Construction of a sequencedClostridium perfringens-Escherichia coli shuttle plasmid

A newClostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from theC. perfringens replicon pIP404 and theE. coli vector pUC18. The multiple cloning site and lacZ gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants inE. coli. Both chloramphenicol and erythromycin resistance can be selected inC. perfringens andE. coli since pJIR418 carries theC. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes fromC. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.     

Clostridium perfringens-Escherichia coli Shuttle Vectors That Carry Single Antibiotic Resistance Determinants

Two versatile Clostridium perfringens-Escherichia coli shuttle vectors were constructed. Each plasmid carried a single antibiotic resistance gene which was expressed in both organisms. The plasmid pJIR750 encoded resistance to chloramphenicol and pJIR751 encoded resistance to erythromycin. Each plasmid contained the pUC18-derived multiple cloning site and the lacZ′ gene which enabled direct screening for recombinants in E. coli . These plasmids should prove invaluable for the genetic manipulation of C. perfringens.     

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     

Electrotransformation of Thermoanaerobacter ethanolicus JW200

Electrotransformation of Thermoanaerobacter ethanolicus JW200 was achieved using the plasmid, pTE16, and a pUC-based suicide vector, pTEA2. The construct pTE16 is based on the Escherichia coli-Clostridium perfringens shuttle vector pJIR715 and contains a thermostable chloramphenicol (Cm) resistance cassette. Evidence supporting transformation was provided by extracting plasmid pTE16 from presumptive transformants of T. ethanolicus and by PCR specific to the chloramphenicol acetyltransferase (cat) gene on the vector pTEA2. Transformation frequencies of plasmid pTE16 and pTEA2 were 50 ± 7.4 and 30 ± 4.2 transformants per μg plasmid DNA. The results provide the first unequivocal gene transfer method functional in T. ethanolicus.     

Plasmid pGB301, a new multiple resistance streptococcal cloning vehicle and its use in cloning of a gentamicin/kanamycin resistance determinant

Summary Streptococcal plasmid pGB301 is an in vivo rearranged plasmid with interesting properties and potential for the molecular cloning of genes in streptococci. Transformation of S. sanguis (Challis) with the group B streptococcal plasmid pIP501 (29.7 kb) gave rise to the deletion derivative pGB301 (9.8 kb, copy number 10) which retained the multiple resistance phenotype of its ancestor (inducible MLS-resistance, chloramphenicol resistance). Among the eight restriction endonucleases used to physically map pGB301 were four that cleaved the plasmid at single sites yielding either sticky (HpaII, KpnI) or bluntends (HpaI, HaeIII/BspRI). Passenger DNA derived from larger streptococcal plasmids (pSF351C61, 69.5 kb; pIP800, 71 kb) was successfully inserted into the HpaII site and, by blunt-end cloning, into the HaeIII/BspRI site. The gentamicin/kanamycin resistance gene of pIP800 was expressed by recombinant plasmids carrying the insert in either orientation. Insertion of passenger DNA into the HaeIII/BspRI site (but not the HpaII site) caused instability of adjacent pGB301 sequences which were frequently deleted, thereby removing the chloramphenicol resistance phenotype. The vector pGB301 has a remarkable capacity for passenger DNA (inserts up to 7 kb) and the property of instability and loss of a resistance phenotype following insertion of passenger DNA into the HaeIII/BspRI site should facilitate the identification of cloned segments of DNA when using this plasmid in molecular cloning experiments.     

谷氨酸棒杆菌/大肠杆菌穿梭型启动子探测载体构建

从质粒pXZ10145和pUC19出发,构建了一个谷氨酸棒杆菌/大肠杆菌穿梭载体pAK6。pAK6的大小为5684bp,带有卡那霉素和氨苄青霉素抗性选择标记,以及多克隆位点。在pAK6基础上,构建了以氯霉素乙酰转移酶为报告基因的启动子探测载体pAKC6,pAKC6的大小为6474bp。采用鸟枪法,将经Sau3AI消化的谷氨酸棒杆菌基因组片段连入pAKC6;根据谷氨酸棒杆菌对氯霉素的抗性,从中分离出两个具有启动子功能的插入片段。通过测定报告基因氯霉素乙酰转移酶的活性,对两个启动子片段在谷氨酸棒杆菌中的强度进行了初步的判断;测序后,用启动子预测软件对其结构进行了预测,证实了启动子序列的存在。     

Insertion of a heterologous gene construct into a non-functional ORF of the Streptococcus thermophilus chromosome

