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A Wegrzyn

Publications and source records attributed to A Wegrzyn.

35 records · Page 2Linked to original sources
PubMed1997

An RNA polymerase alpha subunit mutant impairs N-dependent transcriptional antitermination in Escherichia coli.

We show that the rpoA341 mutation in the gene encoding the alpha subunit of Escherichia coli RNA polymerase results in a decreased level of transcripts originating from the lytic promoters PL and PR of infecting lambda phage. However, using lacZ fusions we demonstrate that initiation of transcription from both PL and PR is not impaired in the rpoA341 host. Rather, it is the level of the longer, antiterminated PL- and PR-derived transcripts which is altered: the activity of beta-galactosidase in bacteria harbouring a source of N and a PL-nutL-tL1-tI-lacZ or PR-nutR-tR1-lacZ fusion is considerably lower in the rpoA341 mutant relative to the rpoA+ strain. In the absence of the antiterminator protein N no difference is observed in the level of longer PR-derived transcripts between wild-type (rpoA+) and mutant (rpoA341) hosts. Although synthesis of N appears to be similar in both phage-infected rpoA+ and rpoA341 cells, overexpression of the N gene leads to restoration of wild-type levels of the longer PL- and PR-derived transcripts in the mutant host. While this mutation does not appear to affect vegetative phage growth in nus+ backgrounds, in combination with certain nus mutations it retards lytic development. Therefore, we conclude that the rpoA341 mutation specifically interferes with the function of the N-antitermination complex, suggesting that the C-terminal domain of the RNA polymerase alpha subunit may play an important role in N-dependent transcriptional antitermination.

Bacterial Proteins↗
PubMed1996

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 the Escherichia coli dnaA gene, which cause temperature sensitivity of initiation of chromosomal replication, on the replication of lambda phage-derived plasmids at 30 degrees C. These mutants showed no allele specificity of dnaA function in replication of either of two lambda pi plasmids studied. On the other hand, the inability of the lambda P+ plasmid to replicate in dnaA508, 46 and 204 cells, in dnaB (groP A15) or in cells that are temperature sensitive for the chaperone genes dnaK756, dnaJ259 and grpE280 at 30 degrees C was suppressible by a single pi mutatation. This suggests that it is a common property of the pi protein, probably its weaker interaction with DnaB helicase, that is responsible for the suppression. One can also conclude that the DnaA-regulated transcriptional activation of ori lambda 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.

Bacterial Proteins↗
PubMed1996

Drastically decreased transcription from CII-activated promoters is responsible for impaired lysogenization of the Escherichia coli rpoA341 mutant by bacteriophage lambda.

It was demonstrated previously that a mutation, rpoA341, in the gene encoding the alpha subunit of Escherichia coli RNA polymerase prevents lysogenization by bacteriophage lambda. The rpoA341 allele is known to be responsible for impaired transcription of some positively regulated E. coli chromosomal operons. Here we demonstrate that the inhibition of lysogenization of the rpoA341 mutant is a result of drastically decreased transcription from positively regulated phage promoters. We were unable to detect any transcripts originating from the CII-activated pE, pI and paQ promoters (important for lysogenic development) in the phage-infected rpoA341 mutant, in contrast to an otherwise isogenic rpoA+ strain. The results are discussed in the light of other reports showing that activation of the pE promoter by CII protein in vitro is decreased only about fivefold when the native alpha subunit is replaced by truncated alpha polypeptides.

Bacteriolysis↗
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↗
PubMed1996

Protein inheritance: lambda plasmid replication perpetuated by the heritable replication complex.

