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G Wegrzyn

Publications and source records attributed to G Wegrzyn.

At least 19 recordsLinked to original sources
PubMed1999

The presence of two DnaA-binding sequences is required for an efficient interaction of the Escherichia coli DnaA protein with each particular weak DnaA box region.

Using an electron microscopic method for visualizing interactions of the Escherichia coli DnaA protein with weak DnaA-binding DNA sequences, we found that DnaA binds effectively to two separated weak DnaA box regions located on the same DNA fragment. As expected, no DnaA-DNA interactions were detected when both DnaA box regions were mutagenized to the sequence totally incapable of binding DnaA. However, when only one of these two regions was mutagenized, the lack of interactions between DnaA and DNA was observed not only at the scrambled DnaA box but also at the second weak DnaA box region. These results indicate that for the efficient binding of DnaA to a weak DnaA box region, the presence of at least two such DNA sequences is necessary. Our finding also suggests that binding of DnaA protein to weak DnaA box sequences may be cooperative. In addition, we found that DnaA-mediated transcription termination in vivo requires two DnaA boxes, one of them is a weak one. It seems, therefore, that some mechanisms of regulation of transcription and DNA replication by DnaA, that involve interactions of DnaA with weak DnaA boxes, may be more complicated than initially proposed.

Bacterial Proteins↗
PubMed1999

Replication of plasmids during bacterial response to amino acid starvation.

Amino acid starvation of bacterial cells leads to expression of the stringent (in wild-type strains) or relaxed (in relA mutants) response (also called the stringent or relaxed control, respectively). The stringent control is a pleiotropic response which changes drastically almost the entire cell physiology. Although starvation is a rule rather than an exception in natural environments of bacteria, and DNA replication is a fundamental cell process, until recently our knowledge about regulation of DNA replication in amino acid-starved cells has been unexpectedly poor. Within recent years the stringent control of DNA replication has been investigated mainly on plasmid models. Several plasmid replicons have been studied, including oriC plasmids, ColE1-like replicons, pSC101, F, R1, RK2, and R6K, and plasmids derived from bacteriophages lambda and P1. However, molecular models of replication regulation in amino acid-starved cells have been proposed to date only for lambda plasmids and ColE1-like replicons. Although further extensive studies are necessary in the understanding of molecular mechanisms of the stringent and relaxed control of replication of other plasmids, the results obtained to date (summarized and discussed in this review) show that studies on DNA replication in amino acid-starved cells may provide new insights into the regulatory mechanisms and lead to more general conclusions.

Amino Acids↗
PubMed1998

Differential inhibition of transcription from sigma70- and sigma32-dependent promoters by rifampicin.

Rifampicin is an antibiotic which binds to the beta subunit of prokaryotic RNA polymerases and prevents initiation of transcription. It was found previously that production of heat shock proteins in Escherichia coli cells after a shift from 30 degrees C to 43 degrees C is not completely inhibited by this antibiotic. Here we demonstrate that while activity of a pL-lacZ fusion (pL is a sigma70-dependent promoter) in E. coli cells is strongly inhibited by rifampicin, a p(groE)-lacZ fusion, whose activity is dependent on the sigam32 factor, retains significant residual activity even at relatively high rifampicin concentrations. Differential sensitivity to this antibiotic of RNA polymerase holoenzymes containing either the sigma70 or the sigma32 subunit was confirmed in vitro. Since the effects of an antibiotic that binds to the beta subunit can be modulated by the presence of either the sigma70 or the sigma32 subunit in the holoenzyme, it is tempting to speculate that binding of various sigma factors to the core of RNA polymerase results in different conformations of particular holoenzymes, including changes in the core enzyme.

Bacterial Proteins↗
PubMed1998

Escherichia coli dnaA gene function and bacteriophage lambda replication.

