Department of Microbiology, National University of Ireland, Galway, Ireland1
Author for correspondence: Emer Colleran. Tel: +353 91 750416. Fax: +353 91 525700. e-mail: emer.colleran{at}nuigalway.ie
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ABSTRACT |
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Keywords: IncH plasmids, plasmid replication, Rep protein, iteron
The GenBank accession numbers for the sequences reported in this paper are U62006 (RepHI2A) and U62007 (RepHI1A(R478)).
a Present address: Elan Pharmaceutical Technologies, Biotechnology Building, Trinity College, Dublin 2, Ireland.
b Present address: Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK.
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INTRODUCTION |
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IncHI plasmids are divided into the three subgroups IncHI1, IncHI2 and IncHI3. Each of the plasmids from one particular subgroup shows a high degree of DNA homology with other members of that subgroup, but not with members of the other two subgroups (Whiteley & Taylor, 1983 ).
Two different H-specific iteron-controlled replicon regions have been characterized to date: IncHI1A and IncHI1B (Gabant et al., 1994 ; Newnham & Taylor, 1994
). These replicons are specific to plasmids of the IncHI1 subgroup. A third relicon, RepFIA, is also associated with IncHI1 plasmids (Saul et al., 1988
) but not with other IncH plasmids, which gives rise to one-way incompatibility between IncHI1 subgroup plasmids and members of the FIA plasmid incompatibility group.
This study reports the isolation, mapping and molecular characterization of two different iteron-regulated replicons from the prototype IncHI2 plasmid, R478.
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METHODS |
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Miniplasmids of pKFW141.
pKFW141 is a 286 kb transfer-defective, Tn7 insertion mutant of R478 (Page et al., 1999 ); 65 kb miniplasmids of pKFW141 were generated using the restriction enzyme SalI as previously described (Whelan & Colleran, 1992
). Miniplasmids were electroporated into DH5
cells using a Bio-Rad gene pulser (Dower et al., 1988
) and selected on media supplemented with trimethoprim (Tp). Further miniplasmids of these 65 kb constructs were similarly generated using EcoRI and used to transform DH5
cells by standard calcium chloride/heat pulse methods (Sambrook et al., 1989
). Tn7 is known to contain a single EcoRI recognition site which does not occur within the gene encoding trimethoprim (Tp) resistance (Gosti-Testu et al., 1983
).
DNA sequencing and analysis.
Nucleotide sequences for both strands were determined by the Sanger chain termination method (Sanger et al., 1977 ). Standard M13/pUC13 forward and reverse oligonucleotides and oligonucleotides based on previous sequence analysis were used for sequencing. Sequence analysis was carried out using the DNA Strider 1.0 program. Nucleic acid homologies were determined with the BLAST program of the National Centre for Biotechnology Information (NCBI) (Altschul et al., 1990
). CLUSTAL W (Thompson et al., 1994
) was used for nucleotide alignments. The sequences of the RepHI2A and RepHI1A(R478) replicon regions have been assigned GenBank/EMBL accession numbers U62006 and U62007 respectively.
Plasmid incompatibility assay.
A test plasmid was introduced by transformation into a strain containing a second plasmid. These transformed cells were grown under conditions selecting for both plasmids. Cells containing both plasmids were allowed to grow in liquid media under non-selective conditions for 24 h at 37 °C. Viable counts (in triplicate) were carried out on solid media under the following conditions: (i) non-selective, (ii) selective for the original resident plasmid and (iii) selective for the incoming plasmid. At least 100 colonies from each of (ii) and (iii) above were subsequently replica-plated (in triplicate) on media selective for each plasmid to determine the percentage of cells that contained only one plasmid or both plasmids.
Plasmid stability assay.
Cells containing both plasmids were grown overnight in minimal media under non-selective conditions, then diluted 1:1000 using fresh, sterile minimal medium and grown under non-selective conditions for a further 10 h. Viable counts of these cells were again obtained by spread plating and replica plating as above. This procedure was repeated five times. Plasmid stability was observed as the percentage of cells that retained the resistance determinant encoded by the plasmid(s) at each dilution step.
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RESULTS |
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Eight different 18 bp iterons flank the rep homologue of RepHI2A (four upstream iterons, AD, and four downstream iterons, EH). Nine different 76 bp iterons, IQ, which contain the 18 bp iteron consensus sequence, occur immediately downstream of the smaller tandem repeats (Fig. 2). A further 18 bp iteron, R, occurs further downstream of the larger iterons. The consensus sequence of these iterons is unique to RepHI2A, as determined by nucleotide homology searches and comparison with all known plasmid consensus iterons. This locus was mapped to within co-ordinates 11 and 14 on R478 (R478 plasmid co-ordinates are determined as described by Whelan & Colleran, 1992
), which coincides with a region that was previously shown to hybridize strongly with the IncHI2-specific diagnostic probe pULB2433 (Whelan & Colleran, 1992
; Couturier et al., 1988
).
