苜蓿中华根瘤菌042BMnoeA基因的克隆、敲除与功能的初步分析

通过PCR扩增获得了 0 4 2BM的noeA基因。该基因与苜蓿中华根瘤菌 (Sinorhizobiummeliloti) 10 2 1noeA的同源性为 99% ,而其NoeA与 10 2 1NoeA的相似性为 97%。还发现其NoeA与中慢生根瘤菌 (Mesorhizobiumsp .)BNC1可能的SAM_依赖性的甲基转移酶相似性为 32 % ,而其 30 3～ 36 2氨基酸区域与大肠杆菌 (Escherichiacoli)的核糖体 5 0S亚基的L11蛋白甲基转移酶 (PrmA)的 16 0～ 2 2 0氨基酸区域的相似性达到 4 1%。通过插入卡那盒 ,敲除noeA ,获得突变株 0 4 2BMA_Km。与苜蓿中华根瘤菌 0 4 2BM相比 ,敲除noeA的突变株在普通紫花、保定、宁夏、百发和傲汉苜蓿品种上的结瘤数、根瘤鲜重和植株地上部分的干重都有不同程度的增加 ,而在秘鲁苜蓿品种上的结瘤数和植株地上部分的干重明显下降 ,在皇后和美国杂花苜蓿品种上则没有明显的变化。     

苜蓿中华根瘤菌042BMnoeA基因的克隆、敲除与功能的初步分析

通过PCR扩增获得了042BM的noeA基因。该基因与苜蓿中华根瘤菌(Sinorhizobium meliloti)1021noeA的同源性为99%,而其NoeA与1021NoeA的相似性为97%。还发现其NoeA与中慢生根瘤菌（Mesorhizobium sp.）BNC1可能的SAM_依赖性的甲基转移酶相似性为32%,而其303～362氨基酸区域与大肠杆菌（Escherichia coli）的核糖体50S亚基的L11蛋白甲基转移酶(PrmA)的160～220氨基酸区域的相似性达到41%。通过插入卡那盒,敲除noeA,获得突变株042BMA_Km。与苜蓿中华根瘤菌042BM相比,敲除noeA的突变株在普通紫花、保定、宁夏、百发和傲汉苜蓿品种上的结瘤数、根瘤鲜重和植株地上部分的干重都有不同程度的增加,而在秘鲁苜蓿品种上的结瘤数和植株地上部分的干重明显下降,在皇后和美国杂花苜蓿品种上则没有明显的变化。     

苜蓿中华根瘤菌042BM noeAB基因的转录调控

对苜蓿中华根瘤菌(Sinorhizobiummeliloti) 0 4 2BMnoeAB基因的表达调控进行研究。结果发现,葫芦巴碱不能使noeAB的表达水平提高,证明它们的转录不受nodD2的调控。当nodD3和syrM同时存在时,noeAB的表达水平没有明显的变化,表明它们也不受nodD3 syrM系统的调控。在FY基本培养基上,毛地黄黄酮的诱导使noeAB基因的表达水平提高16倍,而在不添加该诱导物的TY培养基上,noeAB基因的表达水平也能够提高30倍以上,说明noeAB是受nodD1控制的,但除受毛地黄黄酮诱导外,noeAB还可能受到其他因子的调节     

苜蓿中华根瘤菌042B nodD基因的克隆、序列分析及其表达

苜蓿中华根瘤菌０４２Ｂ是一株能在苜蓿和大豆上结瘤的菌株。将０４２Ｂ的ｎｏｄＳＤ基因克隆到时载体ｐＢＢＲ１ＭＣＳ－５,并在豌豆根瘤菌ＬＲＲ５０４５系统中进行功能分析,发现０４２Ｂ的ＮｏｄＤ蛋白能与大豆的类黄酮化合物ｇｅｎｉｓｔｅｉｎ结合,也怀苜蓿原类黄酮化合物ｌｕｔｅｏｌｉｎ反应。     

苜蓿中华根瘤菌042B共同结瘤基因nodABC的克隆与序列分析

根瘤菌的ｎｏｄＡＢＣ和ｎｏｄＤ在结构和功能上保守,在不同的菌种之间能够互换［１］,是目前所有供试的豆科植物结瘤所必不可少的,称为共同结瘤基因。另一类是寄生专一性结瘤基因,如苜蓿中华根瘤菌（Ｓｉｎｏｒｈｉｚｏｂｉｕｍｍｅｌｉｌｏｔｉ）的ｎｏｄＰＱ等［２...     

