BacA is indispensable for successful Mesorhizobium-Astragalus symbiosis.

A homologue of Sinorhizobium meliloti bacA was isolated from Mesorhizobium huakuii 7653R, which is capable of fixing atmospheric nitrogen in symbiotic association with leguminous Astragalus sinicus (Chinese milk vetch). Inactivation of the bacA gene abolished the ability of M. huakuii 7653R to establish a successful symbiosis with its host plant. Simultaneously, compared with wild-type M. huakuii 7653R, the bacA mutant was more sensitive to cell envelope-disrupting agents (acidic solution, ethanol, SDS, and crystal violet). Mass spectrometry analysis revealed that the very-long-chain fatty acid (27-OHC-28:0 and 29-OHC-30:0) contents of lipid A was reduced in the M. huakuii 7653R bacA mutant. Taken together, our data suggest that the cell envelope was altered in the M. huakuii 7653R bacA mutant, which might deteriorate bacterial adaption to acute environmental changes encountered in host cells and ultimately result in the failure of Mesorhizobium-legume symbiosis.

The symbiosis phenotype and expression patterns of five nodule-specific genes of Astragalus sinicus under ammonium and salt stress conditions.

In previous works, we isolated 14 nodule-specific or nodule-enhanced genes from Astragalus sinicus by suppressive subtractive hybridization. In this study, we have further identified the expression patterns of five nodule-specific genes of A. sinicus under salt and ammonium stress. Transcription levels of genes tested were quantified by quantitative fluorescence real-time RT-PCR. Results showed that: (1) About 80 mM NaCl and all stress treatments containing (NH(4))(2)SO(4) significantly inhibited nitrogen-fixing capacity of inoculated plants. About 40 mM NaCl showed relative lighter inhibition. (2) Compare with positive control at normal conditions, the expressions of all genes were significantly reduced by all ammonium stress. (3) Under salt stress without exogenous nitrogen, transcription levels of AsIIA255 and AsE246 were significantly increased after treatment for 3 days. But expressions of AsG2411, AsIIC2512, and AsB2510 were suppressed by 80 mM NaCl and not significantly affected by 40 mM NaCl. (4) Under salt stress with exogenous nitrogen, expressions of AsG2411, AsIIC2512, AsB2510, and AsIIA255 were significantly suppressed. While, the transcription level of AsE246 under 80 mM NaCl containing 1 mM (NH(4))(2)SO(4) was still higher than that of positive control. The correlation of the expression profiles of three cysteine cluster protein (CCP) genes (AsG2411, AsIIC2512, AsIIA255) and one lipid transfer protein (LTP) gene (AsE246) with the nitrogen-fixing capacities of nodules in each treatments may explain the molecular mechanisms of their supposed functions in symbiosis and nitrogen-fixing process. Our results also implied that AsIIA255 and AsE246 might play a role in the response of A. sinicus to salt stress to facilitate the nitrogen-fixation process.

A novel nodule-enhanced gene encoding a putative universal stress protein from Astragalus sinicus.

A nodule-enhanced gene, AsD243, was identified from infected roots of Astragalus sinicus using suppressive subtractive hybridization (SSH). It encodes a 20-kD protein related to the bacterial universal stress protein family (Usp). Sequence analysis showed that AsD243 is highly similar to the bacterial MJ0577-type of ATP-binding Usp proteins, which have been proposed to function as a molecular switch. Expression analyses revealed that AsD243 was transcribed in all plant organs, and progressively during all stages of nodulation. Its transcripts increased significantly at 7 days after inoculation, which is 2 days later than the onset of leghemoglobin expression in A. sinicus nodules. AsD243 was expressed more strongly in mature roots than in young roots regardless of inoculation status. We suggest that the AsD243 may have other functions in plant processes besides nodulation.

Genotypic characteristics of the rrn operon and genome of indigenous soybean Bradyrhizobia in cropping zones of China.

Four genetic assays, 16S rRNA restriction fragment length polymorphism (RFLP), 16S rRNA sequencing, 16S-23S rRNA intergenetic spacer (IGS) RFLP, and amplified fragment length polymorphism (AFLP), were conducted to determine the genotypic characteristics of 44 indigenous strains of Bradyrhizobium from soybean (Glycine max L.) cropping zones of China. The results generated from different assays showed that soybean bradyrhizobial isolates comprised four genomic groups. Group I was composed of strains mainly isolated from the North and Northeast plains of China. All four assays confirmed this group as phylogenetically divergent from all the reference strains. Strains of the group may represent a new species. Strains in Group II isolated from a variety of geographic regions were ascribed to B. liaoningense. Strains in Group III, mainly isolated from Central and East China, were closely related to the reference strains of B. japonicum. Strains in Group IV belonged to B. elkanii.

Thirteen nodule-specific or nodule-enhanced genes encoding products homologous to cysteine cluster proteins or plant lipid transfer proteins are identified in Astragalus sinicus L. by suppressive subtractive hybridization.

