Degradation of 2-methylaniline in Rhodococcus rhodochrous: cloning and expression of two clustered catechol 2,3-dioxygenase genes from strain CTM

Rhodococcus rhodochrous strain CTM degrades 2-methylaniline mainly via the meta-cleavage pathway. Conversion of the metabolite 3-methylcatechol was catalysed by an Mr 156,000 catechol 2,3-dioxygenase (C23OI) comprising four identical subunits of Mr 39,000. The corresponding gene was detected by using an oligonucleotide as a gene probe. This oligonucleotide was synthesized on the basis of a partial amino acid sequence obtained from the purified enzyme from R. rhodochrous. The structural gene of C23OI was located on a 3.5 kb BglII restriction fragment of plasmid pTC1. On the same restriction fragment the gene for a second catechol 2,3-dioxygenase, designated C23OII, was found. This gene coded for the synthesis of the Mr 40,000 polypeptide of the Mr 158,000 tetrameric C23OII. More precise mapping of the structural genes showed that the C23OI gene was located on a 1.2 kb BglII-SmaI fragment and the C23OII gene on the adjacent 1.15 kb SmaI fragment. Comprehensive substrate range analysis showed that C23OII accepted all the substrates that C23OI did, but additionally cleaved 2,3-dihydroxybiphenyl and catechols derived from phenylcarboxylic acids. C23OI exhibited highest activity towards methylcatechols, whereas C23OII cleaved unsubstituted catechol preferentially.     

Mutagenesis of a gene encoding a cytochrome o-like terminal oxidase of Azotobacter vinelandii: A cytochrome o mutant is aero-tolerant during nitrogen fixation

Abstract The amino acid sequence obtained by translating the nucleotide sequence of a 0.55 kb fragment, amplified from Azotobacter vinelandii chromosomal DNA by PCR, was 57% identical to part of the Escherichia coli cyoB gene, encoding subunit I of the cytochrome bo -type quinol oxidase. This fragment was mutated in vitro by insertion of a kanamycin-resistance cassette and introduced into the chromosome of A. vinelandii by homologous recombination. The mutant contained no spectrally detectable cytochrome o . However, in the stationary phase of growth, the level of the alternative oxidase (cytochrome bd ) was 11-fold higher than in the wild-type strain. Respiration of the mutant was insensitive to chlorpromazine, an inhibitor thought to act specifically on cytochrome o . Cytochrome o -deficient mutants fixed nitrogen in air, clearly distinguishing the role of this oxidase from that of cytochrome bd , which is required for respiratory protection of oxygen-labile nitrogenase.     

大肠杆菌青霉素G酰化酶基因及其邻近区域的核苷酸全序列

Some of microorganisms have been known to possess penicillin G acylase activity. The E. coli derived penicillin G acylase (PGA) can catalyze the conversion of penicillin G into phenylacetic acid and 6-amino-penicillanic acid, the latter is used as the starting compound for the industrial formation of semi-synthetic penicillins. Apart from its industrial importance, the enzyme PGA displays a number of interesting properties. Catalytically active enzyme is localized in the periplasmic space of E. coli cells and composed of two dissimilar subunits. The two subunits are apparently produced from a precursor protein, via a processing pathway hitherto unique in its features for a prokaryotic enzyme. The studies on processing of the precursor and on the relationship between structure and function of the mature enzyme are important theoretically. Previously we cloned a 3.5 kb DNA fragment from a strain (E. coli AS 1.76), which displays PGA activity. In this paper, we report a nucleotide sequence of the 3.5 kb DNA fragment containing PGA gene. After insertion of the DNA fragment into EcoR I and Hind III sites in pWR 13, pPGA 20 had been obtained. We subcloned the Hind III and Bg1 II treated fragment of 1.6 kb in length from pPGA 20 into Hind III and BamH I sites of pWR 13 to get a pPGA 1.6, and Bg1 II and EcoR I treated fragment of 1.9 kb in length into BamH I and EcoR I sites of pWR 13 to get a pPGA 1.9. The linearized pPGA 1.9 which were digested with appropriate restriction enzymes were progressively shortened from both ends respectively by digestion with Bal 31 nuclease, followed by cleavage of shortened target DNA off vector DNA molecules with appropriate restriction enzymes. The series of the DNA fragments shortened from EcoR I end were then cloned into plasmid pWR 13 which had previously digested with Hind III and Sma I enzymes (Fig. 1). The DNA fragment cloned in pWR 13 were directly sequenced on the resulted plasmids by using primer I and primer II. Thus we have obtained the complete nucleotide sequence of the 3.5 kb DNA fragment. The 3.5 kb fragment contains an intact PGA gene which is 2.6 kb.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lipoamide dehydrogenase from Azotobacter vinelandii. Molecular cloning, organization and sequence analysis of the gene

