Construction of shuttle vectors derived fromAcetobacter xylinum for cellulose-producing bacteriumAcetobacter xylinum

Summary Shuttle vector pUF106 was constructed by ligation ofAcetobacter xylinum plasmid pFF6 toEscherichia coli plasmid pUC18. It had unique restriction sites suitable for insertion of a foreign DNA fragment and conferred ampicillin resistance to a host. pUF106 transformed cellulose-producingA. xylinum ATCC10245 as well asE. coli JM109.     

A Clostridium perfringens Vector for the Selection of Promoters

A promoter selection vector for Clostridium perfringens genes was constructed from a C. perfringens-Escherichia coli shuttle vector, pJIR418. The plasmid carries a promoterless chloramphenicol acetyltransferase gene (catP), derived from pIP401, downstream of the multiple cloning sites of pUC18. When a promoter region of the phospholipase C gene was inserted into one of the cloning sites, derivatives of C. perfringens strain 13 carrying the resultant plasmid acquired resistance to chloramphenicol. This plasmid should be a useful reporter system for C. perfringens genes.     

Construction of hybrid plasmid vectors for cloning inEscherichia coli,Bacillus subtilis,and the cyanobacteriumAnacystis nidulans

Two hybrid plasmids capable of acting as shuttle cloning vectors inAnacystis nidulans andBacillus subtilis were constructed by in vitro ligation. One construct, pMG202, consists of theB. subtilis vector pNN101 and the endogenous cyanobacterial plasmid pUH24. This 14.6 kb plasmid confers chloramphenicol resistance in both hosts and tetracycline resistance inB. subtilis. A second vector, pMG101, consists of pNN101 linked to theA. nidulans-Escherichia coli chimeric plasmid pCB4 and is 12.9 kb in size. The pCB4 portion of the vector enables pMG101 to replicate in the third host,E. coli, and confers ampicillin resistance in this bacterium as well as inA. nidulans. Both plasmids possess identical uniqueStu I sites which permit insertional inactivation of the chloramphenicol resistance gene; and, in addition, identical uniqueXho I sites are present on both vectors. Each vector also has a third unique site:Sma I on pMG101 andXba I on pMG202.     

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     

Stabilization of a miniderivative of the broad-host-range IncW plasmid pSa by insertion of plasmid R1parB region

The plasmid vector pEM100 (13.5 kb) constructed from pGV1106, a miniderivative of the broad-host-range IncW pSa plasmid, and the pAM330 plasmid ofBrevibacterium lactofermentum is not stably maintained inEscherichia coli host cells under nonselective growth conditions. By insertion of a 0.9 kb DNA fragment containing theparB locus (responsible for the maintenance of plasmid R1 inE. coli cells) to plasmid pEM100, plasmid pEM110 was prepared which is maintained in a population ofE. coli cells growing without a selection pressure very stably. Translated by Č. Novotny     

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.     

Construction of a promoter-probe vector for the methanol-utilizing bacterium acetobacter methanolicus MB 58

We report here the construction of a promoter-probe vector, pRS2, which can be utilized in either Acetobacter methanolicus MB 58 or Escherichia coli due to the presence of broad-host-range replicon RSF 1010. The vector provides several unique restriction sites for promoter cloning as well as resistance markers for the selection of transformants. The promoter-probe vector was constructed by inserting an EcoRI-SalI-polylinker fragment of pUC 19 into EcoRI/SalI digested pMK 16. The resulting plasmid, pRS1, was cloned into the unique EcoRI site of the broad-host-range plasmid RSF 1010. The vector was used to clone promoter-containing sequences derived from the A. methanolicus MB 58 chromosome as well as the E. coli lac-promoter.     

Plasmid shuttle vector with two insertionally inactivable markers for coryneform bacteria

A new shuttle vector pCEM500 replicating inEscherichia coli and inBrevibacterium flavum was constructed. It carries two antibiotic resistance determinants (Km^r/Gm^r from plasmid pSa of Gram-negative bacteria and Sm^r/Sp^r from plasmid pCG4 ofCorynebacterium glutamicum) which are efficiently expressed in both hosts and can be inactivated by insertion of DNA fragments into the unique restriction endonuclease sites located within them. This vector was found to be stably maintained inB. flavum and can be used for transfer of the cloned genes into this amino-acid-producing coryneform bacterium.     

