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G Wegrzyn

Publications and source records attributed to G Wegrzyn.

At least 19 recordsLinked to original sources
PubMed1996

Disassembly of the coliphage lambda replication complex due to heat shock induction of the groE operon.

We have found previously that, in contrast to the free O initiator protein of lambda phage or plasmid rapidly degraded by the Escherichia coli ClpP/ClpX protease, the lambda O present in the replication complex (RC) is protected from proteolysis. In amino acid-starved E. coli relA cells, a temperature shift from 30 to 43 degrees did not affect RC integrity, as judged from the unchanged level of stable lambda O observed; however, the same temperature shift in a complete medium resulted in the decay of this lambda O fraction, which suggested disassembly of the RC. Examination of this phenomenon revealed that for lambda RC disassembly, heat shock induction of the groE operon, coding for molecular chaperones of the Hsp60 class, is indispensable. Heat shock induction of the groE operon present on a multicopy plasmid inhibited the growth of infecting phage.

Bacterial Proteins↗
PubMed1995

Amplification of lambda plasmids in Escherichia coli relA mutants.

It was previously demonstrated that, contrary to wild-type stringent (rel+) strains of Escherichia coli, in amino acid-starved relaxed (relA) mutants the replication of lambda plasmid proceeds for several hours. The replication leads to amplification of lambda plasmid DNA. Here, the conditions for this amplification have been optimized. The amplification efficiency depends on the temperature as well as on the nature of amino acid starvation, but it is only little or totally not dependent on the pH value of the medium in a range from 6.0 to 8.0. It seems that the most efficient amplification can be achieved by overnight cultivation of E. coli relA arg strain harbouring lambda plasmid at 36-39 degrees C in minimal medium containing Casamino acids. Under these conditions, the copy number of lambda plasmid increases from about 40 to about 300 per cell giving greater than 7-fold amplification.

Bacteriophage lambda↗
PubMed1995

Transcriptional activation of ori lambda regulates lambda plasmid replication in amino acid-starved Escherichia coli cells.

Replication of lambda plasmid DNA is inhibited in amino acid-starved wild type Escherichia coli cells (i.e., during the stringent response), whereas it proceeds for several hours in relA mutants (i.e., during the relaxed response). It was demonstrated previously that ppGpp-mediated inhibition of transcription starting from the pR promoter is responsible for inhibition of lambda plasmid replication; RNA polymerase function is indispensable for replication of lambda plasmid DNA during the relaxed response. The replication is carried out by the heritable replication complex containing the lambda O protein which is protected from proteases by other elements of this complex. Here we demonstrate that the replication is dependent on DnaG (primase) function. Thus, in amino acid-starved cells, lambda plasmid replication requires RNA polymerase function only for transcriptional activation of ori lambda. We also present evidences that the replication is dependent on the function of DNA gyrase. On the basis of these findings and other recent reports, we demonstrate a model of the regulation of lambda plasmid replication driven by the inherited replication complex. It seems that transcriptional activation of ori lambda indirectly triggers the initiation of lambda plasmid DNA replication at least during the relaxed response.

Amino Acids↗
PubMed1995

Plasmid and host functions required for lambda plasmid replication carried out by the inherited replication complex.

We have shown previously that in amino acid-starved, relaxed (rel-) mutants of Escherichia coli replication of the lambda plasmid occurs via the lambda O-containing replication complex (RC) that was assembled prior to the onset of amino acid starvation and is inherited by one of the two daughter plasmid circles in each replication cycle. This replication is regulated neither by binding of the lambda O initiator to ori lambda, nor by the lambda Cro-mediated repression. Here we show that it is dependent on both RNA polymerase and DnaA functions, which is consistent with our recent finding that transcriptional activation of ori lambda is under the control of DnaA. In the system studied, DnaA-regulated transcriptional activation of ori lambda seems to be the only rate-limiting process. The lambda plasmid replication mediated by the inherited RC appeared to be independent of the functions of lambda P and DnaJ required in RC assembly In vitro experiments performed by others suggest that DnaJ first binds to the ori lambda-bound lambda O-lambda P-DnaB pre-primosome and subsequently lambda P complexed with DnaJ is preferentially recognized by DnaK-GrpE; chaperone-mediated rearrangement of this structure relieves DnaB helicase of lambda P inhibition. Recently we proposed that this process is directly coupled to the insertion of the pre-primosome between DNA strands transiently separated by transcription. This last-mentioned process may be required in lambda plasmid replication mediated by the inherited RC, which appeared in turn to be dependent on DnaK and GrpE functions.