The gene encoding lipoamide dehydrogenase from Azotobacter vinelandii has been cloned in Escherichia coli. Fragments of 9-23 kb from Azotobacter vinelandii chromosomal DNA obtained by partial digestion with Sau3A were ligated into the BamHI site of plasmid pUC9. E. coli TG2 cells were transformed with the resulting recombinant plasmids. Screening for clones which produced A. vinelandii lipoamide dehydrogenase was performed with antibodies raised against the purified enzyme. A positive colony was found which produced complete chains of lipoamide dehydrogenase as concluded form SDS gel electrophoresis of the cell-free extract, stained for protein or used for Western blotting. After subcloning of the 14.7-kb insert of this plasmid the structural gene could be located on a 3.2-kb DNA fragment. The nucleotide sequence of this subcloned fragment (3134 bp) has been determined. The protein-coding sequence of the gene consists of 1434 bp (478 codons, including the AUG start codon and the UAA stop codon). It is preceded by an intracistronic region of 85 bp and the structural gene for succinyltransferase. A putative ribosome-binding site and promoter sequence are given. The derived amino acid composition is in excellent agreement with that previously published for the isolated enzyme. The predicted relative molecular mass is 50223, including the FAD. The overall homology with the E. coli enzyme is high with 40% conserved amino acid residues. From a comparison with the three-dimensional structure of the related enzyme glutathione reductase [Rice, D. W., Schultz, G. E. & Guest, J. R. (1984) J. Mol. Biol. 174, 483-496], it appears that essential residues in all four domains have been conserved. The enzyme is strongly expressed, although expression does not depend on the vector-encoded lacZ promoter. The cloned enzyme is, in all the respects tested, identical with the native enzyme.     

Identification and Characterization of two nitrogen fixation regulatory regions, nifA and nfrX, in Azotobacter vinelandii and Azotobacter chroococcum

Five Tn5-induced Nif- mutants of Azotobacter vinelandii were characterized as regulatory mutants because they were restored to Nif+ by the introduction of constitutively expressed nifA from Klebsiella pneumoniae. The mutants fell into two different classes on the basis of hybridization to a Rhizobium leguminosarum nifA gene probe and by complementation with cosmids isolated from pLAFRI gene banks of A. vinelandii and Azotobacter chroococcum. One mutant, MV3, was located in or near a nifA gene. The others, MV12, MV16, MV18 and MV26, defined a new regulatory gene, which has been called nfrX. The lack of expression of different nif-lacZ fusions confirmed the regulatory phenotype of all five mutant strains. The ability of both nifA and nfrX mutants to grow on nitrogen-free medium with vanadium, but not on medium with molybdenum, suggests that neither gene is required for expression of the alternative V-containing nitrogenase of A. vinelandii. A fragment carrying Tn5 and flanking DNA from MV3 was used as a probe to isolate the nifA region of A. chroococcum. Ligation of two adjacent EcoRI fragments of A. chroococcum yielded an intact nifA gene that activated expression of nifH-lac fusions and also restored MV3 to Nif+. The four nfrX mutants were complemented by pLAFR1 cosmids pLV163 and pLC121. The nfrX gene was subcloned from pLV163 and located within a 3.2 kb fragment. To determine whether nfrX might be found in other nitrogen-fixing organisms, DNA from 13 different species was hybridized to an nfrX probe. The failure to observe hybridization suggests that nfrX may be specific to nif regulation in Azotobacter.     

