Research On The Regulatory Functions Of KatG And A SRNA MS2 In Mycobacterium Smegmatis

Tuberculosis is caused by Mycobacterium tuberculosis.Tuberculosis epidemic is not fully controlled,Nowadays it is still the most terrible infectious disease in the world.With the discovery and application of antibiotics for tuberculosis,there are more and more drug resistance tuberculosis in clinical treatment.This is a great challenge for Tuberculosis Treatment.It is particularly important and urgent for us to research on drug resistance tuberculosis.Mycobacterium smegmatis is widely used as a model organism in mycobacterial research works because of its similar genetic background,and its rapid growth rate and hardly cause diseases.（1）Research on regulatory functions of KatG in Mycobacterium smegmatis.kat G produce functional peroxidase-catalase,which protects mycobacteria from reactive oxygen species and nitrogen intermediates in macrophage phagolysosomes.Another major characteristic of mycobacterial catalase-peroxidases is activation of isoniazid,which can kill mycobacteria.Therefore,kat G plays an important role in mycobacterial isoniazid resistance.Appearance of isoniazid resistance in Mycobacterium tuberculosis is often associated with a loss of catalase-peroxidase activity through mutations in kat G.Three different kat G were identified in the Mycobacterium smegmatis mc²155genome,kat G1（MSMEG＿6384）,kat G2（MSMEG＿3461）and kat G3（MSMEG＿3729）.In this study,we analyzed the transcription of three kat G in M.smegmatis.The transcription of kat G was induced by hydrogen peroxide（H₂O₂）through q RT-PCR.We got a series of kat G knockout strains,and conducted a series of experiments such as hydrogen peroxide tolerance experiments,catalase activity and peroxidase activity staining.The result suggested that KatG1 and KatG2 possess hydrogen catalase-peroxidase activity,which plays a major role in the resistence of oxidative stress in M.smegmatis,and KatG3 does not have any hydrogen catalase-peroxidase activity.The isoniazid tolerance assay and the isoniazid oxidation assay in vitro showed that KatG1 act as major role in isoniazid activation,KatG2 donates little in isoniazid activation,and KatG3 has no effect in isoniazid activation.We studied the function of three kat G genes in M.smegmatis,which helps us to understand the mechanism of isoniazid resistance in mycobacteria better and provide a basis for the treatment of isoniazid-resistant tuberculosis.（2）Research on regulatory functions of Ms2 sRNA in Mycobacterium smegmatisNon-coding RNA（nc RNA）is a type of RNA that does not encode protein.Because of its short length,it is also called small RNA.nc RNA is widely distributed in many organisms and is involved in various biological metabolism such as regulation of gene transcription,m RNA stability,RNA molecular processing and modification.In this study,we found a highly expressed sRNA between MSMEG＿0373 and MSMEG＿0374 according to transcriptome data.which we named it Ms2.After overexpression of Ms2,two different sizes of colonies appeared,the surface of the small colonies was smooth,and the surface of the large colonies was rough.Small colonies and big colonies have significant differences in motility,sedimentation,biofilm formation,and antibiotics resistance.The phenotype difference between small colonies and big colonies does not disappear with the elimination of overexpression plasmid.Possible targets of Ms2 were predicted by analyzing differential transcriptome data.In this study,we investigated the phenotypes of large and small colonies produced by Ms2 overexpression in M.smegmatis.This provides a basis for the identification of the Ms2 target,enriching the regulatory network of sRNA.