Site-specific deletions in the recombinant plasmid pSC101 containing the redB-ori region of phage lambda

Large deletions occur in the hybrid plasmid formed by pSC101 and the EcoRI fragment f2 of phage lambda (redB-ori region) under well defined growth conditions (Bernardi and Bernardi, 1980). We have sequenced the novel joints of the four deletions so obtained and shown that they have one endpoint in pSC101, identical in all four cases, the other endpoint being located in four different lambda sequences. Furthermore, the nucleotide sequences of the novel joints show homologies between the conserved pSC101 sequence and the lambda sequences both conserved and deleted. The presence of an IS-type element in pSC101 is postulated; however, this element is unrelated to the 200 bp element already described in pSC101 (Ravetch et al., 1976).     

Overinitiation of chromosome and plasmid replication in a dna Acos mutant of Escherichia coli K12. Evidence for dnaA-dnaB interactions

The dnaAcos mutations are phenotypic suppressors of dnaAts46 that are co-transduced with dnaA, render the cell cold sensitive, and cause an excess of chromosome replication relative to cell mass when the cells are shifted from 42 degrees C to 32 degrees C. We have used pulse labelling and DNA-DNA hybridization to follow the effect of a temperature shift on the replication of the chromosome and of the plasmids pSC101, RTF-Tc, and lambda dv in such strains. After a shift of a dnaAcos strain from 42 degrees C to 32 degrees C (non-permissive temperature), initiation of the chromosome and replication of the plasmid pSC101 are stimulated, while the dnaA-independent plasmid RTF-Tc is not affected. The presence of pSC101 does not affect the level of overinitiation of the chromosome. The presence of lambda dv suppresses the cold sensitivity of dnaAcos mutants and allows the cells to grow at both 32 degrees C and 42 degrees C. The presence of lambda dv suppresses the overinitiation of chromosome and of pSC101 replication at 32 degrees C. Previous reports had shown that these suppressions involve an interaction between the dnaA product and the lambda P protein, which is also known to interact with dnaB. We show here that the mutant prophage P1 bac-crr, which produces high levels of a dnaB analogue, suppresses the dnaAcos phenotype, while wild type P1 does not. These results suggest that initiation involves interactions between the dnaA and dnaB products.     

Effect of dna mutations on the replication of plasmid pSC101 in Escherichia coli K-12.

Escherichia coli strains with mutations in genes dnaB, dnaC, and dnaG were tested for their capacity to replicate pSC101 deoxyribonucleic acid (DNA) at a nonpermissive temperature. Only a small amount of radioactive thymine was incorporated into pSC101 DNA in the dna mutants at 42 degrees C, whereas active incorporation into plasmid DNA took place in wild-type strains under the same conditions. The effects of the dnaB and dnaC mutations were greater on plasmid DNA synthesis than on host chromosomal DNA synthesis, suggesting that these gene products are directly involved in the process of pSC101 DNA replication. In dnaG mutants, both plasmid and chromosomal DNA synthesis were blocked soon after the shift to high temperature; although the extent of inhibition of the plasmid DNA synthesis was greater during the early period of temperature shift to 42 degrees C as compared with that of the host DNA synthesis, during the later period it was less. It was found that the number of copies of pSC101 per chromosome in dnaA and dnaC strains, grown at 30 degrees C, was considerably lower than that in wildtype strains, suggesting that the replication of pSC101 in these mutant strains was partially suppressed even under the permissive conditions. No correlation was found between the number of plasmid copies and the tetracycline resistance level of the host bacterium.     

In vitro deletions in the partition locus of plasmid pSC101.

Deletion mutants in the 375-base-pair EcoRI-AvaI fragment carrying the partition locus of plasmid pSC101 were formed by the combined action of exonuclease III and nuclease S1. Six deletion mutants were isolated, and the endpoints of the deletions were sequenced. One of the deletions extended 69 base pairs from the EcoRI site without impairing plasmid stability. The other five deletions caused the plasmid to be unstable and extended 199 to 251 base pairs from the EcoRI site.     

Physiological consequences of mutations in Escherichia coli heat shock dnaK and dnaJ genes.

