巴西固氮螺菌 Yu62 draTG 基因启动子区域的核苷酸序列及其功能分析

对巴西固氮螺菌ｄｒａＴＧ上游区域进行了全序列分析,结果表明该区域除了编码部分ｎｉｆＨ基因外（ｎｉｆＨ与ｄｒａＴＧ转录方向相反）,不编码任何其它已知的基因。但在该区域发现了一些可能的调控序列,它们包括上游激活序列（ＵＡＳ）、下游启动子组份（ＤＰＥ）和富Ａ＋Ｔ区。这说明：ｎｉｆＨ与ｄｒａＴ间的区域可能主要起调控功能而非编码功能;ｄｒａ操纵元的启动子很可能是ＲｐｏＮ－依赖型。用ｐＡＦ３００做载体,构建了     

巴西固氮螺菌 Yu62 draTG 基因启动子区域的核苷酸序列及其功能分析

对巴西固氮螺菌ｄｒａＴＧ上游区域进行了全序列分析,结果表明该区域除了编码部分ｎｉｆＨ基因外（ｎｉｆＨ与ｄｒａＴＧ转录方向相反）,不编码任何其它已知的基因。但在该区域发现了一些可能的调控序列,它们包括上游激活序列（ＵＡＳ）、下游启动子组份（ＤＰＥ）和富Ａ＋Ｔ区。这说明：ｎｉｆＨ与ｄｒａＴ间的区域可能主要起调控功能而非编码功能;ｄｒａ操纵元（ｏｐｅｒｏｎ）的启动子很可能是ＲｐｏＮ－依赖型。用ｐＡＦ３００做载体,构建了ｄｒａＴ∷ｃａｍ转录融合质粒ｐＡＴ１,并通过检测Ｃｍｒ以检测ｄｒａＴ在大肠杆菌和巴西固氮螺菌中的表达,结果表明ｄｒａＴ在ＬＤ丰富培养基上,好氧条件下,只在巴西固氮螺菌中才表达。这说明,ｄｒａＴ的转录需要某种大肠杆菌中没有的因子,同时也表明ｄｒａＴＧ上游区域有启动子功能。利用启动子探针质粒载体ｐＣＢ１８２,构建了ｄｒａＴ∷ｌａｃＺ转录融合质粒ｐＣＴ１。在大肠杆菌中测定肺炎克氏杆菌ＮｉｆＡ对ｄｒａＴ∷ｌａｃＺ的转录激活作用。结果表明ｎｉｆＡ并不参与ｄｒａＴ的转录调控。     

巴西固氮螺菌Yu62 draTG基因及其下游区域的定位诱变分析*

用卡那霉素盒(Kmr-cassette)插入法,对巴西固氮螺菌(Azospirillum brasilense)Yu62的draTG基因及其下游区域进行了诱变,并获得相应的突变株。研究表明：draT变突株的固氨酶活性不再受铵抑制,而draG突变株在有铵时则丧失固氮酶活性,但当铵耗尽后却不能像野生型菌株那样恢复活性。draTG下游区域突变株YZ4(突变位点距draG约2kb)在无氮及限铵条件下,其固氮酶活性比野生型菌株的高,但其nifH-lacZ转录融合子的表达并不受影响,说明该区域可能有参与固氮酶活性水平调控的基因。     

固氮螺菌耐高铵突变株的生物学特性

本文报道9株固氮螺菌耐铵突变株,在培养基中有30—120m mol/L NH₄⁺存在时,具有强弱不等的固氮酶活性。而出发菌株在有微量NH_4~+存在时,固氮酶活性完全被抑制。耐铵突变株的其它特性与出发菌株相似。     

