The Vitreoscilla hemoglobin gene: Molecular cloning, nucleotide sequence and genetic expression in Escherichia coli

Summary Vitreoscilla hemoglobin is involved in oxygen metabolism of this bacterium, possibly in an unusual role for a microbe. We have isolated the Vitreoscilla hemoglobin structural gene from a pUC19 genomic library using mixed oligodeoxy-nucleotide probes based on the reported amino acid sequence of the protein. The gene is expressed in Escherichia coli from its natural promoter as a major cellular protein. The nucleotide sequence, which is in complete agrecment with the known amino acid sequence of the protein, suggests the existence of promoter and ribosome binding sites with a high degree of homology to consensus E. coli upstream sequences. In the case of at least some amino acids, a codon usage bias can be detected which is different from the biased codon usage pattern in E. coli. The down-stream sequence exhibits homology with the 3 end sequences of several plant leghemoglobin genes. E. coli cells expressing the gene contain greater than fivefold more heme than controls.     

The cloning of the rorB gene of Escherichia coli

Summary A segment of the Escherichia coli genome which complements the ionising radiation sensitivity of the rorB mutation was cloned into pBR322. This DNA segment also complements the mitomycin C sensitivity of the rorB mutation. The gene was subcloned until defined in a fragment of 1.05 kb. Only one gene product, a protein of approximately 16.5 kDa, was found on maxicell analysis of the various subclones. Iso-electric focusing of this gene product suggests it may function in a complex.     

Molecular cloning of protease gene(s) from Xanthomanas campestris pv. campestris: Expression in Escherichia coli and role in pathogenicity

Summary The recombinant plasmid pIJ3070 isolated from a genomic library of Xanthomonas campestris pv. campestris constructed in the conjugal cosmid pLAFR3 contains protease gene(s) which can be expressed in Escherichia coli. Tn5 mutagenesis and subcloning revealed that the protease structural gene(s) is(are) located in a ca. 10 kb EcoRI fragment. Several protease-minus mutants of X. c. campestris were obtained by Tn5 mutagenesis of pIJ3070 and marker exchange techniques. Studies of pathogenicity of these Tn5 mutants showed that the protease is not critically important for the pathogenicity of X. c. campestris on turnip plants but may play a minor role in disease development.Abbreviations Gm gentamicin - Km kanamycin - Rif rifampicin - Spc spectinomycin - Sm streptomycin - Tc tetracycline     

Genetic mapping of phe V, an Escherichia coli gene for tRNAPhe

Summary We report the physical and genetic mapping of pheV, an Escherichia coli gene for phenylalanine tRNA, to 64 min on the chromosomal map in the near vicinity of speC coding for ornithine decarboxylase.     

Escherichia coli cis- and trans-acting mutations that increaseglyA gene expression

We used anEscherichia coli strain blocked in serine biosynthesis and carrying a partialglyA deletion to isolate strains with altered regulation of theglyA gene. TheglyA deletion results in 25% of the normal serine hydroxymethyltransferase activity. Three classes of mutants with increasedglyA expression were isolated on glycine supplemented plates. One class of mutations increasedglyA expression 10-fold by directly altering the – 35 consensus sequence of theglyA promoter. The two other classes increasedglyA expression about 2- and 6-fold, respectively. The latter two classes of mutations also affected regulation of themetE gene of the folate branch of the methionine pathway, but notmetA in the nonfolate branch of the methionine pathway, or thegcv operon, encoding the glycine cleavage enzyme system. The mutations were mapped to about minute 85.5 on theE. coli chromosome.     

A new Escherichia coli gene, fdrA, identified by suppression analysis of dominant negative FtsH mutations

An Escherichia coli membrane protein, FtsH, has been implicated in several cellular processes, including integration of membrane proteins, translocation of secreted proteins, and degradation of some unstable proteins. However, how it takes part in such diverse cellular events is largely unknown. We previously isolated dominant negative ftsH mutations and proposed that FtsH functions in association with some other cellular factor(s). To test this proposal we isolated multicopy suppressors of dominant negative ftsH mutations. One of the multicopy suppressor clones contained an N-terminally truncated version of a new gene that was designated fdrA. The FdrA fragment suppressed both of the phenotypes — increased abnormal translocation of a normally cytoplasmic domain of a model membrane protein and retardation of protein export — caused by dominant negative FtsH proteins. The intact fdrA gene (11.9 min on the chromosome) directed the synthesis of a 60 kDa protein in vitro.     

Cloning and expression of a Bacillus cellulase gene in Escherichia coli

A Bacillus cellulase gene coding for carboxymethylcellulase (CMCase) has been cloned in Escherichia coli using pBR 322 as a vector. The gene was expressed independently of its orientation in the cloning vector showing enzyme activity 40 times greater than that produced by the original Bacillus species. The high production of CMCase in E. coli by the foreign gene did not impede growth of the host cells and the E. coli produced CMCase responded to various pH values and temperatures in the same way as that produced by the gene donor cells.     

