Bacteriophage Resistance Of Klebsiella Pneumoniae Based On CRISPR-Cas System:bioinformatics Analysis And Construction Of Engineering Strains

Bacteriophage infection is a thorny issue in microbial fermentation industry,and there are still no good strategies to control.This study is dedicated to exploring solutions to phage infection problems based on CRISPR-Cas technology.In this paper,Klebsiella pneumoniae was used as an example.Starting from the isolation and analysis of phage,the interaction between natural CRISPR-Cas system and(pro)phage was evaluated.The CRISPR-Cas9 based phage genome editing technology was established.Based on CRISPR-Cas9,the methods for enhancing phage resistance were explored.The research includes the bioinformatics analysis of K.pneumoniae and phages using the public database,as well as the exploration and demonstration of relevant issues from the experimental point of view.The main findings are as follows:Firstly,the isolation,physiological characteristics and genomic analysis of a phage infecting an industrial fermentation strain of K.pneumoniae were completed,as well as the effect of phage infection on 1,3-propanediol fermentation.The phylogenetic tree analysis showed that phiKpS2 belongs to the Kp34 genus phage,which is a kind of widely distributed Klebsiella phage in nature.It is proved that phage infection causes the fermentation to stagnate for a long time,the fermentation cycle is prolonged,and the production efficiency is lowered.In addition,it was found that the effect of phage infection on cell growth and metabolism is closely related to the growth stage of the cells at the time of infection.Secondly,the distribution of prophage in K.pneumoniae and its effect on bacterial adaptation and evolution were evaluated.A new subtype I CRISPR was identified in K.pneumoniae,named I-E*,and its basic features include location on the chromosome,gene organization,Cas protein,tracrRNA structure,PAM,evolutionary sources,etc.were identified.The CRISPR spacer sequences are mainly derived from phages,prophages and plasmids,suggesting that its main function is to prevent invasion of foreign genetic elements.It is revealed that the prophage is one of the important reasons for the genomic plasticity of K.pneumoniae.The CG258 strains contain highly conserved prophages,including its two unique P2-P4 phage systems.The aARGs on the chromosome of K.pneumoniae are concentrated in CG258 and CRISPR-positive strains.Of which 60%of the aARGs of the CG258 strain are located in the prophage region,while only 10%of the aARGs in the CRISPR-positive strain are located in the prophage region.It was shown that the CRISPR-Cas system and the phage of the CG258 may independently participate in the chromosomal integration of ARGs.It was found that the distribution of CRISPR-Cas system and prophage were dependent on the MLST typing of the strain,and there was a high proportion of self-targeting spacers in CRISPR.It may be the reason for the limited impact of the CRISPR-Cas system on the presence of prophage in K.pneumoniae.Thirdly,the prophage inducibility of five strains of K.pneumoniae was examined.The prophage induction process of one strain was quantitatively analyzed,and two induced prophages were analyzed by genome-wide analysis.The results showed that bacteriophage proliferation and cell lysis were prone to occur in K.pneumoniae under MMC induction.Among them,there are two inducible prophage in the genome of K.pneumoniae W3,and there is a spontaneous induction phenomenon.The induced phages PKPW3.1 and PKPW3.3 have no homologous phages in the NCBI phage database,indicating a relatively new phage type.Analysis of the tRNA carried by the prophage showed that the three tRNAs(tRNA-Arg,tRNA-Asn,tRNA-Thr)contained in PKPW3.1 and PKPW3.3 were the most common tRNA types in K.pneumoniae phage.The high abundance,inducibility and spontaneous induction of the prophage in the genome indicated that the prophage is a risk factor of phage infection and must be noted in the fermentation industry based on K.pneumoniae.Fourthly,a CRISPR-Cas9-based phage genome editing tool was established.It was confirmed that a 30-60 bp short homologous arm can efficiently implement a variety of genetic operations including point mutation,gene deletion and insertion,and frameshift mutation,thereby greatly simplifying the cumbersome processes of template construction.By analyzing the correlation between the predicted activity and actual measured activity of the 177 sgRNAs,it was confirmed that the sgRNA prediction model derived from eukaryotes could not be used to predict sgRNA activity against phages.It was confirmed that weak sgRNA can also be used for phage genome editing based on homologous recombination.It was confirmed that the necessity of phage genes can be quickly determined by frameshift mutation.Taking phage phiKpS2 as an example,the essential of 17%genes was preliminarily verified by frameshift mutation and gene deletion.Finally,the activity distribution of sgRNA against phage was analyzed,and the mechanism of phage escape from single target and multi-targets CRISPR-Cas system was examined.It was revealed that CRISPR-Cas-based phage resistance can be significantly enhanced by expressing Mu Gam.Among the sgRNAs against phage,the number of sgRNAs with low activity was high.Among the 177 sgRNAs we analyzed,the sgRNA with the lowest activity accounted for 49.7%,while the sgRNA with the highest activity accounted for only 4.5%.Phages have a variety of ways to escape the CRISPR-Cas system,including non-homologous end joining,homologous recombination,and large-scale deletion of targeted regions.The multi-targets CRISPR-Cas system with 5 sgRNAs in tandem promoted large fragment deletion of the targeted region,but did not significantly enhance the phage resistance.It was confirmed that Mu Gam can significantly enhanced the CRISPR-Cas9-based phage resistance.And Mu Gam's enhanced effect on bacteriophage resistance of CRISPR-Cas system is applicable to all kinds of sgRNA with different active level.The manner in which phage escapes after the expression of Mu Gam has changed,where the wild type phages and the mutants with large fragment deletion of the targeted region are increased.In the future,in the process of breeding K.pneumoniae fermentation strains,it is necessary to carry out inducible detection of prophage,or to develop a phage-clearing technology based on CRISPR-Cas system.In addition,a better interference strategy can be designed to prevent phage escape through in-depth study of the mechanism of phage DNA double-strand break repair.