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.

Distinct Cell Surface Appendages Produced by Sinorhizobium fredii USDA257 and S. fredii USDA191, Cultivar-Specific and Nonspecific Symbionts of Soybean

“…NopX, NopL, NopP, Omp22, NopB, NopC, and NopA were identified in the pilus preparations from both USDA191 and USDA257 (Table 3). This observation is consistent with our earlier report, in which we demonstrated these proteins in the pilus preparation by immunoblot analysis using antibodies raised against Nops (21,23,26). In addition to these proteins, USDA257 also contained prominent flagellin subunits (Table 3).…”

“…The type III secretion apparatus of USDA257 is clustered within a 32-kb region (23). Both USDA257 and USDA191 utilize the TTSS to secrete several Nops (nodulation outer proteins) into the extracellular medium (19,21,23,26). T3SS is under the control of the transcriptional regulator TtsI (18,45), and putative regulatory motifs termed tts boxes are found upstream of ttsI, nopB, nopC, nopL, nopP, and nopX (27,45).…”

Regulation and symbiotic significance of nodulation outer proteins secretion in Sinorhizobium fredii HH103

“…Genes responsible for the biosynthesis of the rhizobial T3SS are organized in the tts region, which also contains genes that encode secreted proteins collectively known as nodulation outer proteins (Nops). To date, eight secreted proteins have been identified: NopA, NopB, NopC, NopD, NopL, NopM, NopP and NopX (Ausmees et al, 2004;Bartsev et al, 2003;Deakin et al, 2005;Krishnan, 2002;Lorio et al, 2004;Rodrigues et al, 2007;Saad et al, 2005;Skorpil et al, 2005). Recently, eight proteins have been found to be type III-secreted in B. japonicum; one of them, GunA2, has been identified as an endoglucanase (Süss et al, 2006).…”

The Sinorhizobium (Ensifer) fredii HH103 Nodulation Outer Protein NopI Is a Determinant for Efficient Nodulation of Soybean and Cowpea Plants

“…HH103 secretes at least eight Nops through the T3SS in response to genistein: NopA (ϳ7 kDa), NopB (ϳ21 kDa), NopC (ϳ11 kDa), NopD (ϳ180 kDa), NopL (ϳ37 kDa), NopM (ϳ60 kDa), NopP (ϳ32 kDa), and NopX (ϳ60 kDa) (14). Three of these proteins, NopA, NopB, and NopX, are components of T3SS extracellular appendages or T3SS pili (15)(16)(17), and the rest can be considered putative effectors, with the exception of NopP and NopC, whose delivery to the interior of the root cells of Vigna unguiculata and Glycine max, respectively, has been confirmed (18,19). In the case of NopL, translocation studies have been performed in Nicotiana benthamiana and Arabidopsis leaves using heterologous Pseudomonas systems (19,20).…”