Bacterial Proteins↗
PubMed1995

Transcriptional activation of the origin of coliphage lambda DNA replication is regulated by the host DnaA initiator function.

The initiator of phage lambda DNA replication, the lambda O protein, is considered to be an analogue of the initiator of DNA replication (DnaA) of its host, Escherichia coli. Both specifically recognize their origins of replication, ori lambda and oriC, respectively, and organize the assembly of specific replication complexes. However, DnaA has an additional activation function, acting on oriC-proximal DnaA-boxes, and regulating transcription initiated at promoters in and around oriC. Here, we demonstrate that lambda plasmid replication can be synchronized by a temperature shift-down that caused renaturation of the previously denatured DnaAts protein. Moreover, we show that elimination of the activating DnaA function affects transcriptional activation at ori lambda. DnaA may act by binding to DnaA-boxes, situated around the lambda pR promoter; there are no such sequences in ori lambda. Our results being to explain in molecular terms why lambda plasmid replication is DnaA-dependent [Kur et al., J. Mol. Biol. 198 (1987) 203-210] and why the initiation of phage lambda DNA replication is blocked (in E. coli devoid of prophage Rac) after inactivation of DnaA [Wegrzyn et al., Genetics (1995) in press].

Bacterial Proteins↗
PubMed1995

Protection of coliphage lambda O initiator protein from proteolysis in the assembly of the replication complex in vivo.

We have shown previously that, in contrast to the free coliphage lambda O initiator protein rapidly degraded by ClpP/ClpX protease, the lambda present in the replication complex (RC) is protected from proteolysis. Now we asked at which step of the pathway of RC assembly in vivo does the stabilization of lambda O occur. In accordance with the in vitro established order we found that lambda P and DnaB helicase functions are, but those of DnaJ and GrpE chaperones are not, required for the protection of lambda O from proteolysis. Therefore, our results suggest that the first lambda O protecting structure of the pathway of RC assembly is the lambda O-lambda P-DnaB preprimosome. The next step of the pathway, the chaperone-mediated rearrangement of the preprimosome, is not essential for lambda O stabilization. However, in contrast to other chaperones, the DnaK function was required for the protection of lambda O from proteolysis, suggesting an earlier access of DnaK to the pathway of RC assembly in vivo, in accordance with current models by which molecular chaperones facilitate protein assembly.

Bacterial Proteins↗
PubMed1995

Effect of increased ppGpp concentration on DNA replication of different replicons in Escherichia coli.

The plasmids harbouring the relA gene under an inducible promoter allowed us to increase the guanosine 5'-diphosphate-3'-diphosphate (ppGpp) concentration in Escherichia coli cells without any starvation and thus, to directly investigate the effect of ppGpp on DNA replication. We studied all types of replicons which were investigated previously in amino acid-starved bacteria and found that ColE1, oriC, lambda plasmid and pSC101 but not RK2 replicons are sensitive to high ppGpp level. To our knowledge, this paper presents the first direct evidence that replication of most, but not all, replicons is dependent on ppGpp concentration and thus, is under stringent control.

DNA Replication↗
PubMed1995

Involvement of the host initiator function dnaA in the replication of coliphage lambda.

We demonstrate that the initiation of coliphage lambda DNA replication is dependent on the host initiator function dnaA, provided that the lambdoid prophage Rac is absent. Presence of Rac compensated the absence of dnaA function, causing initiation of replication. In dnaAts rac+ cells at 43 degrees, most of parental phage DNA molecules, after one round of theta replication, switched to a replication with features of the sigma mode and produced progeny at high yield. Initiation of replication of the lambda Pts1 mutant at 43 degrees was blocked by dnaA function; however, under dnaA-rac+ conditions all parental phage DNA molecules, after one round of theta replication, switched to the sigma mode and produced progeny at high yield. Taking into account our recent finding that transcriptional activation of ori lambda seems to be dnaA-regulated (to be published elsewhere), we suggest that the DnaA-lambda Pts1 incompatibility occurs at the insertion of the ori lambda-bound lambda O-lambda P-DnaB preprimosome between the complementary lambda DNA strands. The role of Rac and the mechanism of the switch from theta to sigma mode of lambda phage DNA replication are discussed.

