Identification Of Phage Resistant Mechanisms Of Phage-resistant Strain Of Lactobacillus Casei

Lactobacillus is recognized as a food-grade microorganism and has various probiotic functions such as regulating immunity,lowering cholesterol,anti-tumor,and anti-hypertension,and plays an important role in immune regulation and maintenance of intestinal microecology.Lactobacillus casei(L.casei)is often used as a genetically engineered vaccine live carrier for the preparation of mucosal vaccines and production of microbial agents;because of its fermentation produces windy flavors,L.casei is also commonly used in milk,meat,feed and vegetable fermentation industries.Vaccine production and food fermentation require the cultivation of a large number of bacteria,However,infection of Lactobacillus phages,the natural enemies of bacteria,would result in the death of bacteria,decreasing the number of viable cells,and even production failure,which has brought great harm to the vaccine industry and the fermentation industry.Lactobacillus phage contamination limits the use of Lactobacillus.In order to effectively prevent Lactobacillus phage contamination,it is of great theoretical and practical significance to screen anti-phage strains and to study the mechanism of bacterial resistance to phage infection and lysis.In this study,the phage of L.casei W56 named LJ isolated by the laboratory was used as the research material to carry out genome-wide analysis to understand the phage gene characteristics;Phage LJ was used as the screening pressure,and secondary infection methods were used to screen for spontaneous mutations.The phage-resistant mutants were identified: the lysogenicity of the phage resistant strains was analyzed;the process of phage-resistant strains resistant to phage infection were determined by analyzing the interactions between the phage-resistant strains and the phage;Identification of differentially expressed genes by phage-infected phage-resistant strains and susceptible strains by transcriptome sequencing.Overexpression of differential genes,verification of differential gene function,and identification of resistance mechanisms.The main research contents and results are as follows:(1)The phage gene was amplified by PCR from L.casei phage LJ and the phage genome was identified as linear double-stranded DNA.The sequence length was 44260 bp and the percentage of GC content was 44.83%.Bioinformatics analysis showed that there are 65 open reading frames in the LJ genome.The phage LJ genome was sorted by functional gene modules like other phages,including the replication module(LJ1-LJ9),the regulatory module(LJ10-LJ33),the DNA assembly module(LJ34-LJ36),and the structural gene module(LJ37-LJ52),lysis Module(LJ53-LJ56)and lysogenic Module(LJ57-LJ65).LJ may hasve lysis cycle or lysogenic cycle.(2)Using phage LJ as screening pressure,20 clonal strains of spontaneous mutated L.casei W56 were isolated and named BIM1-20.The cell morphology,biochemical characteristics,culture characteristics,fermentation characteristics and genetype of BIM1-20 colonies are consistent with the sensitive bacteria.Both of two were L.casei.The results of resistance characteristics determination indicated that the BIM1-20 was resistant to phage LL and Lcb infection,and was completely resistant to phage LJ,and the resistance was genetically stable.(3)Analysis of the lysogenicity of the resistant strain BIM1-20 revealed that BIM1-20 was spontaneously release the lytic phage of L.casei W56,and that mitomycin and UV rays could not induce BIM1-20 to release phage particles.BIM1 was selected for phage gene detection and integration site analysis.The results showed that the resistant strain BIM1 was a strain of lysogenic bacteria.The phage LJ DNA was circumscribed and cleaves at 39625 bp to 39626 bp,and then LJ DNA was integrated into the L.casei W56 genome at 1982081-1982082 bp site.The integration site sequence has a 14 bp core sequence ATGCCGGCTGCAGG,attP is located between the phage integrase and lysogen,and attB is located near the L.casei W56 genomic tRNA-Thr gene.(4)Analysis of the interaction between the phage and the resistant bacteria showed that the resistant bacteria did not use CRISPR,restriction modification and plasmid defense system to resist phage infection;when phage reacted with resistant bacteria,the phage could still adsorb resistant bacteria.The phage-resistant strain can block phage DNA injection into the cells The proliferation of phage in resistant bacteria was inhibited,and the phage does not lyse the resistant bacteria.The resistant bacteria regulated the transcription of the phage gene at 15 min.In summary,the preliminary analysis of the resistant mechanism is that the resistant bacteria can inhibit the phage lysis bacteria by blocking phage DNA injection and influencing phage replication-regulated gene expression.