苜蓿中华根瘤菌042BMnoeAB基因的转录调控

对苜蓿中华根瘤菌（Sinorhizobium meliloti）042BM noeAB基因的表达调控进行研究。结果发现,葫芦巴碱不能使noeAB的表达水平提高,证明它们的转录不受nodD2的调控。当nodD3和syrM同时存在时,noeAB的表达水平没有明显的变化,表明它们也不受nodD3syrM系统的调控。在FY基本培养基上,毛地黄黄酮的诱导使noeAB基因的表达水平提高16倍,而在不添加该诱导物的TY培养基上,noeAB基因的表达水平也能够提高30倍以上,说明noeAB是受nodD1控制的,但除受毛地黄黄酮诱导外,noeAB还可能受到其他因子的调节。     

冻融法转化苜蓿中华根瘤菌

对于苜蓿中华根瘤菌转化方法的研究仅限于三亲杂交法和电激转化,本实验首次将冻融法用于转化苜蓿中华根瘤菌。将携带CUS片段并且含有壮观霉素抗性的质粒用冻融法转移至野生型苜蓿中华根瘤菌菌株,得到的抗性克隆用GUS引物进行PCR扩增鉴定。经过计算,冻融法的转化率为2．2×10^5／μg。与传统方法比较,冻融法操作简单,转化过程快速,转化率较高,所需时间较短,转化成本低,是适合用于苜蓿中华根瘤菌的转化方法。     

苜蓿中华根瘤菌与耐盐有关的DNA片段的克隆

以耐盐的苜蓿中华根瘤菌(\%Sinorhizobium meliloti) \%042B为材料,制备其总DNA,经过限制性内切酶\%Eco\%RⅠ的部分酶解,利用电洗脱方法回收15～25kb大小的DNA片段。以碱法制备载体质粒pLAFRⅠ,用\%Eco\%RⅠ将其切成线状,然后用T\-4DNA连接酶将回收片段与线状载体连接,利用包装蛋白进行包装后,感染大肠杆菌(Escherichia coli)S17\|1,构建了042B的基因文库。以固体亚硝基胍作为诱变剂处理出发菌株,在05mol/LNaCl的条件下,从2000个菌落中筛选得到12株042B的盐敏感突变株,以其中稳定的盐敏突变株GZ17为受体菌,利用两亲本杂交将含有042B的DNA片段的pLAFRⅠ重组质粒转移到GZ17中,在含有四环素和05mol/LNaCl的基本培养基上筛选出能够耐盐的阳性克隆,获得了与耐盐有关的7kb长的DNA片段。对该片段进行亚克隆,最终获得了4kb与耐盐有关的片段。     

苜蓿中华根瘤菌fabA和fabB基因功能的鉴定

在大肠杆菌(Escherichia coli)脂肪酸合成酶体系中,fabA基因编码有双功能的3-羟基脂酰ACP脱水异构酶,其异构产物能被fabB基因编码的3-酮基脂酰ACP合成酶Ⅰ延伸,合成不饱和脂肪酸,该FabA-FabB途径被认为是缺氧条件下不饱和脂肪酸合成的经典途径．生物信息学分析发现,苜蓿中华根瘤菌(Sinorhizobium meliloti)的SmFabA与EcFabA相似性达到60.6%,具有相同的保守活性位点和两个保守的α螺旋结构;SmFabB与EcFabB相似性达到61.1%,具有相同的Cys-His-His活性中心．用携带SmfabA和SmfabB的质粒载体遗传互补大肠杆菌温度敏感突变株CY57和CY242,在添加三氯森(TCL)抑制烯脂酰ACP还原酶活性的条件下,转化子能在42℃恢复生长,且放射性薄层层析能检测到转化子中不饱和脂肪酸棕榈油酸(Δ9C16:1)和十八碳烯酸(Δ11C18:1)的合成．体外重建脂肪酸合成反应表明,SmFabA能催化羟脂酰ACP的脱水反应且能够使反-2-癸烯酰ACP异构化,SmFabB能催化不同链长的脂酰ACP和丙二酸单酰ACP的聚合反应．另外,未得到SmFabA和SmFabB的突变株,表明SmFabA和SmFabB可能是苜蓿中华根瘤菌脂肪酸合成酶系中必不可少的关键蛋白．上述结果证实了苜蓿中华根瘤菌fabA和fabB两个基因在不饱和脂肪酸合成中的功能．     

