外源基因在苏云金杆菌染色体上的定点整合及表达

[目的]研究构建稳定表达外源基因、无抗性标记基因的苏云金杆菌(Bacillus thuringiensis简称Bt)工程菌的方法.在构建Bt工程菌时,高拷贝外源质粒的转入导致Bt芽孢数量减少,芽孢形成期延滞,影响Bt菌株的杀虫活力.而且,外源质粒在Bt中的稳定性较差,外源基因容易丢失.将基因整合人染色体是一种构建遗传性状稳定、杀虫活力高的Bt工程菌的有效方法.[方法]本研究采用PCR技术,分两段扩增定位于Bt无晶体突变株XBU001染色体上的trigger factor基因片段作为同源臂,克隆入温度敏感型载体pKSV7,构建了定点整合载体pKTF12.并利用pKTF12质粒将crylAc基因定点整合入XBU001染色体上.[结果]利用载体pKTF12将crylAc定点插入triggerfactor位点,对宿主菌XBU001的正常生长没有影响.重组菌株KCTF12中的crylAc基因能够稳定遗传、表达并形成菱形晶体.与携带高拷贝外源质粒的Bt菌株HTX42相比较,KCTF12具有芽孢数量增多、芽孢形成期提前的优势.[结论]定点整合法是一种构建稳定表达外源基因、无抗性标记基因Bt工程菌的有效方法.     

Molecular cloning and characterization of a chemotactic transducer gene in Pseudomonas aeruginosa.

A Pseudomonas aeruginosa mutant, defective in taxis toward L-serine but responsive to peptone, was selected by the swarm plate method after N-methyl-N'-nitrosoguanidine mutagenesis. The mutant, designated PCT1, was fully motile but failed to show chemotactic responses to glycine, L-serine, L-threonine, and L-valine. PCT1 also showed weaker responses to some other commonly occurring L-amino acids than did the wild-type strain PAO1. A chemotactic transducer gene, denoted pctA (Pseudomonas chemotactic transducer A), was cloned by phenotypic complementation of PCT1. Nucleotide sequence analysis showed that the pctA gene encodes a putative polypeptide of 629 amino acids with a calculated mass of 68,042. A hydropathy plot of the predicted polypeptide suggested that PctA may be an integral membrane protein with two potential membrane-spanning regions. The C-terminal domain of PctA showed high homology with the enteric methyl-accepting chemotaxis proteins (MCPs). The most significant amino acid sequence similarity was found in the region of MCPs referred to as the highly conserved domain. The pctA gene was inactivated by insertion of a kanamycin resistance gene cassette into the wild-type gene, resulting in the same observed deficiency in taxis toward L-amino acids as PCT1. In vivo methyl labeling experiments with L-[methyl-3H]methionine showed that this knockout mutant lacked an MCP with a molecular weight of approximately 68,000.     

Molecular cloning and characterization of the rfc gene of Pseudomonas aeruginosa (serotype O5)

Previous work from our laboratory has shown that cosmid clone pFVl00, containing a 26 kb insert, is able to restore O-antigen synthesis in serotype O5 rough mutants of Pseudomonas aeruginosa. Mobilization of pFV100 into two P. aeruginosa semi-rough (SR) mutants, AK14O1 and rd7513, resulted in O-antigen expression, indicating that pFV100 may contain an O-polymerase (rfc) gene. pFV.TK6, a subclone of pFVl00 that contains a 5.6 kb chromosomal insert, was able to complement O-antigen expression in these SR mutants. Mutagenesis of pFV.TK6 using Tn1000 exposed a 1.5 kb region that was essential for complementing O-antigen expression in AK14O1. A 2.0 kb Xhol-HindIII fragment, containing this region, was cloned into vector pUCP26 and the resulting plasmid called pFV.TK8. In Southern analysis of the 20 P aeruginosa serotypes using a probe generated from the 1.5 kb Xhol fragment of pFV.TK8, the rfc probe hybridized to a common fragment of the cross-reactive O2-O5-O16-O18-O20 serogroup, suggesting that these serotypes may share a common O-polymerase gene. In functional studies of the rfc gene, the PAOl (serotype O5) chromosomal rfc was mutated using a gene-replacement strategy. These knockout mutants expressed the SR lipopolysaccharide (LPS) phenotype, which indicated that they were no longer producing a functional O-polymerase enzyme. Nucleotide sequence analysis of the insert DNA of pFV.TK8 revealed one open reading frame (ORF), designated ORF48.9, which could code for a 48.9 kDa protein. In comparisons of the P. aeruginosa rfc nucleotide and amino acid sequences with DNA and protein databases, no significant homology was found. However, the deduced structure of the P. aeruginosa Rfc protein indicated that it is very hydrophobic and contains 11 putative membrane-spanning domains. Therefore, the predicted structure is similar to that of other reported Rfc proteins. Furthermore, comparison of the amino acid composition and codon usage of the P. aeruginosa Rfc with other Rfc proteins revealed significant similarity between them.     

