The double mechanism of incompatibility between {lambda} plasmids and Escherichia coli dnaA(ts) host cells

Monika Glinkowska1, Grazyna Konopa1, Alicja Wegrzyn2, Anna Herman-Antosiewicz1, Christoph Weigel3, Harald Seitz3, Walter Messer3 and Grzegorz Wegrzyn1,4

Department of Molecular Biology, University of Gdask, Kladki 24, 80-822 Gdask, Poland1
Laboratory of Molecular Biology (affiliated with the University of Gdask), Polish Academy of Sciences, Kladki 24, 80-822 Gdask, Poland2
Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin-Dahlem, Germany3
Marine Biology Centre, Polish Academy of Sciences, w. Wojciecha 5, 81-347 Gdynia, Poland4

Author for correspondence: Grzegorz Wegrzyn. Tel: +48 58 346 3014. Fax: +48 58 301 0072. e-mail: wegrzyn{at}biotech.univ.gda.pl


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
For plasmids derived from bacteriophage {lambda}, the initiation of bidirectional DNA replication from ori{lambda} depends on the stimulation of transcription from the pR promoter by the host replication initiator protein DnaA. Certain Escherichia coli dnaA(ts) mutants cannot be transformed by wild-type {lambda} plasmids even at the temperature permissive to cell growth. This plasmid–host incompatibility appeared to be due to inefficient stimulation of transcription from the pR promoter by the mutant DnaA protein. This paper shows that there is a second mechanism for the incompatibility between {lambda} plasmids and dnaA(ts) hosts, exemplified in this study by the dnaA46 mutant. This is based on the competition between the {lambda} P protein and the host DnaA and DnaC proteins for DnaB helicase. Both mechanisms must be operative for the incompatibility.

Keywords: {lambda} plasmid replication, dnaA mutants, DnaA protein functions, transcriptional activation of origin, DnaB helicase

Abbreviations: aCT, autoclaved chlortetracycline


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plasmids derived from bacteriophage {lambda} (called {lambda} plasmids) bear the replication region of the phage genome and can be maintained in the host (Escherichia coli) cells at a certain copy number (for a review see Taylor & Wegrzyn, 1995 ). There are two {lambda}-encoded replication proteins, O and P. The O protein binds to the ori{lambda} region (located within the O gene) and initiates the assembly of the replication complex. The host-encoded helicase, DnaB, is delivered to ori{lambda} due to its interaction with the P protein, which also has an affinity to the O protein. Since the P–DnaB interaction is very tight, the action of molecular chaperones, DnaK, DnaJ and GrpE, is necessary for remodelling the nucleoprotein complex in such a way that DnaB is liberated as active helicase from P-mediated inhibition. Additionally, a process called transcriptional activation of the origin, i.e. transcription entering this region, is necessary for efficient initiation of DNA replication from ori{lambda}. This transcription starts from the pR promoter located several hundred base pairs upstream of ori{lambda} (Taylor & Wegrzyn, 1995 ).

Recent studies have revealed that although the assembly of the {lambda} replication complex is necessary for initiation of {lambda} plasmid DNA replication, this process plays no regulatory role (for a review see Wegrzyn & Wegrzyn, 2001 ). In fact, after one replication round the {lambda} replication complex is inherited by one of two daughter plasmid copies (Wegrzyn & Taylor, 1992 ). The complex may function in subsequent replication rounds (Wegrzyn et al., 1996a ), but requires a trigger signal which seems to be provided by transcriptional activation of ori{lambda} (Szalewska-Palasz et al., 1994 ; Szalewska-Palasz & Wegrzyn, 1994 ; Wegrzyn et al., 1996a ). Thus, this process is crucial for regulating the frequency of replication initiation at ori{lambda} (Taylor & Wegrzyn, 1995 , 1998 ; Wegrzyn & Wegrzyn, 2001 ). It was demonstrated recently that the pR promoter is stimulated by DnaA protein (Szalewska-Palasz et al., 1998a ), the host replication initiator. Therefore, DnaA controls initiation of {lambda} DNA replication by stimulating the transcriptional activation of ori{lambda}.