An integrative vector was constructed for inserting heterologous genes within a non-functional open reading frame (ORF) on the chromosome of Streptococcus thermophilus. The vector, pINTRS, contained a temperature sensitive origin of replication and an erythromycin resistance gene for initial selection in S. thermophilus. The region of the vector containing unique cloning sites, for insertion of recombinant genes, was flanked by homologous DNA sequences corresponding to a pseudogene in S. thermophilus to facilitate chromosomal integration. The gene encoding green fluorescent protein, regulated by a plasmid borne hsp promoter of S. thermophilus, was cloned into pINTRS to demonstrate proper functioning of the vector. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Identification of a promoter sequence in the plasmid pUL340 of Brevibacterium lactofermentum and construction of new cloning vectors for corynebacteria containing two selectable markers

A strong promoter P1 has been found in plasmid pUL340, a cloning vector used to transform corynebacteria. This promoter is also expressed efficiently in Escherichia coli. A gene (cat) for chloramphenicol acetyltransferase from Streptomyces acrimycini and a gene (hyg) for hygromycin phosphotransferase from Streptomyces hygroscopicus were subcloned in different positions of the Brevibacterium lactofermentum plasmid pUL340. Both resistance genes are expressed in B. lactofermentum from their own promoters or from the endogenous promoter in pUL340. These genes provide useful screening markers for selecting transformants of B. lactofermentum together with the kanamycin-resistance gene from the transposon Tn5.     

Construction ofClostridium butyricum hybrid plasmids and transfer toBacillus subtilis

Summary Small plasmids were isolated from type strains ofClostridium butyricum. Strain NCIB 7423 carries one plasmid (pCBU1) of 6.4 kb, whereas strain NCTC 7423 carries two unrelated plasmids of 6.3 kb (pCBU2) and 8.4 kb (pCBU3). Cleavage sites for 18 restriction endonucleases have been mapped on these plasmids and detailed physical maps are presented. For the purpose of developing vector plasmids for gene cloning in saccharolytic clostridia these crypticC. butyricum plasmids were joined to a selectable marker that will likely be expressed in clostridia. This was achieved by cloning the clostridial plasmids into theE. coli vector pBR322 carrying the chloramphenicol acetyltransferase (CAT) gene from theStaphylococcus aureus plasmid pC194. The recombinant plasmids were tested for their ability to confer chloramphenicol resistance toBacillus subtilis. Hybrid plasmids (pHL105, pHL1051) derived from pCBU2 were identified, which are capable of replication and expression of theS. aureus drug resistance marker in bothE. coli andB. subtilis. No structural instability was detected upon retransformation of pHL105 fromB. subtilis intoE. coli. The recombinant plasmids might thus be useful as shuttle vectors for the gene transfer betweenE. coli and a wide range of bacilli and clostridia.     

Transfection of Corynebacterium glutamicum with Temperate Phage ϕ CG1

Versatile plasmid vectors useful for gene cloning in Brevibacterium lactofermentum, a glutamic acid-producing bacterium, have been constructed. The trimethoprim (Tp)-resistant dihydrofolate reductase gene derived from chromosomal DNA of the Tp-resistant mutant of B. lactofermentum was introduced into pAM330, a cryptic plasmid in B. lactofermentum. The constructed cloning vector pAJ228 (7.6kb) exists in 10 to 20 copies in cells of B. lactofermentum and donated Tp-resistance, which is a useful selective marker of transformants. pAJ228 was further improved to a versatile plasmid vector pAJ224 having some profitable characteristics such as smaller size (3.7 kb), higher copy number (60 ~ 80 copies), and additional useful cloning sites (Bam HI, Pst I and Sal I) equipped with two different promoters arranged at both orientations for the expression of passenger DNA without promoter. These plasmids were stably retained in B. lactofermentum even in the absence of Tp over many generations. Thus, they have been found very powerful vectors for gene cloning in Brevibacterium and the related bacteria.     

A Useful Cloning Vector for Bacillus subtilis

We have constructed plasmid pDN1050 a new small cloning vector for Bacillus subtilis . pDN1050 harbors the origin of replication of Staphylococcus aureus plasmid pUB110 and the chloramphenicol resistance gene of S. aureus plasmid pC194. The plasmid is segregationally and structurally stable. Plasmid pDN1370, a low copy number mutant of pDN1050 was isolated and shown to harbor a mutation in the repA gene of the replication protein.     

