Cloning and sequence analysis of gyrA gene of Klebsiella pneumoniae.

The gene gyrA encoding the DNA gyrase A subunit of Klebsiella pneumoniae has been cloned in the plasmid pBR322. Bases of about 3.5 Kb DNA have been sequenced to locate the gyrA gene. An open reading frame of 2628 nucleotides coding for a 97 KD protein has been identified. Homology to the extent of about 85% was detected at the nucleotide level and about 90% at the amino acid level, when the sequences were compared with that of Escherichia coli gyrA. Some very interesting differences have, however, been found in the promoter region.     

克雷伯氏菌甘油脱水酶基因在大肠杆菌中的克隆与表达

利用PCR技术从克雷伯氏菌(Klebsiella pneumoniae ATCC49790)总DNA中扩增得到甘油脱水酶(glycerol dehydratase,DHAB)基因的DNA片段,并将其连接到表达质粒pSE380,携带有重组质粒pSE-dhaB的大肠杆菌JM109实现了dhaB基因的表达;对含有dhaB工程菌进行表达研究,表明工程菌在37℃,以1．0mmol／L IPTG诱导5h酶活力即达到1164．14u／L,比野生菌酶活力(168．69U／L)提高了6．9倍。     

Cloning chromosomal lac genes of Klebsiella pneumoniae

The chromosomal gene for beta-galactosidase from Klebsiella pneumoniae strain T17R1 and associated regulatory genes have been cloned as a 5-kb HindIII fragment in the pBR322 plasmid vector. The beta-galactoside permease gene is not present in a functional form in the 5-kb fragment. The K. pneumoniae genes are expressed in an Escherichia coli host. The synthesis of beta-galactosidase is inducible by isopropyl-beta-D-galactosidase (IPTG) and is sensitive to catabolite repression. There appears to be greater homology between the K. pneumoniae and E. coli structural genes for beta-galactosidase than there is between the respective repressor genes.     

Cloning, sequencing and expression of the amylase isozyme gene from Pseudomonas sp. KFCC 10818

Summary A gene coding for amylase was cloned and sequenced from an alkalophilic Pseudomonas sp. KFCC 10818. The coding region for the amylase precursor contained 1,692 nucleotides. The presumed Shine-Dalgarno sequence, AAGG, was located at 8 nucleotides upstream from the ATG initiation codon. The precursor protein had a putative signal peptide of 25 amino acid residues at its amino terminus. The Pseudomonas amylase had four highly conserved regions as other -amylases. The amylase expressed from E. coli harboring the Pseudomonas gene produced maltose and maltotriose from soluble starch.The nucleotide sequence reported in this paper has been submitted in the GenBank/EMBL database with accession number(s) U40056.     

腺苷钴胺素合成酶基因cobs的克隆及其在产1,3-丙二醇菌中的应用

甘油脱水酶是催化由甘油到1,3-丙二醇过程中的关键酶,它需要在辅酶B_(12)存在的情况下才能有效的进行催化;而在此催化过程中甘油脱水酶会出现失活现象,研究表明辅酶B_(12)可以有效的促使甘油脱水酶复活。因此,辅酶B_(12)在由甘油生物催化生产1,3-丙二醇过程中起到非常重要的作用。本研究利用PCR扩增技术,从Escherichia K-12菌株中扩增出产VB_(12)关键酶—腺苷钴胺素合成酶基因cobs,其序列与NCBI上已经公布的序列比对,同源性为99.6%,将基因cobs与产1,3-丙二醇关键酶基因dhaB、yqhD在Klebsiella pneumoniae中共表达,发酵结果显示重组菌所需额外添加的VB_(12)由原始菌株的0.01 g/L下降到0.004 g/L。     

Genome sequencing and functional characterization of the non-pathogenic Klebsiella pneumoniae KpGe bacteria

Klebsiella pneumoniae is an extensively studied human pathogen responsible for a wide variety of infections. Dictyostelium discoideum is a model host organism employed to study many facets of the complex interactions between phagocytic cells and bacteria. Historically, a non-pathogenic strain of K. pneumoniae has been used to feed Dictyostelium amoebae, and more recently to study cellular mechanisms involved in bacterial recognition, ingestion and killing. Here we provide the full genome sequence and functional characterization of this non-pathogenic KpGe strain.     

