Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/5/2011
Publication Date: 9/1/2011
Citation: Krishnan, H.B., Natarajan, S.S., Kim, W. 2011. Distinct cell surface appendages produced by Sinorhizobium fredii USDA257 and S. fredii USDA191, cultivar-specific and nonspecific symbionts of soybean. Applied and Environmental Microbiology. 77(17):6240-6248.
Interpretive Summary: Rhizobia are soil-dwelling bacteria that form nodules on the roots of legume plants. The nodules are specialized structures where atmospheric nitrogen is fixed by the bacterium, which in turn, is utilized by legumes for growth and development. This process is termed biological nitrogen fixation and it enables legumes to grow in nitrogen-poor soils. Sinorhizobium fredii USDA257 forms nodules on primitive soybean cultivars such as “Peking” but fails to induce nodules on agronomically improved cultivars. In contrast, S. fredii USDA191 shows no cultivar specificity and initiates nodules on most of the tested agronomically improved North American soybean cultivars. Both theses strains of rhizobia secrete proteins into the rhizosphere when they come into contact with legume root exudates. Some of these proteins are involved in regulating nodulation on soybean and other legumes. Currently, very little is known if there are differences in the proteins produced by USDA191 and USDA257. Since these proteins play an important role in nodule formation it will be important to investigate the differences between proteins secreted by these two soybean symbionts. Such information is required for designing strategies targeted toward improving biological nitrogen fixation. In this study we have revealed not only differences in the protein profile but also morphological and protein compositional changes in surface appendages elaborated by these two strains of soybean symbionts. Information obtained from this basic study will help scientists to better understand the factors that limit the formation of nitrogen-fixing nodules on legumes. Such an understanding should enable scientists to manipulate biological nitrogen fixation so that farmers can increase yields with minimal use of nitrogen fertilizers.
Technical Abstract: Sinorhizobium fredii USDA257 and S. fredii USDA191 are fast-growing rhizobia that form nitrogen-fixing nodules on soybean roots. In contrast to USDA191, USDA257 exhibits cultivar specificity and can form nodules only on primitive soybean cultivars. In response to flavonoids released from soybean roots these two rhizobia secrete nodulation outer proteins (Nop) to the extracellular milieu through a type III secretion system. In spite of the fact that Nop proteins are known to regulate legume nodulation in a host-specific manner, very little is known about the differences in the composition of Nops and surface appendages elaborated by USDA191 and USDA257. In this study we have compared the Nops profile of USDA191 and USDA257 by 1D and 2D gel electrophoresis and identified several of these proteins by MALDI-TOF MS and LC-MS/MS. Examination of the surface appendages elaborated by these two strains of soybean symbionts by transmission electron microscopy revealed distinct differences in their morphology. Even though the flagella produced by USDA191 and USDA257 were similar in their morphology they differed in their flagellin composition. USDA257 pili resembled long thin-filaments while USDA191 pili were short, rod shaped and much thinner than the flagella. 2D gel electrophoresis of pili-like appendages of USDA191 and USDA257 followed by mass spectrometry resulted in the identification of several of the Nops along with some proteins previously undetected in this strain. Some of the newly identified proteins show homology to putative zinc protease and a LabA-like protein from Bradyrhizobium sp. ORS278, fimbrial type-4 assembly proteins from Ralstonia solanacearum and type III effector Hrp-dependent protein from Rhizobium leguminosarum bv. trifolii.