Factors affecting the kinetics of progeny formation with F′lac in Escherichia coli K12

In matings of F′lac donors with an excess of recipient cells, different donor cells mated at different times. The concentration dependence of mating is incompatible with bimolecular reaction kinetics. In exponentially growing cultures, F′lac transfer from each donor cell continues to occur about once per generation. The establishment of F′lac in some recipient cells may take more than five generations.     

Electron microscopy of polar insertions in the lac operon of Escherichia coli

Summary Several strongly polar mutations in the omega region of the z gene of the lac operon result from insertions consisting of only two specific sequences of DNA, one about 870 and the other 1170 nucleotide pairs long (based on single-strand measurements). No sequence homology was detected between the shorter (IS1) and longer (IS3) insertions. The IS1 insertion was shown to possess a specific attachment site, but it can be inserted with either orientation at several sites in the z gene. Four insertion sites in the omega region of gene z were identified and the position of the lac5 substitution and the SR2 deletion in the plac DNA were determined by heteroduplex mapping.     

Curing of Escherichia coli K12 plasmids by coumermycin

Summary Low concentrations of the antibiotic coumermycin A₁, the inhibitor of bacterial DNA gyrase, effectively eliminate pBR322, pMB9 and other ColE1 related plasmids from E. coli K12 strains. The curing action of antibiotic seems to result from the plasmid degradation and not just from the inhibition of replication.     

Temperature dependent survival of UV-irradiated Escherichia coli K12

Summary We have found that several excision deficient derivatives of Escherichia coli K12 survive better after UV irradiation if incubated at 42°C than if incubated at 30°C. The highest survival was observed when incubation at 42°C followed UV irradiation and was maintained for at least 16 h. Our results indicate that this temperature dependent resistance (TDR) requires a functional recA gene, but not uvr A, uvrB, recF, or recB genes, or the recA441 (tif-1) mutation which allows thermoinduction of the recA-lexA regulon. Our data are consistent with the idea that the increase in survival observed at 42°C reflects enhanced daughterstrand gap repair by DNA strand exchange. Although the conditions used to elicit TDR can induce heat shock proteins and thermotolerance in E. coli, the relationship between the two responses remains to be elucidated.     

Biosynthesis of single-carbon units in Escherichia coli K12

Two routes of single-carbon (C₁) biosynthesis have been described in Escherichia coli K12 previously: from serine via serine transhydroxymethylase and from glycine via a little known set of enzymes, the Glycine Cleavage Pathway. Strains deficient in both of these still have a source of C₁. There therefore exists a third pathway of C₁ formation. Serine appears to be the donor for the pathway. No donor of C₁ other than serine of glycine appears to be of quantitative importance in E. coli K12 growing in a synthetic glucose-supplemented medium.     

On the mechanism of conjugation in Escherichia coli K 12

Summary The phenomenon of conjugation consists of many stages. The most important are: the formation of contacts between mating cells, the transfer of DNA from the donor to the recipient, and the integration of the transfered DNA fragments into the chromosome of the recipient. Only after completion of all these stages are recombinants formed. With the aid of specific inhibitiors (nalidixic acid, FUDR), thymine starvation, and use of special thermosensitive mutants it is possible to study the role of DNA synthesis during every stage of conjugation. It was demonstrated that the genetic transfer is due to semiconservative DNA-replication in the donor cell. The fragments of DNA transfered are synthesized in the period of mating by a special replication system (F-replicon). In case of T _DNA ^S mutants unable to grow at 41°, the ability to synthesize DNA during conjugation is preserved.The inhibition of the DNA synthesis in the donor cell by poisons leads to complete inhibition of genetic transfer. The third stage — formation of recombinants requires DNA synthesis in the recipient cell and is inhibited by poisoning, thymine starvation or T _DNA ^S mutations in the recipient. In cases where recombination is not involved (i.e. sexduction) the inhibition of DNA synthesis in the recipient has no significant effect.     

Molecular cloning of the fnr gene of Escherichia coli K12

Summary Mutations in the fnr gene of Escherichia coli have pleiotropic effects leading to deficiencies in the reduction of fumarate and nitrate, hydrogen production and the ability to grow anaerobically with fumarate or nitrate as terminal electron acceptors. Transducing phages (fnr) carrying the wild-type fnr gene were isolated from populations of artificially-constructed recombinant lambda phages by their ability to complement the lesions of fnr mutants. The fnr phages restored anaerobic growth with fumarate and nitrate as electron acceptors and, as prophages, they promoted normal synthesis of fumarate reductase, nitrate reductase and hydrogenase in fnr mutants. Five independently-isolated fnr phages each contained a R.HindIII fragment (11.5 kilobases) that possessed three internal R.EcoRI targets and had inserted with the same orientation relative to the phage. A physical map of the fnr region was constructed by restriction analysis and flanking fragments were identified by DNA:DNA hybridization.     

