The structure of the lipid A from Rhizobium etli and Rhizobium leguminosarum lipopolysaccharides (LPSs) lacks phosphate and contains a galacturonosyl residue at its 4 position, an acylated 2-aminogluconate in place of the proximal glucosamine, and a very long chain -1 hydroxy fatty acid, 27-hydroxyoctacosanoic acid (27OHC28:0). The 27OHC28:0 moiety is common in lipid A's among members of the Rhizobiaceae and also among a number of the facultative intracellular pathogens that form chronic infections, e.g., Brucella abortus, Bartonella henselae, and Legionella pneumophila. In this paper, a mutant of R. leguminosarum was created by placing a kanamycin resistance cassette within acpXL, the gene which encodes the acyl carrier protein for 27OHC28:0. The result was an LPS containing a tetraacylated lipid A lacking 27OHC28:0. A small amount of the mutant lipid A may contain an added palmitic acid residue. The mutant is sensitive to changes in osmolarity and an increase in acidity, growth conditions that likely occur in the nodule microenvironment. In spite of the probably hostile microenvironment of the nodule, the acpXL mutant is still able to form nitrogenfixing root nodules even though the appearance and development of nodules are delayed. Therefore, it is possible that the acpXL mutant has a host-inducible mechanism which enables it to adapt to these physiological changes.Lipopolysaccharides (LPSs) constitute the outer leaflet of the outer membrane of gram-negative bacteria and are important virulence factors for both animal-and plant-pathogenic bacteria as well as for rhizobia, the nitrogen-fixing symbionts of legumes. It has been shown that the O-chain polysaccharide portion of the LPS is absolutely required for establishing nitrogen-fixing symbioses, including that between R. leguminosarum bv. viciae and pea (for a review, see reference 29). Furthermore, it has been shown that subtle structural changes, e.g., addition of methyl groups, occur to the O-chain polysaccharide during symbiosis (3, 16, 17, 24-29, 32, 33, 45). In the case of R. leguminosarum bv. viciae, important structural alterations also occur to the lipid A portion of the LPS during symbiosis (26). This change involves a unique fatty acyl component found in the lipid A of R. leguminosarum bv. viciae as well as in the lipid A of all members of the Rhizobiaceae with the possible exception of Azorhizobium caulinodans (6) and in the lipid A of some intracellular pathogens, e.g., Brucella abortus and Bartonella henselae (i.e., the causal agent of cat scratch disease) (4). This fatty acyl component is a very long chain -1 fatty acid, 27-hydroxyoctacosanoic acid (27OHC28:0), which doubles in amount in R. leguminosarum bv. viciae lipid A during pea symbiosis (26). Also during symbiosis, and during growth under conditions that mimic those that occur during symbiosis, both the LPS and the entire bacterial cell become more hydrophobic (26). Thus, the increase in 27OHC28:0, together with increased O-chain methylation, may be responsible for this increase in hydr...