Thirteen nodule-specific or nodule-enhanced genes have been revealed by suppressive subtractive hybridization (SSH) with two mRNA populations of infected and uninfected control roots of Astragalus sinicus. Eleven of them encode small polypeptides showing homology to cysteine cluster proteins (CCPs) that contain a putative signal peptide and conserved cysteine residues. Among these CCP-like genes, AsG257 codes for a homologue of the defensin 2 family and AsD255 contains a scorpion toxin-like domain at the C-terminus. Sequence analysis of a genomic AsD255 fragment which was isolated revealed that one intron separates the first exon encoding the signal peptide from the second exon encoding the cysteine cluster domain of this nodulin. Another two genes, AsE246 and AsIB259, encode two different products similar to lipid transfer proteins (LTPs). Virtual northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that the other genes except AsIB259 and AsC2411 were expressed exclusively in inoculated roots and that their expression was 2-4 d later than that of the leghaemoglobin (Lb) gene during nodule development. Transcription of AsIB259 was also detected in uninfected control roots but with a significant decline in expression and a temporal expression similar to Lb. AsC2411 had a basal expression in control roots identified by RT-PCR. Sequence alignment showed that the putative proteins AsE246 and AsIB259 show lower homology with LTPs from legumes than with those from other plants.

[Genetic diversity and phylogeny of soybean bradyrhizobia isolated from south and north region of China].

Studies on genetic diversity and phylogeny of soybean bradyrhizobia isolated from south and north region of China were investigated through 16S rRNA gene PCR RFLP, 16S rRNA gene sequencing and 16S-23S rRNA IGS PCR RFLP assays. Results of 16S rRNA gene PCR RFLP and 16S rRNA gene sequencing analysis reveal that strains tested are ascribed into Bradyrhizobium japonicum and B. elkanii species. B. japonicum is the dominant species and accounts for 91% of strains tested, but B. elkanii just up to 9%, which shows poor genetic diversity. Results of 16S-23S rRNA IGS PCR RFLP assays reveal that strains belonged to B. japonicum can be divided into Group I and Group HIIat the 69 similarity. Group I consists of strains from northern China, and Group 1I consists of strains from southern China, which demonstrates geographical effect on genetic diversity of bradyrhzobia. Results of 16S-23S rRNA IGS PCR RFLP further reflects that strains of Group I and Group II are phylogenetically different with type or representative strains USDA6, USDA110 and USDA122 of B. japonicum.

Cloning and identification of opa22, a new gene involved in nodule formation by Mesorhizobium huakuii.

Using Tn5-sacB insertion mutagenesis, 3000 mutants were obtained from Mesorhizobium huakuii 7653R. Eight nodulation-defective mutants were screened by plant nodulation experiments. The DNA sequences of the contiguous region from the Tn5 insertion site were determined by thermal asymmetric interlaced PCR. A new gene was cloned and designated opa22, as judged from its structural and functional homology. Sequence analysis indicated that opa22 was composed of 774 nucleotides and encoded a protein of 257 amino acids. RPS-BLAST analysis of the Opa22 protein showed a sequence similarity (88.9%) to the opacity protein and related to surface antigens of the bacterial outer membrane, which can mediate various pathogen-host cell interactions and promote invasion. Our results from root hair curling experiments suggested that expression of the opa22 gene might occur at the stage of infection thread formation and nodule development. The complement stain HK24 was able to restore the nodule forming ability of the mutant.

Symbiotic abilities of Sinorhizobium fredii with modified expression of purL.

Previous reports showed that a transposon-induced PurL- mutant of Sinorhizobium fredii induced pseudonodules on Glycine max and the addition of 5-aminoimidazole-4-carboxamide-riboside or adenine to the plant could not restore the mutant to establish effective symbiosis. To gain a better understanding of the impact of the purL gene on symbiosis formation, we measured the effect of modified expression of this gene on the symbiotic abilities of S. fredii on soybean (G. max). A 1.98-kb in-frame deletion mutant in the purL gene of S. fredii was constructed. Transcriptional modification of the purL gene was conducted using several promoters such as those of lac, nifH, nifQ, and fixN. It was found that reduced expression of purL gene or suitable symbiotic expression of purL (such as with the promoter nifH or nifQ) can efficiently establish symbiosis of S. fredii on G. max without the exogenous supplementation of any adenine or purine precursor; at least a minimal level of expression of purL is essential for effective symbiosis with soybean.

[Studies on genetic diversity and phylogeny of slow-growing rhizobia isolated from Vigna radiata at main ecotypes of China].

