Construction of plasmid vectors for gene cloning in Escherichia coli and Bacillus subtilis

The construction and some properties of new hybrid plasmids which are able to replicate in both Escherichia coli and Bacillus subtilis are presented. A 5.5 Md hybrid plasmid pJP9 was constructed from pBR322 (Tc, Ap) and pUB110 (Nm) plasmids. pIM1 (7.0 Md) and pIM3 (7.7 Md) plasmids are its different erythromycin resistant derivatives. Tetracycline, ampicillin, neomycin and possibly erythromycin resistance genes are expressed in E. coli while neomycin and erythromycin resistance genes are expressed in B. subtilis. Insertional inactivation of only one gene is possible using the pJP9 plasmid as a vector in B. subtilis. However, insertional inactivation of at least two different genes can be achieved and monitored in E. coli and B. subtilis transformants in cloning experiments with PIM1 and pIM3 plasmids. Insertional inactivation of antibiotic resistance genes present in pJP9 plasmid was achieved by cloning of Streptococcus sanguis DNA fragments generated by appropriate restriction endonucleases. The pJP9 plasmid and its derivatives were found to be stable in both hosts cells.     

Construction and characterization of plasmid vectors for cloning in Staphylococcus aureus and Staphylococcus carnosus

Several plasmid vectors for cloning in Staphylococcus aureus and S. carnosus have been constructed and characterized. The chimeric plasmids are composed of parts of the following parental plasmids: The chloramphenicol-resistance plasmid, pC194, the tetracycline-resistance plasmid, pMK148, and the erythromycin-resistance plasmid, pE12. All the chimeric plasmids confer two selectable antibiotic-resistance markers on host cells. Insertional inactivation of the various antibiotic-resistance markers occurred at the BclI site of pE12, and the Sau96- or AvaII-site of pMK148; only a slight inactivation of the chloramphenicol-resistance marker occurred at the HaeIII-site of pC194. The chimeric plasmids pCT20 and pCE10 are both stable in S. aureus and S. carnosus. In addition, the hybrid plasmids of pCT20 and pCE10, containing lambda-DNA fragments in various restriction sites between 0.4 and 1.2 kb, are stably maintained. The inserted lambda-DNA fragments appear unchanged.     

Characterization and construction of molecular cloning vehicles within Staphylococcus aureus.

Four chloramphenicol resistance (Cm) and four tetracycline resistance (Tc) plasmids from Staphylococcus aureus were characterized by restriction endonuclease mapping. All four Tc plasmids had molecular masses of 2.9 megadaltons (Mdaltons) and indistinguishable responses to seven different restriction endonucleases. The four Cm plasmids (pCW6, pCW7, pCW8, and pC221) had molecular masses of 2.6, 2.8, 1.9, and 2.9 Mdaltons, respectively. The four Cm plasmids also differed both in the level of resistance to Cm and in susceptibility to retriction endonucleases. Single restriction endonuclease sites contained within each plasmid included the following: in pCW6 for HindIII, XbaI, HpaII, and BstEII; in pCW7 for HindIII, BstEII, BglII, HaeIII, and HpaII; in pCW8 for HindIII, HaeIII, and HpaII; in pC221 for HindIII, BstEII, and EcoRI. The molecular cloning capabilities of pCW8 and pC221 were determined. Cm and erythromycin resistance (Em) recombinant plasmids pCW12, PCW13, and pCW14 were constructed and used to transform S. aureus 8325-4. A 2.8-Mdalton HindIII fragment from plasmid pI258 was found to encode Em resistance and contain single sites for the retriction endonucleases BglII, PstI, HaeIII, and HpaII. The largest EcoRI fragment (8 Mdaltons) from pI258 contained the HindIII fragment encoding Em resistance intact. Cloning of DNA into the BglII site of pCW14 did not alter Em resistance. Cloning of DNA into the HindIII site of pCW8 and the HindIII and EcoRI sites of pC221 did not disrupt either plasmid replication of Cm resistance.     

