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A nopA deletion mutant of Sinorhizobium fredii USDA257, a soybean symbiont, is impaired in nodulation

Won-Seok Kim, Hari B. Krishnan
Current Microbiology 2014 v.68 pp. 239-246
starch granules, fimbriae, symbionts, soybeans, type III secretion system, mutants, transmission electron microscopy, nodulation, Sinorhizobium fredii, promoter regions
Sinorhizobium fredii USDA257 employs type III secretion system (T3SS) to deliver effector proteins into the host cells through filamentous surface appendages, called pili. The NopA protein is the major component of USDA257 pili. The promoter region of USDA257 nopA posses a well conserved tts box. Serial deletion analysis revealed that the tts box is absolutely essential for flavonoid induction of nopA. We have expressed USDA257 NopA in Escherichia coli and raised polyclonal antibodies to the purified recombinant protein. SDS-PAGE analysis of extracellular proteins of a 'nopA mutant induced by flavonoids revealed the complete absence of Nop proteins, including NopX, NopP, NopL, NopB, NopC and NopA. Western blot analysis using antibodies raised against these Nop proteins failed to detect their accumulation both in the extracelluar media and the cell lysates. Interestingly, the nopA mutant was also severely affected in flagellar production. Introduction of a plasmid containing the wild-type nopA fragment into the mutant restored the production of flagellum but not the Nops. Electron microscopy observation revealed that the USDA257 'nopA was compromised in both flagellar and pili production. Deletion of nopA drastically lowered the number of nodules formed by USDA257 on cowpea and soybean cultivar Peking. In contrast to the parental strain, the USDA257 'nopA was able to form few nodules on North American soybean cultivars McCall and Williams 82. Light and transmission electron microscopy examination of these nodules revealed numerous starch grains both in the infected and uninfected cells.