Construction of a recombinant plasmid composed of B. subtilis leucine genes and a B. subtilis (natto) plasmid: its use as cloning vehicle in B. subtilis 168.

Summary Recombinant plasmids composed of Bacillus subtilis 168 leucine genes and a B. subtilis (natto) plasmid have been constructed in a recombination deficient (recE4) mutant of Bacillus subtilis 168. The process involved EcoRI fragmentation and ligation of a B. subtilis (natto) plasmid and a composite plasmid RSF2124-B · leu in which B. subtilis 168 leucine genes are linked to the R-factor RSF2124. A constructed plasmid (pLS102) was found to be composed of an EcoRI fragment derived from the vector plasmid and two tandemly repeated EcoRI fragments carrying the leucine genes. A derivative plasmid (pLS101 or pLS103) consisting of one molecule each of the EcoRI fragments was obtained by in vivo intramolecular recombination between the repeated leucine gene fragments in pLS102. pLS103 was cleaved once with BamNI, SmaI and HpaI. Insertion of foreign DNA (Escherichia coli plasmid pBR322) into the BamNI site inactivated leuA but not the leuC function which thus can serve as selective marker if the plasmid is used as vector in molecular cloning. The penicillin resistance carried in pBR322 was not functionally expressed in B. subtilis cells. By partial digestion of pLS103 with HindIII followed by ligation with T4-induced ligase, pLS107 was obtained which contained only one EcoRI site. However, insertion of exogenous DNA (pBR322) into this EcoRI site inactivated both leuA and leuC functions.     

Molecular cloning of the tolC locus of Escherichia coli K-12 with the use of transposon Tn10

Summary We have cloned the tolC gene of E. coli K-12 into pSF2124 by using transposon Tn10 as the marker to first isolate the relevant DNA fragment. The gene is on a 10.5 kb EcoRI fragment, and Tn5 insertion mutagenesis locates the gene near one end of this EcoRI fragment. An EcoRI-PstI fragment has been subcloned into pBR322 to facilitate further analysis of the gene.Abbreviations Tris Tris (hydroxymethyl) aminomethane - EDTA Ethylenediamine tetra-acetic acid - DOC Sodium deoxycholate - DNA Deoxyribonucleic acid - SDS Sodium dodecyl sulphate - kb kilo base pairs     

Construction of recombinant K88 DNA with Ptac promoter

The gene encoding K88ab was localized on the 11.6 kbHindIII-HindIII fragment of 74 kb plasmid DNA ofE. coli 7301. The smallest recombinant DNA producing the K88ab antigen was obtained by excision of the 5.15 kbEcoRI-EcoRI fragment from recombinant DNA composed of the 11.6 kb K88ab fragment in the pBR322 vectro. The size of the smallest fragment was 6.5 kb. Expression of the K88ab antigen was controlled by the P1 promoter of the pBR322 vector. Substitution of promoter P_tac for promoter P1 made it possible to achieve expression of the K88ab antigen byE. coli MT. Substitution of promoter P_L for promoter P1 failed to achieve expression of the K88 ab antigen in the recipient strains used.     

Recombinant plasmids capable to replication in B. subtilis and E. coli.

Summary The plasmid pBC16 (4.25 kbases), originally isolated from Bacillus cereus, determines tetracycline resistance and can be transformed into competent cells of B. subtilis. A miniplasmid of pBC16 (pBC16-1), 2,7 kb) which has lost an EcoRI fragment of pBC16 retains the replication functions and the tetracycline resistance. This plasmid which carries only one EcoRI site has been joined in vitro to pBS1, a cryptic plasmid previously isolated from B. subtilis and shown to carry also a single EcoRI site (Bernhard et al., 1978). The recombinant plasmid is unstable and dissociates into the plasmid pBS161 (8.2 kb) and the smaller plasmid pBS162 (2.1 kb). Plasmid pBS161 retains the tetracycline resistance. It possesses a single EcoRI site and 6 HindIII sites. The largest HindIII fragment of pBS161 carries the tetracycline resistance gene and the replication function. After circularization in vitro of this fragment a new plasmid, pBS161-1 is generated, which can be used as a HindIII and EcoRI cloning vector in Bacillus subtilis.Hybrid plasmids consisting of the E. coli plasmids pBR322, pWL7 or pAC184 and different HindIII fragments of pBS161 were constructed in vitro. Hybrids containing together with the E. coli plasmid the largest HindIII fragment of pBS161 can replicate in E. coli and B. subtilis. In E. coli only the replicon of the E. coli plasmid part is functioning whereas in B. subtilis replication of the hybrid plasmid is under the control of the Bacillus replicon. The tetracycline resistance of the B. subtilis plasmid is expressed in E. coli, but several antibiotic resistances of the E. coli plasmids (ampicillin, kanamycin and chloramphenicol) are not expressed in B. subtilis. The hybrid plasmids seem to be more unstable in B. subtilis than in E. coli.     

