A broad-host-range vector system for cloning and translational lacZ fusion analysis

A broad-host-range vector system for studying translational fusions was constructed. The region that retains the origin of replication, nic, mob, and rep genes of the broad-host-range plasmid RSF1010 was isolated as either an HincII or a PstI-PvuII restriction fragment. These restriction fragments were ligated to tetracycline, kanamycin, or streptomycin/spectinomycin resistance genes to generate plasmids pUI501, pUI511, pUI504, and pUI506. A functional lacZ gene lacking downstream lac operon sequences together with the lac promoter was constructed from plasmids pMC1871 and pUC18. This lacZ gene was inserted into pUI501 and pUI511 to generate plasmids pUI502, pUI503, pUI512, and pUI513. An oligodeoxynucleotide sequence that carries three unique blunt-end restriction sites was synthesized, annealed, and ligated in frame to the amino-terminal end of the lacZ gene in each of these plasmids. This multiple cloning sequence will allow translational fusions to the lacZ gene in all three reading frames. The stability of these plasmids and the expression of the lacZ gene in both Escherichia coli and Rhodobacter sphaeroides were studied.     

Shuttle vectors containing a multiple cloning site and a lacZ alpha gene for conjugal transfer of DNA from Escherichia coli to gram-positive bacteria

The mobilizable shuttle cloning vectors, pAT18 and pAT19, are composed of: (i) the replication origins of pUC and of the broad-host-range enterococcal plasmid pAM beta 1; (ii) an erythromycin-resistance-encoding gene expressed in Gram- and Gram+ bacteria; (iii) the transfer origin of the IncP plasmid RK2; and (iv) the multiple cloning site and the lacZ alpha reporter gene of pUC18 (pAT18) and pUC19 (pAT19). These 6.6-kb plasmids contain ten unique cloning sites that allow screening of derivatives containing DNA inserts by alpha-complementation in Escherichia coli carrying the lacZ delta M15 deletion, and can be efficiently mobilized by self-transferable IncP plasmids co-resident in the E. coli donors. Plasmids pAT18, pAT19 and recombinant derivatives have been successfully transferred by conjugation from E. coli to Bacillus subtilis, Bacillus thuringiensis, Listeria monocytogenes, Enterococcus faecalis, Lactococcus lactis, and Staphylococcus aureus at frequencies ranging from 10(-6) to 10(-9). The presence of a restriction system in the recipient dramatically affects (by three orders of magnitude) the efficiency of conjugal transfer of these vectors from E. coli to Gram+ bacteria.     

Partial characterization of a small, multiple-copy plasmid from Streptomyces espinosus and the derivation of a high copy-number deletion mutant

An organism classified as Streptomyces espinosus was found to carry an approx. 9.2-kb plasmid. This plasmid, designated pUC6, has a copy number of 30-40 per host genome equivalent. Plasmid pUC1061, a copy-number mutant of pUC6, was isolated after in vitro deletion of an approx. 2.0-kb XhoI restriction fragment. Plasmid pUC1061 has a copy number of 500-600. Plasmid pUC1061 appears to be incompatible with pUC6 and will transform a pUC6-containing culture at a frequency of approx. 1%. The sizes, restriction maps and copy numbers of plasmids pUC6 and pUC1061 indicate these may be valuable vectors for gene cloning Streptomyces.     

Construction of mobilizable vectors derived from plasmids RP4, pUC18 and pUC19

Mobilizable narrow-host-range plasmids were constructed from pUC18 and pUC19 by addition of a segment of pSUP2021 bearing the basis of mobilization (bom) site and origin of transfer (oriT) of RP4. One pair of expression vectors, pARO180 and pARO190, retains the beta-lactamase (bla) gene and twelve of the 13 restriction enzyme multiple cloning sites (MCS) of pUC18/19. Another pair was created by replacing the bla gene with the gene encoding kanamycin resistance (kan) from Tn5. The molecules replicate to high copy number in Escherichia coli and Enterobacter aerogenes. They can be transferred efficiently to other Gram- bacteria from the mobilizing strain, E. coli S17-1. In non-enteric strains, the new plasmids can be used as suicide vectors in site-specific insertional mutagenesis.     

Construction of pRES18 and pRES19, Streptomyces-Escherichia coli shuttle vectors carrying multiple cloning sites

Abstract We developed two Streptomyces-Escherichia coli shuttle vectors. The plasmid pRES102, consisting of the essential region of pRES1 and the thiostrepton resistance gene ( tsr ) fragment of pIJ702, was combined with the E. coli plasmid vector pUC18 or pUC19. The resulting shuttle vectors, designated pRES18 and pRES19, respectively, have relatively compact size (6.25 kb), low copy number, multiple cloning sites reciprocally arranged in opposite directions, and selection markers for both Streptomyces ( tsr ) and E. coli (β-lactamase ( bla ) and β-galactosidase ( lacZ )). These shuttle vectors are capable of carrying DNA fragments as long as 10 kb, of being maintained in S. griseus, S. lavendulae and S. lividans , and are compatible with pIJ702.     

