Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.

Summary A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA)·poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid.Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such as thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec ^- recipient strain of B. subtilis was used.Abbreviations Ap^R resistance to ampicillin - Tc^R resistance to tetracycline - Cm^R resistance to chloramphenicol - CCC covalently closed circular duplex - Mdal magadalton     

A colony bank containing synthetic CoI EI hybrid plasmids representative of the entire E. coli genome

Using the poly(dA-dT) “connector” method (Lobban and Kaiser, 1973), a population of annealed hybrid circular DNAs was constructed in vitro; each hybrid DNA circle contained one molecule of poly(dT)-tailed CoI EI-DNA (L_RI) annealed to any one of a collection of poly(dA)-tailed linear DNA fragments, produced originally by shearing total E. coli DNA to an average size of 8.5 × 10⁶ daltons. This annealed DNA preparation (12 μg) was used to transform an F⁺recA E. coli strain (JA200), selecting transformants by their resistance to collcin EI. A collector or “bank” of over 2000 colicin EI-resistant clones was thereby obtained, 70% of which were shown to contain hybrid CoI EI-DNA (E. coli) plasmids. This colony bank is large enough to include hybrid plasmids representative of the entire E. coli genome. Individual plasmids have been readily identified by replica mating the collection onto plates seeded with cultures of various F^? auxotrophic recipients, selecting for complementation of the auxotrophic markers by F-mediated transfer of hybrid plasmids to the F^? recipients. In this manner, over 80 hybrid CoI EI-DNA (E. coli) plasmid-bearing clones have been identified in the colony bank, and about 40 known E. coli genes have been tentatively assigned to these various plasmids. The hybrid plasmids are transferred efficiently from F^? donors to appropriate F^? recipients. The use of this method to establish similar colony banks in E. coli containing hybrid plasmids representative of various simple eucaryotic genomes is discussed.     

A suitable method for construction and cloning hybrid plasmids containing EcoRI-fragments of E. coli genome.

Summary A convenient procedure for the isolation of specificEcoRI-fragments ofE. coli genome and their amplification on Km-resistance plasmid vector CK 11 is described. The hybrid molecules were constructedin vitro usingEcoRI-digestion, followed by ligation. Then appropriatedE. coli strain was transformed with ligated DNA mixture and hybrid plasmids CK 11-arg ⁺, CK 11-his ⁺, CK 11-thr ⁺ and CK 11-leu ⁺ containing loci ofE. coli genome were selected by molecular cloning. The hybrid plasmids obtained consisted of oneEcoRI-fragment of initial plasmid CK 11 and one respective specific portion ofE. coli genome.     

Reshuffling of the Bacillus subtilis 168 genome by multifold inversion

The genome of Bacillus subtilis 168 was modified to yield a genome vector for the cloning of DNA several Mb in size. Unlike contemporary plasmid-based vectors, this 4.2 Mb genome vector requires specific in vivo handling protocols because of its large size. Inversion mutagenesis, a method to modify local genome structure without gain or loss of genes, was applied intensively to the B. subtilis genome; this technique made possible both exchange and translocation of designated regions of the genome. This method not only reshuffles the genome of B. subtilis, but can provide insight into the biologic principles underlying genome plasticity.     

An SfiI restriction map of the Bacillus subtilis 168 genome

A restriction map of 24 SfiI (GGCCN4/NGGCC) restriction fragments has been constructed for the Bacillus subtilis genome. The combined sizes of the fragments indicate a genome size of approx. 4.2 Mb. The SfiI fragments range in size from 7-730 kb. Genetic markers have been located on 19 of the SfiI fragments, and 69 genetic markers have been assigned to the SfiI restriction map.     

Construction and characterization of hybrid plasmids containing the Escherichia coli nrd region.

Recombinant plasmids containing all or part of the genetic region of Escherichia coli coding for the two subunits of ribonucleoside diphosphate reductase (proteins B1 and B2) were constructed with the aid of the multicopy plasmid pBR322. Two of these plasmids (pPS1 and pPS2) appeared to carry both a regulator and the complete structural information for the enzyme and, after transformation of E. coli, directed a 10- to 20-fold overproduction of both proteins B1 and B2. The other plasmids (pPS101 and pPS201) carried structural information for only protein B2. Cells carrying pPS1 and pPS2 showed a 5- to 500-fold increased resistance against the drug hydroxyurea. This establishes that in E. coli the inhibition of deoxyribonucleic acid synthesis by hydroxyurea is fully explained by its action on ribonucleotide reductase.     

Construction and properties of hybrid plasmids carrying the E. coli gal operon.