Physical and genetic structure of the glpK-cpxA interval of the Escherichia coli K-12 chromosome

Summary Mutations at the cpxA locus of Escherichia coli K-12 affect cellular processes that are not otherwise related. We have now determined the physical and genetic structure of the E. coli chromosome in the region of cpxA (87.5 min). Our results indicate that cpxA is a single gene. Previous studies showed cpxA to be linked to tpiA. We therefore isolated two tpiA ⁺ recombinant plasmids, pRA200 and pRA300, from EcoRI and BamHI digests of F133, respectively. By genetic complementation or enzyme overproduction, the 9.5 kb EcoRI fragment in pRA200 was shown to include glpK, tpiA and cdh. The 13.6 kb BamHI fragment of pRA300 lacks glpK, but includes tpiA, pfkA and cpxA. Neither fragment complemented a deletion of the rha operon. These data indicate the chromosomal gene order: 87 min-rha-cpxA-pfkA-cdh-tpiA-glpK-88 min. The EcoRI and BamHI fragments overlap in an interval corresponding to about 8.2 kb of DNA. The total region of the E. coli K12 chromosome covered by the two fragments is about 15 kb. A terminal 2 kb EcoRI-BamHI fragment from pRA300 complemented the chromosomal cpxA2[Ts] allele with respect to isoleucine and valine synthesis, RNA bacteriophage sensitivity and surface exclusion in Hfr strains, and envelope protein composition. Complementation occurred when the fragment was subcloned in pBR325 but not when it was subcloned in pBR322, suggesting that the 2 kb fragment lacks expression sequences that are supplied by cat (chloramphenicol acetyltransferase gene) expression sequences of pBR325. The cpxA locus on the E. coli chromosome was established with respect to two chromosomal Tn10 insertions by a combination of genetic and physical analyses. The locus established by those analyses was consistent with the location of the 2 kb EcoRI-BamHI fragment in the physical map of the region. Physical analyses of (rha-pfkA) and (rha-tpiA) deletion strains showed that they lack cpxA and surrounding genes. Since these strains were viable, cpxA is not essential under all growth conditions.     

Transduction of Plasmid Antibiotic Resistance Determinants with PseudoT-Even Bacteriophages

Transduction of antibiotic resistance determinants of the plasmid pBR322 with pseudoT-even bacteriophages RB42, RB43, and RB49 was studied. It is established that antibiotic resistance determinants of plasmid pBR322 fromEscherichia coli recA ⁺ and recA ^– donor strains do not differ significantly in respect to the efficiency of transduction. Amber mutants RB43-21, RB43-33, and a double amber mutant RB43am21am33 were obtained. These mutants facilitated transduction experiments in some cases. Transduction of antibiotic resistance markers of the vector plasmid pBR325 and recombinant plasmid pVT123, containing a DNA fragment with hoc–segE–uvsW genes of phage T4, was studied. The frequency of appearance of transductants resistant to pseudoT-even bacteriophages used in transduction was determined, and the sensitivity of resistant transductants to 32 RB bacteriophages and also to phages , T2, T4, T5, T6, T7, and BF23 was estimated. The efficiency of plating pseudoT-even bacteriophages RB42 and RB43 on strain E. coli 802 himA hip carrying mutations in genes that encode subunits of the Integration Host Factor (IHF) was shown to be higher than on isogenic strain E. coli 802. The growth of pseudoT-even bacteriophages limitedin vivo by modification–restriction systems of chromosomal (EcoKI, EcoBI), phage (EcoP1I), and plasmid (EcoRI, EcoR124I, and EcoR124II) localization was analyzed. It was shown that these phages were only slightly restricted by the type I modification–restriction systemsEcoBI, EcoR124I, and EcoR124II. Phage RB42 was restricted by systems EcoKI, EcoP1I, and EcoRI; phage RB43, by systems EcoKI and EcoRI; and phage RB49, by the EcoRI modification–restriction system.     

