Transcriptome analysis of Sinorhizobium meliloti nodule bacteria in nifA mutant background

Gene expression profiles of a Si- norhizobium meliloti 1021 nifA mutant and wild type nodule bacteria were compared using whole genome microarrays. The results revealed a large scale al- teration of gene expression (601 genes) in the nifA minus background. The loss of NifA altered the ex- pression of many functional groups of genes (mac- romolecular metabolism, TCA cycle and respiration, nodulation and nitrogen fixation) and may lead to quite different life stages of the nodule bacteria. Upregulation of fixK and its associated genes was observed in the nifA mutant nodule bacteria. Addi- tional quantitative real-time PCR experiments re- vealed that the transcript levels of fixLJ were signifi- cantly upshifted in the nifA mutant nodule bacteria. Putative NifA binding sites were predicted by a statis- tical method in the upstream sequences of 13 differ- entially regulated genes from the nifA- transcriptome.

The enoyl-ACP reductase gene, fabI1, of Sinorhizobium meliloti is involved in salt tolerance, swarming mobility and nodulation efficiency

Our previous work showed that an enoyl-ACP reductase gene fabI1 of Sinorhizobium meliloti was down-regulated in the nifA mutant nodule bacteria. To gain a better understanding of fabI1 gene, a single site insertion mutant was constructed in this study. The fabI1 mutant was retarded in cell growth, and its ability to grow on media with high concentration of NaCl was reduced. In addition, the mutant was completely defective in swarming phenotype. During symbiosis, the fabI1 mutant had delayed nodule formation on host plants. Despite the fact that FabI1 protein showed 66% identity with another enoyl-ACP reductase FabI2 in S. meliloti, defects in fabI1 were not rescued by the plasmidborne version of fabI2. This indicated the different functions of the two FabI proteins in S. meliloti.

Extra-copy nifA enhances the nodulation efficiency of Sinorhizobium fredii

Previous investigations have shown that nifA gene is involved in nodulation and symbiotic nitrogen fixation regulation of Rhizobium. We study the role of nifA on nodulation of leguminous plants. We found that Sinorhizo-bium fredii harboring multi-copy plasmid carrying the con-stitutively expressed Klebsiella pneumoniae nifA exhibited an increase of nodulation activity and nodulation competitiveness on soybean plants. The Nod-factor secreted by the rhizobia cells containing the multi-copied nifA was assayed, and preliminary results showed that S. fredii containing the multi-copy plasmid carrying nifA produced higher strength of Nod-factor than the rhizobia containing the same plasmid carrying the vector did.

Sinorhizobium meliloti nifA gene exerts a pleiotropic effect on nodulation through the enhanced plant defense response

Sinorhizobium meliloti nifA gene is required for the expression of a bunch of nif and fix genes. Here, we report its pleiotropic effects on the nodule formation. Compared with wild type strain, nifA mutant sig- nificantly reduced nodule suppression rate in split-root system. The plants inoculated with mutant strain produced lower amount of daidzein and less necrotic cells on their roots. In addition, the defense genes failed to be evoked by nifA mutant at the early nodulation stage. These findings indicated that host defense response was one of the mechanisms mediated by nifA gene to regulate nodule formation during symbiosis. Even though nifA mutant could increase the number of nodules in host plant, it synthesized lower Nod factors than wild type. This suggested that nifA gene mediated multiple and diverse instances in nodulation formation.

Complementation analyses of Sinorhizobium meliloti nifA mutant with different originated nifA genes

推断的一个以前的工作 Enterobacter 下水道的 nifA 基因没恢复 Sinorhizobium meliloti nifA 的共生显型变异。在现在的学习， Bra-dyrhizobium 的 twonifA 基因日本嗯并且 Mesorhizobium huakuii 也没恢复 S 的共生显型。meliloti nifA 异种。在微数组试验的整个 genomic， 238 基因被发现是在 S 以后表示的差别。meliloti nifA 组成地在它的 nifAmutant 被表示了。相反，当表示 B.japonium 时，仅仅 20， 7 和 9 基因改变了他们的 transcriptional 层次， M。在 Sm nifA 异种的 huakuii 和 Enterobacter 下水道 nifA 基因独立。这些基因包括房子保留，精力和中央中间代谢被分类进几官能基，运输系统和共生。有趣地， nifHoperons，显示出的高表示面对任何一个 B 铺平的基因。japonium 或 M。huakuii NifA，它被随后的紫胶 Z 熔化实验证实。

Functional difference between Sinorhizobium meliloti NifA and Enterobacter cloacae NifA

The nifA gene is an important regulatory gene and its product, NifA protein, regulates the expression of many nif genes involved in the nitrogen fixation process. We introduced multiple copies of the constitutively expressed Sinorhizobium meliloti (Sm) or Enterobacter cloacae (Ec) nifA gene into both the nifA mutant strain SmY and the wild-type strain Sm1021. Root nodules produced by SmY containing a constitutively expressed Sm nifA gene were capable of fixing nitrogen, while nodules produced by SmY containing the Ec nifA gene remained unable to fix nitrogen, as is the case for SmY itself. However, transfer of an additional Sm nifA gene into Sm1021 improved the nitrogen-fixing efficiency of root nodules to a greater extent than that observed upon transfer of the Ec nifA gene into Sm1021. Comparative analysis of amino acid sequences between Sm NifA and Ec NifA showed that the N-terminal domain was the least similar, but this domain is indispensable for complementation of the Fix? phenotype of SmY by Sm NifA. We conclude that more than one domain is involved in determining functional differences between Sm NifA and Ec NifA.

