The Function Of Mycobacterium Smegmatis MSMEG₆402

Mycobacterium tuberculosis (M.TB) was the pathogen of tuberculosis (TB). Ethambutol (EMB), a first line clinical anti-M.TB drug, could inhibit the growth of tuberculosis during bacteria grow phase effectively. Studies showed that cell wall was important to the survival for TB and arabinose was an important element of cell wall. After treated with EMB, arabinose in cell wall of TB was lost greatly, and the structure of the cell wall was destroyed. Studies indicated that it is because EMB disturbs the metabolism of Decaprenyl-phospho-arabinose (DPA), the active donor of arabinosyl, therefore, EMB inhibited the growth of TB. Experiments showed that the synthesis of DPA consisted of three continuous steps, first, Rv3806c, a kind of phosphotransferase of TB, catalyzed ten isoprene poly phosphate (DP) releasing phosphate and the product reacted with 5-phosphoribosyl pyrophosphate (PRPP), which reaction generated 5-ten isoprene ribose phosphate polymer (5-P-DPR); Second, 5-P-DPR released phosphates and changed into ten ribose poly isoprene (DPR) by the catalysis of a kind of phosphates; At last, DPA was generated by the function of Rv3790 and Rv3791, a kind of epimerizes of TB. Among the whole process of the three reactions above, the enzyme catalyzed the second one was not determined. It is because that the promoter of Rv3807c was the same as Rv3806c, and Rv3807c was predicted a phosphatase, Rv3807c might be the enzyme. This experiment took Mycobacterium smegmatis mc2 155 as the object of research, (1) in supernatant, expressed the protein of MSMEG₆402, which was the homologous genes of Rv3807c, in order to lay the groundwork for determining the function of MSMEG₆402, (2) the role of MSMEG₆402 in the metabolism of arabinose in cell wall, (3) and searched for genes related to MSMEG₆402, in order to complete the route of synthesis of DPA.Methods:1. Amplifying MSMEG₆402: First, received nucleotide sequence of MSMEG₆402 from NCBI. Next, designed primers used for PCR, and added BamHI and NdeI site, which were both restricted enzyme sites, to upstream and downstream of the primers. At last, made use of Ex Taq DNA polymerase amplifying MSMEG₆402 from the genome of Mycobacterium smegmatis mc2 155. As a result, the product took along with two restricted enzyme sites.2. Cloning of MSMEG₆402: Ligated the gene amplified above to the EcoRV site of pMD18-T Vector, and transformed the product to the competent cell of Novablue, which was a kind of strain of Escherichia coli. When the strains were growing up, screened the positive recombinant plasmid by restriction endonuclease, and sequenced the fragment of the product to determine if the sequence was the same as the sequence of MSMEG₆402 from genome database.3. Constructing expression plasmid of MSMEG₆402: Connected MSMEG₆402 to pET16b Vector, which was an expression vector used in Escherichia coli by use of T4 DNA Ligase and transformed the product to Novablue.4. Over expression of MSMEG₆402: Transformed the positive recombinant plasmid to the competent cell of BL21(DE3), which was also a kind of strain of Escherichia coli, in order to express MSMEG₆402 gene. Collected interest soluble protein after IPTG inducing.5. Determining the protein of MSMEG₆420: Determined if the supernatant contains the over expression of MSMEG₆402 by SDS-PAGE electrophoresis and Western Blotting.6. Extracted the polysaccharide in cell wall from both wild type M.smegmatis and M.smegmatis with MSMEG₆402 knocked out and hydrolyzed them into monosaccharide.7. Determined the variety of the ratio of arabinose and galactose between the two kinds of monosaccharide received above by HPLC.8. Determined the soluble proteins from both wild type M.smegmatis andΔMSMEG₆402 by 2-D electrophoresis and compare the differences between them by PDQuest 8.0.Result:1. We expressed the protein of MSMEG₆402 in supernatant successfully.2. The quantity of arabinose in cell wall fromΔMSMEG₆402 was smaller than from wild type M.smegmatis.3. We separated the soluble protein from both wild type M.smegmatis and ΔMSMEG₆402, and it showed that there were about 181 protein spots in wild type sample and about 198 protein spots inΔMSMEG₆402 sample.4. There were 5 different protein spots between wild type M.smegmatis andΔMSMEG₆402.Conclusion:MSMEG₆402 gene played an important role in the synthesis and metabolism of arabinose in cell wall of M.smegmatis and it might participated in the synthesis of DPA.Future research:(1) Purify the protein of MSMEG₆402 and determine its function, (2) construct the synthesis route of DPA in vitro, determine if MSMEG₆402 is the key enzyme during this process directly, (3) prepare the antibody of MSMEG₆402 in order to study the expression of MSMEG₆402 in different ways. (4) analyze the proteins, whose expression impacted by MSMEG₆402, in order to complete the function of MSMEG₆402.