nifH promoter activity is regulated by DNA supercoiling in Sinorhizobium meliloti

In prokaryotes, DNA supercoiling regulates the expression of many genes; for example, the expression of Klebsiella pneumoniae nifLA operon depends on DNA negative supercoiling in anaerobically grown ceils, which indicates that DNA supercoiling might play a role in gene regulation of the anaerobic response. Since the expression of the nifH promoter in Sinorhizobium meliloti is not repressed by oxygen, it is proposed that the status of DNA supercoiling may not affect the expression of the nifH promoter. We tested this hypothesis by analyzing nifH promoter activity in wild-type and gyr- Escherichia coli in the presence and absence of DNA gyrase inhibitors. Our results show that gene expression driven by the S.meliloti nifH promoter requires the presence of active DNA gyrase. Because DNA gyrase increases the number of negative superhelical turns in DNA in the presence of ATP, our data indicate that negative supercoiling is also important for nifH promoter activity. Our study also shows that the DNA supercoiling-dependent S. meliloti nifH promoter activity is related to the trans-acting factors NtrC and NifA that activate it. DNA supercoiling appeared to have a stronger effect on NtrC-activated nifH promoter activity than on NifA-activated promoter activity. Collectively, these results from the S. meliloti nifH promoter model system seem to indicate that, in addition to regulating gene expression during anaerobic signaling, DNA supercoiling may also provide a favorable topology for trans-acting factor binding and promoter activation regardless of oxygen status.     

Desiccation responses and survival of Sinorhizobium meliloti USDA 1021 in relation to growth phase, temperature, chloride and sulfate availability

AIMS: To identify physical and physiological conditions that affect the survival of Sinorhizobium meliloti USDA 1021 during desiccation. METHODS AND RESULTS: An assay was developed to study desiccation response of S. meliloti USDA 1021 over a range of environmental conditions. We determined the survival during desiccation in relation to (i) matrices and media, (ii) growth phase, (iii) temperature, and (iv) chloride and sulfate availability. CONCLUSIONS: This study indicates that survival of S. meliloti USDA 1021 during desiccation is enhanced: (i) when cells were dried in the stationary phase, (ii) with increasing drying temperature at an optimum of 37 degrees C, and (iii) during an increase of chloride and sulfate, but not sodium or potassium availability. In addition, we resolved that the best matrix to test survival was nitrocellulose filters. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of physical and physiological factors that determine the survival during desiccation of S. meliloti USDA 1021 may aid in (i) the strategic development of improved seed inocula, (ii) the isolation, and (iii) the development of rhizobial strains with improved ability to survive desiccation. Furthermore, this work may provide insights into the survival of rhizobia under drought conditions.     

Mutations in sit B and sit D genes affect manganese-growth requirements in Sinorhizobium meliloti

Two transposon-induced mutants of Sinorhizobium meliloti 242 were isolated based on their inability to grow on rich medium supplemented with the metal chelator ethylenediamine di-o-hydroxyphenylacetic acid (EDDHA) and either heme-compounds or siderophores as iron sources. Tagged loci of these mutants were identified as sit B and sit D genes. These genes encode components of an ABC (ATP-binding cassette) metal-type permease in several Gram-negative bacteria. In this work, the phenotypes of these two mutants were compared with those of two siderophore-mediated iron transport mutants. The results strongly implicate a role of the sit genes in manganese acquisition when this metal is limiting in S. meliloti.     

转座子挽救法对苜蓿中华根瘤菌与耐盐有关基因的定位

用含Tn5转座子的质粒pRL1063a诱变苜蓿中华根瘤菌(Sinorhizobium meliloti)042BM,得到盐敏感突变株042BML-2。采用转座子挽救法对Tn5插入位点两边的序列进行克隆与测序,获得了1179bp的转座子插入位点侧翼DNA序列。在GenBank中进行序列同源性和基因定位分析,结果表明：转座子插入在一个功能未知的基因内部,此基因长6270bp。研究证明：该基因与042BM的耐盐性有关,并定名为rtsC。氨基酸疏水性分析表明,在RtsC蛋白的N端有两个跨膜区,该蛋白与细菌趋化性相关蛋白的功能域有同源性。并对RtsC蛋白在苜蓿中华根瘤菌042BM耐盐性中的作用进行了讨论。     

Construction of a Sinorhizobium meliloti strain carrying a stable and non-transmissible chromosomal single copy of the green fluorescent protein GFP-P64L/S65T

A single copy of the green fluorescent protein (GFP)-encoding gene gfp-P64L/S65T under the control of the constitutive nptII promoter was introduced in a neutral region of the Sinorhizobium meliloti chromosome, between the genes recA and alaS. Within the same chromosomal region downstream of gfp-P64L/S65T a tetracycline (Tc) resistant cassette was also inserted. Both markers were very stable during at least 40 bacterial generations without any selective pressure. Similarly, the gfp-Tc cassette was stable and functional in all rhizobia that were recovered from alfalfa nodules. The GFP-associated fluorescence derived from the (single copy) chromosomal gfp-P64L/S65T allowed detection of rhizobia during the colonisation of the root, infection thread formation, and nodule development. The gfp-Tc rhizobia showed indistinguishable phenotypes for nodulation, competitiveness, and nitrogen-fixation from the parental strain. The labelling system described here can be used for the stable fluorescent tagging of S. meliloti strains allowing their detection in biologically complex soil environments.     

