浑球红细菌（R.sphaeroides）glnBA基因克隆及其物理图谱

从浑球红细菌的基因文库中筛选到ｐＨＴ３、ｐＨＴ１０及ｐＨＴ３５三个阳性克隆,其中质粒ｐＨＴ１０与ｐＨＴ３５能遗传互补英膜红细菌的谷氨酰胺缺陷型Ｇ２９,使其ＧＳ酶及固氮酶活性得到恢复,对质粒ｐＨＴ１０上的ｇｌｎＡ同源片段进行了亚克隆和限制性内切酶图谱分析,确定了浑球红细菌ｇｌｎＢ与ｇｌｎＡ之间存在连锁关系。     

浑球红细菌谷氨酰胺合成酶基因（glnA）及PⅡ蛋白基因（glnB）的序…

测定了浑球红细菌的ｇｌｎＡ和ｇｌｎＢ基因ＤＮＡ序列,共２７０７ｎｔ。其中ｇｌｎＡ基因编码区为１４０１ｎｔ,编码４６７个氨基酸;ｇｌｎＢ基因编码区为３３６ｎｔ,编码１１２个氨基酸。ＤＮＡ序列的Ｇ＋Ｃ百分含量为６５％,它们的密码子第三位ＧＣ利用率高灰８９．１％。     

在鱼腥藻7120中建立反义glnA系统

应用反义技术对鱼腥藻７１２０的内源ｇｌｎＡ基因的表达进行调控,首次获得了人工反义系统的蓝藻品系。先从编码谷酰胺合成酶的基因ｇｌｎＡ中取得部分结构基因片段,与表达质粒载体ｐＲＬ－４３９及穿梭质粒载体ｐＤＣ－８相连接。通过酶切鉴定筛选出反向克隆的穿梭表达质粒ｐＤＣ－ＡＴＧＳ,然后应用三亲接合转移法把它鱼腥藻７１２０。     

p35Nck5a基因重复性打靶载体的构建和ES细胞基因打靶研究

为了在小鼠胚胎于细胞（ＥＳ）中引起神经细胞ｃｄｃ２类激酶调节亚基ｐ３５Ｎｃｋ５ａ基因的定点 重复,采用常规的分子克隆技术,构建得到长约１２．２ｋｂ的基因重复性打靶载体ｐＧＤＴＶ。用电 穿孔法将线性化的ｐＧＤＴＶ载体转入ＥＳ细胞,经过Ｇ４１８和ＧＡＮＣ分组药物选择,获得２４５个 双药物抗性的细胞克隆,细胞存活率为６．２２ × １０－５。经ＰＣＲ和基因组Ｓｏｕｔｈｅｒｎ杂交鉴定,２个 ＥＳ细胞克隆发生了ｐ３５Ｎｃｋ５ａ基因的重复,同源重组率为５．０８×１０－７、负向选择系统的应用使 同源重组事件的富集效率提高了７倍。为建立Ａｌｚｈｅｉｍｅｒ病的转基因小鼠模型打下了基础。     

浑球红假单胞菌在暗处发酵生长时的固氮酶、吸氢酶以及放氢机制的研究

光合细菌浑球红假单胞菌(Rhodopesudomonas sphaeroides)能在黑暗厌氧条件下生长。首次发现发酵生长的浑球红假单胞菌有固氮酶和吸氧酶的活性。试验比较了在光营养、黑暗厌氧呼吸和好氧呼吸生长方式下该菌的放氢作用,认为黑暗厌氧呼吸过程中的放氢是氢酶催化的放氢。     

人肿瘤坏死因子基因在变铅青链霉菌中的克隆与表达

以质粒ｐＩＪ４８６为载体,将来源于质粒ｐＨＴ１的肿瘤坏死坏因子（ＴＮＦ－ａ）ｃＤＮＡ克隆至变铅青链霉菌,以新霉素（３０μｇ／ｍｌ）为选择标记,获得了数百转化子,实验表明Ｎｏ．７转化子ｓ．ｌｉｖｉｄａｎｓ ＴＫ５４－ＨＴ所含重组质粒ｐＩＪＴ７已克隆有ＴＮＦｃＤＮＡ。Ｌ９２９细胞毒实验结果表明该转化子ＴＮＦ表达量可达１０＾８活性单位／升以上,中和实验确证其表达产物为人ＴＮＦ－ａ,ＳＤＳ－ＰＡＧＥ表明克     

