Karol Taylor

Escherichia coli dnaA initiation function is required for replication of plasmids derived from coliphage lambda

The dnaA gene function, indispensable for the initiation of Escherichia coli replication from oriC is not essential for the growth of phage lambda. The in-vitro replication of plasmids derived from phage lambda does not seem to require DnaA protein either. However, we present evidence that in vivo the normal replication of lambda plasmids is dnaA-dependent. After inactivating the dnaA gene function, half of the plasmid molecules may enter a single round of replication. Rifampicin sensitivity of this abortive, as well as normal, replication indicates involvement of RNA polymerase. The rifampicin resistance of the normal replication of lambda plasmids in E. coli carrying the dnaAts46 or dnaAts5, but not the dnaAts204 allele at 30 degrees C implies the interaction of DnaA protein and RNA polymerase in this process. We propose that DnaA protein co-operates with RNA polymerase in the initiation of replication at ori lambda. The dispensability of DnaA in the growth of phage lambda and in lambda plasmid replication in vitro is discussed.

Inheritance of the replication complex by one of two daughter copies during λ plasmid replication in Escherichia coli

Direct measurement of DNA synthesis confirmed that lambda plasmid replication proceeds for several hours in an amino acid-starved relA mutant of Escherichia coli, leading to plasmid amplification; this replication is lambda cro-independent, but requires the function of lambda O initiator in the absence of its synthesis. This suggests that after the assembly of the replication complex (RC) at ori lambda the lambda O protein remains in this structure and the affinity of lambda O to ori lambda is alleviated in the assembled RC allowing its movement along the DNA. During amino acid starvation the lambda plasmid DNA synthesis per bacterial mass occurs at a constant level, as would be expected if the number of functioning RCs remained constant. This favors the idea that under these conditions the next replication round operates due to the activity of the RC inherited from the preceding round. Density shift experiments reveal indeed that, from two daughter plasmid copies synthesized after the onset of amino acid starvation only one is able to enter into the next round of replication. We infer that this is the plasmid copy that inherits the lambda O-enclosing RC from the previous replication round. Moreover, the same results of density shift experiments were obtained for plasmids synthesized before the onset of amino acid starvation. Therefore, we presume that in lambda plasmid-harboring bacteria growing in nutrient medium, every second plasmid circle bears an RC that originates from the preceding round of replication. This structure has to be assembled de novo only on the daughter plasmid copy that does not inherit the parental RC. In the absence of lambda O initiator synthesis in amino acid-starved relA cells this process cannot occur, leaving as the only replication pathway that driven by the parental RC. Our results are discussed in relation to the model of regulation of lambda plasmid replication.

ALLELE SPECIFICITY OF THE ESCHERICHIA COLI DNAA GENE FUNCTION IN THE REPLICATION OF PLASMIDS DERIVED FROM PHAGE LAMBDA

We demonstrate a variation in the effects of seven alleles of theEscherichia coli dnaA gene, which cause temperature sensitivity of initiation of chromosomal replication, on the replication ofλ phage-derived plasmids at 30° C. These mutants showed no allele specificity ofdnaA function in replication of either of twoλπ plasmids studied. On the other hand, the inability of theλP+ plasmid to replicate indnaA508, 46 and204 cells, indnaB (groP A15) or in cells that are temperature sensitive for the chaperone genesdnaK756, dnaJ259 andgrpE280 and 30° C was suppressible by a singleπ mutation. This suggests that it is a common property of theπ protein, probably its weaker interaction with DnaB helicase, that is responsible for the suppression. One can also conclude that the DnaA-regulated transcriptional activation oforiλ acts at the step, in which all these gene products cooperate, i.e. during preprimosome loading and chaperone-mediated release of DnaB from P protein inhibition.

Stringent control of replication of plasmids derived from coliphage λ

SummaryThe first events of λ plasmid replication in vivo, which probably regulate this process, are the transcriptional activation of the origin of replication by RNA polymerase and the binding of the initiator protein, λO, to this nucleotide sequence. The λO protein is known for its rapid proteolytic degradation; hence amino acid starvation of Escherichia coli should result in inhibiton of λ plasmid replication caused by inhibition of protein synthesis. However, contrary to this prediction, we found that λ plasmid replication, as measured by the increase in plasmid content per bacterial mass, proceeds for hours in an amino acid-starved, relaxed mutant, whereas it is inhibited in its wild-type stringent partner. λ plasmid replication in amino acid-starved, relaxed cells reveals absolute λO dependence and is not inhibited by chloramphenicol at 200 μg/ml. This process also occurs in wild-type cells treated with chloramphenicol. We conclude that λ plasmid replication is under stringent control, probably as a result of the action of ppGpp, the indirect product of the relA gene, on RNA polymerase. The problem of stability of the λO initiator protein is discussed.

