A Sinorhizobium meliloti Lipopolysaccharide Mutant Altered in Cell Surface Sulfation

Abstract

The Rhizobium-legume symbiosis involves the formation of a novel plant organ, the nodule, in which intracellular bacteria reduce molecular dinitrogen in exchange for plant photosynthates. Nodule development requires a bacterial signal referred to as Nod factor, which in Sinorhizobium meliloti is a ␤-(1,4)-linked tetramer of N-acetylglucosamine containing N-acyl and O-acetyl modifications at the nonreducing end and a critical 6-O-sulfate at the reducing end. This sulfate modification requires the action of three gene products: nodH, which catalyzes the sulfonyl transfer, and nodPQ, which produce the activated form of sulfate, 3-phosphoadenosine-5-phosphosulfate. It was previously reported that S. meliloti cell surface polysaccharides are also covalently modified by sulfate in a reaction dependent on NodPQ. We have further characterized this unique form of bacterial carbohydrate modification. Our studies have determined that one of the nodPQ mutant strains used in the initial study of sulfation of cell surface harbored a second unlinked mutation. We cloned the gene affected by this mutation (referred to as lps-212) and found it to be an allele of lpsL, a gene previously predicted to encode a UDP-glucuronic acid epimerase. We demonstrated that lpsL encoded a UDP-glucuronic acid epimerase activity that was reduced in the lps-212 mutant. The lps-212 mutation resulted in an altered lipopolysaccharide structure that was reduced in sulfate modification in vitro and in vivo. Finally, we determined that the lps-212 mutation resulted in a reduced ability to elicit the formation of plant nodules and by altered infection thread structures that aborted prematurely.In order to acquire reduced nitrogen, many leguminous plants enter into symbiotic associations with bacteria, culminating in the formation of a novel plant organ, the nodule, in which intracellular bacteria reduce molecular dinitrogen to ammonia. In a compatible symbiotic interaction, the bacteria trigger an alteration in the growth of the plant root hairs, resulting in a curled structure that entraps a microcolony of the bacteria. This curled root hair is the site for a plant cell wallencapsulated ingrowth of the root hair, referred to as an infection thread. The infection thread, filled with proliferating bacteria, extends to the base of the root hair cell and then penetrates the root, allowing bacterial entry into the plant. Concurrent with the development of the infection thread, the cells in the root cortex dedifferentiate, leading to new cell division and the consequent formation of the nodule. The infection thread branches and penetrates the developing nodule, delivering the bacteria, which are then released into the plant cytoplasm. These intracellular bacteria undergo a series of developmental changes and signal transduction events to induce the expression of nitrogenase, which catalyzes the reduction of molecular dinitrogen to ammonia (4,5,18,20,31,41).Symbiotic nitrogen-fixing relationships between legumes and the genera Rhizobium, Bradyrhizobium, Mesorhi...

The Lipopolysaccharide of Sinorhizobium meliloti Suppresses Defense-Associated Gene Expression in Cell Cultures of the Host Plant Medicago truncatula

“…The death of rhizobial mutants during the infection process was thought to be due either to impairment of bacterial protection against unfavorable conditions or to inability to interfere with the host's defense response. Another mutant study showed that not only the presence, but also the correct modification, of LPS is needed for optimal nodulation activity (Keating et al, 2002). Furthermore, purified LPS of the microsymbiont has been shown to promote infection in the case of the R. leguminosarum bv trifolii-white clover (Trifolium repens) symbiosis-and to cause the production of novel proteins (Dazzo et al, 1991).…”

The Sinorhizobium meliloti MsbA2 protein is essential for the legume symbiosis

“…Additionally, another study had independently identified that disruption of the Rm1021 msbA2 gene with a transposon insertion also prevented a successful legume symbiosis (J. Griffitts & S. Long, unpublished). Unfortunately, we were unsuccessful in our attempts to clone a wild-type copy of the S. meliloti msbA2 gene into either TOPO (Invitrogen) or the broad-host-range vectors pMS03 (Keating et al, 2002) and pJN105 (Newman & Fuqua, 1999) in E. coli. However, we were able to clone a mutated form of the msbA2 gene (pmsbA2G97A) into pJN105 (Newman & Fuqua, 1999), under control of an arabinose-inducible promoter in the presence of 0.1 % (w/v) glucose.…”

Lipopolysaccharides as a communication signal for progression of legume endosymbiosis

“…Another sign of defense, the thickening of infection thread cell walls, is thought to affect ramification and efficient penetration of the developing nodule or to physically block the bacteria within the threads (8,17,33,35,36). However, this response of the plant to ORS571-oac2 does not prevent the progression of the mutant into the nodule primordium and the release of bacteria in plant cells.…”