Lipid A modifications in polymyxin-resistant Salmonella typhimurium - PMRA-dependent 4-amino-4-deoxy-L-arabinose, and phosphoethanolamine incorporation

Lipid A of Salmonella typhimurium can be resolved into multiple molecular s pecies. Many of these substances are more polar than the predominant hexa-a cylated lipid A 1,4'-bisphosphate of Escherichia coli K-12. By using new is olation methods, we have purified six lipid A subtypes (St1 to St6) from wi ld type S. typhimurium. We demonstrate that these lipid A variants are cova lently modified with one or two 4-amino-4-deoxy-L-arabinose (L-Ara4N) moiet ies. Each lipid A species with a defined set of polar modifications can be further derivatized with a palmitoyl moiety and/or a 2-hydroxymyristoyl res idue in place of the secondary myristoyl chain at position 3'. The unexpect ed finding that St5 and St6 contain two L-Ara4N residues accounts for the a nomalous structures of lipid A precursors seen in S. typhimurium mutants de fective in 3-deoxy-D-manno-octulosonic, acid biosynthesis in which only the 1-phosphate group is modified with the L-Ara4N moiety (Strain, S. M., Armi tage, I. M., Anderson, L., Takayama, K., Quershi, N., and Raetz, C. R. H. ( 1985) J. Biol. Chem. 260, 16089-16098). Phosphoethanolamine (pEtN)-modified lipid A species are much less abundant than L-Ara4N containing forms in wi ld type S. typhimurium grown in broth but accumulate to high levels when L- Ara4N synthesis is blocked in pmrA(C)pmrE(-) and pmrA(C)pmrF(-) mutants. Pu rification and analysis of selected compounds demonstrate that one or two p EtN moieties may be present. Our findings show that S. typhimurium contains versatile enzymes capable of modifying both the 1- and 4'-phosphates of li pid A with L-Ara4N and/or pEtN groups. PmrA null mutants of S. typhimurium produce lipid A species without any pEtN or L-Ara4N substituents. However, PmrA is not needed for the incorporation of 2-hydroxymyristate or palmitate .