Molecular cloning and functional analysis of the cysG and nirB genes of Escherichia coli K12, two closely-linked genes required for NADH-dependent nitrite reductase activity

Summary We have cloned two genes, nirB ⁺and cysG ⁺which are required for NADH-dependent nitrite reductase to be active, from the 74 min region of the Escherichia coli chromosome. Restriction mapping and complementation analysis establish the gene order crp-nirB-cysG-aroB. Both genes are trans-dominant in merodiploids and, under some conditions, can be expressed independently. The cysG ⁺gene can be expressed from both high and low copy number plasmids carrying a 3.6 kb PstI-EcoRI restriction fragment. Attempts to sub-clone the nirB ⁺gene into pBR322 on a 14.5 kb EcoRI fragment were unsuccessful, but this fragment was readily sub-cloned into and expressed from the low copy number plasmid pLG338 (Stoker et al. 1982). Overproduction of the 88 kDa nitrite reductase apoprotein by strains carrying a functional nirB ⁺gene suggests that nirB is the structural gene for this enzyme.     

Cloning and expression of the Bacillus subtilis phage SPP1 in E. coli. I. Construction and characterization of lambda/SPP1 hybrids

Summary We have constructed /SPP1 hybrid phages by in vitro ligation of EcoRI fragments of the Bacillus subtilis phage SPP1 DNA to a lambdoid bacteriophage vector. EcoRI digestion of SPP1 generated 15 DNA fragments of which 13 could be cloned. The SPP1 DNA of such hybrids was stably maintained and replicated in Escherichia coli, as indicated by marker rescue experiments in B. subtilis. EcoRI fragment 1 of SPP1 could not be cloned although subfragments of fragment 1 resulting from spontaneous deletions which occurred during the cloning regime were consistently obtained. A region within EcoRI fragment 1 responsible for its incompatibility with replication in E. coli was defined by these experiments.Part of this work was taken from the doctoral thesis of E.P.A. submitted to the Freie Universität, Berlin 1979     

Cloning Vectors for Lactococci Based on a Plasmid Encoding Resistance to Cadmium

An 8.8-kb plasmid (pND302) was identified in Lactococcus lactis spp lactis M71 which encodes cadmium resistance (Cd^R). Most of the commercial lactococcal strains tested were sensitive to cadmium. Therefore, Cd^R should provide a useful selectable marker for constructing cloning vectors in lactococci. pND302 was mapped with a number of restriction enzymes and found to contain a unique EcoRI site suitable for cloning. Two E. coli/L. lactis shuttle cloning vectors, pND304 and pND624, were constructed by subcloning of the E. coli plasmids pBR322 and pGEM-7Zf(+) containing a 1.6-kb gene encoding nisin resistance (Nis^R) of lactococcal origin into the EcoRI site of pND302, separately. The E. coli DNA component of pND624 was removed and the resulting plasmid, pND625, consisted of only lactococcal DNA, expressing Nis^R and Cd^R, with two synthetic polylinkers that contain multiple restriction sites for versatile cloning. Both pND302 and pND625 can be transformed by electroporation into L. lactis LMO230 at 10³/μg DNA and maintained stably in LMO230. The results indicated that pND302 and pND625 are potential food-grade cloning vectors for lactococci. Received: 27 November 1995 / Accepted: 29 December 1995     

