SummaryThe suppressor mutation, named sfhC21, that allows Escherichia coli ftsH null mutant cells to survive was found to be an allele of fabZ encoding R-3-hydroxyacyl-ACP dehydrase, involved in a key step of fatty acid biosynthesis, and appears to upregulate the dehydrase. The ftsH1(Ts) mutation increased the amount of lipopolysaccharide at 42ЊC. This was accompanied by a dramatic increase in the amount of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase [the lpxC (envA) gene product] involved in the committed step of lipid A biosynthesis. Pulse-chase experiments and in vitro assays with purified components showed that FtsH, the AAA-type membrane-bound metalloprotease, degrades the deacetylase. Genetic evidence also indicated that the FtsH protease activity for the deacetylase might be affected when acyl-ACP pools were altered. The biosynthesis of phospholipids and the lipid A moiety of lipopolysaccharide, both of which derive their fatty acyl chains from the same R-3-hydroxyacyl-ACP pool, is regulated by FtsH.

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Salmonella typhimurium and related organisms synthesize lipid A by the same pathway as Escherichia coli K-12 (1, 2), but they usually modify the final product with additional covalent appendages (Fig. 1A), such as 4-amino-4-deoxy-L-arabinose (L-Ara4N) 1 (3-7), phosphoethanolamine (pEtN) (4 -6), (S)-2-hydroxymyristate (8, 9), and palmitate (5, 6, 10 -12). Different combinations of these substituents account for the remarkable heterogeneity of lipid A molecules found in S. typhimurium.The biosynthesis of lipid A modifications is under the control of the PhoP/PhoQ and the PmrA/PmrB two-component signaling systems (13-15). Addition of the L-Ara4N unit is required for resistance to polymyxin (16 -18). Incorporation of the palmitoyl chain confers resistance to certain cationic anti-microbial peptides (11). Modification of lipid A with L-Ara4N, pEtN, and/or palmitate may also occur in E. coli K-12, but only under special circumstances, as in polymyxin-resistant (pmrA constitutive) mutants (17) or in wild type cells exposed to ammonium metavanadate (7,19).With the exception of PagP, the outer membrane enzyme that incorporates palmitate (12, 20), the enzymes responsible for the covalent modifications of lipid A have not been identified. The L-Ara4N residue is attached primarily to the 4Ј-phosphate group of lipid A in wild type S. typhimurium or in metavanadate-treated E. coli, whereas pEtN is usually attached to the 1-phosphate (19). However, in S. typhimurium mutants defective in Kdo biosynthesis, lipid A precursors accumulate in which L-Ara4N is linked exclusively to the 1-phosphate, and pEtN is attached mainly to the 4Ј-phosphate (5,6,19). The enzymatic pathways that account for these interesting and complex structural anomalies are unknown.An important clue to the origin of the L-Ara4N moiety has emerged from the discovery of the pmrE and pmrF genes, which are required for the maintenance of polymyxin resistance and the biosynthesis of L-Ara4N-modified lipid A (18). The pmrE (ugd) gene encodes UDP-glucose dehydrogenase (18), suggesting that L-Ara4N is derived from UDP-glucuronic acid. The pmrF gene encodes a homologue of yeast dolichyl phosphate-mannose synthase and is part of an operon (18) that includes additional open reading frames hypothesized to encode other putative enzymes required for L-Ara4N biosynthesis and attachment to lipid A (7, 21). The operon is regulated directly by PmrA (18), which in turn may be activated by PhoP/PhoQ, low pH, or ferric ions (14,22,23). So far, no in vitro assays have been developed to validate the functions of the proteins encoded by the pmrF operon.Because of its heterogeneity (Fig. 1A), S. typhimurium lipid * This work was supported by National Institutes of Health Grants GM-51310 (to C. R. H.R.), AI-30479 (to S. I. M.), and GM54882 (to R. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.** To w...

The phospholipids of Escherichia coli consist mainly of phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin. PG makes up ϳ25% of the cellular phospholipid and is essential for growth in wild-type cells. PG is synthesized on the inner surface of the inner membrane from cytidine diphosphate-diacylglycerol and glycerol 3-phosphate, generating the precursor phosphatidylglycerol-phosphate (PGP). This compound is present at low levels (ϳ0.1% of the total lipid). Dephosphorylation of PGP to PG is catalyzed by several PGPphosphatases. The pgpA and pgpB genes, which encode structurally distinct PGP-phosphatases, were identified previously. Double deletion mutants lacking pgpA and pgpB are viable and still make PG, suggesting the presence of additional phosphatase(s). We have identified a third PGP-phosphatase gene (previously annotated as yfhB but renamed pgpC) using an expression cloning strategy. A mutant with deletions in all three phosphatase genes is not viable unless covered by a plasmid expressing either pgpA, pgpB, or pgpC. When the triple mutant is covered with the temperature-sensitive plasmid pMAK705 expressing any one of the three pgp genes, the cells grow at 30 but not 42°C. As growth slows at 42°C, PGP accumulates to high levels, and the PG content declines. PgpC orthologs are present in many other bacteria.