Analysis And Detection Of CRISPR-Cas System And Type Ⅱ TA System Gene Structure Of Serratia

Objective Serratia is one of the common clinical opportunistic pathogens.In recent years,Serratia has attracted increasing attention because its clinical strains are prone to develop resistance to commonly used antibiotics and occasionally lead to the outbreak of nosocomial infections.The Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associate protein(CRISPR-Cas)and the Toxin-Antitoxin(TA)system plays an important role in phage defense by cutting and degrading nucleic acid specifically and abortive infection respectively.In addition,the former is thought to affect the horizontal transfer of mobile elements between bacteria,and the latter is related to the tolerance of bacteria to adverse environments.The purpose of this study is to systematically analyze the structure and distribution characteristics of the CRISPR-Cas system and the type Ⅱ TA system of Serratia,and to study the relationship between them and mobile genetic elements,drug-resistant genes and drug-resistant phenotypes of Serratia.At the same time,the influence of partial TA system expression on bacterial growth will be explored.Methods 1.The complete genome sequences of 88 Serratia strains were downloaded from the Nucleotide database,and CRISPR array and cas gene contained in the Serratia genome was analyzed by CRISPRcasfinder.Sequence alignment and phylogenetic tree construction of cas gene was performed by Megaline.AT content and sequence alignment of the leader were analyzed by DNAMAN8,and BPROM was used to predict its promoter.Repeats were compared with CRISPRmap database,and prediction of the minimum free energy(MFE)and the secondary structure of RNA was completed by RNAfold.Prediction of spacer targeting genes was performed by BLASTN and CRISPRTarget respectively.Online software PHASTER,ICEFinder,Ori TFinder and CARD were used to predict the prophage,integrated conjugate element(ICE),the conjugable transfer plasmid and drug resistance genes in Serratia genome,and the correlation between them and the CRISPRCas system was analyzed.2.TAfinder was used to predict the type Ⅱ toxin-antitoxin system contained in the Serratia genomes.The toxin and antitoxin in TA system were constructed into database respectively,and which were grouped according to the E-value of BLASTP.Type Ⅱ TA system was rematched and its operon characteristic was analyzed.Origin software was used to show the distribution of type Ⅱ TA system in Serratia.Flanking genes of Serratia TA system were identified.The positions of genomic islands(GI),ICE,and prophages in chromosome were predicted to analyze the relationship between TA system and mobile elements.3.Based on the above analysis results,primers of Ⅰ-E and Ⅰ-F cas1 genes and 10 pairs of TA system were designed and used to detect cas1 gene and TA system in 98 clinical strains of Serratia marcescens collected from hospitals in Qingdao and Yantai.The relationship between the existence of these systems and the bacterial drug resistance phenotype was analyzed.4.Amplification products of all toxin and part of antitoxin encoding gene in the 10 TA system were linked to p BAD33 and p ET28 expression vectors respectively,which were expressed and co-expressed in Escherichia coli strains Top10 and BL21,so as to verify the biological function preliminarily.5.Statistical analysis was performed by SPSS 20.0.Chi-square test and Kruskal-Wallis Test were used for difference analysis.Results 1.Total of 17 of 88 strains(19.32%)contain 41 confirmed CRISPR arrays,9strains(10.23%)contain both cas gene cluster and CRISPR array,and the gene cluster was mainly Ⅰ-E or Ⅰ-F type.The cas1 gene of Serratia cas cluster is the most conserved,and its phylogenetic branches are consistent with the typing of the CRISPR-Cas system.Leader of Serratia is not conservative and rich in "AAAA" and "TTTT",most of them were predicted to have promoters.41 CRISPR arrays contain 14 kinds of repeats which are highly conserved within the type.It was predicted that they can form stable secondary structures with "stem loop".The spacer of Serratia is 30～34bp in length.Targeted analysis revealed that most spacer sequences are homologous to bacteriophages and a few are homologous to plasmids.Most of these plasmids and phages also belong to the Enterobacteriaceae.The number of prophages of Serratia containing the complete CRISPR-Cas is significantly lower than that containing CRISPR array only and the non-CRISPR array.There was no difference in distribution of the ICE in the above groups.Serratia containing only CRISPR array contain a higher number of conjugable transfer plasmid.Most of the drug resistance genes carried by Serratia chromosome are spontaneous mutation or inherent,and the mobile drug-resistant genes are distributed among the plasmids.There was no connection between the distribution of these plasmids contains drug-resistant genes and the CRISPRCas system.2.TAfinder predicted that 88 Serratia contained 1820 type Ⅱ TA systems,31 type IV TA systems,and 182 protein sequences related to efflux pump components.The number of type Ⅱ TA systems contained in a single Serratia strain ranged from 6 to 32.The toxin sequences in the Serratia can be divided into 16 categories and 50 sub-categories,of which Rel E and GNAT toxins are more abundant.Almost all antitoxins are transcription regulators,and the majority of them are RHH and Xre families,which can be divided into 52 categories,but their protein sequences and domains are more complex and variable than toxins.TypeⅡ TA systems in Serratia are re-divided into 78 groups and further classified into 16 categories.Distribution of type Ⅱ TA systems in Serratia is extremely uneven,and their structures are highly complex and changeable.Most of type Ⅱ TA systems in Serratia are located on their chromosomes.The same system has a similar genetic context within the same species.A few are located in mobile elements such as plasmids,prophages,and GI,and can be transferred between strains.3.Only 1 out of 98 Serratia marcescens strains amplified the cas1 gene,and the detection rate of 10 pairs of TA systems in clinical strains was roughly in line with the database.The number of TA systems in drug-resistant bacteria is significantly lower than that of non-resistant bacteria.This difference is caused by Rel1,Rel2,Ccd AB,Hic AB,GNAT,MNT/HEPN class TA systems.4.10 of type Ⅱ TA system in Serratia marcescens clinical strains were sequenced and found to be similar to the sequence in the database.Preliminary functional verification of TA system revealed that the toxins of Rel1,Rel2,Rel3,PIN1,PIN2,and Hic AB system can inhibit the growth of Top10 strains.The toxins of the Rel1,Rel2,Rel3,and PIN2 systems also showed inhibitory effect on the BL21 strain,and the toxic effects of Rel1 and PIN2 systems can be neutralized by their homologous antitoxins to a certain extent.Conclusion The presence rate of Serratia CRISPR array and CRISPR-Cas system is significantly lower than the average level of bacteria.Type of CRISPR-Cas system are mainly Ⅰ-E and Ⅰ-F,which are consistent with most of Enterobacteriaceae.Repeats are conserved and Spacer sequences are homologous to bacteriophage and plasmid.CRISPRCas system of Serratia could reduce the content of prophage to a certain extent,but had no effect on the conjugable transfer plasmid,ICE and drug resistance genes.The type Ⅱ TA system is widely distributed in Serratia with a large number,most of which are located in the permanent position of the bacterial chromosome,and its sequence and structure are highly complex and variable.The carrying rate of CRISPR-Cas system in clinical Serratia marcescens was low,and the existence rate of 10 pairs of TA system was roughly consistent with that in the database,and there was a certain correlation between the number of TA and drug resistance of the strain.The heterotopic expression preliminarily revealed that 6 toxins of 10 TA systems showed obvious toxicity,which laid a foundation for further characterization and functional exploration.