浑球红细菌谷氨酰胺合成酶基因(glnA)及PⅡ蛋白基因(glnB)的序列分析

测定了浑球红细菌的glnA和／glnB基因DNA序列,共2707nt。其中glnA基因编码区为1401nt,编码467个氨基酸;glnB基因编码区为336nt,编码112个氨基醚。DNA序列的G＋C百分含量为65％,它们的密码子第三位GC利用率高达89．1％。在氨基酸序列上,GS酶和PⅡ蛋白与其它不同属的细菌间有较好的同源性,尤其是固氮类细菌间同源性较高。     

大肠杆菌苯丙氨酸生物合成关键酶的串联表达

ａｒｏＧ基因编码的 ３－脱氧－２－阿拉伯庚酮糖－７－磷酸合成酶（ＤＡＨＰ　Ｓｙｎｔｈｅｔａｓｅ　ＤＳ）和 ｐｈｅＡ基因编码的分支酸变位酶／预苯酸脱水酶（Ｃｈｏｒｉｍａｔｅ ｍｕｔａｓｅ／ Ｐｒｅｐｈｅｎａｔｅ　ｄｅｈｙｄｒａｔａｓｅ,ＣＷ／ＰＤ）都是本丙氨酸合成途径中的关键酶,为了通过基因工程手段来增加本丙氨酸生物的产量,在利用高效的原核表达载体ｐＢＶ２２ ０对ｐｈｅＡ基因编码的ＣＭ／ ＰＤ 酶进行了表达的基础上,采用ＰＣＲ方法扩增了抗反馈抑制的ａｒｃＧ基因,进行克隆表达,并与ｐｈｅＡ基因串联,以ＰＲＰＬ－ａｒｏＧ－ＰＬ－ｐｈｅＡ的形式,实现了２种酶基因在大肠杆菌中的表达, ＳＤＳＰＡＧＥ 图谱显示了新增的４３ｋｕ及３５ｋｕ蛋白带,经酶活性测定ＤＳ、ＣＭ／ＰＤ酶的比活分别提高了 ４．６７倍、８０５／１０．７１倍。     

粘虫核型多角体病毒凋亡抑制基因的定位,序列和启动子结构

以ＡｃＮＰＶ凋亡抑制基因ｐ３５为探针,与ＬｓＮＰＶＤＮＡ的限制性片段和ＬｓＮＰＶＤＮＡＥｃｏＲＶ片段杂交,发现ＥｃｏＲＶ５．５ｋｂ片段有强烈的杂交信号。将此片段亚克隆后,测定了１２４４ｂｐ序列,发现一个完整的ＯＲＦ,推导的３０２个氨基酸与ＡｃＮＰＶｐ３５蛋白有７０．４％的氨基酸同源性,证明所测ＯＲＦ为ＬｓＮＰＶ的ｐ３５基因。结构分析发现其５′端有早期基因启动子元件ＧＣ、ＡＣＧＴ和ＴＡＴＡｂｏｘ。有２２ｂｐ的顺向重复序列,包括由两个重叠的ＴＡＴＡｂｏｘ和上下游两个ＡＣＧＴｍｏｔｉｆ组成的两套启动子元件,这些结构特征与ＡｃＮＰＶ的凋亡抑制基因十分相似。     

表达浑球红细菌hemA基因生产5-氨基乙酰丙酸的研究

5-氨基乙酰丙酸（5-aminolevulinate acid,ALA）在农业,工业,医药业具有广泛的应用。ALA由5-氨基乙酰丙酸合酶（5-aminolevulinate acid synthase, ALAS）催化产生,其生物合成受终产物血红素的反馈抑制。本研究克隆一种浑球红细菌的hemA基因,序列分析其与已报道的基因具有96％的同源性,蛋白质编码区域也发生改变,并利用生物信息学软件进行同源关系的分析。采用大肠杆菌重组技术,构建表达载体pET28a—hemA,表达了有活性的浑球红细菌（Rhodobacter sphaeroides）的ALAS,研究了IPTG诱导和PH对研究ALAS的影响,同时分析了重组菌株合成ALA的能力,测定胞外产量。结果表明,在PH6．5,30mmol／L琥珀酸和60mmol／L甘氨酸培养条件下,胞外ALA的最大合成量达到669mg／L。     

Expression of the nif and ntr genes of Klebsiella pneumoniae in Rhodobacter sphaeroides cells

The genes glnA, ntr, nif or their promoters from Klebsiella pneumoniae cloned on the vectors, based on the plasmid RSF1010, were introduced into Rhodobacter sphaeroides cells. It was found that K. pneumoniae genes glnA, nifB, nifE, nifL and nifH are not expressed in R. sphaeroides. Neither was the glnA gene from cyanobacterium Anabaena 7120 expressed in R. sphaeroides. No functional activity of K. pneumoniae product of ntrA gene which is expressed from its own promoter, and the product of the gene nifA which is expressed from the constitutive promoter of the kanamycin resistance gene of the transposon Tn903, was detected. The implications of these findings are discussed.     

