The F plasmid CcdB protein induces efficient ATP-dependent DNA cleavage by gyrase.

DNA topoisomerases perform essential roles in DNA replication, gene transcription, and chromosome segregation. Recently, we identified a new type of topoisomerase II poison: the CcdB protein of plasmid F. When its action is not prevented by CcdA protein, the CcdB protein is a potent cytotoxin. In this paper, using purified CcdB, CcdA and gyrase, we show that CcdB protein efficiently traps gyrase in a cleavable complex. The CcdA protein not only prevents the gyrase poisoning activity of CcdB but also reverses its effect on gyrase. The mechanism by which the CcdB protein induces DNA strand breakage is closely related to the action of quinolone antibiotics. However, the ATP dependence of the CcdB cleavage process differentiates the CcdB mechanism from quinolone-dependent reactions because the quinolone antibiotics stimulate efficient DNA breakage, whether or not ATP is present. We previously showed that bacteria resistant to quinolone antibiotics are sensitive to CcdB and vice versa. Elucidation of the mechanism of action of CcdB protein may permit the design of drugs targeting gyrase so as to take advantage of this new poisoning mechanism.

Identification of the bacteriophage D108 kil gene and of the second region of sequence nonhomology with bacteriophage Mu.

To identify the second region of sequence nonhomology between the genomes of the transposable bacteriophages Mu and D108 originally observed by electron-microscopic analysis of DNA heteroduplexes and to localize functions ascribed to the 'accessory' or 'semi-essential' early regions of the phages between genes B and C, a 0.9-kb fragment of each genome located immediately beyond the B gene was cloned and sequenced. Three open reading frames (ORFs) were identified in each. The region of nonhomology is located within the 3' portion of the third ORF. D108 is shown to possess a Kil function similar to that previously shown for Mu, and that function is encoded by the first ORF.

Central location of the Mu strong gyrase binding site is obligatory for optimal rates of replicative transposition.

The bacteriophage Mu genome contains a strong DNA gyrase binding site (SGS) near its center, and disruption of the SGS by deletion or by insertion results in long delays in replication following induction of the appropriate lysogen. To determine if the central location of the SGS is obligatory for its function in Mu replication, we pursued two lines of investigation. First, fragments of Mu DNA containing the SGS were inserted into various locations in a Mu prophage lacking the central SGS. Replication following induction was restored in all of the lysogens constructed, but the observed rate of replication for different prophages decreased with increasing distance between the new location of the SGS and the center of the genome. We also deleted different lengths of DNA from within the right half of a wild-type prophage, retaining the SGS and displacing it from a central location. Replication rates of the deleted prophages were reduced, with larger deletions resulting in larger reductions. Pairing deletions in the right half of the prophage with a deletion in the left half resulted in substantially higher rates of replication than observed with the right half deletions alone. We conclude that the SGS must be located centrally between the Mu termini for optimal rates of Mu replication. These results are discussed in terms of a model that proposes that the SGS is involved in organizing the topology of supercoiled prophage DNA to assist in synapsis of the Mu termini.

Lambda excision revisited: testing a model for synapsis of prophage ends.

Excision of lambda prophage was reexamined to test a model for prophage end synapsis. The model proposes that, during in situ prophage replication, following induction, the diverging replication forks are held together. Consequently, prophage DNA is spooled through the replication machinery, drawing the prophage ends together and facilitating synapsis. The model predicts that excision will be slowed if in situ lambda replication is inhibited, and the predicted low rate of excision of a nonreplicating prophage was observed after thermoinduction. However, excision was rapid if additional Int protein was supplied or if the temperature was reduced after induction, showing that (i) Int is partially thermosensitive for excision at 42 degrees C and (ii) in situ replication is not required for rapid excision, a finding that is inconsistent with the model.

Role of ribonucleic acid synthesis in replication of deoxyribonucleic acid.

An experiment previously interpreted to show a ribonucleic acid requirement for propagation of deoxyribonucleic replication is reexamined and the earlier interpretation is shown to be incorrect.

Alterations of the rate of movement of deoxyribonucleic acid replication forks.

