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Sinorhizobium fredii YC4B is a spontaneous mutant derivative of strain YC4 that is unable to nodulate soybeans. The second-largest plasmid of strain YC4B, termed pSfrYC4b (810 kb), was transferred to S. fredii HN01SR, a strain which contains three large indigenous plasmids (pSfrHN01a, pSfrHN01b and pSfrHN01c). Surprisingly, two stable indigenous plasmids (pSfrHN01a and pSfrHN01b) of strain HN01SR were cured simultaneously by the introduction of pSfrYC4b. Furthermore, a novel, unstable plasmid (pHY4) became visible in agarose gels. The electrophoretic mobility of plasmid pHY4 was slower than that shown by the cured plasmids, indicating that the molecular weight of the former is higher than that of plasmids pSfrYC4b and pSfrHN01b. Replication gene repC-like sequences were detected by polymerase chain reaction (PCR) on pSfrHN01a and pSfrYC4b, but not on pSfrHN01b. Sau3AI and PstI restriction patterns of the PCR-amplified repC-like sequences from HN01SR and YC4B were very similar.

Mesorhizobium huakuii strain 7653R harbored two indigenous plasmids named pMH7653Ra and pMH7653Rb. The larger plasmid pMH7653Rb (symbiotic plasmid) was transferred to M. huakuii HN308SR harboring three plasmids: pMHHN308a, pMHHN308b and pMHHN308c, and HN3015SR harboring three plasmids: pMHHN3015a, pMHHN3015b and pMHHN3015c by tri-parent mating. Two stable indigenous plasmids, pMHHN308b and pMHHN308c of HN308SR, were co-eliminated due to the introduction of pMH7653Rb, and the transconjugant was named HN308SRN14. The results implied that pMH7653Rb and pMHHN308b, pMHHN308c were incompatible and might have been ascribed to the same incompatible group. The plasmid profiles of transconjugant HN3015SRN14 showed that the second largest plasmid pMHHN3015b of HN3015SR was cured due to the introduction of pMH7653Rb. The results also implied that pMH7653Rb and pMHHN3015b were incompatible. Results from plant nodulation tests showed that pMH7653Rb could only maintain the nodulation ability in transconjugant HN308SRN14 and its nodule number was more than that of wild strain HN308SR, but could not replace the nitrogen fixation effect of pMHHN308b and pMHHN308c. The plasmid cured mutant HN308SRN14D harboring only pMHHN308a formed null nodules that demonstrated pMHHN308a was relevant to nodulation ability. HN3015SRN14 harboring pMH7653Rb, pMHHN3015a and pMHHN3015c formed null nodules while HN3015SRN14D containing pMHHN3015a and pMHHN3015c lost the nodulation ability. The plasmid replication repC-like gene sequences were detected by a polymerase chain reaction from 7653R, HN308, HN3015, HN308SRN14 and HN3015SRN14. The repC gene sequence similarities of the strains tested attained 99%.

A Mesorhizobium huakuii strain 2020, isolated from a rice-growing field in southern China, contains three indigenous plasmids named p2020a, p2020b and p2020c, respectively. The plasmids were deleted via Tn5-sacB insertion, and two cured derivatives were obtained. Interestingly, the mutant 2020D29 curing of p2020c could significantly enhance the capacity of symbiotic nitrogen fixation. But the mutant 2020D8 curing of p2020b lost the ability to nodulate Astragalus sinicus. Furthermore, the third plasmid p2020a could be hardly eliminated, suggesting that some house-keeping genes necessary for strain growth located on this plasmid. Then the Sym plasmid pJB5JI of R. leguminosarum bv. viciae was transferred into 2020 and its cured derivatives. The pot plant test showed that the ability of competition and symbiotic nitrogen fixation of transconjugant 2020-137 (pJB5JI) was increased evidently in contrast to 2020. pJB5JI could not restore the ability of 2020D8 to nodulate Astragalus sinicus. 2020D8-8 (pJB5JI) could form ineffective nodules on peas, which implied that the symbiotic plasmid pJB5JI could express its function at the chromosomal background of Mesorhizobium huakuii 2020. The plasmid stability was checked in transconjugants under free-living and during symbiosis. The results indicated that pJB5JI failed to be detected in some nodule isolates. That Km resistance gene could be amplified from all transconjugants and nodule isolates suggested that pJB5JI was fully or partially integrated into the chromosome of recipients.

Mesorhizobium huakuii 7653R forms a symbiotic relationship with Astragalus sinicus to produce nitrogen-fixing root nodules under nitrogen-limiting conditions. By screening its transposon-inserted mutant library, a mutation in the lpsH gene was found to form pseudonodules on A. sinicus. Its effect was further confirmed by double-crossover mutant HK242. DNA sequence analysis revealed that the lpsH gene encodes a deduced protein of 397 amino acids with a predicted molecular mass of 43.6 kD. LpsH contains a putative signal peptide, 11 transmembrane helices, and an O-antigen polymerase domain, which locates on the periplasmic membrane and is necessary to lipopolysaccharide synthesis. Plant studies showed that the lpsH gene mutant formed ineffective nodules, and this symbiotic phenotype was linked to abnormal nodule development.

Sinorhizobium fredii YC4 can form nitrogen-fixing nodules on soybean (Glycine max) and wild soybean (G. soja). It can produce unique lipochitooligosaccharide nod factors (LCOs), in comparison with the other four strains of S. fredii. The constitution of LCOs produced by YC4 contained more hydrophobic substitutions detected by Thin-layer chromatography (TLC) of 14C-labeled nod factors. A spontaneous mutant termed YSC3 amplified in the symbiotic plasmid was isolated from YC4, which can produce more amount of LCOs than its parental strain at 28 degrees C, and showed a difference in the construction of LCOs. Nodulation test indicated that YSC3 only formed ineffective nodules on soybean G. soja.

Rhizobium huakuii Chen (Chen et al., 1991) is the rhizobia isolated from nodules of Astragalus sinicus L., which forms nodules mainly on A.sinicus and a few Astragalus spp. in a very narrow host-specific manner (Chen et al., 1944). A.sinicus is an important legume crop for green manure, traditioinally grown in rice fields in Yangtze River Valley and southern China. The biology, physiology and ecology of the A.sinicus Rhuakuii symbiosis, as well as its application in agriculture have been extensively studied for several decades in China (Chen et al., 1992). Studies on its genetics have been conducted only lately. This paper focuses on the identification of symbioitic plasmid and cloning of early symbiotic genes in R.huakuii