Site-directed insertion mutagenesis with cloned fragments in Escherichia coli by P1 phage transduction

Summary A cloned gene with an insertion, which was made by introducing cat, was ligated to the cloning site of the phage gt11. P1 phage grown on cells lysogenized with the recombinant phage could transduce the mutant gene into the original site on the Escherichia coli chromosome.     

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.     

Cloning and characterization of the Escherichia coli chromosomal region surrounding the dnaG gene, with a correlated physical and genetic map of dnaG generated via transposon Tn5 mutagenesis

Summary A 24 kilobase pair region of the E. coli chromosome surrounding the dnaG gene has been cloned and characterized. A phage library was first constructed by ligating a Sau3A (GATC) partial DNA digest of the entire E. coli chromosome into the BamHI (G GATCC) cloning vector charon 28. Partial digestion was performed to generate overlapping chromosomal fragments and to allow one to walk along the chromosome. This library was probed with a nick-translated plasmid (pRRB1) containing the rpoD gene, which maps adjacent to dnaG at 66 min. Four bacteriophages: 3, 4, 5, 6 that hybridized to the probe were isolated from the 2,500 plaques screened. One phage recombinant 4, was shown to contain the dnaG gene. Three recombinant plasmids containing dnaG: pGL444, pGL445, pBS105, were constructed via subcloning of 4 using different restriction fragments. Plasmids pGL444 and pBS105 were subjected to transposon Tn5 mutagenesis and 88 Tn5 inserts into the cloned region were isolated. The location of the Tn5 inserts were mapped by restriction enzyme analysis of the plasmids and the insertion mutations were checked for ability to complement a dnaGts chromosomal marker at nonpermissive 40° C. In this manner a correlated physical and genetic map of dnaG was determined. A large number of Tn5 inserts map to a specific 900 b.p. region which we propose may be involved in the regulation of dnaG gene expression.     

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.     

Inhibition of modification and restriction for phages lambda and T-1 by co-infecting T3

Summary The host controlled modifications of phage -DNA byEscherichia coli B, K, and C (P1) can be suppressed by preinfecting the bacteria with UV-irradiated phage T3. Since UV-irradiated T3 induces an enzyme which cleaves S-adenosylmethionine into homoserine and thiomethyl adenosine, and since S-adenosylmethionine is the only methyl group donor for DNA methylation, we conclude that methylation is a required step in the host controlled modification of -DNA.T3 itself successfully infectsE. coli K and B with its nonmethylated DNA. Also, restricted phage or T1 will be accepted by the restrictive hostsE. coli B, K, and C(P1) if these are preinfected with UV-T3. It thus appears that T3 is capable of blocking the restriction mechanisms in these hosts.The inability of T3 to grow on C(P1) is not understood. Since T3-DNA is restricted but not degraded into nucleotides byE. coli C(P1) we presume that degradation is not the initial step in restriction.Supported by Grant No. GB 1033 R of the National Science Foundation.Postdoctoral fellow of the Deutsche Forschungsgemeinschaft.     

lamB mutations in E. coli K12: Growth of λ host range mutants and effect of nonsense suppressors

Summary Over sixty EMS induced mutations affecting gene lamB, presumably the structural gene for the receptor in Escherichia coli K12, were examined for growth of host range mutants and effect of nonsense suppressors. By the first criterion the mutations could be grouped in three classes. Bacteria with class I mutations allow growth of mutants with extended host range (noted h) of the type already described (Appleyard, Mac Gregor and Baird, 1956). Bacteria with class II mutations allow growth of h mutants with still more extended host range (noted hh ^*). No host range mutants of could be found which would grow on bacteria with class III mutations. Using nonsense suppressors it was found that class I and II consist of missense mutations, while class III consists of nonsense mutations. Exceptions are likely to exist (especially in class III) but were not found among the mutations tested. These observations are briefly discussed in terms of outer membrane protein integration and of phage receptor interaction.     