Allele specificity of the Escherichia coli dnaA gene function in the replication of plasmids derived from bacteriophage lambda has been demonstrated previously. Here, using a series of dnaA temperature-sensitive mutants, we investigated dnaA allele specificity of the replication of phages lambda P+ and lambda Pts 1 pi A66. We found that phage lambda P+ produces its progeny efficiently at 43 degrees C irrespective of the dnaA allele, whereas lambda Pts 1 pi A66, which is unable to develop lytically in the dnaA+ host at this temperature, can replicate with different efficiency in certain dnaA mutants. Since the main role of DnaA in lambda development seems to be stimulation of transcription from the pR promoter, we measured the activity of this promoter (using a pR-lacZ fusion) and the abundance of pR-derived transcripts (by Northern blotting analysis) in dnaA+ host and dnaA(ts) mutants at 30 and 43 degrees C. We found significant differences in the activity of pR in various dnaA(ts) mutants at 30 degrees C, which indicate different levels of stimulation of this promoter by products of particular dnaA alleles at permissive temperature. Differential levels of DnaA-mediated stimulation of pR in various dnaA(ts) mutants were also found at 43 degrees C. Stimulation of the pR promoter by DnaA is necessary for both efficient production of the lambda replication proteins, O and P, and effective transcriptional activation of ori lambda. The differences in the efficiency of pR activation observed in dnaA mutants at 30 and 43 degrees C can explain the mechanisms of allele specificity of dnaA gene function in the replication of bacteriophage lambda and plasmids derived from this phage.

Alleles↗
PubMed1998

DnaA-mediated regulation of phage lambda-derived replicons in the absence of pR and Cro function.

Bacteriophage lambda-derived replicons can replicate in Escherichia coli cells as plasmids. In the control of replication of these plasmids, an important role was ascribed to the lambda Cro repressor autoregulatory loop. However, the oR/pR-cro-tR-cII' region could be replaced by the ptetA promoter under the control of the TetR repressor, producing plasmid pTClambda. Here, we demonstrate that stable maintenance of pTClambda depends on the host DnaA function because deletion of one of DnaA-binding sequences present in pTClambda resulted in a decrease in the plasmid (pTClambda) copy number and poor maintenance of pTClambda in E. coli. Moreover, in contrast to the replication of the wild-type lambda plasmid, previously found to be positively regulated by DnaA (acting on a relaxed DnaA box situated immediately downstream of the pR promoter), the replication of pTC plasmids (devoid of pR) was found to be negatively regulated by DnaA. Contrary to wild-type lambda plasmids, in cells harboring lambda cro[temperature-sensitive (ts)] or pTClambda (but not pTClambda) plasmid, the lambda replication complex was heat shock resistant; this complex, however, disassembled after inactivation of DnaA function. This disassembly was blocked by DNA gyrase inhibitors. According to our model outlined previously, we propose that the heat shock resistance of the replication complex of lambdacro- plasmids depends on the interaction of the DNA-bound DnaA protein with the DNA-bound lambda replication complex. The replication complex-DnaA-lambda DNA structure may be directly related to the role of DnaA as the Cro-replacing negative regulator of lambdacro- plasmid replication.

Bacterial Proteins↗
PubMed1998

Rapid degradation of polyadenylated oop RNA.

The oop RNA is a short (77 nucleotides (nt)) transcript encoded by bacteriophage lambda which acts as an antisense RNA for lambda cII gene expression. Recently we demonstrated that oop RNA is specifically polyadenylated at its 3' end by poly(A) polymerase I (PAP I), the pcnB gene product. Here we demonstrate that the half life of oop RNA is 3 times longer in the pcnB mutant relative to the pcnB+ host, indicating that polyadenylation of this transcript causes its accelerated degradation. Although it was proposed that polyadenylation of RNAs in bacteria leads to their enhanced degradation, in most cases stabilization of these molecules was observed only when other mutations (pnp, rnb and rne) were present in the pcnB- strain. Therefore it seems that oop RNA may serve as a very useful model in further studies on molecular mechanisms of RNA polyadenylation and degradation in bacteria. Analysis of oop RNA and its degradation product isolated from Escherichia coli cells suggests that both polyadenylated and non-modified oop transcripts can act as antisense RNA.

Bacterial Proteins↗
PubMed1998

Replication of lambda plasmid DNA in the Escherichia coli cell cycle.