Use of the plasmid R89S replicon for constructing integration vectors

It has been shown that the plasmid R89S derivatives can be used as integrative vectors for bacteria in which the plasmid is unable to replicate autonomously. The chromosomal and plasmid fragments of phototrophic bacterium Rhodobacter sphaeroides have been cloned in plasmid pVZ365, a SmRKmR-derivative of R89S. The obtained recombinant plasmids were mobilized into R. sphaeroides cells by the I pcP-group conjugative plasmid R751. The frequencies of the SmR-transconjugants formation are 3.7.10(-5) to 5.6.10(-3) per recipient cell. The formation of the SmR-transconjugants has not been revealed in case of the plasmid pVZ365 mobilization. The recombinant molecules containing R. sphaeroides plasmid fragments have been shown to integrate into endogenous plasmids and form cointegrates with them.     

Expression and regulation of Bradyrhizobium japonicum and Xanthobacter flavus CO2 fixation genes in a photosynthetic bacterial host.

Calvin cycle carbon dioxide fixation genes encoded on DNA fragments from two nonphotosynthetic, chemolithoautotrophic bacteria, Bradyrhizobium japonicum and Xanthobacter flavus, were found to complement and support photosynthetic growth of a ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion mutant of the purple nonsulfur bacterium Rhodobacter sphaeroides. The regulation of RubisCO expression was analyzed in the complemented R. sphaeroides RubisCO deletion mutant. Distinct differences in the regulation of RubisCO synthesis were revealed when the complemented R. sphaeroides strains were cultured under photolithoautotrophic and photoheterotrophic growth conditions, e.g., a reversal in the normal pattern of RubisCO gene expression. These studies suggest that sequences and molecular signals which regulate the expression of diverse RubisCO genes may be probed by using the R. sphaeroides complementation system.     

fbc operon, encoding the Rieske Fe-S protein cytochrome b, and cytochrome c1 apoproteins previously described from Rhodopseudomonas sphaeroides, is from Rhodopseudomonas capsulata

Detailed comparison of the 'Rhodopseudomonas sphaeroides GA' strain used by Gabellini et al. (1985) with genuine R. sphaeroides and R. capsulata strains indicated that the previously reported fbc operon of R. sphaeroides (Gabellini and Sebald, 1986) encoding the structural genes for the Rieske Fe-S protein, cytochrome b and cytochrome c1 subunits of the ubiquinol:cytochrome c2 oxidoreductase, is not from R. sphaeroides, but is rather from a strain of R. capsulata. Consequently, the genuine bc1 genes from R. sphaeroides were cloned using corresponding R. capsulata genes as probes, and a partial nucleotide sequence for the Rieske Fe-S protein of R. sphaeroides was determined and compared with that of R. capsulata.     

Non-reciprocal regulation of Rhodobacter capsulatus and Rhodobacter sphaeroides recA genes expression

Abstract The Rhodobacter capsulatus recA gene has been isolated and sequenced. Its deduced amino acid sequence showed the closest identity with the Rhodobacter sphaeroides RecA protein (91% identity). However, the promoter regions of both R. capsulatus and R. sphaeroides recA genes are only 64% similar. An Escherichia coli -like LexA binding site was not present in the upstream region of the R. capsulatus recA gene. Nevertheless, the R. capsulatus recA gene is inducible by DNA damage in both hetero- and phototrophically growing conditions. The R. capsulatus recA gene is poorly induced when inserted into the chromosome of R. sphaeroides , indicating that the recA gene of both bacteria possess different control sequences despite their phylogenetically close relationship.     

Identification and molecular genetic analysis of multiple loci contributing to high-level tellurite resistance in Rhodobacter sphaeroides 2.4.1.

The ability of the facultative photoheterotroph Rhodobacter sphaeroides to tolerate and reduce high levels of tellurite in addition to at least 10 other rare earth metal oxides and oxyanions has considerable potential for detoxification and bioremediation of contaminated environments. We report the identification and characterization of two loci involved in high-level tellurite resistance. The first locus contains four genes, two of which, trgAB, confer increased tellurite resistance when introduced into the related bacterium Paracoccus denitrificans. The trgAB-derived products display no significant homology to known proteins, but both are likely to be membrane-associated proteins. Immediately downstream of trgB, the cysK (cysteine synthase) and orf323 genes were identified. Disruption of the cysK gene resulted in decreased tellurite resistance in R. sphaeroides, confirming earlier observations on the importance of cysteine metabolism for high-level tellurite resistance. The second locus identified is represented by the telA gene, which is separated from trgAB by 115 kb. The telA gene product is 65% similar to the product of the klaB (telA) gene from the tellurite-resistance-encoding kilA operon from plasmid RK2. The genes immediately linked to the R. sphaeroides telA gene have no similarity to other components of the kilA operon. R. sphaeroides telA could not functionally substitute for the plasmid RK2 telA gene, indicating substantial functional divergence between the two gene products. However, inactivation of R. sphaeroides telA resulted in a significant decrease in tellurite resistance compared to the wild-type strain. Both cysK and telA null mutations readily gave rise to suppressors, suggesting that the phenomenon of high-level tellurite resistance in R. sphaeroides is complex and other, as yet uncharacterized, loci may be involved.     