A family of hybrid plasmids carrying the entire gal operon of E. coli and designated pgal was constructed in vitro. In the case of pgal 1 (mol. wt. 16.4 Md), a fragment cut by Bam HI endonuclease from lambda gal phage DNA (lambda D-J-gal-att-int) was joined to pMB9 and cloned in the gal-strain of E. coli, which was grown on selective media with galactose as a sole source of carbon. Plasmid pgal2 was derived from pgal 1 by elimination of the 1.1 Md fragment located between the two EcoRI sites and carrying the lambda att-int region and part of pMB9. To obtain pgal3, the 10.7 Md fragment of lambda DNA located between the two SmaI sites (lambda D-J and part of pMB9) in pgal2 was cut out and the resulting flush-end fragments were sealed by the T4DNA ligase. The mol. wt. of pgal3 containing one SmaI site amounted to 4.6 Md, while several pgal3 variants that had lost their SmaI site were still smaller. Plasmid pgal1 inhibited the growth of the gal- host cells, which effect could be overcome by the accompanying helper pMB9. The presence of pgal2 and pgal3 supported the growth and multiplication of gal- cells on selective media even without the helper plasmid. The total amount of pgal plasmid DNA per cell was constant and equalled 60--70 Md (4 copies of pgal1 or 15--16 copies of pgal3, ColE1 or pMB9). This might explain why the co-presence of pMB9 helper does alleviate the "harmful" effects of the plasmid pgal1 (which carries att-int genes), by reducing the copy number of the latter from four to one.     

Region dependent efficiency for recombinational transfer of the Bacillus subtilis 168 genome

Submega-sized regions of the Bacillus subtilis genome were cloned to plasmid by the B. subtilis Recombinational Transfer (BReT) method. BReT efficiency depends not only on the genome location but also on the choice of sequences for simultaneous homologous recombination during BReT. In an extreme case, a 91-kb region that was unsuccessful on the first attempt was obtained when the slightly shifted 98-kb region was targeted.     

Construction of hybrid plasmids containing the yeast replicator

In Saccharomyces cerevisiae strain 6-1G-P188 about 10 per cent of rRNA genes exist as extrachromosomal copies of rDNA repeating units. These extrachromosomal copies can be isolated as covalently closed molecules with lengths around 3mu. We have constructed a set of hybrid plasmids containing the bacterial vector pBR325, the LEU2 gene of yeast encoding beta-isopropylmalatedehydrogenase and various EcoRI restriction fragments of the 3mu DNA. We have tested the ability of our hybrid plasmids to transform LEU2 strain DC5 to leucine prototrophy. One of the plasmids Rcp21/11 transforms DC5 at the frequency comparable with that obtained with YEp13, containing the 2mu DNA replication origin. The 2400 bp EcoRI-B fragment of the 3mu DNA in Rcp21/11 carries a gene for 5S rRNA and two spacers. Our results on transformation experiments allow un to suggest that this EcoRI fragment also carries the 3mu DNA replication origin. Yeast transformants containing this plasmid are highly unstable but during the prolonged growth in selective conditions the stabilization of the LEU+ phenotype is observed being most likely a result of integration of Rcp21/11 into the yeast chromosome.     

Study of plasmids isolated from saccharolytic Clostridia: Construction of hybrid plasmids and transfer toEscherichia coli andBacillus subtilis

Summary Small plasmids ofClostridium acetobutylicum and related strains were isolated and studied. Their restriction maps were established and different hybrid plasmids were constructed by ligation with plasmid pHV33.     

Autolytic enzyme-deficient mutants of Bacillus subtilis 168.

Mutants of Bacillus subtilis strain 168 have been isolated that are at least 90 to 95% deficient in the autolytic enzymes N-acetylmuramyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase. These mutants grow at normal rates as very long chains of unseparated cells. The length of the chains is directly related to the growth rates. They are nonmotile and have no flagella, but otherwise appear to have normal cell morphology. Their walls are fully sysceptible to enzymes formed by the wild type and have the same chemical composition as the latter. Cell wall preparations from the mutants lyse at about 10% of the rate of those from the isogenic wild type, with the correspondingly small liberation of both the amino groups of alanine at pH 8.0 and of reducing groups at pH 5.6. Likewise, Microcococcus luteus walls at pH 5.6 and B. subtilis walls at pH 8 are lysed only very slowly by LiCl extracts made from the mutants as compared with rates obtained with wild-type extracts. Thus, the activity of both autolytic enzymes in the mutants is depressed. The frequencies of transformation, the isolation of revertants, and observations with a temperature-sensitive mutant all point to the likelihood that the pleiotropic, phenotypic properties of the strains are due to a single mutation. The mutants did not produce more protease or amylase than did the wild type. They sporulate and the spores germinate normally. The addition of antibiotics to exponentially growing cultures prevents wall synthesis but leads to less lysis than is obtained with the wild type. The bacteriophage PBSX can be induced in the mutants by treatment with mitomycin C.     

Transformation of Escherichia coli and Bacillus subtilis with a hybrid plasmid molecule.

A hybrid molecule constructed from Escherichia coli plasmid pMB9 and a fragment of Bacillus subtilis 168 deoxyribonucleic acid functions in cells of leu-E. coli, converting them to leucine prototrophy, but fails to survive in strains of B. subtilis 168.     

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.