Expression of streptococcal plasmid-determined resistance to erythromycin and lincomycin in Escherichia coli

Summary By using recombinant DNA techniques, we have constructed a chimeric plasmid, pSM7322 (10.8 kb), between the streptococcal erythromycin resistance vector plasmid pSM7 (6.4 kb) and the E. coli vector pBR322 (4.4 kb). As judged by the minimum inhibitory concentrations of erythromycin and lincomycin, pSM7-determined resistance to these antibiotics is expressed in E. coli.     

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.     

Requirements for the high-level expression of murine interleukin-3 cDNA inEscherichia coli

Summary Murine interleukin-3 (Mu IL-3) cDNA was previously expressed inEscherichia coli using atac promoter and a constitutive high copy number plasmid vector. We found that significant increases in expression levels could be realized by using thetac promoter for the expression of Mu IL-3 in a plasmid vector possessing a temperature-inducible runaway-replicon. In contrast, use of anlpp promoter under similar conditions did not result in an increase in the Mu IL-3 expression level. Significant differences were observed when the expression levels of IL-3 were monitored in variousE. coli hosts having different genetic backgrounds. A mutant ofE. coli which lacks the protease La was found to increase the level of IL-3 produced. This report describes the effect of a specific protease-deficientE. coli host strain, as well as the effect of different promoters and plasmid replicons on the expression levels and stability of a heterologous gene product.     

Non-oncogenic plant vectors for use in the agrobacterium binary system

Summary Agrobacterium strains harbouring the T-region and the virulence-region of the Ti plasmid on separate replicons still display efficient T-DNA transfer to plants. Based on this binary vector strategy we have constructed T-region derived gene vectors for the introduction of foreign DNA into plants. The vectors constructed can replicate in E. coli, thus the genetic manipulations with them can be performed with E. coli as a host. They can be transferred to Agrobacterium as a cointegrate with the wide host range plasmid R772. Their T-regions are transferred to plant cells from Agrobacterium strains conferring virulence functions.The plasmid pRAL 3940 reported here is 11.5 kb large, contains a marker to identify transformed plant cells and unique restriction sites for direct cloning of passenger DNA, flanked by the left- and right-hand border fragments of the T-region (including the 25 bp border repeats). The plasmid is free of onc-genes. Therefore, is does not confer tumorigenic traits on the transformed plant cells and mature, fertile plants can thus be regenerated from them.     

Site-specific properties of Tn7 transposition into the E. coli Chromosome

Summary A study has been made of the insertional properties of transposon Tn7, a 14 kilobase transposable element encoding resistances to trimethoprim, streptomycin and specitinomycin. It has previously been shown that Tn7 transposes at a low frequency and with low specificity into multiple sites in large transmissible plasmids. However, Tn7 transposes with extrame specificity and at high efficiency into the E. coli chromosome. In all cases we have studied, insertion of Tn7 into the chromosome has occurred at a unique site and with a unique orientation. A combination of genetic and biochemical techniques have been used to precisely locate this site on the E. coli chromosome to minute 82 on the linkage map between markers glmS and uncA.To investigate the nature of this highly specific transpositional event, a small region of the E. coli chromosome that includes the unique site, was cloned into the plasmid vector pBR322. Subsequently a lkb restriction fragment, including the Tn7 insertion site, was sub-cloned from this plasmid into the plasmid pACYC184. We show that Tn7 transposes into both these plasmid recombinants with the frequency and specificity characteristie of the E. coli chromosome.     

Next generation synthetic vectors for transformation of the plastid genome of higher plants

Plastid transformation vectors are E. coli plasmids carrying a plastid marker gene for selection, adjacent cloning sites and flanking plastid DNA to target insertions in the plastid genome by homologous recombination. We report here on a family of next generation plastid vectors carrying synthetic DNA vector arms targeting insertions in the rbcL-accD intergenic region of the tobacco (Nicotiana tabacum) plastid genome. The pSS22 plasmid carries only synthetic vector arms from which the undesirable restriction sites have been removed by point mutations. The pSS24 vector carries a c-Myc tagged spectinomycin resistance (aadA) marker gene whereas in vector pSS30 aadA is flanked with loxP sequences for post-transformation marker excision. The synthetic vectors will enable direct manipulation of passenger genes in the transformation vector targeting insertions in the rbcL-accD intergenic region that contains many commonly used restriction sites. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Construction of New Shuttle Vectors for Acetbacter

