Studies on the regulation of initiation of chromosome replication in Escherichia coli.

The total initiation frequency of chromosome replication in Escherichia coli is dependent on two factors; the timing or time interval between successive initiations on an individual chromosome (initiation pace) and the number of individual chromosomes which are being replicated per cell. We have examined these parameters in a dnaA^ts, conditionally-lethal, “initiation mutant” of an E. coli K12 strain growing at different permissive temperatures. Our results indicate that at temperatures between 30 and 35 °C the gene product of the dnaA167 allele becomes limiting with respect to the number of replicating chromosomes per cell, which decreases from two at 30 °C to one at 35 °C. However, over this same temperature range it is clear that cell growth is balanced and the initiation pace, as determined from the growth rate, increases with temperature and is indistinguishable from that of the dnaA⁺ parent. These results demonstrate that one can alter the total initiation frequency independently of the initiation pace, indicating the involvement of at least two cellular components in the regulation of initiation. They also suggest that while the dnaA product may be involved in determining the total number of initiation events which can occur per cell per doubling time it does not control the timing or pace at which successive initiation events are triggered on each chromosome, i.e. it is not the “pace-maker” for initiation.     

Trans-dominance of dnaA mutants in Escherichia coli.

Summary Temperature sensitivity of growth and DNA synthesis was tested in merogenotes heterozygous for thednaA allele. All combinations tested (FdnaA⁺/dnaA5, FdnaA⁺/dnaA46, FdnaA⁺/dnaA204, FdnaA5/dnaA⁺, FdnaA204/dnaA⁺) were temperature sensitive. The mutantdnaA allele is thus trans-dominant to the wild type allele.     

A cold sensitive dnaA mutant of E. coli which overinitiates chromosome replication at low temperature

Summary A heat resistant mutant of E. coli dnaAts46 was isolated, which grows normally only at temperatures above 39°. After a temperature shift from 42° to 32° the mutant overproduces DNA relative to protein. This is due to overinitiation of rounds of chromosome replication at low temperature, as indicated by hybridization and other experiments. The mutation is cotransduced by Pl with ilv and could not be separated from dnaAts46 by transduction.     

A gene cloning system for the siomycin producer <Emphasis Type="Italic">Streptomyces sioyaensis</Emphasis> NRRL-B5408

Streptomyces sioyaensis NRRL-B5408 produces a siomycin complex (a group of thiopeptide antibiotics structurally related to thiostrepton). Development of genetic tools for the detection of siomycin production and DNA transfer into this strain is described. The existing tipA-based reporter system for determination of siomycin production was modified to achieve its stable integration into actinomycete genomes. Various replicative plasmids (pKC1139, pKC1218E, pSOK101) as well as actinophage ϕC31- and VWB-based vectors pSET152 and pSOK804, respectively, were conjugally transferred from E. coli into the siomycin producer at a frequency ranging from 3.7 × 10⁻⁹ to 1.1 × 10⁻⁵. The transconjugants did not differ from wild type in their ability to produce siomycin. There is one attB site for each integrative plasmid. The utility of temperature sensitive replicon of pKC1139 for insertional gene inactivation in S. sioyaensis has been validated by disruption of putative nonribosomal peptide synthetase gene.     

Increased spontaneous mutation rates in mutants of E. coli with altered DNA polymerase III

Summary Two temperature-sensitive mutants in dnaE, the structural gene for DNA polymerase III of Escherichia coli, show increased spontaneous mutation rates at permissive temperatures. Studies of the reversion of well-characterized trpA mutations in dnaE strains show that the mutagenic effect of altered DNA polymerase III applies to several different base substitution events, but not to frameshifts. The results suggest that DNA polymerase III is involved in base-selection during DNA replication.MRC Molecular Genetics Unit     

Immunological properties of membrane fractions from wild type and dnaA mutants of Escherichia coli

