Bioinformatics Analysis On CRISPR-Cas System And Examination On Bacteriophage Resistance Mechanisms Of Streptococcus Thermophilus

With the development of the economy,fermented dairy food has an increasing market demand in the modern society.However,phage infection would lead to the failure of fermentation.Clustered regularly interspaced short palindromic repeats(CRISPR)together with CRISPR associated genes(cas genes)constitute an adaptive immune system,which provides acquired resistance against viruses and plasmids,in bacteria and archaea.In this article,the homology of CRISPR-Cas systems in Lactic acid bacteria was analyzed,and the signature cas genes were amplified and sequenced to predict the type of CRISPR-Cas systems in unknown S.thermophilus strains.The CRISPR sequences were also amplified and sequenced,the protospacers were predicted.Finally,these BIMs were generated from S.thermophilus ST-I in order to explore the anti-phage mechanisms of CRSPR-Cas systems.Bioinformatics method was used for predicting CRSPR-Cas system in Lactic acid bacteria published in NCBI.The phylogenetic analysis was performed to investigate the level of conservation and divergence between the identified Lactic acid bacteria CRISPR immune systems using the repeats and universal Cas protein.S.thermophilus,Lactobacillus delbrueckii subsp.bulgaricus and Lactobacillus helveticus all contain a different number of CRISPR-Cas systems,the repeats of each type were highly conserved and the Cas proteins demonstrated high similarity.For homology analysis,repeats and Cas proteins showed high similarity with several individual species of distant relationships,which suggested that the CRISPRs of tested strains also had the characteristic of horizontal gene transfer(HGT).The CRSPR-Cas system of six strains of S.thermophilus were detected.Four primers were designed according to the flanking sequences of signature cas genes of standard strains and the CRISPR-Cas system of six strains of S.thermophilus have been detected by PCR method.Among the six strains of S.thermophilus,S.thermophilus S4 has a cas9 gene,others all have cas9 gene,cas10 gene and cas9*gene.In addition,S.thermophilus 79 and S.thermophilus KLDS3.0207 still have cas3 gene.Signature genes amplification of CRSPR-Cas system could predict the type of CRSPR-Cas system in unsequenced strains,these findings establishe the foundation for the studyof CRSPR-Cas system in lactic acid bacteria.Spacers in CRISPR loci can provide specific immunity to invasive elements that carry the same or a similar sequence.Thus,the prospacer of 3 strains of S.thermophilus were detected,to lay the foundation for exploring phage resistance mechanism.The CRISPR of 3 strains of S.thermophilus have been detected by PCR method,and the homology of repeats or spacers were analyzed.Also,we analyzed CRISPR activity across four distinct CRISPR loci in several S.thermophilus strains.Results showed that S.thermophilus S0 had 3 CRISPR,S.thermophilus S4 only had one CRISPR,S.thermophilus 79 had 4 CRISPR.We propose that the activity is highest for CRISPR1,followed by CRISPR3,while CRISPR2 may be the least active locus.Even in closely related strains,spacer content is very dynamic.S.thermophilus 79 had the same CRISPR sequence with S.thermophilus MN-BM-A02 and S.thermophilus ASCC 1275.The spacers derived from bacteriophages or plasmids,and chromosomal sequences,which suggested that the bacteria insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory.There BIMs were generated from S.thermophilus ST-I,the homology of CRISPRs sequence in BIMs of S.thermophilus St-I were analyzed.Secondary structures of the repeats and the PAMs of each CRISPR locus were also predicted.Results showed that CRISPR1 has 27 repeat-spacer units,5 of them had duplicates;CRISPR2 has one repeat-spacer unit;CRISPR3 has 28repeat-spacer units.Only BIM1 had a new spacer acquirtion in CRISPR3,while BIM2 and BIM3 had no new spacers' insertion,thus indicating that while most CRISPR1 were more active than CRISPR3,new spacer acquisition occurred just in CRSPR3 in some situations.The yogurt fermentation properties of sensitive bacterial and BIMs were determined under higher concentrations phage environment,the result showed that the acid production,hardness and viscosity of sensitive bacterial had dropped significantly,prolonged curd.And the fermentation properties of BIMs essentially unchanged.For homology analysis,repeats and Cas proteins showed high similarity with several individual species of distant relationships.S.thermophilus 79 had high similarity with S.thermophilus MN-BM-A02 and S.thermophilus ASCC 1275,which suggested that S.thermophilus79 had the potential to produce acid quickly and high yield of polysaccharide.The acquirtion of new spacers made the CRISPR-Cas system against the infection of bacteriophage.