Wild-type {lambda} plasmids cannot transform certain E. coli temperature-sensitive (ts) mutants in the dnaA gene, dnaA46, dnaA204 and dnaA508, even at a temperature permissive for bacterial growth (Wegrzyn et al., 1996b ). This incompatibility between {lambda} plasmids and dnaA(ts) mutants presumably acts at the level of replication initiation, and therefore is comparable to incompatibility between two plasmid replicons that cannot co-exist in a host cell. It can be abolished by specific mutations in the {lambda} P gene, called {pi} (Kur et al., 1987 ; Wegrzyn et al., 1996b ). However, neither wild-type {lambda} plasmids nor {pi} mutants can replicate in all tested dnaA(ts) mutants at elevated (above 42 °C) temperatures (Kur et al., 1987 ; Wegrzyn et al., 1996b ).

The aim of this work was to investigate the mechanism(s) of the incompatibility between {lambda} plasmids and certain dnaA(ts) mutants, exemplified in this study by the dnaA46 mutant. We suspected that such studies would provide important information about the role of transcription in the control of DNA replication and about the functions of the DnaA protein.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and plasmids.
Escherichia coli wild-type strain MG1655 (Jensen, 1993 ) and a series of its dnaA and/or {Delta}seqA::Tn10 derivatives (described by Wegrzyn et al., 1999 ) were used. The following plasmids were employed: pBR322 (Bolivar et al., 1977 ), pKB2 (wild-type {lambda} plasmid harbouring a kanamycin-resistance gene) (Kur et al., 1987 ), pCB104 (wild-type {lambda} plasmid harbouring a chloramphenicol-resistance gene) (Boyd & Sheratt, 1995 ), pAW6 (a derivative of pCB104 but bearing the {pi}A66 mutation) (Wegrzyn et al., 1995 ), pTC{lambda}1 (an artificial {lambda} plasmid containing the ptet promoter instead of pR) (Herman-Antosiewicz et al., 1998b ), pTC{lambda}1{pi} [a derivative of pTC{lambda}1 but bearing the {pi}A66 mutation; this plasmid was constructed by insertion of the PvuII–PvuII fragment of pAW6, bearing the replication region, into the SmaI site of plasmid pCattTrE18 (Herman-Antosiewicz et al., 1998b ) to produce pAW10, and then by deletion of the NotI–NotI fragment of pAW10 encompassing oripBR322], and a series of pINGK-derived plasmids (pINGK is a cloning vector with the ara promoter) bearing the dnaB (pINB), dnaC (pINC) or dnaB and dnaC genes (pINBC) under control of the ara promoter (Allen & Kornberg, 1991 ).

Culture medium and growth conditions.
All experiments with bacterial cultures were performed using LB medium (Sambrook et al., 1989 ). Cultivations were performed in shake flasks at 30 °C.

Estimation of the efficiency of transformation and plasmid copy number.
The procedures described by Wegrzyn et al. (1996b) were used.

Electron microscopic analysis of protein–DNA interactions.
The interactions between DnaA protein and linear DNA fragments were investigated using electron microscopy as described previously (Szalewska-Palasz et al., 1998c ; Konopa et al., 1999 ). The DNA fragment, biotinylated at one end and encompassing the region of wild-type {lambda} plasmid from pR to ori{lambda}, was prepared by PCR using pKB2 plasmid DNA as a template as described previously (Szalewska-Palasz et al., 1998c ). The analogous DNA fragment encompassing the region of pTC{lambda}1 from ptet to ori{lambda} was also prepared by PCR as described above but using pTC{lambda}1 plasmid DNA as a template and the following primers: 5'-CTG CTC TAC ACC TAG CTT CT-3' and 5'-TTC TCT GAC GAA TAA TCT TT-3' (biotinylated at the 5' end).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Impaired stimulation of the pR promoter is responsible for the incompatibility between {lambda} plasmids and dnaA mutants
To test whether a few-fold decrease in the activity of the pR promoter, as observed in the dnaA46 mutant (Szalewska-Palasz et al., 1998b ), is responsible for the incompatibility between this mutant and {lambda} plasmids, we used an artificial {lambda} plasmid (pTC{lambda}1) which contains the ptet promoter instead of pR (Herman-Antosiewicz et al., 1998b ). It was found previously that activity of a ptetlacZ fusion is independent of the dnaA gene function (Herman-Antosiewicz et al., 1998a ). Here we show, using electron microscopic techniques, that in contrast to the pR promoter region, the DnaA protein does not interact specifically with DNA regions located at or near the ptet promoter. When a fragment of wild-type {lambda} plasmid containing the pR promoter was investigated, a large peak representing a significant number of DnaA molecules bound to the promoter region was observed in a statistical analysis of electron microscopic pictures of several hundred DnaA–DNA complexes (Fig. 1). There was no such a peak at the promoter region when an analogous fragment of plasmid pTC{lambda}1, which bears ptet instead of pR, was investigated (Fig. 1).



