Analysis of the replication region of a mycobacterial plasmid, pMSC262.

We determined the nucleotide sequence of a DNA fragment which contains the replication region of pMSC262, a Mycobacterium scrofulaceum plasmid used to construct the Mycobacterium-Escherichia coli shuttle vector. The complete sequence of the fragment contained 2,504 bp with an overall G+C content of 69.8%. By deletion analysis, we found that the minimum length required for plasmid replication in M. bovis BCG was about 1.6 kb. Within this region, several open reading frames (ORFs) and a putative replication origin (ori) were identified by computer analysis. One of the ORFs, ORF2, which encodes a putative 28.9-kDa basic protein with characteristics of DNA-binding proteins, appeared to be involved in replication of the plasmid in BCG. By separation of ORF2 and the putative ori region, it was revealed that the relative locations of ORF2 and the putative ori region are likely important for replication in BCG. No DNA or amino acid homologies were found between this replication region and that of pAL5000, another mycobacterial plasmid used for vector plasmid construction. In addition, we found that this replicon did not lead to replication in E. coli and was compatible in BCG with pAL5000-derived vector plasmid pYUB75 (R. G. Barletta, D. D. Kim, S. B. Snapper, B. R. Bloom, and W. R. Jacobs, J., J. Gen. Microbiol. 138:23-30, 1992).     

Futile attempts to differentiate provide molecular evidence for individual differences within a population of cells during cellular reprogramming

A cryptic plasmid from Arthrobacter rhombi PRH1, designated as pPRH, was sequenced and characterized. It was 5000 bp in length with a G+C content of 66 mol%. The plasmid pPRH was predicted to encode six putative open reading frames (ORFs), in which ORF2 and ORF3 formed the minimal replicon of plasmid pPRH and shared 55-61% and 60-69% homology, respectively, with the RepA and RepB proteins of reported rhodococcal plasmids. Sequence analysis revealed a typical ColE2-type ori located 45 bp upstream of the gene repA. Sequence and phylogenetic analysis led to the conclusion that pPRH is a representative of a novel group of pAL5000 subfamily of ColE2 family plasmids. Three shuttle vectors pRMU824, pRMU824Km and pRMU824Tc, encoding chloramphenicol resistance, were constructed. The latter two harboured additional antibiotic resistance genes kan and tet, respectively. All vectors successfully replicated in Escherichia coli, Arthrobacter and Rhodococcus spp. The vector pRMU824Km was employed for functional screening of 2-hydroxypyridine catabolism encoding genes from Arthrobacter sp. PY22. Sequence analysis of the cloned 6-kb DNA fragment revealed eight putative ORFs, among which hpyB gene encoded a putative monooxygenase.     

抗辐射茵(Deinococcus radiopugnance)的质粒pUE30的克隆及序列分析

抗辐射菌(Deinococcus radiopugnance)ATCC 19172株中存在约14.6 kb、8.7 kb、7.0 kb、3.65 kb、及2.45 kb等5种以上的隐秘性质粒,对其中的约2.45 kb的小型质粒pUE30进行了序列测定与分析,该质粒由2 467 bp的碱基对组成,其中包含了267 nt及1 068 nt的2个开放阅读框架(open reading frame,ORF)和一个AT-rich领域。经过与GenBank的数据库分析,其中267 nt的ORF(repC)与豆科根瘤菌(Rhizobium leguminosa-rum)及根癌农杆菌(Agrobacterium tumefaciens)由来的质粒的RepC蛋白质具有一定的同源性;1 068 nt的ORF(repD)与抗辐射菌(Deinococcus radiodurans)Sark株的质粒pUE10的RepU蛋白质、嗜热菌(Thermus sp.)ATCC27737株由来的质粒pMY1的RepA蛋白质具有较高的同源性。研究结果对于利用该小型质粒构建大肠杆菌-抗辐射菌属间的穿梭载体,表明抗辐射菌高效正确的DNA损伤修复机理等具有重要的意义。     

Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a "mini" mycobacterium-Escherichia coli shuttle vector

Functional domains of pAL5000 were determined by gene disruption and deletion analysis. Of the five plasmid open reading frames (ORFs), ORF1 to ORF5, and a putative origin of replication previously identified (J. Rauzier, J. Moniz-Pereira, and B. Gicquel-Sanzey, Gene 71:315-321), two of the ORFs (ORF3 and ORF4) were deemed dispensable for plasmid replication. A "mini" mycobacterium-Escherichia coli shuttle plasmid applicable for general recombinant DNA studies in mycobacteria was constructed by using the gene for Kanr (Tn903) as a selective marker. Heterologous expression of the gene for Kanr was confirmed by Western blotting (immunoblotting) analysis.     

