Identification of M. tuberculosis Rv3441c and M. smegmatis MSMEG_1556 and essentiality of M. smegmatis MSMEG_1556.
The normal growth of mycobacteria attributes to the integrity of cell wall core which consists of peptidoglycan (PG), arabinogalactan (AG) and mycolic acids. N-acetyl glucosamine (GlcNAc) is an essential component in both PG and AG of mycobacterial cell wall. The biosynthetic pathway for UDP-N-acetylglucosamine (UDP-GlcNAc), as a sugar donor of GlcNAc, is different in prokaryotes and eukaryotes. The conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, which is catalyzed by phosphoglucosamine mutase (GlmM), is unique to prokaryotes. Bioinformatic analysis showed that Msm MSMEG_1556 and Mtb Rv3441c are homologous to Ec GlmM. In this study, soluble Msm MSMEG_1556 protein and Mtb Rv3441c protein were expressed in E. coli BL21(DE3) and their phosphoglucosamine mutase activity were detected. In order to further investigate the essentiality of MSMEG_1556 for the growth of M. smegmatis, we generated a conditional MSMEG_1556 knockout mutant, which harbored thermo-sensitive rescue plasmid carrying Mtb Rv3441c. As the rescue plasmid was unable to complement MSMEG_1556 deficiency at 42 °C, MSMEG_1556 knockout mutant did not grow. The dramatic morphological changes of MSMEG_1556 knockout mutant after temperature shift from 30 °C to 42 °C have been observed by scanning electron microscope. These results demonstrated that MSMEG_1556 is essential for growth of M. smegmatis. This study provided evidence that GlmM enzyme could be as a potential target for developing anti-tuberculosis drugs.
Department of Biochemistry and Molecular Biology, Dalian Medical Universtiy, Dalian, Liaoning, China.
Shuang
S
Li
Jian
J
Kang
Wendan
W
Yu
Yan
Y
Zhou
Wenli
W
Zhang
Yi
Y
Xin
Yufang
Y
Ma
D000117:Acetylglucosamine / Q000737:chemistry; D000595:Amino Acid Sequence; D000995:Antitubercular Agents / Q000494:pharmacology; D001671:Biochemistry / Q000379:methods; D002473:Cell Wall / Q000378:metabolism; D019295:Computational Biology / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D005685:Galactans / Q000737:chemistry; D008855:Microscopy, Electron, Scanning / Q000379:methods; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D009171:Mycolic Acids / Q000737:chemistry; D010457:Peptidoglycan / Q000737:chemistry; D010733:Phosphoglucomutase / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D000995:Antitubercular Agents; D017931:DNA Primers; D005685:Galactans; D009171:Mycolic Acids; D010457:Peptidoglycan; C097631:phosphoglucosamine mutase; D010733:Phosphoglucomutase; D000117:Acetylglucosamine