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Fig. 1. Electron microscopic analysis of the interaction of DnaA protein with replication regions of plasmids pKB2 and pTC{lambda}1. At the top, a genetic map of the investigated fragment of the wild-type {lambda} plasmid (like pKB2) is shown, including the promoters, terminators and transcripts (long arrows). The positions and orientations of DnaA boxes are marked by short arrows; these boxes are numbered according to Szalewska-Palasz et al. (1998c) . Below, the results of the DNA–DnaA interactions are presented for the DNA fragment described above, and further below, for the analogous fragment of the pTC{lambda}1 plasmid, whose map is also shown. Examples of protein–DNA complexes are presented at the bottom of the figure.

 
Transcription from the ptet promoter in pTC{lambda}1 is induced by autoclaved chlortetracycline (aCT). After autoclaving, chlortetracycline loses its antibiotic activity but retains the ability to induce the ptet promoter (see Herman-Antosiewicz et al., 1998b a nd references therein). Without induction pTC{lambda}1 cannot replicate in E. coli cells (Herman-Antosiewicz et al., 1998b ). We measured the efficiency of transformation of dnaA+ and dnaA46 hosts with pTC{lambda}1 at different concentrations of aCT, resulting in different activities of the ptet promoter. We found that both wild-type and dnaA46 hosts were efficiently transformed by plasmid pTC{lambda}1, which bears the wild-type P gene, in the presence of aCT (Table 1). Above 0·005 µg ml-1, the concentration of aCT has a relatively small effect on the efficiency of transformation (Table 1). These results indicate that when the activity of the promoter responsible for transcription of the {lambda} replication region is independent of dnaA gene function, the incompatibility between the dnaA46 mutant and {lambda} plasmids bearing the wild-type P gene is abolished.


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Table 1. Efficiency of transformation and copy number of plasmids pTC{lambda}1 and pTC{lambda}1{pi} in E. coli dnaA+ and dnaA46 hosts at different concentrations of aCT in the medium

 
It was demonstrated previously that the copy number of pTC{lambda}1 in wild-type cells increases with an increase in the concentration of aCT in the medium (Herman-Antosiewicz et al., 1998a , b ). We found that this is also the case for the dnaA46 host, but at a somewhat lower level (Table 1). Therefore, there is no significant influence of the dna46 allele on the replication initiated at ori{lambda} at 30 °C when transcription of the replication region is DnaA-independent.

We investigated the efficiency of transformation of dnaA+ and dnaA46 hosts with a derivative of plasmid pTC{lambda}1, bearing the {pi} mutation (pTC{lambda}1{pi}). For efficient transformation of the wild-type host by this plasmid, a considerably higher concentration of aCT (and thus considerably more efficient transcription of the replication region) was required relative to the pTC{lambda}1 plasmid (Table 1). This requirement was abolished by the dnaA46 mutation (Table 1). The copy number of pTC{lambda}1{pi} was significantly lower than that of pTC{lambda}1 in both host strains, and this difference was especially pronounced at low aCT concentrations (Table 1). The specific defect of the {pi} mutation in pTC{lambda}1{pi} is a weaker interaction of the mutated P protein with DnaB helicase (Konieczny & Marszalek, 1995 ). The observation of more efficient establishment of this plasmid in the dnaA46 host as compared to the dnaA+ host suggests that a competition for DnaB helicase might play a role in the incompatibility of the wild-type {lambda} plasmid and dnaA(ts) hosts, in addition to impaired stimulation of transcriptional activation of ori{lambda}.

