The novel gene(s) ARD of plasmid pKM101: alleviation of EcoK restriction

Summary The host-controlled K restriction of unmodified phage was 10-100-fold alleviated in the wild-type strain E. coli K12, carrying plasmid pKM101 of incompability group N. pKM101-mediated release of K restriction was also observed in lexA ^-, recA ^-, and recB ^- strains of E. coli K12. By restriction mapping Tn5 insertions in pKM101, which reduced pKM101-mediated alleviation of restriction, were shown to be located within the BglIIB fragment approximately 11 kb anticlockwise from the RI site of pKM101. We have termed the gene(s) promoting the alleviation of K restriction of phage ard (alleviation of restriction of DNA). It was shown (1) that ard function affected only the EcoK restriction system and not the EcoB, EcoRI, EcoRIII, or EcoPI system, (2) ard gene(s) did not mediate EcoK type modification of DNA and did not increase the modification activity of the EcoK system in a way similar to that observed with gene ral of bacteriophage .     

Overproduction of E. coli xylose isomerase

Summary A promoterles DNA fragment containing theE. coli xylose isomerase gene and its ribosome binding site was ligated into a plasmid downstream from the strong P_L promoter. The plasmid was then used to transformE. coli strains containing a temperature-sensitive repressor (cI857). The transformants overproduced xylose isomerase when the repressor was thermally inactivated.     

Use of lambda vehicles to isolate ompC-lacZ gene fusions in Salmonella typhimurium LT2

Summary A novel plasmid vector, pAMH70 carrying both the lamB and nusA genes of Escherichia coli K12 was constructed. Introduction of this plasmid into Salmonella typhimurium LT2 renders this bacterium both sensitive to adsorption and able to sustain growth and lysogenization by . Using this strain as a recipient, stable gene fusions to the gene encoding a major outer membrane porin protein OmpC, were constructed with a vehicle placMu. To confirm the actual site of fusions they were genetically mapped and transducing phages carrying the ompC-lacZ fusion were isolated and relysogenized. The fusions were also shown to be to ompC by their regulatory properties.     

UV-induced allevation of lambda restriction in Escherichia coli K-12: kinetics of induction and specificity of this SOS function

Summary In UV-irradiated cells of Escherichia coli K-12 a partial release of the restriction of non-modified phage is observed when the cells are recA ⁺ lexA ⁺. We show here that the induction of this restriction allevation (RA) also depends on the recBC enzyme and that the expression of RA requires protein synthesis. Maximum expression was reached within 60 to 90 min after irradiation. Experiments are presented which show that upon UV-irradiation a signal is created which triggers the development of RA when protein synthesis is allowed. This signal decayed with a half-life of only a few minutes in cells treated with chloramphenicol. The decay kinetics were similar in uvr ⁺ and uvrA mutants. RA appeared to be specific for EcoK insofar as no allevation of restriction by EcoRI, EcoRII and EcoP1 occurred. During maximum expression of RA no gross reduction of the activities of the recBC enzyme (exonuclease V) and the restriction endonuclease EcoK was observed and no new DNA modifying activity appeared in the cells. Since, in fully expressed cells, up to 75% of the infecting DNA was converted to acid-soluble material within 20 min after infection we suggest that only a small specific fraction of infections may undergo RA.     

Cloning of the replication gene P of bacteriophage lambda: Effects of increased P-protein synthesis on cellular and phage DNA replication

Summary A restriction fragment of DNA carrying the P gene was cloned in the high copy number plasmid RSF2124. Cells harbouring this new plasmid RSF2124/E complement Pam80 phage. A lac promoter-operator region (lacP), produced by EcoRI digestion of plasmid pKB252, was inserted into RSF2124/glE such that induction of the lac promoter by IPTG or lactose leads to increased production of the P gene product. A high amount of P protein in E. coli cells results in a slow inhibition of bacterial DNA synthesis, suggesting that the initiation reaction is blocked by P protein. Synthesis of DNA proceeds normally under these conditions.Nonsuppressing groPA15 mutant bacteria which are unable to support the replication of wild-type (wt), acquire the ability to replicate Pam80 phage but not wt when they are transformed with a plasmid carrying the P gene. When harbouring a plasmid containing the mutant Pamber 80 gene, groPA15 mutants are able to support the replication of wt phage when infected at a high multiplicity. Pam80 phage does not multiply in these cells.     

