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.     

pTn5cat:A Tn5-Derived Genetic Element to Facilitate Insertion Mutagenesis, Promoter Probing, Physical Mapping, Cloning, and Marker Exchange in Phytopathogenic and Other Gram-Negative Bacteria

A Tn5-derived mobile element has been constructed to identify genes and promoters related to pathogenesis and virulence inPseudomonas syringaepv.phaseolicola.To enhance the rate of mutation this Tn5derivative was constructed carrying a mutant transposase which was placed incisto the transposable element, but just outside the inverted repeats, therefore eliminating secondary transposition and increasing the stability of the insertion. The new element also contains a promoterlesscat(chloramphenicol acetyltransferase) gene as reporter to allow for positive selection of promoters being expressed under specific conditions. To facilitate cloning and manipulations inEscherichia coli,a ColE1 origin of replication has been included within the transposable element as well as the Mob region from the broad-host-range plasmid RP4, which allows this element to be efficiently mobilized by a triparental mating or by using anE. colistrain such as S17-1 to provide thetrafunctions. Sites for the rare cuttersPacI andPmeI have also been included to facilitate locating the insertions on aPacI and/orPmeI physical map. This construction combines the properties of both a mobilizable plasmid and a transposon and therefore has been termed pTn5cat.It is almost the same size as the wild-type Tn5, 5877 bp, and has successfully been tested inP.s. phaseolicolaandXanthomonas campestrispv.campestris.     

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     

Development of integrative vectors for Actinomycetes--producers of antibiotics]

The integrative vectors pSU 475 and pSU 476 with variable numbers of copies per genome were developed for antibiotic producing actinomycetes. For this, the amplifying sequence AUD-Sr 1 of Streptomyces rimosus and the BamHIB fragment of the eSA 1 genetic element from Streptomyces antibioticus were used. The eSA 1 fragment was an element required for integration of a vector to the actinomycete chromosomes since it was homologous with the chromosomal DNAs of S. lividans, S. erythraeus and S. antibioticus. At the first stage the AUD-Sr 1 sequence within the actinomycete plastid pSU 23 was cloned by the vector pUC 19 to E coli. In that experiment the 12.4-kb plasmid pSU 449 was isolated. At the second stage the BamHIB-fragment of the eSA 1 element was incorporated into the resultant hybrid plasmid pSU 449. The 16.5-kb hybrid plasmids pSU 475 and pSU 476 were isolated. In these plasmids the BamHIB fragment of eSA 1 was present in two orientations. The developed vectors were useful in cloning DNA to S. lividans and S. erythraeus.     

Modification of in vitro metabolism of T-2 toxin by esterase inhibitors.

A shuttle vector that can replicate in both Streptococcus spp. and Escherichia coli has been constructed by joining the E. coli plasmid pACYC184 (chloramphenicol and tetracycline resistance) to the streptococcal plasmid pGB305 (erythromycin resistance). The resulting chimeric plasmid is designated pSA3 (chloramphenicol, erythromycin, and tetracycline resistance) and has seven unique restriction sites: EcoRI, EcoRV, BamHI, SalI, XbaI, NruI, and SphI. Molecular cloning into the EcoRI or EcoRV site results in inactivation of chloramphenicol resistance, and cloning into the BamHI, SalI, or SphI site results in inactivation of tetracycline resistance in E. coli. pSA3 was transformed and was stable in Streptococcus sanguis and Streptococcus mutans in the presence of erythromycin. We have used pSA3 to construct a library of the S. mutans GS5 genome in E. coli, and expression of surface antigens in this heterologous host has been confirmed with S. mutans antiserum. A previously cloned determinant that specifies streptokinase was subcloned into pSA3, and this recombinant plasmid was stable in the presence of a selective pressure and expressed streptokinase activity in E. coli, S. sanguis (Challis), and S. mutans.     