Identification of genes induced during Medicago sativa nodule development by using the cDNA-AFLP technique

A variety of genes involved in nodule development and function were induced during sym biotic root nodule development in alfalfa. We identi fied those genes by comparing the cDNA-amplified fragment length polymorphism (cDNA-AFLP) pat terns of infected nodules and uninfected roots. Sev enty-one transcript-derived fragments (TDFs) were isolated from about 3000 TDFs, which are enhanced or specifically expressed in nodule. The differentia expression patterns of 61 genes were confirmed b reverse Northern hybridization or Northern hybridiza tion analyses. Among these, 31 exhibited significan similarities to characterized database entries. These results suggest that the genes corresponding to the 31 TDFs may play important roles in signal transduc tion, gene expression and regulation, substance transportation or auto-regulation of nodulation during nodule development. The Northern hybridization analysis also indicates that the gene corresponding to TDF RX89 was not expressed in the nodule elic ited by bacA mutant, presumably it is specially in duced at the stage of nodule development when bacteroids begin to be differentiated.

Systematic insertion mutagenesis of GntR family tran-scriptional regulator genes in Sinorhizobium meliloti

GntR-type transcriptional regulators regulate the most diverse biological processes in bacteria. Al- though GntR-type transcriptional regulators consist of the second largest family of transcriptional regulators in Sinorhizobium meliloti, little is known about their functions. In this study, we investigated 54 putative genes encoding GntR family of transcriptional regulators in S. meliloti Rm1021. Secondary structure analysis of the C-terminal domain of these putative transcriptional regulators indicated that thirty-seven were members of the FadR subfamily, ten of the HutC subfamily and five of the MocR subfamily. The remaining two did not fall into any specific subfamily category, and may form two new subfamilies. The 54 gntR genes were mutagenized by plasmid insertion mutagenesis to investigate their roles. We found that, of the 54 mutants, only the gtrA1 and gtrB1 mutants had slower growth rates and cell maximal yields on both rich medium and minimal medium, and lower cell motility on swarming plate than wild type Rm1021. All mutants, with the exception of gtrA1 and gtrB1, can establish effective symbioses with alfalfa. Plants inoculated with gtrA1 and gtrB1 mutants grew shorter than those in- oculated with wild type, and formed relatively smaller, round and light pink nodules, which were mainly located on lateral roots. And there was an abnormal increase in the number of nodules induced by both mutants. These results suggested that the gtrA1 and gtrB1 mutants were symbiotically deficient. Our work presents a global overview of GntR-like transcriptional regulators involved in symbiosis in S. meliloti, and provides new insight into the functions of GntR-like transcriptional regulators.

Molecular studies of the Medicago truncatula MtAnn3 gene involved in root hair deformation

The full-length annexin gene,MtAnn3,of Medicago truncatula was cloned by 5' RACE.Compared with typical annexins,which contain a head domain and four homologous repeats in the conserved core domain,the MtAnn3 protein has only one repeat in the core domain.MtAnn3 can bind cell membranes when transiently expressed in onion epidermal cells.Agrobacterium rhizogenes-mediated transformation of MtAnn3 into Medicago roots revealed that overexpression of the gene can change the polarity of root hair growth in Ca2+-free medium.The plant hormone cytokinin was able to upregulate the expression of MtAnn3.While MtAnn3 transcripts were detected in young nodules,expression was not nodule-specific,and could be detected at high levels in the roots,stems and leaves as well.

Identification and characterization of two gcvA genes in Sinorhizobium meliloti

GcvA is a member of the LysR family of transcriptional regulators that mediates the expression of the glycine cleavage (GCV) operon (gcvTHP) in response to glycine in Escherichia coli. In our previous work, 90 putative regulator genes of the LysR family in Sinorhizobium meliloti were mutagenized to determine their phenotype. In the present study, we found that the S. meliloti genome had two gcvA genes, gcvA1 and gcvA2. Both gcvA1 and gcvA2 were required for full activation of the gcvTHP operon in the presence of exogenous glycine. The gcvA1-mediated activation of gcvTHP operon was gly- cine-inducible, while gcvA2-mediated activation was not. We speculate that the regulatory mechanism for gcvTHP expression in S. meliloti differed from E. coli. Evolutionary analysis showed that GcvA were distributed in many genera of Proteobacteria and the distances between GcvA1 and GcvA2 in S. meliloti and GcvA in E. coli were large, which may explain the different regulatory mechanisms for gcvTHP expression. These findings could provide new clues to the role of the LysR gene family.