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.     

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.     

Genetic selection of lambda phage clones from a gene clonotheque

Summary A genetic procedure for selection of specific clones, by homologous recombination between clones from a gene clonotheque and sequences cloned into a plasmid, was developed. Resulting clones are isolated in transduction experiments by plating infected Escherichia coli cells under conditions selecting for the antibiotic resistance marker carried by the plasmid. The feasibility of the method was demonstrated in a model test system as well as by isolation of -interferon-specific sequences from the human gene clonotheque.     

Integration of bacteriophages lambda and phi 80 in wild-type Escherichia coli at secondary attachment sites. II. Genetic structure and mechanism of polylysogen formation for lambda, phi 80 and the lambda att80 hybrid

Summary The frequency of occurrence and the genetic structure of polylysogens were studied for phages , 80 and att80. In the case of , frequency of polylysogenization is high (0.20 to 0.41) with a tandem integration of prophages at the primary att site (att). With 80 and att80, this frequency is about 10 times lower, and usually one prophage becomes integrated at the primary att site (att80-I) while another (sometimes two others) integrates at one of the secondary sites. At least four secondary att80 sites have been found in wild-type Escherichia coli , two of which (near the his and tolC loci) are preferred. The frequency of secondary integration of 80 and att80 does not differ significantly in the wild-type host and in that deleted for the primary att site (0.041 and 0.045, respectively, among surviving cells at an MOI of 10).Homoimmune superinfection has revealed a constitutive cI-independent expression of the 80 int gene in the prophage state. The only 80 tandem detected proved to be unstable. With the 80int ^- mutant, we observed stabilization of 80 tandems; as a consequence, their frequency of occurrence during coinfection with 80int ⁺ was up to the level and no nontandem insertions were found. A model is proposed for the 80 and att80 nontandem integration.Abbreviations TP transducing phage(s) - PFU plaque-forming units - PC pink clear-resistant colonies on EMBO plates - MOI multiplicity of infection - O origin of Hfr transfer

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Bacteriophage lambda replication proteins: formation of a mixed oligomer and binding to the origin of lambda DNA

Summary The purified bacteriophage replication proteins O and P sediment separately in metrizamide gradients of low ionic strength as dimers. Together they interact with each other forming an oligomer, composed of two molecules of O and one molecule of P. The O-P oligomer is active in the in vitro replication of ori-containing DNA.Equilibrium sedimentation in preformed metrizamide density gradients under conditions that separate DNA-protein complexes from free proteins was employed in order to study possible interactions among the replication proteins and ori DNA. It was found that the P protein binds specifically to ori-containing plasmid DNA only in the presence of O protein. About 100 molecules of O and 10 molecules of P form a complex with the ori DNA. The DNA-O-P complex was shown to be active in an in vitro replication system.Since the physical interactions between ori and O and between P and the Escherichia coli dnaB replication protein are well documented, the evidence for a O-P interaction presented in this paper provides the missing link in the molecular mechanism that enables to direct the host replication machinery to the replication of its own DNA.     

Structure and function of the repressor of bacteriophage lambda

Summary By mutagenizing a cIts (cI857) lysogen, a mutant has been isolated with a wild-type phenotype. This mutant phage lysogenizes with low efficiency and produces a low burst. Though the initial rates of repressor synthesis in Escherichia coli after infection with wild-type and mutant are the same, the maximum level of repressor that is synthesized in the latter case is only about 30% of that synthesized in the former. Virulent plates on the lysogen of mutant with slightly less efficiency producing very tiny plaques. Operator-binding studies made in vitro with purified mutant and wild-type repressors show that the binding curve of the former repressor is a rectangular hyperbola while that of the latter is sigmoid. The half-lives of the complexes of mutant and wild-type repressors with right operator are 133 and 27 min, respectively. All these results suggest that the mutant repressor possibly has a higher affinity for the operators. This mutant has been named cIha (ha=high affinity).     

Requirement of protein and RNA synthesis for lambda repressor inactivation by tif-1: effects of chloramphenicol, neomycin and rifampicin.

Summary The inactivation of repressor was followed by the specific DNA binding assay during the course of lysogenic induction provoked by incubation at 42°C of an E. coli tif-1 lysogenic strain. The presence of up to 400 g/ml chloramphenicol during the inducing treatment did not impair the loss of repressor binding activity, whilst concentrations of 200 g/ml neomycin and 100 g/ml rifampicin effectively inhibited the inactivation of repressor.Residual protein synthesis in the presence of chloramphenicol, neomycin and rifampicin was 5%, 5% and 27% respectively of that observed in the drug-free control. This residual synthesis did not appear to involve amplification of the X-protein. These results suggest that tif-mediated inactivation of the repressor requires the activation of some specific gene(s), the translation of which appears to be resistant to chloramphenicol.     