巴西固氮螺菌Yu62glnB基因的克隆及其功能分析

通过原位杂交从巴西固氮螺菌Yu62的基因文库中,筛选到glnB基因的阳性克隆,将3.7kb/EcoRI PstI的阳性克隆亚克隆到pUC19中,进行了全序列分析,其在GenBank中的登记号是AF323960。DNA序列分析表明该阳性克隆含有完整的glnB基因,glnB基因下游是编码谷氨酰胺合成酶（GS）的glnA基因,glnB基因上游是一个编码未知蛋白的ORF。glnB基因编码区长336bp,编码112个氨基酸,与肺炎克氏杆菌、大豆慢生根瘤菌、豌豆根瘤菌及大肠杆菌在氨基酸顺序的同源性分别高达71%、77%、79%和69%。将卡那霉素抗性片段（Km-cassette)插入glnB基因的BglII位点,通过三亲杂交法将其引入到巴西固氮螺菌Yu62中,通过同源重组,获得GlnB^-突变株（glnB::Km）。为进一步分析glnB基因的功能,将glnB基因的编码区（339bp）构建在pVK100中,置于Km启动子下组成型表达,形成重组质粒pVK-II。将重组质粒pVK-II转入到GlnB^-突变株,构建成互补株C-glnB(glnB:Km/glnB)。对GlnB^-突变株和互补株的固氮酶活性和生长性能的测定表明,GlnB^-突变体无固氮酶活性,即表型为Nif^-;而互补株像野生型菌株一样具有固氮酶活性。突变株、互补株及野生型在菌落生长速度上基本相同。将含有glnB基因的重组质粒pVK-II分别转移到野生型Yu62菌株和具有一定抗铵能力的DraT^-突变标中,使glnB基因的拷贝数增加,并进一步比较它们的固氮酶活性,结果表明多拷贝的glnB基因能显著提高固氮酶活性。     

巴西固氮螺菌中PⅡ和Pz在固氮调节中的不同使用

在巴西固氮螺菌（Azospirillum brasilense)中,glnB和glnZ是两个高度同源基因,分别位于3.7kgb/EcoRI PstI和3.7kb/SalI的两个不同的染色体片段上。用卡那霉素盒（Km^r-cas-sette)插入法,对glnB和glnZ分别进行定位诱变,并获得相应的突变株,即glnB^-和glnZ^-。研究表明,glnB^-突变株丧失固氮酶活性,表现为Nif^-,glnZ^-象野生型菌株一样具有固氮酶活性。为了进一步研究这两个基因的功能,将glnB和glnZ分别构建在pVK100载体上形成重组质粒pVK-Ⅱ和pVK-Z,对glnB^-和glnZ^-突变株进行互补实验,进一步证明了glnB与固氮酶活直接相关性,而glnZ无此作用。同时,通过三亲接合法将pVK-Ⅱ和pVK-Z分别转移到巴西固氮螺菌野生型Yu62和具有一定抗铵能力的draT^-突变株中,使glnB和glnZ的拷贝数增加,进一步比较它们的固氮酶活性。结果表明多拷贝的glnB基因,能显著提高固氮酶活性,而多拷贝的glnZ对固氮酶活性无影响。同时,将pVK-Ⅱ和pVK-Z分别转移到nifA^-突变株中,结果表明glnB和glnZ均不能恢复nifA^-的固氮酶活性。     

固氮螺菌耐高铵突变株的选育

应用亚硝基脏(N-nitrosoguanidine,NTG)诱变剂对固氮螺菌菌株Ma241、Ma99、Sp7和G14进行诱变处理后,在掭加了铵的类似物乙撑二胺(ethyleae diamina)的D6bcreiner无氯培养基中进行筛选．反复纯化,获得了在4 5 n、M NH}浓度以上,保持固氯酶活性的耐铵突变株共9株。突变株22的耐铵固氮酶活性最强,在75mM NH+4浓度下,固氮酶活性达到464n mol乙烯／mg蛋白·小时,在200m M NH+4浓度下,固氯酶话性仍有32nmol/mg蛋白·小时。     

