Functions And Behavior Of CglI Gene Complex In Corynebacterium Cretanum

Coryneform bacteria are important industrial producers of amino acid.The starter cultures are very susceptable to phage infection because of the large-scale and continuity of the fermentation process.As a result,the problem of phage infection has serious impact on the manufacture process and causes severe economic loss.Methods trditionally used to prevent phage infection in fermentation industry involve in ameliorating production technology and sanitation conditions,controlling manufacture links and so on.Though infection degree is alleviated,the problem still occasionally arises.Screening resistant mutants was usually adapted in order to solve the problem. The mutants often have the problems of lagged growth rate or other physiological character changes,low production capacity or narrow resistance range and reversible mutation.So it is not an easy thing to obtain fine mutation strains which are suitable for industrial manufacture.For the reasons above,the problem of phage infection has always been the threat to amino acid manufacture.In this paper,in order to explore the feasibility of applying cglI gene complex to construct engineered phage-resistant strains,and further,try to radically solve the phage infection in fermentation industry, cglI gene complex,the restriction-modification system from Corynebacterium glutamicum,was transformed into Corynebacterium crenatum to construct recombinant strain based on exploring the available approach of cloning cglI gene complex into E.coli.At the same time,the stability and influence of recombinant plasmid carrying cglI gene complex on the recombinant strain growth and metabolism were studied. PCR primers were designed according to the sequence of cglI gene complex in GenBank,and the BamHI digestion site sequences were added to their 5' terminal. Using Corynebacterium glutamicum CICC10226 genome as template DNA,standard PCR amplified the cglI gene fragment.The recombinant plasmid pMD18-T-cglI was constructed by connecting the PCR products with cloning vector pMD18-T.The transformants were identified by colonial PCR,and the cglI gene fragment was sequenced.The recombinant cloning vector pMD18-T-cglI and coryneform bacteria shuttle vector pJL23 were both digested by BamHI.The recovered cglI gene fragment and pJL23 big fragment were connected by DNA ligase,and the products were transformed into E.coli HB 101.The recombinant plasmid was extracted,the size and ligation orientation of cglI gene fragment were identified by enzyme digestion and PCR amplification.The plasmid DNA of large-scale preparation was transformed into Corynebacterium crenatum by means of electrotransformation and the transformants were screened on LB plate with Km.After identified the recombinant plasmid by enzyme digestion and PCR amplification,the phage infection experiment was performed to identify the function activity of recombinant Corynebacterium crenatum. The recombinant Corynebacterium crenatum was continuously transferred 100 times on LB plate without Km.The plasmid segregation stability was measured by inoculating 100 colonies selected from each transferred time to LB plate with and without Km.The colonies of different transferred times were inoculated to LB liquid culture media without Km,and then phage infection experiment was carried out to measure the plasmid structure stability.The influence of the recombinant plasmid carrying cglI gene complex on the growth and metabolism of recombinant Corynebacterium crenatum was studied by measuring the growth carve and the glutamate production of recombinant strains,respectively.ResultsThe result of cloning cglI gene complex indicated that the product of amplification by PCR using P1/P2 as primers was the same as had expected(2177bp).Sequence analysis showed the homology between the cloned gene fragment and the known gene sequence was 100%.Positive clones containing the recombinant expression vector pJL23-cg/I were obtained after cglI gene fragment and coryneform bacteria shuttle vector pJL23 were ligated and then transformed into E.coli HB 101.The gene size and ligation oritentaion are correct according to enzyme digestion and PCR amplification identification.It was found that the cglI restriction enzyme alone had the function activity in E.coli HB 101,but no activity was found for the whole cglI gene complex in E.coli HB101.The recombinant Corynebacterium crenatum was constructed by transforming recombinant expression plasmid pJL23-cg/I into Corynebacterium crenatum.No changes were observed in the gene size and ligation oritation according to the identification results.The research of function activity of cglI gene complex in Corynebacterium crenatum showed that the recombinant Corynebacterium crenatum had strong capacity of restriction activity to 5 kind of relative phages.It was indicated that the phages infecting the recombinant Corynebacterium crenatum failed to propagate inside the host cell.The stability research of recombinant plasmid revealed that the segregation and structural stabilities of recombinant plasmid in the Corynebacterium crenatum reached to 100%after 100 times of culture transferred continuously.The measure of growth rate demonstrated that,compared with wild Corynebacterium crenatum,the time of recombinant Corynebacterium crenatum reaching to logarithmic phase lagged about 1 h,but the time reaching to stationary phase tended to be identical.The measure of glutamate production manifested that recombinant Corynebacterium crenatum and wild Corynebacterium crenatum had the same tendency in glutamate accumulation.Both of them had the same top peak of glutamate accumulation at 32 h with 277.74mg/L and 261.39mg/L,respectively.This indicates that the introduction of recombinant plasmid pJL23-cg/I has no impact on the metabolism of Corynebacterium crenatum with respect to the acid production. Using McrBC restriction system deficiency E.coli as host,the restriction-modification system cglI gene complex from Corynebacterium glutamicum was successfully cloned in this paper.Phage resistance strains were successfully constructed by transforming the recombinant plasmid carrying cglI gene complex into Corynebacterium crenatum.The function activity and broad resistance spectrum that cglI gene complex had in the Corynebacterium crenatum was confirmed.In the Corynebacterium crenatum,the recombinant plasmids carrying cglI gene complex were not only structural stability,but also segregation stability.Though the introduction of recombinant plasmid pJL23-cg/I into Corynebacterium crenatum had some impacts on the early growth of the recombinant strains,no impact was found on the production of glutamate.Thus,it was confirmed that cytosine methylation in the host genome which was methylated by the methyltransferases encoded by the cglI gene complex had no impact on the growth and metabolism of Corynebacterium crenatum.In this paper, an effective method was provided to radically solve the phage infection problem in fermentation industry.Also,theoretical foundation was established for further application of cglI gene complex.