Cloning and Expression of <Emphasis Type="Italic">cyt</Emphasis>2Ba7 Gene from a Soil-Isolated <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Bacillus thuringiensis (Bt) cyt genes coding hemolytic and cytolytic toxins constitute a gene family, which are divided into two groups: cyt1 and cyt2. A novel cyt2 gene was detected from a soil-isolated Bt strain T301, which was highly homologous to cyt2Ba1 and finally designated cyt2Ba7. Until now, Cyt2Ba has not been expressed alone in Bt or other hosts. In this study, the cyt2Ba7 gene was cloned into the vector pQE30 and expressed as a fusion protein with 6×Histidine residues in Escherichia coli. Unlike cyt1A, cyt2Ba7 was freely expressed and formed cytoplasmic inclusions without the need for a “helper” protein. The 6×His-tagged Cyt2Ba7 was purified in one step by Ni-NTA affinity chromatography, examined cytolytic activity on Sf9 cells, and developed as an antigen to obtain the antiserum against Cyt2Ba by subcutaneous injection into rabbits. This gene was also cloned into the Bt–E. coli shuttle vector pHT3101 and expressed in Bt strain 4Q7. Immunoblotting analysis revealed that the antiserum was remarkably selective and specific to Cyt2Ba. Received: 21 December 2001 / Accepted: 28 January 2002     

Development of a Plasmid Vector for Easy Selection of Strong Promoters

A promoter vector pACPR33 for Escherichia coli based on the promotorless ampicillin-resistance gene from pBR322 has been constructed. The promoter of the ampicillin-resistance gene was deleted and replaced by a suitable multiple cloning site. Molecular cloning of promoters into the polylinker resulted in activation of the ampicillin resistance in E. coli. The plasmid contains a functional origin of DNA replication and a tetracycline resistance gene for E. coli, and a chloramphenicol resistance gene for S. aureus. The vector permitted direct detection of promoter activity, especially strong promoters, by easy iodometric determination of β-lactamase activity in liquid or solid media. Received: 26 July 1999 / Accepted: 22 November 1999     

Highly efficient yeast-based in vivo DNA cloning of multiple DNA fragments and the simultaneous construction of yeast/ Escherichia coli shuttle vectors

In vivo recombinational cloning in yeast is a very efficient method. Until now, this method has been limited to experiments with yeast vectors because most animal, insect, and bacterial vectors lack yeast replication origins. We developed a new system to apply yeast-based in vivo cloning to vectors lacking yeast replication origins. Many cloning vectors are derived from the plasmid pBR322 and have a similar backbone that contains the ampicillin resistance gene and pBR322-derived replication origin for Escherichia coli. We constructed a helper plasmid pSUO that allows the in vivo conversion of a pBR322-derived vector to a yeast/E. coli shuttle vector through the use of this backbone sequence. The DNA fragment to be cloned is PCR-amplified with the addition of 40 bp of homology to a pBR322-derived vector. Cotransformation of linearized pSU0, the pBR322-derived vector, and a PCR-amplified DNA fragment, results in the conversion of the pBR322-derived vector into a yeast/E. coli shuttle vector carrying the DNA fragment of interest. Furthermore, this method is applicable to multifragment cloning, which is useful for the creation of fusion genes. Our method provides an alternative to traditional cloning methods.     

Expression of the larvicidal gene of Bacillus sphaericus 1593M in the cyanobacterium Anacystis nidulans R2

Summary A 3.6 kb HindIII DNA fragement from Bacillus sphaericus 1593M was cloned and expressed in Escherichia coli and B. subtilis using pHV33 as shuttle vector and in the cyanobacterium Anacystis nidulans R2 with pUC303 as shuttle vector. The level of toxin activity of the respective recombinant plasmids pGsp04 and pGsp12 against Culex mosquito larvae was found to be the same in Escherichia coli and in the cyanobacterium.     

