Fate of lambda DNA in a bacterial host defective in DNA synthesis.
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Chromosomes, Bacterial↗
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Activation of mutant forms of DnaA protein of Escherichia coli by DnaK and GrpE proteins occurs prior to DNA replication.
Mutant forms of DnaA protein, inert in a replication system composed of other purified proteins, are "activated" by DnaK and GrpE heat shock proteins (Hupp, T. R., and Kaguni, J. M. (1993) J. Biol. Chem. 268, 13137-13142). The effect of these heat shock proteins on DnaA5 and DnaA46 protein was separated from the event of DNA synthesis by incubation in two stages. Components necessary during the first stage for "activation" included GrpE and DnaK proteins, ATP at 0.2 mM or greater, and polyvinyl alcohol (8%) or glycerol, optimal at concentrations between 20 and 30%. An ATP regenerating system provided by creatine kinase and creatine phosphate was stimulatory. Addition of the activated form of DnaA5 or DnaA46 protein to a reconstituted system containing other purified replication proteins during the second stage of incubation resulted in DNA replication. Activation of DnaA5 or DnaA46 protein by heat shock proteins was thermolabile, suggesting that the temperature sensitivity of dnaA5 and dnaA46 mutants is related to this thermolabile interaction. A third heat shock protein, DnaJ protein, interfered with the activation of DnaA5 protein if present during the first stage of incubation. This inhibitory effect was less striking if included during the second stage of incubation. These results suggest a mechanism for regulation of the activity of DnaA protein.
Adenosine Triphosphate↗
[Ability of lectins to model the action of antibiotics on growth of mutants of Bacillus subtilis].
Using the bacterium B. subtilis as a model and antibiotics as metabolic inhibitors which can suppress replication (mitomycin), transcription (rifampycin) or translation (streptomycin), it is shown that carbohydrate-binding proteins (lectins of plant origin) have different action on intracellular processes under study. The effect depends on lectin's structure and on the condition of bacterial reparation/recombination system. Lectins with chitin-binding domain (STA, WGA) is characterized by the most expressed effect on the repair-proficient strain when the inhibitor of template function of DNA (mitomycin C) was used. The absence of such effect on mutants recP and polC may prove that the corresponding bacterial proteins play the role of mediators in transmission of the signal influence of lectins. The representative of ribosome-inactivating lectins--SNA-I--could increase the streptomycin effect. It is proposed that intracellular effects of lectins have complex mechanisms which need participation of repair functions.
Anti-Bacterial Agents↗
Mini-F plasmid-induced SOS signal in Escherichia coli is RecBC dependent.
Dispensable replicons such as F plasmid [95 kilobases (kb)] or its mini-derivatives such as mini-F (9.3 kb) or lambda mini-F efficiently induced cellular SOS genes such as sfiA (sulA) when they were damaged by UV irradiation and then introduced into a recipient bacterium. To generate an SOS signal, UV light-damaged mini-F or mini-F conditional mutants deficient in replication required that the bacterial RecBC enzyme retained some activity different from the nuclease activity that was dispensable. In contrast, UV light-damaged F plasmid produced an SOS signal independently of the activity of the RecBC enzyme and of the expression of the mini-F, -H, and -G proteins. Our findings are consistent with a picture in which the SOS signal is constituted by stretches of single-stranded DNA on a replicon. Moreover, our present data combined with other data previously published lead to the hypothesis that the SOS signal induced by mini-F plasmid is located in trans on the host chromosome, whereas the one generated by UV light-damaged F plasmid is in cis on the transferred DNA.
Chromosomes, Bacterial↗
The mechanism of integration of phage Mu in the chromosome of Escherichia coli.
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Chromosomes, Bacterial↗
Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: primase as the sole priming enzyme.