{lambda} plasmid–dnaA46 incompatibility at different levels of dnaB and dnaC expression
The {lambda} P protein interacts with DnaB to form a replication complex at ori{lambda} (for a review see Taylor & Wegrzyn, 1995 ). DnaA also interacts with DnaB, and this interaction is required for the delivery of the helicase to oriC (Marszalek & Kaguni, 1994 ; Sutton et al., 1998 ; Seitz et al., 2000 ). Therefore, we suspected that the P protein might compete with the host replication initiator DnaA for DnaB, in addition to the previously reported competition between P and DnaC (Taylor & Wegrzyn, 1995 and references therein).

To test this, we measured the efficiency of transformation of dnaA+ and dnaA46 hosts with {lambda} plasmids bearing either wild-type or {pi} alleles of the P gene under conditions of normal expression of dnaB and dnaC, and in cells overexpressing one or both of these genes. As expected, we found that wild-type {lambda} plasmid (pCB104) could not transform the dnaA46 mutant, in contrast to the wild-type host (Table 2). Corroborating previous reports, both hosts were transformed efficiently with the {lambda}{pi} mutant plasmid (Table 2). We found that overexpression of dnaC or dnaB and dnaC together (but not dnaB alone) resulted in effective transformation of the dnaA46 host by a wild-type {lambda} plasmid, although the efficiency of this transformation was lower than that measured for dnaA+ bacteria (Table 2). We checked that these relatively rare dnaA46 transformants contained wild-type {lambda} plasmids rather than {pi} mutants because plasmid DNA isolated from these clones could transform wild-type cells but not the dnaA46 mutant (data not shown).


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Table 2. Efficiency of transformation of E. coli dnaA+ and dnaA46 strains by {lambda}P+ and {lambda}{pi} plasmids under conditions of dnaB and/or dnaC overexpression

 
{lambda} plasmid–dnaA(ts) incompatibility is abolished in {Delta}seqA dnaA(ts) double mutant strains
E. coli {Delta}seqA cells lack a protein which binds preferentially to hemimethylated DNA containing GATC sites (Lu et al., 1994 ). In wild-type cells, oriC remains hemimethylated for one-third of the cell cycle and is sequestered from initiation for this time, termed the eclipse period. In seqA mutants the temperature-sensitive growth phenotype of the dnaA46 mutant is suppressed (Lu et al., 1994 ).

We measured the efficiency of transformation of dnaA+ seqA+, dnaA(ts) seqA+, dnaA+ {Delta}seqA, and dnaA(ts) {Delta}seqA hosts with {lambda} plasmids bearing either wild-type or {pi} alleles of the P gene (Table 3). Efficient transformation was found in all combinations tested, with the exceptions of single-mutant dnaA46, dnaA204 and dnaA508 hosts and the wild-type {lambda} plasmid. Despite a high number of transformants, the colonies obtained in the dnaA46 {Delta}seqA host were rather small, and those of the dnaA508 {Delta}seqA host were tiny (Table 3). These results show that the incompatibility between {lambda} plasmids and dnaA(ts) hosts is abolished in the {Delta}seqA background.


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Table 3. Transformation of E. coli dnaA+ seqA+, dnaA+ {Delta}seqA, dnaA(ts) seqA+, and dnaA(ts) {Delta}seqA strains by {lambda} plasmids

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The aim of this study was to investigate the mechanism of the incompatibility between {lambda} plasmids and certain host temperature-sensitive mutants in the dnaA gene at a temperature permissive for cell growth. The experiments presented here not only provided the answer to this question, but also shed some light on the functions of DnaA and the role of the transcriptional activation of the origin in the regulation of {lambda} DNA replication. Our results indicate that there are two mechanisms leading to the incompatibility between {lambda} plasmids and dnaA(ts) mutants, and a combination of both these processes is lethal for the host cells (under antibiotic selection pressure) due to inhibition of replication of either plasmid (bearing an antibiotic-resistance gene) or host chromosome or both.