Membrane vesicles from Escherichia coli wild type and an otherwise isogenic dnaA mutant were used to immunize rabbits. In addition, a membrane protein fraction, containing the material found deficient in dnaA mutants, was purified by preparative polyacrylamide gel electrophoresis in sodium dodecylsulfate, and used for immunization. The antisera produced were analyzed by immunoelectrophoresis and immunofluorescence microscopy. The antisera obtained by immunization with membrane vesicles from either wild type or dnaA mutant membrane preparations were qualitatively similar in the precipitin bands seen after immunoelectrophoresis. The antisera obtained by immunization with the purified protein fraction contained a subset of the antibodies seen when whole vesicles were used for immunization. In a semiquantitative precipitin assay, the antisera prepared against whole membrane vesicles or the isolated protein fraction both caused the precipitation of more protein from sodium dodecylsulfate-solubilized membranes of wild type than of dnaA mutants. No difference was seen by immunoelectrophoresis between the protein composition of wild type or dnaA membrane preparations. Thus, the dnaA mutant appears to differ from the wild type in the quantitative composition of its membrane proteins, whereas no qualitative differences were detected.Fluorescein-conjugated antiserum preparations were employed to assess the reactivity of intact cells, spheroplasts and membrane vesicles with the antisera studied above. Wild type cells of E. coli have a barrier to reaction with the antisera; this barrier is removed when the cells are converted to spheroplasts or to membrane vesicle. Similarly, a highly permeable mutant of E. coli permits reaction of the antisera with unaltered cells. Antisera to both whole membrane vesicles and to the isolated protein fraction react identically with the cellular and subcellular preparations. Thus, antisera prepared from membrane proteins isolated after sodium dodecylsulfate-polyacrylamide gel electrophoresis can still recognize some antigens present in membrane vesicle preparations.     

Evidence for the involvement of unsaturated fatty acids in initiating chromosome replication in Escherichia coli

The analogue 3-decynoyl-N-acetylcysteamine inhibits the synthesis of unsaturated fatty acids in Escherichia coli, resulting in the accumulation of saturated fatty acids in the membrane (Kass, 1968).In the presence of this analogue, DNA, RNA and protein synthesis continue at a linear rate for approximately two doubling times, and then cease. On the other hand, the analogue will inhibit the formation of new replication forks (premature initiation), which normally arise as a result of thymine starvation.Unlike other temperature-sensitive DNA mutants, mutants that are defective in initiating DNA replication (dnaA or dnaC) are unable to replicate DNA at a permissive temperature if they terminate replication at 42 °C in the presence of 3-decynoyl-N-acetylcysteamine.When replication is terminated at 42 °C, cultures of dnaA or dnaC mutants normally will reinitiate replication upon lowering the temperature to 30 °C. For each mutant this reinitiation is characterized by a particular temperature sensitivity. Such mutants become more temperature sensitive if the temperature is lowered in the presence of 3-decynoyl-N-acetylcysteamine. All the effects of this analogue can be reversed by the addition of unsaturated fatty acids.These results are interpreted using a model in which replication is initiated at a particular lipid site on the membrane. In the absence of unsaturated fatty acids functional lipid sites are not made. Functional sites, however, can be used again provided they are not inactivated by interaction with an inactive dnaA or dnaC product.     

Regulation of the dnaA product in Escherichia coli.

Summary When an E. coli mutant (CRT46, dnaA46), thermosensitive in the initiation of DNA replication, grows at intermediate temperatures its DNA/mass ratio is somewhat lower than normal, but the cells possess an excess of initiation capacity, which can be expressed in the absence of proteins synthesis and lead to the accumulation of anomalously high amounts of DNA. A shift-up in temperature causes inhibition of initiation, and at the same time the production of initiation capacity is accelerated. After a shift-down in temperature initiation is released but the production of capacity is inhibited. The initiation capacity is thermolabile.The simplest explanation of these observations is that the dnaA product has a dual role: a positive function as an initiator of replication and a negative control function in its own synthesis.     

The initiation of chromosome replication in a dnaAts46 and a dnaA+ strain at various temperatures

Summary The regulation of chromosome replication initiation was studied at various temperatures with an E. coli dnaA46 strain and its dnaA ⁺ parent. We find that, in both strains, the initiation mass varies depending upon growth temperature while the replication time remains constant relative to the cell doubling time. In the permissive temperature range, the initiation mass of the dnaA46 mutant strain is larger by a constant factor than that for a dnaA ⁺ strain. We conclude that, even at temperatures permissive for growth of the dnaA46 strain, the activity of the dnaA46 product is lower than that of the wild-type protein. The dnaA gene product, therefore, plays an important role in regulating initiation.     

Completion of the replication and division cycle in temperature-sensitive DNA initiation mutants of Bacillus subtilis 168 at the non-permissive temperature.