Studies on lambda virulent mutants

Summary Lambda virC mutant, presumably an operator mutant for the operon including x, y, CII and O genes (Fig. 1), produce clearish plaque on a sensitive bacteria.Four revertants producing turbid plaques were isolated from virC and the mutational sites of which were studied. One (tw ₁) is located very close to and on the left side of virC₃₄, and another (tw ₃₂) is at the almost same site of virC₃₄. The others (tx ₆ and tx ₅₃) are located on the right side of virC₃₄. tx recombinants have been isolated and characterized. These recombinants produce very turbid plaques and the rate of the repressor formation in the presence of CIts repressor is somewhat higher than that of wild type. tx develops very poorly after infection to sensitive cells but CItx develops normally. tx lysogens synthesize two to three times more exonuclease than the wild type lysogen. On a function of x region for the repressor formation and on a presence of a possible anti-repressor were discussed. The mutant tw ₁ might be a promotor mutation of the CI-rex operon.This material has been published as an abstract in Jap. J. Genetics 45, 474 (1970).     

Structure and function of the repressor of bacteriophage lambda

Summary The high-affinity mutant cI gene of cIha (Nag et al. 1984) was cloned in the multicopy plasmid pBR322. In the resulting plasmid, pMD 102, a lacUV5 promoter was inserted giving the lacUV5-cIha fusion plasmid pMD 205. Bacteria carrying pMD 102 and pMD 205 contain 2.5 and 15 times, respectively, the level of repressor in a monolysogen of cIha. Results of the study of certain properties of the bacteria carrying these plasmids suggest that the ha repressor also has a higher affinity for the virulent mutant operators as well as the prm promoter of .     

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.     

Normal expression of the viral gene N interferes with growth of bacteriophage lambda in Escherichia coli 15T-.

Summary Growth of and of some lambdoid phages is considerably inhibited on strain 3057 derived from E. coli 15T^-. Mutants of which overcome this inhibition map in gene N. Some of these hty mutants are temperature sensitive for growth on E. coli K12. Thus plating of on strain 3057 allows one to isolate temperature sensitive N mutants. The hty mutants produce less than normal N activity as judged by their low efficiency of plating on a nus ^- host and by the extended latent period of some of them on normal hosts. The inability of strain 3057 to propagate can be at least partially reversed by addition of thymidine to the medium and the growth difference between hty and in 3057 increases with decreasing thymidine concentration. The amount of DNA produced by in 3057 at low thymidine concentration is lower than that produced by hty under the same conditions. Only a small percentage of the DNA produced by in 3057 is packaged into viable phage particles. This suggests that not only produces less DNA in 3057 than hty but that an important part of the DNA in 3057 is in a form which can not be packaged or which is noninfective for other reasons. A hypothesis is discussed that hty mutations enable to grow on E. coli 15T^- at low thymidine concentration because they lead to reduction in the number of single strand nicks in the DNA by reducing the intracellular endonuclease activity. Under permissive conditions conditional lethal N mutants are favored for growth on 3057 over N ⁺ which confirms the idea that N activity or the activity of a gene under N control interferes with growth in 3057 at low thymidine concentration.     

Cloning bacterial genes with plasmid λdv

Summary Fragments ofEscherichia coli DNA carrying genes for -galactosidase, or for biosynthesis of guanine or biotin were recombined in vitro with dv DNA. The cloned recombinant molecules recovered from transformedE. coli cells consisted of a biologically functional bacterial DNA fragment and, except for dv-bio30-7, two dv monomer units: one of the dv units was used as the insertion site for the bacterial DNA, whereas the other was intact, and seemed to be responsible for the replication of the recombinant plasmid. The process which gives rise to these recombinant molecules at high frequency from mixtures of monomeric dv DNA's and bacterial DNA fragments is discussed.     

A method for the isolation of phage mutants altered in their response to lysogenic induction

Summary Phage cl ⁺ gives clear plaques whereas phage cIind ^- gives turbid plaques on a lawn of a mutant strain of E. coli K12. This strain, called STS, carries mutation spr in a tif sfi genetic background. I hypothesize that upon temperate phage infection, STS bacteria spontaneously inactivate phage repressor by the same mechanism involved in normal lysogenic induction which results in obligatory lytic growth of ⁺. The use of the STS mutant facilitates the isolation and genetic analysis of phage mutants with an abnormal response to lysogenic induction.     