狐、貉源大肠杆菌分离鉴定及毒力基因检测

[背景]狐、貉肺炎频发导致养殖户遭受巨大经济损失.[目的]调查黑龙江省、吉林省、河北省、山东省等地狐、貉肺炎原因,对病原菌进行分离、鉴定及部分生物学特性研究.[方法]无菌采集患病狐、貉肺组织进行细菌分离培养;对分离菌进行革兰氏染色、生化试验、特异性基因PCR鉴定、药物纸片扩散法敏感性试验、毒力基因检测及小鼠致病性试验....     

Regulation of the phosphate regulon of Escherichia coli: Characterization of the promoter of the pstS gene

Summary The pstS gene belongs to the phosphate regulon whose expression is induced by phosphate starvation and regulated positively by the PhoB protein. The phosphate (pho) box is a consensus sequence shared by the regulatory regions of the genes in the pho regulon. We constructed two series of deletion mutations in a plasmid in vitro, with upstream and downstream deletions in the promoter region of pstS, which contains two pho boxes in tandem, and studied their promoter activity by connecting them with a promoterless gene for chloramphenicol acetyltransferase. Deletions extending into the upstream pho box but retaining the downstream pho box greatly reduced promoter activity, but the remaining activity was still regulated by phosphate levels in the medium and by the PhoB protein, indicating that each pho box is functional. No activity was observed in deletion mutants which lacked the remaining pho box or the-10 region. Therefore, the pstS promoter was defined to include the two pho boxes and the-10 region. The PhoB protein binding region in the pstS regulatory region was studied with the deletion plasmids by a gelmobility retardation assay. The results suggest the protein binds to each pho box on the pstS promoter. A phoB deletion mutant was constructed, and we demonstrated that expression of pstS was strictly dependent on the function of the PhoB protein.     

SlyA, a regulatory protein from Salmonella typhimurium, induces a haemolytic and pore-forming protein in Escherichia coli

A chromosomal fragment from Salmonella typhimurium, when cloned in Escherichia coli, generates a haemolytic phenotype. This fragment carries two genes, termed slyA and slyB. The expression of slyA is sufficient for the haemolytic phenotype. The haemolytic activity of E. coli carrying multiple copies of slyA is found mainly in the cytoplasm, with some in the periplasm of cells grown to stationary phase, but overexpression of SlyB, a 15 kDa lipoprotein probably located in the outer membrane, may lead to enhanced, albeit unspecific, release of the haemolytic activity into the medium. Polyclonal antibodies raised against a purified SlyA-HlyA fusion protein identified the over-expressed monomeric 17 kDa SlyA protein mainly in the cytoplasm of E. coli grown to stationary phase, although smaller amounts were also found in the periplasm and even in the culture supernatant. However, the anti-SlyA antibodies reacted with the SlyA protein in a periplasmic fraction that did not contain the haemolytic activity. Conversely, the periplasmic fraction exhibiting haemolytic activity did not contain the 17 kDa SlyA protein. Furthermore, S. typhimurium transformed with multiple copies of the slyA gene did not show a haemolytic phenotype when grown in rich culture media, although the SlyA protein was expressed in amounts similar to those in the recombinant E. coli strain. These results indicate that SlyA is not itself a cytolysin but rather induces in E. coli (but not in S. typhimurium) the synthesis of an uncharacterised, haemolytically active protein which forms pores with a diameter of about 2.6 nm in an artificial lipid bilayer. The SlyA protein thus seems to represent a regulation factor in Salmonella, as is also suggested by the similarity of the SlyA protein to some other bacterial regulatory proteins. slyA- and slyB-related genes were also obtained by PCR from E. coli, Shigella sp. and Citrobacter diversus but not from several other gram-negative bacteria tested.     

米曲霉（Aspergillus oryzae）果胶酸内切水解酶（PGA）在原核系统中重组表达

果胶酶具有广阔的商业用途,在食品工业上主要用于果汁和酒类的澄清、提高植物油的提取率、提高水果的硬度和植物纤维脱胶。米曲霉（Aspergillus oryzae）一直用于传统发酵食品的生产,自然条件下其果胶酶的产量较低。文献报道的果胶酶的重组表达成功的例子较少,且活性较低。通过RT-PCR 的方法,获得不含信号肽的果胶酸内切水解酶A(polygalacturonase A, PGA) 的cDNA,PGA cDNA 连入pET-28a (+)载体, 构建 pET-28a (+)-pga 质粒。pET-28a (+)-pga 转化Turner (DE3) placⅠ细胞,得到转化子pET-28a (+)-pga-Turner (DE3) placⅠ,首次实现了米曲霉PGA在大肠杆菌系统中过表达,进一步对PGA在大肠杆菌系统中表达的条件进行了研究。在37℃、220 r/min条件培养pET-28a (+)-pga-Turner (DE3) placⅠ细胞,OD₆₀₀至 0.8左右时,用500μmol/L isopropyl β-D-thiogalactogalactopyranoside (IPTG)进行诱导表达,在15℃和170r/min条件下继续培养24 h,表达效果最好,相对于每毫升培养基而言,产酶可达到70u/mL,是米曲霉自然条件产酶量的87.5倍,远优于文献报道的重组表达的PGA酶活     