Characterization of the ksgA gene of Escherichia coli determining kasugamycin sensitivity

In the plasmid pUC8ksgA7, the coding region of the ksgA gene is preceded by the lac promoter (Plac) and a small open reading frame (ORF). This ORF of 15 codons is composed of nucleotides derived from the lacZ gene, a multiple cloning site and the ksgA gene itself. The reading frame begins with the ATG initiation codon of lacZ and ends a few nucleotides beyond the ATG start codon of ksgA. The ksgA gene is not preceded by a Shine-Dalgarno (SD) signal. Cells transformed with pUC8ksgA7 produce active methylase, the product of the ksgA gene. Introduction of an in-phase TAA stop codon in the small ORF abolishes methylase production in transformed cells. On the plasmid pUC8ksgA5, which contains the entire ksgA region, the promoter of the ksgA gene was found to reside in a 380 base pair Bgl1-Pvu2 restriction fragment, partly overlapping the ksgA gene, by two independent methods. Cloning of this fragment in front of the galK gene in plasmid pKO1 stimulates galactokinase activity in transformants and its insertion into the expression vector pKL203 makes beta-galactosidase synthesis independent of the presence of Plac. The sequence of the Bgl1-Pvu2 fragment was determined and a putative promoter sequence identified. An SD signal could not be distinguished at a proper distance upstream from the ksgA start codon. Instead, an ORF of 13 codons starting with ATG in tandem with an SD signal and ending 4 codons ahead of the ksgA gene was identified. This suggests that translation of the ORF is required for expression of the ksgA gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development and use of cloning systems for Bacteroides fragilis: cloning of a plasmid-encoded clindamycin resistance determinant.

Chimeric plasmids able to replicate in Bacteroides fragilis or in B. fragilis and Escherichia coli were constructed and used as molecular cloning vectors. The 2.7-kilobase pair (kb) cryptic Bacteroides plasmid pBI143 and the E. coli cloning vector pUC19 were the two replicons used for these constructions. Selection of the plasmid vectors in B. fragilis was made possible by ligation to a restriction fragment bearing the clindamycin resistance (Ccr) determinant from a Bacteroides R plasmid, pBF4;Ccr was not expressed in E. coli. The chimeric plasmids ranged from 5.3 to 7.3 kb in size and contained at least 10 unique restriction enzyme recognition sites suitable for cloning. Transformation of B. fragilis with the chimeric plasmids was dependent upon the source of the DNA; generally 10(5) transformants micrograms-1 of DNA were recovered when plasmid purified from B. fragilis was used. When the source of DNA was E. coli, there was a 1,000-fold decrease in the number of transformants obtained. Two of the shuttle plasmids not containing the pBF4 Ccr determinant were used in an analysis of the transposon-like structure encoding Ccr in the R plasmid pBI136. This gene encoding Ccr was located on a 0.85-kb EcoRI-HaeII fragment and cloned nonselectively in E. coli. Recombinants containing the gene inserted in both orientations at the unique ClaI site within the pBI143 portion of the shuttle plasmids could transform B. fragilis to clindamycin resistance. These results together with previous structural data show that the gene encoding Ccr lies directly adjacent to one of the repeated sequences of the pBI136 transposon-like structure.     

Construction and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivatives.

A family of cloning vectors derived from plasmid pACYC184 and, therefore, compatible with pBR322 and its derivatives (especially the pUC family of vectors), is described. They all contain a multiple cloning site (MCS) and the lacZ alpha reporter gene for easy cloning. They have been grouped in three sets: (i) six of the vectors contain a chloramphenicol-resistance (CmR)-encoding gene and each a different MCS with 16 unique restriction sites overall; (ii) another six vectors contain a kanamycin-resistance (KmR)-encoding gene and the same six MCS; and (iii) two CmR vectors that contain the SP6 and T7 promoters flanking the MCS and lacZ alpha reporter gene of pUC18/19.     

Design of recombinant-stable plasmids of the pFH series

By cloning synthetic oligonucleotides into pUC18 plasmid, pFH123--pFH127 plasmids have been constructed. Their polylinker area, along with sites of widely used restriction endonucleases, contains two pairs each of FokI and HgaI sites in the opposite orientation to provide subfragment with unique predetermined 5'-ends. Comparative stability of the new plasmids and their derivatives has been studied and compared with that of the earlier constructed pMB plasmids.     