Mutations in the heat shock genes, dnaK and dnaJ, cause severe defects of several cellular functions. Null dnaJ and dnaKdnaJ mutations can be transduced in a restricted range of temperature. The efficiency of transformation with three unrelated plasmids, viz pACYC184, pBR322 and pSC101, is two times lower in dnaK mutants while the dnaJ mutant is characterized by slightly impaired transformation with pSC101 only. The lack of DnaJ function negatively influences the stability of pSC101 at 42 degrees C, and this plasmid cannot be stably maintained at 30 degrees C in the delta dnaKdnaJ mutant. The double deletion mutant, delta dbaKdnaJ, is characterized by impaired osmoadaptation. The galactokinase content is lower in both mutants tested compared with wild-type strains even at 30 degrees C. The efficient complementation of some of these defects by the wild-type alleles present on low-copy number plasmid was achieved.     

Deletions within E. coli plasmids carrying yeast rDNA.

Deletions occur in recombinant DNA plasmids that contain yeast ribosomal DNA (rDNA) inserted into the E. coli plasmids pSC101 and pMB9. Deletions within a pMB9 plasmid containing an insert longer than one tandem rDNA repeat apparently are due to homologous recombination because (1) all of the independently derived deletion products of this plasmid lost one complete rDNA repeat (8.6 kb) and retained only a single copy of the segment repeated at the ends of the original insert and (2) deletions were detected only when the insert had terminal redundancy. Deletions also occur within a pSC101 plasmid containing a tandem duplication of a segment (4.7 kb) including both pSC101 DNA and rDNA. Once again these deletions appear to be due to the presence of a duplicated region because all deletion products have lost one complete repeat. Deletions within both of these plasmids took place in both rec+ and recA- host cells, but occurred more frequently in rec+ cells. Oligomerization of the deletion products also occurred in both hosts and was more frequent in rec+ cells.     

Specific-purpose plasmid cloning vectors. I. Low copy number, temperature-sensitive, mobilization-defective pSC101-derived containment vectors

Two cloning vector plasmids, pHSG415 (7100 bp) and a lambda phage cos site-containing derivative (cosmid) thereof, pHSG422 (8760 bp), were constructed from a low copy number plasmid (pSC101) replicon to permit the propagation of cloned DNA segments at low gene dosage levels. Two features of the vectors, namely temperature sensitivity of replication and inability to be mobilized by conjugative plasmids, cause them to exhibit a high level of "biological containment". The essential characteristics of pHSG415 and pHSG422 may be summarized as follows: (1) their genome copy number is low (4--6 copies/chromosome); (2) their replication ceases at high temperature and they are rapidly lost from host cells grown at temperatures of 37 degrees C and above; (3) the relaxation nick site of pSC101, which is thought to be synonymous with its origin of transfer replication, is absent from the vectors; as a consequence, they are not mobilized to a significant extent by co-existing conjugative plasmids that are able to mobilize wild-type pSC101; (4) they contain unique insertion sites for DNA fragments generated by the following restriction endonucleases: EcoRI, XhoI, XmaI, HindIII and PstI; pHSG415 additionally contains single BamHI, BstEII and HincII sites and may also be used to clone PvuI-generated fragments; (5) the plasmids confer upon their host cells resistance to chloramphenicol, kanamycin and ampicillin, and every unique cloning site, except those of BamHI and BstEII, is located within one of these antibiotic-resistance genes.     

Mutations of temperature sensitivity in R plasmid pSC101.

Temperature-sensitive (Ts) mutant plasmids isolated from tetracycline resistance R plasmid pSC101 were investigated for their segregation kinetics and deoxyribonucleic acid (DNA) replication. The results fit well with the hypothesis that multiple copies of a plasmid are distributed to daughter cells in a random fashion and are thus diluted out when a new round of plasmid DNA replication is blocked. When cells harboring type I mutant plasmids were grown at 43 degrees C in the absence of tetracycline, antibiotic-sensitive cells were segregated after a certain lag time. This lag most likely corresponds to a dilution of plasmids existing prior to the temperature shift. The synthesis of plasmid DNA in cells harboring type I mutant plasmids was almost completely blocked at 43 degrees C. It seems that these plasmids have mutations in the gene(s) necessary for plasmid DNA replication. Cells haboring a type II mutant plasmid exhibited neither segregation due to antibiotic sensitivity nor inhibition of plasmid DNA replication throughout cultivation at high temperature. It is likely that the type II mutant plasmid has a temperature-sensitive mutation in the tetracycline resistance gene. Antibiotic-sensitive cells haboring type III mutant plasmids appeared at high frequency after a certain lag time, and the plasmid DNA synthesis was partially suppressed at the nonpermissive temperature. They exhibited also a pleiotrophic phenotype, such as an increase of drug resistance level at 30 degrees C and a decrease in the number of plasmid genomes in a cell.     