Bacterial Proteins↗
PubMed1995

Replication of coliphage lambda DNA.

A general scheme of lambda phage and plasmid DNA replication in Escherichia coli is presented, and results of in vivo experiments from the authors' laboratory are superimposed. The initiator lambda O functions in the assembly of the replication complex (RC) at ori lambda, making it a stable component of this structure. ClpP/ClpX protease-specific action on lambda O does not affect the regulation of replication; it only degrades the surplus of synthesized lambda O. The initiator lambda O becomes protected from proteolysis at a distinct step of the pathway of RC assembly. The host DnaA initiator-regulated transcriptional activation of ori lambda seems to be coupled with RC assembly at the step of chaperone-mediated rearrangement of the pre-primosome. The once-assembled RC is inherited by one of two lambda plasmid daughter copies at each round of circle-to-circle (theta) replication. The inherited, old RC-driven replication is also dependent on RNA polymerase and DnaA functions. It seems that DnaA licenses lambda plasmid DNA for only one replication round, resembling the putative eukaryotic licensing factor in this respect. The lambda O binding to ori lambda does not seem to play any role in regulation of lambda plasmid replication, and the Cro-autoregulatory loop may be deleted. The emerging picture shows lambda plasmid circles with RCs bound to their ori, awaiting a signal triggering initiation of replication. The host DnaA initiator-regulated transcriptional activation of ori lambda may be involved in signal transmission. Inactivation of DnaA function blocks initiation of lambda phage DNA replication, but the lambdoid prophage Rac compensates this defect and all parental phage DNA molecules, after one round of theta replication switch to the sigma mode and produce progeny in high yield. We suspect that DnaA-regulated transcriptional activation is involved in installation and adequate positioning of two RCs, required for bidirectional replication, but in the Rac-promoted process only one RC may be installed, leading to unidirectional replication continued in the sigma mode. In wild-type cells consumption of DnaA function by the rapidly replicating lambda phage DNA may switch replication from bidirectional theta to unidirectional theta, and later to the sigma mode; the lambda circles produced earlier may play the role of Rac, which is required only when DnaA function has been inactivated prior to phage infection.

Bacteriophage lambda↗
PubMed1995

Inhibition of transcription starting from bacteriophage lambda pR promoter during the stringent response in Escherichia coli: implications for lambda DNA replication.

Replication of lambda plasmid DNA is halted in amino acid-starved wild type (stringent) strains whereas it proceeds in relA (relaxed) mutants. The only transcription which could be important in lambda plasmid DNA replication in amino acid-starved Escherichia coli cells is that starting from the pR promoter. Using a fusion which consists of the lacZ gene under the control of bacteriophage lambda pR promoter we found that transcription starting from this promoter was inhibited during the stringent, but not the relaxed, response in E. coli. We confirmed our conclusion by estimating the relative level of the pR transcript by RNA-DNA hybridization. We propose that decreased transcription from the pR promoter which serves as transcriptional activation of ori lambda is responsible for inhibition of lambda plasmid replication during the stringent response. The results presented in this paper, combined with our recent findings (published elsewhere), indicate that the transcriptional activation of ori lambda may be a main regulatory process controlling lambda DNA replication not only during the relaxed response but also in normal growth conditions.

Bacteriophage lambda↗
PubMed1994

The mechanism of the stringent control of lambda plasmid DNA replication.

Lambda plasmid DNA replication is inhibited in amino acid-starved wild type Escherichia coli strains (stringent response) but not in amino acid-starved relA mutants (relaxed response). This replication is perpetuated by the replication complex containing the lambda O protein (which is protected from proteases by other elements of the complex) and inherited by one of two daughter copies after a replication round. Since a fraction of stable lambda O protein was observed in relA- and relA+ strains, and negative regulation by the lambda Cro repressor does not seem to be important in the stringent or relaxed response of lambda plasmid replication to amino acid starvation, the inhibition of lambda plasmid replication in amino acid-starved wild type strains was investigated. lambda plasmids were unable to replicate in amino acid-starved relA- bacteria treated with rifampicin. Moreover, transcription from pR, which produces mRNA for replication protein synthesis and serves as transcriptional activation of ori lambda, was significantly decreased during the stringent response as well as in non-starved cells containing increased levels of ppGpp. However, it was little or totally not affected by the relaxed response. The replacement of pR with plac (which is known to be uninhibited by ppGpp) in a lambda plasmid resulted in its DNA replication during relaxed and stringent responses as well as during overproduction of ppGpp in unstarved bacteria. We conclude that ppGpp-mediated inhibition of transcriptional activation of ori lambda is responsible for inhibition of lambda plasmid DNA replication in amino acid-starved wild type strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage lambda↗
PubMed1994

An additional role of transcriptional activation of ori lambda in the regulation of lambda plasmid replication in Escherichia coli.

