Cloning of Klebsiella pneumoniae pqq genes and PQQ biosynthesis in Escherichia coli

We have cloned genes from Klebsiella pneumoniae which are required for pyrroloquinoline quinone (PQQ) biosynthesis. The cloned 6.7 kb fragment can complement several chromosomal pqq mutants. Escherichia coli strains are unable to synthesize PQQ but E. coli strains containing the cloned 6.7 kb K. pneumoniae fragment can synthesize PQQ in large amounts and E. coli pts mutants can be complemented on minimal glucose medium by this clone.     

Characterization and nucleotide sequence of pqqE and pqqF in Methylobacterium extorquens AM1.

Methylobacterium extorquens AM1 pqqEF are genes required for synthesis of pyrroloquinoline quinone (PQQ). The nucleotide sequence of these genes indicates PqqE belongs to an endopeptidase family, including PqqF of Klebsiella pneumoniae, and M. extorquens AM1 PqqF has low identity with the same endopeptidase family. M. extorquens AM1 pqqE complemented a K. pneumoniae pqqF mutant.     

寡聚核苷酸诱导突变法改造φ×174噬菌体A基因蛋白的DNA识别序列

 为了进一步研究φX174噬菌体A基因蛋白的复制功能与其所识别的30核苷酸保守序列的关系,我们采用寡聚核苷酸诱导的定点突变法成功地改造了这30核苷酸保守序列。将此保守序列重组到M_(13)mp9噬菌体后,以其单链为模板,在14或16寡聚核苷酸的诱导下,合成共价闭环DNA。经转化到E.coli JM103菌株,用点印迹(Dot blot)杂交法筛选,得到两种重组突变株。一种突变株其30核苷酸保守序列正链的第22碱基由A改为G。另一突变株为其第10碱基A改为C,第11碱基T改为A。突变效率约为5％。制备了此突变株单链及双链DNA,分别做了双脱氧末端终止法及Maxam和Gilbert法序列分析鉴定。     

Cloning, sequencing and analysis of a gene encoding Escherichia coli proline dehydrogenase

Abstract Using a genomic subtraction technique, we cloned a DNA sequence that is present in wild-type Escherichia coli strain CSH4 but is missing in a presumptive proline dehydrogenase deletion mutant RM2. Experimental evidence indicated that the cloned fragment codes for proline dehydrogenase (EC 1.5.99.8) since RM2 cells transformed with a plasmid containing this sequence was able to survive on minimal medium supplemented with proline as the sole nitrogen and carbon sources. The cloned DNA fragment has an open reading frame of 3942 bp and encodes a protein of 1313 amino acids with a calculated M _r of 143 808. The deduced amino acid sequence of the E. colli proline dehydrogenase has an 84.9% homology to the previously reported Salmonella typhimurium putA gene but it is 111 amino acids longer at the C-terminal than the latter.     

Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene.

A 5.9-kb DNA fragment was cloned from Pseudomonas aeruginosa PA103 by its ability to functionally complement a fur mutation in Escherichia coli. A fur null mutant E. coli strain that contains multiple copies of the 5.9-kb DNA fragment produces a 15-kDa protein which cross-reacts with a polyclonal anti-E. coli Fur serum. Sequencing of a subclone of the 5.9-kb DNA fragment identified an open reading frame predicted to encode a protein 53% identical to E. coli Fur and 49% identical to Vibrio cholerae Fur and Yersinia pestis Fur. While there is extensive homology among these Fur proteins, Fur from P. aeruginosa differs markedly at its carboxy terminus from all of the other Fur proteins. It has been proposed that this region is a metal-binding domain in E. coli Fur. A positive selection procedure involving the isolation of manganese-resistant mutants was used to isolate mutants of strain PA103 that produce altered Fur proteins. These manganese-resistant Fur mutants constitutively produce siderophores and exotoxin A when grown in concentrations of iron that normally repress their production. A multicopy plasmid carrying the P. aeruginosa fur gene restores manganese susceptibility and wild-type regulation of exotoxin A and siderophore production in these Fur mutants.     

Mutants ofMethylobacterium organophilum unable to synthesize PQQ

The phenotype of mutants unable to synthesize PQQ is analyzed for different categories of methylotrophic bacteria. The advantages offered by strains dissimilating methylamine through methylated amino-acids are discussed. InM.organophilum, 40% of the mutants unable to grow in methanol medium but with normal methylamine utilization, were affected in PQQ metabolism. The genetic properties ofM.organophilum useful to study PQQ mutants are discussed, mainly the use of pSUP106 to create insertion mutations in the bacterial chromosome and to replace wild-type genes by modified genes. An example is given of the possibility to create R plasmids containing large fragments ofM.organophilum DNA. Some physiological properties of a PQQ mutant are described, regarding growth kinetics, PQQ uptake and accumulation.     