Tn5-mediated integration of the delta-endotoxin gene from Bacillus thuringiensis into the chromosome of root-colonizing pseudomonads

Gene replacement mediated by Tn5 sequences was used to integrate the Bacillus thuringiensis subsp. kurstaki HD-1 delta-endotoxin gene (tox) into the chromosome of two corn root-colonizing strains of Pseudomonas fluorescens. A Tn5 transposase deletion element containing the tox gene (delta Tn5-tox) was substituted for a Tn5 element previously present in the P. fluorescens chromosome. Two classes of delta Tn5-tox elements were made. The first class encodes kanamycin resistance in addition to the Tox protein, whereas the second class encodes only the Tox protein. Both classes of delta Tn5-tox elements can no longer transpose, owing to a 324-base-pair deletion in the transposase gene of IS50R, minimizing the potential for horizontal gene transfer of the tox gene to other bacterial species. A frameshift mutation in the transposase gene of IS50L was also constructed to eliminate the possibility of suppression or of a spontaneous reversion at the ochre termination codon that would create an active transposase. Expression of the Tox protein in P. fluorescens strains 112-12 and Ps3732-3-7 was demonstrated by an immunological assay (Western blot) and toxicity against larvae of the tobacco hornworm (Manduca sexta).     

棉花黄萎病真菌Verticillium dahliae木聚糖酶基因的克隆、表达和酶学性质分析

[目的]从棉花黄萎病真菌Verticillium dahliae中克隆木聚糖酶基因,并在毕赤酵母中进行异源表达,研究酶学性质.[方法]通过多序列比对设计简并引物,扩增出真菌V. dahliae木聚糖酶基因片段,再采用基因组步行PCR技术获得全长木聚糖酶基因序列.经BLAST比对并结合GT-AG原则分析,该基因含有一个大小为63 bp的内含子,利用DpnI介导的缺失方法对含内含子的全长木聚糖酶基因进行剪接,获得该基因的全长cDNA.将克隆到的cDNA在毕赤酵母GS115进行了表达,重组酶经纯化后进行酶学性质分析.[结果]BLAST比对显示,该cDNA推测的氨基酸序列和已知木聚糖酶的最高一致性为72%.测得该酶最适反应温度为45℃,最适反应pH值为6,在pH5-9维持50%以上的活性,对山毛榉材木聚糖具有最好的水解效果.Mg2 和Ca2 对酶有激活作用,分别提高了33.7%和16.6%,EDTA,β-巯基乙醇和NaN3对酶的活性基本没有影响,Tween-80和DMSO使酶活性提高了28.4%和12.8%.[结论]本文从引起棉花黄萎病的真菌V. dahliae中克隆到的木聚糖酶基因是在GenBank上登录的第一个来自棉花黄萎病真菌的木聚糖酶基因序列.本文所用的克隆方法可以高效的从植物病原真菌和白腐真菌克隆只含一个内含子的11家族的新木聚糖酶基因,避免了摸索原始菌株酶表达诱导条件,检测酶的活性等繁琐的操作.酶学性质分析显示该酶在低聚木糖的制备,面包改良上有潜在的应用价值.     

Molecular cloning and heterologous expression of a Klebsiella pneumoniae gene encoding alginate lyase

The alginate lyase (Aly; guluronate specific)-coding gene of Klebsiella pneumoniae was cloned using the cosmid vector pMMB33, transduced into Escherichia coli and expressed in this host. Four Aly-positive clones with unstable phenotypes were identified out of 700 kanamycin-resistant transductants. A stable derivative of one of the clones was studied further and contained 12.1-kb of insert DNA. The Aly-coding gene (aly), still partially under the control of its native promoter, was localised within a 1.95-kb HindIII fragment by transposon gamma delta mutagenesis and sub-cloning. Most of the Aly produced was secreted into the medium by both the original K. pneumoniae strain (71.7%) and the E. coli recombinant clones (85.1%). The enzyme from both K. pneumoniae and the E. coli clones had a pI of 8.9 and comprised a single 28-kDa polypeptide chain. Other minor bands were also observed on isoelectric focusing and these were attributed to processing intermediates of a single gene product. It is concluded that E. coli can recognise and process the signal peptide of Aly to produce a mature polypeptide that is identical to that synthesised by K. pneumoniae.     

