Study M. Smegmatis Phage Resistance Mechanisms For SWU1and Preliminary Function Of MSMEG₃705Gene

Phages are viruses that invade bacterial cells, are ubiquitous in nature and more than1030phages have been recognized. Bacteriophages are central components in the development of molecular tools for microbial genetics. It is also a kind of valuable biological resources. Phage treats bacterial infection, rapid detection of drug resistant bacteria, bacterial genotyping, control of bacteria in food, control of environmental pollution, control the spread of pathogens and molecular biology research is a very promising tool. Especially, routine examine of Mycobacterium tuberculosis take long time in the clinic, but using the new kit Mycobacterium phage developed have greatly shorten the time of diagnosis of tuberculosis, to win a valuable treatment time for the patient. However, pathogenic bacteria phage resistant to gradually, which gives the original promising phage therapy and diagnosis of cast a shadow. In this study we used Mycobacterium smegmatis as model strains to explore the phage resistance mechanism of Mycobacterium tuberculosis.We used the lytic mycobacterium phage TM4as the screening conditions, successfully screened a TM4and SWU1phage resistant M. smegmatis mc2155Tn5mutant strain, according to the order of the mutant library we named M12. We found that SWU1genome injected into M12mutant strain by electrotransformation led turbid plaque on the phage cultivating plate. Next, we analyzed the colonial morphology of M12mutant strain and wild type Mycobacterium smegmatis. We found M12single colony morphology was smooth, shiny, neat edge, no fold, whereas, the wild type Mycobacterium smegmatis colony was shown irregular edge, matt surface, and fold. This finding suggests morphological changing of M12may due to changes of cell wall component which might lead to SWU1phage resistance. Therefore, we examined the M12and wild type Mycobacterium smegmatis surface fatty acid composition by GC-MS. We found no differences in fatty acid composition M12mutant strain compared with the wild-type strain; however, there is larger difference in content of each component. Subsequently, we used Thermal Asymmetric Interlaced PCR （Tail-PCR） to identify M12mutant strain transposon5（Tn5） insertion sites located in Mycobacterium smegmatis genome MSMEG₃705gene. Through the Xer-cise system, we construct a MSMEG3705knockout mutant strain, and used the PCR method to amplify specific fragment, through sequencing of the specific fragment, results show the successful construction of MSMEG₃705gene knockout strain. Then we examined the sensitivity of MSMEG3705knockout strains to SWU1, found that MSMEG3705knockout mutant strains did not resist to SWU1phage and have not M12transposon mutant strain SWU1phage resistant phenotype. Accordingly, to the above, we draw the conclusion initially, M12Tn5mutant strain phage resistance phenotype have not direct relation with Tn5insert in the MSMEG₃705gene causes this gene function lose. We speculate that M12tolerance mechanism of TM4, SWU1may be due to spontaneous mutations in M12genome, leading host phage receptors are missing or changed to form the resistant phenotype.On the other hand, we get the MSMEG₃705knockout strain, through alignment of MSMEG₃705protein sequences by NCBI Blast, further analysis the function of MSMEG3705gene. We found that have seven Mycobacterium hypothesis proteins with MSMEG₃705protein homology. All the seven putative protein are major facilitator superfamily. We further analysis and prediction transmembrane domains and three-dimensional structures, it was found that MSMEG₃705has12alpha helix domains and it is typical transmembrane domain in MFS. Bioinformatics analysis showed that MSMEG3705belongs to the MFS. MSMEG₃705mutant strain minimum inhibitory concentration （MIC） of10kinds of common mycobacterial antibiotics and five metal ions, we determined that the MSMEG₃705knockout strains capreomycin sensitivity increased2times, but showed RIF tolerance, MIC is2times higher than that of control group. On most other antibiotic MIC compared with the wild type control had no significant change, and this result indicating that MSMEG3705plays an important role in Mycobacterium smegmatis efflux capreomycin. The ethidium bromide efflux analysis, we found that the MSMEG₃705gene encoding efflux pump protein, it also plays an important role in Mycobacterium smegmatis EB efflux. In this study, we also found that MSMEG₃705knockout strains than wild type Mycobacterium smegmatis advance into the logarithmic growth phase under the same condition, the reverse transcription PCR and real time PCR results indicated that MSMEG₃705gene knockout increased expression of downstream MSMEG₃706gene.