Abstract
Lipid A is the hydrophobic anchor of lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria. Lipid A of all Rhizobiaceae is acylated with a long fatty acid chain, 27-hydroxyoctacosanoic acid. Biosynthesis of this long acyl substitution requires a special acyl carrier protein, AcpXL, which serves as a donor of C28 (omega-1)-hydroxylated fatty acid for acylation of rhizobial lipid A (Brozek, K.A., Carlson, R.W., and Raetz, C. R. (1996) J. Biol. Chem. 271, 32126-32136). To determine the biological function of the C28 acylation of lipid A, we constructed an acpXL mutant of Sinorhizobium meliloti strain 1021. Gas-liquid chromatography and mass spectrometry analysis of the fatty acid composition showed that the acpXL mutation indeed blocked C28 acylation of lipid A. SDS-PAGE analysis of acpXL mutant LPS revealed only a fast migrating band, rough LPS, whereas the parental strain 1021 manifested both rough and smooth LPS. Regardless of this, the LPS of parental and mutant strains had a similar sugar composition and exposed the same antigenic epitopes, implying that different electrophoretic profiles might account for different aggregation properties of LPS molecules with and without a long acyl chain. The acpXL mutant of strain 1021 displayed sensitivity to deoxycholate, delayed nodulation of Medicago sativa, and a reduced competitive ability. However, nodules elicited by this mutant on roots of M. sativa and Medicago truncatula had a normal morphology and fixed nitrogen. Thus, the C28 fatty acid moiety of lipid A is not crucial, but it is beneficial for establishing an effective symbiosis with host plants. acpXL lies upstream from a cluster of five genes, including msbB (lpxXL), which might be also involved in biosynthesis and transfer of the C28 fatty acid to the lipid A precursor.
Highlights
Lipid A is the hydrophobic anchor of lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria
The acpXL-msbB Gene Cluster May Direct the Biosynthesis of the C28 Fatty Acid Chain and Its Incorporation into the S. meliloti Lipid A—All genes of the acpXL-msbB cluster are oriented in the same direction and probably comprise three transcriptional units: (i) acpXL; (ii) SMc04277, SMc04275, and SMc04270; and (iii) SMc04270 and msbB (Fig. 2)
A similar gene cluster was recently identified in R. leguminosarum [39], accession AF510733. acpXL of R. leguminosarum is a specialized acyl carrier protein for a C28 acyl chain and is involved in attachment of this long fatty acid chain to (Kdo)2-lipid IVA, a key lipid A precursor common to different Gram-negative bacteria [21]
Summary
This work of an acyloxyacyl residue and in the heterogeneity of lipid A species concerning the presence or absence of an acyl substitution at position 3 [56]. Studies with rhizobial mutants producing modified lipid A may shed more light on mechanisms of LPS interaction with plant cells. In addition to the conserved lipid A biosynthesis genes, a gene cluster containing the acpXL gene was found This cluster may be responsible for the formation of the C28 acyl substitution of lipid A. The acpXL gene was first identified because of the partial sequencing of a protein purified from R. leguminosarum. This protein mediated the incorporation of a C28 fatty acid chain into a lipid A precursor in vitro [21]. Based on the results of LPS composition analysis as well as results of electrophoretic and immunological comparisons of mutant and wild type LPS, we reconsider here the structure of S. meliloti LPS
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