巴西固氮螺菌中PⅡ和Pz在固氮调节中的不同作用

在巴西固氮螺菌(Azospirillum brasilense)中,glnB和glnZ是两个高度同源基因,分别位于3.7kb/EcoRI+PstI和3.7kb/SalI的两个不同的染色体片段上.用卡那霉素盒(Kmr-cas-sette)插入法,对glnB和glnZ分别进行定位诱变,并获得相应的突变株,即glnB-和glnZ-.研究表明,glnB-突变株丧失固氮酶活性,表现为Nif-,而glnZ-象野生型菌株一样具有固氮酶活性.为了进一步研究这两个基因的功能,将glnB和glnZ分别构建在pVK100载体上形成重组质粒pVK-Ⅱ和pVK-Z,对glnB-和glnZ突变株进行互补实验,进一步证明了glnB与固氮酶活有直接相关性,而glnZ无此作用.同时,通过三亲接合法将pVK-Ⅱ和pVK-Z分别转移到巴西固氮螺菌野生型Yu62和具有一定抗铵能力的draT-突变株中,使glnB和glnZ的拷贝数增加,进一步比较它们的固氮酶活性.结果表明多拷贝的glnB基因,能显著提高固氮酶活性,而多拷贝的glnZ对固氮酶活性无影响.同时,将pVK-Ⅱ和pVK-Z分别转移到nifA-突变株中,结果表明glnB和glnZ均不能恢复nifA-的固氮酶活性.     

腔孢纲真菌的三个新种

在调查香龙血树Ｄｒａｃａｅｎａ ｆｒａｇｒａｎｓ（Ｌ．）Ｋｅｒ－Ｇａｎｌ．的病害中,发现了腔孢纲真菌的３个新种,即：Ｐｈｌｍｏｐｓｉｓ ｄｒａｃａｅｎｉｃｏｌａＺ．Ｄ．Ｊｉａｎｇ,Ｐ．Ｇ．Ｘｉ ｅｔ Ｐ．Ｋ．Ｃｈｉ,Ｂａｒｔａｌｉｎｉａ ｄｒａｃａｅｎａｅ Ｐ．Ｇ．Ｘｉ,Ｚ．Ｄ．Ｊｉａｎｇ ｅｔ Ｐ．Ｋ．Ｃｈｉ及Ｓｐｈａｅｒｏｐｓｉｓ ｄｒａｃａｅｎａｅ Ｐ．Ｇ．Ｘｉ ｅｔ Ｐ．Ｋ．Ｃｈｉ,模式标     

联合固氮菌Enterobactergergoviae57—7泌铵突变株的分离和特性

经 Tn5转座子诱变从野生型菌株 ( Enterobacter gergoviae 5 7- 7)筛选到抗甲胺 ( 0 .3mol/L )的泌铵突变株 MG61 ,该突变株在固氮生长时能分泌铵 2 .0 mmol/L。由于泌铵 MG61的生长比野生型菌株慢 ,在培养基中含铵 ( 5 mmol/L或 30 mmol/L)条件下 ,MG61的生长速率与对照相同 ,而远比野生型菌株慢 ,表明 MG 61不能很好地利用铵。在含 2 0 mmol/L铵的培养基中 MG 61仍表达 86%的固氮活性 ,而野生型菌株完全丧失了固氮活性。在谷氨酸存在下 MG61的生长速率及固氮酶活性都比对照高。在硝酸盐存在下 MG61的生长速率与对照相同 ,但泌铵量达 7.8mmol/L。     

Cloning, sequencing, mutagenesis, and functional characterization of draT and draG genes from Azospirillum brasilense.

