Varying the Abundance of O Antigen in Rhizobium etli and Its Effect on Symbiosis with Phaseolus vulgaris

Abstract

Judged by migration of its lipopolysaccharide (LPS) in gel electrophoresis, the O antigen of Rhizobium etli mutant strain CE166 was apparently of normal size. However, its LPS sugar composition and staining of the LPS bands after electrophoresis indicated that the proportion of its LPS molecules that possessed O antigen was only 40% of the wild-type value. Its LPS also differed from the wild type by lacking quinovosamine (2-amino-2,6-dideoxyglucose). Both of these defects were due to a single genetic locus carrying a Tn5 insertion. The deficiency in O-antigen amount, but not the absence of quinovosamine, was suppressed by transferring into this strain recombinant plasmids that shared a 7.8-kb stretch of the R. etli CE3 lps genetic region ␣, even though this suppressing DNA did not carry the genetic region mutated in strain CE166. Strain CE166 gave rise to pseudonodules on legume host Phaseolus vulgaris, whereas the mutant suppressed by DNA from lps region ␣ elicited nitrogen-fixing nodules. However, the nodules in the latter case developed slowly and were widely dispersed. Two other R. etli mutants that had one-half or less of the normal amount of O antigen also gave rise to pseudonodules on P. vulgaris. The latter strains were mutated in lps region ␣ and could be restored to normal LPS content and normal symbiosis by complementation with wild-type DNA from this region. Hence, the symbiotic role of LPS requires near-normal abundance of O antigen and may require a structural feature conferred by quinovosamine.Rhizobium etli bacteria induce bean plants to form root nodules in which they fix nitrogen. As nodules develop, the bacteria penetrate to interior plant cell layers by means of an infection thread, in which a linear bacterial colony is surrounded by a tubular wall and plant plasma membrane that separates the bacteria from plant cytoplasm (24).Mutants of R. etli that lack the O-antigen portion of the lipopolysaccharide (LPS) are defective in infection (6,10,30,35). Fewer nodules are elicited by such mutants per region of susceptible root, and in the nodules that are formed, very few bacteria can be recovered. Microscopic examination reveals that the infection threads in these nodules are distended and cease development within the root hair or the subjacent cell layer (35). Closely resembling the development of nodules elicited by mutants that do not infect at all (46), the resulting nodule structure lacks most features of a normal mature nodule and has been described as a pseudonodule (27,35). Oantigen-deficient mutants of Rhizobium leguminosarum and Bradyrhizobium japonicum also exhibit defects in infection and cause aberrant development of nodules on their respective host legumes (4,12,22,23,37,38,39,44).Although these studies demonstrate that the presence of O antigen is important in the symbiosis, the specific structural features that are required are still not established (30). However, there is evidence that addresses the question of how much O antigen each bacterium must possess in order to be symbioti...

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

“…Midnight Black Turtle Soup supplied by Idaho Seed Bean, Twin Falls, ID). R. etli CE3 was grown in medium (8) to which the crude anthocyanin extract had been added as described (10). The LPS was isolated by hot phenol/water extraction as described above and purified by Sepharose 4B chromatography after dialysis and treatment with nucleases and proteinase K (21,26).…”

“…etli CE3 was grown as described (8), and the pellets of two 300-ml overnight late exponential phase cultures were extensively washed with nutrient solution and added to the AGC after seedlings were transferred. The nutrient solution, including the CE3 inoculum, was refreshed every other day for 4 weeks.…”

“…It was furthermore demonstrated that not only the presence of the O-chain polysaccharide on the LPS but also the abundance of O-chain polysaccharide was important for nodulation. For example, mutant strain R. etli CE166 produced, based on PAGE analysis of the LPS, only 40% LPS containing the O-chain polysaccharide compared with the parent strain, and the symbiotic phenotype of this mutant was the same as that observed for a mutant that entirely lacks the O-chain polysaccharide (7,8).…”

The Determinants of the Actinorhizal Symbiosis

“…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.…”

Signals in Soybean's Inoculants

“…Some of them are secreted to the media, others are exposed on the surface or present in the periplasmic space ). EPS appears to be essential for the successful invasion of indeterminate nodules (Pellock et al, 2000), whereas LPS are involved principally in the formation of determinate nodules, especially during the initiation and elongation of the infection thread (Lerouge & Vanderleyden, 2001;Noel et al, 2000). Both, in determinate and indeterminate nodule formation, the absence of cyclic glucan synthesis affect the invasion capacity of the bacteria .…”