枯草芽孢杆菌耐盐突变株proA基因克隆及proBA基因渗透压调节功能研究

利用TaKaRaLAPCRTM试剂盒扩增枯草芽孢杆菌 931 5 1耐盐突变株proA基因的未知下游序列。根据测序结果 ,设计引物 ,克隆出发菌株和突变株全长proBA基因。将出发菌株和突变株的proBA基因分别转化大肠杆菌JM83(proBA- ) ,均能够与其功能互补。SDS PAGE分析其表达产物 ,有两条分子量分别约为 4 0kD和 4 5kD的新蛋白带出现。测定 4种转化子 (分别含有出发菌株和突变株proB基因的大肠杆菌 1 1 2 5 2转化子及proBA基因的大肠杆菌JM83转化子 )的耐盐能力。发现含有突变株proB或proBA基因转化子的耐盐能力 ,均比相应的含有出发菌株proB或proBA基因的转化子高。另外含有出发菌株和突变株的proBA基因转化子的耐盐能力 ,也均比相应的仅含proB基因的转化子高 ,表明枯草芽孢杆菌的ProA比大肠杆菌的ProA更为有效。测定所有JM83转化子胞内自由脯氨酸 ,发现其含量随盐浓度的上升而提高 ,其中含突变菌株proBA基因的转化子提高更为显著     

A 'phase-shift' fusion system for the regulation of foreign gene expression by lambda repressor in gram-negative bacteria

A 'phase-shift' translation fusion vector was constructed in which mutually compatible restriction sites BamHI, BclI and BglII are positioned in such a manner that the cut point is in a different reading frame, immediately following the ATG start codon and ribosome-binding site of the lambda cro gene. The lambda cro gene is expressed from promoter pR and controlled by a thermosensitive (cI857) lambda repressor. The usefulness of the expression vector was demonstrated using a galK gene lacking the ATG start codon and fusing this to the pR promoter and ATG start codon of the lambda cro gene, resulting in cI857-regulated expression of galactokinase. The vector is of general use for foreign gene expression in Escherichia coli when the target gene has a compatible cohesive end (5'-GATC-3') at the N terminus (provided, for example, by a BamHI linker). The lambda cI857-pR-cro-galK cassette was cloned into pJRD215, a wide-host-range plasmid and transferred by conjugation to a variety of Gram-negative bacteria. In all cases, thermosensitive regulation of galactokinase could be demonstrated, though the levels of induction varied considerably. These results show that the powerful lambda pR promoter and the efficient lambda repressor can be used to regulate expression of foreign genes in Gram-negative organisms other than E. coli.     

Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase. Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product

The Escherichia coli gene for folylpolyglutamate synthetase-dihydrofolate synthetase was localized to plasmids pLC22-45, 24-31, and 28-44 of the Clarke-Carbon E. coli colony bank (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99) by screening the bank by replica mating with an E. coli folC mutant. The folC gene was subcloned from pLC22-45 and inserted into a high copy number plasmid containing the lambda replication control region under the control of the temperature-sensitive cI857 repressor and into a high expression plasmid containing the lambda PL promoter and the cI857 repressor. The folC structural gene was located on a 1.52-kilobase PvuI fragment, sufficient to code for a protein of maximum Mr 55,000. E. coli transformants containing the recombinant plasmids, when induced by culturing at 42 degrees C, had folylpolyglutamate synthetase and dihydrofolate synthetase levels that were 100- to 400-fold higher than in wild type strains and which represented up to 4% of the soluble cell protein. The E. coli folylpolyglutamate synthetase-dihydrofolate synthetase has been purified to homogeneity from the transformants. Both activities are catalyzed by a single protein of Mr 47,000. Some kinetic properties of the enzymes and a new spectrophotometric method for assaying dihydrofolate synthetase activity are described.     

渗透压调节基因proBA的融合表达对大肠杆菌耐高渗胁迫能力的影响

枯草芽孢杆菌 9315 1的渗透压调节基因proB和proA以重叠基因的方式组织 ,但是表达两个单独的蛋白质ProB和ProA。通过引物设计 ,在抗脯氨酸反馈抑制耐盐突变菌株 9315 1 14的proBA基因重叠区引入一个限制性酶切位点 ,分别扩增出proB和proA基因 ,并构建融合的proBA基因。SDS PAGE分析显示有一条新的分子质量约为85kD的蛋白带出现。相对于表达未融合的proB和proA ,proBA融合基因的表达明显提高了宿主菌大肠杆菌JM83胞内游离脯氨酸含量和其耐高渗胁迫能力     

