Disruption of Cg-Ppm1, a Polyprenyl Monophosphomannose Synthase, and the Generation of Lipoglycan-less Mutants in Corynebacterium glutamicum

Abstract

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.Corynebacterial strains are widely distributed throughout nature and represent an important branch of the Actinomycetales family. The human pathogen Corynebacterium diphtheriae is the causal agent of diphtheria, and serious economic losses occur from the infection of animals by corynebacterial strains, such as Corynebacterium pseudotuberculosis and Corynebacterium matruchotti (2, 3). In addition, Corynebacterium glutamicum is of industrial importance producing large quantities of amino acids worldwide (4). Corynebacteria belong to a suprageneric actinomycete taxon termed the Corynebacterianeae (5), which includes mycobacteria, rhodococci, nocardiae, and other closely related genera. Therefore, it is of no surprise to find that corynebacteria and mycobacteria share a similar cell envelope ultrastructure, which comprises a core mycolyl-arabinogalactan peptidoglycan complex, termed mAGP (6, 7).An important class of components, found within the cell envelope, are the biosynthetically related glycolipids, phosphatidyl-myo-inositol mannosides (PIMs), 1,2 and lipoglycans, termed lipomannan (LM) and lipoarabinomannan (LAM). These glycolipids and lipoglycans are present within the cell envelope of both mycobacteria (8) and corynebacteria (7, 9). The basic structural features of mycobacterial LAM are well described elsewhere and reveal that LAM possess an amphipathic tripartite structure (10 -13). The two common domains include a mannosyl-phosphatidyl-myo-inositol anchor (MPI), with a polysaccharide backbone consisting of mannose and arabinose that are further elaborated by either mannooligosaccharide or phosphoinositol caps, representing the third domain, and...

Biosynthesis of mycobacterial lipoarabinomannan: Role of a branching mannosyltransferase

“…Clearly, MSMEG4250 is not essential in M. smegmatis but is required for growth at elevated temperatures. Electron microscopy analyses of the mutant strain as compared with the WT did not reveal any marked ultrastructural changes, in accord with the observation that a LAM-like deficient mutant of Corynebacterium did not display any altered ultrastructural morphology (19).…”

“…MSMEG4250 and PIG-M are of similar size and contain 10 predicted transmembrane-spanning domains with the DID motif located in the first extracytoplasmic loop and both the N and C termini within the cytoplasm, all of which are indicative of analogous functions. Moreover, a homologous ORF is present in the known genomes of other actinomycetes, all with the ability to synthesize PIMs and related products (19).…”

Ethambutol, a cell wall inhibitor of Mycobacterium tuberculosis, elicits l-glutamate efflux of Corynebacterium glutamicum

“…for the free lipids, or in petroleum ether/acetone (95 : 5, v/v) for the bound lipids. Autoradiograms were produced by 2-3 days' exposure to Kodak X-Omat AR film to reveal 14 C-labelled lipids, 14 C-labelled fatty acid methyl esters (FAMES) and 14 C-labelled mycolic acid methyl esters (MAMES), which were compared with known standards (Puech et al, 2000(Puech et al, , 2001Gibson et al, 2003).…”

Fluorescent Phospholipid Analogs as Microscopic Probes for Detection of the Mycolic Acid-Containing Layer inCorynebacterium glutamicum: Detecting Alterations in the Mycolic Acid-Containing Layer Following Ethambutol Treatment

“…[16][17][18] A C. glutamicum homolog of mycobacterial polyprenyl monophosphomannose synthetase, which is involved in mycolate-layer formation, was also identified. 19) Recently, an emb gene encoding a possible target enzyme of EMB was cloned from C. glutamicum. The effects of EMB on C. glutamicum cells were investigated using strains in which expression of emb could be controlled.…”