[Diversity of antagonistic bacteria isolated from rhizosphere of several cash crops].

By adopting antimicrobial spectrum test, BOXAIR-PCR, physiological and biochemical, and 16S rDNA sequencing analysis, this paper analyzed the diversity of 55 antagonistic bacterial strains isolated from the rhizosphere of 10 cash crops. There was a high diversity of the antagonism of the strains. Based on BOXAIR-PCR, all the strains were clustered into 7 groups at the similarity level of 72.1%, and divided into 25 groups at the similarity level of 85.0%. All the strains belonged to Bacillus, Paenibacillus, Brevibacillus, Pseudomonas and Alcaligenes, respectively. The antagonistic bacteria isolated from the rhizosphere had high genetic diversity and high diversity in antagonistic activity.

[Cloning and functional analysis of cysI gene involved in siderophores biosynthesis in Pseudomonas mosselii E1].

A siderophores-producing strain E1 was isolated from the rhizosphere of cotton. Its 16S rDNA is identical to that of Pseudomonas mosselii sp. nov. at 100% level. The suicide plasmid pRL1063a carrying Tn5-1063 was introduced into E1 by triparental mating and 1000 transposon insertion mutants were subsequently screened using CAS assay. One mutant deficiency in siderophores production was obtained, namely, E1-185. DNA sequences flanking Tn5-1063 of E1-185 was amplified by TAIL-PCR. According to the DNA sequencing results, it is found that Tn5-1063 was inserted into cysI gene. The cysI of E1 is identical to that of Pseudomonas entomophila. L48 at 96% level, and similarity of amino acid sequences of their CysI is 97% . The cysI gene is required for the synthesis of cysteine. However, The ability in siderophores production of E1-185 on the plate of CAS with cysteine was recovered. It is indicated that cysI play an important role during the synthesis of siderophores. It was supposed that cysI is involved in the synthesis of acyl-S-PCPs, which is the key protein in the synthesis pathway of siderophores.

[Transcriptional regulation of noeAB from Sinorhizobium meliloti 042BM].

The expression regulation of S. meliloti 042BM noeAB was studied. The results showed that trigonelline could not elevate the level of noeAB expression, which indicated that these genes are not regulated by nodD2. Since association of nodD3 and syrM could not change the level of the genes expression, they aren't also controlled by nodD3-syrM system. However, induction of luteolin resulted in 16 times increase of noeAB expression, which indicated that noeAB was regulated by nodD1. Most interestingly, more than 30 times increase in its expression was observed on TY medium without any flavonoid. Thus, it was suggested that noeAB may be controlled by other unknown factors.

[Cloning, deletion and functional analysis of noeA from Sinorhizobium meliloti 042BM].

042BM noeA was obtained by PCR. It is identical to that of S. meliloti 1021 at 99% level, and similarity of their NoeA is 97%. In addition, it was found that this protein shares significant homology with the SAM-dependent methyltransferase of Mesorhizobium sp. BNC1 (32% similarity), and the similarity of its 303-362 region to the 160- 220 region of Ll11 methyltransferases of E. coli (PrmA) is 41%. Compared to 042BM, the noeA deletion mutant 042BMA-Km showed different degrees of increase in number of nodule, fresh weight of nodule and plant top dry weight on alfalfa cultivars of Putong Zihua, Baoding, Ningxia, Baifa and Aohan, but decrease on Milu. However, this mutant has no significant change in ability to nodulate cultivars of Huanghou and Zahua. Hence, noeA is involved in alfalfa cultivar-specific nodulation.

[Positional analysis of a gene related to salt tolerance in Sinorhizobium meliloti by transposon rescue].

Salt sensitive mutant 042BML-2 was obtained by mutating Sinorhizobium meliloti 042BM with transposon Tn5 carried on the plasmid pRL1063a. By transposon rescue, a 1.179 kb of DNA sequence of S. meliloti flanking the Tn5 insertion site was obtained. The sequence similarity analysis through BLAST analysis in GenBank revealed the transposon was inserted into a functionally unknown gene, which is 6 270 bp in length, of S. meliloti, and the gene was named rtsC. This study indicated that rtsC was associated with salt tolerance in S. meliloti 042BM. Hydrophobicity profile analysis of the putative amino acid sequence showed that two predicted transmembrane domains existed in N-terminal portion of RtsC. The significance of RtsC protein in the salt-tolerance in S. meliloti was discussed.

Salt-tolerance genes involved in cation efflux and osmoregulation of Sinorhizobium fredii RT19 detected by isolation and characterization of Tn5 mutants.

Salt-tolerance genes of Sinorhizobium fredii RT19 were identified by the construction and screening of a transposon Tn5-1063 library containing over 30,000 clones. Twenty-one salt-sensitive mutants were obtained and five different genes were identified by sequencing. Eight mutants were found with disruptions in the phaA2 gene, which encodes a cation efflux system protein, while mutations in genes encoding other cation effux system proteins were found in seven (phaD2), two (phaF2) and two (phaG2) mutants. A mutation in the metH gene, encoding 5' methyltetrahydrofolate homocysteine methyltransferase, was found in two of the salt sensitive strains. Growth experiments showed that phaA2, phaD2, phaF2 and phaG2 mutants were hypersensitive to Na+/Li+ and slightly sensitive to K+ and not sensitive to sucrose and that metH mutants were highly sensitive to any of Na+, Li+, K+ and sucrose. Na+ intracellular content measurements established that phaA2, phaD2, phaF2 and phaG2 are mainly involved in the Na+ efflux in S. fredii RT19. Recovery of growth of the metH mutants incubated with different concentrations of NaCl could be obtained by additions of methionine, choline and betaine, which showed that the metH gene is probably involved in osmoregulation in S. fredii RT19.

[A Sinorhizoboium meliloti strain that can nodulate soybean plants].

Sinorhizobium meliloti XJ96077 was isolated from root nodules of alfalfa (Medicago sativa) in Xinjiang Region of China. Nodulation experiments showed that both soybean and alfalfa were effectively nodulated by XJ96077. The DNA (G+ C) mol% of strain XJ96077 was 61.9%. The DNA homologies of strain XJ96077 were 93% and 80% with S. meliloti USDA1002T and 042BM, respectively. These results showed that XJ96077 belongs to Sinorhizobium meliloti. To prove the capability of XJ96077 to nodulate both soybean and alfalfa, constitutively expressed green fluorescence protein gene gfp was introduced to XJ96077, and the recombinant strain XJ96077(G) was obtained. Root nodules of the soybean and alfalfa inoculated with XJ96077(G) and the expression of gfp were observed using the confocal laser scanning microscope. XJ96077 showed various nodulation capacities with different soybean cultivars.