导入额外拷贝nifA基因对费氏中华根瘤菌共生固氮作用的影响

以广宿主、稳定性质粒pTR102为载体构建重组质粒pHN306,其上克隆有来自肺炎克氏杆菌（Klebsiellapneumoniae）的nifA基因和来自pDB30所含的发光酶标记基因（luxAB）。经三亲本接合转移,将pHN306导入费氏中华根瘤菌（Sinorhizobiumfredii）HN01,GR3和YC4。与出发菌相比较的盆栽试验结果表明：HN01（pHN306）和GR3（pHN306）分别在大豆渝豆一号和黑龙33上能显著提高瘤数,瘤重,植株地上部分干重和总氮量,YC4（pHN306）在大豆渝豆一号上也能显著提高瘤数,癌重和总氮量,对植株地上部分干重表现出一定的促进作用。结果表明：nifA基因对固氮效率和结瘤能力的促进作用与受体根瘤菌和大豆品种等因素有关。以luxAB为报告基因进行的菌落和根瘤发光检测结果表明：pHN306可在供试根瘤菌中稳定遗传。     

费氏中华根瘤菌HN01DL在大豆根圈能定殖动态与结瘤研究

以发光酶基因luxAB为标记,在根盒缩影条件下研究了费氏中华根瘤菌HN01DL在大豆根圈的定殖动态、分布范围及其结瘤情况．结果表明,HN01DL在根盒．灭菌土壤和非灭菌土壤缩影中的大豆全根系定殖动态与水平明显不同,前者在第12d时达到最高定殖密度(8．65log cfu·g^-1),而后者的早期定殖数量下降较快,且于第15d时达到最高定殖密度(6．88log cfu·g^-1)．HN01DL在大豆播种5d后在大豆根部的A(0～4cm)区根段上达到最高定殖密度(7．05log cfu·g^-1),然后开始缓慢下降,至第19d时仍维持相对稳定,在第33d时又开始回升．至播种后第46d时HN01DL可散布至种子下方16cm处的根段部位．HN01DL在A区根段的定殖密度持续较高,所形成的发光根瘤总数(16．3个)及发光比例(68．8％)最高,且发光根瘤主要集中于该区段主根上．发光根瘤比例沿A-E区段逐渐下降,在E区段未检测到发光根瘤．     

消除共生质粒的费氏中华根瘤菌HND29SR在大豆根圈的定殖动态

比较研究了费氏中华根瘤菌（Sinorhizobium fredii)HN01(出发菌）、发光酶基因标记菌HNO1L（参照菌）、消除HN01共生质粒的菌株HND29SR在无菌砂培条件下的大豆根圈定殖动态。供试菌单独接种时：HN01、HN01L和HND29SR的定殖动态基本一致,其早期定殖密度下降较快,播种后第16天时HN01和HN01L分别达到较高的定殖水平6.49logcfu/g鲜根和6.78logcfu/g鲜根,然后维持相对稳定的定殖水平。但HND29SR的定殖密度持续下降到播种后第16天时才开始上升,至第35天时仍维持相对稳定的定殖密度6.94logcfu/g鲜根。等量混合接种时供试菌在根圈定殖群体中各自定殖密度在测定过程中基本相等。结果表明消除HN01的共生质粒对其在大豆根圈中定殖能力无显著影响。     

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。     

Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family

A repetitive element (IS986), previously isolated from Mycobacterium tuberculosis and shown to detect multiple restriction fragment-length polymorphisms (RFLPs), has been sequenced. It consists of a potential insertion sequence of 1358bp, with 30-bp inverted repeat ends. IS986 has four potentially significant open reading frames (ORFs): ORFa1, ORFa2 and ORFb on one strand and ORFc on the complementary strand. The sequences of the potential translated products identify IS986 as a member of the IS3 family, with an apparent frameshift between ORFa1 and ORFa2. IS986 has potential as a highly specific probe for detection and typing of M. tuberculosis, as well as for transposon mutagenesis of mycobacteria. The sequence of IS986 is virtually identical to that of another recently described element, IS6110 (Thierry et al., 1990).     