Characterization of two genes (hupD and hupE) required for hydrogenase activity in Azotobacter chroococcum

In Azotobacter chroococcum the hydrogenase structural genes (hupSL) cover about 2.8 kb of a 15-kb region associated with hydrogen-uptake (Hup) activity. Two other genes in this region, hupD and hupE, were located 8.9 kb downstream of hupL and were shown to be essential for hydrogenase activity by insertion mutagenesis. A fragment of DNA beginning 3.4 kb downstream of hupL was able to complement the hupE mutant, supporting earlier evidence for a promoter downstream of hupSL. Hybridization experiments showed that hupD and hupE share some similarity with a region of Alcaligenes eutrophus DNA which is apparently involved in the formation of catalytically active hydrogenase. The hupD gene encodes a 379-amino acid, 41.4-kDa polypeptide while hupE codes for a 341-amino acid, 36.1-kDa product. The predicted amino acid sequences of the hupD and hupE genes are homologous to the Escherichia coli hypD and hypE gene products, respectively. A polar mutation in hupD had no effect on beta-galactosidase activity in a strain also carrying a hupL-lacZ fusion, indicating that hupD and hupE are probably not involved in regulating hydrogenase structural gene expression.     

Cloning the modification methylase gene of Bacillus sphaericus R in Escherichia coli

The gene coding for the sequence-specific modification methylase methM . BspI of Bacillus sphaericus R has been cloned in Escherichia coli by means of plasmid pBR322. The selection was based on the expression of the cloned gene which rendered the recombinant plasmid resistant to BspI restriction endonuclease cleavage. The gene is carried by a 9 kb BamHI fragment and by a smaller 2.5 kb EcoRI fragment derived from the BamHI fragment. The Bsp-specific methylase level was found to be higher in the recombinant clones than in the parental strain. The methylase gene is probably located on the Bacillus sphaericus chromosome, and not on a plasmid known to be carried by this strain. The recombinant clones do not exhibit an BspI restriction endonuclease activity.     

Phenotypic characterisation and genetic complementation of dimethylsulfoxide respiratory mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus

Abstract Two chlorate resistant mutants of Rhodobacter sphaeroides were isolated which were deficient in dimethylsulfoxide reductase activity. Immunoblotting experiments showed that the phenotype of these mutants and that of Rhodobacter capsulatus strain DK9, a mutant unable to reduce dimethylsulfoxide, was correlated with low or undetectable levels of the dimethylsulfoxide reductase apoprotein. All three mutants were complemented by a cosmid from a library of Rhodobacter sphaeroides genomic DNA. Further genetic complementation analysis revealed that functions required for restoration of dimethylsulfoxide reductase activity in the Rhodobacter sphaeroides mutants were encoded on an 9 kb EcoR1 DNA fragment derived from this cosmid. Expression of this 9 kb DNA fragment in Escherichia coli showed that it encoded the dimethylsulfoxide reductase structural gene of Rhodobacter sphaeroides .     

Cloning of the gene for phosphoenolpyruvate carboxylase from Azotobacter chroococcum, an enzyme important in aerobic nitrogen fixation

Summary Azotobacter chroococcum Fos 189 is a Tn1-induced mutant which, unlike the parent strain MCD1, does not fix nitrogen in air when provided with glucose or pyruvate as sole carbon sources. Fos 189 showed 5% of parental activity for phosphoenolpyruvate carboxylase though PEP synthetase activity was normal. The A. chroococcum phosphoenolpyruvate carboxylase (ppc) gene was isolated after complementation of an appropriate Escherichia coli mutant using a broad host range gene bank prepared from A. chroococcum genomic DNA. The gene was localised by transposon mutagenesis and subcloning on a minimum DNA fragment of 6.6 kb. Broad host range plasmids containing the A. chroococcum ppc gene complemented the mutation in Fos 189 thereby restoring aerotolerant nitrogen fixation.     

