Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/7/2001
Publication Date: N/A
Technical Abstract: Sinorhizobium fredii USDA257 nodulates 'Peking', a primitive soybean [Glycine max (L.) Merr.] cultivar, but fails to nodulate improved cultivars such as 'McCall'. The cultivar specificity is dependent on the nol XWBTUV locus. Mutations in any member of this locus allow S. fredii USDA257 to nodulate McCall and abolish the flavonoid-dependent export of at least five eextracellular proteins. We have earlier demonstrated that the flavonoid- dependent secretion of these five extracellular proteins by S. fredii USDA257 is also dependent on the Type III secretion system. Two of these extracellular proteins from S. fredii USDA257 are physically associated with pilus-like fibrillar appendages, which may be involved in cell-to-cell contact to deliver proteins into the host. From a flavonoid-induced culture of S. fredii USDA257, a 21-kD extracellular protein was purified. The N-terminal sequence of the purified protein was determined to be MMLPVTSISNSLPRVASS, which was identical to the published sequence of NolB. We have cloned the coding region of nolB into the protein expression vector pET 28a(+) and over-expressed NolB in Escherichia coli. Western blot analysis using polyclonal antibodies against the extracellular proteins from S. fredii USDA257 showed cross-reactivity with the recombinant NolB protein. Efforts are under way to perform functional studies with a NolB protein fused to the green fluorescent protein. Polyclonal antibodies against NolB and other Type III-dependent extracellular proteins are being generated. These antibodies will be used for subcellular localization of the extracellular proteins and to explore their possible involvement in pili formation.