米曲霉（Aspergillus oryzae）果胶酸内切水解酶（PGA）在原核系统中重组表达

果胶酶具有广阔的商业用途,在食品工业上主要用于果汁和酒类的澄清、提高植物油的提取率、提高水果的硬度和植物纤维脱胶。米曲霉（Aspergillus oryzae）一直用于传统发酵食品的生产,自然条件下其果胶酶的产量较低。文献报道的果胶酶的重组表达成功的例子较少,且活性较低。通过RT-PCR 的方法,获得不含信号肽的果胶酸内切水解酶A(polygalacturonase A, PGA) 的cDNA,PGA cDNA 连入pET-28a (+)载体, 构建 pET-28a (+)-pga 质粒。pET-28a (+)-pga 转化Turner (DE3) placⅠ细胞,得到转化子pET-28a (+)-pga-Turner (DE3) placⅠ,首次实现了米曲霉PGA在大肠杆菌系统中过表达,进一步对PGA在大肠杆菌系统中表达的条件进行了研究。在37℃、220 r/min条件培养pET-28a (+)-pga-Turner (DE3) placⅠ细胞,OD₆₀₀至 0.8左右时,用500μmol/L isopropyl β-D-thiogalactogalactopyranoside (IPTG)进行诱导表达,在15℃和170r/min条件下继续培养24 h,表达效果最好,相对于每毫升培养基而言,产酶可达到70u/mL,是米曲霉自然条件产酶量的87.5倍,远优于文献报道的重组表达的PGA酶活     

Cloning of the histidine, pyrimidine and cysteine genes of Azospirillum brasilense: Expression of pyrimidine and three clustered histidine genes in Escherichia coli

Summary A gene bank from Azospirillum brasilense, Sp6 strain, was constructed in Escherichia coli in plasmid pRK290 and was used to identify Azospirillum genes. Clones carrying the his1, his2, pyr and cys1 genes were identified by genetic complementation and the expression of A. brasilense his1, his2 and pyr genes in E. coli was demonstrated. By E. coli complementation experiments, a cluster of three genes for the histidine biosynthetic pathway in A. brasilense has also been found, suggesting the existence of an operon-like unit.     

Expression of the recA gene of Escherichia coli in several species of gram-negative bacteria

Summary A broad host range plasmid containing an operon fusion between the recA and lacZ genes of Escherichia coli was introduced into various aerobic and facultative gram-negative bacteria — 30 species belonging to 20 different genera — to study the expression of the recA gene after DNA damage. These included species of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Vibrionaceae, Neisseriaceae, Rhodospirillaceae and Azotobacteraceae. Results obtained show that all bacteria tested, except Xanthomonas campestris and those of the genus Rhodobacter, are able to repress and induce the recA gene of E. coli in the absence and in the presence of DNA damage, respectively. All these data indicate that the SOS system is present in bacterial species of several families and that the LexA-binding site must be very conserved in them.     

Molecular cloning of a maltose transport gene from Bacillus stearothermophilus and its expression in Escherichia coli K-12

Genes responsible for maltose utilization from Bacillus stearothermophilus ATCC7953 were cloned in the plasmid vector pBR325 and functionally expressed in Escherichia coli. The 4.2 kb Bacillus DNA insert in clone pAM1750 suppressed the growth defects on maltose caused by mutations in E. coli maltose transport genes (malE, malK or complete malB deletion) but not mutations in genes affecting intracellular maltose metabolism (malA region). Transport studies in E. coli and B. stearothermophilus suggested that pAM1750 codes for a high affinity transport system, probably one of two maltose uptake systems found in B. stearothermophilus ATCC7953. Nucleotide sequence analysis of a 3.6 kb fragment of pAM 1750 revealed three open reading frames (ORFs). One of the ORFs, malA, encoded a putative hydrophobic protein with 12 potential transmembrane segments. MalA showed amino acid sequence similarity to proteins in the superfamily containing LacY lactose permease and also some similarity to MaIG protein, a member of a binding protein-dependent transport system in E. coli. The products of two other ORFs were not hydrophobic, did not show similarity to other known sequences and were found not to be essential for maltose utilization in transport-defective E. coli mutants. Hence MalA protein was the only protein necessary for maltose transport, but despite giving a detectable but low level of transport function in E. coli, the protein was very poorly expressed and could not be identified.