Molecular cloning of the T4 genome; organization and expression of the frd--DNA ligase region

Summary derivatives including the thymidylate synthetase (td) gene of T4 were selected by their ability to substitute for the thyA gene of E. coli. Two HindIII fragments of T4 DNA, but only one EcoRI fragment, are required for a functional td gene; one of the HindIII fragments includes a functional frd gene. The organisation of the EcoRI and HindIII fragments in the td region and their orientation with respect to the T4 genome have been deduced from genetic, physical, and functional evidence. The T4 genes can be transcribed from phage promoters and the T4td derivatives include genes specifying five T4 polypeptides. Three of these are identified as the products of the frd, td, and nrdA genes; two, neither of which appears to be the nrdB gene product, remain to be identified. Some td phages yield lysogens of thyA bacteria which are thymine-independent and some frd phages yield trimethoprim-resistant lysogens, indicating that the td and frd genes can be transcribed from included T4 DNA sequences. EcoRI fragments of DNA from the td and lig regions, used as probes, identified a single large HindIII fragment that joins the HindIII fragment carrying the DNA ligase gene to that carrying the td gene. Since this fragment, which must include genes coding for RNA ligase and polynucleotide kinase, could not be recovered in either phage or plasmid vectors, a derivative of it was used to identify the EcoRI fragments located between the td and DNA ligase genes. The order of these fragments within the T4 genome was deduced and all but one of them cloned in a vector. As none of these recombinants rescued T4 phage having mutations within the RNA ligase gene, the missing fragment may include this gene. Three adjacent EcoRI fragments, each of which has been cloned, are missing in a mutant of T4 deleted for the polynucleotide kinase gene.     

Cloning of the ugp region containing the structural genes for the pho regulon-dependent sn-glycerol-3-phosphate transport system of Escherichia coli

Summary Using a novel positive selection method for G3P transport activity, phages that carry either all or part of ugp, the genes of the pho regulon-dependent G3P transport system of Escherichia coli were isolated from a library of EcoRI fragments of Escherichia coli established in gt7. By subcloning EcoRI fragments carried by the different phages into the multicopy plasmids pACYC184 and pUR222, it was shown that two chromosomal fragments of 6.0 and 6.6 kb are required for the expression of ugp, whereas all the structural information is located on the 6.6 kb EcoRI fragment. A restriction map of the cloned DNA was established and the extent of ugp genes determined by Tn5 insertions. Using ugp-lacZ fusions, it could be shown that the ugp region consists of at least two different operons that are transcribed in the same direction (counterclockwise) on the E. coli chromosome.Abbreviations DHBP 3,4-dihydroxibutyl-1-phosphonate - G3P sn-glycerol-3-phosphate - G3PBP glycerol-3-phosphate binding protein - IPTG isopropyl--d-thiogalactopyranoside - XG 5-bromo-4-chloro-3-indolyl--d-galactopyranoside     

Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis.

Summary Hybrid ColE1 plasmids called ColE1-cos-guaA or ColE1-cos-gal can be efficiently transduced into various E. coli K-12 cells through packaging into phage particles. Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E. coli K-12 mutants. (1) The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-cos-guaA. (2) Pre-existing hybrid ColE1 plasmids had no effect on the frequency of phage-mediated transduction of another differentially marked hybrid ColE1 DNAs. (3) ColE1-cos-guaA and ColE1-cos-gal DNAs could temporarily but not stably co-exist in E. coli K-12 recA cells. (4) The presence of ColE1-cos-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-cos-guaA about 7-fold. (5) The same ColE1-cos-gal plasmid in a uvrB recA double mutant did not have this promoting effect. These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA ⁺ gen function(s) and suggest that ColE1 plasmid itself provides no recA ⁺-like functions.     

Cloning of an EcoRI fragment carrying E. coli tufA gene.