Construction of a shuttle vector for Zymomonas mobilis

Summary An Escherichia coli-Zymomonas mobilis shuttle vector was constructed from a 15.5 kb native plasmid of ZM6 00 and the E. coli plasmid, pBR329. Integrative transfer of this shuttle vector from E. coli to Z. mobilis was achieved with the aid of the mobilizing plasmid, pRK2013. The shuttle vector was stable in Z. mobilis for at least 300 generations without antibiotic selection.Offprint requests to: S. F. Delaney     

Cloning and expression of <Emphasis Type="Italic">Bacillus thuringiensis cry</Emphasis>11 crystal protein gene in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The six most toxic Pakistani isolates of Bacillus thuringiensis (SBS Bt-23, 29, 34, 37, 45 and 47), which were previously characterized for their toxicity against larvae of mosquito, Anopheles stephensi, and the presence of cry4 gene, were used for cry11 (cry4D) gene amplification. A 1.9-kb DNA fragment of cry11 gene was PCR-amplified, cloned in expression vector pT7-7, and then used for transformation of E. coli BL21C. The optimum expression was obtained with 1 mM IPTG at 37°C for 3 h. This gene showed different percentage homologies at protein level with scattered mutations in the toxic region. Biotoxicity assay of recombinant protein showed that Cry11 of SBS Bt 45 (DAB Bt 5) was the most toxic protein against third instar larvae of mosquito, A. stephensi, and has potentiality of a bioinsecticide against mosquitoes.     

Gene cloning,expression and characterization of a new cold-active and salt-tolerant endo-β-1,4-xylanase from marine <Emphasis Type="Italic">Glaciecola mesophila</Emphasis> KMM 241

A new plasmid pCASE1 was isolated from Gram-positive Corynebacterium casei JCM 12072. It comprised a 2.4-kb nucleotide sequence with three ORFs, two of which were indispensable for autonomous replication in Corynebacterium glutamicum. Homology search identified these two ORFs as repA and repB, areas coding proteins involved in plasmid replication. repA sequence showed high similarity to theta-replicating Escherichia coli ColE2-P9 plasmids and even higher similarity to plasmids derived from Gram-positive bacteria belonging to a subfamily of this ColE2-P9 group. An E. coli–C. glutamicum shuttle vector was constructed with pCASE1 fragment including repA and repB to transform C. glutamicum and showed compatibility with corynebacterial plasmids from different plasmid families. The copy number of the shuttle vector in C. glutamicum was 13 and the vector showed stability for 102 generations with no selective pressure.     

Development of a Native Plasmid as a Cloning Vector in Clavibacter xyli subsp. cynodontis

A 50-kilobase (kb) cryptic plasmid was found in three geographic isolates of Clavibacter xyli subsp. cynodontis (Cxc). The DNA region essential for replication of the plasmid was cloned in an Escherichia coli vector. The resulting vector, which functions as a shuttle vector between E. coli and Cxc, was characterized further with respect to its stability and copy number.     

Structure and function of the region of the replication origin of the Bacillus subtilis chromosome

Summary A BamHI restriction endonuclease fragment, B7, which is replicated first among all other fragments derived from the Bacillus subtilis chromosome, was cloned in Escherichia coli using as vector a hybrid plasmid pMS102 that can replicate both in E. coli and B. subtilis. Digestion of pMS102 with BamHI produced two fragments and the smaller one was replaced by the B7 fragment.The cloned plasmid pMS102-B7 exhibited some peculiar properties that were not observed with plasmids containing other fragments from the B. subtilis chromosome. (1) E. coli cells harboring this plasmid stuck to each other and to glass. This property was more apparent when cells were grown in poor media. (2) E. coli cells tended to lose the plasmid spontaneously when they were grown without the selective pressure favorable to the plasmid. (3) The frequency of transformation of B. subtilis by pMS102-B7 was less than 1/1,000 of that by the vector plasmid pMS102. The number of copies of pMS102-B7 present in the transformants was also markedly reduced, although the pUB110 origin of replication on the vector was intact and should be functional in B. subtilis. This inhibitory effect of the B7 fragment on plasmid replication was confirmed more directly by developing a semi in vitro replication system using protoplasts.Both in E. coli and B. subtilis the B7 fragment affected replication of its own molecule but not that of the coexisting plasmid with an identical replication system. The implication of the function of the B7 fragment in the initiation of the B. subtilis chromosome will be discussed.     

Cloning and expression of Citrobacter freundii β-isopropylmalate dehydrogenase gene in both Escherichia coli and Bacillus subtilis