Isolation and Characterization of a ColE1-like Plasmid fromEnterobacter agglomeranswith a Novel Variant ofromGene

Complete nucleotide sequence of a plasmid isolated fromEnterobacter agglomeranshas been determined. The plasmid, called pPIGDM1, consists of 2495 base pairs. The analysis of its nucleotide sequence suggested that pPIGDM1 may be a ColE1-like replicon. We confirmed this hypothesis by constructing a pPIGDM1-derived plasmid harboring thecatgene (pBW4), which could be introduced intoEscherichia colicells, and demonstrating that pBW4 cannot replicate in the absence of thepolAfunction and that its copy number is significantly decreased in thepcnBmutant. Like some other ColE1-type replicons (e.g., pBR322), pPIGDM1-derived plasmids can be amplified both by chloramphenicol method and in isoleucine-starvedrelAmutants but not inrelA⁺bacteria. Inactivation of the putativeromgene by insertion of an ampicillin-resistance gene resulted in significant increase in pPIGDM1-derived plasmid copy number inE. colidespite the fact that amino acid sequence of the putative RNA I modulator (Rom) protein is only 55.7% identical to the ColE1 analog. The pPIGDM1-derivedrom-like coding sequence is also homologous to therom-like gene present in theProteus vulgarisplasmid pPvu1. We suggest to group all these gene products into a new family called ROMS (RNA one modulators). Since a pPIGDM1-derived plasmid is compatible with other ColE1-like replicons (pMB1-, p15A, RSF1030-, and CloDF13-derived) inE. coli,one may consider pPIGDM1 as a progenitor of new cloning vehicles compatible with most (if not all) of currently used plasmid vectors. Moreover, this plasmid may serve as a source of the newrom-like gene coding for a protein useful in investigation of RNA–protein interactions. A role for the pPIGDM1 plasmid in the host strain is not known.     

Molecular cloning and expression ofBacillus subtilis bglS gene inSaccharomyces cerevisiae

A 2.7-kb EcoRI DNA fragment carrying aBacillus subtilis endo--1,3-1,4-glucanase gene (bglS) from theE. coli plasmid pFG1 was cloned into anEscherichia coli/yeast shuttle vector to construct a hybrid plasmid YCSH. The hybrid plasmid was used to transformSaccharomyces cerevisiae, and thebglS gene was expressed. Variation between levels ofbglS gene expression inS. cerevisiae was about 2.3-fold, depending on the orientation of the 2.7-kb DNA fragment. Assay of substrate specificity and optimal pH of the enzyme demonstrated that the enzyme encoded by YCSH (bglS) was identical with that found inB. subtilis, but the expression level ofbglS gene inS. cerevisiae (YCSH) was much lower than that inE. coli (YCSH).     

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.     

TheisfA mutation inhibits mutator activity and processing of UmuD protein inEscherichia coli recA730 strains

Further studies on theisfA mutation responsible for anti-SOS and antimutagenic activities inEscherichia coli are described. We have previously shown that theisfA mutation inhibits mutagenesis and other SOS-dependent phenomena, possibly by interfering with RecA coprotease activity. TheisfA mutation has now been demonstrated also to suppress mutator activity inE. coli recA730 andrecA730 lexA51(Def) strains that constitutively express RecA coprotease activity. We further show that the antimutator activity of theisfA mutation is related to inhibition of RecA coprotease-dependent processing of UmuD. Expression of UmuD' from plasmid pGW2122 efficiently restores UV-induced mutagenesis in therecA730 isfA strain and partially restores its mutator activity. On the other hand, overproduction of UmuD'C proteins from pGW2123 plasmid markedly enhances UV sensitivity with no restoration of mutability.     

Stable multicopy integration of vector sequences inHansenula polymorpha

Plasmids without an origin of replication, but bearing theURA3 gene ofSaccharomyces cerevisiae as a selective marker for transformation, are shown to replicate autonomously inHansenula polymorpha, indicating that parts of theS. cerevisiae URA3 gene can fulfil an autonomous replication and stabilization function inH. polymorpha. Such plasmids, replicated in low copy number in monomeric conformation, could be rescued inE. coli, and showed a low mitotic stability under selective and non-selective conditions. Selective propagation of such transformants, however, led to the integration of plasmid sequences into theH. polymorpha genome. The integration event usually occurred in high copy number (approx. 30–50) at a single non-homologous site of the genome. The plasmid sequences were found to be present in tandem array and stable under non-selective conditions. It contrast, the use of homologousURA3 gene under similar conditions led to low-copy-number transformants.     

