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...

Lipopolysaccharides from pea-nodulating strain Rhizobium leguminosarum by. viciae 3841, as all other members of the family Rhizobiaceae with the possible exception of Azorhizobium caulinodans, contains a very long chain fatty acid; 27-hydroxyoctacosanoic acid (27OHC28:0) in its lipid A region. The exact function and importance of this residue, however, is not known. In this work, a previously constructed mutant, Rhizobium leguminosarum by. viciae 22, deficient in the fatty acid residue, was analyzed for its symbiotic phenotype. While the mutant was able to form nitrogen-fixing nodules, a detailed study of the timing and efficiency of nodulation using light and electron microscopy showed that there was a delay in the onset of nodulation and nodule tissue invasion. Further, microscopy showed that the mutant was unable to differentiate normally forming numerous irregularly shaped bacteroids, that the resultant mature bacteroids were unusually large, and that several bacteroids were frequently enclosed in a single symbiosome membrane, a feature not observed with parent bacteroids. In addition, the mutant nodules were delayed in the onset of nitrogenase production and showed reduced nitrogenase throughout the testing period. These results imply that the lack of 27OHC28:0 in the lipid A in mutant bacteroids results in altered membrane properties that are essential for the development of normal bacteroids.

Members of the Rhizobiaceae contain 27-hydroxyoctacosanoic acid (27OHC 28:0 ) in their lipid A. A Rhizobium leguminosarum 3841 acpXL mutant (named here Rlv22) lacking a functional specialized acyl carrier lacked 27OHC 28:0 in its lipid A, had altered growth and physiological properties (e.g., it was unable to grow in the presence of an elevated salt concentration [0.5% NaCl]), and formed irregularly shaped bacteroids, and the synchronous division of this mutant and the host plant-derived symbiosome membrane was disrupted. In spite of these defects, the mutant was able to persist within the root nodule cells and eventually form, albeit inefficiently, nitrogen-fixing bacteroids. This result suggested that while it is in a host root nodule, the mutant may have some mechanism by which it adapts to the loss of 27OHC 28:0 from its lipid A. In order to further define the function of this fatty acyl residue, it was necessary to examine the lipid A isolated from mutant bacteroids. In this report we show that addition of 27OHC 28:0 to the lipid A of Rlv22 lipopolysaccharides is partially restored in Rlv22 acpXL mutant bacteroids. We hypothesize that R. leguminosarum bv. viciae 3841 contains an alternate mechanism (e.g., another acp gene) for the synthesis of 27OHC 28:0 , which is activated when the bacteria are in the nodule environment, and that it is this alternative mechanism which functionally replaces acpXL and is responsible for the synthesis of 27OHC 28:0 -containing lipid A in the Rlv22 acpXL bacteroids.Rhizobium leguminosarum cells have an envelope similar to that of other gram-negative bacteria. Lipopolysaccharide (LPS) is the primary component of the bacterial outer leaflet and is comprised of three structural regions: the O-chain polysaccharide, the core oligosaccharide, and lipid A. The lipid A region is anchored in the bacterial outer membrane, and the carbohydrate portion projects from the outer surface into the surrounding milieu and is the primary immunogenic determinant. Correlating LPS structure with function has been difficult as LPS is a very complex molecule and LPS preparations consist of structurally heterogeneous mixtures of molecules. There are pronounced variations in LPS structure from strain to strain, and even within a strain there are different sizes and compositions of LPS (23). Interestingly, there are marked differences between LPSs from free-living cultures and LPSs from nitrogen-fixing bacteroids in terms of size, composition, and antigenic properties (10,32,33). It has been shown that the rhizobial LPS undergoes structural modifications during the formation of bacteroids and that there are composition differences between the bacterial and bacteroid LPSs (10, 32).Variation in LPS structure due to environmental changes has been studied in cultured rhizobia by altering the growth conditions, such as lowering the oxygen level, lowering the pH, altering the carbon source, or adding plant-derived compounds (2,14,21,23,27). Such studies have shown that cues from the environment play an import...