导入dctABD和nifA基因对费氏中华根瘤菌共生固氮的影响研究

以pTR102为载体构建重组质粒pHN307,其上克隆有来自昔蓿中华根瘤菌（Sinorthizobium meliloti）的四碳二羧酸转移酶基因dctABD、来自肺炎克氏杆菌(Klebsiella pneumoniae）的nifA基因和来自pDB30所含的发光酶基因lux-AB。经三亲本接合转移,将pHN307导入费氏中华根瘤菌（S.fredii)NH01、YC4和GR3,并考察了转移接合子中pHN307在传代培养和共生条件下的稳定性。与出发菌相比较的植物盆栽试验结果表明,在与大豆黑农33共生时,导入pHN307后的转移接合子均可显著提高结瘤植株的瘤重、地上部分干重和地上部分总氮量。在与大豆川早一号共生时,转移接合子HN01（pHN307）可显著提高结瘤植株的瘤数和瘤重;GR3（pHN307）可显著提高结瘤植株的瘤数、瘤重、地上部分干重和地上部分总氮量;导入pHN307的YC4却呈现出负作用。本研究表明,导入dctABD可提高固氮效率     

Phylogeny of fast-growing soybean-nodulating rhizobia support synonymy of Sinorhizobium and Rhizobium and assignment to Rhizobium fredii.

We determined the sequences for a 260-base segment amplified by the polymerase chain reaction (corresponding to positions 44 to 337 in the Escherichia coli 16S rRNA sequence) from seven strains of fast-growing soybean-nodulating rhizobia (including the type strains of Rhizobium fredii chemovar fredii, Rhizobium fredii chemovar siensis, Sinorhizobium fredii, and Sinorhizobium xinjiangensis) and broad-host-range Rhizobium sp. strain NGR 234. These sequences were compared with the corresponding previously published sequences of Rhizobium leguminosarum, Rhizobium meliloti, Agrobacterium tumefaciens, Azorhizobium caulinodans, and Bradyrhizobium japonicum. All of the sequences of the fast-growing soybean rhizobia, including strain NGR 234, were identical to the sequence of R. meliloti and similar to the sequence of R. leguminosarum. These results are discussed in relation to previous findings; we concluded that the fast-growing soybean-nodulating rhizobia belong in the genus Rhizobium and should be called Rhizobium fredii.     

应用发光酶基因对快生型大豆根瘤菌HN01结瘤作用进行检测

含发光酶基因luxAB的Tn5转座子自杀质粒pHNC3,在辅助质粒pRK2013的帮助下,转入快生型大豆根瘤菌HN01中小,pHNC3经自杀重组,其Tn5－luxAB转座插入HN01基因组中,从而赋予HN01以发光活性。挑取具有发光活性的HN01杂交单菌落,进行质粒快检和以luxAB为探针的分子杂交,选取Tn5-luxAB分别插入到HN01染色体上和不同质粒上的标记菌株,进行灭菌盆栽实验,并对一株Tn5-luxAB标记于染色体上的菌株HN01LC02进行了模拟大豆栽培条件下的有菌盆栽实验,包括对发光根瘤菌占瘤率的测定和发光根瘤在根系上分布情况的测定。     

The pha2 gene cluster involved in Na+ resistance and adaption to alkaline pH in Sinorhizobium fredii RT19 encodes a monovalent cation/proton antiporter

Sinorhizobium fredii RT19 can tolerate up to 0.6 M NaCl, whereas all its pha2-disrupted mutants, constructed by Tn5 mutagenesis, failed to grow in even the presence of 0.1 M NaCl. No growth difference was detected in pha2 mutants at a pH<7.5 in the presence or absence of K+, but growth reduction was observed in the presence of K+ when pH>7.5. The pha2 gene cluster was able to completely restore the growth of the pha2 mutants of S. fredii RT19 in 0.6 M NaCl. Measurement of monovalent cation intracellular content suggested that pha2 was involved in both Na+ (Li+) and K+ efflux. The pha2 mutants exhibited K+/H+, but no apparent Na+(Li+)/H+ antiporter activity in everted membrane vesicles. Taken together, these results indicated that the pha2 cluster of S. fredii RT19 encodes a monovalent cation/proton antiporter involved in resistance to Na+ and adaption to pH, which was very different from the pha1 cluster of Sinorhizobium meliloti, which encodes a K+/H+ antiporter.     