(5)The samples of phage-infected bacterium 15 min were subjected to transcriptome sequencing to search for differentially expressed genes of sensitive strains and resistant strains,and further analysis the mechanisms that resistant bacteria blocked phage DNA injection and affected phage replication-regulated gene expression.S-VS-R,There were a total of 95 bacterial genes were differentially expressed,of which 59 were up-regulated genes and 36 were down-regulated genes.There were a total of 156 differentially expressed bacterial genes in SA-VS-RA,of which 112 were up-regulated genes and 44 were down-regulated genes.The gene expression levels of bacterial mannose permease gene and PTS system mannose-specific EIAAB unit were significantly decreased when phages were added to the resistant bacteria;The expression levels of bacterial HTH type transcriptional regulatory factors,redox-sensitive transcriptional regulators,GntR family transcriptional regulators,and operon genes were significantly up-regulated;the expression levels of lysogenic genes such as phage repressor proteins and anti-repressor proteins were significantly up-regulated,and the expression levels of phage-replicating genes and other pre-expressed genes were significantly down-regulated.Themajor biochemical metabolic pathways and signal transduction pathways in which the differentially expressed genes KEGG are enriched are metabolic pathways,environmental information processing pathways and cellular process pathways,mainly including fructose-mannose metabolism,fatty acid metabolism,purine metabolism,pyrimidine metabolism,PTS Phosphotransferase system and two-component system.The GO functional analysis results of significant differentially expressed genes indicated that GO functional items such as metabolic processes,single biological processes,cell membrane components,transcription factor activities,and catalytic activities played an important role in fighting against phage infection.(6)The differentially expressed genes were subjected to real-time fluorescence quantitative PCR to analyze the gene expression.The qPCR results were consistent with the transcriptome sequencing results.Overexpression methods were used to verify the role of the bacterial PTS phosphotransferase system and phage lysogenic genes in the phage-resistant strains.A recombinant Lactobacillus expression system was constructed.The resistant bacteria were used to express the pts gene and the susceptible bacteria were used to express the phage lysogen gene.Western-bolt identification and indirect immunofluorescence demonstrated that the recombinant bacteria expressed bacterial PTS protein,phage repressor protein CI,membrane protein M and anti-repressor protein Cro-ant.The susceptibility of recombinant L.casei to phage was tested,and the growth curve of the bacteria and the one-step growth curve of phage when the phage invaded the recombinant bacteria were determined.The results showed that the susceptibility of susceptible strains expressing CI and M protein to phage was changed from sensitivity to insensitivity;the susceptibility of PTS-expressing bacteria and Cro-ant-expressing strains to phage did not change significantly.However,the Cro-ant-expressing strains grow slowly.RT-qPCR detected the expression levels of bacterial and phage gene when phage was inoculated with recombinant L.casei.The results showed that compared with phage infection of pPG-PFT/S,when phage infects pPG-PFT-CI-M,the expression of phage replication regulatory genes and lytic genes was significantly decreased,the expression levels of integrase were significantly increased,the expression levels of related genes in bacterial PTS systems changed significantly,and the expression level of bacterial transcription regulator genes was significantly increased.The expression level of metabolism-related genes was significantly increased,and the expression level of cell cycle regulatory genes was significantly increased.The over-expression of CI and M protein can help bacteria resist phage lysis by improving metabolism,regulating transcription,regulating cell cycle and changing PTS system signal transduction and membrane transport,which proves that cI and m genes are the key genes of resistant bacterium resistant to phage infection.In this study,the identification of phage and the study of the characteristics and resistance mechanisms of the isolated resistant bacteria provided the conditions for the construction of excellent phage-resistant strains,and provided a theoretical basis for the development of an effective phage control strategy in industrial production.