The dioxygenase-encoding olsD gene from Burkholderia cenocepacia causes the hydroxylation of the amide-linked fatty acyl moiety of ornithine-containing membrane lipids.

Burkholderia cenocepacia is an important opportunistic pathogen, and one of the most striking features of the Burkholderia genus is the collection of polar lipids present in its membrane, including phosphatidylethanolamine (PE) and ornithine-containing lipids (OLs), as well as the 2-hydroxylated derivatives of PE and OLs (2-OH-PE and 2-OH-OLs, respectively), which differ from the standard versions by virtue of the presence of a hydroxyl group at C2 (2-OH) of an esterified fatty acyl residue. Similarly, a lipid A-esterified myristoyl group from Salmonella typhimurium can have a 2-hydroxy modification that is due to the LpxO enzyme. We thus postulated that 2-hydroxylation of 2-OH-OLs might be catalyzed by a novel dioxygenase homologue of LpxO. In B. cenocepacia, we have now identified two open reading frames (BCAM1214 and BCAM2401) homologous to LpxO from S. typhimurium. The introduction of bcam2401 (designated olsD) into Sinorhizobium meliloti leads to the formation of one new lipid and in B. cenocepacia of two new lipids. Surprisingly, the lipid modifications on OLs due to OlsD occur on the amide-linked fatty acyl chain. This is the first report of a hydroxyl modification of OLs on the amide-linked fatty acyl moiety. Formation of hydroxylated OLs occurs only when the biosynthesis pathway for nonmodified standard OLs is intact. The hydroxyl modification of OLs on the amide-linked fatty acyl moiety occurs only under acid stress conditions. An assay has been developed for the OlsD dioxygenase, and an initial characterization of the enzyme is presented.

Identification of a gene required for the formation of lyso-ornithine lipid, an intermediate in the biosynthesis of ornithine-containing lipids.

Under phosphate-limiting conditions, some bacteria replace their membrane phospholipids by lipids not containing any phosphorus. One of these phosphorus-free lipids is an ornithine-containing lipid (OL) that is widespread among eubacteria. In earlier work, we had identified a gene (olsA) required for OL biosynthesis that probably encodes an O-acyltransferase using acyl-acyl carrier protein (acyl-AcpP) as an acyl donor and that converts lyso-ornithine lipid into OL. We now report on a second gene (olsB) required for OL biosynthesis that is needed for the incorporation of radiolabelled ornithine into OL. Overexpression of OlsB in an olsA-deficient mutant of Sinorhizobium (Rhizobium) meliloti leads to the transient accumulation of lyso-ornithine lipid, the biosynthetic intermediate of OL biosynthesis. Overexpression of OlsB in Escherichia coli is sufficient to cause the in vivo formation of lyso-ornithine lipid in this organism and is the cause for a 3-hydroxyacyl-AcpP-dependent acyltransferase activity forming lyso-ornithine lipid from ornithine. These results demonstrate that OlsB is required for the first step of OL biosynthesis, in which ornithine is N-acylated with a 3-hydroxy-fatty acyl residue in order to obtain lyso-ornithine lipid. OL formation in a wild-type S. meliloti is increased upon growth under phosphate-limiting conditions. Expression of OlsB from a broad host range vector leads to the constitutive formation of relatively high amounts of OL (12-14% of total membrane lipids) independently of whether strains are grown in the presence of low or high concentrations of phosphate, suggesting that in S. meliloti the formation of OlsB is usually limiting for the amount of OL formed in this organism. Open reading frames homologous to OlsA and OlsB were identified in many eubacteria and although in S. meliloti the olsB and olsA gene are 14 kb apart, in numerous other bacteria they form an operon.