The Cro repressor autoregulatory loop has long been considered the main regulatory process in controlling lambda plasmid replication initiation in Escherichia coli. However, we found recently that lambda plasmids can be maintained at a constant copy number in the absence of Cro function. Here we demonstrate that shortly after inactivation of the Cro repressor, the synthesis of lambda plasmid DNA increases significantly but is then stabilized at a level similar to that observed in the presence of the Cro function. We found that replication initiation of lambda plasmids carrying a functional cro gene proceeds randomly in the host cell cycle, but in the absence of Cro function the replication initiation of lambda plasmid DNA appears to be cell cycle dependent. The host DnaA protein appears to be at least one of the factors involved in the cell-cycle-specific control of lambda cro- plasmid replication. Therefore, it seems that the lambda cro- plasmid may serve as an amazingly simple model for studies on the regulation of DNA replication in the cell cycle.

Bacterial Proteins↗
PubMed1998

Random inheritance of the replication complex by one of two daughter lambda plasmid copies after a replication round in Escherichia coli.

There are two pathways for replication of plasmids derived from bacteriophage lambda (so-called lambda plasmids) in Escherichia coli. One pathway is based on the assembly of the new replication complex at ori lambda, and the second requires activity of the replication complex inherited by one of two daughter plasmid copies after each replication round. Although these two replication pathways proceed at the same time in the host cell, we previously found conditions for specific elimination of the pathway based on the assembly of the new replication complex; thus, replication is restricted to that carried out by the heritable replication complex. These conditions are (i) the relaxed response to amino acid starvation and (ii) temperature upshift of the culture of cells harboring the lambda crotsPts1 plasmid. Here we asked whether the replication complex is inherited randomly by one of two daughter plasmid copies or whether the inheritance is preferred by one particular copy, that containing the parental DNA r strand or that bearing the l strand. We performed density shift experiments which allowed us to separate plasmid DNA molecules replicated by the heritable replication complex from those devoid of the replication complex and therefore not able to replicate. Then, [3H]thymidine-labelled plasmid DNA strands were separated and hybridized to membrane-bound ssDNA containing a fragment of either the r or l strand of lambda DNA. We found roughly equal efficiency of hybridization to both r and l strands in all experimental systems used. Therefore, we conclude that the lambda replication complex is randomly inherited by one of two daughter plasmid copies rather than preferentially inherited by either the copy carrying the parental r strand or that containing the l strand.

Bacteriophage lambda↗
PubMed1998

Polyadenylation of oop RNA in the regulation of bacteriophage lambda development.

We have shown that Escherichia coli pcnB mutants are lysogenized by bacteriophage lambda with lower efficiency as compared to the pcnB+ strains. Our genetic analysis revealed that expression of the lambda cII gene is decreased in the pcnB mutants. However, using various lacZ fusions we demonstrated that neither activities of pL and pR promoters nor transcription termination at tR1 were significantly impaired in the pcnB- host. On the other hand, we found that oop RNA, an antisense RNA for cII expression, is involved in this regulation. Primer protection experiments revealed that oop RNA was polyadenylated and that this polyadenylation was impaired in the pcnB mutant. We found that the oop RNA was more abundant in the pcnB mutant than in the pcnB+ strain. Furthermore, we showed that activity of the pO promoter was not stimulated in the pcnB mutant. Such findings indicated that degradation of oop RNA in the pcnB strain was slower because of inefficient polyadenylation, which could lead to more effective inhibition of cII expression by the antisense oop RNA, resulting in less efficient lysogenization of the host. The oop RNA was found previously to play a role in phage lambda development only under conditions of overproduction of this transcript. Here we demonstrate for the first time, the physiological function of oop RNA in lambda development, confirming that this short transcript plays an important role in the negative regulation of cII gene expression during lambda infection. Moreover, polyadenylation of oop RNA is one of very few known examples of specific RNA polyadenylation by PAP I in prokaryotic cells and its role in gene expression regulation.

Bacterial Proteins↗
PubMed1998

DnaA-stimulated transcriptional activation of orilambda: Escherichia coli RNA polymerase beta subunit as a transcriptional activator contact site.