Spores of the temperature-sensitive DNA initiation mutants of Bacillus subtilis 168, TsB134 and dna-1(Ts), were allowed to germinate at 34 °C in the presence of [³H]thymine until after the start of the first round of replication. The [³H]-thymine was then replaced by non-radioactive thymine and the outgrowing spores transferred to a higher temperature (49 °C for TsB134, 45 °C for dna-1(Ts)) which had been shown to block completely the initiation of a second round of replication. Autoradiography of the colonies which developed under such conditions showed the majority to contain two grain clusters. In most cases the clusters were separated by a division septum. Thus, it appears that the temperature sensitive activity of the dna gene product in each case is not needed for either replication through the termination region of the chromosome or the ensuing segregation of the daughters.Further studies of the septation process showed that, when replication of the first round after germination was allowed to proceed to termination at the non-permissive temperature, a centrally located septum appeared readily in both mutants. On the other hand, at levels of thymine which prevented progress of the round to termination within the time of the experiment, central septation did not occur in colonies of the same length. Rather, asymmetrical septation occurred at a relatively low frequency. It appears that the formation of the central septum is coupled to termination and reflects normal division septation at the non-permissive temperature. It is concluded that in neither mutant does such septation require the action of the temperature-sensitive dna gene product at a late stage in the overall cycle.     

Control of the initiation of DNA replication in Escherichia coli. II. Function of the dnaA product.

Summary General growth parameters and the kinetics of DNA replication have been determined in merogenotes carrying different combinations of the dnaA⁺ and the dnaA5 allele. The strain which is homozygous diploid for dnaA5 is different from all other combinations in cell volume, DNA per mass ratio, number of replication points per chromosome, and polymerization rate of DNA. From this we deduce that the dnaA product is a positively acting regulatory protein in initiation.In an appendix we show that in combinations between the dnaA5 and dnaA204 alleles the phenotype of dnaA5 is dominant.     

Two types of temperature sensitivity in DNA replication of an Escherichia coli dnaB mutant

In the Escherichia coli dnaB mutant BT165/70 were observed two types of temperature sensitivity of DNA replication: one slow but irreversible, occurring before the initiation of DNA replication, and the other instant but reversible, occurring during replication. These two types of temperature sensitivity appear to result from the single dnaB mutation. The observation suggests two different states of the dnaB gene product within the cell. Interaction of the dnaB protein with other components of the hypothetical replication complex is suggested. A temperature-insensitive revertant (second site mutation) of BT165/70 was isolated whose phenotype suggests an alteration in the interacting ability of the revertant protein.     

Initiation of DNA replication in Bacillus subtilis. IV. The effect of an intercalating dye, ethidium bromide

Summary The effects of an intercalating dye, ethidium bromide (EtBr), on the initiation of chromosome replication in Bacillus subtilis were studied. Spores of a thymine requiring mutant acquired the ability to initiate one round of replication in the absence of RNA and protein synthesis (initiation potential) during germination in a thymine starved medium. When EtBr was added after the initiation potential was fully established, initiation of replication was completely inhibited. This inhibition was reversible, and initiation was resumed when the drug was removed. The recovery of initiation occurred in the absence of protein synthesis but did require RNA synthesis and an active dna gene product.During germination both a DNA-protein complex and a DNA-membrane complex were formed at the replication origin in parallel with the establishment of initiation potential. EtBr destroyed both of these complexes at the concentration which inhibited initiation.The first round of replication of a plasmid DNA, pSL103, during spore germination was also prevented by EtBr. However a higher concentration was required to inhibit plasmid replication. It was found that the plasmid formed two complexes identical to the S- and M-complex of the chromosome origin. Compared to the chromosome complexes the plasmid complexes were less sensitive to EtBr. The loss of sensitivity was equivalent to that for the initiation of the plasmid compared to the chromosome. These results indicate that the target of EtBr is the DNA in the S- and M-complexes whose conformation is essential for the initiation of chromosome and plasmid replication.III of this series is Murakami et al. 1976     

Strand-specific DNA degradation in a mutant of Escherichia coli

Summary A dna B mutant of Escherichia coli which is thermosensitive for DNA synthesis at 42° C degrades DNA at the restrictive temperature. The degradation specifically affects newly synthesized DNA, begins at the replication forks and proceeds toward the replication origin, and is limited to 10–15% of one chromosome. The parameters of DNA degradation, as well as DNA-DNA annealing experiments on newly synthesized DNA which is resistant to degradation, indicate a specific strand of newly synthesized DNA is degraded.     