Antibiotics that inhibit ribonucleic acid (RNA) or protein synthesis are often used in studies of deoxyribonucleic acid (DNA) synthesis. The experiments presented here demonstrate that the rate of movement of DNA replication forks can be influenced by such antibotics. Addition of either chloramphenicol, which inhibits movement of ribosomes along messenger RNA, or streptolydigin, which inhibits movement of RNA polymerase, leads to a decrease in the rate of fork movement. Rifampin, which inhibits initiation of RNA synthesis, reverses the effects of chloramphenicol or streptolydigin. The slowed movement of DNA replication forks is discussed in terms of obstruction of fork movement by transcription complexes temporarily immobilized on the DNA template.

Alterations of deoxyribonucleoside triphosphate pools in Escherichia coli: effects on deoxyribonucleic acid replication and evidence for compartmentation.

Inhibition of ribonucleic acid synthesis in Escherichia coli 15 TAU bar with rifampin or streptolydigin leads to large increases in the sizes of cellular ribonucleoside and deoxyribonucleoside triphosphate pools. Inhibition of protein synthesis leads to increases in the sizes of all nucleoside triphosphate pools except the guanosine triphosphate and deoxyguanosine triphosphate pools; a decrease in the size of the latter pool may be responsible for the slowing of deoxyribonucleic acid replication fork movement observed in this strain in the absence of protein synthesis. Analysis of the kinetics of incorporation of labeled precursors into deoxyribonucleic acid and into cellular pools suggests that functional compartmentation of nucleotide pools exists, allowing the incorporation of exogenously supplied precursors into deoxyribonucleic acid without prior equilibration with the cellular pools.

Tetracycline inhibits propagation of deoxyribonucleic acid replication and alters membrane properties.

Tetracycline, at concentrations greater than required for inhibition of protein synthesis, rapidly and completely inhibits replication of deoxyribonucleic acid (DNA) in Escherichia coli and Bacillus subtilis. At these concentrations of tetracycline, synthesis of ribonucleic acid is not appreciably altered. In addition to inhibiting DNA replication, tetracycline causes alterations of the cytoplasmic membrane resulting in leakage of intracellular pools of nucleotides, amino acids, and the non-metabolizable sugar analogue, thiomethylgalactoside. As DNA is synthesized at a site on the membrane, alterations of membrane structure by tetracycline may be responsible for the observed inhibition of DNA replication.

Cellular location of Mu DNA replicas.

To ascertain the form and cellular location of the copies of bacteriophage Mu DNA synthesized during lytic development, DNA from an Escherichia coli lysogen was isolated at intervals after induction of the Mu prophage. Host chromosomes were isolated as intact, folded nucleoids, which could be digested with ribonuclease or heated in the presence of sodium dodecyl sulfate to yield intact, unfolded nucleoid DNA. Almost all of the Mu DNA in induced cells was associated with the nucleoids until shortly before cell lysis, even after unfolding of the nucleoid structure. We suggest that the replicas of Mu DNA are integrated into the host chromosomes, possibly by concerted replication-integration events, and are accumulated there until packaged shortly before cell lysis. Nucleoids also were isolated from induced lambda lysogens and from cells containing plasmid DNA. Most of the plasmid DNA sedimented independently of the unfolded nucleoid DNA, whereas 50% or more of the lambda DNA from induced lysogens cosedimented with unfolded nucleoid DNA. Possible explanations for the association of extrachromosomal DNA with nucleoid DNA are discussed.

Replacement of the bacteriophage Mu strong gyrase site and effect on Mu DNA replication.

The bacteriophage Mu strong gyrase site (SGS) is required for efficient replicative transposition and functions by promoting the synapsis of prophage termini. To look for other sites which could substitute for the SGS in promoting Mu replication, we have replaced the SGS in the middle of the Mu genome with fragments of DNA from various sources. A central fragment from the transposing virus D108 allowed efficient Mu replication and was shown to contain a strong gyrase site. However, neither the strong gyrase site from the plasmid pSC101 nor the major gyrase site from pBR322 could promote efficient Mu replication, even though the pSC101 site is a stronger gyrase site than the Mu SGS as assayed by cleavage in the presence of gyrase and the quinolone enoxacin. To look for SGS-like sites in the Escherichia coli chromosome which might be involved in organizing nucleoid structure, fragments of E. coli chromosomal DNA were substituted for the SGS: first, repeat sequences associated with gyrase binding (bacterial interspersed mosaic elements), and, second, random fragments of the entire chromosome. No fragments were found that could replace the SGS in promoting efficient Mu replication. These results demonstrate that the gyrase sites from the transposing phages possess unusual properties and emphasize the need to determine the basis of these properties.