A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjacent to Tn917 insertions

Summary A rapid and general procedure has been devised for the pBR322-mediated cloning in Escherichia coli of Bacillus subtilis chromosomal DNA extending in a specified direction from any Tn917 insertion. Derivatives of Tn917 have been constructed that contain a pBR322-derived replicon, together with a chloramphenicol-resistance (Cm^r) gene of Gram-positive origin (selectable in B. subtilis), inserted by ligation in two orientations into a SalI restriction site located near the center of the transposon. When linearized plasmid DNA carrying such derivatives was used to transform to Cm^r B. subtilis bacteria already containing a chromosomal insertion of Tn917, the pBR322 sequences efficiently became integrated into the chromosomal copy of the transposon by homologous recombination. It was then possible to clone chromosomal sequences adjacent to either transposon insertion junction into E. coli, using a selection for ampicillin-resistance, by transforming CaCl₂-treated cells with small amounts of insert-containing DNA that had been digested with various restriction enzymes and then ligated at a dilute concentration. Because pBR322 sequences may be inserted by recombination in either orientation with respect to the transposon arms, a single restriction enzyme (such as EcoRi or SphI) that has a unique recognition site in pBR322 DNA may be used to separately clone chromosomal DNA extending in either direction from the site of any transposon insertion. A family of clones generated from the region of an insertional spo mutation (spoIIH::Tn917) was used in Southern hybridization experiments to verify that cloned material isolated with this procedure accurately reflected the arrangement of sequences present in the chromosome. Strategies are discussed for taking advantage of certain properties inherent in the structure of clones generated in this way to facilitate the identification and study of promoters of insertionally mutated genes.     

Cloning of the cyclomaltodextrinase gene fromBacillus subtilis high-temperature growth transformant H-17

The cyclomaltodextrinase gene fromBacillus subtilis high-temperature growth transformant H-17 was cloned on separatePstI,BamHI, andEcoRI fragments into the plasmid vector pUC18, but was expressed in an inactive form in the host,Escherichia coli DH5. High level constitutive expression of the gene product was also detrimental to theE. coli host, which led to structural instability of the recombinant plasmid. The cyclomaltodextrinase gene was cloned on a 3-kbEcoRI fragment into the plasmid vector pPL708, and the fragment was structurally maintained in the hostB. subtilis YB886. The cloned gene product was synthesized in an enzymatically active form in theB. subtilis host; however, expression was at a low level. Subcloning of the 3-kbEcoRI fragment into pUC18 and transformation intoE. coli XL1-Blue (FlacI^q) indicated that the cyclomaltodextrinase gene was cloned with its own promoter, since expression of the gene occurred in the absence of IPTG. Subcloning of the cyclomaltodextrinase gene downstream from theBacillus temperate phage SPO2 promoter of pPL708 may increase expression of this gene.Florida Agricultural Experiment Station Journal Series No. R-02177     

Construction and physical mapping of plasmids containing the MetA gene of Escherichia coli K-12

Summary Plasmids containing the metA gene of E. coli K-12 were constructed in vitro using pBR322 as the cloning vehicle and metA transducing phage as the source of metA DNA. EcoRI digests of pBR322 and metA20 were joined by ligase and plasmids carrying the metA gene were selected after transformation in a metA deletion strain. Recombinant DNA molecules contained one pBR322 fragment and one metA20 fragment of 12.2 kb which was present in either of two possible orientations. Plasmids constructed by BamH1 digestion of metA2 contained a single bacterial DNA fragment of 5.8 kb inserted in the tet gene. Insertion of the metA fragment led to loss of resistance to tetracycline in one orientation and partial resistance in the opposite orientation.     

New cloning vectors using benomyl resistance as a dominant marker for selection inNeurospora crassa and in other filamentous fungi

We describe five new plasmid vectors derived from pBR322 that carry theNeurospora crassa β-tubulin gene conferring resistance to benomyl. The benomyl resistance gene has been modified to eliminate an internalEcoRI site to facilitate the cloning ofEcoRI restriction fragments. These plasmids allow rapid subcloning of fragments from one replicon to another without insert fragment purification due to the presence of different drug resistance markers (resistance to kanamycin, tetracycline, or chloramphenicol) carried on the plasmids. These vectors will allow rapid transformation ofN. crassa and other filamentous fungi to allow phenotypic characterization of subcloned fragments.     