苜蓿中华根瘤菌与耐盐有关基因rstA在大肠杆菌中的高效表达及其产物的纯化

苜蓿中华根瘤菌(Sinorhizobiummeliloti) 0 4 2BM与耐盐有关的1 9kbDNA片段含有两个开放阅读框,采用PCR方法分别将它们扩增,连接到穿梭质粒上,并进行了耐盐功能检测,证明其中的ORF2具有耐盐性,定名为rstA基因。将它分别克隆到表达载体pThio HisA、B和C上,构建成重组质粒pGSA、pGSB和pGSC ,转化大肠杆菌(Escherichiacoli)Top10后,经IPTG诱导,pGSA获得高效表达。表达蛋白占菌体总蛋白的36 % ,但大多数以包涵体形式存在。对表达产物依次进行ProBondTM树脂亲和纯化、饱和硫酸铵盐析,最后得到纯度为95 %的融合蛋白。SDS PAGE显示纯化的蛋白质为分子量4 3kD的单一蛋白带,经Westernblot检测证实了表达结果     

Functional Analysis of Sinorhizobium meliloti Genes Involved in Biotin Synthesis and Transport

External biotin greatly stimulates bacterial growth and alfalfa root colonization by Sinorhizobium meliloti strain 1021. Several genes involved in responses to plant-derived biotin have been identified in this bacterium, but no genes required for biotin transport are known, and not all loci required for biotin synthesis have been assigned. Searches of the S. meliloti genome database in combination with complementation tests of Escherichia coli biotin auxotrophs indicate that biotin synthesis probably is limited in S. meliloti 1021 by the poor functioning or complete absence of several key genes. Although several open reading frames with significant similarities to genes required for synthesis of biotin in gram-positive and gram-negative bacteria were found, only bioB, bioF, and bioH were demonstrably functional in complementation tests with known E. coli mutants. No sequence or complementation evidence was found for bioA, bioC, bioD, or bioZ. In contrast to other microorganisms, the S. meliloti bioB and bioF genes are not localized in a biotin synthesis operon, but bioB is cotranscribed with two genes coding for ABC transporter-like proteins, designated here bioM and bioN. Mutations in bioM and bioN eliminated growth on alfalfa roots and reduced bacterial capacity to maintain normal intracellular levels of biotin. Taken together, these data suggest that S. meliloti normally grows on exogenous biotin using bioM and bioN to conserve biotin assimilated from external sources.     

Functional analysis of nine putative chemoreceptor proteins in Sinorhizobium meliloti

The genome of the symbiotic soil bacterium Sinorhizobium meliloti contains eight genes coding for methyl-accepting chemotaxis proteins (MCPs) McpS to McpZ and one gene coding for a transducer-like protein, IcpA. Seven of the MCPs are localized in the cytoplasmic membrane via two membrane-spanning regions, whereas McpY and IcpA lack such hydrophobic regions. The periplasmic regions of McpU, McpV, and McpX contain the small-ligand-binding domain Cache. In addition, McpU possesses the ligand-binding domain TarH. By probing gene expression with lacZ fusions, we have identified mcpU and mcpX as being highly expressed. Deletion of any one of the receptor genes caused impairments in the chemotactic response toward most organic acids, amino acids, and sugars in a swarm plate assay. The data imply that chemoreceptor proteins in S. meliloti can sense more than one class of carbon source and suggest that many or all receptors work as an ensemble. Tactic responses were virtually eliminated for a strain lacking all nine receptor genes. Capillary assays revealed three important sensors for the strong attractant proline: McpU, McpX, and McpY. Receptor deletions variously affected free-swimming speed and attractant-induced chemokinesis. Noticeably, cells lacking mcpU were swimming 9% slower than the wild-type control. We infer that McpU inhibits the kinase activity of CheA in the absence of an attractant. Cells lacking one of the two soluble receptors were impaired in chemokinetic proficiency by more than 50%. We propose that the internal sensors, IcpA and the PAS domain containing McpY, monitor the metabolic state of S. meliloti.     