Use of β-galactosidase (lacZ) gene α-complementation as a novel approach for assessment of titanium oxide nanoparticles induced mutagenesis

The mutagenic potential of titanium dioxide nanoparticles (TiO(2)-NPs) of an average size 30.6nm was investigated using β-galactosidase (lacZ) gene complementation in plasmid pUC19/lacZ(-)Escherichia coli DH5α system. Plasmid pUC19 was treated with varying concentrations of TiO(2)-NPs and allowed to transfect the CaCl(2)-induced competent DH5α cells. The data revealed loss in transformation efficiency of TiO(2)-NPs treated plasmids as compared to untreated plasmid DNA in DH5α host cells. Induction of multiple mutations in α-fragment of lacZ gene caused synthesis of non-functional β-galactosidase enzyme, which resulted in a significant number of white (mutant) colonies of transformed E. coli cells. Screening of mutant transformants based on blue:white colony assay and DNA sequence analysis of lacZ gene fragment clearly demonstrated TiO(2)-NPs induced mutagenesis. Multiple alignment of selectable marker lacZ gene sequences from randomly selected mutants and control cells provided a gene specific map of TiO(2)-NPs induced mutations. Mutational analysis suggested that all nucleotide changes were point mutations, predominantly transversions (TVs) and transitions (TSs). A total of 32 TVs and 6 TSs mutations were mapped within 296 nucleotides (nt) long partial sequence of lacZ gene. The region between 102 and 147nt within lacZ gene sequence was found to be most susceptible to mutations with nine detectable point mutations (8 TVs and 1 TSs). Guanine base was determined to be more prone to TiO(2)-NPs induced mutations. This study suggested the pUC19/E. coli DH5αlacZ gene α-complementation system, as a novel genetic approach for determining the mutagenic potential, and specificity of manufactured NPs and nanomaterials.     

pACYC184-derived cloning vectors containing the multiple cloning site and lacZ alpha reporter gene of pUC8/9 and pUC18/19 plasmids

A new series of vectors, pSU2716, pSU2717, pSU2718, and pSU2719, has been constructed. The plasmids contain (i) the P15A replicon, (ii) the chloramphenicol acetyl transferase (CAT)-coding gene from Tn9, and (iii) the HaeII fragment which carries the multiple cloning site and the lacZ alpha reporter gene of pUC8, pUC9, pUC18 and pUC19, respectively. These vectors allow rapid and simple transfer of inserts from pUC plasmids, have an intermediate copy number (which allows regulated expression from the lac promoter), and are compatible with ColE1-derived vectors (and, therefore, can be used in studies requiring the joint expression of two genes, for example, in genetic complementation analysis). Furthermore, the accumulation of CAT instead of beta-lactamase, allows an easy visualization in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of proteins of 28-35 kDa, which can otherwise be obscured by the beta-lactamase.     

New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments

Two new shuttle vectors have been constructed by fusing the Escherichia coli plasmid pUC9 with the Staphylococcus aureus plasmids pU110 and pC194. The resulting hybrids replicate in both E. coli and Bacillus subtilis and contain seven restriction sites within a part of the lacZ gene. Insertion of foreign DNA into those sites can be easily detected in E. coli and hybrid plasmids can subsequently be transformed into B. subtilis.     

Cloning and analysis of the Clostridium perfringens tetracycline resistance plasmid, pCW3

Clostridium perfringens strain CW92 carries pCW3, a conjugative 47-kb plasmid that confers inducible resistance to tetracycline. The plasmid was examined by restriction endonuclease analysis and by cloning each of the five ClaI fragments of pCW3 in Escherichia coli, using pBR322. Analysis of the recombinant plasmids allowed the deduction of a detailed restriction map of pCW3. The tetracycline resistance determinant of pCW3 was mapped by examining the phenotype of recombinant E. coli clones derived from the cloning, into pUC vector plasmids, of EcoRI fragments from pCW3. The C. perfringens tetracycline resistance determinant was expressed in E. coli and was shown to be located on two juxtaposed EcoRI fragments which together encompass a 4-kb region of pCW3. Deletion experiments showed that the tetracycline resistance gene, and/or its control regions, contained internal EcoRI and SphI sites. E. coli strains that carried recombinant plasmids with only the 4-kb region were found to express tetracycline resistance constitutively. In contrast, recombinant plasmids harboring a 10.5-kb ClaI fragment of pCW3, that included the 4-kb region, coded for an inducible tetracycline resistance phenotype. The existence of a negatively regulated resistance gene, similar to that proposed for several other bacteria is postulated.     

Plasmid vectors with a semi-synthetic beta-galactosidase gene of E. coli

A partial synthesis of a structural gene for beta-galactosidase and construction of a series of pLZ plasmids for quantitative study of E. coli promoters are reported. The gene was assembled of two short synthetic DNAs and of a 3000 bp long EcoRI fragment (comprising the lacZ sequence 16-3013) isolated from plasmid p198/1 of B. Gronenborn. Among the plasmids constructed, pLZ4 is a promoter-probe vector that contains the semi-synthetic gene fused with a synthetic Shine-Dalgarno sequence and preceded by unique EcoRI and KpnI cleavage sites. On cloning a promoter into these sites, its signal strength in vivo could easily be measured by assaying beta-galactosidase activity. The use of pLZ4 vector was demonstrated by quantifying the effect of T7 early promoters A1 and A2, the latter being found 4,5 times more active under the conditions employed.