using-enhanced-green-fluorescence-protein-expressing-escherichia-coli

Using Enhanced Green Fluorescence Protein-expressing <em>Escherichia Coli</em> to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

The second hospital of Dalian Medical University

The mycobacterial peptidoglycan has structure and biosynthetic pathways to similar those of other bacteria. UDP-N-acetylglucosamine enolpyruvyle transferase (MurA) catalyzes the first reaction in the biosynthesis of peptidoglycan. The MurA enzyme has been identified from various bacterial species, but the in-depth biochemical properties of mycobacterial MurA have not been characterized. In this study, both Mycobacterium tuberculosis MurA protein and Mycobacterium smegmatis MurA protein were overexpressed in Escherichia coli and purified by affinity chromatography. MurA activity was detected by HPLC. A colorimetric assay of MurA activity was also developed and the kinetic properties of Mtb MurA and Msm MurA were determined using this colorimetric assay. A conditional murA gene knockout strain was constructed by DNA homologous recombination. The disruption of murA in the genome of M. smegmatis led to loss of viability at a non-permissive temperature. Drastic morphological and structural alterations in the M. smegmatis murA knockout strain were observed by scanning electron microscopy and transmission electron microscopy. These results demonstrated that murA was an essential gene for growth of M. smegmatis. Therefore, MurA is a potential target for developing new anti-tuberculosis drugs. 

Li-ming Xu

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