Functional and topological analysis of phosphatidylcholine synthase from Sinorhizobium meliloti

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of eubacteria. It can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation pathway or the phosphatidylcholine synthase (Pcs) pathway. Pcs belongs to the CDP-alcohol phosphotransferase superfamily and synthesizes PC and CMP in one step from CDP-diacylglycerol and choline. In this study, we aligned sequences of characterized Pcs enzymes to identify conserved amino acid residues. Alanine scanning mutagenesis was performed on 55 of these conserved residues. The mutation of nine residues caused a drastic to complete loss (<20% of wild type activity) of Pcs activity. Six of these essential residues were subjected to further mutagenesis studies replacing them by amino acids with similar properties or size. A topological analysis of sinorhizobial Pcs showed the presence of eight transmembrane helices, with the C- and N-terminus located in the cytoplasm. The majority of the conserved residues is predicted to be either located within the cytoplasmic loops or on the cytoplasmic side of the membrane which can be expected for an enzyme using one membrane-associated and one soluble substrate.     

Functional genomic analysis of global regulator NolR in Sinorhizobium meliloti

NolR is a regulator of nodulation genes present in species belonging to the genera Rhizobium and Sinorhizobium. The expression of the nolR gene in Sinorhizobium meliloti AK631 was investigated in relation to stage of growth, availability of nutrients, and different environmental stimuli using the nolR::lacZ fusion report system. It has been shown that the nolR gene is regulated in a population-density-dependent fashion and influenced by a number of environmental stimuli, including nutrients, pH, and oxygen. Exploration of the physiological functions of NolR under various laboratory conditions has shown that NolR is required for the optimal growth of the bacteria on solid media, optimal survival of the bacteria in carbon-starved minimal medium, and after heat shock challenge. NolR also is involved in recipient-induced conjugative transfer of a plasmid. Proteome analysis of strain AK631 and its Tn5-induced nolR-deficient mutant EK698 revealed that a functional NolR induced significant differences in the accumulation of 20 polypeptides in peptide mass fingerprinting early-log-phase cultures and 48 polypeptides in stationary-phase cultures. NolR acted mainly as a repressor in the early-log-phase cultures, whereas it acted as both repressor and activator in the stationary-phase cultures. The NolR protein and 59 NolR-associated proteins have been identified by peptide mass fingerprinting. The NolR protein was differentially expressed only in the NolR+ wild-type strain AK631 but not in its NolR- derivative EK698, confirming that no functional NolR was produced in the mutant. The NolR-associated proteins have diverse functions in amino acid metabolism, carbohydrate metabolism, lipid metabolism, nucleotide metabolism, energy metabolism, metabolism of Co-factors, and cellular adaptation and transportation. These results further support our previous proposal that the NolR is a global regulatory protein which is required for the optimization of nodulation, bacterial growth and survival, and conjugative transfer of a plasmid.     

Functional analysis of Sinorhizobium meliloti genes involved in biotin synthesis and transport

External biotin greatly stimulates bacterial growth and alfalfa root colonization by Sinorhizobium meliloti strain 1021. Several genes involved in responses to plant-derived biotin have been identified in this bacterium, but no genes required for biotin transport are known, and not all loci required for biotin synthesis have been assigned. Searches of the S. meliloti genome database in combination with complementation tests of Escherichia coli biotin auxotrophs indicate that biotin synthesis probably is limited in S. meliloti 1021 by the poor functioning or complete absence of several key genes. Although several open reading frames with significant similarities to genes required for synthesis of biotin in gram-positive and gram-negative bacteria were found, only bioB, bioF, and bioH were demonstrably functional in complementation tests with known E. coli mutants. No sequence or complementation evidence was found for bioA, bioC, bioD, or bioZ. In contrast to other microorganisms, the S. meliloti bioB and bioF genes are not localized in a biotin synthesis operon, but bioB is cotranscribed with two genes coding for ABC transporter-like proteins, designated here bioM and bioN. Mutations in bioM and bioN eliminated growth on alfalfa roots and reduced bacterial capacity to maintain normal intracellular levels of biotin. Taken together, these data suggest that S. meliloti normally grows on exogenous biotin using bioM and bioN to conserve biotin assimilated from external sources.     

苜蓿中华根瘤菌nifA基因突变影响多种细胞学过程

苜蓿中华根瘤菌nifA基因在共生固氮过程中担负着调控功能,nifA突变株Rm1354在宿主植物的根部诱导白色无效根瘤。本文报道Rm1354在自生状态下的表型变化。nifA的突变导致根瘤菌在半固体培养基上泳动变慢,胞外蛋白含量降低。有趣的是,Rm1354在延宕期间高丝氨酸内酯含量比野生型低,在指数期和静止期却比野生型高。另外,突变株Rm1354的竞争结瘤能力也大大减弱。这些结果揭示了苜蓿中华根瘤菌nifA基因对许多细胞学过程都有调控作用。     

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。