Function of cloned T4 recombination genes, uvsX and uvsY, in cells of Escherichia coli

Summary Genes uvsX and uvsY of bacteriophage T4 both control genetic recombination and repair of damaged DNA, and their mutant phenotypes bear a striking resemblance to each other. It has been shown recently that the uvsX gene product is analogous to the recA gene product of Escherichia coli (Yonesaki et al. 1985; Yonesaki and Minagawa 1985; Formosa and Alberts 1986), but the function of the uvsY gene is unknown. To obtain further insight into the function of these genes we introduced plasmid-borne copies of the two genes separately or together into E. coli. The uvsX gene rendered recA ^- cells more resistant to UV and raised the recombination frequency of phage and E. coli, but hampered induction of the prophage and the SOS function of E. coli. The uvsY gene had no detectable function when introduced alone into E. coli but significantly enhanced the function of the uvsX gene when the two plasmid-borne genes were introduced together.     

Mutants of phage HK239 defective in excluding phages lambda T4rII, P1, and P2

Summary Escherichia coli cells lysogenic for temperate phage HK239 exclude phages , HK022, P1 vir, P2, and rII mutants of phage T4. After mutagenic treatment, four isolates were obtained for their inability to exclude T4rII. It is shown that this mutation, designated exc, is located in the prophage HK239, and that, it also abolishes the exclusion of phages , HK022, P1 vir, and P2.     

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 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.     

Cleavage of lambda repressor and synthesis of RecA protein induced by transferred UV-damaged F sex factor

Summary Transfer of a UV-damaged F sex factor to a recipient lysogen induces prophage development. Under these conditions RecA protein synthesis was induced and repressor cleaved, as observed upon direct induction, that is, when the recipient lysogen was directly exposed to UV-light. The efficiency of induction of RecA protein synthesis in recipient bacteria which had received an irradiated F-lac factor was about 80% of that measured upon direct induction. We observed the simultaneous disappearance of repressor and a slight production of cleavage fragments; quantitation by densitometric scanning of the autoradiogram after correction for the efficiency of transfer indicated that 55% of repressor was cleaved. Transfer of UV-damaged Hfr DNA failed to induce RecA protein synthesis. A phage vector carrying oriF, the cloned origin of F plasmid replication, after exposure to UV-light and infection of a recipient lysogen, induced RecA protein synthesis and a moderate but significant cleavage of repressor. Indirect induction by UV-damaged F sex factor or phage oriF resulted in biochemical cellular reactions similar to those observed upon direct induction. LexA repressor that negatively controls RecA protein synthesis appeared more susceptible to cleavage than did repressor.     

Localization of the metJBLF gene cluster of Escherichia coli in lambda met transducing phage

Summary The position of the metJBLF gene cluster in the transducing phage dmet102 was determined by ligation of its leftmost EcoRI fragment (102-1) to the BCDEF (nin5) EcoRI fragment of gtl (BC) and characterization of the resultant recombinant phage. The new transducing phage carries about 6kb of bacterial DNA which contains the entire met gene cluster including the promoter of its rightmost member metF. Reasonable estimates of the coding capacity required for the four genes indicate that most of the bacterial DNA of the recombinant phage is occupied by the met gene cluster.     

A hotspot of spontaneous and UV-induced illegitimate recombination during formation ofλbio transducing phage

To study the mechanism of spontaneous and UV-induced illegitimate recombination, we examined the formation of thebio specialized transducing phage inEscherichia coli. Because mostbio transducing phages have double defects in thered andgam genes and have the capacity to form a plaque on anE. coli P2 lysogen (Spi^– phenotype), we selectedbio transducing phage by their Spi^– phenotype, rather than using thebio marker. We determined sequences of recombination junctions ofbio transducing phages isolated with or without UV irradiation and deduced sequences of parental recombination sites. The recombination sites were widely distributed onE. coli bio and DNAs, except for a hotspot which accounts for 57% of UV-inducedbio transducing phages and 77% of spontaneously inducedbio transducing phages. The hotspot sites onE. coli and DNAs shared a short homology of 9 bp. In addition, we detected direct repeat sequences of 8 by within and near both thebio and hotspots. ArecA mutation did not affect the frequency of the recombination at the hotspot, indicating that this recombination is not a variant ofrecA-dependent homologous recombination. We discuss a model in which the short homology as well as the direct repeats play essential roles in illegitimate recombination at the hotspot.     