The Mu strong gyrase-binding site promotes efficient synapsis of the prophage termini.

A strong DNA gyrase-binding site (SGS) is located midway between the termini of the bacteriophage Mu genome and is required for efficient replicative transposition. We have proposed that the SGS promotes the efficient synapsis of the Mu prophage ends (an obligate early step in replicative transposition), and that it does so by helping to organize the prophage DNA into a supercoiled loop with the SGS at the apex of the loop and the prophage termini at the base. The positioning of the synapsing termini equidistant from the SGS is a key element in the proposed model. To test this proposal, we have constructed prophages with a second, internal right end and asked whether the natural, external right end or the internal right end is used for synapsis with the left end in the presence and absence of the SGS. In the presence of the central SGS, the natural, or outside, right end was used exclusively and very efficiently. In the absence of the central SGS, the internal right end was used preferentially and inefficiently: the efficiency of transposition decreased with increasing distance between the internal right end and the left end. Repositioning the SGS midway between the left end and an internal right end allowed highly efficient use of the internal right end. These results support a model in which gyrase can influence long-range DNA interactions to promote efficient synapsis of Mu prophage ends.

Early events in the replication of Mu prophage DNA.

To determine whether the early replication of Mu prophage DNA proceeds beyond the termini of the prophage into hose DNA, the amounts of both Mu DNA and the prophage-adjacent host DNA sequences were measured using a DNA-DNA annealing assay after induction of the Mu vegetative cycle. Whereas Mu-specific DNA synthesis began 6 to 8 min after induction, no amplification of the adjacent DNA sequences was observed. These data suggest that early Mu-induced DNA synthesis is constrained within the boundaries of the Mu prophage. Since prophage Mu DNA does not undergo a prophage lambda-like excision from its original site after induction (E. Ljungquist and A. I. Bukhari, Proc. Natl. Acad. Sci. U.S.A. 74:3143--3147, 1977), we propose the existence of a control mechanism which excludes prophage-adjacent sequences from the initial mu prophage replication. The frequencies of the Mu prophage-adjacent DNA sequences, relative to other Escherichia coli genes, were not observed to change after the onset of Mu-specific DNA replication. This suggests that these regions remain associated with the host chromosome and continue to be replicated by the chromosomal replication fork. Therefore, we conclude that both the Mu prophage and adjacent host sequences are maintained in the host chromosome, rather than on an extrachromosomal form containing Mu and host DNA.

Instability of transposase activity: evidence from bacteriophage mu DNA replication.

Transposition of genetic elements involves coupled replication and integration events catalyzed in part by a class of proteins called transposases. We have asked whether the transposase activity of bacteriophage Mu (the Mu A protein) is stable and capable of catalyzing multiple rounds of coupled replication/integration, or whether its continued synthesis is required to maintain Mu DNA replication. Inhibition of protein synthesis during the lytic cycle with chloramphenicol inhibited Mu DNA synthesis with a half-life of approximately 3 min, demonstrating a need for continued protein synthesis to maintain Mu DNA replication. Synthesis of specific Mu-encoded proteins was inhibited by infecting a host carrying a temperature-sensitive suppressor, at permissive temperature, with Mu amber phages, then shifting to nonpermissive temperature. When Aam phages were used, Mu DNA replication was inhibited with kinetics essentially identical to those with chloramphenicol addition; hence, it is likely that continued synthesis of the Mu A protein is required to maintain Mu DNA replication. The data suggest that the activity of the Mu A protein is unstable, and raise the possibility that the Mu A protein and other transposases may be used stoichiometrically rather than catalytically.

Genetic analysis of the strong gyrase site (SGS) of bacteriophage Mu: localization of determinants required for promoting Mu replication.