Regulatory mutants of dihydrofolate reductase in Escherichia coli K12

Summary Trimethoprim inhibits dihydrofolate reductase. Mutations conferring trimethoprim-resistance on E. coli K12 result in either an altered reductase with decreased affinity for the drug, or in 2–30 fold higher levels of the enzyme. Studies of the latter class of mutants indicate that dihydrofolate reductase is regulated by a diffusible molecule, and is probably under negative control. The regulatory mutants, some of which are temperature-sensitive, act cis.     

Female-specific phages and F-minus strains of Escherichia coli K12

Summary The female-specific phages, I, II, W31 and H (but not T3 and T7) show a low efficiency of plating on all F-minus strains of Escherichia coli K12 except for the thr leu thi mutants descended from strain Y53. The locus responsible is linked to the histidine region and was presumably eliminated from the Y53 line of mutants in the course of mutagenesis.     

Functional expression of Aquaspirillum magnetotacticum genes in Escherichia coli K12

Summary Gene libraries from the magnetotactic bacterium, Aquaspirillum magnetotacticum were constructed in Escherichia coli with cosmids pLAFR3 and c2RB as vectors. Recombinant cosmids able to complement the thr-1, leuB, and proA mutations of the host were identified. The Pro⁺ recombinant cosmid restored wild-type phenotype in proA and proB but not in the proC mutants of E. coli. The results of restriction endonuclease digestion and Southern hybridization analysis indicate that the relevent leu and pro biosynthetic genes of A. magnetotacticum are not closely linked on the chromosome.     

Uvm mutants of Escherichia coli K 12 deficient in UV mutagenesis

Summary Selection for defective reversion induction, after UV treatment of E. coli K 12, yielded uvm mutants. These mutants exhibited highly reduced or no UV mutability for all loci tested although they were moderately and normally mutable by X-rays and EMS, respectively. Uvm mutations confer only a slight sensitivity to killing by UV and X-rays and no clear sensitivity to the lethal effect of HN2, EMS or MMS. Growth and viability of untreated uvm cells were normal. The properties of uvm mutants are discussed in relation to those of other relevant mutant types and to some actual problems of induced mutagenesis.     

Uvm mutants of Escherichia coli K12 deficient in UV mutagenesis

Summary Uvm mutants of Escherichia coli K12 selected for defective UV reversion induction have previously been reported to differ considerably from the UV-reversion-less recA and lexA mutants with regard to survival or mutagenic response to UV, X-rays and alkylating agents. In the present study, the phenotypic characterization of uvm mutants was extended to investigate several cellular processes which also may be related to or involved in UV mutagenesis. Like recA and lexA mutations, the uvm mutations exhibit highly reduced Weigle reactivation and normal host cell reactivation of UV irradiated phage . But unlike recA and lexA, the uvm mutations do not impair genetic recombination, UV induction of prophage or R plasmid-mediated UV resistance and mutagenesis. These phenotypical characteristics and preliminary results of genetic mapping lend further support to the assumption that the uvm site may be a novel locus affecting, apart from the recA and lexA loci, the error-prone repair pathway in E. coli.     

Nucleotide sequence and analysis of the mgl operon of Escherichia coli K12

Summary The nucleotide sequence of the Escherichia coli K12 -methylgalactoside transport operon, mgl, was determined. Primer extension analysis indicated that the synthesis of mRNA initiates at guanine residue 145 of the determined sequence. The operon contains three open reading frames (ORF). The operator proximal ORF, mglB, encodes the galactose binding protein, a periplasmic protein of 332 amino acids including the 23 residue amino-terminal signal peptide. Following a 62 nucleotide spacer, the second ORF, mglA, is capable of encoding a protein of 506 amino acids. The amino-terminal and carboxyl-terminal halves of this protein are homologous to each other and each half contains a putative nucleotide binding site. The third ORF, mglC, is capable of encoding a hydrophobic protein of 336 amino acids which is thought to generate the transmembrane pore. The overall organization of the mglBAC operon and its potential to encode three proteins is similar to that of the ara FGH high affinity transport operon, located approximately 1 min away on the E. coli K12 chromosome.     

Intramolecular homologous recombination of linearized plasmids in Escherichia coli K12

Summary The efficacy of linear DNA as a substrate for general homologous recombination was demonstrated using BamHI-linearized pKLC8.5, a plasmid that carries internal direct repeats flanking the unique BamHI site. An analogous plasmid, pKLC2.31, was used in a parallel and comparative study of intramolecular homologous recombination in circular DNA substrates. When the rec ⁺ wild-type strain, AB1157, and its isogenic rec ^– derivatives were transformed with linear pKLC8.5 DNA, intramolecular homologous recombination was independent of recA, recB, recN, recO and exonuclease III (xth-1) functions. Although the recBCsbcA and recBCsbcBC cells were both very recombination proficient, only linear but not circular DNA was used as substrate for intramolecular homologous recombination in the recBCsbcA cells. In both the recBCsbcA and recBCsbcBC genetic backgrounds, the recombination frequencies for linearized pKLC8.5 DNA were 100%. A notable difference between the two strains was that none of the recBCsbcA transformants obtained with circular pKLC8.5 DNA were Tc^s recombinants, whereas 11% of the corresponding recBCsbcBC transformants were Tc^s recombinants. The sbcB mutation was responsible for the recombination proficiency of the recBCsbcBC cells. Unlike the case in recBCsbcA cells, intramolecular homologous recombination of linear DNA in the recBCsbcBC cells was dependent on recA and recF as well as recN and recO gene functions, but was independent of recJ and reeL gene functions.     