We present evidence that Escherichia coli RNA polymerase beta subunit may be a transcriptional activator contact site. Stimulation of the activity of the pR promoter by DnaA protein is necessary for replication of plasmids derived from bacteriophage lambda. We found that DnaA activates the pR promoter in vitro. Particular mutations in the rpoB gene were able to suppress negative effects that certain dnaA mutations had on the replication of lambda plasmids; this suppression was allele-specific. When a potential DnaA-binding sequence located several base pairs downstream of the pR promoter was scrambled by in vitro mutagenesis, the pR promoter was no longer activated by DnaA both in vivo and in vitro. Therefore, we conclude that DnaA may contact the beta subunit of RNA polymerase during activation of the pR promoter. A new classification of prokaryotic transcriptional activators is proposed.

Bacterial Proteins↗
PubMed1998

Replication regulation of ColE1-like plasmids in amino acid-starved Escherichia coli.

Differential replication of various ColE1-type plasmids in stringent (relA+) and relaxed (relA-) strains of Escherichia coli starved for particular amino acids was reported previously. A role for the plasmid-encoded Rom protein in the stringent control of ColE1 replication has also been demonstrated. Here we have studied the efficiency of replication of five ColE1-type plasmids in E. coli relA+ and relA- strains starved for five amino acids to find the differential replication of each plasmid in cells starved for each amino acid. The efficiency of replication was found to be in positive correlation with the homology between nucleotide sequences of particular loops of RNA I or RNA II and anticodon loops of tRNA molecules corresponding to the kind of the amino acid deprived. Efficient plasmid DNA replication was observed under conditions for which we predicted (on the basis of theoretical calculations) relatively strong interactions between tRNA molecules, expected to occur in high concentrations in an uncharged from, and RNA I or RNA II. When the theoretical possibility of the tRNA-RNA I or tRNA-RNA II interactions was very small, the observed plasmid DNA replication was negligible. Replication of ColE1-like plasmids during the stringent response was observed only in the absence of a functional rom gene. We observed plasmid replication in the amino acid-starved pcnB relA double mutant. We propose a model for regulation of ColE1 replication in the amino acid-starved E. coli cells based on interactions between uncharged tRNA molecules and RNA I or RNA II. During starvation for different amino acids, different kinds of uncharged tRNA molecules appear in cells (they are much more abundant, however, in relA- mutants than in relA+ hosts) leading to various efficiencies of replication initiation. The Rom protein may modulate the effect of tRNA(s) by enhancing RNAI-RNA II, but not tRNA-RNA I and tRNA-RNA II, interactions.

Amino Acids↗
PubMed1998

Replication and maintenance of lambda plasmids devoid of the Cro repressor autoregulatory loop in Escherichia coli.

Plasmids derived from bacteriophage lambda are known as lambda plasmids. These plasmids contain the ori lambda region and lambda replication genes O and P. Typical lambda plasmids also contain the cro gene, the product of which is a repressor of the pR promoter when present at relatively high concentrations. These genes stably maintain the plasmid in Escherichia coli at copy numbers of 20 to 50 per cell. According to a generally accepted model, stable maintenance of lambda plasmids is possible due to the Cro repressor autoregulatory loop (the cro gene is under control of pR). Here we demonstrate that lambda plasmids devoid of the Cro autoregulatory loop can also be stably maintained in E. coli strains. We present data for two such plasmids: pTC lambda 1 in which the pR-cro region has been replaced by the ptetA promoter and the tetR gene (coding for the TetR repressor), and a standard lambda plasmid with inactivated cro gene (lambda cro-null plasmid). Thus, the presence of the Cro repressor autoregulatory loop does not appear to be essential to the maintenance of lambda plasmids in vivo.

Bacteriophage lambda↗
PubMed1998

Guanosine tetraphosphate (ppGpp)-mediated inhibition of the activity of the bacteriophage lambda pR promoter in Escherichia coli.