Expression of cDNA fragment encoding sperm membrane peptide inE. coli

A secretory high-level expression cloning vector designated as pSBC-20 was constructed by inserting a DNA fragment encoding the signal peptide of ompA protein into pBV 220 vector. Any foreign DNA fragment can be inserted into the polylinker cloning sites located after the secretion signal sequence. The cloned foreign gene is under the control of the P _R -P _L promoter while the expression of the gene is regulated by the cI-gene product. The products are secreted into the periplasmic space of bacteria or into the medium. A recombinant plasmid (pRSD-220) was constructed by inserting the 210 bp from RSD-2, a cDNA encoding a peptide fragment of human sperm protein, into the EcoRI site of pSBC-20. TheE. coli cells transformed with pRSD-220 were propagated at 30 °C, then incubated at 42 °C for several hrs. The cloned gene product was secreted into the culture medium at a high rate. The yield was about 60 mg of gene product per liter of cultured medium.     

Analysis of the Phospholipase C Gene of Clostridium perfringens KZ1340 Isolated from Antarctic Soil

Clostridium perfringens KZ1340 isolated from Antarctic soil was first classified as Clostridium plagarum and later as a lecithinase-negative variant of C. perfringens. Although the strain produced no detectable lecithinase (phospholipase C, PLC) activity in the culture supernatant, it was shown by Southern blot hybridization to possess a PLC-encoding gene (plc). To determine the cause of the PLC deficiency, we cloned and sequenced the plc gene from KZ1340. The deduced amino acid sequence consists of 398 amino acid residues, coinciding with those of the plc genes previously determined. Tyrosine was substituted for histidine at amino acid position 148, which is thought to bind a zinc ion essential for PLC activity. Northern blot analysis revealed that KZ1340 expressed the plc gene at an extremely low level. Furthermore, the plc gene cloned from C. perfringens strain 13 into a plasmid was expressed weakly in KZ1340, compared to that in strain 13. This indicates that the former strain represses plc gene expression in trans. When a phylogenetic tree of plc genes was constructed, the KZ1340 plc gene formed a monophyletic branch along with those of various other C. perfringens strains, supporting the classification of the strain as a variant of C. perfringens.     

The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector

Summary A plant gene vector cassette to be used in combination with various Escherichia coli gene-cloning vectors was constructed. This cassette contains a replication and mobilization unit which allows it to be maintained and to be transferred back and forth between E. coli and Agrobacterium tumefaciens hosts provided these hosts contain plasmid RK2 replication and mobilization helper functions. The cassette also harbors a transferable DNA unit with plant selectable marker genes and cloning sites which can be combined with different bacterial replicons, thus facilitating the reisolation of transferred DNA from transformed plants in E. coli. The vector cassette contains two different promoters derived from the T-DNA-encoded genes 5 and nopaline synthase (NOS). By comparing the levels of expression of the marker enzymes linked to each of these promoter sequences, it was found that the gene 5 promoter is active in a tissue-specific fashion whereas this is not the case for the NOS promoter. This observation provides the first documented instance of a gene derived from a procaryotic host the expression of which is apparently regulated by plant growth factors.Abbreviations OCS octopine synthase (gene) - NOS nopaline synthase (gene) - NPT-II neomycin phosphotransferase (gene) of transposon Tn5 - vir Ti-plasmid region encoding virulence functions - Cb carbenicillin - Gm gentamycin - Km kanamycin - Cm chloramphenicol - Sm streptomycin - Sp spectinomycin - Rif rifampicin - Ery erythromycin - bom basis of mobilization - ori _r origin of conjugational plasmid transfer - Tra, Mob functions required for conjugational transfer of plasmids - BAP N⁶-benzylaminopurine - NAA -naphthaleneacetic acid - CTAB N-cetyl-N,N,N-trimethyl-ammonium bromide     

A novel vector allowing the expression of genes in a wide range of gram-negative bacteria

The construction and use of a novel vector allowing the expression of genes in a wide range of Gram-negative bacteria is described. The vector utilizes the regulatory region from IS50. The 70-bp promoter region was isolated from one of the terminal inverted repeats of Tn5 by creating EcoRI and Sa/I or PstI restriction sites by in vitro mutagenesis. This 70-bp region was shown to direct the expression of cat and lacZ genes in different bacterial genera including Alcaligenes, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas stutzeri, Pseudomonas fluorescens, and Serratia marcescens. Different strains containing the cat gene behind the regulatory elements of IS50 were able to tolerate high concentrations (300 micrograms/ml) of chloramphenicol in the medium. The 70-bp promoter region was cloned into a broad-host-range plasmid behind multiple cloning sites to create pAV10, which has unique restriction sites for BamHI, KpnI, SstI, and XbaI. Genes cloned into pAV10 can be expressed in a variety of Gram-negative bacteria.