Department of Molecular Biology, University of Gdask, Kladki 24, 80-822 Gda
sk, Poland1
Laboratory of Molecular Biology (affiliated with the University of Gdask), Polish Academy of Sciences, K
adki 24, 80-822 Gda
sk, 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 Wgrzyn. Tel: +48 58 346 3014. Fax: +48 58 301 0072. e-mail: wegrzyn{at}biotech.univ.gda.pl
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ABSTRACT |
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Keywords: plasmid replication, dnaA mutants, DnaA protein functions, transcriptional activation of origin, DnaB helicase
Abbreviations: aCT, autoclaved chlortetracycline
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INTRODUCTION |
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Recent studies have revealed that although the assembly of the replication complex is necessary for initiation of
plasmid DNA replication, this process plays no regulatory role (for a review see W
grzyn & W
grzyn, 2001
). In fact, after one replication round the
replication complex is inherited by one of two daughter plasmid copies (W
grzyn & Taylor, 1992
). The complex may function in subsequent replication rounds (W
grzyn et al., 1996a
), but requires a trigger signal which seems to be provided by transcriptional activation of ori
(Szalewska-Pa
asz et al., 1994
; Szalewska-Pa
asz & W
grzyn, 1994
; W
grzyn et al., 1996a
). Thus, this process is crucial for regulating the frequency of replication initiation at ori
(Taylor & W
grzyn, 1995
, 1998
; W
grzyn & W
grzyn, 2001
). It was demonstrated recently that the pR promoter is stimulated by DnaA protein (Szalewska-Pa
asz et al., 1998a
), the host replication initiator. Therefore, DnaA controls initiation of
DNA replication by stimulating the transcriptional activation of ori
.
Wild-type 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 (W
grzyn et al., 1996b
). This incompatibility between
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
P gene, called
(Kur et al., 1987
; W
grzyn et al., 1996b
). However, neither wild-type
plasmids nor
mutants can replicate in all tested dnaA(ts) mutants at elevated (above 42 °C) temperatures (Kur et al., 1987
; W
grzyn et al., 1996b
).
The aim of this work was to investigate the mechanism(s) of the incompatibility between 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.
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METHODS |
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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 Wgrzyn et al. (1996b)
were used.
Electron microscopic analysis of proteinDNA interactions.
The interactions between DnaA protein and linear DNA fragments were investigated using electron microscopy as described previously (Szalewska-Paasz et al., 1998c
; Konopa et al., 1999
). The DNA fragment, biotinylated at one end and encompassing the region of wild-type
plasmid from pR to ori
, was prepared by PCR using pKB2 plasmid DNA as a template as described previously (Szalewska-Pa
asz et al., 1998c
). The analogous DNA fragment encompassing the region of pTC
1 from ptet to ori
was also prepared by PCR as described above but using pTC
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).
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RESULTS |
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We investigated the efficiency of transformation of dnaA+ and dnaA46 hosts with a derivative of plasmid pTC1, bearing the
mutation (pTC
1
). 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
1 plasmid (Table 1
). This requirement was abolished by the dnaA46 mutation (Table 1
). The copy number of pTC
1
was significantly lower than that of pTC
1 in both host strains, and this difference was especially pronounced at low aCT concentrations (Table 1
). The specific defect of the
mutation in pTC
1
is a weaker interaction of the mutated P protein with DnaB helicase (Konieczny & Marsza
ek, 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
plasmid and dnaA(ts) hosts, in addition to impaired stimulation of transcriptional activation of ori
.
plasmiddnaA46 incompatibility at different levels of dnaB and dnaC expression
The P protein interacts with DnaB to form a replication complex at ori
(for a review see Taylor & W
grzyn, 1995
). DnaA also interacts with DnaB, and this interaction is required for the delivery of the helicase to oriC (Marsza
ek & 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 & W
grzyn, 1995
and references therein).
To test this, we measured the efficiency of transformation of dnaA+ and dnaA46 hosts with plasmids bearing either wild-type or
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
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
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
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
plasmids rather than
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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We measured the efficiency of transformation of dnaA+ seqA+, dnaA(ts) seqA+, dnaA+ seqA, and dnaA(ts)
seqA hosts with
plasmids bearing either wild-type or
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
plasmid. Despite a high number of transformants, the colonies obtained in the dnaA46
seqA host were rather small, and those of the dnaA508
seqA host were tiny (Table 3
). These results show that the incompatibility between
plasmids and dnaA(ts) hosts is abolished in the
seqA background.
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DISCUSSION |
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The first mechanism is based on the finding that moderately decreased transcription of the replication region, observed in the dnaA46 mutant, as well as in other dnaA mutants which cannot be transformed by wild-type
plasmids (Szalewska-Pa
asz et al., 1998b
), results in a dramatic impairment of the initiation of replication from ori
. 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
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
plasmids and certain dnaA mutants exists. This mechanism is based on the competition between the
P protein and the host DnaA and DnaC proteins for DnaB helicase. The PDnaB and DnaCDnaB 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 (Marsza
ek & 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 DnaADnaB 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 2486 and 130148 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 DnaA46DnaB 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
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
plasmid replication. This hypothesis is supported by the fact that wild-type
plasmids can transform
seqA dnaA double mutants (Table 3
). It seems that extension of the period for potential effective DnaADnaB interactions, observed in the seqA mutants, increases the probability of a successful competition of mutated DnaA with
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
seqA host, whereas a lack of seqA gene function did not stabilize DnaA46 protein (Torheim et al., 2000
).
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ACKNOWLEDGEMENTS |
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Received 19 January 2001;
revised 20 March 2001;
accepted 27 March 2001.