Disruption of Cg-Ppm1, a polyprenyl monophosphomannose synthase, and the generation of lipoglycan-less mutants in Corynebacterium glutamicum.
The glycosyl donor, polyprenyl monophosphomannose (PPM), has been shown to be involved in the biosynthesis of the mycobacterial lipoglycans: lipomannan and lipoarabinomannan. The mycobacterial PPM synthase (Mt-ppm1) catalyzes the transfer of mannose from GDP-mannose to polyprenyl phosphates. Based on sequence homology to Mt-ppm1, we have identified the PPM synthase from Corynebacterium glutamicum. In the present study, we demonstrate that the corynebacterial synthase is composed of two distinct domains; a catalytic domain (Cg-ppm1) and a membrane domain (Cg-ppm2). Through the inactivation of Cg-ppm1, we observed a complex phenotype that included altered cell growth rate and inability to synthesize PPM molecules and lipoglycans. When Cg-ppm2 was deleted, no observable phenotype was noted, indicating the clear organization of the two domains. The complementation of the inactivated Cg-ppm1 strain with the corresponding mycobacterial enzyme (Mt-Ppm1/D2) led to the restoration of a wild type phenotype. The present study illustrates, for the first time, the generation of a lipoglycan-less mutant based on a molecular strategy in a member of the Corynebacterianeae family. Lipoglycans are important immunomodulatory molecules involved in determining the outcome of infection, and so the generation of defined mutants and their subsequent immunological characterization is timely.
Gibson
Kevin J C
KJ
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
Eggeling
Lothar
L
Maughan
William N
WN
Krumbach
Karin
K
Gurcha
Sudagar S
SS
Nigou
Jérôme
J
Puzo
Germain
G
Sahm
Hermann
H
Besra
Gurdyal S
GS
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D002855:Chromatography, Thin Layer; D003352:Corynebacterium / Q000201:enzymology*; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D008070:Lipopolysaccharides / Q000378:metabolism; D008358:Mannose / Q000096:biosynthesis* / Q000737:chemistry; D008364:Mannosyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008957:Models, Genetic; D009154:Mutation; D010641:Phenotype; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013997:Time Factors
D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D004247:DNA; D008364:Mannosyltransferases; C479135:polyprenyl monophosphomannose synthase, bacteria; D008358:Mannose