Development of real-time PCR assay for detection and quantification of Sinorhizobium meliloti in soil and plant tissue

Aims:  Sinorhizobium meliloti is a nitrogen-fixing alpha-proteobacterium present in soil and symbiotically associated with root nodules of leguminous plants. To date, estimation of bacterial titres in soil is achieved by most-probable-number assays based on the number of nodules on the roots of test plants. Here, we report the development of two real-time PCR (qPCR) assays to detect the presence of S. meliloti in soil and plant tissues by targeting, in a species-specific fashion, the chromosomal gene rpo E1 and the pSymA gene nod C.
Methods and Results:  rpo E1 and nod C primer pairs were tested on DNA extracted from soil samples unspiked and spiked with known titres of S. meliloti and from plant root samples nodulated with S. meliloti . Results obtained were well in agreement with viable titres of S. meliloti cells estimated in the same samples.
Conclusions:  The developed qPCR assays appear to be enough sensitive, precise and species-specific to be used as a complementary tool for S. meliloti titre estimation.
Significance and Impact of the Study:  These two novel markers offer the possibility of quick and reliable estimation of S. meliloti titres in soil and plant roots contributing new tools to explore S. meliloti biology and ecology including viable but nonculturable fraction.     

Ribosomal protein gene cluster analysis in eubacterium genomics: homology between Sinorhizobium meliloti strain 1021 and Bacillus subtilis

The first whole genome sequence of a symbiotic soil bacterium, Sinorhizobium meliloti (formely named Rhizobium meliloti) strain 1021, is due in 2001. As an active participant in the European and North American consortium that has completed this work, our group has sequenced a region on the chromosome containing clusters rpoBC, str, S10, spc and alpha corresponding to 30 protein genes. The structural organization and function of these genes were compared with those of orthologs in another 15 complete eubacterial genomes available in databases. This study, involving the DNA and amino acid sequences as well as the organization of the whole region (gene order, cluster order, etc.), has shown that the phylogenetic tree resulting from a comparison of the amino acid sequence is rather similar to that derived from 16S rRNA sequence data. However, the tree achieved by aligning DNA sequences groups the organisms with a high GC content (>60% GC), while that based on a comparison of gene cluster orientation and organization reveals a greater level of correspondence between the α-proteobacteria S.meliloti and the firmicute Bacillus subtilis.     

Genetic and biochemical characterization of a pathway for the degradation of 2-aminoethylphosphonate in Sinorhizobium meliloti 1021

A variety of microorganisms have the ability to use phosphonic acids as sole sources of phosphorus. Here, a novel pathway for degradation of 2-aminoethylphosphonate in the bacterium Sinorhizobium meliloti 1021 is proposed based on the analysis of the genome sequence. Gene deletion experiments confirmed the involvement of the locus containing phnW, phnA, and phnY genes in the conversion of 2-aminoethylphosphonate to inorganic phosphate. Biochemical studies of the recombinant PhnY and PhnA proteins verified their roles as phosphonoacetaldehyde dehydrogenase and phosphonoacetate hydrolase, respectively. This pathway is likely not limited to S. meliloti as suggested by the presence of homologous gene clusters in other bacterial genomes.     

Biochemical characterization of Sinorhizobium meliloti mutants reveals gene products involved in the biosynthesis of the unusual lipid A very long-chain fatty acid

Sinorhizobium meliloti forms a symbiosis with the legume alfalfa, whereby it differentiates into a nitrogen-fixing bacteroid. The lipid A species of S. meliloti are modified with very long-chain fatty acids (VLCFAs), which play a central role in bacteroid development. A six-gene cluster was hypothesized to be essential for the biosynthesis of VLCFA-modified lipid A. Previously, two cluster gene products, AcpXL and LpxXL, were found to be essential for S. meliloti lipid A VLCFA biosynthesis. In this paper, we show that the remaining four cluster genes are all involved in lipid A VLCFA biosynthesis. Therefore, we have identified novel gene products involved in the biosynthesis of these unusual lipid modifications. By physiological characterization of the cluster mutant strains, we demonstrate the importance of this gene cluster in the legume symbiosis and for growth in the absence of salt. Bacterial LPS species modified with VLCFAs are substantially less immunogenic than Escherichia coli LPS species, which lack VLCFAs. However, we show that the VLCFA modifications do not suppress the immunogenicity of S. meliloti LPS or affect the ability of S. meliloti to induce fluorescent plant defense molecules within the legume. Because VLCFA-modified lipids are produced by other rhizobia and mammalian pathogens, these findings will also be important in understanding the function and biosynthesis of these unusual fatty acids in diverse bacterial species.     