The Azospirillum brasilense draT gene, encoding dinitrogenase reductase ATP-ribosyltransferase, and draG gene, encoding dinitrogenase reductase activating glycohydrolase, were cloned and sequenced. Two genes were contiguous on the A. brasilense chromosome and showed extensive similarity to the same genes from Rhodospirillum rubrum. Analysis of mutations introduced into the dra region on the A. brasilense chromosome showed that mutants affected in draT were incapable of regulating nitrogenase activity in response to ammonium. In contrast, a mutant with an insertion in draG was still capable of ADP-ribosylating dinitrogenase reductase in response to ammonium but was no longer able to recover activity after ammonium depletion. Plasmid-borne draTG genes from A. brasilense were introduced into dra mutants of R. rubrum and restored these mutants to an apparently wild-type phenotype. It is particularly interesting that dra mutants of R. rubrum containing draTG of A. brasilense can respond to darkness and light, since A. brasilense is a nonphotosynthetic bacterium and its dra system does not normally possess that regulatory response. The nifH gene of A. brasilense, encoding dinitrogenase reductase (the substrate of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase), is located 1.9 kb from the start of draT and is divergently transcribed. Two insertion mutations in the region between draT and nifH showed no significant effect on nitrogenase activity or its regulation.     

Comparison studies of dinitrogenase reductase ADP-ribosyl transferase/dinitrogenase reductase activating glycohydrolase regulatory systems in Rhodospirillum rubrum and Azospirillum brasilense.

Reversible ADP ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase activating glycohydrolase (DRAG) regulatory system, has been characterized in both Rhodospirillum rubrum and Azospirillum brasilense. Although the general functions of DRAT and DRAG are very similar in these two organisms, there are a number of interesting differences, e.g., in the timing and extent of the regulatory response to different stimuli. In this work, the basis of these differences has been studied by the heterologous expression of either draTG or nifH from A. brasilense in R. rubrum mutants that lack these genes, as well as the expression of draTG from R. rubrum in an A. brasilense draTG mutant. In general, these hybrid strains respond to stimuli in a manner similar to that of the wild-type parent of the recipient strain rather than the wild-type source of the introduced genes. These results suggest that the differences seen in the regulatory response in these organisms are not primarily a result of different properties of DRAT, DRAG, or dinitrogenase reductase. Instead, the differences are likely the result of different signal pathways that regulate DRAG and DRAT activities in these two organisms. Our results also suggest that draT and draG are cotranscribed in A. brasilense.     

Cloning and expression of draTG genes from Azospirillum lipoferum

A genomic library of Azospirillum lipoferum was constructed with phage lambda EMBL4 as vector. From this library, the genes encoding dinitrogenase reductase ADP-ribosyltransferase (DRAT), draT, and dinitrogenase reductase-activating glycohydrolase (DRAG), draG, were cloned by hybridization with the heterologous probes of Rhodospirillum rubrum. As in R. rubrum, draT is located between draG and nifH, the gene encoding dinitrogenase reductase (a substrate for the DRAG/DRAT system). In the crude extract of Escherichia coli harboring the expression vector for this region, DRAT and DRAG enzyme activities were detected, confirming the identity of the cloned genes. Southern hybridization with genomic DNA from different Azospirillum spp., demonstrated a correlation between observable draTG hybridization and the biochemical demonstration of this covalent modification system.     

Posttranslational regulatory system for nitrogenase activity in Azospirillum spp.

The mechanism for "NH4+ switch-off/on" of nitrogenase activity in Azospirillum brasilense and A. lipoferum was investigated. A correlation was established between the in vivo regulation of nitrogenase activity by NH4Cl or glutamine and the reversible covalent modification of dinitrogenase reductase. Dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was detected in extracts of A. brasilense with NAD as the donor molecule. Dinitrogenase reductase-activating glycohydrolase (DRAG) activity was present in extracts of both A. brasilense and A. lipoferum. The DRAG activity in A. lipoferum was membrane associated, and it catalyzed the activation of inactive nitrogenase (by covalent modification of dinitrogenase reductase) from both A. lipoferum and Rhodospirillum rubrum. A region homologous to R. rubrum draT and draG was identified in the genomic DNA of A. brasilense as a 12-kilobase EcoRI fragment and in A. lipoferum as a 7-kilobase EcoRI fragment. It is concluded that a posttranslational regulatory system for nitrogenase activity is present in A. brasilense and A. lipoferum and that it operates via ADP-ribosylation of dinitrogenase reductase as it does in R. rubrum.     

