Kaspar von Meyenburg

The DnaA protein determines the initiation mass of Escherichia coli K-12

DNA replication was studied in a dnaA(Ts) strain containing a plasmid with the dnaA+ gene under plac control. At 42 degrees C, initiation of DNA replication was totally dependent upon the gratuitous inducer isopropyl beta-D-thiogalactopyranoside (IPTG). Flow cytometric measurements showed that at 13% induction of the lac promoter the growth rate, cell size, DNA content, and timing of initiation of DNA replication were indistinguishable from those observed in a wild-type control cell. Higher levels of induction resulted in initiations earlier in the cell cycle and a corresponding increase in the time from initiation to termination. We conclude that the concentration of DnaA protein determines the time of initiation and thereby the initiation mass. With an induction level equal to or above 13%, the synchrony of multiple initiations within one cell was close to that found in a wild-type control cell, showing that a cyclic variation in DnaA content is not necessary for a high degree of synchrony.

Promoters of the atp operon coding for the membrane-bound ATP synthase of Escherichia coli Mapped by Tn 10 insertion mutations

SummaryTransposon Tn10 insertion mutations in the oriC (replication origin) — atp (operon for the subunits of the membrane-bound ATP synthase) region of the Escherichia coli K12 chromosome were used to define promoters of the atp operon. The Tn10 insertions were first isolated and physically mapped on the specialized transducing phage λasn20 with a genome size of 23.5 MD, the insertion into which of the 6 MD Tn10 did not reduce packaging or stability. After transfer by recombination into the chromosome, a class of six Tn10 insertions was found to reduce growth yield and expression of the atp operon determined by quantitizing synthesis of the c-subunit of the ATP synthase. These six Tn10 insertions mapped within a 400 basepair segment of chromosomal DNA in front of the atpB gene. This segment contains the coding sequence for a 14KD polypeptide designated atpI. Complementation tests showed that the mutations are cis dominant and revealed that the 14 KD atpI gene product is not essential for biosynthesis and activity of the membrane bound ATP synthase. The insertions appear to block transcription into the atp genes located downstream. The insertions had different effects on atp operon expression: the ones closest to the atpB gene gave a 80%–90% decrease in c-subunit synthesis while those 150–200 basepairs further upstream only gave a 60%–70% decrease. These differential effects are taken to indicate the presence of three transcription start sites for the atp operon, a major promoter, designated atpIp, in front of the atpI gene and two minor ones, atpB1p and atpB2p, within the coding sequence atpI in front of the atpB gene. The allocation of atpIp coincides with potential transcription start signals found by DNA sequence analysis.

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

SummarySpecialized transducing phages λtna (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 couR. 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 λtna. 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”).

The nucleotide sequence of the atp genes coding for the F0 subunits a, b, c and the F1 subunit δ of the membrane bound ATP synthase of Escherichia coli

SummaryThe nucleotide sequence has been determined of a 2.500 base pair segment of the E. coli chromosome located between 3.75 and 6.25 kb counterclockwise of the origin of replication at 83.5 min. The sequence contains the atp genes coding for subunits a-, b-, c-, δ- and part of the α-subunit of the membrane bound ATP synthase. The precise start positions of the atpE (c), atpF (b), atpH (δ) and atpA (α) genes have been defined by comparison of the potential coding sequences with the known amino acid sequence of the c-subunit and the determined N-terminal amino acid sequences of the respective subunits. The genes are expressed in the counterclockwise direction. Their order (counterclockwise) is: atpB (a), atpE (c), atpF (b), atpH (δ) and atpA (α). The coding sequences for subunits b and δ yield polypeptides of 156 and 177 amino acids, respectively, in accordance with the established sizes of these subunits; the one for the c-subunit, the DCCD binding protein, fits perfectly with its known sequence of 79 amino acids. The a-subunit is comprised within a coding sequence yielding a polypeptide of 271 amino acids. It is suggested, however, that the a-subunit (atpB) contains only 201 amino acids, in accordance with its known size, starting from a translation initiation site within the larger coding sequence. The stoichiometry of the F0 sector subunits is discussed and a model is proposed for the functioning of the highly charged b-subunit of the F0 sector as the actual proton conductor.

Origin of replication, oriC, of the Escherichia coli chromosome on specialized transducing phages λasn

SummarySpecialized transducing phages λasn harboring chromosomal DNA and genetic markers on either side of the asn gene were isolated. Phages carrying chromosomal DNA counterclockwise of the asn gene can upon infection establish themselves as self-replicating plasmids in asn, recA hosts lysogenic for lambda. It is concluded that this bypassing of normal lambda immunity is due to the presence of the chromosomal replication origin, oriC, in this class of phages. Genetic analysis and the determination of restriction endonuclease cleavage patterns of the different λasn lead to the allocation of oriC within 1.5 megadaltons of the asn gene towards the uncA, uncB genes at 82 min on the genetic map of E. coli, The clockwise order of genes on the chromosome is found to be: bglB, (pst, glmS), (uncA, uncB), criC, asn, trkD, rbs, rrnC, ilv.