Sinorhizobium fredii HH103 mutants affected in capsular polysaccharide (KPS) are impaired for nodulation with soybean and Cajanus cajan

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharides (KPS) production, was isolated and sequenced. The organization of the S. fredii genes identified, rkpUAGHIJ and kpsF3, was identical to that described for S. meliloti 1021 but different from that of S. meliloti AK631. The long rkpA gene (7.5 kb) of S. fredii HH103 and S. meliloti 1021 appears as a fusion of six clustered AK631 genes, rkpABCDEF. S. fredii HH103-Rif(r) mutants affected in rkpH or rkpG were constructed. An exoA mutant unable to produce exopolysaccharide (EPS) and a double mutant exoA rkpH also were obtained. Glycine max (soybean) and Cajanus cajan (pigeon pea) plants inoculated with the rkpH, rkpG, and rkpH exoA derivatives of S. fredii HH103 showed reduced nodulation and severe symptoms of nitrogen starvation. The symbiotic capacity of the exoA mutant was not significantly altered. All these results indicate that KPS, but not EPS, is of crucial importance for the symbiotic capacity of S. fredii HH103-Rif(r). S. meliloti strains that produce only EPS or KPS are still effective with alfalfa. In S. fredii HH103, however, EPS and KPS are not equivalent, because mutants in rkp genes are symbiotically impaired regardless of whether or not EPS is produced.     

Identification of fast and slow growing rhizobia nodulating soybean (Glycine max [L.] Merr) by a multiplex PCR reaction

Two DNA fragments, a 730-bp and a 900-bp fragment, one homologous to host cultivar specificity genes nolBT of Sinorhizobium fredii and the other one homologous to RSalpha, an insertion-like sequence present in Bradyrhizobium japonicum, were generated by polymerase chain reaction (PCR) with two pairs of primers. The amount of each fragment generated by the multiplex PCR was proportional to the amount of template DNA present. The amplification of the 900-bp RSalpha fragment was more sensitive, since it was amplified from a smaller amount of template DNA than the 730-bp nolBT fragment. By running the multiplex reaction in the presence of template DNA isolated from different sources, we confirmed that the reaction can discriminate between S. fredii, Bradyrhizobium japonicum and Sinorhizobium xinjiangensis.     

Expression of a Serratia marcescens chitinase gene in Sinorhizobium fredii USDA191 and Sinorhizobium meliloti RCR2011 impedes soybean and alfalfa nodulation.

A gene encoding chitinase from Serratia marcescens BJL200 was cloned into a broad-host-range vector (pRK415) and mobilized into Sinorhizobium fredii USDA191. Chitinolytic activity was detected in S. fredii USDA191 transconjugants that carried the S. marcescens chiB gene. Chitinase-producing S. fredii USDA191 formed nodules on soybean cultivar McCall. However, there was a delay in nodule formation and a marked decrease in the total number of nodules formed by the chitinase-producing S. fredii in comparison with the wild-type strain. Expression of chitinase in S. meliloti RCR2011 also impeded alfalfa nodulation. Thin-layer chromatography of 14C-labeled Nod factors from chitinase-producing S. fredii USDA191 revealed hydrolysis of lipochitooligosaccharides.     

Reduction of adenosine-5'-phosphosulfate instead of 3'-phosphoadenosine-5'-phosphosulfate in cysteine biosynthesis by Rhizobium meliloti and other members of the family Rhizobiaceae.

We have cloned and sequenced three genes from Rhizobium meliloti (Sinorhizobium meliloti) that are involved in sulfate activation for cysteine biosynthesis. Two of the genes display homology to the Escherichia coli cysDN genes, which code for an ATP sulfurylase (EC 2.7.7.4). The third gene has homology to the E. coli cysH gene, a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase (EC 1.8.99.4), but has greater homology to a set of genes found in Arabidopsis thaliana that encode an adenosine-5'-phosphosulfate (APS) reductase. In order to determine the specificity of the R. meliloti reductase, the R. meliloti cysH homolog was histidine tagged and purified, and its specificity was assayed in vitro. Like the A. thaliana reductases, the histidine-tagged R. meliloti cysH gene product appears to favor APS over PAPS as a substrate, with a Km for APS of 3 to 4 microM but a Km for PAPS of >100 microM. In order to determine whether this preference for APS is unique to R. meliloti among members of the family Rhizobiaceae or is more widespread, cell extracts from R. leguminosarum, Rhizobium sp. strain NGR234, Rhizobium fredii (Sinorhizobium fredii), and Agrobacterium tumefaciens were assayed for APS or PAPS reductase activity. Cell extracts from all four species also preferentially reduce APS over PAPS.     

pH对土壤中土著快、慢生大豆根瘤菌结瘤的影响

1　引　　言土壤 ｐＨ对根瘤菌结瘤的影响一直是微生物学和微生物生态学研究的内容之一[4] .在对大豆根瘤菌的研究中 ,早期的研究主要集中于生长慢、产碱的大豆慢生根瘤菌 (Ｂｒａｄｙｒｈｉｚｏｂｉｕｍｊａｐｏｎｉｃｕｍ) [1,2 ] .1982年 ,Ｋｅｙｓｅｒ等[3] 报道了一类生长快、产酸的大豆根瘤菌 ,并命名为费氏中华根瘤菌 (Ｓｉｎｏｒｈｚｏｂｉｕｍ ｆｒｅｄｉ ｉ) .由于它们在生理特性方面存在着明显的差异 ,其结瘤能力以及环境的生物、物理和化学等因素对结瘤的影响一直受到广泛的重视 .本文研究了偏酸、偏碱的 ｐＨ对费氏中华根瘤菌…     

A functional myo-inositol dehydrogenase gene is required for efficient nitrogen fixation and competitiveness of Sinorhizobium fredii USDA191 to nodulate soybean (Glycine max [L.] Merr.)