Molecular cloning and characterization of the recA gene of Pseudomonas aeruginosa PAO.

The recA gene of Pseudomonas aeruginosa PAO has been isolated and introduced into Escherichia coli K-12. Resistance to killing by UV irradiation was restored in several RecA-E. coli K-12 hosts by the P. aeruginosa gene, as was resistance to methyl methanesulfonate. Recombination proficiency was also restored, as measured by HfrH-mediated conjugation and by the ability to propagate Fec-phage lambda derivatives. The cloned P. aeruginosa recA gene restored both spontaneous and mitomycin C-stimulated induction of lambda prophage in lysogens of a recA strain of E. coli K-12.     

Multiplicity of the integration sites of Mu-like bacteriophages into Pseudomonas aeruginosa chromosome and plasmids

It has been shown that D3112 prophage can be integrated into different chromosomal sites of Pseudomonas aeruginosa. The other Mu-like phages (B3, B39, PM69) are capable to insert their genomes during infection process into the plasmids RPL11, RMS148, RMS163. Their integration is occasionally accompanied by formation of mutations in plasmid genes. The certain types of auxotrophic and morphological mutants (thi, met, pigmented, met - pigmented) can be found at a frequency about 10% among survivors after a long (48 h) incubation at 42 degrees C of PAO (D3112cts15) or PAO (B39cts1) lysogens. The spectrum of mutants might depend on the time of heat induction. After a short exposure (10-20 min), arg and pigmented mutants can be found. Accumulation of certain kinds of mutants after heat induction is quite a specific phenomenon for Mu-like phages; heat induction of PAO (F116ts245) does not lead to selection of these specific bacterial mutants (F116 is unrelated to Mu-like phages and has extrachromosomal location).     

Protocols for yTREX/Tn5-based gene cluster expression in Pseudomonas putida

Bacterial gene clusters, which represent a genetic treasure trove for secondary metabolite pathways, often need to be activated in a heterologous host to access the valuable biosynthetic products. We provide here a detailed protocol for the application of the yTREX ‘gene cluster transplantation tool’: Via yeast recombinational cloning, a gene cluster of interest can be cloned in the yTREX vector, which enables the robust conjugational transfer of the gene cluster to bacteria like Pseudomonas putida, and their subsequent transposon Tn5-based insertion into the host chromosome. Depending on the gene cluster architecture and chromosomal insertion site, the respective pathway genes can be transcribed effectively from a chromosomal promoter, thereby enabling the biosynthesis of a natural product. We describe workflows for the design of a gene cluster expression cassette, cloning of the cassette in the yTREX vector by yeast recombineering, and subsequent transfer and expression in P. putida. As an example for yTREX-based transplantation of a natural product biosynthesis, we provide details on the cloning and activation of the phenazine-1-carboxylic acid biosynthetic genes from Pseudomonas aeruginosa in P. putidaKT2440 as well as the use of β-galactosidase-encoding lacZ as a reporter of production levels.     

Tn7 and Tn501 Insertions into Pseudomonas aeruginosa plasmid R91-5: mapping of two transfer regions.

We constructed a restriction endonuclease map of the Pseudomonas aeruginosa narrow-host-range plasmid R91-5. Insertions of transposons Tn7 and Tn501 into the plasmid DNA were characterized physically and genetically. The distribution of sites of insertion showed some regional specificity for the insertion of these transposons, especially TN501. The insertion of Tn7 was unusual in that all 42 of 43 insertions were in the same orientation. By relating phenotypic changes to the site of insertion, the Tn1 transposon that was already present on R91-5 and coded for carbenicillin resistance was mapped, and its orientation was determined. Two major transfer regions were identified. We believe that Tra1 is involved in conjugal DNA metabolism, whereas Tra2 is involved mainly in production of the sex pili.     

Molecular cloning, characterization, and regulation of a Pseudomonas pickettii PKO1 gene encoding phenol hydroxylase and expression of the gene in Pseudomonas aeruginosa PAO1c.