Studies on genetic diversity and phylogeny of slow-growing rhizobia isolated from Vigna radiata at main ecotypes of China were conducted by using 16S rRNA gene PCR-RFLP, 16S rRNA gene sequencing and 16S-23S rRNA IGS PCR-RFLP assays. Results of 16S rRNA gene PCR RFLP analysis reveal that all the strains tested are clustered into three groups at the similarity of 76%. Group I contains 13 slow-growing rhizobia tested including LYG1; Group II consists of 21 strains tested and the type strains of B. japonicum and B. liaoningense, and 10 tested strains isolated from Guangdong, Guangxi and the type strain of B. elkanii compose Group III . The results of 16S-23S rRNA IGS PCR-RFLP show that strains tested could be divided into A and B groups, which could be correspondently subdivided into A I , A II , AIII , BI and B II subgroups at the similarity of 85% . Compared with 16S rRNA PCR-RFLP, IGS RFLP assay show higher resolution, strains and reference strains tested can be divided into 21 IGS RFLP patterns. The strains isolated from Xinjiang, Guangdong and Guangxi regions show obvious geographical effect on genetic diversity.

[Effects of bkdAB interruption on avermectin biosynthesis].

In this study, Streptomyces avermitilis Bjbm0006 which produces four avermectin B components was used as an original test strain. A replacement plasmid containing a gene cluster bkdAB (branched-chain alpha-keto acid dehydrogenase gene) involved in the biosynthesis of avermectin B in S. avermitilis Bjbm0006 was constructed by means of PCR technique and then named as pHJ5821 (pHZ1358::bkdAB&erm). A recombinant strain Bjbm5821 was obtained after the gene cluster was interrupted by double crossover. This strain was tested in laboratory conditions and analysed by PCR using the total DNA as template. The HPLC analysis showed that the strain Bjbm5821 synthesized the same 'a' components Bla and B2a as the original strain did. However, It lost the ability for the production of 'b'components for example B1b and B2b. A novel compound was detected in fermentation products. The results of present study suggests that the production of gene cluster bkdAB may play a main role similar to alpha-ketoisovaleric acid dehydrogenase in the pathway of avermectin synthesis.

[A study of the effects on the symbiotic nitrogen fixation of Sinorhizobium fredii with the introduction of dctABD and nifA genes].

A recombinant plasmid pHN307 containing C4-dicarboxylic acid transport genes (dctABD) from Sinorhizobium meliloti, nifA genes from Klebsiella pneumoniae and reporter genes luxAB from pDB30 was constructed by using pTR102 as the vector. The pHN307 was then introduced into the S. fredii HN01, YC4 and GR3 by tri-parental mating, and the stability of pHN307 in the transconjugants under free-living and symbiotic condition was also investigated. The results of plant pot experiment indicated that the introduction of pHN307 in the transconjugants could significantly increase the nodule fresh weight, shoot dry weight (biomass) and total nitrogen content of the symbiotic plants with soybean variety of Heilong 33. When the transconjugants were in symbiotic with soybean variety of Chuanzao No. 1, HN01 (pHN307) could significantly increase its root nodule number and fresh weight; GR3 (pHN307) could significantly increase its root nodule number, nodule fresh weight, shoot dry weight and total nitrogen content, but YC4(pHN307) demonstrated negative effect under the same condition. The results of this study suggested that the introduction of dctABD and nifA could improve the symbiotic nitrogen fixation efficiency and nodulation ability of the rhizobia tested, respectively, and its effect was relevant to the characteristics of recipient rhizobia and soybean variety.

[Genetic diversity and phylogeny of rhizobia isolated from peanut (Arachis hypogaea)].

Forty three rhizobium strains isolated from peanut (Arachis hypogaea) and 15 reference strains from other genus and species were analyzed by the method of 16S rRNA RFLP, 16S rRNA sequencing and 16S-23S IGS PCR RFLP. The results of the 16S rRNA RFLP shown that 43 strains tested were all ascribed to the genus of Bradyrhizobium phylogenetically. Strains tested were adjacent to the B. japonicum and far from B. elkanii 16S rRNA genotype. The genotypes generated by the 4 restriction endonucleases, Mbo I, Dde I, Hae III and Msp I, were same as the representatives of B. japonicum. The dendrogram generated by 16S rRNA sequence and Neighbor-joining method shown that peanut rhizobia clustered into the subcluster represented by B. japonicum and B. liaoningense, were more close to B. liaoningense genetically, and the sequence difference between them was less than 1%. High sequence similarity was also determined between B. liaoningense and B. japonicum. JZ1, representative strain of peanut rhizobia were systematically far from the B. elkanii, and the sequence divergence about 2%. The results from IGS RFLP analysis indicated that although they were phylogenetically close to B. japonicum and B. elkanii, peanut rhizobia forming an independent group at the similarity of 71% could be further divided into four subgroups, A, B, C and D. Subgroup A consisted of strains from different region, subgroup B was composed of strains from Wuchang, Qianjiang and Jingzhou, subgroup C was mainly composed of strains from Jingzhou and starins of subgroup D mainly from Neijiang. Reference strains from B. japonicum and B. elkanii were independently clustered into the subgroup E at the similarity of 71%. The geographical factor effect on genetic diversity of rhizobia was found.