Inositol derivative compounds provide a nutrient source for soil bacteria that possess the ability to degrade such compounds. Rhizobium strains that are capable of utilizing certain inositol derivatives are better colonizers of their host plants. We have cloned and determined the nucleotide sequence of the myo-inositol dehydrogenase gene (idhA) of Sinorhizobium fredii USDA191, the first enzyme responsible for inositol catabolism. The deduced IdhA protein has a molecular mass of 34,648 Da and shows significant sequence similarity with protein sequences of Sinorhizobium meliloti IdhA and MocA; Bacillus subtilis IolG, YrbE, and YucG; and Streptomyces griseus StrI. S. fredii USDA191 idhA mutants revealed no detectable myo-inositol dehydrogenase activity and failed to grow on myo-inositol as a sole carbon source. Northern blot analysis and idhA-lacZ fusion expression studies indicate that idhA is inducible by myo-inositol. S. fredii USDA191 idhA mutant was drastically affected in its ability to reduce nitrogen and revealed deteriorating bacteroids inside the nodules. The number of bacteria recovered from such nodules was about threefold lower than the number of bacteria isolated from nodules initiated by S. fredii USDA191. In addition, the idhA mutant was also severely affected in its ability to compete with the wild-type strain in nodulating soybean. Under competitive conditions, nodules induced on soybean roots were predominantly occupied by the parent strain, even when the idhA mutant was applied at a 10-fold numerical advantage. Thus, we conclude that a functional idhA gene is required for efficient nitrogen fixation and for competitive nodulation of soybeans by S. fredii USDA191.     

湖北潜江灰潮土中费氏中华根瘤菌(Sinorhizobium fredii)多样性的研究

用5种代表性大豆为捕集植物从湖北潜江灰潮土中经盆栽试验分离筛选出50株费氏中华根瘤菌,对它们的生长速度、耐盐性、pH生长范围、天然抗药性、碳氮源利用和共生效应等同时进行了比较研究,证实了同一土壤环境中费氏中华根瘤菌群体的多样性。     

提高大豆根瘤菌质粒稳定性的研究

以发光酶基因luxAB作为报告基因,将广谱稳定性质 粒pTR102的parCBA/DE基因导入含37kb增效片段的pLARF3并去除该质粒的cos序列,构建成重组质粒pHN115和pHN156。同时,构建只带有cos序列和luxAB的参比质粒pHN157和pHN158。将上述4种质粒通过三亲本杂交分别导入费氏中华根瘤菌(Sinorhizobiu m fredii) HN01,将pHN155和pHN158通过两亲本杂交分别导入大豆慢生根瘤菌(Bradyrhizobium japonicum)TA11,在人工继代培养条件下比较测定其质粒保持率。结果表明;经连续转接培养7次后,pHN155、pHN156、pHN157和pHN158在HN01中的质粒保持率分 别为100%、67%、72%和92% 。连续转接培养4次后,pHN155和pHN158在TA11中的质粒保持率分别为98%和92%。说明parCBA/DE基因能显著提高质粒在快、慢生型大豆根瘤菌中的遗传稳定性,cos序列的去除也有一定的作用。     

Sinorhizobium fredii isolates can be specifically identified by a 260 bp fragment from the nolXWBTUV locus

A pair of primers homologous to the nolXWBTUV locus generated a 260 bp fragment by PCR only in the presence of Sinorhizobium fredii template DNA of different quality. This resulted in a fast and accurate method for the identification of S. fredii either from pure DNA, whole bacterial cells or nodule extracts. By means of two PCR fragments, one specific for S. fredii (260-bp) and the other specific for Bradyrhizobium japonicum (RSalpha), we found that S. fredii strain SMH12 and B. japonicum E109 were equally efficient at developing nodules on soybean plants grown under controlled environmental conditions.     

Apparent incompatibility of plasmid pSfrYC4b of <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis> with two different plasmids in another strain

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.