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.     

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.     

Identification in vivo of promoter activity in the left site of the att region of bacteriophage lambda DNA

The promoter-probing vector (pSK plasmid) was explored for cloning of the fragments from lambda cI857 and lambda b2 DNAs containing different regions of the att site. We have constructed all-tet fusions where the fusions are: 1) HindIII/BamHI-491 base pairs (b. p.) fragment of lambda cI857 DNA containing POP' site (plasmid pSK-PP'); 2) AluI-242 b. p. fragment of lambda cI857 DNA containing the left arm of the POP' site (plasmid pSK-P); 3) AluI-242 b. p. fragment of lambda cI857 DNA with opposite orientation (plasmid pSK-P); 4) EcoRI/BamHI-750 b. p. fragment of lambda b2 DNA containing the right arm of the POP' site (plasmid pSK-P'). These fusions permit us to analyse the effect of various pieces of the attachment site on the expression tet gene as the result of reparation of this gene promoter. We find that expression of tet (tetracycline resistant phenotype) takes place in the pSK-PP' and pSK-P but not in the pSK-P' and pSK-P. These facts permit us to conclude that the left arm of the att site contains a rightward promoter functioning in vivo. We postulate that this promoter activity might correspond to the promoter patt, which was described in previous experiments in vitro.     

pHG276: a multiple cloning site pBR322 copy number vector expressing a functional lac alpha peptide from the bacteriophage lambda PR promoter

The bacteriophage lambda early promoter PR has been used to direct the synthesis of lac alpha in a plasmid containing the multiple cloning site of pUC8. The copy number of the plasmid is controlled by the rop(rom) gene and the plasmid incorporates the cI857 gene for temperature regulation of lac alpha expression. Isolation of recombinant derivatives with DNA inserts in the multiple cloning region can be identified by insertional inactivation of lac alpha and consequently, a Lac- phenotype in a host carrying the M15 deletion of lac. The potential of pHG276 as a fully regulated expression vector is examined.     

Cloning of xylanase gene of Streptomyces flavogriseus in Escherichia coli and bacteriophage lambda-induced lysis for the release of cloned enzyme.

The xylanase gene of Streptomyces flavogriseus was cloned in pUC8 plasmid and expressed in Escherichia coli lysogenic for lambda cI857. lambda-Induced lysis of E. coli at 42 degrees C allowed efficient release of cloned enzyme activity in extracellular environment. The xylanase gene was located in the 0.8-kb HindIII fragment and coded for 18,000 Mr xylanase.     

Temperature-inducible amber suppressor: construction of plasmids containing the Escherichia coli serU- (supD-) gene under control of the bacteriophage lambda pL promoter.

An Escherichia coli DNA fragment containing the structural gene serU132 for the nonsense suppressor tRNASer2am was identified and purified by being cloned into a plasmid vector. Information obtained from DNA sequence analysis was used to select a serU132 fragment for insertion downstream from the bacteriophage lambda pL promoter in two pBR322-lambda derivatives. In nonsense mutant strains bearing the resulting serU132 hybrid plasmids, the presence of the lambda cI857 repressor gene carried on the same plasmid or in a prophage genome permits thermal regulation of suppressor synthesis.     

New cloning vectors for integration in the lambda attachment site attB of the Escherichia coli chromosome.

A set of plasmid cloning vectors has been constructed, allowing the integration of any DNA fragment into the bacteriophage lambda attachment site attB of the Escherichia coli chromosome. The system is based upon two components: (i) a number of cloning vectors containing the lambda attachment site attP and (ii) a helper plasmid, bearing the lambda int gene, transcribed from the lambda PR promoter under the control of the temperature-sensitive repressor cI857. The DNA fragment of interest is cloned into the multicloning site of one of the attP-harboring plasmids. Subsequently, the origin of the plasmid, located on a cloning cassette, is cut out and the DNA becomes newly ligated, resulting in a circular DNA molecule without replication ability. The strain of choice, containing the int gene carrying helper plasmid, is transformed with this DNA molecule and incubated at 42 degrees C to induce int gene expression. Additionally, the temperature shift leads to the loss of the helper plasmid after a few cell generations, because the replication ability of its replicon is blocked at 42 degrees C. These vectors have been successfully used for integration of several promoter-lacZ fusions into the chromosome. The ratio between integration due to homologous recombination and Int protein-mediated integration has been determined.     

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.     

Efficient production of native, biologically active human cystatin C by Escherichia coli

A cDNA encoding the mature human cysteine proteinase inhibitor cystatin C was fused to the coding sequence for the Escherichia coli outer membrane protein A signal peptide, and the recombinant gene was expressed in E. coli under the control of the lambda PR promoter, an optimized Shine-Dalgarno sequence and the lambda cI 857 repressor. When induced at 42 degrees C, such cells expressed large amounts of recombinant cystatin C. The recombinant protein was isolated in high yield and characterized. All physicochemical properties investigated, including the positions of disulfide bonds, indicated that the E. coli derived cystatin C was identical to cystatin C isolated from human biological fluids, except that the proline residue in position three was not hydroxylated. The recombinant protein displayed full biological activity against papain, cathepsin B and dipeptidyl peptidase I.     

