Biomedical subjects

G Wegrzyn

Publications and source records attributed to G Wegrzyn.

At least 73 records · Page 4Linked to original sources

PubMed1997

Replication of plasmids derived from P1, F, R1, R6K and RK2 replicons in amino acid-starved Escherichia coli stringent and relaxed strains.

Replication of mini-plasmids derived from bacteriophage P1 and naturally existing plasmids F, R1, R6K and RK2 in otherwise isogenic relA+ and relA- Escherichia coli strains during amino acid starvation and limitation was investigated. Since it was previously demonstrated that inhibition of DNA synthesis or amplification of plasmid DNA may depend on the nature of deprived amino acid, we starved bacteria for five different amino acids. We found differential replication of all these plasmids but RK2 (which did not replicate at all in amino acid-starved bacteria) during the stringent and relaxed response. While in almost all cases plasmid DNA replication was inhibited during the stringent response irrespective of the nature of deprived amino acid, wild-type or copy-up mini-P1, mini-F and mini-R1 plasmids replicated in relA- bacteria depending on the kind of starvation. R6K-derived plasmids harbouring ori beta and gamma (but not those containing ori alpha, beta and gamma or only ori gamma) were able to replicate in relA- bacteria starved for all tested amino acids. Possible explanations for the mechanisms of regulation of replication of plasmids derived from P1, F, R1, R6K and RK2 during amino acid starvation are discussed. Our results also indicate that, like in the case of some other replicons, appropriate amino acid starvation or limitation may be used as a method for efficient amplification of plasmids derived from P1, F, R1 and R6K.

Amino Acids↗

PubMed1997

Isolation and characterization of a ColE1-like plasmid from Enterobacter agglomerans with a novel variant of rom gene.

Complete nucleotide sequence of a plasmid isolated from Enterobacter agglomerans has been determined. The plasmid, called pPIGDM1, consists of 2495 base pairs. The analysis of its nucleotide sequence suggested that pPIGDM1 may be a ColE1-like replicon. We confirmed this hypothesis by constructing a pPIGDM1-derived plasmid harboring the cat gene (pBW4), which could be introduced into Escherichia coli cells, and demonstrating that pBW4 cannot replicate in the absence of the polA function and that its copy number is significantly decreased in the pcnB mutant. Like some other ColE1-type replicons (e.g., pBR322), pPIGDM1-derived plasmids can be amplified both by chloramphenicol method and in isoleucine-starved relA mutants but not in relA+ bacteria. Inactivation of the putative rom gene by insertion of an amplicillin-resistance gene resulted in significant increase in pPIGDM1-derived plasmid copy number in E. coli-despite the fact that amino acid sequence of the putative RNA 1 modulator (Rom) protein is only 55.7% identical to the ColE1 analog. The pPIGDM1-derived rom-like coding sequence is also homologous to the rom-like gene present in the Proteus vulgaris plasmid pPvul. We suggest to group all these gene products into a new family called ROMS (RNA one modulators). Since a pPIGDM1-derived plasmid is compatible with other ColE1-like replicons (pMB1-, p15A, RSF1030-, and CloDF13-derived) in E. coli, one may consider pPIGDM1 as a progenitor of new cloning vehicles compatible with most (if not all) of currently used plasmid vectors. Moreover, this plasmid may serve as a source of the new rom-like gene coding for a protein useful in investigation of RNA-protein interactions. A role for the pPIGDM1 plasmid in the host strain is not known.

Amino Acid Sequence↗

PubMed1997

Differential amplification efficiency of pMB1 and p15A (ColE1-type) replicons in Escherichia coli stringent and relaxed strains starved for particular amino acids.

It was demonstrated previously that ColE1-type plasmids, the most commonly used vectors in molecular cloning, can be amplified in amino acid-starved relA mutants of Escherichia coli. Subsequent studies demonstrated that replication of at least some plasmids during amino acid starvation depends not only on the host relA allele but also on temperature and on the nature of deprived amino acid. Therefore, we investigated efficiency of amplification of two types of ColE1 plasmids (pMB1- and p15A-derived replicons) in E. coli relA+ and relA- hosts starved for different amino acids at 30 degrees C, 37 degrees C and 43 degrees C. We found differential amplification efficiency of plasmids pBR328 (pMB1-derived replicon) and pACYC184 (p15A-derived replicon) in the relA mutant during starvation for particular amino acids. Although amplification of pBR328 was negligible in the relA+ host, significant increase in pACYC184 content was observed in this strain starved for some (but not all) amino acids. The amplification efficiency of pBR328 and pACYC184 was found to be dependent on temperature. These results indicate that for maximal amplification of particular plasmid appropriate amino acid starvation and optimal temperature should be chosen. Our findings are in agreement with recently proposed model of the regulation of ColE1-type plasmid replication in amino acid-starved E. coli cells.

Amino Acids↗

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↗

PubMed1997

Stability of CII is a key element in the cold stress response of bacteriophage lambda infection.

Bacteria are known to adapt to environmental changes such as temperature fluctuations. It was found that temperature affects the lysis-lysogeny decision of lambda such that at body temperature (37 degrees C) the phage can select between the lytic and lysogenic pathways, while at ambient temperature (20 degrees C) the lytic pathway is blocked. This temperature-dependent discriminatory developmental pathway is governed mainly by the phage CII activity as a transcriptional activator. Mutations in cII or point mutations at the pRE promoter lead to an over-1,000-fold increase in mature-phage production at low temperature while mutations in cI cause a smaller increase in phage production. Interference with CII activity can restore lytic growth at low temperature. We found that at low temperature the stability of CII in vivo is greatly increased. It was also found that phage DNA replication is blocked at 20 degrees C but can be restored by supplying O and P in trans. It is proposed that CII hampers transcription of the rightward pR promoter, thus reducing the levels of the lambda O and P proteins, which are necessary for phage DNA replication. Our results implicate CII itself or host proteins affecting CII stability as a "molecular thermometer".

Bacteriophage lambda↗

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

Synthesis of the bacteriophage lambda P protein in amino acid-starved Escherichia coli cells.

It was demonstrated previously that in isoleucine-starved Escherichia coli relA mutants harboring a plasmid derived from bacteriophage lambda the lambda O protein is not synthesized. However, a protein which coprecipited with the lambda O during immunoprecipitation with anti-lambda O serum was synthesized during the relaxed response. Here we found that this protein is the lambda P gene product. Despite significant inhibition of transcription from the pR promoter (which produces mRNA for the lambda P protein synthesis) during the stringent response, the lambda P protein was efficiently synthesized in relA- as well as relA+ strains starved for isoleucine, threonine and histidine, whereas the synthesis was negligible during starvation for arginine and leucine. The synthesis of the lambda P protein in amino acid-starved cells is sensitive to rifampicin. Thus we presume that this phenomenon is not caused by eventual increased stability of the lambda P mRNA but rather is an effect of preferential translation of this mRNA and incorporation of limited amount of amino acids arising in the starved cells as a result of intracellular proteolysis. One of possible explanations of the mechanism of this phenomenon may suggest that the same signals can be recognized in both prokaryotic and eukaryotic cells during initiation of translation at non-AUG codons.

Amino Acid Sequence↗

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

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↗