Enhancement of transfecting activity of bacteriophage P22 DNA upon exonucleolytic erosion

175

A plasmid pC194, encoding resistance to chloramphenicol, can serve as a cloning vector in Bacillus subtilis 168 for other HindIII-cleaved DNA segments. Replicons constructed by linking pC194 to several Escherichia coli plasmids can be used to introduce and compare the expression of the same genes in these two bacterial hosts.Since the pioneering experiments of Cohen and his colleagues (1), the technology of DNA cloning in Escherichia coli has become a major tool of molecular genetics. The main extensions beyond E. coli experiments are with plasmid RP4 and its wide range of Gram-negative hosts (2).Bacillus subtilis is a Gram-positive, sporulating, aerobic bacterium phylogenetically very distant from E. coli. A B. subtilis cloning system (3) parallel to that developed for E. coli permits studies of the interaction of the same genes with two, possibly very different, cellular environments, which would offer greater insight into the diverse processes that convert genetic information to the corresponding phenotype.The development of the B. subtilis cloning system has been mainly hindered by the absence of suitable vector replicons. Several plasmids can be introduced and maintained in the highly transformable B. subtilis strain 168 (4, 5); however, they lacked genetic markers for selection and DNA cloning. More recently, plasmids originally isolated from Staphylococcus aureus that code for resistance to tetracycline or chloramphenicol have been transfected into B. subtilis (6). These can be tested for their ability to accept inserted DNA segments, while still maintaining the capacity to replicate and express their genetic information in this host. The present experiments show that DNA segments can be inserted into the chloramphenicol resistance plasmid pC194, which then constitutes a DNA cloning vector for B. subtilis 168.MATERIALS AND METHODS Bacterial Strains. E. coli strains used were W5443 thr-1 leu-6 thi-l supE44 lacYl r-m-strR (ton B tryp delta). The following plasmids were carried in the C600 strain: pSC105 (1), pBR322 (7), pBR313 (8), pMB9 (8), and pWL7 (from W. Goebel). B. subtilis strains included SB202 trypC2 his-2 tyr-1 aroB and SB634 thy aroB tyr-l, which were deposited in the collection of this laboratory. The plasmids pT127 and pC194 (6) were carried by SB634.DNAs and Enzymes. Plasmid DNAs were prepared by the cleared lysis procedure (9). The HindIII, BamHI, and Pst restriction endonucleases were commercial preparations (Biolabs, Beverly, MA). Hae III was a gift from V. Sgaramella. The EcoRI endonuclease and the T4 DNA ligase were isolated and used as described (10,11).Transformation Procedure. Competence-induction and

Section: Resultsmentioning

confidence: 99%

DNA cloning in Bacillus subtilis.

Ehrlich¹

1978

175

“…Horizontal agarose slab gels were used as described (8). Electron microscopy was also performed (9).…”

Section: Methodsmentioning

confidence: 99%

“…EcoRI endonuclease and T4 ligase were purified and used as described (5,6). HindIII was a commercial preparation (BioLabs).…”

Section: Methodsmentioning

confidence: 99%

Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis.

Ehrlich¹

1977

260

One S. aureus plasmid coding for tetracycline resistance, pT127, and four plasmids (pC194, pC221, pC223, and pUB112) coding for chloramphenic resistance have been introduced by transformation into B. subtifis. The plasmids replicate in-and confer antibiotic resistance upon-their new host. These experiments show that the potential for genetic exchange between diverse bacterial species is greater than has been commonly assumed.Most of the plasmids studied so far have a narrow host range. Some, however, can replicate in a wider host range. For example, plasmid RP4 from Pseudomonas aeruginosa can be transferred to other Gram-negative bacteria such as Escherichia coli, Salmonella typhimurium, Klebsiella aerogenes, Rhizobium leguminosarum, and Agrobacterium tumefaciens (1).Another instance of plasmid-replicon transfer among bacterial species, perhaps even more widely separated, is reported in this work. Five Staphylococcus aureus plasmids, coding for tetracycline or chloramphenicol resistance, have been introduced by direct DNA transformation into Bacillus subtilis. The plasmids can replicate and express their genetic information (antibiotic resistance) in this new host. MATERIALS AND METHODSBacterial Strains. S. aureus strains used were SA231 (pC194) Cmr, RN154 (pC223) Cmr, RN1305 (pC221) Cmr, RN1777 (pS177) Smr, RN1801 (pT127) Tcr, RN1953 (pK545) Kmr/ Nmr, and RN2438 (pUB112) Cmr from R. Novick (2). B. subtills strains SB634 thy-aroB tyr-1 and SB748 his-2 trypC2 thy-, derived from SB168, are from the Stanford collection.Media. B. subtilis was grown in L and Penassay liquid media. Resistant bacteria were selected on L-agar plates supplemented with antibiotics [tetracycline (Tc), 15 ,ug/ml; chloramphenical (Cm), 5 ,ug/ml; streptomycin (Sm), 30 jig/ml; kanamycin (Km), 3 jig/ml] S. aureus cells were grown in CY liquid media or on GL-agar plates (3).DNAs and Enzymes. Plasmid DNAs were prepared from S. aureus strains essentially as described by Novick (2). Low-salt lysates of stationary phase cultures were clarified by centrifugation, concentrated with polyethylene glycol (molecular weight; 6000), and centrifuged to equilibrium in CsCl density gradients containing ethidium bromide. Cleared lysis treatment of B. subtilis strains was essentially as described for E. coli (4). Lysates were then processed similarly to the S. aureus ones (see above).EcoRI endonuclease and T4 ligase were purified and used as described (5, 6). HindIII was a commercial preparation (BioLabs).Transformation Procedure. Induction of competence and transformation of B. subtilis were as described (7).Electrophoresis and Electron Microscopy. Horizontal agarose slab gels were used as described (8). Electron microscopy was also performed (9). RESULTSTetracycline resistance plasmid pT127 S. aureus strain RN1801 carries the plasmid pT127 that confers tetracycline resistance on its host (3). The plasmic DNA can transform B. subtilis strains to tetracycline resistance: 107 cells of SB634 (competence level 0.2%) exposed to 0.1 Ag of pT127 DNA yielded 10...