Evidence of abortive recombination in ruv mutants of Escherichia coli K12

Summary Genetic recombination in Escherichia coli was investigated by measuring the effect of mutations in ruv and rec genes on F-prime transfer and mobilization of nonconjugative plasmids. Mutation of ruv was found to reduce the recovery of F-prime transconjugants in crosses with recB recC sbcA strains by about 30-fold and with recB recC sbcB sbcC strains by more than 300-fold. Conjugative plasmids lacking any significant homology with the chromosome were transferred normally to these ruv mutants. Mobilization of the plasmid cloning vectors pHSG415, pBR322, pACYC184 and pUC18 were reduced by 20- to 100-fold in crosses with ruv rec ⁺ sbc ⁺ strains, depending on the plasmid used. Recombinant plasmids carrying ruv ⁺ were transferred efficiently. With both F-prime transfer and F-prime cointegrate mobilization, the effect of ruv was suppressed by inactivating recA. It is proposed that the failure to recover transconjugants in ruv recA ⁺strains is due to abortive recombination and that the ruv genes define activities which function late in recombination to help convert recombination intermediates into viable products.     

High efficiency temperature-sensitive amber suppressor strains of Escherichia coli K12

Summary A set of eight strains combining the supD43,74 ts¹ suppressor gene with alleles of three suppressor-enhancing (sue) genes have been constructed and characterized. The sue mutations work cooperatively to raise suppressor activity and together raise the activity of the supD43, 74-encoded suppressor 40-fold. These strains further expand the utility of the ts suppressor system by providing as much as 100% suppressor activity at temperatures at or below 20°C to as little as 0.015% suppressor activity at 43°C.     

Biosynthesis of RNA polymerase in Escherichia coli

Summary Effect of temperature-sensitive, assembly-defective mutations in Escherichia coli RNA polymerase (rpoB) or subunit gene (rpoC) was investigated on the expression of wild-type rpoB ⁺C⁺operon, which was introduced by infection of a lambda transducing phage drif ⁺ (rpoB ⁺)-6 after UV-irradiation of the mutant cells. In rpoB2·rpoB7 strain which accumulates assembly-intermediates, free , ₂ complex and premature core, the expression of rpoB ⁺C⁺operon measured by the rate of subunit synthesis was considerably inhibited whereas that of EF(translation elongation factor)-Tu, ribosomal proteins L1 and L7/L12, and some -coded proteins remained unaffected. On the other hand, the expression was enhanced specifically for only rpoB ⁺C⁺operon in either rpoC4 or rpoC1 mutants, which are defective in the association of ₂ complex and subunit or the activation of premature core enzyme, respectively. Upon preincubation of the mutant cells at 42° C prior to phage infection, during which assembly intermediates degraded rapidly, the rate of subunit synthesis relative to other phage-corded proteins increased remarkably in rpoB2·rpoB7 mutant as well as in rpoC4 and rpoC1 mutants. These observations strongly suggested the autogenous regulation for at least (rpoB ⁺C⁺) operon by some trans-active diffusible protein complexes built of RNA polymerase subunits. Nature of the regulatory molecules is discussed.Paper VI in this series is Saitoh and Ishihama (1977)     

Biosynthesis of RNA polymerase in Escherichia coli

Summary In spite of the generally well-coordinated synthesis of RNA polymerase core enzyme subunits (, and ) in Escherichia coli, a situation was found during the growth transition from exponential to stationary phase in which this coordination was broken (the order of differential repression being ; Kawakami et al. (1979)). The present study indicates that, during a certain period of the growth transition, twice as much subunit is synthesized as subunit and the overproduced subunit accumulates as the assembly intermediate ₂ complex, which is rapidly and preferentially degraded.Two independent factors, i.e., carbon source down-shift and oxygen depletion, were examined separately for their influence on the coordinated regulation of the synthesis of RNA polymerase subunits. The depletion of glucose added as a sole carbon source was accompanied by repression of the synthesis of all core enzyme subunits, while under the same conditions the differential rate of subunit synthesis increased. In contrast, the sudden ending of the oxygen supply resulted in specific repression of the synthesis of only and subunits but not of and subunits. The latter result may be explained by the autogenous repression of the rpoBC genes by a temporal increase in the amount of unused cytoplasmic RNA polymerase.Paper XI in this series is Kawakami and Ishihama (1980)