A transducing lambda phage carrying grpE, a bacterial gene necessary for lambda DNA replication, and two ribosomal protein genes, rpsP (S16) and rplS (L19).

Summary A grpE mutation of Escherichia coli K12, which blocks DNA replication of the phage (Saito and Uchida, 1977), was mapped at 56 min on the standard genetic map. A transducing phage, grpE22, carrying the wild type allele of the grpE gene was constructed in vitro. Structures of grpE22 and its viable deletion derivatives were determined by electron microscopic analyses of appropriate heteroduplexes. Proteins coded by the bacterial DNA incorporated into the transducing phages were detected by two-dimensional gel electrophoresis. The results showed that the product of the grpE gene is a weakly acidic protein of molecular weight 24,000. Structural genes for two ribosomal proteins, rplS (L19) and rpsP (S16) were also shown to be carried by grpE22.     

Molecular cloning of the gene for the beta-lactamase of Bacillus licheniformis and its expression in Escherichia coli

Summary The structural gene, pen, for the -lactamase of B. licheniformis has been cloned into a vector and shown to be expressed at a low rate in E. coli. The cloned pen gene appears to be expressed from a promoter within the fragment of B. licheniformis DNA, since its rate of expression is not affected by the presence of the phage repressor, the absence of the phage's positive-control functions, or the position or orientation of the gene within the phage genome.     

The bacteriophage T4 dexA gene: sequence and analysis of a gene conditionally required for DNA replication

Summary We have cloned and sequenced a bacteriophage T4 EcoRI fragment that complements T4 del (39-56) infections of an optA defective Escherichia coli strain. Bacteria containing this recombinant plasmid synthesize two new proteins with molecular weights of 9 and 26 kilodaltons. We have identified the gene encoding the 26 kilodalton protein as essential for T4 infections of optA defective E. coli. Genetic and biochemical results are consistent with the identification of this protein as the product of the dexA gene, which encodes a 3 to 5 exonuclease.     

Identification of the protein products of the rrnC, ilv, rho region of the Escherichia coli K-12 chromosome

Summary Two methods have been used to identify the protein products of the Escherichia coli K-12 ilv region at 84 min and the flanking rrnC (counterclockwise) and rho (clockwise) loci. First, a set of dilv specialized transducing phages, including some phages that carry rho and others that carry part of rrnC, was used to infect UV irradiated cells. The proteins produced by the infecting dilv phage were selectively labelled with radioactive amino acids and identified by SDS gel electrophoresis and autoradiography. Second, restriction enzyme fragments were cloned from the dilv phage into pBR322 and the plasmid specific gene products produced in maxicells were similarly identified by SDS gel electrophoresis and autoradiography. The proteins produced were correlated with specific genes and restriction enzyme fragments present in the dilv phage and the pBR322 derivatives. Several ilv gene products that have previously been refractory to protein purification attempts have been identified for the first time by this technique. The presence of mutations at the ilvO site is shown to activate the cryptic ilvG gene and to result in the production of a 62,000 dalton protein. A 15,000 dalton protein of unknown function is synthesized from a DNA segment between ilv and rrnC. The rho gene was cloned from dilv phage into pBR322 and shown to be dominant to a rho mutation on the host cromosome. The rho gene product and four additional proteins coded by genes near or between rho and ilv have been detected.