“…Competence induction and transformation of E. coli and B. subtilis strains was done according to published procedures (8,9). Cautions appropriate for a P2 level of containment were adopted for the relevant parts of this investigation.…”

mentioning

confidence: 99%

Expression of the thymidylate synthetase gene of the Bacillus subtilis bacteriophage Phi-3-T in Escherichia coli.

Ehrlich

Bursztyn-Pettegrew

Stroynowski³

et al. 1976

The thymidylate synthetase gene of B. subtifis bacteriophage Phi-3-T, when cloned in plasmids pSC101 or pMB9, is expressed in E. colt. Phi-3-T is a Bacillus subtilis temperate bacteriophage capable of lysogenic conversion of thy-B. subtilis clones to prototrophy (1): we describe here the cloning of the synthetase gene carried by Phi-3-T on the Escherichia coli plasmids pSC101 (2) and pMB9. The gene complements thymine-deficiency mutation in E. coli which indicates its correct transcription and translation in this new host. The promoter of the gene, cloned in pSC101, is likely to be contained within the inserted segment. Hybrid plasmid DNA transforms B. subtilis, although about 10fold less efficiently than the intact phage DNA. B. subtilis clones transformed with the hybrid plasmid, DNA do not contain detectable pSC101 sequences, but do show sequences homologous with part of the Phi-3-T genome.MATERIALS AND METHODS Bacterial Strains. E. coli strains used were W5443 thr-1 leu-6 thi-1 supE44 lacYl r-m-thy-str (tonB tryp-delta) (SB2 of D. Finnegan), W5469 thy-hs-argA metB leu-xyl-lacY strA polAts214 from D. Helinski, C600(pSCIOI) from S. Cohen, HB129 (pMB9) endo" rB+ mB+ gal-lac-strepr leu-prothi-from H. Boyer, and C600 (ColElAmp) (3) from V. Hershfield. B. subtilis strains included SB168 trypC2, SB591 thy-, SB1158 thyA (thyB ilvD6 delta) from this laboratory and SB168(Phi-3-T) from D. Dean.DNAs and Enzymes. Phage, purified by differential centrifugation followed by two CsCl density bandings, was used to prepare Phi-3-T DNA by phenol extraction. Plasmid DNAs were prepared by the clear lysis procedure (4).EcoRI nuclease was prepared according to an unpublished procedure (T. Landers, personal communication). T4 ligase was prepared and used as described (5). EcoRI cleavage was done as described (6). RNA Isolation of Tcr thy+ E. coli transformants Cleavage of the Phi-3-T DNA with EcoRI enzyme decreases the transforming activity of the thy gene about 1000 times (not shown). We have, therefore, attempted the cloning of that gene from an incompletely digested DNA sample, in which the transforming activity has been decreased only 10-fold. The sample was ligated to EcoRI cleaved pSC101 DNA and used to transform thymine-requiring E. coli cells by selecting either for thymine independence or tetracycline resistance. About 400 Tce clones were inspected for the presence of hybrid plasmids by colony hybridization (11). Nearly 8% of the clones contained sequences complementary to Phi-3-T cRNA. Two of the Tcr clones showed a thy + phenotype. Two more thy + clones have been obtained by selecting that phenotype directly: these were also Tcr. The frequency of phenotype occurrence for the di-4145 Abbreviation: pFT, a cloned plasmid chosen on the basis of its ability to hybridize with Phi-3-T cRNA.