Transcriptomic Analysis of csn2 Gene Mutant Strains of Streptococcus mutans CRISPR-Cas9 System

HE Xiao-ya, ZHANG An-qi, GONG Tao, LI Yu-qing


To explore the differences in transcriptional levels between mutant strains of csn2 gene of CRISPR-Cas9 system of Streptococcus mutans (S. mutans) and wild-type strains.  Methods  The S. mutans UA159, csn2-gene-deleted strains (Δcsn2) and csn2-gene-covering strains (Δcsn2/pDL278-csn2) of S. mutans were cultivated. Total RNA was extracted, and high-throughput sequencing technology was used for transcriptome sequencing. Based on the GO analysis and the KEGG analysis of the differentially expressed genes, the biological processes involved were thoroughly examined. The qRT-PCR method was used to verify the transcriptome sequencing results.  Results  The transcriptome results showed that, compared with UA159, there were 176 genes in Δcsn2 whose gene expression changed more than one fold (P<0.05), of which 72 were up-regulated and 104 were down-regulated. The GO enrichment analysis and the KEGG enrichment analysis revealed that both the up-regulated and down-regulated differentially expressed genes (DEG) were involved in amino acid transport and metabolism. In addition, the biological processes that up-regulated DEGs participated in were mainly related to carbohydrate metabolism, energy production and conversion, and transcription; down-regulated DEGs were mainly related to lipid metabolism, DNA replication, recombination and repair, signal transduction mechanisms, nucleotide transport and metabolism. The functions of some DEGs were still unclear. Results of qRT-PCR verified that the expressions of leuA, leuC and leuD (genes related to the formation of branched-chain amino acids) were significantly down-regulated in Δcsn2 when compared with UA159 and Δcsn2/pDL278-csn2.  Conclusion  Through transcriptome sequencing and qRT-PCR verification, it was found that the expression of genes related to branched-chain amino acid synthesis and cell membrane permeability in Δcsn2 changed significantly.


Keywords: Streptococcus mutans, CRISPR-Cas, csn2, Transcriptome


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WIEDENHEFT B, STERNBERG S H, DOUDNA J A. RNA-guided genetic silencing systems in bacteria and archaea. Nature,2012, 482(7385): 331–338.

KOONIN E V,MAKAROVA K S. CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes. RNA Biol,2013,10(5): 679–686.

BARRANGOU R, MARRAFFINI L A. CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity. Mol Cell,2014,54(2): 234–244.

VAN DER OOST J, WESTRA E R, JACKSON R N, et al. Unravelling the structural and mechanistic basis of CRISPR-Cas systems. Nat Rev Microbiol,2014,12(7): 479–492.

BARRANGOU R, FREMAUX C, DEVEAU H, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science,2007, 315(5819): 1709–1712.

VAN DER PLOEG J R. Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages. Microbiology (Reading),2009,155(Pt 6): 1966–1976.

SERBANESCU M A, CORDOVA M, KRASTEL K, et al. Role of the Streptococcus mutans CRISPR-Cas systems in immunity and cell physiology. J Bacteriol,2015,197(4): 749–761.

BURMISTRZ M, DUDEK B, STANIEC D, et al. Functional analysis of Porphyromonas gingivalis W83 CRISPR-Cas systems. J Bacteriol,2015, 197(16): 2631–2641.

BURLEY K M, SEDGLEY C M. CRISPR-Cas, a prokaryotic adaptive immune system, in endodontic, oral, and multidrug-resistant hospital-acquired Enterococcus faecalis. J Endod,2012,38(11): 1511–1515.

TONG Z, DU Y, LING J, et al. Relevance of the clustered regularly interspaced short palindromic repeats of Enterococcus faecalis strains isolated from retreatment root canals on periapical lesions, resistance to irrigants and biofilms. Exp Ther Med,2017,14(6): 5491–5496.

CHEN J, LI T, ZHOU X, et al. Characterization of the clustered regularly interspaced short palindromic repeats sites in Streptococcus mutans isolated from early childhood caries patients. Arch Oral Biol, 2017,83: 174–180.