"Green-like" and "red-like" RubisCO cbbL genes in Rhodobacter azotoformans

We found that Rhodobacter azotoformans IFO 16436T contains two different cbbL genes coding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunits. One gene is located within a "green-like" group of the RubisCO phylogenetic tree, and the other is located within a "red-like" group. This is the first report that one organism contains both green-like and red-like RubisCO genes. Moreover, by PCR using primers which amplify two green-like and red-like cbbL genes alternatively and dot blot hybridization, we demonstrated that Rhodobacter blasticus, Rhodobacter capsulatus, and Rhodobacter veldkampii possess only green-like cbbL genes, and Rhodobacter sphaeroides possesses only a red-like cbbL gene. In the cbbL phylogenic analysis, R. spaeroides and R. azotoformans 1 (red-like) formed a cluster within the red-like group, and R. capsulatus, R. azotoformans 2 (green-like), R. blasticus, and R. veldkampii formed a cluster within the green-like group. This suggests that red-like cbbL genes of Rhodobacter species were derived from one ancestor, and green-like cbbL genes were derived from another ancestor. On the other hand, molecular phylogeny of the bacteria indicates that R. veldkampii, which has only a green-like cbbL gene, is the earliest evolved Rhodobacter species and that R. azotoformans and R. sphaeroides, which have red-like cbbL genes, are the latest evolved. Consequently, the following hypothesis is proposed: the common ancestor of Rhodobacter had a green-like cbbL gene, the common ancestor of R. azotoformans and R. sphaeroides subsequently obtained a red-like cbbL gene by a horizontal gene transfer, and the ancestor of R. sphaeroides later lost the green-like cbbL gene.     

Cloning of poly(3-hydroxybutyric acid) synthase genes of Rhodobacter sphaeroides and Rhodospirillum rubrum and heterologous expression in Alcaligenes eutrophus

From genomic libraries of the purple non-sulfur bacteria Rhodospirillum rubrum Ha and Rhodobacter sphaeroides ATCC 17023 in the broad-host range cosmid pVK100, we cloned a 15- and a 14-kbp HindIII restriction fragment, respectively. Each of these fragments restored the ability to accumulate poly(3-hydroxybutyrate) (PHB), in the PHB-negative mutant Alcaligenes eutrophus PHB-4. These hybrid cosmids also complemented PHB-negative mutants derived from wild-type R. rubrum or R. sphaeroides. Both fragments hybridized with the PHB synthase structural gene of A. eutrophus H16 and conferred the ability to express PHB synthase activity. Only the 15-kbp HindIII fragment from R. rubrum conferred on the mutant PHB-4 the ability to form large PHB granules (length up to 3.5 microns).     

Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. The coxII/coxIII operon codes for structural and assembly proteins homologous to those in yeast.

The coxII/coxIII operon of Rhodobacter sphaeroides cytochrome c oxidase has been sequenced and characterized by insertional inactivation/complementation analysis. The organization of the genes in this locus (coxII.orf1.orf3.coxIII) is the same as that of the equivalent operon of Paracoccus denitrificans (ctaC.ctaB.ctaG.ctaE), but unlike that of other bacteria whose cytochrome oxidase genes have been characterized so far. The predicted amino acid sequence homology with eukaryotic oxidases is also higher for Rb. sphaeroides (and P. denitrificans) than for other bacterial versions of the enzyme. The inactivation of coxII results in loss of the characteristic cytochrome oxidase spectrum from membranes of the mutant strain. Full recovery requires introduction into the bacterium of the complete operon containing coxII.orf1.orf3.coxIII; partial complementation yielding a spectrally altered enzyme is achieved with a plasmid containing coxII or coxII.orf1.orf3. These results indicate that the peptides ORF1, ORF3, and COXIII are all required for assembly of native cytochrome c oxidase, suggesting an oxidase-specific assembly or chaperonin function for the ORFs in Rb. sphaeroides similar to that observed for the homologous gene products in yeast, COX10 and COX11.