接种量对费氏中华根瘤菌HN01DL根圈定殖与结瘤的影响

在灭菌土和未灭菌土盆栽系统中 ,研究了大豆种子的表面初始接种量对费氏中华根瘤菌HN0 1DL在大豆根圈中的定殖动态与结瘤的影响。结果表明 ,与 3个接种量对应的早期根圈定殖动态和水平有明显差异 ,但随着宿主植物根系的生长其差异逐渐减小 ,整个定殖动态曲线的变化和定殖密度趋向一致 ,并且发现 3个不同的初始接种量对HN0 1DL在大豆黑农 33根系上的结瘤数量和占瘤率没有显著影响。     

Mutation in the <Emphasis Type="Italic">cobO</Emphasis> gene generates auxotrophy for cobalamin and methionine and impairs the symbiotic properties of <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis> HH103 with soybean and other legumes

We report here the isolation of a methionine and cobalamin mutant strain (SVQ336) of Sinorhizobium fredii HH103 obtained by Tn5-lacZ mutagenesis. Sequence analysis showed that the transposon was inserted into a gene homologous to cobO. This gene codes for a cobalamin adenosyltransferase which is involved in the biosynthesis of vitamin B₁₂. Another HH103 cobO mutant (strain SVQ524), was constructed by the insertion of Ω interposon. Both cobO mutants required the addition of methionine because cobalamin acts as a cofactor of the enzyme MetH, which catalyses the last step of the methionine biosynthesis. Mutant SVQ524 failed to nodulate on Vigna radiate but was able to nodulate on Glycine max cvs. Williams and Peking and Cajanus cajan, although the total number of nodules formed was highly reduced in comparison with that of plants inoculated with the wild-type strain HH103. The roots of these plants did not seem to secrete enough cobalamin and/or methionine to support growth of cobalamin/methionine auxotrophs in the rhizosphere. In all cases, the phenotype of SVQ524 was nearly overcome by the addition of methionine or cobalamin to the plant growth media or by the presence of a copy of the cobO gene in cosmid pMUS756. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Roundabout gene family functions during sensory axon guidance in the drosophila embryo are mediated by both Slit-dependent and Slit-independent mechanisms

roundabout (robo) family genes play key roles in axon guidance in a wide variety of animals. We have investigated the roles of the robo family members, robo, robo2, and robo3, in the guidance of sensory axons in the Drosophila embryo. In robo(-/-), slit(-/-), and robo(-/+) slit(-/+) mutants, lateral cluster sensory neurons misproject to cells and axons in the nearby ventral' (v') cluster. These phenotypes, together with the normal expression pattern of Slit and Robo, suggest that Slit ligand secreted from the epidermis interacts with Robo receptors on lateral cluster sensory growth cones to limit their exploration of nearby attractive substrates. The most common sensory axon phenotype seen in robo2(-/-) mutants was misprojection of dorsal cluster sensory axons away from their normal growth substrate, the transverse connective of the trachea. slit appears to play no role in this aspect of sensory axon growth. Robo2 is expressed, not on the dorsal sensory axons, but on the transverse connective. These results suggest a novel, non-cell-autonomous mechanism for axon guidance by robo family genes: Robo2 expressed on the trachea acts as an attractant for the dorsal sensory growth cones.     

Locus-specific DNA methylation in the placenta is associated with levels of pro-inflammatory proteins in cord blood and they are both independently affected by maternal smoking during pregnancy

We investigated the impact of maternal smoking during pregnancy on placental DNA methylation and how this may mediate the association between maternal smoking and pro-inflammatory proteins in cord blood. The study population consisted of 27 individuals exposed to maternal smoking throughout pregnancy, 32 individuals exposed during a proportion of the pregnancy, and 61 unexposed individuals. Methylation of 11 regions within 6 genes in placenta tissue was assessed by pyrosequencing. Levels of 7 pro-inflammatory proteins in cord blood were assessed by electrochemiluminescence. Differential methylation was observed in the CYP1A1 promoter and AHRR gene body regions between women who smoked throughout pregnancy and non-smokers on the fetal-side of the placenta and in the GFI1 promoter between women who quit smoking while pregnant and non-smokers on the maternal-side of the placenta. Maternal smoking resulted in elevated levels of IL-8 protein in cord blood, which was not mediated by DNA methylation of our candidate regions at either the maternal or the fetal side of the placenta. Placental DNA methylation was associated with levels of inflammatory proteins in cord blood. Our observations suggest that maternal smoking during pregnancy affects both placental DNA methylation and the neonate's immune response.