- and SecYEG translocon-dependent pathway. Together, these data indicate that the capacity of a TMD to promote the biogenesis of E. coli IMPs is strongly dependent upon the polypeptide context in which it is presented. IMPORTANCE A subset of membrane proteins is targeted to and inserted into the membrane via a hydrophobic transmembrane domain (TMD) that is positioned at the very C terminus of the protein. The biogenesis of these so-called tail-anchored proteins (TAMPs) has been studied in detail in eukaryotic cells. Various partly redundant pathways were identified, the choice for which depends in part on the hydrophobicity of the TMD. Much less is known about bacterial TAMPs. The significance of our research is in identifying the role of TMD hydrophobicity in the routing of E. coli TAMPs. Our data suggest that both the nature of the TMD and its role in routing can be very different for TAMPs versus “regular” membrane proteins. Elucidating these position-specific effects of TMDs will increase our understanding of how prokaryotic cells face the challenge of producing a wide variety of membrane proteins.

scholarly journals Novel Probiotic Mechanisms of the Oral Bacterium Streptococcus sp. A12 as Explored with Functional Genomics

2019 ◽  
Vol 85 (21) ◽  
Author(s):  
K. Lee ◽  
A. R. Walker ◽  
B. Chakraborty ◽  
J. R. Kaspar ◽  
M. M. Nascimento ◽  
...  
Keyword(s):  
Oral Health ◽  
Functional Genomics ◽  
Streptococcus Mutans ◽  
Molecular Mechanisms ◽  
Oral Microbiota ◽  
Physiological Factors ◽  
Content Type ◽  
Oral Bacterium ◽  
Probiotic Mechanisms ◽  
Colicin V

ABSTRACT Health-associated biofilms in the oral cavity are composed of a diverse group of microbial species that can foster an environment that is less favorable for the outgrowth of dental caries pathogens, like Streptococcus mutans. A novel oral bacterium, designated Streptococcus A12, was previously isolated from supragingival dental plaque of a caries-free individual and was shown to interfere potently with the growth and virulence properties of S. mutans. In this study, we applied functional genomics to begin to identify molecular mechanisms used by A12 to antagonize, and to resist the antagonistic factors of, S. mutans. Using bioinformatics, genes that could encode factors that enhance the ability of A12 to compete with S. mutans were identified. Selected genes, designated potential competitive factors (pcf), were deleted. Certain mutant derivatives showed a reduced capacity to compete with S. mutans compared to that of the parental strain. The A12 pcfO mutant lost the ability to inhibit comX-inducing peptide (XIP) signaling by S. mutans, while mutants with changes in the pcfFEG locus were impaired in sensing of, and were more sensitive to, the lantibiotic nisin. Loss of PcfV, annotated as a colicin V biosynthetic protein, resulted in diminished antagonism of S. mutans. Collectively, the data provide new insights into the complexities and variety of factors that affect biofilm ecology and virulence. Continued exploration of the genomic and physiological factors that distinguish commensals from truly beneficial members of the oral microbiota will lead to a better understanding of the microbiome and new approaches to promote oral health. IMPORTANCE Advances in defining the composition of health-associated biofilms have highlighted the important role of beneficial species in maintaining health. Comparatively little, however, has been done to address the genomic and physiological bases underlying the probiotic mechanisms of beneficial commensals. In this study, we explored the ability of a novel oral bacterial isolate, Streptococcus A12, to compete with the dental pathogen Streptococcus mutans using various gene products with diverse functions. A12 displayed enhanced competitiveness by (i) disrupting intercellular communication pathways of S. mutans, (ii) sensing and resisting antimicrobial peptides, and (iii) producing factors involved in the production of a putative antimicrobial compound. Research on the probiotic mechanisms employed by Streptococcus A12 is providing essential insights into how beneficial bacteria may help maintain oral health, which will aid in the development of biomarkers and therapeutics that can improve the practice of clinical dentistry.

scholarly journals Contribution of the Interaction of Streptococcus mutans SerotypekStrains with Fibrinogen to the Pathogenicity of Infective Endocarditis

2014 ◽  
Vol 82 (12) ◽  
pp. 5223-5234 ◽  
Author(s):  
Ryota Nomura ◽  
Masatoshi Otsugu ◽  
Shuhei Naka ◽  
Noboru Teramoto ◽  
Ayuchi Kojima ◽  
...  
Keyword(s):  
Infective Endocarditis ◽  
Streptococcus Mutans ◽  
Heart Valve ◽  
Heart Valves ◽  
Binding Assays ◽  
Bacterial Dna ◽  
Content Type ◽  
Fibrinogen Binding ◽  
Level Of Aggregation

ABSTRACTStreptococcus mutans, a pathogen responsible for dental caries, is occasionally isolated from the blood of patients with bacteremia and infective endocarditis (IE). Our previous study demonstrated that serotypek-specific bacterial DNA is frequently detected inS. mutans-positive heart valve specimens extirpated from IE patients. However, the reason for this frequent detection remains unknown. In the present study, we analyzed the virulence of IE fromS. mutansstrains, focusing on the characterization of serotypekstrains, most of which are positive for the 120-kDa cell surface collagen-binding protein Cbm and negative for the 190-kDa protein antigen (PA) known as SpaP, P1, antigen I/II, and other designations. Fibrinogen-binding assays were performed with 85 clinical strains classified by Cbm and PA expression levels. The Cbm+/PA−group strains had significantly higher fibrinogen-binding rates than the other groups. Analysis of platelet aggregation revealed that SA31, a Cbm+/PA−strain, induced an increased level of aggregation in the presence of fibrinogen, while negligible aggregation was induced by the Cbm-defective isogenic mutant SA31CBD. A rat IE model with an artificial impairment of the aortic valve created using a catheter showed that extirpated heart valves in the SA31 group displayed a prominent vegetation mass not seen in those in the SA31CBD group. These findings could explain why Cbm+/PA−strains are highly virulent and are related to the development of IE, and the findings could also explain the frequent detection of serotypekDNA inS. mutans-positive heart valve clinical specimens.

Sign in / Sign up

Export Citation Format

Share Document