Self-transmissible IncP R995 plasmids with alternative markers and utility for Flp/FRT cloning strategies

The IncP plasmid R995 has been a useful self-transmissible, broad-host-range vector for a number of applications including the recombinase/conjugation-based cloning of large genomic DNA segments. However, R995 derivatives (or related plasmids) expressing a wide range of different resistance markers and Flp recombinase target sites do not exist in the literature. In addition, documented strategies for applying such plasmids in cloning applications that take advantage of conjugation for the convenient isolation and recovery of constructs are extremely limited. Here, we report a new series of R995 plasmids with alternative markers to increase options for applications in backgrounds already expressing resistance to a particular antibiotic(s). These R995 plasmids have been engineered to contain FRT sites that can be used for recombinase-based cloning. We demonstrate the utility of this approach by cloning 20 kb regions from the Salmonella Typhimurium and Escherichia coli genomes and by cloning DNA from an exogenous plasmid source. To our knowledge, this represents the first systematic engineering of an intact, self-transmissible IncP plasmid with a series of alternative antibiotic markers and FRT sites.     

Construction of improved vectors for gene cloning inMicromonospora melanosporea

Two vectors forMicromonospora melanosporea have been constructed with theStreptomyces plasmid pIJ702 along with thesgm gene (sisomicin-gentamicin resistance gene fromM. zionensis) as the second antibiotic resistance marker. These plasmids, containingsgm gene as the second selectable marker, may be an attractive alternative to pIJ702, which is incapable of conferring melanin production inM. melanosporea and consequently is not useful for insertional inactivation in this bacterium. The constructions remove restriction site forM. melanosporea restriction endonuclease and provide additional unique sites for the insertional inactivation of selectable markers, which enhance the use of these plasmids as general cloning vectors in bothM. melanosporea andS. lividans. On the other side, inS. lividans, plasmid pMK33-1 facilitates isolation and studies of promoters based on detection of extremely convenient phenotype of melanin production. This has been proved by shotgun cloning of chromosomal DNA fragments ofM. melanosporea and chromogenic identification ofS. lividans transformants which were capable of producing a melanin.     

Cloning vectors, mutagenesis, and gene disruption (ermR) for the erythromycin-producing bacterium Aeromicrobium erythreum.

Genetic systems for study of Aeromicrobium erythreum, a gram-positive, G + C-rich (72%) bacterium with the capacity for erythromycin biosynthesis, are described. High-copy-number plasmids suitable as gene cloning vectors include derivatives of the Streptomyces plasmids pIJ101, pVE1, and pJV1. pIJ101 derivatives with missense substitutions at the rep gene BamHI site do not replicate in A. erythreum. Ethyl methanesulfonate treatment generated several amino acid auxotrophs and non-erythromycin-producing (Ery-) strains. Using the Ery- strain AR1807 as a recipient for plasmid-directed integrative recombination, the chromosomal ermR gene (encoding 23S rRNA methyltransferase) was disrupted. Phenotypic characterizations demonstrated that ermR is the sole determinant of macrolide antibiotic resistance in A. erythreum.     

Cloning vectors, mutagenesis, and gene disruption (ermR) for the erythromycin-producing bacterium Aeromicrobium erythreum.

Genetic systems for study of Aeromicrobium erythreum, a gram-positive, G + C-rich (72%) bacterium with the capacity for erythromycin biosynthesis, are described. High-copy-number plasmids suitable as gene cloning vectors include derivatives of the Streptomyces plasmids pIJ101, pVE1, and pJV1. pIJ101 derivatives with missense substitutions at the rep gene BamHI site do not replicate in A. erythreum. Ethyl methanesulfonate treatment generated several amino acid auxotrophs and non-erythromycin-producing (Ery-) strains. Using the Ery- strain AR1807 as a recipient for plasmid-directed integrative recombination, the chromosomal ermR gene (encoding 23S rRNA methyltransferase) was disrupted. Phenotypic characterizations demonstrated that ermR is the sole determinant of macrolide antibiotic resistance in A. erythreum.     

