A temperature-sensitive Escherichia coli mutant defective in DNA replication: dnaA, a new gene adjacent to the dnA, gene

Summary An Escherichia coli mutant defective in replication of the chromosome has been isolated from temperature-sensitive mutants that cannot support colicin E1 plasmid DNA synthesis in the presence of chloramphenicol. Cellular DNA synthesis of the mutant ceases almost immediately after transfer to the nonpermissive temperature. The defect is due to a single mutation, dna-59, which is located close to the sites of dnaA mutations and a cou ^R mutation conferring DNA gyrase with resistance to coumermycin. The dna-59 mutant is not able to support DNA synthesis of phage at the high temperature. The mutant also restricts growth of X174 phage at the high temperature, but permits formation of supercoiled closedcircular duplex replicative intermediates. T7 phage can grow on the mutant even at the high temperature.A specialized transducing phage imm ²¹[tna dnaA]#2 (Miki et al., 1978) supports growth of dna-59, dnaA46 and dna-167 cells at the high temperature. Some of the EDTA-resistant derivatives of the phage have lost part or all of the dnaA gene, but carry gene function complementing the defect of dna-59 cells, as judged by conversion of the above dna strains to wild type cells by phage infection, and by suppression of the loss of viability of dna-59 cells at the high temperature by phage infection. The gene containing the dna-59 mutation site is thus distinct from the dnaA gene. Since the dna-59 mutation does not affect expression of the cou ^r gene of DNA gyrase, which is another known gene involved in DNA synthesis near the dnaA gene, this mutation is probably in a new gene, dnaN. From analysis of the suppression activities of imm ²¹[tna dnaA]#2 phage and its deletion derivatives against dnaN59 cells, it is suggested that the expression of the dnaN gene function is reduced by deletion in the dnaA region.     

Characterization of dnaA gene carried by lambda transducing phage

Summary Specialized transducing phages dnaA were obtained by inducing lysogens in which tna was integrated at the tnaA region of the Escherichia coli chromosome; the tnA region is located in the vicinity of the dnaA gene. The dnaA ^- deletion derivatives of dnaA were isolated from the lysate of dnaA grown on bacteria carrying a transposon Tn3.The structures of various transducing phages thus obtained were determined by heteroduplex DNA mapping. From these results, the transducing fragment of 13.8-kb-long was divided into nine domains. Upon infection of UV-irradiated cells with the phage, production of polypeptides of 49 kD and 42 kD was specifically associated with infections by the dnaA and recF transducing phages. Polypeptides of 49 kD and 42 kD appeared to be coded for by dnaA and recF genes, respectively. The dnaA gene was assigned to the region of 2.8-kb-long which extends by 2.4 kb in the counterclockwise direction on the E. coli genetic map and 0.4 kb in the opposite direction, as measured from the nearest HindIII site close to the tnaA gene. The recF gene was also discovered to lie very close to dnaA in the order of tnaA-dnaA-recF.Merogenotes heterozygous for the dnaA gene were constructed by introducing F100-12 carrying dnaA into the recipients with different mutations at or near dnaA. For combinations, F(dnaA ⁺)/dnaA46 and F(dna ⁺)/dna-83, dnaA ⁺ was trans-dominant, whereas the dnaA ⁺ was recessive for F(dnaA ⁺)/dna-5. For F(dnaA ⁺)/dna-167, the result of the transdominance test was affected by the growth media employed; dnaA ⁺ was dominant on a -broth plate, and dna-167 was dominant on an M9-minimal plate. Thus, transdominance of dnaA ⁺ in heterozygotes is affected by difference in mutations and growth media.     

Requirement of the Escherichia coli dnaA gene function for integrative suppression of dnaA mutations by plasmid R100-1

Summary The phenotype of Escherichia coli dnaA missense and nonsense mutations was integratively suppressed by plasmid R100-1. The suppressed strains, however, could not survive when the dnaA function was totally inactivated. This was demonstrated by the inability of replacing the dnaA allele in the suppressed strain by a dnaA::Tn10 insertion using phage P1-mediated transduction. When the intact dnaA ⁺ allele was additionally supplied by a specialized transducing phage, imm ²¹ dnaA ⁺, which integrated at the att site on the E. coli chromosome, then the dnaA::Tn10 insertion, together with a oriC deletion, were able to be introduced into the suppressed strain. Thus, the mechanisms of dnaA function for oriC and for the replication origin of R100-1 may not be quite the same.     

