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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.

The Rhizobium leguminosarum biovar viceae host-range plasmid pJB5JI was transferred into Rhizobium huakuii strains, both wild-type 7653R and its sym plasmid-cured mutant 7653R-1. Transconjugant 7653R-1 (pJB5JI) acquired the ability to form ineffective nodules on pea plants, whereas transconjugant 7653R (pJB5JI) could not do so, indicating that the indigenous symbiotic plasmid could restrict the functional expression of pJB5JI. On the other hand, transconjugant 7653R (pJB5JI) showed higher nitrogenase activity on A. sinicus and higher shoot dry weight than the recipient strain 7653R. The alien plasmid pJB5JI in both kinds of transconjugants remained stable during frequent transfer on culture media, but in part of the isolates from nodules formed by them the pJB5JI was not visualized on gel by the Eckhardt procedure. Southern hybridization with Tn5 and nod gene probes showed that these isolates still reserved, at least in part, DNA of pJB5JI, which was probably intergrated onto the chromosome of cells.

Purine pathway in Rhizobium is important during the nodulation processes. The purL gene in Sinorhizobium fredii (S. fredii) has been identified to be required for the whole establishment of a nitrogen-fixing nodule. To get a better understanding of the purL gene’s impacts on Rhizobium–plant interaction, the competitive nodulation abilities of S. fredii containing different purL expression plasmids were studied. Several kinds of coinoculations were performed, including using different bacterial concentration ratios, with or without the supplementation of purine source in the plant nutrient solution, and the delayed coinoculation tests. The results indicated that the competitive nodule occupancy of S. fredii was affected significantly by the purL expression level during the early nodulation periods. The mutant strain containing no purL expression could not elicit competitive nodules both in the presence and absence of purine source. A positive linear correlation within certain limits was observed between strain’s competitive nodule occupancy and purL gene expression level. All these results suggested that the purL gene played a role in the competitive nodulation of S. fredii.

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%.

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA intergenic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S-23S IGS RFLP patterns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. leguminosarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.

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.

A novel microcalorimetric technique based on the bacterial heat output was applied to evaluate the biological effect of nisin on the growth of Staphylococcus aureus. The thermogenic curves of S. aureus in the presence of nisin were studied by an LKB-2277 Thermal Activity Monitor. The thermokinetic parameters, such as the growth rate constant (k), the generation times (G), the inhibitory ratio (I), and the half inhibitory concentration (IC 50 ), for the growth of S. aureus at different nisin concentrations were determined. The relationship between the growth rate constant (k) and the concentration of nisin (c) is nearly linear, which can be modeled by the formula k = 0.03794 - 4.005 x 10 -4 × c, with a correlation coefficient of -0.9971. Based on this model, we obtained the critical inhibitory concentration of nisin on the growth of S. aureus at 94.73 IU/mL. We proposed that this microcalorimetric method could be a useful tool in monitoring the biological effect of nisin on microorganisms, and providing valuable information on the study of microorganism metabolisms.

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.

Rhizobox-soil microcosms studies on the colonization, dispersal and nodulation of Sinorhizobium fredii HN01DL marked with luxAB gene in Glycine max rhizosphere showed that the colonization dynamics and the density of HN01DL in non-sterilized rhizobox-soil microcosms were different from those in sterilized rhizobox-soil microcosms. The colonization density of the former reached the maximum (8.65 log cfu.g-1 root) 12 days after the coated seeds planted, and that of the latter decreased rapidly at the early stage and achieved the maximum (6.88 log cfu.g-1 root) 15 days afterwards. Furthermore, the colonization density of HN01DL reached the maximum (7.05 log cfu.g-1 root) in section A (0-4 cm) of root system 5 days after seeds planted, decreased slowly and kept a relative stable level until 19 days, and began to rise up again 33 days afterwards. The strain could also disperse to the place of 16 cm from seed to root tip by 46 days after seed planted. HN01DL maintained a constantly higher colonization density level in section A of root system, formed the largest number of luminescent nodules (total 16.3, dominantly located in main root of section A), and had the highest luminescent percentage (68.8%). The luminescent nodule percentage decreased gradually along section A to E of root system, and no luminescent nodule was detected in section E of root system.

With gene marking technique, green fluorescent protein (GFP) and nodule bacteria gene has been linked together toform a single fusion gene expression vector. Then the vector is transferred into the nodule bacteria and the astragalus sinicusis invaded by the vector. With laser conlocal scanning microscope (LCSM), some important morphological information inplant nitrogen fixation research about the invading of nodule bacteria and the formation process of root nodule has beenobtained through the three-dimensional imaging of GFP reporting fluorescence. Keywords: laser confocal scanning microscope, three-dimensional imaging, GF1 astragalus sinicus, DNA reconstruction, GFP marking technique, nodule bacteria, root nodule, invading line, nitrogen fixation 1. INTRODUCTIONTo clarify the diverse biological activity process and its regularity in intact living cells in molecular level is the objective of the study of molecular biology. But before the appearance of GFP, almost all the study methods demand fixingor destroying the cell. Even the macromolecular fluorescence marking technique can only study some specific molecularprocesses under specific conditions in living cells. And its experiment operation is very complex and can only meet thedemand of short time observation.The appearance of the GFP marking system overcomes the above defects thoroughly'23. With DNA reconstructiontechnique, the GFP gene and the objective protein gene is linked together. Then a single fusion gene expression vector isformed. If the objective protein gene is expressed, the GFP gene can be expressed simultaneously. So we can trace theexpression and the time-space features of the objective protein gene through detecting the fluorescence of GFP GFP nowbecomes a very important method in genetics research. Also the generation of GFP fluorescence need not any auxiliaryelements or substrates. So with the GFP marking system, researchers can do direct observations and studies to the diverseprocesses carried out in living cells or living biological tissues and its molecular mechanisms under normal physiological

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