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Mutation in GDP-Fucose Synthesis Genes of Sinorhizobium fredii Alters Nod Factors and Significantly Decreases Competitiveness to Nodulate Soybeans

¹Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Spain; ²Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Apdo. 1095, 41080-Sevilla, Spain; ³Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Apdo. de Correos 553, 41071-Sevilla, Spain; ⁴Department of Plant Pathology, University of Missouri, Columbia 65211, U.S.A.; ⁵Institute of Molecular Plant Sciences, Leiden University, 2333 AL, Leiden, The Netherlands; ⁶Bijvoet Centre for Biomolecular Research, Department of Mass Spectrometry, Utrecht University, 3584 CA Utrecht, The Netherlands

We mutagenized Sinorhizobium fredii HH103-1 with Tn5- B20 and screened about 2,000 colonies for increased β-galactosidase activity in the presence of the flavonoid naringenin. One mutant, designated SVQ287, produces lipochitooligosaccharide Nod factors (LCOs) that differ from those of the parental strain. The nonreducing N-acetylglucosamine residues of all of the LCOs of mutant SVQ287 lack fucose and 2-O-methylfucose substituents. In addition, SVQ287 synthesizes an LCO with an unusually long, C20:1 fatty acyl side chain. The transposon insertion of mutant SVQ287 lies within a 1.1-kb HindIII fragment. This and an adjacent 2.4-kb HindIII fragment were sequenced. The sequence contains the 3′ end of noeK, nodZ, and noeL (the gene interrupted by Tn5-B20), and the 5′ end of nolK, all in the same orientation. Although each of these genes has a similarly oriented counterpart on the symbiosis plasmid of the broad-host-range Rhizobium sp. strain NGR234, there are significant differences in the noeK/nodZ intergenic region. Based on amino acid sequence homology, noeL encodes GDP-D-mannose dehydratase, an enzyme involved in the synthesis of GDP-Lfucose, and nolK encodes a NAD-dependent nucleotide sugar epimerase/dehydrogenase. We show that expression of the noeL gene is under the control of NodD1 in S. fredii and is most probably mediated by the nod box that precedes nodZ. Transposon insertion into noeL has two impacts on symbiosis with Williams soybean: nodulation rate is reduced slightly and competitiveness for nodulation is decreased significantly. Mutant SVQ287 retains its ability to form nitrogen-fixing nodules on other legumes, but final nodule number is attenuated on Cajanus cajan.