Cloning and characterisation of nifLA regulatory mutations from Klebsiella pneumoniae

Summary A total of nine regulatory mutations in the nifLA operon of Klebsiella pneumoniae were cloned in the high copy-number plasmid vector pACYC184. The regulatory phenotypes of the resultant clones were then correlated with their restriction maps and their ability to synthesise nifL and nifA polypeptides in vivo. One mutation, nifL2401, was identified as a 400 bp. deletion within the nifL gene. This mutation was non-polar and caused a Nif⁺ phenotype which showed escape from repression by oxygen and low levels of fixed nitrogen. Identification of this deletion allows the first definitive allocation of a mutation with this phenotype to the nifL gene and provides further evidence for the role of the nifL gene product in nif-specific repression.     

克雷伯杆菌甘油脱氢酶基因的克隆表达与纯化

以克雷伯杆菌(Klebsiella pneumoniae)基因组DNA为模板, 运用PCR扩增得到编码甘油脱氢酶(GDH)的基因(gldA), 并克隆到pMD-18T载体上, 构建克隆载体pMD-gldA。经测序正确后, 将gldA亚克隆至表达载体pET-32a(+)上构建表达质粒pET-32gldA。在乳糖诱导下, 携带pET-32gldA的E. coli BL21 (DE3)高效表达分子量约为54 kD的可溶性蛋白。表达产物带有His6-tag标记, 选用Ni柱对表达产物进行纯化, 纯化后酶液的比活为188 u/mg, 纯化倍数和回收率分别为3倍和67.5%。     

克雷伯杆菌甘油脱氢酶基因的克隆表达与纯化

以克雷伯杆菌(Klebsiella pneumoniae)基因组DNA为模板, 运用PCR扩增得到编码甘油脱氢酶(GDH)的基因(gldA), 并克隆到pMD-18T载体上, 构建克隆载体pMD-gldA。经测序正确后, 将gldA亚克隆至表达载体pET-32a(+)上构建表达质粒pET-32gldA。在乳糖诱导下, 携带pET-32gldA的E. coli BL21 (DE3)高效表达分子量约为54 kD的可溶性蛋白。表达产物带有His6-tag标记, 选用Ni柱对表达产物进行纯化, 纯化后酶液的比活为188 u/mg, 纯化倍数和回收率分别为3倍和67.5%。     

克雷伯肺炎杆菌1,3-丙二醇氧化还原酶基因克隆及表达条件研究

1,3-丙二醇氧化还原酶是甘油歧化为1,3-丙二醇的一种关键酶。本研究从克雷伯肺炎杆菌(Klebsiella pneumoniae)基因组中,用PCR方法克隆了其编码基因dhaT。TA克隆测序正确后,构建胞内表达载体pET-28a-dhaT和分泌表达载体pET-22b-dhaT,然后转化E.coliBL21(DE3)进行原核表达。表达部位确定、SDS-PAGE和酶活分析表明,该酶得到了高水平表达。其中使用pET-22b表达的目的蛋白大都是不溶的包涵体;而使用pET-28a表达的目的蛋白胞内可溶,占胞内可溶总蛋白的45%,占菌体总蛋白的25%。常规(30℃)诱导表达即呈现1,3-丙二醇氧化还原酶活性,但低温(20℃)14 h诱导显示3.7倍的酶活性。     

Cloning and sequencing of the bovine gastrin gene

In order to deduce the primary structure of bovine preprogastrin we therefore sequenced a gastrin DNA clone isolated from a bovine liver cosmid library. Bovine preprogastrin comprises 104 amino acids and consists of a signal peptide, a 37 amino acid spacer-sequence, the gastrin-34 sequence followed by an amidation-site (Gly-Arg-Arg), and a C-terminal nonapeptide. Comparison with human, porcine, and rat cDNA sequences revealed extensive homology in the coding region as well as in short noncoding structures.     

Biochemical and crystallographic analyses of maltohexaose-producing amylase from alkalophilic Bacillus sp. 707

Maltohexaose-producing amylase, called G6-amylase (EC 3.2.1.98), from alkalophilic Bacillus sp.707 predominantly produces maltohexaose (G6) from starch and related alpha-1,4-glucans. To elucidate the reaction mechanism of G6-amylase, the enzyme activities were evaluated and crystal structures were determined for the native enzyme and its complex with pseudo-maltononaose at 2.1 and 1.9 A resolutions, respectively. The optimal condition for starch-degrading reaction activity was found at 45 degrees C and pH 8.8, and the enzyme produced G6 in a yield of more than 30% of the total products from short-chain amylose (DP = 17). The crystal structures revealed that Asp236 is a nucleophilic catalyst and Glu266 is a proton donor/acceptor. Pseudo-maltononaose occupies subsites -6 to +3 and induces the conformational change of Glu266 and Asp333 to form a salt linkage with the N-glycosidic amino group and a hydrogen bond with secondary hydroxyl groups of the cyclitol residue bound to subsite -1, respectively. The indole moiety of Trp140 is stacked on the cyclitol and 4-amino-6-deoxyglucose residues located at subsites -6 and -5 within a 4 A distance. Such a face-to-face short contact may regulate the disposition of the glucosyl residue at subsite -6 and would govern the product specificity for G6 production.     

