Structural and topological studies on the lipid-mediated assembly of a membrane-associated lipomannan in Micrococcus luteus

Abstract

The biosynthesis of three mannolipids and the presence of a membrane-associated lipomannan in Micrococcus luteus (formerly Micrococcus lysodeikticus) were documented over 30 years ago. Structural and topological studies have been conducted to learn more about the possible role of the mannolipids in the assembly of the lipomannan. The major mannolipid has been purified and characterized as alpha-D-mannosyl-(1 --> 3)-alpha-D-mannosyl-(1 --> 3)-diacylglycerol (Man2-DAG) by negative-ion electrospray-ionization multistage mass spectrometry (ESI-MSn). Analysis of the fragmentation patterns indicates that the sn-1 position is predominantly acylated with a 12-methyltetradecanoyl group and the sn-2 position is acylated with a myristoyl group. The lipomannan is shown to be located on the exterior face of the cytoplasmic membrane, and not exposed on the surface of intact cells, by staining of intact protoplasts with fluorescein isothiocyanate (FITC)-linked concanavalin A (Con A). When cell homogenates of M. luteus are incubated with GDP-[3H]mannose (GDP-Man), [3H]mannosyl units are incorporated into Man1-2-DAG, mannosylphosphorylundecaprenol (Man-P-Undec) and the membrane-associated lipomannan. The addition of amphomycin, an inhibitor of Man-P-Undec synthesis, had no effect on the synthesis of Man1-2-DAG, but blocked the incorporation of [3H]mannose into Man-P-Undec and consequently the lipomannan. These results strongly indicate that GDP-Man is the direct mannosyl donor for the synthesis of Man1-2-DAG, and that the majority of the 50 mannosyl units in the lipomannan are derived from Man-P-Undec. Protease-sensitivity studies with intact and lysed protoplasts indicate that the active sites of the mannosyltransferases catalyzing the formation of Man1-2-DAG and Man-P-Undec are exposed on the inner face, and the Man-P-Undec-mediated reactions occur on the outer surface of the cytoplasmic membrane. Based on all of these results, a topological model is proposed for the lipid-mediated assembly of the membrane-bound lipomannan.

Inactivation of Corynebacterium glutamicum NCgl0452 and the Role of MgtA in the Biosynthesis of a Novel Mannosylated Glycolipid Involved in Lipomannan Biosynthesis

“…It was previously established that these glycosylated diacylglycerols function as precursors/anchors for hyperglycosylated variants, such as the lipomannans, as found in the case of dimannosyl diacylglycerols in Micrococcus and lipoteichoic acids (47)(48)(49). Moreover, related di-and monoacylglycerols containing glucuronosyl residues have been well reported in Pseudomonas spp.…”

Altered lipid composition in Streptococcus pneumoniae cpoA mutants

“…Deletion mutants of S. aureus ypfP produced LTA which was probably attached directly to DAG [34,35]. In the GC-rich organism M. luteus , dimannosyl-DAG is the lipid anchor of the essential lipomannan cell wall polymer [40]. Therefore, temperature sensitive mutants defective in lipomannan assembly were isolated of M. luteus , and one of them (mms1) contained a reduced amount of dimannosyl-DAG whereas the amount of monomannosyl-DAG was increased [41].…”

Structural elucidation of diglycosyl diacylglycerol and monoglycosyl diacylglycerol from Streptococcus pneumoniae by multiple‐stage linear ion‐trap mass spectrometry with electrospray ionization

“…A few electrospray ionization (ESI) tandem mass spectrometric approaches toward identification of DGDG molecules have been reported . Among them, Guella et al .…”

“…A few electrospray ionization (ESI) tandem mass spectrometric approaches toward identification of DGDG molecules have been reported. [12][13][14] Among them, Guella et al employed MS 2 on the [M + Na] + adduct ions with an ion-trap instrument for assignments of the fatty acid moieties on the glycerol backbone of galactolipids. The determination is based on the findings that loss of the fatty acid substituent at sn-1 is more facile than that at sn-2.…”