The enzymatic replication of plasmids containing the unique (245 base pair) origin of the Escherichia coli chromosome (oriC) can be initiated with any of three enzyme priming systems: primase alone, RNA polymerase alone, or both combined (Ogawa, T., Baker, T. A., van der Ende, A. & Kornberg, A. (1985) Proc. Natl. Acad. Sci. USA 82, 3562-3566). At certain levels of auxiliary proteins (topoisomerase I, protein HU, and RNase H), the solo primase system is efficient and responsible for priming synthesis of all DNA strands. Replication of oriC plasmids is here separated into four stages: (i) formation of an isolable, prepriming complex requiring oriC, dnaA protein, dnaB protein, dnaC protein, gyrase, single-strand binding protein, and ATP; (ii) formation of a primed template by primase; (iii) rapid, semiconservative replication by DNA polymerase III holoenzyme; and (iv) conversion of nearly completed daughter molecules to larger DNA forms. Optimal initiation of the leading strand of DNA synthesis, over a range of levels of auxiliary proteins, appears to depend on transcriptional activation of the oriC region by RNA polymerase prior to priming by primase.
Chromosomes, Bacterial↗
Suppression of temperature-sensitivity of a dnaA46 mutant by excessive DNA supercoiling.
We report here that the high-temperature sensitivity of a dnaA46 mutant was suppressed by addition of high concentrations of NaCl into the culture medium. This suppression was also observed with other high-temperature-sensitive dnaA mutants, except dnaA167 and dnaA508 mutants, which have mutations in the N-terminal region of DnaA protein. Since high concentrations of NaCl in the medium increased negative DNA supercoiling in a dnaA46 mutant, we hypothesized that the increase in DNA supercoiling is involved in the suppression of the temperature-sensitivity of the dnaA46 mutant by high concentrations of NaCl. This hypothesis was supported by in vitro and in vivo results as follows. A low DNA replication activity of purified DnaA46 protein at high temperatures was increased in line with an increase in DNA supercoiling of template DNA. The dnaA46 mutant showed higher sensitivity to nalidixic acid, a DNA-relaxing drug, than did the wild-type cells under the conditions of high temperatures and high concentrations of NaCl.
Bacterial Proteins↗
The bacteriophage lambda DNA replication protein P inhibits the oriC DNA- and ATP-binding functions of the DNA replication initiator protein DnaA of Escherichia coli.
Under the condition of expression of lambda P protein at lethal level, the oriC DNA-binding activity is significantly affected in wild-type E. coli but not in the rpl mutant. In purified system, the lambda P protein inhibits the binding of both oriC DNA and ATP to the wild-type DnaA protein but not to the rpl DnaA protein. We conclude that the lambda P protein inhibits the binding of oriC DNA and ATP to the wild-type DnaA protein, which causes the inhibition of host DNA synthesis initiation that ultimately leads to bacterial death. A possible beneficial effect of this interaction of lambda P protein with E. coli DNA initiator protein DnaA for phage DNA replication has been proposed.
Adenosine Triphosphate↗
dam methylation and the initiation of DNA replication on oriC plasmids.
Plasmid DNA containing the replication origin of the Escherichia coli chromosome (oriC) has been shown to be inefficient as a template for DNA synthesis in vitro when isolated from dam mutants. Here, we extend this study to hemimethylated oriC plasmids and to replication in dam-3 mutant enzyme extracts. The results show that: (1) hemimethylated oriC plasmids replicate with the same low efficiency as nonmethylated DNA; (2) DNA synthesis starts at oriC regardless of the methylated state of the template; (3) replication in dam-3 enzyme extracts is inefficient because this strain is deficient in DnaA protein; and (4) consistent with this observation, the copy number of the oriC plasmid pFH271 is reduced in the dam-3 mutant. However, we have found that low DnaA protein levels in dam-3 mutants are not sufficient to explain the reduced transformation efficiency of oriC plasmids. We suggest that there must exist in vivo inhibitory factors not present or present in low quantities in vitro which specifically recognize the hemimethylated or nonmethylated forms of the oriC region.
Bacterial Proteins↗
Conditional-lethal deoxyribonucleic acid ligase mutant of Escherichia coli.
A new Escherichia coli deoxyribonucleic acid (DNA) ligase mutant has been identified among a collection of temperature-sensitive DNA replication mutants isolated recently (Sevastopoulos, Wehr, and Glaser, Proc. Natl. Acad. Sci. U.S.A. 74:3485-3489, 1977). At the nonpermissive temperature DNA synthesis in the mutant stops rapidly, the DNA is degraded to acid-soluble material, and cell death ensures. This suggests that the mutant may be among the most ligase-deficient strains yet characterized.