Summary -Isopropylmalate (IPM) dehydrogenase gene of Citrobacter freundii was cloned in both Escherichia coli and Bacillus subtilis. Plasmid pCBL 1 containing C. freundii -IPM dehydrogenase gene was isolated using E. coli (leuB) as a host, pBR 322 as a vector and Hind III as an enzyme. The molecular weight (mol.wt.) of pCBL 1 was 7.7 megadalton (Md) and the plasmid was restricted at two sites by Hind III or Sal I, at three sites by BamH I and at four sites by Pst I. The second hybrid plasmid pCBL 2 containing -IPM dehydrogenase gene was reconstructed from 2.1 Md Pst I fragment of pCBL 1 and pBR 322. -IPM dehydrogenase activities of E. coli transformants with pCBL 1 or pCBL 2 were 2–7-fold higher than those of the present strains. The -IPM dehydrogenase gene was transferred from pBR 322 to pLS 353, a shuttle vector between E. coli and B. subtilis. The third plasmid, pCBL 3 (mol.wt. 5.6Md), was cloned in B. subtilis (leuC) and expressed the enzyme activity which complemented the Leucharacter. The enzyme activities of B. subtilis transformants with pCBL 3 were about 5-fold higher than those of present strains. Thus, the C. freundii gene was effectively expressed in both E. coli and B. subtilis.     

The replication origin of <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis>

The putative replication origin of Azotobacter vinelandii was cloned as an autonomously replicating fragment after ligation to an antibiotic resistance cartridge. The resulting plasmids could be isolated and labelled by Southern hybridisation with the antibiotic resistance cartridge as probe and also visualised by electron microscopy. These plasmids integrated into the chromosome after a few generations, even in the recA mutant of A. vinelandii. The integrated copy of the plasmid was re-isolated from the chromosome and the DNA and its subfragments were cloned in the plasmid vector pBR322. A 200-bp DNA fragment was sufficient to allow the replication of pBR322 in an Escherichia coli polA strain. Electron microscopic analysis of this plasmid showed that replication initiated mostly within the A. vinelandii DNA fragment. The nucleotide sequence of the putative replication origin and its flanking regions was determined. In the sequence of the 200-bp fragment many of the distinctive features found in other replication origins are lacking. A greater variation from the consensus DnaA binding sequence was observed in A. vinelandii. Direct sequencing of the relevant genomic fragment was also carried after amplifying it from A. vinelandii chromosomal DNA by PCR. This confirmed that no rearrangements had taken place while the cloned fragment was resident in E. coli. It was shown by hybridisation that the 200-bp chromosomal origin fragment of A. vinelandii was present in three other field strains of Azotobacter spp.     

Cloning of cybB, the gene for cytochrome b561 of Escherichia coli K12

Summary A 37 kb fragment of DNA from an F-prime factor, F100-12, which showed a gene dosage effect on b-type cytochromes, was cloned with a cosmid vector, pHC79. Gel filtration of cytochromes and product analysis of the hybrid plasmids indicated that this fragment contained cybB, the structural gene for cytochrome b561. A chromosomal DNA fragment carrying the cybB gene was cloned by the plaque hybridization technique with Charon 4A as a vector. The gene was subcloned into pBR322 and was located in a 1.3 kb DNA fragment. It was concluded that the cybB gene is located on the chromosome of Escherichia coli K12.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - HPLC high performance liquid chromatography - NADH reduced form of nicotinamide adenine dinucleotide     

Ultra-Low Background DNA Cloning System

Yeast-based in vivo cloning is useful for cloning DNA fragments into plasmid vectors and is based on the ability of yeast to recombine the DNA fragments by homologous recombination. Although this method is efficient, it produces some by-products. We have developed an “ultra-low background DNA cloning system” on the basis of yeast-based in vivo cloning, by almost completely eliminating the generation of by-products and applying the method to commonly used Escherichia coli vectors, particularly those lacking yeast replication origins and carrying an ampicillin resistance gene (Amp^r). First, we constructed a conversion cassette containing the DNA sequences in the following order: an Amp^r 5′ UTR (untranslated region) and coding region, an autonomous replication sequence and a centromere sequence from yeast, a TRP1 yeast selectable marker, and an Amp^r 3′ UTR. This cassette allowed conversion of the Amp^r-containing vector into the yeast/E. coli shuttle vector through use of the Amp^r sequence by homologous recombination. Furthermore, simultaneous transformation of the desired DNA fragment into yeast allowed cloning of this DNA fragment into the same vector. We rescued the plasmid vectors from all yeast transformants, and by-products containing the E. coli replication origin disappeared. Next, the rescued vectors were transformed into E. coli and the by-products containing the yeast replication origin disappeared. Thus, our method used yeast- and E. coli-specific “origins of replication” to eliminate the generation of by-products. Finally, we successfully cloned the DNA fragment into the vector with almost 100% efficiency.     