圈卷产色链霉菌尼可霉素生物合成基因：—sanJ的克隆及功能研究

以尼可霉素生物合成相关的基因片段为探针,从圈卷产色链霉菌cosmid基因文库中筛选到１个大约７．５kb的ＤＮＡ片段,交ＤＮＡ片段克隆到载体pBluescripM13-的KpnⅠ位点,得到了重组质粒pNL2200.对pNL2200中外源ＤＮＡ片段进行了一系列的亚克隆及部分核苷酸序列分析。结果表明,２．３kb的SalⅠ－ＢaｍＨⅠＤＮＡ片段中含有１个完整的开放阅读框,起始密码子为２７１位的ＧＴＧ,终止密码子为１９５４位的ＴＧＡ,该基因的大小为１６８６bp,编码１个大小为５６１个氨基酸的蛋白质产物。利用blastx程序的蛋白质数据库中进行同源比较,结果揭示此基因产物与腺苷酸形成酶超家族的连接酶有４４％的一致性,此外,该基因的破坏导致圈卷产色链霉菌尼可霉素生物合成能力的丧失,证明它是尼可霉素生物合成所必需的,命名为其为sanＪ。     

一种来源于链霉菌的纤溶酶的纯化及其基因的克隆

链霉菌C3662的发酵液上清经 80 %硫酸铵沉淀 ,DEAE Sepharose和CM Sepharose层析分离后纯化出一种纤溶酶。SDS PAGE显示为单一的条带 ,分子量约为 30kD。以 pIJ699为载体 ,S .lividansTK2 4为宿主菌 ,鸟枪法克隆纤溶酶基因 ,从 30 0 0个转化子中挑选到 1个具活性转化子 ,经亚克隆 ,序列测定得到一个 90 3bp的完整ORF ,其GC %为 68.33% ,密码子第三位GC %为 95.6% ,符合链霉菌基因的典型特征。与多种蛋白酶具有较高的同源性     

Nucleotide sequence of sporulation locus spoIIA in Bacillus subtilis

We have determined a sequence of 2073 bp from two recombinant plasmids carrying the whole spoIIA locus from Bacillus subtilis, the expression of which is required for spore formation. The sequence contains three long open reading frames (ORFs), each of them being preceded by a ribosome binding site. These three putative proteins (mol. wts 13100, 16300 and 22200) are likely to be expressed and are probably encoded on the same mRNA. The stop codon of ORF1 overlaps with the start codon of ORF2 suggesting that there might be translational coupling between the two ORFs. Although some known promoter sequences were found, the only one upstream from the first open reading frame is about 260 bp from it.     

Conserved reiterated domains in Clostridium thermocellum endoglucanases are not essential for catalytic activity

The complete nucleotide sequence of the Clostridium thermocellum celE gene, coding for an endo-beta-1,4-glucanase (endoglucanase E; EGE) with xylan-hydrolysing activity has been determined. The structural gene consists of an open reading frame (ORF) of 2442 bp commencing with a GTG start codon and followed by a TAA stop codon. The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that derived by N-terminal amino acid sequencing of the purified protein. The EGE sequence contains a region homologous to the reiterated domain found at the C terminus of other endoglucanases from the same organism. BAL 31 deletions of the structural gene have revealed the extent to which this conserved sequence is necessary for endoglucanase and xylanase activity. A region of DNA, upstream from the structural gene has also been sequenced and a ribosome-binding site and putative promoter sequences have been identified. A second ORF which ends 349 bp 5' to the GTG start codon of the celE gene has also been identified. The encoded product contains a C terminus homologous to other C. thermocellum endoglucanases.     

Comparative sequence analysis of plasmids pME2001 and pME2200 of methanothermobacter marburgensis strains Marburg and ZH3

Comparison of the updated complete nucleotide sequences of the two related plasmids pME2001 and pME2200 from the thermophilic archaeon Methanothermobacter marburgensis (formerly Methanobacterium thermoautotrophicum) strains Marburg and ZH3, respectively, revealed an almost identical common backbone structure and five plasmid-specific inserted fragments (IFs), four of which are flanked by perfect or nearly perfect direct repeats 25-52 bp in length. A 4354-bp minimal replicon was derived from the alignment of the two plasmids, which encodes one putative antisense RNA related to replication control and five open reading frames (ORFs) organized in two operons. The first operon consists of four ORFs, the third of which, i.e. ORF3, contains a helix-turn-helix motif and a purine NTP-binding motif often found in proteins involved in DNA metabolic processes. The database search results suggest that ORF3 might function as a replication initiator protein. The large putative Rep protein encoded by pME2001 was overexpressed in Escherichia coli as an N-terminal His-tagged version using pET28a and a compatible helper plasmid that coexpresses minor tRNAs, argU and ileX to compensate for codon usage difference. ORFs 1, 2, and 3 are organized in a sequence reminiscent of that described in E. coli plasmids of the R1 family, cop-tap-rep. ORF6 encoded by IF1, one of the pME2200-specific elements, showed significant similarity to ORF6 encoded by archaeal phage psiM2 of M. marburgensis strain Marburg and may confer the apparent immunity of its host strain ZH3 to infection by phage psiM2. Our data indicate that M. marburgensis plasmids may evolve by a series of gene duplication and excision events.     