A 26-kilobase BamHI restriction endonuclease DNA fragment was cloned from Pseudomonas pickettii PKO1, a strain isolated from a soil microcosm that had been amended with benzene, toluene, and xylene. This DNA fragment, cloned into vector plasmid pRO1727 and designated pRO1957, allowed Pseudomonas aeruginosa PAO1c to grow on phenol as the sole source of carbon. Physical and functional restriction endonuclease maps have been derived for the cloned DNA fragment. Two DNA fragments carried in trans and derived from subclones of pRO1957 show phenol hydroxylase activity in cell extracts of P. aeruginosa. Deletion and subcloning analyses of these fragments indicated that the gene encoding phenol hydroxylase is positively regulated. Phenol and m-cresol were shown to be inducers of the enzyme. o-Cresol and p-cresol did not induce enzymatic activity but could be metabolized by cells that had been previously exposed to phenol or m-cresol; moreover, the enzyme exhibited a rather broad substrate specificity and was sensitive to thiol-inhibiting reagents. A novel polypeptide with an estimated molecular mass of 80,000 daltons was detected in extracts of phenol-induced cells of P. aeruginosa carrying plasmid pRO1959.     

Tn5 insertion mutants of Pseudomonas aeruginosa deficient in surface expression of ferripyochelin-binding protein.

Transposon (Tn5) insertion mutants were isolated in Pseudomonas aeruginosa PAO. These mutants were screened for expression of the ferripyochelin-binding protein with monoclonal antibody in a whole-cell immunoblot assay. Fourteen mutants were identified which did not express ferripyochelin-binding protein on the cell surface. These mutants did not take up 59Fe-labeled pyochelin and grew slowly in the presence of iron chelators.     

Molecular cloning and heterologous expression of progesterone 5beta-reductase from Digitalis lanata Ehrh

A full-length cDNA clone that encodes progesterone 5beta-reductase (5beta-POR) was isolated from Digitalis lanata leaves. The reading frame of the 5beta-POR gene is 1170 nucleotides corresponding to 389 amino acids. For expression, a Sph1/Sal1 5beta-POR fragment was cloned into the pQE vector and was transformed into Escherichia coli strain M15[pREP4]. The recombinant gene was functionally expressed and the recombinant enzyme was characterized. The K(m) and v(max) values for the putative natural substrate progesterone were calculated to be 0.120 mM and 45 nkat mg(-1) protein, respectively. Only 5beta-pregnane-3,20-dione but not its alpha-isomer was formed when progesterone was used as the substrate. Kinetic constants for cortisol, cortexone, 4-androstene-3,17-dione and NADPH were also determined. The molecular organization of the 5beta-POR gene in D. lanata was determined by Southern blot analysis. The 5beta-POR is highly conserved within the genus Digitalis and the respective genes and proteins share considerable homology to putative progesterone reductases from other plant species.     

Cyclohexadienyl dehydratase from Pseudomonas aeruginosa. Molecular cloning of the gene and characterization of the gene product.

The gene encoding cyclohexadienyl dehydratase (denoted pheC) was cloned from Pseudomonas aeruginosa by functional complementation of a pheA auxotroph of Escherichia coli. The gene was highly expressed in E. coli due to the use of the high-copy number vector pUC18. The P. aeruginosa cyclohexadienyl dehydratase expressed in E. coli was purified to electrophoretic homogeneity. The latter enzyme exhibited identical physical and biochemical properties as those obtained for cyclohexadienyl dehydratase purified from P. aeruginosa. The activity ratios of prephenate dehydratase to arogenate dehydratase remained constant (about 3.3-fold) throughout purification, thus demonstrating a single protein having broad substrate specificity. The cyclohexadienyl dehydratase exhibited Km values of 0.42 mM for prephenate and 0.22 mM for L-arogenate, respectively. The pheC gene was 807 base pairs in length, encoding a protein with a calculated molecular mass of 30,480 daltons. This compares with a molecular mass value of 29.5 kDa determined for the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since the native molecular mass determined by gel filtration was 72 kDa, the enzyme probably is a homodimer. Comparison of the deduced amino acid sequence of pheC from P. aeruginosa with those of the prephenate dehydratases of Corynebacterium glutamicum, Bacillus subtilis, E. coli, and Pseudomonas stutzeri by standard pairwise alignments did not establish obvious homology. However, a more detailed analysis revealed a conserved motif (containing a threonine residue known to be essential for catalysis) that was shared by all of the dehydratase proteins.     

Characteristics of Tn3611 and Tn3613 transposons discovered in the chromosome of the BS205 strain of Pseudomonas aeruginosa

The transposons Tn3611 (HgR, 9.0 kb) and Tn3613 (SmRCmRKmRSuRHgR, 24.0 kb) were discovered in the chromosome of Pseudomonas aeruginosa BS205. The physical and genetic maps of these transposons were constructed using restriction endonucleases EcoRI, BamHI, SalI, HindIII. Genes of the mer operon, tnpA and tnpR were localized in the Tn3611 transposon, the genes DHPS-II, aad, tnpA, tnpR being situated in the map of the Tn3613 transposon. It was established that the Tn3611 belongs to the class of Tn3-like transposons and the Tn3613 is a complex transposon flanked by inverted repeats 2.2 kb in size, the transposon comprising both Tn3611 and a migrating sequence.     