Stability of coliphage λ DNA replication initiator, the λO protein☆

The initiator of coliphage λ DNA replication, λO protein, may be detected among other 35S-labeled phage and bacterial proteins by a method based on immunoprecipitation. This method makes it possible to study λO proteolytic degradation in λ plasmid-harboring or λ phage-infected cells; it avoids ultraviolet (u.v.)-irradiation of bacteria, used for depression of host protein synthesis, prior to λ phage infection. We confirm the rapid decay of λO protein (half-time of 80 s), but we demonstrate the existence of a stable λO fraction. In the standard five minute pulse-chase experiments, 20% of synthesized λO is stable. The extension of the [35S]methionine pulse, possible in λ plasmid-harboring cells, leads to a linear increase of this fraction, as if a part of the synthesized λO was constantly made resistant to proteolysis. Less than 5% of λO protein synthesized during one minute is transformed into a stable form. We presume that the stable λO is identical with λO present in the normal replication complex and thus protected from proteases. We cannot find any stable λO inEscherichia coli recA+ cells that were irradiated with u.v. light prior to λ phage infection, but their recA− counterparts behave normally, suggesting that recA function interferes in the assembly of a normal replication complex in u.v.-irradiated bacteria. The stable λO found in λ plasmid-harboring, amino acid-starved relA cells is responsible for the λO-dependent λ plasmid replication that occurs in this system in the absence of λO synthesis. The existence of stable λO raises doubt concerning its role as the limiting initiator protein in the control of replication. Another significance of λO rapid degradation is proposed.

Bacteriophage λ replication proteins: Formation of a mixed oligomer and binding to the origin of λ DNA

SummaryThe purified bacteriophage λ replication proteins O and P sediment separately in metrizamide gradients of low ionic strength as dimers. Together they interact with each other forming an oligomer, composed of two molecules of λO and one molecule of λP. The λO-P oligomer is active in the in vitro replication of oriλ-containing DNA.Equilibrium sedimentation in preformed metrizamide density gradients under conditions that separate DNA-protein complexes from free proteins was employed in order to study possible interactions among the λ replication proteins and oriλ DNA. It was found that the λP protein binds specifically to oriλ-containing plasmid DNA only in the presence of λO protein. About 100 molecules of λO and 10 molecules of λP form a complex with the oriλ DNA. The λ DNA-λO-λP complex was shown to be active in an in vitro replication system.Since the physical interactions between oriλ and λO and between λP and the Escherichia coli dnaB replication protein are well documented, the evidence for a λO-P interaction presented in this paper provides the missing link in the molecular mechanism that enables λ to direct the host replication machinery to the replication of its own DNA.

Synthesis and decay of λ DNA replication proteins in minicells

Abstract The coliphage λ DNA replication proteins, the O - and P -gene products, have been identified by infection of nonpermissive Escherichia coli minicells with the appropriate λ amber mutants as proteins of a molecular weight of about 34000 and 23000, respectively. Proteins of exactly the same size were found in minicells harbouring the plasmid λ dv . Both proteins seem to be synthesized at the same rate. In λ-infected minicells, as well as in lambda; dv -harbouring minicells the pulse-and-chase experiments have shown an exceptionally rapid decay of the O-protein.

INTERACTION OF THE ESCHERICHIA COLI DNAA PROTEIN WITH BACTERIOPHAGE LAMBDA DNA

Abstract Interaction of the Escherichia coli DnaA (replication initiator) protein with restriction fragments of phage λ DNA demonstrated differential binding of DnaA along the whole λ DNA. Interaction of DnaA with the λ replication region (from the promoter pR to the origin of replication, oriλ) demonstrated a strong binding of DnaA to the region around the po promoter where synthesis of a short antisense oop RNA is initiated. The four sequences protected by DnaA (two 9mers and two 5mers) are not related even to a relaxed DnaA box. The pattern of protection of these four sequences and the location of three DNase I hypersensitive sites in the λ DNA r strand, together with results of mobility shift assays and electron microscopy studies, may indicate an interaction involving DnaA monomers bound to different DNA positions on one side of the helix and the formation of higher-order nucleoprotein structures. Therefore, it is tempting to suggest that DnaA, in addition to its activity in regulation of replication and transcription, could be considered as a factor which structures certain chromosomal regions.

Regulation of Bacteriophage λ Replication

Bacteriophage λ is both a model for basic biological studies on the molecular level and a commonly used tool in molecular cloning. In this review we summarise the control of bacteriophage λ development, placing particular attention to the mechanisms involved in the “lysis or lysogeny” decision and the regulation of replication of phage λ DNA, as well as of plasmids derived from this phage.

Escherichia coli dnaJ- and dnaK-gene products: Synthesis in minicells and membrane-affinity

Escherichia coli dnaJ- and dnaK-gene products have been identified in a system of minicells infected with the appropriate transducing lambda phages. The molecular weights of these polypeptides in dodecyl sulphate/acrylamide electrophoresis amounted to 39,000 and 77,000, respectively. Equilibrium sedimentation of minicell lysates in metrizamide density gradients has revealed that both these host proteins, indispensable for lambda DNA replication, are membrane-bound.