A new approach for molecular cloning in cyanobacteria: cloning of an Anacystis nidulans met gene using a Tn901-induced mutant

A new strategy for molecular cloning in the cyanobacterium Anacystis nidulans R-2 is described. This strategy involved the use of a transposon and was developed for the cloning of a gene encoding methionine biosynthesis. A met::Tn901 mutant was isolated. Chromosomal DNA fragments were cloned in the Escherichia coli plasmid vector pACYC184. A recombinant plasmid carrying the inactivated met::Tn901 gene was selected after transformation to E. coli. The cloned met::Tn901 DNA fragment was used as a probe to select the corresponding A. nidulans R-2 wild-type met gene from a gene library prepared in E. coli, using the newly constructed shuttle cosmid vector pPUC29. When transformed into A. nidulans Met- mutants, this cloned gene allowed the mutants to grow prototrophically.     

肠出血性大肠杆菌毒力相关基因突变株的构建

目的:利用Red重组系统敲除肠出血性大肠杆菌O157∶H7的毒力基因espA、espB、espD,构建3株突变株。方法:以肠出血性大肠杆菌O157∶H7为模板,PCR扩增基因两翼的同源序列;将PCR产物插入pEASY-T1载体并测序,将测序正确的上、下游同源序列分步酶切,构建于pUC19-kan质粒上,经PCR获得两端同源序列中间嵌合卡那霉素抗性基因标记的线性片段,利用质粒pKD46介导的重组技术,敲除espA、espB、espD基因,之后转入pCP20质粒以去除抗性标记,最后测定突变株及野生菌株的生长曲线。结果:敲除了肠出血性大肠杆菌O157∶H7的毒力基因espA、es pB、espD,获得3株突变株,突变株与野生株的生长曲线相近。结论:为进一步研究espA、espB、espD基因在肠出血性大肠杆菌O157∶H7致病过程中的作用奠定了基础。     

Localization of a pyrroloquinoline quinone biosynthesis gene near the methanol dehydrogenase structural gene in Methylobacterium organophilum DSM 760

A partial Sau3AI genomic bank of Methylobacterium organophilum DSM 760 was constructed in the cosmid pSUP106 and moxF, the structural gene for methanol dehydrogenase, was isolated. In M. organophilum, pSUP106 behaves as a suicide plasmid. This property was used to insert Tn5 into the bacterial chromosome, in the vicinity of moxF, by marker exchange. Mobilization of the Tn5-labelled chromosomal region by a broad-host-range plasmid, pJB3J1 (an R68-45 derivative), allowed the selection of several large R' hybrid plasmids in Escherichia coli HB101. Most of them were able to complement both mutants of the moxF region and mutant MTM1, the first mutant of the pyrroloquinoline quinone (PQQ) biosynthesis pathway in M. organophilum. The gene involved, pqqA, was subcloned and localized.     

大肠杆菌ptsG和ptsM突变体的构建及特性研究

目的：敲除大肠杆菌DH5α中与葡萄糖磷酸化转运相关的ptsG、ptsM基因,考察缺陷株生长特性及其可能的应用。方法：PCR扩增靶基因,构建两翼带有靶基因序列并嵌合抗药基因标记的线性片段,利用Red同源重组技术敲除靶基因。结果：成功敲除了大肠杆菌DH5α的ptsG和ptsM基因;在含有葡萄糖的LB培养基中,DH5αΔptsG最高菌密度是亲本的2.8倍,添加吡咯喹啉醌或导入其生物合成基因后能够产酸;DH5αΔptsM最高菌密度是亲本的4/10,有明显的产酸现象。结论：DH5αΔptsG可用于大肠杆菌高密度发酵和吡咯喹啉醌生物合成基因缺陷株筛选。     

Cloning the gyrA gene of Bacillus subtilis.

We have isolated an eight kilobase fragment of Bacillus subtilis DNA by specific integration and excision of a plasmid containing a sequence adjacent to ribosomal operon rrn O. The genetic locus of the cloned fragment was verified by linkage of the integrated vector to nearby genetic markers using both transduction and transformation. Functional gyrA activity encoded by this fragment complements E. coli gyrA mutants. Recombination between the Bacillus sequences and the E. coli chromosome did not occur. The Bacillus wild type gyrA gene, which confers sensitivity to nalidixic acid, is dominant in E. coli as is the E. coli gene. The cloned DNA precisely defines the physical location of the gyrA mutation on the B. subtilis chromosome. Since an analogous fragment from a nalidixic acid resistant strain has also been isolated, and shown to transform B. subtilis to nalidixic acid resistance, both alleles have been cloned.     