Somatic homologous recombination in planta: The recombination frequency is dependent on the allelic state of recombining sequences and may be influenced by genomic position effects

The Escherichia coli sodA gene encoding the antioxidant enzyme Mn-containing superoxide dismutase (MnSOD), was cloned in the expression vector pMG36e. This vector has a multiple cloning site down-stream of a promoter and Shine-Dalgarno sequences derived from Lactococcus. The protein-coding region of sodA from E. coli was amplified by the polymerase chain reaction, using a thermocycler and Taq DNA polymerase before cloning into pMG36e. When introduced into E. coli, the recombinant plasmid expressed the predicted fusion protein, both in the presence and absence of oxygen. The expression of the fusion protein in E. coli was verified by SOD assays, activity gels and Western blots. The recombinant plasmid was also introduced into Lactococcus lactis, which contains a resident SOD, and into Lactobacillus gasseri, which is devoid of SOD. Transformed lactococci expressed an active SodA fusion protein plus an active hybrid protein composed of subunits of the Lactococcus and the recombinant E. coli enzymes. Transformants of L. gasseri expressed only the fusion SodA protein, which was enzymatically active.     

Elimination of plasmids pKM 101 and F'lac fromSalmonella typhimurium andEscherichia coli by bisammonium salt

Plasmid-curing activity of N,N′-bis(decyldimethyl)-1,6-hexanediammonium dibromide, BDHD, was tested on six different plasmids inE. coli and plasmid pKM 101 inS. typhimurium. BDHD eliminated theFlac plasmid fromE. coli cells only with a low efficiency. Plasmid pKM 101 was eliminated fromS. typhimurium cells significantly and this effect was dependent on an outer membrane pattern. A deep-rough mutant ofS. typhimurium is completely resistant to curing activity of BDHD, while part-rough and smooth cells are susceptible to it. In contrast to pKM 101, a cryptic plasmid being present inS. typhimurium cells was not eliminated by BDHD. The curing activity of sodium dodecyl sulfate, acridine orange, crystal violet, and promethazine was also affected by the outer membrane pattern ofS typhimurium cells.     

Construction of a Shuttle Vector for Use between Pasteurella multocida and Escherichia coli

The isolation of a small plasmid from Pasteurella multocida has enabled to construction of a shuttle vector for use between P. multocida and Escherichia coli. The vector pBAC64 contains the origin of replication from P. multocida, an antibiotic resistance gene which functions in P. multocida, and the E. coli vector pUC18. The presence of the pUC18 multiple cloning site together with the lacZ′ gene provides a screening method and allows cloning and manipulation in E. coli as well as cloning in P. multocida.     

Molecular cloning and sequence of theBacillus stearothermophilus translational initiation factor IF2 gene

Summary The structural gene for theBacillus stearothermophilus initiation factor IF2 was localized to a 6 kbHindIII restriction fragment by cross-hybridization with theSstI-SmaI fragment of theEscherichia coli infB gene. This fragment corresponds to the central region of the molecule containing the GTP-binding domain which is homologous inE. coli IF2, EF-Tu, EF-G and the humanras1 oncogene protein. After cloning into pACYC177, theHindIII fragment was further analysed by restriction mapping and cross-hybridization. A smaller (2.2 kb)SphI-HindIII fragment, which showed cross-hybridization, was subcloned into M13 phage and sequenced by the dideoxy chain-terminating method. This fragment was found to contain the entire IF2 gene except for the region coding for the N-terminus. This remaining region, coding for 45 amino acids, was located by homologous hybridization on an overlappingClaI-SstI fragment which was also subcloned and sequenced. Overall, theB. stearothermophilus IF2 gene codes for a protein of 742 amino acids (M_r=82,043) whose primary sequence displays extensive homology with the C-terminal two-thirds (but little or no homology with the N-terminal one-third) of the correspondingE. coli IF2 molecule. When cloned into an expression vector under the control of the λP_L promoter, theB. stearothermophilus IF2 gene, reconstituted by ligation of the two separately cloned pieces, could be expressed at high levels inE. coli cells.     

Increased Expression ofBrevibacterium sterolicumCholesterol Oxidase inEscherichia coliby Genetic Modification

To improve expression ofBrevibacterium sterolicumcholesterol oxidase inEscherichia coli,we utilized theT7lacpromoter and modified the gene to encode the first 21 amino acids with high-expressionE. colicodons. These changes resulted in a 60-fold improvement of expression level. N-terminal sequencing revealed that theE. coliproduced cholesterol oxidase signal peptide is cleaved 6 amino acids closer to the N-terminus than inB. sterolicum.The recombinantE. coliproduced protein is composed of 513 amino acids with a calculatedM_rof 55,374. The kinetic rate constants of the recombinant protein and theB. sterolicumproduced cholesterol oxidase are identical.     

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