Plasmid pSC101 replication mutants generated by insertion of the transposon Tn1000

A derivative of pSC101, pLC709, was constructed by ligation of the HincII-A fragment of pSC101 to the mini-colEI plasmid pVH51 and to a DNA fragment encoding resistance to the antibiotics streptomycin and spectinomycin. Insertions of the transposon Tn1000 (gamma-delta) into the pSC101 replication region of pLC709 were isolated following cotransfer of the plasmid with the sex factor F. The sites of insertion of the transposon were determined by restriction enzyme analysis and the replication and incompatibility properties of the insertion plasmids and DNA fragments cloned from them were analysed. The insertion mutations defined a locus, inc, of approximately 200 base-pairs that is responsible for pSC101-specific incompatibility. Two mutations adjacent to this region inactivate pSC101 replication but can be complemented in trans by a wild-type pSC101 plasmid, and thus define a trans-acting replication function, rep. The inc locus is within a larger region of some 450 base-pairs that is essential for pSC101 replication and that includes the origin of replication. This 450 base-pair segment can replicate in the presence of a helper plasmid that supplies the rep function in trans.     

New method for generating deletions and gene replacements in Escherichia coli.

We describe a method for generating gene replacements and deletions in Escherichia coli. The technique is simple and rapid and can be applied to most genes, even those that are essential. What makes this method unique and particularly effective is the use of a temperature-sensitive pSC101 replicon to facilitate the gene replacement. The method proceeds by homologous recombination between a gene on the chromosome and homologous sequences carried on a plasmid temperature sensitive for DNA replication. Thus, after transformation of the plasmid into an appropriate host, it is possible to select for integration of the plasmid into the chromosome at 44 degrees C. Subsequent growth of these cointegrates at 30 degrees C leads to a second recombination event, resulting in their resolution. Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retain the original copy of the gene. The procedure can also be used to effect the transfer of an allele from a plasmid to the chromosome or to rescue a chromosomal allele onto a plasmid. Since the resolved plasmid can be maintained by selection, this technique can be used to generate deletions of essential genes.     

Identification of an Rts1 DNA fragment conferring temperature-dependent instability to vector plasmids

The multiphenotypic drug resistance factor Rts1 expresses a temperature-dependent instability characteristic. This plasmid was digested with the restriction enzyme BamHI. A DNA fragment with a molecular weight of 5.6 MDa (the H fragment) was inserted into plasmid pBR322 (pFK896) or into pSC105 (pYH156) at the BamHI site. These plasmids were unstable at 42 degrees C but stable at 32 degrees C. A restriction-enzyme map of the H fragment was constructed and the instability phenotype (Tdi) was localized to a DNA fragment with 0.5 MDa molecular weight. The temperature-dependent loss of the unstable plasmid pFK896 is abrupt and no gradual plasmid loss of this multicopy recombinant plasmid is observed. The possibility that the Tdi phenotype is due to overgrowth of R- cells was eliminated.     

Selection of lambda Spi- transducing phages using the P2 old gene cloned onto a plasmid

The old gene product of the P2 prophage interferes with plaque formation by lambda wild type phage but allows lambda phages whose red and gam genes have been deleted to form small, visible plaques (the lambda Spi- phenotype). The old gene product also kills Escherichia coli recB or recC mutants. We have cloned the old gene into the high-copy-number plasmid pBR322, where it prevents plaque formation by both lambda Spi+ and lambda Spi- phages. We transferred a DNA fragment that carries the old gene to the low-copy-number plasmid pSC101 and found that lambda Spi- phages can be selected on strains that carry this plasmid. The plasmid-borne old gene kills E. coli recB mutants, providing a selection for old- mutants.     

A deletion analysis of hybrid phage carrying the US region of Herpes simplex virus type 1 (Patton). I. Isolation of deletion derivatives and identification of chi-likes sequences

The EcoRI-H fragment (15.4 kb) of Herpes simplex virus type 1 (HSV-1) has been cloned in lambda gtWES in both orientations. This fragment contains the entire US region and has about 900 bp of terminal redundant sequences derived from the internal and terminal repeats of the S region. 56 independent plaque-forming deletion derivatives of the lambda gt/WES::EcoRI-H hybrid phage were isolated using either EDTA resistance or ability to grow on Escherichia coli(P2) lysogens as selective methods. The endpoints of these deletions were located using nine restriction enzymes that cleave within the EcoRI-H fragment. All of the deletions have at least one endpoint within the cloned fragment. Several unusual features of the lambda hybrids, including heterogeneity of a particular region in the HSV-1 EcoRI-H fragment and the presence of chi-like sequences in the US region of HSV-1, are discussed.     