肺炎克氏杆菌NifA对巴西固氮螺菌nifH启动子的转录激活作用

用PR方法克隆了巴西固氮螺菌Yu62 nifH的启动子片段,DNA序列分析表明菌株Yu62与标准菌株sp7之间的DNA序列差异很小。利用启动子探针质粒载体pcBl82,构建了3个不同的nifH：：lacz转录融合质粒,在大肠杆菌中分别测定肺炎克氏杆菌NifA对它们的转录激活作用。结果表明巴西固氮螺菌nifH启动子的转录是依赖于NifA的,缺失了上游激活序列的启动子不能被NifA激活转录,肺炎克氏杆菌NifA对其自身nifH及巴西固氮螺菌nifH启动子的转录激活作用并无很大差异。     

Posttranslational regulation of nitrogenase in Rhodospirillum rubrum strains overexpressing the regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase activating glycohydrolase.

Rhodospirillum rubrum strains that overexpress the enzymes involved in posttranslational nitrogenase regulation, dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG), were constructed, and the effect of this overexpression on in vivo DRAT and DRAG regulation was investigated. Broad-host-range plasmid constructs containing a fusion of the R. rubrum nifH promoter and translation initiation sequences to the second codon of draT, the first gene of the dra operon, were constructed. Overexpression plasmid constructs which overexpressed (i) only functional DRAT, (ii) only functional DRAG and presumably the putative downstream open reading frame (ORF)-encoded protein, or (iii) all three proteins were generated and introduced into wild-type R. rubrum. Overexpression of DRAT still allowed proper regulation of nitrogenase activity, with ADP-ribosylation of dinitrogenase reductase by DRAT occurring only upon dark or ammonium stimuli, suggesting that DRAT is still regulated upon overexpression. However, overexpression of DRAG and the downstream ORF altered nitrogenase regulation such that dinitrogenase reductase did not accumulate in the ADP-ribosylated form under inactivation conditions, suggesting that DRAG was constitutively active and that therefore DRAG regulation is altered upon overexpression. Proper DRAG regulation was observed in a strain overexpressing DRAT, DRAG, and the downstream ORF, suggesting that a proper balance of DRAT and DRAG levels is required for proper DRAG regulation.     

Regulation of nitrogenase in the photosynthetic bacterium Rhodobacter sphaeroides containing draTG and nifHDK genes from Rhodobacter capsulatus

The photosynthetic bacteria Rhodobacter capsulatus and Rhodospirillum rubrum regulate their nitrogenase activity by the reversible ADP-ribosylation of nitrogenase Fe-protein in response to ammonium addition or darkness. This regulation is mediated by two enzymes, dinitrogenase reductase ADP-ribosyl transferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG). Recently, we demonstrated that another photosynthetic bacterium, Rhodobacter sphaeroides, appears to have no draTG genes, and no evidence of Fe-protein ADP-ribosylation was found in this bacterium under a variety of growth and incubation conditions. Here we show that four different strains of Rba. sphaeroides are incapable of modifying Fe-protein, whereas four out of five Rba. capsulatus strains possess this ability. Introduction of Rba. capsulatus draTG and nifHDK (structural genes for nitrogenase proteins) into Rba. sphaeroides had no effect on in vivo nitrogenase activity and on nitrogenase switch-off by ammonium. However, transfer of draTG from Rba. capsulatus was sufficient to confer on Rba. sphaeroides the ability to reversibly modify the nitrogenase Fe-protein in response to either ammonium addition or darkness. These data suggest that Rba. sphaeroides, which lacks DRAT and DRAG, possesses all the elements necessary for the transduction of signals generated by ammonium or darkness to these proteins.