Initiation of replication of plasmids derived from coliphage lambda in vivo is dependent on transcription at or near the replication origin, ori lambda. However, this transcriptional activation is dispensable for lambda plasmid DNA replication reconstituted in vitro from purified lambda and Escherichia coli proteins. It was proposed previously that histone-like protein HU interferes with the assembly or function of the pre-primosomal complex, and transcription at or near ori lambda abolishes HU-mediated inhibition of lambda DNA replication. We found that during lambda plasmid replication driven by the previously assembled replication complex (in amino acid-starved relA mutants), when the inhibition by HU protein should not be observed, the synthesis of lambda DNA was still dependent on transcriptional activation. Moreover, in hupA hupB double mutants the transcription is necessary for the initiation of lambda plasmid DNA replication perpetuated by the replication complex inherited by one of two daughter copies after a replication round. We conclude that transcriptional activation of ori lambda has an additional role in the initiation of lambda plasmid DNA replication beside the abolition of HU-mediated inhibition.

Bacterial Proteins↗
PubMed1994

Regulation of replication of plasmid pBR322 in amino acid-starved Escherichia coli strains.

The stringent response causes inhibition of replication of plasmid pBR322 in amino acid-starved Escherichia coli cells whereas in relaxed mutants the replication of this plasmid proceeds for several hours. On the basis of density shift experiments and pulse-labelling experiments we showed that most of the pBR322 molecules begin replication during the relaxed response and the rate of plasmid DNA synthesis in unstarved and isoleucine-starved relA- bacteria is similar. We found that the Rom function plays a key role in the stringent control of plasmid pBR322 replication, as insertional inactivation of the rom gene causes amplification of pBR322rom- in both relA- and relA+ strains during amino acid starvation. Moreover, pUC19, which is a pBR322-derived plasmid lacking the rom gene, behaves like pBR322rom-, whereas introduction of the rom gene into the pUC19 replicon drives it into the pBR322 mode of replication in amino acid-starved bacteria. A model for the regulation of pBR322 plasmid DNA replication by Rom protein in amino acid-starved Escherichia coli strains is proposed.

Adaptation, Physiological↗
PubMed1994

Differential replication of plasmids during stringent and relaxed response of Escherichia coli.

Stringent control of DNA replication has been demonstrated for a few replicons like oriC, pBR322, and plasmids derived from coliphage lambda. In this study we investigated the replication of other plasmids harboring a well defined origin (orip15A, oripSC101, and oriRK2 = oriV) in amino-acid-starved stringent and relaxed strains of Escherichia coli. We found differential replication of plasmids during stringent and relaxed response. Inhibition of DNA synthesis or amplification of plasmid DNA in amino acid-starved relA+ and relA- cells depends on the kind of replicon and, surprisingly, on the nature of deprived amino acid. We conclude that there are no general rules for stringent control of DNA replication and each replicon must be considered separately. There are, however, possible explanations for the differences shown between replicons in their response to stringent and relaxed conditions.

Amino Acids↗
PubMed1994

Neither absence nor excess of lambda O initiator-digesting ClpXP protease affects lambda plasmid or phage replication in Escherichia coli.

Owing to rapid proteolysis of the coliphage lambda-coded initiator protein, lambda O, this protein is considered to carry a rate-limiting step in lambda DNA replication. The discovery of ClpXP protease responsible for lambda O protein turnover allowed an opportunity to verify this hypothesis. However, neither absence nor excess of this protease significantly affected the transformation efficiency and copy number of lambda plasmid, or the kinetics of the lambda phage growth. These results are also incompatible with the hypothesis that the stabilization of lambda O plays a role in the switch from early (circle-to-circle) to late (rolling-circle) lambda phage DNA replication. Transcriptional activation of ori lambda, probably assisted by the Escherichia coli DnaA function, remains as the possible rate-limiting step in lambda DNA replication.

ATP-Dependent Proteases↗