Shuttle vector pMV301 was constructed by ligation of pMV102 found in A. aceti subsp. xylinum NBI 1002 to E. coli plasmid pACYC177. It is 6.0 kb in size, has unique restriction sites suitable for insertion of a foreign DNA fragment and confers ampicillin resistance to the Acetobacter host. This vector transforms A. aceti subsp. aceti 10-80S1 and industrial vinegar producer A. aceti subsp. xylinum NBI 1002 as well as E. coli. Various chimeric plasmids were also constructed by ligation of pMV102 to E. coli plasmids to examine the expression of drug resistance genes. In addition to the ampicillin resistance gene, resistance genes for kanamycin, chloramphenicol and tetracycline derived from E. coli plasmids were expressed in Acetobacter. Most of the constructed shuttle vectors were stably maintained in Acetobacter.     

pUB307 mobilizes resistance plasmids from Escherichia coli into Neisseria gonorrhoeae

Summary The plasmid pUB307, a derivative of RP1, is a conjugative, broad-host-range plasmid. We have shown that this element mobilizes gonococcal resistance plasmids from Escherichia coli to Neisseria gonorrhoeae, thus providing evidence that extrachromosomal elements can efficiently enter gonococci by conjugation. Furthermore, pUB307 can also be used as a helper element to mobilize the cloning vector pLES2 into N. gonorrhoeae. This finding significantly increases the usefulness of pLES2 as a shuttle vector between E. coli and gonococcus.     

Cloning vector system forNocardia spp.

An electro-transformation system has been developed forNocardia asteroides andNocardia corallina by using aNocardia-Escherichia coli shuttle vector. The shuttle vector, named pCY104, was constructed by joining a 2.5-kb crypticN. asteroides plasmid pCY101 with theE. coli plasmid pIJ4625. The resistance genes for kanamycin, chloramphenicol, and thiostrepton on plasmid pCY104 were expressed inN. asteroides andN. corallina. The transformation method was optimized forN. asteroides, and transformation efficiency of 8×10⁴ transformants per g plasmid DNA was achieved routinely.     

The Streptomyces ghanaensis low copy plasmid pSG2 and its use for vector construction

A plasmid, pSG2, was isolated from Streptomyces ghanaensis and characterized by electron microscopy, buoyant density measurement, and restriction enzyme analysis. The length of 13.8 kb, single restriction sites for HindIII, EcoRV and PvuII and the possibility of deleting non-essential regions of the plasmid made pSG2 a suitable basic replicon for vector development. pSG2 has a copy number of about four. Plasmid pSG2 was fused to a pACYC184 derivative modified to harbour a thiostrepton resistance gene. The resulting plasmid, designated pSW1, is a 16.6 kb shuttle plasmid which replicates in Escherichia coli and in several Streptomyces strains, including S. ghanaensis, S. lividans and S. viridochromogenes. Replacement of a Bg/II-fragment of plasmid pSG2 by a fragment encoding thiostrepton resistance resulted in a low copy 12.2 kb Streptomyces plasmid. This plasmid, designated pSW2, is a Streptomyces broad host range plasmid.     

A newEscherichia coli: Bacteroides shuttle vector,pRRI207, based on theBacteroides ruminicola plasmid replicon pRRI2

A 3.4kb cryptic plasmid, pRRI2, was isolated fromBacteroides ruminicola 223/M2/7 and used as the basis for a newBacteroides/Escherichia shuttle vector. Constructs were made incorporating pRRI2, aBacteroides erythromycin/clindamycin resistance marker and theE. coli pUC8-derived vector pHG165. One of these, pRRI207 (11 kb), was capable of introduction into strains belonging to four different species ofBacteroides (B. uniformis, B. distasonis, B. thetaiotaomicron, orB. ruminicola) either by conjugal transfer fromE. coli or by electrotransformation. pRRI207 carries several unique restriction sites derived from the pUC8 multiple cloning site. Only one of sixB. ruminicola strains tested was used successfully as a recipient for pRRI207, indicating that further modifications to transfer procedures or marker genes may be needed for wider application in this species.     

Construction of a new shuttle vector pSTE33 and its stabilities in Bacillus stearothermophilus,bacillus subtilis,and Escherichia coli

Summary A shuttle vector that could replicate in B. stearothermophilus, B. subtilis, and E. coli was constructed from B. stearothermophilus cryptic plasmid pSTK1, E. coli vector pUC19, and a thermostable kanamycin-resistance marker. This new vector was stably maintained in B. stearothermophilus at 67°C without selective pressure.