Gene disruption by plasmid integration in Listeria monocytogenes: insertional inactivation of the listeriolysin determinant lisA

Summary A plasmid integration technique was developed for insertional inactivation of chromosomal Listeria monocytogenes genes. A Listeria-Escherichia coli shuttle vector (pLSV1) was constructed which carried the temperature-sensitive gram-positive replication origin from plasmid pTV32(Ts). An internal fragment of the listeriolysin gene (IisA) was cloned into pLSV1 to create pLSV2. In L. monocytogenes pLSV2 transformants, plasmid pLSV2 integrated into the L. monocytogenes chromosome at a frequency of 2 × 10^–3 via lisA homology and these cells could be selected at 42° C using a plasmid-encoded erythromycin resistance. Plasmid integration resulted in disruption of the lisA gene, production of a truncated, immunologically cross-reactive listeriolysin protein and loss of the hemolytic phenotype. An improved Listeria protoplast transformation method is also described which facilitates genetic manipulation of Listeria species.     

Construction and characterization of new cloning vehicles V. Mobilization and coding properties of pBR322 and several deletion derivatives including pBR327 and pBR328

A DNA sequence essential for the R64drd11 + ColK-mediated conjugal transfer of pBR322 has been located in a 540 bp HaeIII fragment (HaeIII-2) between the vegetative origin of replication and the tetracycline resistance (Tc^r) gene of this vector. The pBR322 derivatives pBR327 and pBR328 lack this DNA sequence and are not mobilized by conjugation. Two derivatives of pBR328 were constructed by re-inserting the HaeIII-2 fragment in both orientations into the chloramphenicol-resistance gene of the same vector. One orientation of the HaeIII-2 fragment permitted mobilization by conjugation while the opposite orientation prevented mobilization. Further examination of pBR322 and derivatives revealed that the region between the origin of replication and Tc^r gene also plays a role in regulating plasmid copy number.     

Expression of chitinase-encoding genes from Aeromonas hydrophila and Pseudomonas maltophilia in Bacillus thuringiensis subsp. israelensis

Fifty isolates of chitinase (Cts)-producing bacteria were collected from soil samples and tested for their ability to degrade chitin using colloidal chitin agar as the primary plating medium. The results indicated that three isolates could degrade chitin at high pH. Further studies also demonstrated that crude Cts preparations from Bacillus circulans (Bc) No. 4.1 could enhance the toxicity of Bacillus thuringiensis subsp. kurstaki (Bt-k) toward diamondback moth larvae. Thus, it might be useful to increase the toxicity of B. thuringiensis (Bt) toward target insects by introducing a Cts-encoding gene (cts) into BtTo investigate the expression of cts in Bt, cloned cts from Aeromonas hydrophila (pHYA1) and Pseudomonas maltophilia (pHYB1, pHYB2 and pHYB3) were cloned into the shuttle vector pHY300PLK and transformed into Escherichia coli DH5α using 4-methylumbelliferyl β-d-N,N′-diacetylchitobioside (4-MUF GlcNAc) as the detecting substrate. The four plasmids were then introduced into B. thuringiensis subsp. israelensis (Bt-i) strain c4Q272 by electroporation. Various transformants harboring cloned cts were selected, and expression and stability of the plasmids in Bt were studied.     

Molecular cloning of the N-acetylneuraminate lyase gene in Escherichia coli K-12

Summary The gene for N-acetylneuraminate lyase [N-acetylneuraminate pyruvate-lyase; NPL] of Escherichia coli C600 was cloned onto pBR322 as a 9.8 kilobase HindIII fragment of chromosomal DNA and the hybrid plasmid was designated pMK2. The gene in the hybrid plasmid was subcloned in pBR322 as a 1.2 kilobase HindIII-EcoRI fragment and the resultant hybrid plasmid was designated pMK6. NPL activity level was increased more than 5-fold in the pMK6-bearing strain compared with that of the wild type, when the cells were grown on a medium containing inducer (N-acetylneuraminate: NANA). The transformants harbouring pMK6 also showed higher activity even in the absence of inducer. The NPL produced by pMK6-bearing cells was structurally and immunologically the same as that purified from E. coli C600.     

Nucleotide Sequence of the Gene for an Alkaline Endoglucanase from an Alkalophilic Bacillus and Its Expression in Escherichia coli and Bacillus subtilis

The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an M_r of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M_r of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGTAAGC-GGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms.     

The cloning of Aspergillus nidulans mitochondrial DNA in Escherichia coli on plasmid pBR322

Summary We report the cloning of almost the entire mitochondrial DNA of Aspergillus nidulans on plasmid pBR322 in Escherichia coli. Only one fragment containing about 11% of the mitochondrial genome has not been cloned. We believe that this fragment cannot be maintained in E. coli on the pBR322 plasmid.