Identification of the dnaA and dnaN gene products of Escherichia coli

Summary A specialized transducing lambda phage carrying the dnaN and dnaA genes of Escherichia coli specifies two proteins of about 41 and 48 kilodaltons (kd). The temperature-sensitive mutations, dnaN59 and dnaA167, were found to result in altered isoelectric points of the 41 and 48 kd proteins, respectively. Thus the dnaN gene product was identified as a weakly acidic 41 kd protein, and the dnaA gene product as a weakly basic 48 kd protein. The synthesis of the dnaN gene product is greatly reduced by insertion of a transposon Tn3 in the dnaA gene and by deletion in the gene at the distal end to the dnaN gene. Temperature-sensitive dnaA mutations, on the other hand, do not affect the synthesis of the dnaN gene product. These results indicate that the synthesis of the dnaN gene product is dependent on the structural integrity of the dnaA gene.Abbreviations kd kilodaltons - SDS sodium dodecyl sulfate     

Effect of the dnaN mutation on the growth of small DNA phages

Summary The effect of the dnaN mutation on the growth of single-stranded DNA phages was studied by burst experiments. In HC138 dnaN cells exposed to 42.5° C at 5 min before infection, growth of spherical (microvirid or isometric) phages such as 3, Kh-1 and X174 was partially reduced at the nonpermissive temperature. When infection was performed at 30 min after temperature shift-up, viral replication was completely inhibited at 42.5° C in the dnaN strain but not in a dna ⁺ revertant. At 41° C, multiplication of filamentous (inovirid) phages M13 and fd was restricted specifically in HC138 F⁺ dnaN bacteria. When dnaN cells lysogenic for i²¹ were grown at 42.5° C for 60 min and then shifted down to 33° C, a burst of i²¹ occurred with concomitant cellular lysis, manifesting induction of the prophage development.     

Genetic and physical mapping of recF in Escherichia coli K-12

Summary Two factor transductional crosses place recF at approximately 82 min on the E. coli chromosome; recF is highly cotransducible with dnaA and gyrB (cou). Transductional analysis with a series of tna specialized transducing phages carrying chromosomal DNA from the tnaA region place recF between dnaA and gyrB. This analysis also indicates that a gene lying in the same region and producing an easily detectable protein (estimated MW of 45 kD) is dnaN and not recF.     

Explanations accounting for transduction by bacteriophage lambda in maltose negative bacteriophage lambda resistant mutants of Escherichia coli K-12.

Summary Entry of DNA from phages particles into rMal^- mutants of Escherichia coli K-12 is shown to be due to two distinguishable processes. One, residual transduction, results from a low level expression of lamB. The other one, background transduction, is independent of gene lamB.Interpretations are presented for these results. It is proposed that residual transduction is due to a weak promoter pB3 located within or near the distal part of the gene preceeding lamB in the same operon. It is proposed that background transduction is due to a secondary receptor structure for phage . Finally a tentative hypothesis relating pB3 to insertion sequences is presented.     

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.     

Studies on radiation-sensitive mutants of E. coli. 3. Participation of the rec system in induction of mutation by ultraviolet irradiation

Summary The bacterial recA gene participates in the induction by UV irradiation of the clear mutation of phage and the Lac^- mutation of bacteria. The necessary function is induced by irradiation of Rec⁺ bacteria and acts upon DNA irradiated with UV light.     

Establishment of Escherichia coli cells with an integrated high copy number plasmid

Summary The dnaA46 cells can grow at high temperature when a high copy number plasmid pKY31, a derivative of pBR322 carrying a segment of the E. coli chromosome, integrates into the bacterial chromosome. In contrast, the dnaA46 polA ^- cells with the integrated plasmid can not grow at high temperature. Therefore, integration of the plasmid can suppress the dnaA mutation and this suppression requires DNA polymerase I which has been known to be required for plasmid replication. Full reversion of polA or lysogenization of polA ⁺ is lethal for the dnaA46polA ^- bacteria that carry the plasmid only in integrated state. Partial reversion of polA allows these cells to grow at both low and high temperatures. Introduction of the plasmid pBR322 into cytoplasm of these bacteria suppresses the lethal effect caused by full reversion of polA or lysogenization of polA ⁺. This lethal effect expresses independent of the presence or absence of the dnaA mutation. In partial revertants of polA which have only integrated plasmid, the number of copies of a region near the replication origin of integrated plasmid increases. The number is reduced by the presence of extrachromosomal pBR322. It is suggested that the lethal effect of normal levels of DNA polymerase I in strains that carry only the integrated plasmid is due to excessive initiation of replication of the bacterial chromosome from the plasmid origin and high potential of initiation can be absorbed in many copies of cytoplasmic plasmid, probably, in their replication origins.Abbreviations Amp^r ampicillin resistant (resistance) - Tet^s tetracycline sensitive - Tet^r tetracycline resistant - MMS^r methyl methane sulfonate resistant (resistance) - ts temperature sensitive - Kb kilobase pairs     

The cis-specificity of the Q-gene product of bacteriophage lambda

Summary A rtrp/lac W205 substitution, fused to the late ragion of bacteriophage lambda, provided a convenient assay for phage late gene expression in the presence or absence of lambda pQ. Comparison of lacZ expression from Q ⁺ and Q ^- phages showed that late gene expression was markedly Q-dependent (263-fold difference). A cis/trans comparison of lambda pQ action showed a 180-fold difference in lacZ expression. The results suggest that pQ is only significantly active when supplied in cis to its site of action.     