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Replication and stability properties of RepHI2A and RepHI1A(R478)
Similar studies were carried out with both pDTP61 (RepHI2A) and pDTP71 (RepHI1A(R478)). These two constructs independently supported growth of the transformed DH5 host on media supplemented with chloramphenicol, indicating that each contained a functional origin of replication. E. coli SF800 is a mutant strain which lacks expression of DNA polymerase I. pDTP61 and pDTP71 were each independently maintained within SF800, showing replication of both RepHI2A and RepHI1A(R478) to be polymerase I independent. However, the presence of consensus DnaA-binding domains upstream of the large ORFs of both replicons indicates the requirement for at least some host replication mechanisms.
pDTP61 and pDTP71 were each tested for stability within DH5 cells. Both of these constructs were not stably maintained, with loss rates of up to 5% per generation, indicating that neither RepHI2A nor RepHI1A(R478) expresses plasmid-maintenance characteristics.
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DISCUSSION |
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RepHI2A occurs within co-ordinates 11 and 14 on R478. A 1·9 kb EcoRI fragment subcloned from this region of R478 was previously shown to hybridize strongly with the IncHI2-specific diagnostic probe pULB2433 (Whelan & Colleran, 1992 ). DNA sequence analysis has revealed that this 1·9 kb region occurs within RepHI2A and contains all but one of the downstream iteron elements of RepHI2A but neither the large ORF nor the upstream replicon elements of RepHI2A. Since pULB2433 is recognized as a diagnostic probe for IncHI2 plasmids (Couturier et al., 1988
), it is likely that RepHI2A is common to all plasmids of this subgroup. Upstream iteron sequences normally specify the incompatibility characteristic of any particular iteron-controlled replicon (del Solar et al., 1998
). In contrast to most previously identified iteron-controlled replicons, RepHI2A contains an unusually large number of iterons located downstream of the rep gene. This probably explains why the IncHI2-specific incompatibility probe, pULB2433, hybridizes so strongly with the downstream region of RepHI2A.
Our results failed to show incompatibility between R478 and the cloned RepHI2A replicon even though RepHI2A (pDTP60) exhibited strong incompatibility with an alternatively cloned copy of itself (pDTP61). Similar observations have been reported between the RepHI1A replicon and its parent IncHI1 subgroup plasmid, R27 (Newnham & Taylor, 1994 ). This previous study suggested that the observed compatibility between R27 and RepHI1A was due to the presence of an additional functional replicon on R27, RepHI1B, which was known to function independently of RepHI2A (Gabant et al., 1994
). Our work has shown that R478 does contain a second functional replicon, RepHI1A(R478), which may explain the observed compatibility between R478 and RepHI2A. However, we have also shown that the cloned RepHI1A(R478) replicon (pDTP70) is strongly incompatible with both R478 and R27, which reflects the incompatibility between R478 and R27. These observations indicate that the mechanisms governing replication initiation from specific replicons of a multireplicon plasmid are complex and may involve preferential initiation from dominant replicons. Such preferential mechanisms have been suggested to govern the replication of the F plasmid under different metabolic states of the host cell (Lane & Gardner, 1979
; Gardner et al., 1980
).
RepHI1A(R478) is closely related to the RepHI1A replicon of the IncHI1 subgroup plasmid, R27 (RepHI1A(R27)). The repA gene of RepHI1A(R27) and its R478 homologue encode 85·9% amino acid identity (Fig. 4). The consensus iteron sequences (upstream of the repA ORFs) of both replicons are also very similar and are nearly perfectly aligned with respect to the repA sequence (Fig. 5
). The conserved regions within the consensus iteron sequence of these replicons may highlight motifs that are necessary for the binding of the Rep protein. cis-acting elements of an iteron-regulated replicon which occur upstream of the repA gene are essential for replicon function and are responsible for the specific incompatibility phenotype exhibited by a particular replicon (Helinski et al., 1996
). It is likely that the similarity between the upstream iteron sequences of RepHI1A(R478) and RepHI1A(R27) (Fig. 4b
) provides the molecular basis for the incompatibility between the plasmids R478 and R27.
It was demonstrated that IncHI1 and IncHI2 plasmids share little DNA homology (Grindley et al., 1973 ; Roussel & Chabbert, 1978
). The striking similarities between RepHI1A(R478) and RepHI1A(R27) suggest that genetic exchange occurred between these heterologous plasmids at some stage in their evolutionary development similar to that between IncHI1 plasmids and the F plasmid (Saul et al., 1988
). A greater understanding of the molecular mechanisms that give rise to such apparent replicon exchange events will increase our understanding of how these medically and environmentally important plasmids evolve and expand into new bacterial host environments.
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ACKNOWLEDGEMENTS |
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Received 4 December 2000;
revised 15 February 2001;
accepted 19 February 2001.
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