费氏中华根瘤菌与耐盐有关的DNA片段的亚克隆和测序

将费氏中华根瘤菌（Ｓｉｎｏｒｈｉｚｏｂｉｕｍ ｆｒｅｄｉｉ）ＫＴ１９与耐盐有关的２３ｋｂ ＤＮＡ片段用ＢａｍＨⅠ酶切成大小不同的长度,分别与质粒ｐＭＬ１２２连接,然后转化大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）Ｓ１７－１,筛选出３个转化子。以这些转化子为供体,ＲＴ１９的盐敏感突变株ＲＣ３－３为受体,分别进行二亲本杂交,筛选到接合子ＢＲ２,得到４．４ｋｂ与耐盐有关的ＤＮＡ片段。根据其物理图谱,酶     

生米卡链霉菌丙酰化酶基因定位及其核苷酸序列测定

我们已往的工作已经确定在重组质粒ｐＩＪＭ９中,生米卡链霉菌来源的４．１６ｋｂ插入ＤＮＡ片段带有丙酰化酶基因,为了进一步确定该基因在４．１６ｋｂ插入片段中的位置,我们以ＢａｍＨＩ对ｐＩＪＭ９进行酶切,在此基础上进行缺失重组亚克隆,在所获得的转化子中,Ｎｏ．５转化子含重组质粒ｐＩＪＭ９５,分子量为５．０ｋｂ,插入ＤＮＡ片段为０．５３ｋｂ,以Ｎｏ．５转化子对螺旋霉素进行生物转化实验,其转化产物经薄层层析     

The metabolic pathway of 2,4-dichlorophenoxyacetic acid degradation involves different families of tfdA and tfdB genes according to PCR-RFLP analysis

Abstract: Twenty-five 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacteria from geographically diverse locations and presenting various degrees of similarity or no similarity to the tfdA and tfdB genes from Alcaligenes eutrophus JMP134 were analysed by PCR-RFLP (restriction length fragment polymorphism). Primers for the 2,4-D etherase gene were derived by sequence alignment of the tfdA genes from A. eutrophus JMP134 and Burkholderia sp. RASC. Primers for the 2,4-dichlorophenolhydroxylase gene were based on the tfdB gene sequence from A. eutrophus JMP134 by taking codon degeneration and variations in amino acid residue sequences into consideration. PCR amplification using the tfdA primer set produced fragments of 0.3 kb from 17 strains which showed varying degrees of similarity to the tfdA gene probe from A. eutrophus JMP134. Significant variations in the gene sequences were confirmed by PCR-RFLP analysis. DNA amplification using the tfdB primer set produced a 1.1 kb fragment from 19 strains. Amongst them, two did not show any similarity to the tfdB gene probe. The size and restriction pattern of the products obtained from A. eutrophus JMP134 were in accordance with the expected size calculated from the A. eutrophus tfdA and tfdB gene sequence and their theoretical PCR-RFLP patterns. Some strains which did not amplify using the tfdA primer set did however amplify with the tfdB primer set. These results suggest the independent evolution of these two genes in the construction of the 2,4-D metabolic pathway. Our tfdA and tfdB primer sets could be used for the detection of similar sequences in bacteria and soils. Moreover, PCR-RFLP patterns could also be used to select subsets of strains for sequencing to study the phylogeny of the tfdA and tfdB genes.     

Cloning of the promoter region of plasma membrane aquaporin BnPIP1 from Brassica napus and its functional analysis

Aquaporins make water-selective channels in plants, facilitating the permeation of water through membranes and adjusting water fast transport during seed germination, cell elongation, stoma movement, fertilization and responses to environmental stresses. They belong to the MIP (major intrinsic protein) family with molecular weight of 2629 kD and are characterized by six membrane-spanning a-helixes connected by five loops and short N-terminal and C-terminal domains in the cytoplasm[13]. The p…     