Interaction of alleles of therelA, relC andspoT genes inEscherichia coli: Analysis of the interconversion of GTP, ppGpp and pppGpp

SummaryMutants in thespoT gene have been isolated as stringent second site revertants of therelC mutation. These show varying degrees of the characteristics associated with thespoT1 gene,viz relative amount and absolute levels of both pppGpp and ppGpp and the decay rate of the latter. The entry of3H-guanosine into GTP and ppGpp pools inspoT+ andspoT1 cells either growing exponentially or during amino acid starvation was determined, and the rate of ppGpp synthesis and its decay constant calculated. During exponential growth the ppGpp pool is 2-fold higher, its decay constant 10-fold lower, and its synthesis rate 5-fold lower inspoT- than inspoT+ cells; during amino acid starvation the ppGpp pool is 2-fold higher, its decay constant 20-fold lower, and its synthesis rate 10-fold lower inspoT than inspoT+ cells. In one of the “intermediate”spoT mutants the rate of entry of3H-guanosine into GTP, ppGpp and pppGpp was measured during amino acid starvation. The data form the basis of a model for the interconversion of the guanosine nucleotides in which the flow is:GDP→GTP→pppGpp→ppGpp→Y. Calculations of the rates of synthesis and conversion of pppGpp and ppGpp under various conditions in variousspoT+ andspoT- strains indicate that the ppGpp concentration indirectly controls the rate of pppGpp synthesis.ThespoT1 allele was introduced into various relaxed mutants. It was shown that many phenomena associated with the relaxed response ofrelC and “intermediate”relA mutants were phenotypically suppressed when thespoT1 allele was introduced into these mutants. These double mutants exhibit ppGpp accumulation, rate of RNA accumulation, rate of β-galactosidase synthesis, and heat lability of β-galactosidase synthesized during amino acid starvation similar to the stringent wild-type. It is concluded that the relaxed response is due directly to the lack of ppGpp and that the stringest response is due directly to ppGpp.

Initiation of DNA replication in Escherichia coli after overproduction of the DnaA protein

SummaryFlow cytometry was used to study initiation of DNA replication in Escherichia coli K12 after induced expression of a plasmid-borne dnaA+ gene. When the dnaA gene was induced from either the plac or the λpL promoter initiation was stimulated, as evidenced by an increase in the number of origins and in DNA content per mass unit. During prolonged growth under inducing conditions the origin and DNA content per mass unit were stabilized at levels significantly higher than those found before induction or in similarly treated control cells. The largest increase was observed when using the stronger promoter λpL compared to plac. Synchrony of initiation was reasonably well maintained with elevated DnaA protein concentrations, indicating that simultaneous initiation of all origins was still preferred under these conditions. A reduced rate of replication fork movement was found in the presence of rifampin when the DnaA protein was overproduced. We conclude that increased synthesis levels or increased concentrations of the DnaA protein stimulate initiation of DNA replication. The data suggest that the DnaA protein may be the limiting factor for initiation under normal physiological conditions.

Origin of replication, oriC, of the Escherichia coli chromosome: Mapping of genes relative to R.EcoRI cleavage sites in the oriC region

SummaryA precise genetic-physical map of the tnailv region at 82 min on the genetic map of E. coli is obtained through deletion mapping and analysis by restriction endonuclease EcoRI of plasmids, derived from an F′ carrying the genes between aroE and ilv.A locus, designated het, which in its diploid state results in slow growth and heterogeneity of cell size due to distorted cell division, maps between bglB and asn, 30–45 kb counterclockwise of ilv.The pattern of R.EcolRI cleavage sites in the het region is identical with the pattern obtained by Marsh and worcel (1977) who analyzed DNA labeled preferentially in the region of the DNA replication origin (oriC). We suggest that oriC is identical with the het site and that it can be allocated to a position 32 kb counterclockwise of the ilv operon.

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

SummaryTwo 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 λtna 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.

The genes for the eight subunits of the membrane bound ATP synthase of Escherichia coli

SummaryThe genes for the eight subunits of the membrane bound ATP synthase of Escherichia coli (Ca++, Mg++ dependent ATPase, EC 3.6.1.3) were mapped through genetic, physical and functional analysis of specialized transducing phages λasn (von Meyenburg et al. 1978). The ATP synthase genes, designated atp1, are located at 83.2 min in a segment of the chromosome between 3.5 and 11.3 kb left (counterclockwise) of the origin of replication oriC. The counterclockwise order of the genes for the eight subunits, the expression of which starts from a control region at 3.5 kb-L, was found to be: a, (c, b, δ), α, γ, (ε, β) which in the notation of Downie et al. (1981) reads atpB (E F H) A G (C D). The analysis was in part based on the isolation of new types of atp (unc, Suc-) mutations. We made use of the fact that specialized transducing phages λasn carrying oriC can establish themselves as minichromosomes rendering asnA cells Asn+, and that the resulting Asn+ cells grow slowly if the λasn carries part or all of the atp operon. Selecting for fast growing strains mutations were isolated on the λasn which either eliminated atp genes or affected their expression (“promoter” mutations). The relationship between these atp mutations and the cop mutations of Ogura et al. (1980), which also appear to map in front of or within the atp genes, is discussed.