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A Wegrzyn

Publications and source records attributed to A Wegrzyn.

At least 19 recordsLinked to original sources
PubMed2000

Bacteriophage and host mutants causing the rolling-circle lambda DNA replication early after infection.

There are two modes of bacteriophage lambda DNA replication during its lytic development in Escherichia coli cells. The circle-to-circle (theta) replication predominates at early stages of the phage growth, whereas rolling-circle (sigma) replication occurs late after infection to produce long concatemers that serve as substrates for packaging of lambda DNA into phage proheads. The mechanism regulating the switch from theta to sigma replication remains unknown. Our previous genetic studies indicated that the bacteriophage lambda Pts1piA66 mutant cannot replicate at 43 degrees C in the wild-type E. coli host, but it can replicate in the dnaA46(ts) mutant. Density shift experiments suggested that the parental DNA molecules of the infecting phage enter sigma replication. Here, using electron microscopy, we demonstrate that as soon as 5 min after infection of the dnaA46(ts) mutant by the lambdaPts1piA66 phage at 43 degrees C, the sigma replication intermediates are highly predominant over theta replication intermediates, contrary to the wild-type conditions (wild-type bacteria infected with the lambdaP(+) phage). The initiation of replication of the lambdaPts1piA66 mutant at 43 degrees C was strongly inhibited in the dnaA(+) host, as demonstrated by electron microscopy and by pulse-labeling of the phage-derived plasmid replicon. Implications for the mechanism of the regulation of the switch from theta to sigma replication mode are discussed.

Bacterial Proteins↗
PubMed2000

Replication of oriJ-based plasmid DNA during the stringent and relaxed responses of Escherichia coli.

The oriJ-based plasmids contain the origin of DNA replication from the cryptic Rac prophage, present in the chromosomes of most Escherichia coli K-12 strains. The organization of the oriJ replication region resembles that of the bacteriophage lambda, although sequence similarity is small. Here we investigated the regulation of replication of the oriJ-based plasmid in E. coli relA(+) and relA(-) hosts during amino acid starvation and limitation, i.e., during the stringent and relaxed responses. We found that, contrary to plasmids derived from phage lambda, replication of the oriJ-based plasmid proceeds efficiently during both stringent and relaxed responses. On the other hand, density shift experiments and measurement of the stability of a putative replication initiator protein (the lambda O protein homologue) suggest that this replication may be carried out by the heritable replication complex, as previously demonstrated for lambda plasmids. We demonstrate that contrary to bacteriophage lambda p(R) promoter, an analogous promoter from the oriJ region is activated rather than inhibited at increased ppGpp levels. We propose that various responses of these promoters (p(R) and p(R-Rac), which are necessary for transcriptional activation of orilambda and perhaps oriJ, respectively) to ppGpp are responsible for differences in the replication regulation between orilambda- and oriJ-based plasmids during the stringent response.

Artificial Gene Fusion↗
PubMed2000

Formation and stability of the bacteriophage lambda replication complexes in UV-irradiated Escherichia coli.

Bacteriophage lambda replication complex, containing the phage-encoded O initiator protein protected from proteases by other elements of this complex, is a stable structure that can be inherited by one of the two daughter lambda DNA copies after a replication round in Escherichia coli. In normal growth conditions in bacteria bearing a plasmid derived from bacteriophage lambda, such a complex may be stable for many cell generations. However, it was found that this stable structure is disassembled under certain conditions, namely, after heat shock. Therefore, we asked whether other environmental stresses may cause disassembly of the lambda replication complex. We found that UV irradiation of the host cells prevented formation of the stable lambda replication complex (though not preventing phage replication), while the same UV doses did not affect the stability of the replication complex assembled prior to the irradiation. These results indicate that the stable lambda replication complex, although sensitive to heat shock, is resistant to some other environmental stresses and that formation of at least two types of lambda replication complexes is possible. Both stable and unstable lambda replication complexes are functional because replication of lambda DNA under conditions preventing formation of the stable complex proceeds efficiently.

Bacteriolysis↗
PubMed1999

The role of DnaK/DnaJ and GroEL/GroES systems in the removal of endogenous proteins aggregated by heat-shock from Escherichia coli cells.