Stabilization of anE. coli plasmid by a mini-F fragment of DNA

Summary In anEscherichia coli K-12 strain (trpA trpE tnaA) cultured in LB broth without selective pressure, a pBR322 derivative containing the gene for tryptophan synthase (pBR322-trpBA) was found to be unstable. After 70 cell-number doublings, only 50% of the host cells retained the gene for ampicillin resistance (Ap^r). Insertion of the mini-F fragment of F factor DNA into this plasmid could effectively reduce the plasmid loss. Partial derepression of the tryptophan promotor-operator by 3-indopleacrylic acid further decreased the stability of the pBR322-trpBA but not that of the mini-F inserted plasmid (pBR322F-trpBA) The vector pBR322F-trpBA could be maintained at high copy number in the culture after 100 generations of growth; the culture was able to overproduce tryptophan synthase in the presence of 3-indoleacrylic acid.l-Tryptophan was produced from indole andl-serine using andE. coli host transformed with.pBR322F-trpBA DNA. After 8 h of incubation, the expression level was approximately 180 g/l.     

Cloning of the replication and incompatibility regions of a plasmid derived from Rts1

A replication region, consisting of a 1.1-megadalton (Md) EcoRI/HindIII fragment, was isolated from an Rts1 derivative plasmid. This 1.1-Md fragment, designated as mini-Rts1, was ligated to either pBR322 or a nonreplicating DNA fragment specifying a drug resistance, and its replication properties were investigated. The mini-Rts1 plasmid was cured at a high frequency at 42 °C, while it was maintained stably at 37 °C despite it existed in low copy number. These behaviors are quite similar to those of Rts1. By dissecting the pBR322:mini-Rts1 chimeric plasmid with AccI endonuclease, an inc region of 0.34 Md in size was cloned, which expressed incompatibility toward Rts1. Proteins encoded on the mini-Rts1 genome were examined in the minicell system, and one specific product of 35,000 daltons in molecular weight was identified. Any polypeptides specific for the 0.34-Md inc⁺ region within mini-Rts1 were not detected.     

Selective Excretion of Alkaline Xylanase by Escherichia coli Carrying pCX311

Plasmid pCX311, which we constructed, has two HindlU DNA fragments (2.6 kbp and 2.0 kbp) of alkalophilic Bacillus sp. strain C-125 in the HindlU site of pBR322.

These two fragments were essential not only for the xylanase production but also for the excretion of periplasmic proteins. The cloned 4.6 kbp fragment encodes some components that made the outer membrane of E. coli permeable. Some proteins such as xylanase and ²-lactamase were excreted, but alkaline phosphatase was not excreted into the culture broth.     

The cloning of Aspergillus nidulans mitochondrial DNA in Escherichia coli on plasmid pBR322

Summary We report the cloning of almost the entire mitochondrial DNA of Aspergillus nidulans on plasmid pBR322 in Escherichia coli. Only one fragment containing about 11% of the mitochondrial genome has not been cloned. We believe that this fragment cannot be maintained in E. coli on the pBR322 plasmid.     

Escherichia coli growth and plasmid copy numbers in continuous cultivations

Summary AnEscherichia coli K-12 strain harbouring either the plasmid pBR322, or the recombinant plasmid pKTH1220, a 14 kb derivative of pBR322, or no plasmid was grown in a chemostat. The cultivations were continued for 300–400 bacterial generations.E. coli hosts harbouring pBR322 or no plasmid grew in a similar way, but the growth of the host containing the big recombinant plasmid was slower. The plasmid copy numbers increased up to 2–3 fold as the dilution rate was increased from 0 to ca. 1 h^–1. After this point the increase in dilution rate seemed to induce a rapid decrease in the plasmid copy numbers. High copy numbers could be maintained using dilution rates resulting in good productivity of the cell mass.