The long term goal of this research is to gain insights into the molecular basis of development and cell-cell interactions through an exploration of the Rhizobium meliloti/alfalfa symbiosis. Work supported by this grant has demonstrated the importance of particular bacterial exopolysaccharides in nodule invasion and nodule development. Genetic and biochemical characterizations of one of these exopolysaccharides, succinoglycan, have made it one of the best-characterized bacterial exopolysaccharides. This work is highly relevant to studies of human pathogens since exopolysaccharides are important determinants of bacterial virulence and also to studies of the mechanism of synthesis of complex polysaccharides, such as those involved in protein glycosylation. A specific oligosaccharide derived from succinoglycan has been shown to be critical for nodule invasion and analyses of the molecular basis of this phenomenon are relevant to the studies of the physiological actions of specific oligosaccharides in humans. This work has also led to the identification of a gene, bacA/sbmA, that is required for R. meliloti to invade the plant cytoplasm and is highly conserved in other bacteria, including invasive mammalian pathogens. Genetic and biochemical characterizations of polysaccharide biosynthesis will be continued and will offer insights into such issues as the biosynthetic origins of the modifications on succinoglycan and the nature of the interactions between the various proteins involved in succinoglycan biosynthesis. Since the active oligosaccharide appears to be derived from degradation of high molecular weight succinoglycan, R. meliloti-encoded glucanases that degrade succinoglycan, including ExoK, will be characterized and their roles in the production of the active oligosaccharide determined. Several lines of experimentation will address the roles of the exopolysaccharides in symbiosis by such experiments as identifying plant genes induced by exopolysaccharide-deficient bacteria and studying the recently-discovered aberrant attachment of exopolysaccharide-deficient R. meliloti to roots. The bacA/sbmA gene, whose product is critical for events immediately after release of R. meliloti into the plant cytoplasm, transports compounds containing thiazole rings. The range of compounds transported by this system will be determined and screens will be developed for possible plant compounds transported by this system. The possible requirement for bacA/sbmA function for invasion of mammalian cells by invasive mammalian pathogens will be explored. The synthesis of bacterial and plant carbon- storage polymers that are correlated with nodule formation will also be analyzed.
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