NopB, a Soybean Cultivar-Specificity Protein from Sinorhizobium fredii USDA257, Is a Type III Secreted Protein

Abstract

The type III secretion system (TTSS) of plant- and animal-pathogenic bacteria is involved in translocation of virulence factors into the host cell cytosol where they modulate cellular processes. Sinorhizobium fredii USDA257 is a gram-negative soil bacterium that forms nitrogen-fixing nodules on specific soybean cultivars (Glycine max (L.) Merr.). This microsymbiont is known to secrete at least five nodulation outer proteins (Nops) in response to flavonoid induction. Some of these Nops have been shown to be secreted by TTSS in this symbiotic bacterium. We have isolated and purified an 18-kDa extracellular protein from flavonoid-induced cultures of USDA257. The N-terminal amino acid sequence of this purified protein was identical to the published sequence of the soybean cultivar-specificity protein, NopB (formerly NoIB). Inactivation of rhcN, which encodes an ATPase, abolished secretion of NopB. Similarly, a nonpolar nopB deletion mutant was compromised in its ability to secrete several Nops. A construct containing the coding region of nopB under control of a T7 promoter was expressed successfully in Escherichia coli and, subsequently, the recombinant NopB was purified by nickel-affinity column chromatography. Polyclonal antibodies raised against purified recombinant NopB were used in Western blot analysis to demonstrate the association of NopB with pilus-like surface appendages. Deletion analysis indicated that the first 33 N-terminal residues of NopB were necessary and sufficient to mediate the secretion of a green fluorescent protein reporter. Introduction of plasmid-borne extra copies of nopB into USDA257 resulted in lower accumulation of native NopB. We also show that USDA257 and its nonpolar nopB deletion mutant exhibited discernible differences in their ability to nodulate legume hosts.