Thymidine salvage in Pseudomonas stutzeri and Pseudomonas aeruginosa provided by heterologous expression of Escherichia coli thymidine kinase gene.

Unlike enteric bacteria, Pseudomonas spp. generally lack thymidine phosphorylase and thymidine kinase activities, thus preventing their utilization of exogenous thymine or thymidine and precluding specific radioactive labeling of their DNA in vivo. To overcome this limitation, a DNA fragment encoding thymidine kinase (EC 2.7.1.21) from Escherichia coli was cloned into pKT230, a small, broad-host-range plasmid derived from plasmid RSF1010. From transformed E. coli colonies, the recombinant plasmid bearing the thymidine kinase gene was conjugally transferred to Pseudomonas stutzeri, Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes. Thymidine kinase activity was expressed in all of these species, and all gained the ability to incorporate exogenous [2-14C]thymidine into their DNA. Thymidine incorporation into P. stutzeri was enhanced 12-fold more in mutants lacking thymidylate synthetase activity. These mutants produced higher levels of thymidine kinase and were thymidine auxotrophs; thymineless death resulted from removal of thymidine from a growing culture.     

Molecular cloning and expression of the beta-hydroxysteroid dehydrogenase gene from Pseudomonas testosteroni.

The structural gene (hsd) of the Pseudomonas testosteroni encoding the 17 beta-hydroxysteroid dehydrogenase has been cloned using the cosmid vector pVK102. Escherichia coli carrying recombinant clones of hsd, isolated by immunological screening, were able to express the biologically active enzyme, as measured by the conversion of testosterone into androstenedione. Subcloning experiments, restriction and deletion analysis, and site-directed insertion mutagenesis showed that the hsd gene is located within a 1.3-kb HindIII-PstI restriction fragment. A 26.5-kDa protein encoded by a recombinant plasmid containing this Ps. testosteroni DNA restriction fragment was detected by SDS-PAGE analysis of in vitro [35S]methionine-labeled polypeptides.     

Molecular cloning and expression of perhydrolase genes from Pseudomonas aeruginosa and Burkholderia cepacia in Escherichia coli

Two bacterial perhydrolase genes, perPA and perBC, were cloned from Pseudomonas aeruginosa and Burkholderia cepacia, respectively, using PCR amplification with primers designed to be specific for conserved amino acid sequences of the already-known perhydrolases. The amino acid sequence of PerPA was identical to a putative perhydrolase of P. aeruginosa PAO1 genome sequences, whereas PerBC of B. cepacia was a novel bacterial perhydrolase showing similarity of less than 80% with all other existing perhydrolases. Most importantly, the perPA gene was expressed as a soluble intracellular form to an extent of more than 50% of the total protein content in Escherichia coli. Two perhydrolase enzymes were confirmed to exhibit the halogenation activity towards Phenol Red and monochlorodimedone. These results suggested that we successfully obtained the newly identified members of the bacterial perhydrolase family, expanding the pool of available perhydrolases.     

pPSX: a novel vector for the cloning and heterologous expression of antitumor antibiotic gene clusters

A cosmid cloning vector has been constructed that demonstrates high levels of segregational stability in Escherichia coli K12. pPSX is a 14-kilobase vector derived from the IncW plasmid pR388. pPSX is highly stable in E. coli in the absence of antibiotic selection, even while expressing the toxic indolocarbazole antitumor antibiotic violacein. The incorporation of the lambdacos sequence enables construction of cosmid libraries with inserts ranging from 24 to 36kb. The inclusion of a lacZalpha multiple cloning site (MCS) allows blue/white screening. pPSX cosmids can be extracted from the host cell with commercial plasmid extraction kits facilitating downstream analysis, sequencing and sub-cloning. pPSX can be transferred to a variety of heterologous hosts by either electroporation or mobilization from E. coli S17-1. While it is unstable in non-E. coli hosts without antibiotic selection, heterologous host strains such as Rhodobacter sphaeroides and Pseudomonas stutzeri will maintain the plasmid under antibiotic selection to allow screening of expressed inserts. pPSX provides the benefits of large insert sizes with high stability to allow cloning of chemotherapeutic gene clusters in E. coli and a range of other heterologous hosts.