Chromosome Mapping↗
DNA damage-inducible replication of the Escherichia coli chromosome is initiated at separable sites within the minimal oriC.
When Escherichia coli cells are subjected to genetic stress by exposure to agents or conditions that transiently block DNA replication, the mode of DNA replication is profoundly altered. One of the alterations is the induction of inducible stable DNA replication (iSDR) that does not require the initiator protein, DnaA, and occurs despite the presence of rifampin and chloramphenicol, which inhibit the initiation of usual chromosome replication at oriC. It has been demonstrated that iSDR starts primarily from both the oriC and terC regions of the chromosome. To precisely map the iSDR origin (oriM1) located in the oriC region, various oriC fragments were inserted into a plasmid vector derived from pSC101, and the copy number of these plasmid constructs was measured in the presence of rifampin and chloramphenicol after cells were induced for the SOS response by thymine starvation. The results indicated that there are at least two origins for iSDR within the minimal oriC; one (oriM1A) is located between the BamHI (coordinate +1) and the AvaII(155) sites, and the other (oriM1B) between the AvaII(155) and the HindIII(244) sites. Furthermore, a 263 bp fragment containing oriM1, which was placed at the att lambda site of the chromosome, was found to initiate chromosome replication in the presence of the drugs when cells were starved of thymine. Introduction of additional copies of oriM1 into a cell stimulated initiation of iSDR at oriM1 on the chromosome. The result supported the model that iSDR starts from D-loops created between oriM1 sequences and that the amount of D-loops determines the level of the iSDR activity.
Bacterial Proteins↗
Novel SOS phenotypes caused by second-site mutations in the recA430 gene of Escherichia coli.
E coli recA430 mutants are recombination-proficient, extremely UV sensitive, UV nonmutable and partially deficient in RecA-mediated proteolysis and in RecA-dependent 'induced replisome reactivation' (IRR), the ability to recover DNA replication activity after UV irradiation. To determine how this pleiotropic phenotype can be altered by mutation, we isolated 10 independent derivatives of a recA430 strain, selecting for increased UV resistance. Eight of the 10 owed their resistance to altered recA alleles. We here describe the phenotypes conferred by two of the new recA alleles (recA720 and recA727), each of which contains the original recA430 mutation (G662 to A) and a second-site transition: T167 to C in recA720, and G103 to A in recA727. The second-site change in recA720 suppresses all the defects caused by recA430, and causes RecA720 to exhibit greater activity than RecA+ in some respects. Some, but not all, of the recA430 defects are partially corrected by the second-site mutation in recA727.
Bacterial Proteins↗
Cell reproduction and morphological changes in Mycoplasma capricolum.
The cell reproduction of Mycoplasma capricolum was studied. The velocity of DNA replication fork progression was about 6 kb/min, which is 10 times slower than that of Escherichia coli. The time required for one round of DNA replication accorded with the doubling time. The origin/terminus ratio was 2.0. M. capricolum cell morphology was classified into two types, rod and branched. In the ordinary-growth phase, the rod cells accounted for about 90% of the total population, with branched cells comprising the remaining 10%. The proportion of branched cells increased to 90% following inhibition of DNA replication by nucleoside starvation. An increase in the proportion of branched cells was induced by transfer of a temperature-sensitive mutant deficient in DNA replication to the restrictive temperature. The rod cells had a regular structure, a fixed cell length, and constrictions in the center. The DNA contents of individual rod cells were distributed with a standard deviation of 0.40 of average. The branched cells had irregular structures and a wide distribution of DNA contents. Counting of viable cells revealed that the cells ceased division upon cell type conversion; however, branched cells maintained a reproductive capacity. A model for the reproduction process is proposed.
Bacterial Proteins↗
Amber-mutants of plasmid mini-F defective in replication.