Identification by deletion analysis of an inducible protein required for plasmid pSC101-mediated tetracycline resistance.

Plasmids containing small deletions within a tetracycline (Tc) resistance gene(s) of plasmid pHA121 were isolated. Plasmid pHA121 was formed by ligating the EcoRI-digested Tc resistance plasmid pSC101 and similarly digested mini-ColE1 plasmid pHA105. The DNA deletion plasmids were constructed by digesting plasmid pHA121 DNA with the restriction endonucleases BamH1 and Sal1 and, in addition, λ exonuclease. Two plasmids, designated pJT131 and pJT133, had small deletions of approximately 0.64 to 0.8 kb and a comparison of the radioactive polypeptides synthesized in plasmid-containing minicells revealed that a 34-kdal polypeptide was not specified by either pJT131 or pJT133. We conclude that the 34-kdal polypeptide is required for the expression of Tc resistance and that its structural gene probably maps in the deleted region of pSC101 DNA.     

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.     

Low-copy-number plasmid-cloning vectors amplifiable by derepression of an inserted foreign promoter

By insertion of a DNA fragment, containing the phage lambda pR promoter and the pM-promoted cI857 allele of the lambda repressor gene, in plasmid R1 upstream of the replication control genes, cloning vectors have been constructed which are present in one copy per chromosome at temperatures below 37 degrees C, and which display uncontrolled replication at 42 degrees C. Derivatives have been made which carry the R1 par region, stabilizing the plasmid at low temperature when grown in the absence of selection pressure. Cells harbouring these plasmids stop growing after 1-2 h incubation at 42 degrees C, and at this time 50% of the total DNA in the cells is plasmid DNA corresponding to more than 1000 plasmid molecules per cell. Concomitant with plasmid amplification at the high temperature, synthesis of plasmid-coded gene products is amplified, and these vectors can therefore be utilized for obtaining greatly enhanced yields of gene products that may be detrimental to the host cell when present in large amounts.     

Molecular cloning of the C-terminal domain of Escherichia coli D-mannitol permease: expression, phosphorylation, and complementation with C-terminal permease deletion proteins.

We have subcloned a portion of the Escherichia coli mtlA gene encoding the hydrophilic, C-terminal domain of the mannitol-specific enzyme II (mannitol permease; molecular mass, 68 kilodaltons [kDa]) of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system. This mtlA fragment, encoding residues 379 to 637 (residue 637 = C terminus), was cloned in frame into the expression vector pCQV2 immediately downstream from the lambda pr promoter of the vector, which also encodes a temperature-sensitive lambda repressor. E. coli cells carrying a chromosomal deletion in mtlA (strain LGS322) and harboring this recombinant plasmid, pDW1, expressed a 28-kDa protein cross-reacting with antipermease antibody when grown at 42 degrees C but not when grown at 32 degrees C. This protein was relatively stable and could be phosphorylated in vitro by the general phospho-carrier protein of the phosphotransferase system, phospho-HPr. Thus, this fragment of the permease, when expressed in the absence of the hydrophobic, membrane-bound N-terminal domain, can apparently fold into a conformation resembling that of the C-terminal domain of the intact permease. When transformed into LGS322 cells harboring plasmid pGJ9-delta 137, which encodes a C-terminally truncated and inactive permease (residues 1 to ca. 480; molecular mass, 51 kDa), pDW1 conferred a mannitol-positive phenotype to this strain when grown at 42 degrees C but not when grown at 32 degrees C. This strain also exhibited phosphoenolpyruvate-dependent mannitol phosphorylation activity only when grown at the higher temperature. In contrast, pDW1 could not complement a plasmid encoding the complementary N-terminal part of the permease (residues 1 to 377). The pathway of phosphorylation of mannitol by the combined protein products of pGJ9-delta 137 and pDPW1 was also investigated by using N-ethylmaleimide to inactivate the second phosphorylation sites of these permease fragments (proposed to be Cys-384). These results are discussed with respect to the domain structure of the permease and its mechanism of transport and phosphorylation.