Construction of a novel gene bank of Bacillus subtilis using a low copy number vector in Escherichia coli

Low copy number vector plasmid pCT571 was constructed to clone Bacillus subtilis genomic fragments in Escherichia coli. pCT571 confers KmR, TcR and CmR in E. coli and CmR in B. subtilis. It has unique restriction sites within the KmR and TcR markers to allow screening for recombinant plasmids by insertional inactivation of these genes. It contains the pSC101 replicon and replicates normally at six to eight copies per chromosome equivalent in E. coli. It also contains oriVRK2, which when supplied with the product of the trfA gene of RK2 in trans, allows pCT571 to replicate at 35-40 copies per chromosome equivalent. A B. subtilis gene bank was created by cloning partially Sau3A-digested and size-fractionated fragments of B. subtilis chromosomal DNA into the BamHI site of pCT571. DNA from 1097 KmR TcS transformants was extracted and analysed electrophoretically as supercoiled DNA and after digesting with EcoRI or EcoRI and SalI. Approximately 1000 hybrid plasmids were found with reasonably sized B. subtilis fragments. The mean size of the inserts in pCT571 is 8 kb, ranging from 4 to 20 kb in different plasmids. The gene bank covers most of the B. subtilis chromosome, as demonstrated by the results of screening the gene bank for selectable nutritional markers in E. coli and B. subtilis. Hybrid plasmids which complement E. coli mutants for arg, his, lys, met, pdx, pyr and thr markers were identified from the gene bank. In B. subtilis the presence of argC, cysA, dal, hisA, ilvA, leuA, lys, metB, metC, phe, purA, purB, thr and trpC was established by transformation experiments. The effects of copy number on cloning and long-term maintenance in the bacterial strains were also investigated. At high copy number some hybrid plasmids cannot be maintained at all, while others show an increased rate of structural deletions and rearrangements.     

双抗性质粒pSC33、pSC48的构建及其特性研究

截短的短小芽孢杆菌质粒pCJ3与去除了复制功能的金黄色葡萄球菌质粒PUB110经EcoRI酶切,DNA连接酶连接后组建T_o~r及K_m~r的双抗性的重组质粒pSC33和和PSC48。根据电泳迁移率估算pSC33及pSC48的大小分别为6.7及6.27Kb。具有BamHⅠ、AVaⅠ、XbaⅠ及BgLⅡ等限制酶的单切点,其中BgLⅡ切点位于卡那霉素抗性基因内。pSC33及pSC48能转化枯草杆菌各种突变体的感受态细胞,转化率比亲本质粒高一个数量级,也能转化枯草杆菌的原生质体。pSC33及pSC48在枯草杆菌BR151中表现稳定,以PSC48和载体克隆了滑鼠蛇肝线粒体DNA片段。     

Cloning vehicles for the homologous Bacillus subtilis host-vector system

A series of Bacillus subtilis plasmids was constructed which carry either the leu region or both the leu and the dihydrofolate reductase (DHFR) regions of the B. subtilis chromosome. The DHFR-coding gene was derived from a trimethoprim resistant (Tmpr) B. subtilis strain, and cells harboring the DHFR plasmid showed resistance to trimethoprim (Tmp). One such leu+tmpr plasmid, pTL12, was found to be useful for cloning DNA fragments at the BamHI, EcoRI, BglII and XmaI sites. It was also shown that insertion of DNA fragments at the BamHI and XmaI sites of pTL12 inactivated the leuA gene function (insertional inactivation) but not tmpr, indicating that cells carrying recombinant plasmids can be detected easily by selecting Leu-Tmpr colonies. Combination of B. subtilis 168 and plasmid pTL12 should serve as an efficient homologous cloning system in B. subtilis.     