Molecular characterization of pmbA, an Escherichia coli chromosomal gene required for the production of the antibiotic peptide MccB17

Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains harbouring plasmid pMccB17. We have isolated two mutations that strongly reduce the production of MccB17. These mutations, which map at 96 min on the E. coli chromosome, define a new gene that we have called pmbA. A chromosomal DNA fragment of about 13 kb, including the wild-type pmbA allele, was cloned into a mini-Mu plasmid vector. pmbA was located within the cloned DNA fragment by insertional mutagenesis and deletion analysis. The nucleotide sequence of a 1.7 kb DNA region containing the gene was determined. pmbA encodes a hydrophilic protein of 450-amino-acid residues with a predicted molecular size of 48375D, which was visualized in polyacrylamide gels. Protein profiles of cellular envelope and soluble fractions from cells with plasmids overproducing PmbA indicated that it is cytoplasmic. Physiological experiments suggested that pmbA mutants synthesize a molecule (pro-MccB17) able to inhibit DNA replication but unable to be released from cells. We propose that PmbA facilitates the secretion of the antibiotic by completing its maturation.     

Physiological properties of a pyrroloquinoline quinone mutant of Methylobacterium organophilum

Abstract The grwoth of MTMl, a mutant of methylobacterium organophilum) blocked in the use of methanol as a carbon and energy source, was restored by addition of pyrroloquinoline quinone (PQQ) in the culture medium. No PQQ could be detected in crude medium. No PQQ could be of MTMl. Therefore, MTMl can be regarded as a mutant blocked in the biosynthesis of PQQ. Under the conditions of growth employed, growth rates of MTMl on methanol, comparable to those of the wild type, occured at a PQQ concentration of 1 μM. Since lower amounts of methanol dehydrogenase (MDH) wer found in cell-free extracts of PQQ-supplemented MTMl, the wild type strain synthesizes a surplus of MDH under these conditions. Growth of M. organophilum on ethanol proceeds via MDH as a catalyst for the first step, since (NAD(P) -dependent etanol. dehydrogenase was absent in cell-free extracts and growth of MTMl on ethanol only took place in the presence of PQQ. On the hand, growth of MTMl on mthylamine was unimpaired. This is in accordance with the fact that methylamine dehydrogenase was absent and N -methylamine mate dehydrogenase was present in cell-free extracts     

大肠杆菌嘌呤核苷磷酸化酶基因的克隆与真核表达载体的构建

根据Ｇｅｎｂａｎｋ中大肠杆菌嘌呤核苷磷酸化酶（ＰＮＰ）基因的核苷酸序列,设计并合成了一对引物,以大肠杆菌基因组ＤＮＡ为模板,进行ＰＣＲ扩增,并将扩增产物定向连接到克隆、测序及真核表达载体ＰＣＤＮＡ３中,进行酶切鉴定、测序及序列分析。结果表明ＰＣＲ扩增出７４１ｂｐ大小的片段,通过酶切和序列分析证明含完整的ＰＮＰ基因序列且基因插入方向正确,此序列与文献报道的ＰＮＰ基因的同源性为９９．７％。说明克隆的ＰＮＰ基因与文献报道的基本一致,ｐｃＤＮＡ３－ＰＮＰ的构建成功为今后用其进行基因转染来研究ＰＮＰ／Ｍｅｐ－ｄＲ自杀基因系统在肿瘤基因治疗中的应用打下了基础。     

Novel structure of the recA locus of Mycobacterium tuberculosis implies processing of the gene product.

A fragment of Mycobacterium tuberculosis DNA containing recA-like sequences was identified by hybridization with the Escherichia coli recA gene and cloned. Although no expression was detected from its own promoter in E. coli, expression from a vector promoter partially complemented E. coli recA mutants for recombination, DNA repair, and mutagenesis, but not for induction of phage lambda. This clone produced a protein which cross-reacts with antisera raised against the E. coli RecA protein and was approximately the same size. However, the nucleotide sequence of the cloned fragment revealed the presence of an open reading frame for a protein about twice the size of other RecA proteins and the cloned product detected by Western blotting (immunoblotting). The predicted M. tuberculosis RecA protein sequence was homologous with RecA sequences from other bacteria, but this homology was not dispersed; rather it was localized to the first 254 and the last 96 amino acids, with the intervening 440 amino acids being unrelated. Furthermore, the junctions of homology were in register with the uninterrupted sequence of the E. coli RecA protein. Identical restriction fragments were found in the genomic DNAs of M. tuberculosis H37Rv and H37Ra and of M. bovis BCG. It is concluded that the ancestral recA gene of these species diversified via an insertional mutation of at least 1,320 bp of DNA. Possible processing mechanisms for synthesizing a normal-size RecA protein from this elongated sequence are discussed.     