The first mechanism is based on the finding that moderately decreased transcription of the {lambda} replication region, observed in the dnaA46 mutant, as well as in other dnaA mutants which cannot be transformed by wild-type {lambda} plasmids (Szalewska-Palasz et al., 1998b ), results in a dramatic impairment of the initiation of replication from ori{lambda}. Since replacement of the DnaA-stimulated pR promoter by a DnaA-independent ptet promoter resulted in abolition of the incompatibility (Table 1), it seems that the main function of DnaA in the regulation of {lambda} plasmid replication is the control of the efficiency of transcription of the replication region.

The results presented here (Table 2) suggest that a second mechanism leading to the incompatibility between {lambda} plasmids and certain dnaA mutants exists. This mechanism is based on the competition between the {lambda} P protein and the host DnaA and DnaC proteins for DnaB helicase. The P–DnaB and DnaC–DnaB interactions have been well known for a long time (for a review see Kornberg & Baker, 1992 ), and it was demonstrated that DnaA interacts with DnaB at the stage of the delivery of the helicase to oriC (Marszalek & Kaguni, 1994 ). DnaB is present at only about 20 molecules per cell (Kornberg & Baker, 1992 ), making it a likely candidate for competitive effects. It seems that P competes with DnaC for DnaB significantly more successfully in combination with DnaA46 than with wild-type DnaA.

The reason for this preference is at present not known. However, we believe that it is only indirectly due to DnaA–DnaB interaction. The interaction domains of the DnaA and DnaB proteins have recently been mapped (Seitz et al., 2000 ). The physical contacts between these proteins involve residues 24–86 and 130–148 of DnaA. The product of the dnaA46 allele contains two amino acid substitutions, A184V and H252Y (Hansen et al., 1992 ), both located outside of the regions involved in interactions with DnaB. Therefore, a decreased efficiency of the DnaA46–DnaB interaction is unlikely. Moreover, the product of the dnaA46 allele is not defective in the loading of DnaB at oriC (Seitz et al., 2000 ; H. Seitz, C. Weigel & W. Messer, unpublished data). Therefore, we propose another explanation. The DnaA46 protein may have problems with oriC unwinding due to a reduced capacity for domain 3-mediated homo-oligomerization (Messer et al., 2001 ). A partial sequestering of DnaB by the {lambda} P protein in these mutants may cause severe problems for host chromosome replication as there are simply too few DnaB molecules available for interaction with DnaC and DnaA, preventing efficient formation of the replication complexes at oriC. This may lead to cell death irrespective of {lambda} plasmid replication. This hypothesis is supported by the fact that wild-type {lambda} plasmids can transform {Delta}seqA dnaA double mutants (Table 3). It seems that extension of the period for potential effective DnaA–DnaB interactions, observed in the seqA mutants, increases the probability of a successful competition of mutated DnaA with {lambda} P for DnaB. In addition, the level of DnaA was found to be increased twofold in seqA mutants (von Freiesleben et al., 1994 ; Torheim et al., 2000 ). On the other hand, DnaA204 protein was reported to be stabilized in the {Delta}seqA host, whereas a lack of seqA gene function did not stabilize DnaA46 protein (Torheim et al., 2000 ).


   ACKNOWLEDGEMENTS
 
We thank Kirsten Skarstad for providing plasmids. This work was supported by Polish State Committee for Scientific Research (project 6 P04A 016 16) and by Volkswagen-Stiftung (project I/74 639). G.W. acknowledges also a financial support from the Foundation for Polish Science (subsidium 14/2000).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 19 January 2001; revised 20 March 2001; accepted 27 March 2001.