title: The effect of MSMEG_6402 gene disruption on the cell wall structure of Mycobacterium smegmatis.
abstract: Arabinogalactan (AG) of mycobacterial cell wall consists of arabinan region, galactan region and disaccharide linker. The arabinan is composed of D-arabinofuranose residues, and decaprenyphosphoryl-D-arabinose (DPA) is the donor of the D-arabinofuranose residues. DPA is formed from phosphoribose diphosphate (PRPP) in a four-step process catalyzed by transferase, phosphatase and epimerase, respectively. Mycobacterium tuberculosis Rv3806c has been identified as PRPP: decaprenyl-phosphate 5-phosphoribosyltransferase, and heteromeric Rv3790/Rv3791 has epimerase activity. Rv3807c is putative phospholipid phosphatase. However, there is no direct biochemical evidence since expression of Rv3807c has been unsuccessful. Mycobacterium smegmatis MSMEG_6402 is ortholog of Rv3807c. To investigate the function of MSMEG_6402 on AG biosynthesis, a conditional MSMEG_6402 gene knock out (M. sm-ΔM_6402) strain was constructed through homologous recombination technique. The morphological and compositional changes of cell wall were examined in the M. sm-ΔM_6402 strain. The M. sm-ΔM_6402 strain grew at non-permissive temperature slower than that at permissive temperature, indicating that MSMEG_6402 is non-essential for growth of M. smegmatis. The change of cell shape and detectable bulging on the cell surface of M. sm-ΔM_6402 strain were observed by scanning electron microscopy, and curled as well as deformed cell wall of M. sm-ΔM_6402 strain was revealed by transmission electron microscopy. Analysis of sugar composition in the cell wall by HPLC indicated that the ratio of arabinofuran to galactofuran in M. sm-ΔM_6402 strain was changed to 1.7:1 comparing with 2:1 in the wild type. It demonstrates that the lacking MSMEG_6402 interferes the biosynthesis of arabinan. Analyzing 5' P-DPR and DPR from both M. sm-ΔM_6402 strain and wild type M. smegmatis is undergoing in this lab.
authors.affiliation: Department of Biotechnology, Dalian Medical University, Dalian 116044, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Jiang
authors.forename: Lianqi
authors.initials: L
authors.lastname: He
authors.forename: Yaoyao
authors.initials: Y
authors.lastname: Zhan
authors.forename: Shizhu
authors.initials: S
authors.lastname: Zang
authors.forename: Yufang
authors.initials: Y
authors.lastname: Ma
authors.forename: Xiaojiao
authors.initials: X
authors.lastname: Zhao
authors.forename: Cuili
authors.initials: C
authors.lastname: Zhang
authors.forename: Yi
authors.initials: Y
authors.lastname: Xin
mesh_terms: D001089:Arabinose / Q000031:analogs & derivatives / Q000032:analysis; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D002851:Chromatography, High Pressure Liquid; D005685:Galactans / Q000378:metabolism*; D005690:Galactose / Q000032:analysis; D055786:Gene Knockout Techniques; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D020102:Mycobacterium smegmatis / Q000166:cytology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D005685:Galactans; C431714:arabinofuranose; D001089:Arabinose; D010744:Phosphoric Monoester Hydrolases; C005653:arabinogalactan; D005690:Galactose