BACKGROUND: Replication of a plasmid derived from the Escherichia coli phage lambda initiates by binding of the lambda O protein initiator to the origin of lambda DNA replication, ori lambda. The lambda P protein participates in subsequent steps of assembly of the lambda replication complex. A function of lambda P required for replication complex assembly is inactivated at 43 degrees C by the ts1 mutation. RESULTS: We found that the lambda replication complex assembled at 30 degrees C survives the temperature upshift in lambda crotsPts1 plasmid-harbouring bacteria. We present several lines of evidence that in this system (in which the replication complex assembly does not occur), the replication complex assembled prior to the temperature upshift is inherited by one of two daughter plasmid copies at each replication round for more than 30 cell generations. The 'old' replication complex-driven replication is chloramphenicol-resistant and rifampicin-sensitive. This replication is dependent on lambda O and host dnaK, dnaJ and grpE chaperone gene functions. CONCLUSIONS: The lambda O-containing replication complex is inherited together with DNA and bears information how to initiate the next round of replication at ori lambda; thus, we consider that this phenomenon deserves to be called protein inheritance.

Bacterial Proteins↗
PubMed1996

The cbpA chaperone gene function compensates for dnaJ in lambda plasmid replication during amino acid starvation of Escherichia coli.

We found previously that lambda plasmid DNA replication in amino acid-starved Escherichia coli relA mutants (i.e., during the relaxed response), which is carried out by the inherited replication complex, is dependent on functions of DnaK and GrpE molecular chaperones but proceeds in a dnaj mutant at a nonpermissive temperature. Here we demonstrate that this replication is inhibited when functions of both dnaJ and cbpA are impaired. In complete media, the growth of the lambda pi A66 phage (capable of replicating in E. coli dnaJ, dnaK, and grpE missense mutants at 30 degrees C), as well as efficiency of transformation by the lambda pi A66 plasmid, is significantly decreased in a dnaJ259 cbpA::kan double mutant. These results strengthen the proposal of other authors (C. Ueguchi, M. Kakeda, H. Yamada, and T. Mizuno, Proc. Natl. Acad. Sci. USA 91:1054-1058, 1994; C. Ueguchi, T. Shiozawa, M. Kakeda, H. Yamada, and T. Mizuno, J. Bacteriol. 177:3894-3896, 1995; and T. Yamashino, M. Kakeda, C. Ueguchi, and T. Mizuno, Mol. Microbiol. 13:475-483, 1994) that the cbpA gene product is a functional analog of the DnaJ chaperone in E. coli.

Amino Acids↗
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

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↗
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

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↗
PubMedDate not supplied

ClpP/ClpX-mediated degradation of the bacteriophage lambda O protein and regulation of lambda phage and lambda plasmid replication.

The O protein is a replication initiator that binds to the orilambda region and promotes assembly of the bacteriophage lambda replication complex. This protein, although protected from proteases by other elements of the replication complex, in a free form is rapidly degraded in the host, Escherichia coli, by the ClpP/ClpX protease. Nevertheless, the physiological role of this rapid degradation remains unclear. Here we demonstrate that the copy number of plasmids derived from bacteriophage lambda is significantly higher in wild-type cells growing in rich media than in slowly growing bacteria. However, lambda plasmid copy number in bacteria devoid of the ClpP/ClpX protease was not dependent on the bacterial growth rate and in all minimal media tested was comparable to that observed in wildtype cells growing in a rich medium. Contrary to lambda plasmid replication, the efficiency of lytic growth of bacteriophage lambda was found to be dependent on the host growth rate in both wild-type bacteria and clpP and clpX mutants. The activities of two major lambda promoters operating during the lytic development, p(R) and p(L), were found to be slightly dependent on the host growth rate. However, when p(R) activity was significantly decreased in the dnaA mutant, production of phage progeny was completely abolished at low growth rates. These results indicate that the O protein (whose level in E. coli cells depends on the activity of ClpP/ClpX protease) is a major limiting factor in the regulation of lambda plasmid replication at low bacterial growth rates. However, this protein seems to be only one of the limiting factors in the bacteriophage lambda lytic development under poor growth conditions of host cells. Therefore, it seems that the role of the rapid ClpP/ClpX-mediated proteolysis of the O protein is to decrease the efficiency of early DNA replication of the phage in slowly growing host cells.

ATPases Associated with Diverse Cellular Activitie↗