Directed integration of an F'' plasmid by integrative suppression: isolation of plaque forming lambda transducing phage for the dnaC gene.

Summary A new approach for isolation of a plaque forming specialized transducing phage is described. It consists of directed transposition of an F plasmid into the gal region of a dnaAts galE ^- Escherichia coli strain by integrative suppression and deletion of the chlD region in order to shorten the distance between the marker of interest on the F and the prophage serving to prepare an LFT¹ lysate.An F danC ⁺ thr ⁺ plasmid was used here and dthr and ddnaC phages were isolated. In addition, pdnaC was obtained from a double lysogen for ddnaC and b2.     

Inactivation of DNA-binding activity of repressor in extracts of lambda-lysogen treated with mitomycin C

Summary Crude extracts from -lysogens treated with mitomycin C were prepared, and immunity repressor levels in the extracts were assayed by the binding activity specific to DNA immunity region. It has been shown that while the repressor levels in the extracts from C600(+) are reduced after mitomycin C treatment, the levels in C600recA(+) and C600 C72(+) which have defects in lifting the immunity are not affected by the treatment. The repressor levels in the extracts prepared from C600 T44(+) after temperature shift up, whose prophage is inducible at high temperature, are also reduced. From the study of chloramphenicol effect, it was indicated that de novo protein synthesis is required for the inactivation of the repressor in C600(+) by mitomycin C, but not in T44(+) by high temperature.     

RexAB proteins of bacteriophage lambda enhance the effect of photolyase-dimer complexes on lacZ gene expression in Escherichia coli.

Summary Expression of the lacZ gene in Escherichia coli is inactivated by exposure to ultraviolet light (UV). Inactivation is exceptionally effective when cells contain amplified levels of DNA photolyase (which forms complexes with pyrimidine dimers in the absence of light for actual photoreversal) and a prophage. Without amplified photolyase, the prophage or both, inactivation rates are similar and much lower. UV-inactivation of lacZ gene expression in the presence of both amplified photolyase and is even more effective if cI857 is used in place of the wildtype prophage but is wholly unexceptional if the prophage carries defects in the genes rexA or rexB. When Rex AB proteins are provided by expression from a plasmid and the cell also contains amplified photolyase, exceptional inactivation rates again obtain; in fact inactivation is most effective under these conditions. The data are considered to reveal a role for Rex AB proteins, which mediate superinfection exclusion, in the exceptional inactivation of gene expression by photolyase bound to pyrimidine dimers in DNA. Photolyase-dimer complexes may mimic the structure of certain complexes that arise during phage development and thus influence Rex A and/or B proteins, thereby shutting down cell metabolism.     

Reduced expression of a distal gene of the prn gene cluster in deletion mutants of Aspergillus nidulans: genetic evidence for a dicistronic messenger in an eukaryote.

Summary Temperature sensitive dnaAts46 mutants, in which initiation of chromosome replication is blocked at 42° C, are unable to maintain a dv plasmid at the permissive temperature unless the plasmid carries a mutation in gene P of the type permitting phage to grow in groP (dnaB) bacteria. The growth rate of dnaAts46 mutants seems to be impaired by the presence of the dvP mutant plasmid.Cold sensitive dnaAcos mutants which overinitiate replication at low temperature and grow normally only at 40° and above, can maintain efficiently dvP ⁺ plasmids as well as dvP mutants. Cold sensitivity of dnaAcos mutants is suppressed by the presence of the plasmid dvP ⁺ and by certain dvP mutants, but not by others.The gene P product seems to act by reducing the initiation potential of both types of dnaA mutants, aggravating the initiation defect in dnaAts46 and correcting the overinitiation of dnaAcos.     

Construction in vitro of "phage-plasmid" chimerae: a new tool to analyse the mechanism of plasmid maintenance.

Summary In this paper, we report the construction in vitro of chimerae between lambdoid replacement vectors (Murray et al., 1977) and the miniF Ap^r plasmid: pSC138 (Timmis et al., 1975). F recombinants were shown to be chimerae between the and the F replicons. By genetical tests, we have demonstrated that both and F replication mechanisms are functional: the F recombinant behaves as a non defective plaque forming phage on sensitive bacteria and establishes itself as a stable plasmid on recA F^- homoimmune bacteria. In the extra-chromosomal state, the F recombinant apparently retains the controlled autonomous replication and the FI incompatibility characteristics of the F plasmid. The potential experimental uses of these phages are discussed.