A broad-host-range expression vector based on the pL promoter of coliphage lambda: regulated synthesis of human interleukin 2 in Erwinia and Serratia species.

We report the construction of a broad-host-range expression vector based on an RSF1010-derived replicon. The vector carries the strong leftward promoter (pL) of coliphage lambda as well as the cI857 allele, which codes for a thermolabile repressor protein. The coding region of mature human interleukin 2, which is preceded by the ner ribosome binding site of phage Mu, was cloned downstream from the pL promoter. The plasmid was introduced into Erwinia and Serratia species by means of mobilization. Heat-inducible synthesis of interleukin 2 protein was obtained, showing that the pL promoter is functional in these genera. As in Escherichia coli, the bulk of the overproduced protein was present in an insoluble form.     

Analysis of the Escherichia coli proBA locus by DNA and protein sequencing

A 2.9 kb DNA fragment carrying the Escherichia coli proBA region, which encodes the first two enzymes of the proline biosynthetic pathway, was subcloned onto an expression plasmid carrying both the bacteriophage lambda PL promoter (lambda PL) and the lambda gene encoding a thermolabile cI repressor protein (cI857). Derepression of the lambda PL promoter by thermal inactivation of the cI857 repressor protein resulted in the simultaneous overproduction of the proB (gamma-glutamyl kinase) and proA (gamma-glutamyl phosphate reductase) gene products. Nucleotide sequence analysis of the proBA locus allowed gene assignments consistent with the NH2 and COOH-terminal analyses and amino acid compositions of homogeneous preparations of the proB and proA proteins. The contiguous nature of the proB and proA genes suggests that the two genes constitute an operon in which proB precedes proA.     

Heat-inducible autolytic vector for high-throughput screening

In directed evolution, a high-throughput screening system is often a prerequisite for sampling the enzyme variants. When the target enzyme is expressed intracellularly, for example when Escherichia coli is used as the host, chemical or enzymatic disruption of cell membrane is often required in many cases, which can be tedious, time-consuming, and costly. In this study, a set of heat-inducible autolytic vectors were constructed to solve this problem, in which the SRRz lysis gene cassette from bacteriophage lambda was placed downstream of heat-inducible promoters, lambda cI857/pR promoter and its mutant, c1857/pR(M). The artificial autolytic units were inserted into the backbone of pUC18 (away from the multiple cloning sites). For the wild promoter; cI857/pR, the SRRz lysis cassette was expressed by temperature up-shift from 28 degrees to 38 degrees C, and the lysis efficiency of transformed bacterial cells was found to be consistent and could reach 96.3% as measured by the reporter beta3-galactosidase assay. In order to obtain a higher cell growth rate, the mutant promoter cI857/pR(M) was utilized to allow bacteria growth at 35 degrees C and lysis at 42 degrees C. However; this heat-inducible system showed significant inconsistency in terms of lysis efficiency. Bacillus subtilis 168 lipase A gene was further inserted into the multiple cloning sites of the autolytic vector containing cI857/pR, and 93.7% of the expressed lipase activity was found in the culture medium upon heat induction, demonstrating the utility of the vector for expression and rapid extracellular assay of heterologous enzymes.     

Specialized lambda transducing bacteriophage which carries hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase.

A number of specialized lambda transducing bacteriophages which carry the Escherichia coli gene guaB were isolated from E. coli. One of these bacteriophages, lambda cI857 Sam7 d guaB-2, also carries hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase (EC 6.1.1.21). Histidyl-transfer ribonucleic acid synthetase activities in induced and uninduced lysogens carrying lambda d guaB-2 indicate that the phage carries the entire structural gene and that the gene is under the control of an E. coli promoter. These conclusions were confirmed by the in vivo production of a protein encoded by the phage which comigrates with authentic histidyl-transfer ribonucleic acid synthetase on two-dimensional polyacrylamide gels.     

嗜盐古菌启动子DNA片段的功能检测

将来源于嗜盐古菌染色体DNA的启动子片段RM07或RM13插入到启动子探针载体pYLZ_2的报告基因lacZ之前,通过β_半乳糖苷酶酶活性的检测,进一步确证RM07和RM13片段在大肠杆菌(Escherichia coli)中的启动功能。同时用微量热技术检测了大肠杆菌DH5α及其重组菌株在LB培养基中37℃生长过程的热输出功率。T2(pYLZ_2)、TE07(pYL726)、TE07_2(pYL702)、TE131(pYL131)和TE132(pYL132)菌株的生长速率分别比大肠杆菌DH5α降低了6.5%、11%4、1.1%4、7.5%和42.7%。当启动子启动了基因表达时,菌株的生长速率显著降低,热力学参数与酶活性检测结果有较好的一致性。微量热结果表明基因的表达比质粒DNA的复制过程需要消耗更多的能量,对细菌的生理代谢有较大改变。微量热技术为检测基因的表达和转录调控提供了新的方法和思路。