Sequence analysis and expression of the aspartokinase and aspartate semialdehyde dehydrogenase operon from rifamycin SV-producing amycolatopsis mediterranei.

A approximately 4.8 kb KpnI fragment, from the upstream region of the methylmalonyl-CoA mutase gene (mutAB) of rifamycin SV-producing Amycolatopsis mediterranei, was cloned and partially sequenced. Codon preference analysis showed three complete ORFs. ORF2 is internal to ORF1, and encodes a polypeptide corresponding to 172 amino acids, whereas ORF1 encodes a polypeptide of 421 amino acids. They were identified as the encoding genes of aspartokinase alpha- and beta-subunits by comparing the amino acid sequences with those in the database. The downstream ORF3, whose start codon was overlapped with the stop codon of both ORF1 and ORF2 by 1 bp, was identified as the aspartate semialdehyde dehydrogenase gene (asd), encoding a polypeptide of 346 amino acids. Subclones containing either the ask gene or the asd gene were constructed, in which the genes could be expressed under Lac promoters. Two subclones could transform E. coli CGSC 5074 (ask-) and E. coli X6118 (asd-) to prototrophy, supporting the functional assignments. Southern hybridisation indicated that the approximately 4.8 kb sequenced region represented a continuous segment in the A. mediterranei chromosome. It is concluded that ask and asd genes are present in an operon in A. mediterranei, and therefore that organisation of these two genes is the same as in most gram-positive bacteria, such as Mycobacteria, Corynebacterium glutamicum and Bacillus subtilis, but is different from Streptomyces akiyoshiensis.     

圈卷产色链霉菌尼可霉素生物合成基因-sanK的克隆及功能研究

利用染色体步移策略,以尼可霉素生物合成相关的基因片段为探针,从圈卷产色链霉菌中克隆到了一个大约１０ｋｂ的ＤＮＡ片段。对其中１．８ｋｂ的ＰｖｕⅡ－ＳａｃⅡ片段进行了序列分析,结果表明：此片段中含有一个具有１１７０个核苷酸的完整开放阅读框,起始密码子为４４７位的ＡＴＧ,终止密码子为１６１４位的ＴＧＡ,推测其编码一个３８９个氨基酸的蛋白质产物。利用ＢＬＡＳＴＸ程序进行了分析揭示,此基因编码一个肌氨酸单体     

Characterization of a novel plasmid from extremely halophilic Archaea: nucleotide sequence and function analysis

We determined the complete nucleotide sequence of the 16341 bp plasmid pHH205 of the extremely halophilic archaeon Halobacterium salinarum J7. The plasmid has a G+C content of 61.1%. A number of direct and inverted repeat sequences were found in pHH205, while no insertion sequences were found. Thirty-eight large open reading frames (ORFs) were identified in both strands, and most of them had no significant similarities to known proteins. A putative protein encoded by ORF31 showed 20-41% homology to some hypothetical proteins, which are annotated in several archaeal genome databases as predicted nucleic acid-binding proteins containing PIN domain. Sequence analysis using the GC skew procedure predicted a possible origin of replication. A 4.8 kb PvuII-SnaBI fragment containing both this region and ORF31 was shown to be able to restore replicate of pWL102, a replicon-deficient plasmid in Haloferax volcanii and in H. salinarum R1. Several methods failed to completely cure H. salinarum J7 of pHH205, suggesting that the plasmid probably played an important role in the growth and metabolism of the host. Our work describes a novel haloarchaeal replicon, which may be useful in the construction of cloning and shuttle vectors.     

Molecular cloning and nucleotide sequence of a gene for alkaline cellulase from Bacillus sp. KSM-635

A gene for alkaline cellulase from the alkalophilic Bacillus sp. KSM-635 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. Although the recombinant plasmid contained two HindIII inserts of 2.6 kb and 4.0 kb, the inserts were found to be contiguous in the Bacillus genome by hybridization analysis. Nucleotide sequences of a 2.4 kb region which was indispensable for the production of cellulase, and the flanking, 1.1 kb region, were determined. There was an open reading frame (ORF) of 2823 bp in the 3498 bp sequence determined, which encoded 941 amino acid residues. Two putative ribosome-binding sites and a sigma 43-type, promoter-like sequence were found upstream from an initiation codon in the ORF. The deduced amino-terminal sequence resembles the signal peptide of extracellular proteins. A region of amino acids, 249 to 568, of the deduced amino acid sequence of the cellulase from this organism is homologous with those of alkaline and neutral enzymes of other micro-organisms, but nine amino acid residues were found to be conserved only in the alkaline enzymes.