It was previously demonstrated that the activity of bacteriophage lambda promoter pR is decreased in wild-type Escherichia coli cells starved for amino acids (during the stringent response). Since pR activity is necessary for the transcriptional activation of ori lambda, this leads to inhibition of the replication of plasmids derived from phage lambda. These results led to the proposal that the pR promoter susceptible to control by the stringent response. However, subsequent studies demonstrated that this promoter is activated by the host dnaA gene product and since the dnaA promoter was reported to be controlled by the stringent response, it is possible that the inhibition of pR activity in amino acid-starved cells is indirect, and results from the impairment of DnaA-mediated transcriptional activation. Here we present evidence that pR is negatively regulated by ppGpp, even when DnaA protein is provided in excess as well as in cells devoid of DnaA function. We have checked that the level of ppGpp is increased during prolonged (up to 4 h) starvation for isoleucine in relA+ cells but not in the relA- mutant. At the same time we observed inhibition of lambda plasmid replication during the stringent, but not relaxed, response, even when DnaA was overproduced. Finally, we found that the activity of a pR-lacZ fusion is inhibited after gratuitously induced overproduction of ppGpp in unstarved cells, irrespective of the status of the dnaA gene product. We conclude that the activity of the pR promoter is inhibited directly by ppGpp.

Bacterial Proteins↗
PubMed1998

Regulation of replication of lambda phage and lambda plasmid DNAs at low temperature.

It was previously demonstrated that while lysogenic development of bacteriophage lambda in Escherichia coli proceeds normally at low temperature (20-25 degrees C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the pE promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage lambda cIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37 degrees C, while no phage progeny are observed at 20 degrees C. Contrary to previous reports, it is possible to demonstrate that pE promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20 degrees C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage lambda is neither inhibited at 20 degrees C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between lambda phage and lambda plasmid DNA at low temperature.

Bacteriophage lambda↗
PubMed1998

Interaction of the Escherichia coli DnaA protein with bacteriophage lambda DNA.

Interaction of the Escherichia coli DnaA (replication initiator) protein with restriction fragments of phage lambda DNA demonstrated differential binding of DnaA along the whole lambda DNA. Interaction of DnaA with the lambda replication region (from the promoter pR to the origin of replication, orilambda) demonstrated a strong binding of DnaA to the region around the p(o) 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 lambda 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.

Bacterial Proteins↗
PubMed1998

Molecular mechanism of heat shock-provoked disassembly of the coliphage lambda replication complex.

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 lambdaO present in the replication complex (RC) is protected from proteolysis. However, in cells growing in a complete medium, a temperature shift from 30 to 43 degrees C resulted in the decay of the lambdaO fraction, which indicated disassembly of RC. This process occurred due to heat shock induction of the groE operon, coding for molecular chaperones of the Hsp60 system. Here we demonstrate that an increase in the cellular concentration of GroEL and GroES proteins is not in itself sufficient to cause RC disassembly. Another requirement is a DNA gyrase-mediated negative resupercoiling of lambda plasmid DNA, which counteracts DNA relaxation and starts to dominate 10 min after the temperature upshift. We presume that RC dissociates from lambda DNA during the negative resupercoiling, becoming susceptible to the subsequent action of GroELS and ClpP/ClpX proteins. In contrast to lambda cro+, in lambda cro- plasmid-harboring cells, the RC reveals heat shock resistance. After temperature upshift of the lambda crots plasmid-harboring cells, a Cro repressor-independent control of lambda DNA replication and heat shock resistance of RC are established before the period of DNA gyrase-mediated negative supercoiling. We suggest that the tight binding of RC to lambda DNA is due to interaction of RC with other DNA-bound proteins, and is related to the molecular basis of the lambda cro- plasmid replication control.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1998

A method for isolation of plasmid DNA replication intermediates from unsynchronized bacterial cultures for electron microscopy analysis.

Electron microscopy is a powerful technique for analysis of DNA replication intermediates. However, isolation of replicating DNA molecules from living cells is tricky and difficult, especially in the case of small DNA molecules (such as bacterial plasmids) whose initiation of replication is not easily synchronized. Here a relatively simple and rapid method for efficient isolation of replicating plasmid molecules from unsynchronized Escherichia coli cultures is described. The efficiency of this procedure is high enough for electron microscopy analysis of plasmid replication intermediates appearing in living cells in normal growth conditions. Under optimal conditions, using standard procedures of isolation of plasmid DNA, it is possible to achieve a content of only as few as 0.02 percent of replication intermediates in a plasmid DNA sample. The described method allowed us to enrich up to 100-fold the fraction of replication intermediates suitable for microscopic analysis among all plasmid molecules.

Cell Cycle↗