Expression of Bacillus subtilis proBA genes and reduction of feedback inhibition of proline synthesis increases proline production and confers osmotolerance in transgenic Arabidopsis

Proline accumulation has been shown to correlate with tolerance to drought and salt stresses in plants. We attempt to introduce the wild-type, mutant, and fusion proBA genes derived from Bacillus subtilis into Arabidopsis thaliana under the control of a strong promoter cauliflower mosaic virus 35S (CaMV35S). The transgenic plants produced higher level of free proline than control and the overproduction of proline resulted in the increased tolerance to osmotic stress in transgenic plants. Besides, the mutation in proBA genes, which were proved to lead gamma-glutamyl kinase (gamma-GK) reduces sensitivity to the end-product inhibition and the fusion of proB and proA also result in increasing proline production and confer osmotolerance in transgenic lines.     

Genetic and physical characterization of proBA genes of the marine bacterium Vibrio parahaemolyticus.

Intracellular proline pools have been implicated in the halotolerance of many organisms. To examine this relationship in a moderately halotolerant marine bacterium, Vibrio parahaemolyticus, proline biosynthesis genes were cloned in various plasmids. Some genetic and structural properties of those genes were examined. Subcloning showed that about 3.1 kilobases of V. parahaemolyticus DNA could complement proA and proB but not proC mutations of Escherichia coli. The same fragment would also complement some Pro- mutants of V. parahaemolyticus. Gamma-delta insertion mutagenesis of this subcloned fragment indicated that proB and proA genes of V. parahaemolyticus might be transcribed from different promoters. Two other genes, phoE and gpt, which map closely to the proBA genes in E. coli, were also found to be in close proximity to the proBA genes of V. parahaemolyticus.     

Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene E expression in Escherichia coli

Cell lysis of Gram-negative bacteria can be efficiently achieved by expression of the cloned lysis gene E of bacteriophage PhiX174. Gene E expression is tightly controlled by the rightward lambda pR promoter and the temperature-sensitive repressor cI857 on lysis plasmid pAW12. The resulting empty bacterial cell envelopes, called bacterial ghosts, are currently under investigation as candidate vaccines. Expression of gene E is stringently repressed at temperatures up to 30 degrees C, whereas gene E expression, and thus cell lysis, is induced at temperatures higher than 30 degrees C due to thermal inactivation of the cI857 repressor. As a consequence, the production of ghosts requires that bacteria have to be grown at 28 degrees C before the lysis process is induced. In order to reflect the growth temperature of pathogenic bacteria in vivo, it seemed favorable to extend the heat stability of the lambda pR promoter/cI857 repressor system, allowing pathogens to grow at 37 degrees C before induction of lysis. In this study we describe a mutation in the lambda pR promoter, which allows stringent repression of gene E expression at temperatures up to 36 degrees C, but still permits induction of cell lysis at 42 degrees C.     

Genetic and physical characterization of proBA genes of the marine bacterium Vibrio parahaemolyticus

Intracellular proline pools have been implicated in the halotolerance of many organisms. To examine this relationship in a moderately halotolerant marine bacterium, Vibrio parahaemolyticus, proline biosynthesis genes were cloned in various plasmids. Some genetic and structural properties of those genes were examined. Subcloning showed that about 3.1 kilobases of V. parahaemolyticus DNA could complement proA and proB but not proC mutations of Escherichia coli. The same fragment would also complement some Pro- mutants of V. parahaemolyticus. Gamma-delta insertion mutagenesis of this subcloned fragment indicated that proB and proA genes of V. parahaemolyticus might be transcribed from different promoters. Two other genes, phoE and gpt, which map closely to the proBA genes in E. coli, were also found to be in close proximity to the proBA genes of V. parahaemolyticus.     