GONG T, ZENG J, TANG B, et al. CRISPR-Cas systems in oral microbiome: from immune defense to physiological regulation. Mol Oral Microbiol,2020,35(2): 41–48.

ZHANG A, CHEN J, GONG T, et al. Deletion of csn2 gene affects acid tolerance and exopolysaccharide synthesis in Streptococcus mutans. Mol Oral Microbiol,2020,35(5): 211–221.

ZHENG X, ZHANG K, ZHOU X, et al. Involvement of gshAB in the interspecies competition within oral biofilm. J Dent Res,2013,92(9): 819–824.

AJDIC D, CHEN Z. A novel phosphotransferase system of Streptococcus mutans is responsible for transport of carbohydrates with α-1,3 linkage. Mol Oral Microbiol,2013,28(2): 114–128.

WEBB A J, HOMER K A, HOSIE A H. Two closely related ABC transporters in Streptococcus mutans are involved in disaccharide and/or oligosaccharide uptake. J Bacteriol, 2008, 190(1), 168–178.

SALZER R, KERN T, JOOS F, et al. The Thermus thermophilus comEA/comEC operon is associated with DNA binding and regulation of the DNA translocator and type Ⅳ pili. Environ Microbiol,2016,18(1): 65–74.

LOUWEN R, STAALS R H, ENDTZ H P, et al. The role of CRISPR-Cas systems in virulence of pathogenic bacteria. Microbiol Mol Biol Rev, 2014,78(1): 74–88.

LEBLANC D J, LEE L N, ABU-AL-JAIBAT A. Molecular, genetic, and functional analysis of the basic replicon of pVA380-1, a plasmid of oral streptococcal origin. Plasmid,1992,28(2): 130–145.

LEMOS J A, ABRANCHES J, BURNE R A. Responses of cariogenic streptococci to environmental stresses. Curr Issues Mol Biol,2005,7(1): 95–107.

MATSUI R, CVITKOVITCH D. Acid tolerance mechanisms utilized by Streptococcus mutans. Future Microbiol,2010,5(3): 403–417.

COTTER P D, HILL C. Surviving the acid test: responses of gram-positive bacteria to low pH. Microbiol Mol Biol Rev, 2003, 67(3): 429–453.

FOZO E M, QUIVEY R G. Shifts in the membrane fatty acid profile of Streptococcus mutans enhance survival in acidic environments. Appl Environ Microbiol,2004,70(2): 929–36.

CROSBY H A, HEINIGER E K, HARWOOD C S, et al. Reversible N epsilon-lysine acetylation regulates the activity of acyl-CoA synthetases involved in anaerobic benzoate catabolism in Rhodopseudomonas palustris. Mol Microbiol,2010,76(4): 874–888.

KOHLI G S, JOHN U, VAN DOLAH F M, et al. Evolutionary distinctiveness of fatty acid and polyketide synthesis in eukaryotes. ISME J,2016,10(8): 1877–1890.

BOYD D A, CVITKOVITCH D G, BLEIWEIS A S, et al. Defects in D-alanyl-lipoteichoic acid synthesis in Streptococcus mutans results in acid sensitivity. J Bacteriol,2000,182(21): 6055–6065.

LU P, MA D, CHEN Y, et al. L-glutamine provides acid resistance for Escherichia coli through enzymatic release of ammonia. Cell Res,2013, 23(5): 635–644.

BAKER J L, ABRANCHES J, FAUSTOFERRI R C, et al. Transcriptional profile of glucose-shocked and acid-adapted strains of Streptococcus mutans. Mol Oral Microbiol,2015,30(6): 496–517.

KANAPKA J A, KLEINBERG I. Catabolism of arginine by the mixed bacteria in human salivary sediment under conditions of low and high glucose concentration. Arch Oral Biol,1983,28(11): 1007–1015.

HUANG X, ZHANG K, DENG M, et al. Effect of arginine on the growth and biofilm formation of oral bacteria. Arch Oral Biol,2017,82: 256–262.

WELIN-NEILANDS J, SVENSÄTER G. Acid tolerance of biofilm cells of Streptococcus mutans. Appl Environ Microbiol,2007,73(17): 5633–5638.


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