嗜热菌Aquifex pyrophilus中mbh 2基因簇的鉴定和部分基因的克隆及测序

基于嗜热菌Aquifex aeolicus的mbhS2基因,设计合成特异性引物,以Aquifex pyrophilus 的染色体DNA为模板,应用PCR技术扩增出目的片段.序列测定结果显示,其推导出的氨基酸序列与A.aeolicus的相应序列具有85%的同源性.以此PCR片段为探针,从A.pyrophilus 的Nco I部分基因组文库(4～6kb)中筛选出含5kb大小插入片段的阳性克隆,进而对其进行了亚克隆及测序.结果表明,该插入子包含A.pyrophilus mbj2基因簇的小亚基基因mbhS2、orf1基因和部分orf2基因.所推导出的氨基酸序列与A.aeolicus的MbhS2和Orf963相比较的同源性分别为81%和60%.     

麦迪霉素产生菌酮基还原酶基因的研究

将麦迪霉素产生菌基因文库中与放线紫红索酮基还原酶基因actⅢ有同源性的4·0kb DNA片段克隆到质粒载体pWHM3中,构成重组质粒pCB4。将质粒pCB4转入酮基还原酶基因缺陷菌株——加利利链霉菌ATCC3167l中,得到转化子。转化子发酵产物经TLC和HPLC分析证明是阿克拉菌酮,与加利利链霉菌原株ATCC31133的产物相同,说明麦迪霉素产生菌酮基还原酶基因互补了加利利链霉菌ATCC31671中缺陷的酮基还原酶基因,使其恢复了产生阿克拉菌酮的能力。4．Okb DNA片段插入方向相反的重组质粒pCBR4在加利利链霉菌ATCC31671中发酵产物经TLC分析证明也是阿克拉菌酮,这说明4．0kbDNA片段中麦迪霉素产生菌酮基还原酶基因具有自身的启动子。对4．0kb DNA片段进行了限制酶酶切分析,建立了其酶切图谱。以actⅢ基因为探针,经分子杂交以及亚克隆和DNA转化实验,将麦迪霉索产生菌酮基还原酶基因定位于BssH Ⅱ—BamH Ⅰ 1．3kb DNA片段上。对1．3kb DNA片段核苷酸序列分析结果表明：此1．3kbDNA片段中含有一个独立的ORF,起始密码ATG,终止密码TAG,含783bp;在起始密码上游有GGAGG5个核苷酸SD序列;此ORF编码260个氨基酸,与actⅢ基因编码的261个氨基酸相似性为77．4%,相同性为66．7％,对麦迪霉素产生苗酮基还原酶基因的可能作用进行了讨论。     

Physical and genetic map of the Methanococcus voltae chromosome

A physical map of the Methanococcus voltae chromosome was constructed on the basis of restriction mapping and cross-hybridization experiments, employing total and partial digests obtained with rarely cutting restriction enzymes. On the basis of the sum of the fragment sizes of digests with seven enzymes the chromosome length was calculated to be approximately 1900 kb. The derived map is circular. Hybridization of gene probes to mapped restriction fragments has led to a genetic map of genes for structural RNAs as well as proteins, including enzymes involved in the methanogenic pathway.     

Molecular cloning of nif DNA from Azotobacter vinelandii.

Two clones which contained nif DNA were isolated from a clone bank of total EcoRI-digested Azotobacter vinelandii DNA. The clones carrying the recombinant plasmids were identified by use of the 32P-labeled 6.2-kilobase (kb) nif insert from pSA30 (which contains the Klebsiella pneumoniae nifK, nifD, and nifH genes) as a hybridization probe. Hybridization analysis with fragments derived from the nif insert of pSA30 showed that the 2.6-kb insert from one of the plasmids (pLB1) contains nifK whereas the 1.4-kb insert from the other plasmid (pLB3) contains nifD. Marker rescue tests using genetic transformation indicated that the 2.6-kb A. vinelandii nif fragment contains the wild-type alleles for the nif-6 and nif-38 mutations carried by Nif- strains UW6 and UW38. The 1.4-kb insert contains the wild-type allele for the nif-10 mutation carried by Nif- strain UW10.