Summary EcoRI fragments of the transducing phage fus3 DNA have been linked to the ColEl derivative plasmid RSF2124 (ColEl-Ap^r) DNA using bacteriophage T4 ligase. Among the plasmids formed, one designated pTUAl was found to contain the E. coli tufA gene. The proof for the presence of tufA gene in pTUAl is based on the following observations: (1) ability of pTUAl DNA and its EcoRI fragments to direct synthesis of EF-Tu in a cell-free protein synthesizing system; and (2) RNA·DNA hybridization of RNA transcribed from phage rif ^d18 carrying tufB with DNA from pTUAl.     

Construction of recombinant lambda phages that carry the E. coli recB and recC genes

Summary A fragment of the E. coli chromosome including the recC gene has been cloned by in vitro recombinant DNA techniques into a phage vector to give the recombinant phage drecC. This was used to derive the phage drecBC by in vivo recombination. On lysogenisation of recB and recC mutants with drecBC wild type levels of UV-resistance and RecBC DNase activity were restored. Infection of E. coli with drecBC led to the synthesis of phage-coded proteins of 125 kilodaltons (kd) and 135 kd that were not synthesised on infection with the original vector, whereas a 125 kd protein but not a 135 kd protein was synthesised in similar experiments with drecC. The recombinant phages, which are unable to form plaques, presumably due to the deletion of essential phage genes during their construction, provide useful starting points from which to subclone the recB, recC, and the neighbouring thyA and argA genes individually into multiple copy plasmid vectors.     

Cosmid cloning and transposon mutagenesis in Salmonella typhimurium using phage lambda vehicles

Summary We have constructed a strain of Salmonella typhimurium which contains the malB region from Escherichia coli and carries the bacteriophage receptor protein in its outer membrane. Phage adsorbs to this strain but cannot grow, thus providing a very useful system for transposon mutagenesis of S. typhimurium using vehicles carrying transposons. This system can also be used for cosmid cloning.     

Alleviation of EcoK DNA restriction in Escherichia coli and involvement of umuDC activity.

Summary The activity of the EcoK DNA restriction system of Escherichia coli reduces both the plating efficiency of unmodified phage and the transforming ability of unmodified pBR322 plasmid DNA. However, restriction can be alleviated in wild-type cells, by UV irradiation and expression of the SOS response, so that 10³-to 10⁴-fold increases in phage growth and fourfold increases in plasmid transformation occurred with unmodified DNA. Restriction alleviation was found to be a transient effect because induced cells, which initially failed to restrict unmodified plasmid DNA, later restricted unmodified phage . Although the SOS response was needed for restriction alleviation, constitutive SOS induction, elicited genetically with a recA730 mutation, did not alleviate restriction and UV irradiation was still needed. A hitherto unsuspected involvement of the umuDC operon in this alleviation of restriction is characterized and, by differential complementation, was separated from the better known role of umuDC in mutagenic DNA repair. The need for cleavage of UmuD for restriction alleviation was shown with plasmids encoding cleavable, cleaved, and non-cleavable forms of UmuD. However, UV irradiation was still needed even when cleaved UmuD was provided. The possibility that restriction alleviation occurs by a general inhibition of the EcoK restriction/modification complex was tested and discounted because modification of was not reduced by UV irradiation. An alternative idea, that restriction activity was competitively reduced by an increase in EcoK modification, was also discounted by the lack of any increase in the modification of Ral^–, a naturally undermodified phage. Other possible mechanisms for restriction alleviation are discussed.     