The Mu strong gyrase site (SGS), located in the centre of the Mu genome, is required for efficient Mu replication, as it promotes synapsis of the prophage termini. Other gyrase sites tested, even very strong ones, were unable to substitute for the SGS in Mu replication. To determine the features required for its unique properties, a deletion analysis was performed on the SGS. For this analysis, we defined the 20 bp centred on the midpoint of the 4 bp staggered cleavage made by gyrase to be the 'core' and the flanking sequences to be the 'arms'. The deletion analysis showed that (i) approximately 40 bp of the right arm is required, in addition to core sequences, for both efficient Mu replication and gyrase cleavage; and (ii) the left arm was not required for efficient Mu replication, although it was required for efficient gyrase cleavage. These observations implicated the right arm as the unique feature of the SGS. The second observation showed that strong gyrase cleavage and Mu replication could be dissociated and suggested that even weak gyrase sites, if supplied with the right arm of the SGS, could promote Mu replication. Hybrid sites were constructed with gyrase sites that could not support efficient Mu replication. The SGS right arm was used to replace one arm of the strong pSC101 gyrase site or the weaker pBR322 site. The pSC101 hybrid site allowed efficient Mu replication, whereas the pBR322 hybrid site allowed substantial, but reduced, replication. Hence, it appears that optimal Mu replication requires a central strong gyrase site with the properties imparted by the right arm sequences. Possible roles for the SGS right arm in Mu replication are addressed.

Stoichiometric use of the transposase of bacteriophage Mu.

The transposase of bacteriophage Mu (gene A protein) mediates the coupled replication and integration processes that constitute transposition during the lytic cycle. Our previous results showed that the activity of the A protein is unstable, as its continued synthesis is required to maintain Mu DNA replication throughout the lytic cycle. We present here the results of experiments in which the A protein is used stoichiometrically and must be synthesized de novo for each round of Mu DNA replication. Induction of a Mu lysogen in the absence of DNA replication allows accumulation of potential for a single round of Mu DNA replication. Once achieved, this potential is stable even in the absence of further protein synthesis. Release of inhibition of DNA replication leads to a single semi-conservative replicative transposition event, followed by later rounds only if additional synthesis of the A protein is allowed.

Fate of plasmids containing Mu DNA: chromosome association and mobilization.

The fluorescent dye, diamidinophenylindole-dihydrochloride (DAPI) can be added to CsCl gradients to enhance the density resolution of DNA species, independent of their topological configurations. When Proteus mirabilis and Escherichia coli strains carrying an RP4::Mucts plasmid were examined with the use of such a technique, it was found that after thermal induction of the prophage essentially al of the plasmid DNA became associated with the chromosome. This quantitative association is detergent-RNase- and pronase-resistant and dependent on the expression of Mu genes. The association is temporally, and probably functionally, correlated with the onset of Mu DNA replication. Genetic studies with F'::mini Mu plasmids indicate that some of the association results in stable Hfr formation, and does not require the product of Mu gene B.

Messenger ribonucleic acid synthesis and degradation in Escherichia coli during inhibition of translation.

Various aspects of the coupling between the movement of ribosomes along messenger ribonucleic acids (mRNA) and the synthesis and degradation of mRNA have been investigated. Decreasing the rate of movement of ribosomes along an mRNA does not affect the rate of movement of some, and possibly most, of the RNA polymerases transcribing the gene coding for that mRNA. Inhibiting translation with antibiotics such as chloramphenicol, tetracycline, or fusidic acid protects extant mRNA from degradation, presumably by immobilizing ribosomes, whereas puromycin exposes mRNA to more rapid degradation than normal. The promoter distal (3') portion of mRNA, synthesized after ribosomes have been immobilized by chloramphenicol on the promoter proximal (5') portion of the mRNA, is subsequently degraded.

Pattern of replication of a colicin factor during the cell cycle of Escherichia coli.

The ColBM, trp, lac episome was transferred to a lacZ derivative of Escherichia coli B/r, and the manner of replication of this colicinogenic factor was followed through the cell cycle. The results suggest a pattern of replication not connected with any particular stage in the cell cycle.

Re-examination of F plasmid replication in a dnaC mutant of Escherichia coli.

The replication of an F' plasmid in a dnaC mutant, thermolabile for initiation of chromosomal replication, has been re-examined using a novel DNA-DNA annealing assay. Plasmid replication ceases rapidly at non-permissive conditions, consistent with a direct role for the dnaC product in the replication of F.