Polymorphism of Rv3303c−Rv3304 of Mycobacterium tuberculosis modern Beijing genotype strains and physiological significance in China

Objective To analyze the polymorphism of gene spacer region of Rv3303c−Rv3304 of 141 strains of Mycobacterium tuberculosis in China and discuss the physiological significance of Rv3303c−Rv3304 gene spacer.

Methods The sequence of Rv3303c−Rv3304 gene spacer in 141 strains of M. tuberculosis modern Beijing genotype was analyzed by polymerase chain reaction (PCR) and sequencing, and the recombinant plasmid of Rv3303c−Rv3304 gene spacer was constructed, and the recombinant plasmid and empty plasmid were electroporated into Mycobacterium smegmatis mc²155. β-galactosidase (lacZ gene expression product) activity assay was used to detect the expression level of reporter gene lacZ. Software SPSS 25.0 was used to conduct a one-way analysis of variance.

Results There were 376 bp, 260 bp and 144 bp sequences in the spacer region of the Rv3303c−Rv3304 gene of 141 strains of the modern Beijing genotype, which contained 4, 2 and 0 copy repeats, respectively. Two copy repeats were the predominant sequence, accounting for 95.74%. Based on these three different copy repeats, recombinant plasmids pSD5B-334, pSD5B-332, and pSD5B-330 were constructed, and the recombinant plasmids and empty plasmids were transferred into Mycobacterium smegmatis. The results of β-galactosidase activity detection showed that the β-galactosidase activity of recombinant strains MSMEG-334, MSMEG-332 and MSMEG-330 increased compared with strains MSMEG-pSD5B, and the differences of β-galactosidase activity among strains were significant (P<0.001).

Conclusion In China, the gene spacer region of Rv3303c−Rv3304 of the modern Beijing genotype strain of M. tuberculosis, has polymorphism, which can significantly affect the transcription of reporter gene lacZ indicated by model strain verification.