Cloning and expression of the Thiobacillus ferrooxidans glutamine synthetase gene in Escherichia coli.

The glutamine synthetase (GS) gene glnA of Thiobacillus ferrooxidans was cloned on recombinant plasmid pMEB100 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as the sole source of nitrogen. High levels of GS-specific activity were obtained in the E. coli glnA deletion mutants containing the T. ferrooxidans GS gene. The cloned T. ferrooxidans DNA fragment containing the glnA gene activated histidase activity in an E. coli glnA glnL glnG deletion mutant containing the Klebsiella aerogenes hut operon. Plasmid pMEB100 also enabled the E. coli glnA glnL glnG deletion mutant to utilize arginine or low levels of glutamine as the sole source of nitrogen. There was no detectable DNA homology between the T. ferrooxidans glnA gene and the E. coli glnA gene.     

Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase of Pseudomonas aeruginosa.

Nitrosoguanidine-induced Pseudomonas aeruginosa mutants which were unable to utilize glycerol as a carbon source were isolated. By utilizing PAO104, a mutant defective in glycerol transport and sn-glycerol-3-phosphate dehydrogenase (glpD), the glpD gene was cloned by a phage mini-D3112-based in vivo cloning method. The cloned gene was able to complement an Escherichia coli glpD mutant. Restriction analysis and recloning of DNA fragments located the glpD gene to a 1.6-kb EcoRI-SphI DNA fragment. In E. coli, a single 56,000-Da protein was expressed from the cloned DNA fragments. An in-frame glpD'-'lacZ translational fusion was isolated and used to determine the reading frame of glpD by sequencing across the fusion junction. The nucleotide sequence of a 1,792-bp fragment containing the glpD region was determined. The glpD gene encodes a protein containing 510 amino acids and with a predicted molecular weight of 56,150. Compared with the aerobic sn-glycerol-3-phosphate dehydrogenase from E. coli, P. aeruginosa GlpD is 56% identical and 69% similar. A similar comparison with GlpD from Bacillus subtilis reveals 21% identity and 40% similarity. A flavin-binding domain near the amino terminus which shared the consensus sequence reported for other bacterial flavoproteins was identified.     

Cloning of an Erwinia herbicola gene necessary for gluconic acid production and enhanced mineral phosphate solubilization in Escherichia coli HB101: nucleotide sequence and probable involvement in biosynthesis of the coenzyme pyrroloquinoline quinone.

Escherichia coli is capable of synthesizing the apo-glucose dehydrogenase enzyme (GDH) but not the cofactor pyrroloquinoline quinone (PQQ), which is essential for formation of the holoenzyme. Therefore, in the absence of exogenous PQQ, E. coli does not produce gluconic acid. Evidence is presented to show that the expression of an Erwinia herbicola gene in E. coli HB101(pMCG898) resulted in the production of gluconic acid, which, in turn, implied PQQ biosynthesis. Transposon mutagenesis showed that the essential gene or locus was within a 1.8-kb region of a 4.5-kb insert of the plasmid pMCG898. This 1.8-kb region contained only one apparent open reading frame. In this paper, we present the nucleotide sequence of this open reading frame, a 1,134-bp DNA fragment coding for a protein with an M(r) of 42,160. The deduced sequence of this protein had a high degree of homology with that of gene III (M(r), 43,600) of a PQQ synthase gene complex from Acinetobacter calcoaceticus previously identified by Goosen et al. (J. Bacteriol. 171:447-455, 1989). In minicell analysis, pMCG898 encoded a protein with an M(r) of 41,000. These data indicate that E. coli HB101(pMCG898) produced the GDH-PQQ holoenzyme, which, in turn, catalyzed the oxidation of glucose to gluconic acid in the periplasmic space. As a result of the gluconic acid production, E. coli HB101(pMCG898) showed an enhanced mineral phosphate-solubilizing phenotype due to acid dissolution of the hydroxyapatite substrate.