Amber mutants of the mini-F plasmid pML31 were isolated with the mutagen hydroxylamine. Under non-permissive conditions amber mutants segregate and show no incorporation of label into supercoiled plasmid DNA in double-label experiments. Wild-type and one mutant of mini-F were integrated by recombinant DNA techniques into the single EcoRI site of plasmid vector pBR322. Plasmid specific proteins were labeled in minicells and analysed by SDS-PAGE. A 34,000 dalton molecular weight protein was identified to be missing in the amber mutant of plasmid mini-F.
Bacterial Proteins↗
Attempts to purify a membrane attached chromoid of bacteriophage lambda.
Using methods which proved successful for the isolation of E. coli chromosome in a folded form (the E. coli chromoid), we have attempted to purity the "native" form of bacteriophage gamma chromosome from gamma infected cells. Upon sedimentation of lysates we find that phage DNA separates into two fractions, one of which cosediments with the bacterial chromoid ; the other sediments nearer to the top of gradient. Both fractions probably contain membrane-bound phage DNA, and both support the in vivo synthesis of phage DNA. The heavier fraction contains more closed circular parental DNA molecules than the lighter fraction. Formation of the latter is blocked by certain phage mutations. Being relatively free of bacterial DNA, the lighter fraction is suitable for further analysis.
Cell Membrane↗
The Bacillus subtilis replication terminator system functions in Escherichia coli.
The Bacillus subtilis DNA terminators, IRI + IRII, were inserted into the Escherichia coli plasmid pACYC184 such that the IRI terminator would be in its active orientation with respect to the approaching unidirectionally moving replication fork. When this new plasmid was transferred into E. coli, harbouring an expression plasmid producing the B. subtilis terminator protein RTP, fork arrest was observed to occur at the position of the inserted terminator region. Thus, the B. subtilis replication terminator system can function in E. coli. It was shown that the B. subtilis system operated with approximately 30% of the efficiency of the E. coli system utilizing the R6K TerR2 DNA terminator and the E. coli Tus terminator protein. Assuming that RTP and Tus have quite different folded structures these results suggest that fork arrest in B. subtilis is not dependent upon a highly specific recognition and interaction between RTP positioned on the DNA terminator and a component(s) of the approaching replisome.
Bacillus subtilis↗
[Analysis of chromosomal and extrachromosomal functions involved in the replication of bacterial plasmids].
A variety of functions codified by both the host chromosome and the plasmid genome are necessary for plasmid replication. We have found that dna B and dna F are essential for replication of both types of plasmids: R144 and Sa which have a restricted control of replication, and R6K with a relaxed control; on the other hand plasmids belonging to the first group absolutely depend on dna A and dna E gene products. Plasmid Sa, which shares with the 1st group a restricted control of replication but appears on a number of intermediate copies, shows a partial requirement to the dna A gene product. Location studies of plasmid-coded genes, involved in the replications of plasmids, were carried out in plasmid pSC102, a derivative of the resistance plasmic R6-5. All the genes required for replication are contained into a DNA segment which consists of two Pst I fragments: P-4 and P-6. The origin of replication On V is not itself sufficient for autonomous replication but needs in addition the presence of the Rep A gene. Genes responsible for copy control and incompatibility Cop/Inc are located on fragment P-6, i.e. they are physically distinct from Ori V. This indicates that the origin of replication is not responsible for, although may participate in, the incompatibility phenomenon.
Chromosomes, Bacterial↗
Initiation of plasmid pC194 replication and its control in Bacillus subtilis.
By deletion analysis we have defined a 1.1 kb segment required for driving autonomous replication of the plasmid pC194. The minimal replicon specifies a positive, RepH, and a negative, Inc8A, trans-acting product and their target sites. The RepH product has a Mr of 34.1 kDa, could be overproduced, and binds specifically to the pC194 origin region. By trans complementation studies we have shown that pC194 replication is indirectly controlled at the level of RepH synthesis by a negative product, IncA, that is transcribed within the repH transcription unit in the opposite direction ("antisense RNA"). The antisense RNA regulates the RepH synthesis by a mechanism that seems to involve RNA/RNA interaction in a manner that interferes with translation. In addition, an autoregulatory control might be operative.
Bacillus subtilis↗