title: Y4xP, an open reading frame located in a type III protein secretion system locus of Sinorhizobium fredii USDA257 and USDA191, encodes cysteine synthase.
abstract: Sinorhizobium fredii USDA257, a soybean symbiont, exports several nodulation outer proteins (Nops) into the rhizosphere. These proteins, which are exported by a type III secretion system (TTSS), have a pivotal role in host-specific nodulation. The entire TTSS of S. fredii lies within a 31-kb region that includes conserved genes that code for secretion machinery proteins, Nops, and several open reading frames (ORF) of unknown function. Identifying the functions of these ORF is essential to understand fully the role of TTSS in nodulation. Here, we report the characterization of y4xP, an ORF of previously unknown function. Southern blot analysis revealed that USDA257 contains two copies of y4xP, while a sibling, USDA191, contains a single copy. The amino acid sequence of Y4XP is homologous to both eukaryotic and prokaryotic cysteine synthase, a key enzyme in sulfur assimilation. The coding region of USDA257 y4xP under control of T7 promoter was expressed in Escherichia coli, and the recombinant protein was purified by nickel-affinity chromatography. Antibodies generated against soybean cysteine synthase cross-reacted with the recombinant protein. A nonpolar mutant of y4xP of USDA191 showed a marked reduction in cysteine synthase activity. Enzyme activity was completely restored when the mutant was complemented with a plasmid containing the y4xP sequence. Cysteine synthase activity was confined to the cell cytosol. Extracellular protein fraction from genistein-induced USDA191 showed no cysteine synthase activity. This observation indicates that cysteine synthase, which is located in the TTSS locus, is not a type III secreted protein. A nonpolar cysteine synthase mutant was able to export all the Nops to the rhizosphere, albeit in reduced amounts compared with the wild-type USDA191. Interestingly, USDA191 cysteine synthase mutant was able to initiate nodules on 'McCall' soybean more efficiently than the wild-type. Our results demonstrate that y4xP encodes a cysteine synthase and inactivation of this gene enhances the ability of USDA191 to form nodules on 'McCall' soybean by regulating Nops production.
authors.affiliation: Department of Plant Microbiology and Pathology, United States Department of Agriculture-Agricultural Research Service, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Julio C
authors.initials: JC
authors.lastname: Lorio
authors.forename: Demosthenis
authors.initials: D
authors.lastname: Chronis
authors.forename: Hari B
authors.initials: HB
authors.lastname: Krishnan
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003547:Cysteine Synthase / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D019833:Genistein / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000032:analysis; D016415:Sequence Alignment; D042181:Sinorhizobium fredii / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity; D013025:Glycine max / Q000382:microbiology; D013559:Symbiosis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D019833:Genistein; D003547:Cysteine Synthase