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Sinorhizobium fredii HH103 cgs Mutants Are Unable to Nodulate Determinate- and Indeterminate Nodule--Forming Legumes and Overproduce an Altered EPS

¹Departamento de Microbiología and ²Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes 6, C.P. 41012, Sevilla, Spain; ³Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Apdo. 553, 41071-Sevilla, Spain; ⁴Servicio de Espectrometría de Masas, Centro de Investigación Tecnológica e Investigación (CITIUS), Universidad de Sevilla, Avda. Reina Mercedes 4, C.P. 41012, Sevilla, Spain; ⁵Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Darwin 2, 28049-Madrid, Spain; ⁶Department of Chemistry, University of York, Heslington, York, YO10 5DD, U.K.; ⁷Department of Chemistry, University of Durham, University Science Labs, South Road, Durham, DH1 3LE, U.K.; ⁸Institute of Biology, Leiden University, Wassenaarseweg 64, 2333AL Leiden, The Netherlands

Sinorhizobium fredii HH103 produces cyclic β glucans (CG) composed of 18 to 24 glucose residues without or with 1-phosphoglycerol as the only substituent. The S. fredii HH103-Rif^r cgs gene (formerly known as ndvB) was sequenced and mutated with the lacZ-gentamicin resistance cassette. Mutant SVQ562 did not produce CG, was immobile, and grew more slowly in the hypoosmotic GYM medium, but its survival in distilled water was equal to that of HH103-Rif^r. Lipopolysaccharides and K-antigen polysaccharides produced by SVQ562 were not apparently altered. SVQ562 overproduced exopolysaccharides (EPS) and its exoA gene was transcribed at higher levels than in HH103-Rif^r. In GYM medium, the EPS produced by SVQ562 was of higher molecular weight and carried higher levels of substituents than that produced by HH103-Rif^r. The expression of the SVQ562 cgs∷lacZ fusion was influenced by the pH and the osmolarity of the growth medium. The S. fredii cgs mutants SVQ561 (carrying cgs∷Ω) and SVQ562 only formed pseudonodules on Glycine max (determinate nodules) and on Glycyrrhiza uralensis (indeterminate nodules). Although nodulation factors were detected in SVQ561 cultures, none of the cgs mutants induced any macroscopic response in Vigna unguiculata roots. Thus, the nodulation process induced by S. fredii cgs mutants is aborted at earlier stages in V. unguiculata than in Glycine max.