Construction of compatible wide-host-range shuttle vectors for lactic acid bacteria and Escherichia coli

A new collection of shuttle cloning vectors has been constructed that can be used in a broad host range, because they carry replication origins which are functional in Escherichia coli (p15A, pWV01, ColE1), Lactococcus lactis, lactobacilli, and Bacillus subtilis (pAMbeta1, pWV01). These plasmids contain the lacZ-T1T2 cassette from pJDC9, which allows the X-gal selection and cloning of DNA fragments that could cause plasmid instability in E. coli. In addition, they have been proved to be structurally and segregationally stable in Lactobacillus casei, in which their copy number has been determined by real-time quantitative PCR. Furthermore, the antibiotic resistance markers (beta-lactamase, chloramphenicol acetyl transferase, and erythromycin transacetylase) and the theta and rolling circle replicating origins have been combined to obtain this set of compatible plasmids (pIA family) that can be cotransformed, both in lactic acid bacteria and in E. coli.     

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.     

Genetic and molecular analysis of pIP417 and pIP419: Bacteroides plasmids encoding 5-nitroimidazole resistance.

This report describes a genetic and molecular analysis of two transferable Bacteroides plasmids, pIP417 and pIP419, which carry genetic determinants conferring low-level resistance to 5-nitroimidazoles. The restriction endonuclease cleavage sites for each plasmid were localized. The NiR genetic determinants of pIP417 and pIP419 plasmids have been cloned into the Bacteroides cloning vector pBI191 (C.J. Smith, J. Bacteriol. 164, 294-301, 1985) as PvuII and Sau3A fragments, respectively. Both inserts had different restriction sites and did not cross-hybridize by Southern blot analysis. Genetic data obtained by cloning into pBI191 clearly show that the PvuII-generated fragments A (Rep) and B (Mob) of pIP417 are involved in plasmid replication and transfer, respectively. Although encoding resistance to the same antibiotic, both plasmids appeared different with regard to the 5-nitroimidazole resistance and replication genetic determinants. However, they share a homology in a region involved, at least in one case, in plasmid transfer. Considering the spontaneous high level of resistance to 5-nitroimidazole in Escherichia coli, this work, based on direct gene cloning into Bacteroides, demonstrates the value of such an approach.     

Transformation of Acholeplasma laidlawii with streptococcal plasmids pVA868 and pVA920

One limitation with studying mycoplasma genetics is the lack of cloning vectors. Studies were undertaken to determine whether streptococcal plasmids could replicate in Acholeplasma laidlawii, for the purpose of identifying potential vectors. Plasmids pVA868 and pVA920 contain the same origin of replication and tetracycline resistance determinant. pVA920 also contains an erythromycin resistance determinant not present in pVA868. A. laidlawii was transformed with plasmids pVA868 (13.7 kb) and pVA920 (12.2 kb), and isolated from the transformants were deletion derivatives of the parent plasmids having sizes of 3.7 and 10.3 kb, respectively. The tetracycline and erythromycin resistance markers functioned in A. laidlawii, and the deletion derivatives may be useful for development of mycoplasma vectors. However, difficulties may arise due to plasmid instability.     

Instability of plasmid DNA sequences: Macro and micro evolution of the antibiotic resistance plasmid R6-5

Summary Detailed examination of the structure of cloned DNA fragments of the R6-5 antibiotic resistance plasmid has revealed a substantial degree of polynucleotide sequence heterogeneity and indicates that sequence rearrangements in plasmids and possibly other replicons occur more frequently than has hitherto been appreciated. The sequence changes in cloned R6-5 fragments were shown in some instances to have occurred prior to cloning, i.e. existed in the original population of R6-5 molecules that was obtained from a single bacterial clone and by several different criteria judged to be homogeneous,and in others to have occurred either during the cloning procedure or during subsequent propagation of hybrid molecules. The molecular changes that are described involved insertion/deletion of the previously characterized IS2 insertion element, formation of a new inverted repeat structure probably by duplication of a preexisting R6-5 DNA sequence, sequence inversion, and loss and gain of restriction endonuclease cleavage sites.     