Cloning and characterization of the gene cluster encoding type 3 (MR/K) fimbriae of Klebsiella pneumoniae

Abstract The gene cluster encoding the type 3 fimbriae of a Klebsiella pneumoniae isolate was cloned using the cosmid-cloning technique. Escherichia coli transformants, expressing type 3 fimbriae, were selected by reactivity with a monoclonal antibody directed against an epitope of the purified type 3 fimbriae. The phenotypic expression of type 3 fimbriae by transformants possessing the parental plasmid was dependent upon the host strain used. However, subcloning of this plasmid resulted in the construction of a chimeric molecule which imparted a stable phenotype regardless of the host strain. In addition, subcloning of the parental recombinant plasmid suggested that the minimal size of DNA necessary for production and expression of fimbriae was approximately 5.5 kb.     

Cloning and sequencing of a gene involved in the synthesis of the capsular polysaccharide of Streptococcus pneumoniae type 3

A 4.5 kb ScaI chromosomal DNA fragment of a clinical isolate of Streptococcus pneumoniae serotype 3 was cloned in Escherichia coli. Combined genetic and molecular analyses have allowed the localization, in a 781 by EcoRV subfragment, of a gene (cap3-1) directly responsible for the transformation of an unencapsulated, serotype 3 mutant to the capsulated phenotype. Comparison of the deduced amino acid sequence of CAP3-1 with the protein sequences compiled in the data banks revealed that the CAP3-1 polypeptide was highly similar to the amino-terminus of the GDP-mannose dehydrogenase of Pseudomonas aeruginosa, an enzyme that participates in the synthesis of the mucoid polysaccharide of this species. In addition, the 32 N-terminal amino acids of CAP3-1 perfectly matched structures common to NAD⁺-binding domains of many dehydrogenases. Our results indicate that the 4.5 kb ScaI fragment might also contain genes common to 13 different pneumococcal serogroups or scrotypes tested. To the best of our knowledge, this is the first time that a gene of the capsular complex of S. pneumoniae has been cloned and sequenced. The findings reported here provide new insights for the study of the molecular biology of the main virulence factor responsible for the pathogenesis of pneumococcal infections and might represent a basic step in the identification of cross-reactive antigens that should allow the preparation of new and improved vaccines.     

Expression of the Klebsiella pneumoniae nifH gene in Saccharomyces cerevisiae

Dinitrogenase reductase, a component of a complex and highly regulated prokaryotic enzyme, nitrogenase, is expressed in the eukaryote Saccharomyces cerevisiae. A plasmid pH-ADH-1 was constructed that directs the expression of the Klebsiella pneumoniae nifH gene, encoding dinitrogenase reductase, from the yeast alcohol dehydrogenase I promoter. In addition to being polyadenylated, yeast nifH-specific RNA is shown to be under the regulation of the alcohol dehydrogenase I promoter and is the size predicted by the nifH nucleotide sequence. Yeast transformed with the pH-ADH-1 plasmid synthesizes a polypeptide that reacts with antisera raised against dinitrogenase reductase and which, on two-dimensional polyacrylamide gels, co-migrates with dinitrogenase reductase isolated from K. pneumoniae.     

Nucleotide sequence of the maltohexaose-producing amylase gene from an alkalophilic Bacillus sp. #707 and structural similarity to liquefying type alpha-amylases

The nucleotide sequence of the gene for maltohexaose-producing amylase from an alkalophilic Bacillus sp. #707 was determined. Starting at an ATG initiation codon, an open reading frame was composed of 1554 bp (518 amino acids). The NH2-terminal portion encoded a 33 amino acid-long signal peptide. The deduced amino acid sequence of the extracellular mature enzyme was more than 60% homologous to those of the liquefying type alpha-amylases but not to those of the saccharifying type alpha-amylases. The sequence of its signal peptide was completely different from those of other alpha-amylases.