Transduction of nitrate reductase loci of Escherichia coli by phages P-1 and lambda

Summary A lactate-nitrate medium suitable for genetic studies with nitrate reductase mutants (nar ^-) of Escherichia coli was devised. This permitted the selection of nar ^- strains by their failure to use nitrate as terminal electron acceptor during anaerobic growth, in addition to the selection procedure based on the chlorate resistance of nar ^- mutants. Transduction studies with phage P1 and nar ^- mutants from both sources demonstrated the existence of at least three nar genes in the gal region of the E. coli linkage map, their relative positions being: gal .... narF .... bio .... narD .... narE. Using phage cotransduction of narD with bio was observed and several independently-isolated defective -transducing phages were examined. Phage also transduced the narF gene with gal linkage but the narE gene was not -transducible.     

Identification of the E. coli groNB(nusB) gene product

Summary The E. coli groNB(nusB) gene product has been previously shown to be necessary for bacteriophage N protein function. The product of the groNB gene has been identified on SDS polyacrylamide gels after infection of UV-irradiated E. coli cells with various groNB ⁺ transducing phage derivatives. It is a polypeptide with an apparent molecular weight of 14,000 daltons. Transducing phage carrying either a deletion or an amber mutation in the groNB gene fail to synthesize the 14,000-M_r polypeptide chain upon infection of a sup ⁺ host. However, am ⁺ revertants of the groNBam phage do induce the synthesis of the polypeptide.     

Characterization of the dnaA, gyrB and other genes in the dnaA region of the Escherichia coli chromosome on specialized transducing phages lambda tna.

Summary Specialized transducing phages tna (tryptophanase) harboring chromosomal DNA and genetic markers from the dnaA region of the Escherichia coli chromosome were isolated. Transductional analysis showed that some of these tnaA transducing phages carry two genes important in DNA replication, namely the dnaA gene (initiation of chromosome replication) and the gyrB gene (subunit B of DNA gyrase), formerly designated cou ^R. The following clockwise order of genetic markers was found: uhp, gyrB, dnaA, rimA, tnaA, bglB.The gene-protein relationship was established by the determination of the gene products encoded on the chromosomal DNA of the different tna. A 54 kD and a 91 kD polypeptide appear to be coded for by the dnaA and gyrB genes, respectively; the 91 kD protein is encoded on a region in which coumermycin sensitivity maps and is with respect to electrophoretic behavior identical to subunit B of DNA gyrase. The 54 kD protein is encoded on the region in which different independently isolated dnaA(Ts) mutations (dnaA5, dnaA46, dnaA167, dnaA203, dnaA204, dnaA205, dnaA211, dnaA508) are located. Additional genes which code for polypeptides with hitherto unknown functions were identified and mapped. The acriflavin sensitivity mutation acrB1 was found to be an allele of the gyrB gene (see Note Added in Proof).     

O0 and strong-polar mutations in the gal operon are insertions

Summary Three dg phages carrying strong-polar mutations in the gal operon are denser than the corresponding phages carrying the wildtype gal operon or reversions of the mutations to the Gal ⁺ phenotype. The latter phages have the same density. It is concluded that these strong-polar mutations are insertions of DNA into the gal operon.The amount of inserted DNA is different in the three mutations and is calculated to be 450, 1,080 and 1,800 nucleotide pairs respectively.The strong-polar phenotype is also found in a mutant supplied by A. Taylor which carries a Mu-1 phage integrated into the transferase gene.     

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.     

An improved selection method for λlacZ − phages based on galactose sensitivity

The determination of thelacZ mutant frequency in gt10lacZ phage vectors isolated from the transgenic mouse strain 40.6 (MutaMouse), requires the screening of large numbers of phages on -galactosidase activity. Existing methods rely on distinguishing a few white plaques on X-gal containing plates amongst a multide of blue ones which is both time-consuming and expensive. The new screening method described here employs the galactose sensitiveEscherichia coli C lacZ recA galE strain into which a multicopy plasmid has been introduced, which results in over-expression of thegalK andgalT genes. In the presence of phenyl--d-galactopyranoside, a substrate for -galactosidase, this leads to the suppression of lacZ ⁺ phage propagation without affecting the ability of lacZ ^– phages to form plaques. With this method it is possible to screen 1.5×10⁶ phages on a single 9-cm Petri dish. Furthermore, the need for blue/white screening has been eliminated.