Characterization of a cryptic plasmid from Lactobacillus fermentum KC5b and its use for constructing a stable Lactobacillus cloning vector

Lactobacillus fermentum KC5b, a strain originally isolated from the human vagina, contains a cryptic plasmid pKC5b. The sequence and genetic organization of the 4392-bp plasmid were determined. It contains two convergently oriented replicons, which are homologous to each other and to the stable replicon of the Enterococcus faecium plasmid pMBB1. The two replicons of pKC5b were used either individually or together to construct Lactobacillus–Escherichia coli shuttle plasmids. Only the plasmid pSP1 that carried both replicons transformed lactobacilli, suggesting a complementary function between the two replicons. Since the replicons had a high homology to those of other plasmids that replicate via a theta-like mechanism and no detectable single-stranded intermediates were found for the plasmid, it is possible that pKC5b may replicate via a theta-like mechanism. The new shuttle plasmid pSP1 has been transformed and stably maintained in several Lactobacillus strains. As an initial application, pSP1 was used to clone the S-layer protein gene (slpA) of Lactobacillus acidophilus ATCC 4356 into a heterologous vaginal Lactobacillus strain and achieved surface-bound expression of the protein.     

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.     

SeqA limits DnaA activity in replication from oriC in Escherichia coli

A mutant Escherichia coli that transforms minichromosomes with high efficiency in the absence of Dam methylation has been Isolated and the mutation mapped to 16.25 min on the E. coli map. The mutant strain containing seqA2 is defective for growth in rich medium but not in minimal medium. A similar mutation In this gene, named seqA1, has also been isolated. Here we show that the product of the seqA gene, SeqA, normally acts as an inhibitor of chromosomal initiation. In the seqA2-containing mutant, the frequency of initiation increases by a factor of three. Introduction of the wild-type seqA gene on a low-copy plasmid suppresses the cold sensitivity of a dnaAcos mutant known to overinitiate at temperatures below 39°C. In addition, the seqA2 mutation is a suppressor of several dnaA (Ts) alleles. The seqA2 mutant overinitiates replication from oriC and displays the asynchronous initiation phenotype. Also the seqA2 mutant has an elevated level of DnaA protein (twofold). The introduction of minichromosomes or a low-copy-number plasmid carrying five DnaA-boxes from the oriC region increases the growth rate of the seqA2 mutant in rich medium to the wild-type level, reduces overinitiation but does not restore synchrony. We propose that the role of SeqA is to limit the activity level of the E. coli regulator of chromosome initiation, DnaA.     

Mutational analysis and properties of the msbA gene of Escherichia coli, coding for an essential ABC family transporter

The htrB gene was discovered because its insertional inactivation interfered with Escherichia coli growth and viability at temperatures above 32.5°C, as a result of accumulation of phospholipids. The msbA gene was originally discovered because when cloned on a low-copy-number plasmid vector it was able to suppress the temperature-sensitive growth phenotype of an htrB null mutant as well as the accumulation of phospholipids. The msbA gene product belongs to the superfamily of ABC transporters, a universally conserved family of proteins characterized by a highly conserved ATP-binding domain. The msbA gene is essential for bacterial viability at all temperatures. In order to understand the physiological role of the MsbA protein, we mutated the ATP-binding domain using random PCR mutagenesis. Six independent mutants were isolated and characterized. Four of these mutations resulted in single-amino-acid substitutions in non-conserved residues and were able to support cell growth at 30°C but not at 43°C. The remaining two mutations behaved as recessive lethals, and resulted in single-amino-acid substitutions in Walker motif B, one of the two highly conserved regions of the ATP-binding domain. Despite the fact that neither of these two mutant proteins can support E. coli growth, they both retained the ability to bind ATP in vitro. In addition, we present evidence to show that W-acetyl [³H]-glucosamine, a precursor of lipopolysaccharides, accumulates at the non-permissive temperature in the inner membrane of either htrB null or msbA conditional lethal strains. Translocation of the precursor to the outer membrane is restored by transformation with a plasmid containing the wild-type msbA gene. A possible role for MsbA     

Characterization of plasmids from Haemophilus parainfluenzae and Haemophilus haemolyticus.

The laboratory strains of Haemophilus parainfluenzae and Haemophilus haemolyticus that are commonly used as restriction enzyme sources carry several small multicopy plasmids. H. parainfluenzae carries plasmids pKC1, pKC2, and pKC3 of sizes 1.50, 2.86, and 3.84 kb, respectively, as determined by gel electrophoresis and electron microscopy. H. haemolyticus carries plasmids pKC4 and pKC5 of sizes 1.3 and 1.7 kb as determined by gel electrophoresis. At least two of the plasmids pKC1 and pKC4 were successfully transferred into E. coli by cotransfection with plasmid pBR322. They are compatible with pBR322 and have a comparable copy number.