Cloning and characterization of the alkaline phosphatase positive regulator gene (phoB) of Escherichia coli

Summary The positive regulator gene (phoB) for alkaline phosphatase of Escherichia coli was cloned into the EcoRI site of pBR322 from the E. coli chromosome by a shotgun method. phoB was then constructed in vitro by replacing the C fragment of gtC by the phoB chromosomal fragment obtained from the hybrid plasmid. When the phoB mutant was lysogenized by phoB, the lysogen became PhoB⁺. The integration site of the phage was identified by P1 phage transduction to be around phoB site on the chromosome. From these results, we conclude that the cloned gene is phoB and not a gene which suppresses phenotypically phoB mutation when it is in a multi-copy state. The restriction map was constructed. Based on this information, several PhoB deletion plasmids and smaller PhoB⁺ plasmids were constructed in vitro. By examining PhoB phenotype when these plasmids were introduced into phoB mutant, we could define the phoB gene locus in 2 kb on the restriction map of the cloned chromosomal fragment. Cells carrying the multi-copy phoB gene produced alkaline phosphatase qualitatively under normal phosphate regulation. The phoB gene product was identified by the maxicell method as a protein with a molecular weight of approximately 31,000 daltons.     

In vitro insertion of the lambda attachment site into the plasmid RP4.

Summary The region of the phage lambda chromosome containing the attachment site (P · P) and the genes int and xis, excised by the action of endonuclease R.EcoRI, has been inserted into the unique site for that enzyme on the promiscuous conjugative plasmid, RP4, generating the recombinant plasmid RP4att. Transformants containing the hybrid plasmid were recognised by their ability to allow efficient lysogenization by phage b2 (Weil and Signer, 1968; Echols et al., 1968) containing the mutant attachment site · P. The construction and properties of the hybrid plasmid RP4att are described.     

Map location of the Escherichia coli origin of replication.

Summary Working with restriction fragments obtained directly from the Escherichia coli K12 chromosome, the EcoRI-HindIII restriction map of the section of the chromosome containing the replication origin has been extended by 14 kilobase pairs (kb) to cover 56kb. Within this newly mapped portion, the liv and rrnC cistrons have been identified by (1) hybridization of individual restriction fragmenents to the ilv-transducing phage dilv5 and (2) a comparison of the restriction map of this region with the EcoRI map of dilv5 and the HindIII map of the plasmid pJC110, a ColE1-ilv hybrid. The replication origin is located approximately 30 kb from the ilvE gene and 20 kb from the rrnC 16S rRNA cistron. This places the origin near 82.7 min on the genetic map, close to uncA.     

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.     

Cloning of the dnaB gene of Escherichia coli: the dnaB gene of groPB534 and groPB612 and the replication of phage lambda

Summary Fragments of the E. coli chromosome that carry the dnaB groPB534 or groPB612 alleles have been cloned into a cosmid vector. The resulting recombinant plasmids contained the genes uvrA, groP (B534 or B612), and lexA. Further subcloning into high copy number plasmids, during which the uvrA and lexA genes were removed successively, yielded a groPB534 and groPB612 DNA fragment of about 2.4 kb each. Both fragments contained an overlapping 1.8 kb segment of DNA in which the sites of all restriction enzymes tested were identical. The size of these dnaB gene fragments were further delimited by deletion analysis.In E. coli groPB534 in which wild-type and A mutants do not replicate (Georgopoulos and Herskowitz 1971) phage replication is rescued if the strain contains the groPB534 gene on high copy number plasmids. On the contrary, in E. coli groPB612, which is temperature-sensitive for its groP character, replication of and A is abolished at 30° C if the strain contains the groPB612 recombinant plasmid. On the other hand, replication of B remains unaffected whether or not the groP strains harbor the isogenic dnaB gene-containing plasmid. The results suggest that within the cell not only the quality but also the relative amounts of dnaB and P protein are crucial for phage replication.     

Lysogenic induction of lambdoid phages in lexA mutants of Escherichia coli

Summary UV irradiation of lexA3 mutants of E. coli caused lysogenic induction of prophage , _i21, _i434 and 80. Maximal induction in lexA3 lysogens needed less UV than in lexA ⁺ bacteria and gave 25–100% of the normal levels of infective centres induced. Assays of gene expression arising from derepression of a defective prophage showed at least 40% of the normal levels of induction by mitomycin C in lexA3 bacteria. The need for post-irradiation protein synthesis for lysogenic induction in lexA3 lysogens was reduced by increasing the basal level of recA protein with a recA ⁺ plasmid. It is concluded that in lexA E. coli some recA protein synthesis, too small to be detected by physical means, is needed for UV induced lysogenic induction.     

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