The submission of Escherichia coli cells to heat-shock (45 degrees C, 15 min) caused the intracellular aggregation of endogenous proteins. In the wt cells the aggregates (the S fraction) disappeared 10 min after transfer to 37 degrees C. In contrast, the S fraction in the dnaK and dnaJ mutant strains was stable during approximately one generation time (45 min). This demonstrated that neither the renaturation nor the degradation of the denatured proteins was possible in the absence of DnaK and DnaJ. The groEL44 and groES619 mutations stabilised the aggregates to a lesser extent. It was shown by the use of cloned genes, dnaK/dnaJ or groEL/groES, producing the corresponding proteins in about 4-fold excess, that the appearance of the S fraction in the wt strain resulted from a transiently insufficient supply of the heat-shock proteins. Overproduction of the GroEL/GroES proteins in dnaK756 or dnaJ259 background prevented the aggregation, however, overproduction of the DnaK/DnaJ proteins did not prevent the aggregation in the groEL44 or groES619 mutant cells although it accelerated the disappearance of the aggregates. The properties of the aggregated proteins are discussed from the point of view of their competence to renaturation/degradation by the heat-shock system.

Bacterial Proteins↗
PubMed1999

Altered biological properties of cell membranes in Escherichia coli dnaA and seqA mutants.

We present evidence that biological properties of cell membranes are altered in dnaA and seqA mutants of Escherichia coli relative to wild-type bacteria. We found that bacteriophage lambda forms extremely large plaques on the dnaA seqA double mutants. On the single mutants, dnaA and seqA, the plaques are also bigger than those formed on the wild-type host. However, no significant differences in intracellular phage lambda development were observed between wild-type and mutant hosts, indicating that differences in burst size do not account for the observed differences in plaque size. On the other hand, more efficient release of the phage lytic proteins and/or higher sensitivity of the cell membranes to these proteins may result in more efficient cell lysis. We found that the efficiency of adsorption of bacteriophage lambda to the dnaA seqA mutant cells is decreased at 0 degrees C , but not at 30 degrees C, relative to the wild-type strain. A considerable increase in the permeability of membranes of the mutant cells for beta-galactosidase is demonstrated. The dnaA and seqA mutants are more sensitive to ethanol (an organic solvent) than wild-type bacteria, and the seqA strain and the double mutant dnaA seqA are very sensitive to deoxycholate (a detergent). We conclude that lesions in the genes dnaA and seqA result in alterations in cell membranes, such that the permeability and possibly also other properties of the membranes are significantly altered relative to wild-type bacteria.

Bacterial Outer Membrane Proteins↗
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

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

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

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

The Escherichia coli RNA polymerase alpha subunit and transcriptional activation by bacteriophage lambda CII protein.

Bacteriophage lambda is not able to lysogenise the Escherichia coli rpoA341 mutant. This mutation causes a single amino acid substitution Lys271Glu in the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). Our previous studies indicated that the impaired lysogenisation of the rpoA341 host is due to a defect in transcriptional activation by the phage CII protein and suggested a role for alphaCTD in this process. Here we used a series of truncation and point mutants in the rpoA gene placed on a plasmid to investigate the process of transcriptional activation by the cII gene product. Our results indicate that amino-acid residues 265, 268 and 271 in the a subunit may play an important role in the CII-mediated activation of the pE promoter (most probably residue 271) or may be involved in putative interactions between alphaCTD and an UP-like element near pE (most probably residues 265 and 268). Measurement of the activity of pE-lacZ, pI-lacZ and p(aQ)-lacZ fusions in the rpoA+ and rpoA341 hosts demonstrated that the mechanism of activation of these CII-dependent promoters may be in each case different.

Artificial Gene Fusion↗
PubMed1997

Impaired lysogenisation of the Escherichia coli rpoA341 mutant by bacteriophage lambda is due to the inability of CII to act as a transcriptional activator.

The C-terminus of the alpha subunit of Escherichia coli RNA polymerase is known to function in transcriptional activation at certain promoters. This region was previously shown to be necessary for full activation of the pE promoter by the phage lambda CII protein in vitro. In this work we investigated the inability of phage lambda to follow the lysogenic pathway in cells carrying the point mutation rpoA341 (a change of lysine 271 to glutamic acid). We found that neither overexpression of the cII gene nor stabilisation of the CII protein by the can1 mutation or by cIII gene overexpression was able to suppress the block in lysogenisation. In contrast, the lambda cin1 phage, which carries a CII-independent promoter for the expression of the cI gene, was able to efficiently lysogenise the rpoA341 mutant strain. Furthermore, the rpoA341 mutation prevented the activation of pE-lacZ and pI-lacZ transcriptional fusions by CII. Therefore we conclude that transcriptional activation by the cII gene product is abolished by the rpoA341 mutation, most probably due to impaired interaction between the CII activator and mutant RNA polymerase. The inability of RNA polymerase to respond to CII results in the impairment of lysogenisation of the rpoA341 mutant by phage lambda.

Bacteriophage lambda↗
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↗