ARhizobium leguminosarumAcpXL Mutant Produces Lipopolysaccharide Lacking 27-Hydroxyoctacosanoic Acid

Abstract

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...

Rhizobium etli CE3 Bacteroid Lipopolysaccharides Are Structurally Similar but Not Identical to Those Produced by Cultured CE3 Bacteria

“…In the case of indeterminate nodules, it is possible that the maintenance of single occupancy symbiosomes results from more intimate contact between the dividing bacterial cell and the PM, whereas the latter determinate process involves detachment of the bacterial and PM cell. In the former case, we have reported that an R. leguminosarum biovar viciae mutant that is defective in the synthesis of the very long fatty acid moiety of its lipid A is also defective in bacteroid formation, and results in multiple occupancy symbiosomes (52,53). It is important to investigate the characteristics of the surface polysaccharides of bacteroids in these and other symbiotic systems in order to be able to fully understand the mechanism of symbiosis.…”

Lipid A Modification Systems in Gram-Negative Bacteria

“…These mutant bacteria grow slowly, and they are hypersensitive to detergents and low pH (234,235). However, these mutants are nevertheless able to form partially functional nodules in their host plants.…”

“…results in the failure to incorporate the long secondary acyl chain (Figure 13) (234,235). These mutant bacteria grow slowly, and they are hypersensitive to detergents and low pH (234,235).…”

The Determinants of the Actinorhizal Symbiosis

“…Up to now, the most severe impairments in nodulation were reported in mutants deficient in O-chain (73,98). Significance of lipid A in symbiosis remains unclear because most of lipid A mutants show weak phenotypes such as delay and reduced competitiveness for nodulation (93,112) or sometimes no apparent symbiotic lesion (46,111). LPS may contribute to the establishment of symbiosis as a suppressor of plant defense responses because LPS from S. meliloti have been found to suppress defense responses in a host plant Medicago sativa cell culture induced by a yeast elicitor (1).…”

“…The O-antigen is the most variable domain of the LPS, showing strain by strain diversity (35). LPS mutants of rhizobia exhibited extensive levels of symbiotic impairments ranging from complete lack of nodule formation (98), formation of non-nitrogen-fixing nodules devoid of bacteroids (73,74), nodules with less bacteroids and nitrogen fixation (17), abnormally developed bacteroids (19,85), delay in nodulation (50,93,112) to decreased competitiveness (31,50,93). Nevertheless, the majority of the LPS mutants can invade plant tissue more or less, suggesting that LPS is particularly important in later stage of symbiosis.…”