Construction and characterization of new cloning vectors derived from Streptomyces griseobrunneus plasmid pBT1 and containing amikacin and sulfomycin resistance genes

Three cryptic plasmids, designated pBT1 (5.6 kb), pBT2 (9.7 kb), and pBT3 (16.6 kb), were isolated from Streptomyces griseobrunneus ISP5066 and physically characterized. pBT1 and pBT2, which differ by a 4.1-kb segment, are high copy-number plasmids (40-100 copies per chromosome) that coexist with each other. pBT3 is a low copy-number plasmid. Vectors containing amikacin (or kanamycin) and sulfomycin (or thiostrepton) resistance genes from Streptomyces litmocidini ISP5164 and Streptomyces viridochromogenes subsp. sulfomycini ATCC 29776, respectively, were constructed from pBT1. One such vector, pBT37, has unique restriction sites for cloning, including BglII, XhoI, PvuII, ClaI, and SacI, with the PvuII and ClaI sites allowing clone recognition by insertional inactivation of sulfomycin resistance. Since many Streptomyces species were very sensitive to amikacin and sulfomycin, these resistance genes serve as useful selective markers. pBT37 could transform several Streptomyces strains that produce antibiotics such as tetracyclines, macrolides, beta-lactams, and aminoglycosides. This plasmid is a potentially useful vector for cloning antibiotic biosynthetic genes.     

Aminocyclitol resistance in Staphylococcus aureus: presence of plasmids and aminocyclitol-modifying enzymes

Aminocyclitol resistance in Staphylococcus aureus has been investigated by the analysis of the plasmids and aminocyclitol-modifying enzymes present in several clinical staphylococcal isolates. All of the strains tested were resistant to a broad range of aminocyclitols and contained large plasmids which encoded a variety of aminocyclitol-modifying enzymes in addition to other antibiotic resistances. All strains expressed multiple aminocyclitol-modifying enzymes. The plasmids present in these strains appear to be related by virtue of their similar restriction endonuclease digestion patterns. The plasmids are related and differ by the gain or loss of small DNA segments, one of which encodes erythromycin and kanamycin resistance.     

Comparative study of the symbiotic plasmid DNA in free living bacteria and bacteroids of Rhizobium leguminosarum

Plasmids pIM13, pT127 and pBC16 delta 1, introduced by transformation into Clostridium acetobutylicum N1-4081, were shown to replicate in, and to confer antibiotic resistance upon this new host. Recombinant plasmids were constructed by inserting erythromycin-resistant plasmid pIM13 into the unique ClaI site of pBR322 or by ligating a tetracycline-resistant determinant of plasmid pT127 to HindIII-linearized pIM13. The hybrid plasmids replicated and expressed erythromycin resistance in C. acetobutylicum strain N1-4081 and in Escherichia coli or Bacillus subtilis, indicating that they might be useful as shuttle vectors for transferring genes between these strains. The efficiency and stability of different replicons in C. acetobutylicum were compared.     

球孢链霉菌质粒pSGL1的性质研究

链霉菌质粒pSGL1是本实验室在球孢链霉菌（Streptomycelglobisporus）中发现的新的质粒,它可以在变铅青链霉菌中复制。用缺失分析确定了pSGL1的基本复制区位于一个2．0kb的片段上。pSGL1能够与pIJ101相容。pSGL1是一个高拷贝质粒,拷贝数约为70～250。在对质粒进行分析的过程中得到的一些衍生质粒如pSGLN和pSGLS3等可以作为新的链霉菌基因克隆载体,并可用于构建新的高效分泌表达的链霉菌载体。     

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.