Cloning, expression, and purification of gene 3 endonuclease from bacteriophage T7

The structural gene for the single-stranded endonuclease coded for by gene 3 of bacteriophage T7 has been cloned in pGW7, a derivative of the plasmid pBR322, which contains the lambda PL promoter and the gene for the temperature-sensitive lambda repressor, cI857. The complete gene 3 DNA sequence has been placed downstream of the PL promoter, and the endonuclease is overproduced by temperature induction at mid-log phase of Escherichia coli carrying the recombinant plasmid pTP2. Despite the fact that cell growth rapidly declines due to toxic effects of the excess endonuclease, significant amounts of the enzyme can be isolated in nearly homogeneous form from the induced cells. An assay of nuclease activity has been devised using gel electrophoresis of the product DNA fragments from DNA substrates. These assays show the enzyme to have an absolute requirement for Mg(II) (10 mM), a broad pH optimum near pH 7, but significant activity from pH 3 to pH 9, and a 10-100-fold preference for single-stranded DNA (ssDNA). The enzyme is readily inactivated by ethylenediaminetetraacetic acid or high salt. The differential activity in favor of ssDNA can be exploited to map small single-stranded regions in double-stranded DNAs as shown by cleavage of the melted region of an open complex of T7 RNA polymerase and its promoter.     

Construction of an E. coli strain overproducing the Tn10-encoded TET repressor and its use for large scale purification.

Overproduction of the repressor protein from the Tn10-encoded tetracycline resistance operon is achieved by placement of the respective gene under control of bacteriophage lambda promoter PL in a vector-host system. All cloning steps have to be carried out under repressed conditions to assure survival of the cell. The cI 857 mutation is used to control expression of the tetR gene in large scale fermentation. After induction, the overproducing Escherichia coli strain continues to grow for 2.5 generations before growth terminates. In the expression phase, active TET repressor comprises up to 13% of the total soluble protein. A procedure is described to purify the TET repressor protein to homogeneity on a large scale. Starting from a 10 litre culture, approximately 250 mg of homogeneous, active TET repressor are obtained. The amino acid sequence of the N and C termini are in agreement with the gene start and stop determined from the nucleotide sequence of the Tn10 tetR gene.     

Identification and disruption of the proBA locus in Listeria monocytogenes: role of proline biosynthesis in salt tolerance and murine infection

Intracellular accumulation of the amino acid proline has previously been linked to the salt tolerance and virulence potential of a number of bacteria. Taking advantage of the proBA mutant Escherichia coli CSH26, we identified a listerial proBA operon coding for enzymes functionally similar to the glutamyl kinase (GK) and glutamylphosphate reductase (GPR) enzyme complex which catalyzes the first and second steps of proline biosynthesis in E. coli. The first gene of the operon, proB, is predicted to encode GK, a 276-residue protein with a calculated molecular mass of 30.03 kDa and pl of 5.2. Distal to the promoter and overlapping the 3' end of proB by 17 bp is proA, which encodes GPR, a 415-residue protein with a calculated molecular mass of 45.50 kDa (pl 5.3). Using this information, we created a chromosomal deletion mutant by allelic exchange which is auxotrophic for proline. This mutant was used to assess the contribution of proline anabolism to osmotolerance and virulence. While inactivation of proBA had no significant effect on virulence in mouse assays (either perorally or intraperitoneally), growth at low (2 to 4% NaCl) and high (>6% NaCl) salt concentrations in complex media was significantly reduced in the absence of efficient proline synthesis. We conclude that while proline biosynthesis plays little, if any, role in the intracellular life cycle and infectious nature of Listeria monocytogenes, it can play an important role in survival in osmolyte-depleted environments of elevated osmolarity.     

Nucleotide sequence of bglC, the gene specifying enzymeIIbgl of the PEP:sugar phosphotransferase system in Escherichia coli K12, and overexpression of the gene product

The EnzymeIIbgl of the phosphoenolpyruvate- (PEP-) dependent phosphotransferase system catalyses the uptake and concomitant phosphorylation of beta-glucosides by Escherichia coli; it is specified by the gene bglC. The nucleotide sequence of a 3.6 kb HindIII restriction fragment spanning bglC, cloned on a plasmid, was determined. DNA analysis strongly suggests that the published order of this and other genes involved in beta-glucoside utilization, bgl C, S, B, is incorrect, and that the regulatory gene bglS may be located upstream of the structural genes bglC and bglB. From the deduced amino acid sequence it is predicted that the membrane protein specified by bglC consists of 625 amino acid residues (66.48 kDa). The protein has the hydropathic profile expected of an integral membrane protein (average hydropathy = 0.62). Comparisons between the amino acid sequences deduced for the EnzymeIIbgl and for the mannitol-specific EnzymeIImtl show that these proteins are related, and a little direct homology is apparent. A 2.3 kb AluI fragment spanning bglC was subcloned into an expression vector which carries the lambda PL promoter and then transformed into a host strain which produces thermolabile cI857 repressor and the anti-terminator N; thermoinduction resulted in the overproduction of a membrane protein and the appearance of Bgl activity.