Characterization of two autoreplicative regions of the IncHI2 plasmid R478: RepHI2A and RepHI1A(R478)

David T. Pagea,1, Kenneth F. Whelanb,1 and Emer Colleran1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plasmids of the incompatibility groups HI and HII (IncH plasmids) generally confer multiple antibiotic resistances upon their host pathogenic strain. IncHI group plasmids are distinguished by their property of optimal transfer by conjugation at temperatures below 30 °C, allowing for the spread of multiple antibiotic resistance outside their host natural environment, the gut. Plasmids of the IncHI1 subgroup encode multiple replicons. In the present study it is shown that the prototype IncHI2 subgroup plasmid, R478, contains at least two iteron-controlled autoreplicative regions, RepHI2A and RepHI1A(R478). The DNA sequence and the molecular characteristics of each replicon region are described. RepHI2A is unique to plasmids of the IncHI2 subgroup and contains an unusually large number of iteron sequences downstream of the replication initiator gene. The nucleotide sequence of the replication initiator gene and of the iterons within RepHI1A(R478) show very close similarity with those of the previously reported RepHI1A replicon of the IncHI1 subgroup plasmid R27. The presence of RepHI1A(R478) on R478 most likely accounts for the observed incompatibility between R478 and plasmids of the IncHI1 subgroup. These are the first autoreplicative regions from an IncHI2 subgroup plasmid to be described.

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.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plasmids belonging to the incompatibility group H (IncH plasmids) encode multiple resistance determinants and have been associated with many Salmonella-related epidemics and pandemics throughout the world over the last 30 years (Anderson, 1975 ; Goldstein et al., 1986 ; Cordano & Virgilio, 1996 ). Two different H incompatibility groups, IncHI and IncHII, are recognized (Bradley et al., 1982 ). The main defining characteristic of plasmids from the IncHI group is their ability to preferentially transfer by conjugation at temperatures below 30 °C, allowing for the spread of multi-resistance under environmental conditions (Rodriguez-Lemoine et al., 1975 ; Maher & Colleran, 1987 ).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains, plasmids, media and antibiotics.
The bacterial strains and plasmids used in this study are listed in Table 1. The Escherichia coli strain DH5{alpha} was used for all cloning experiments. Plasmids R478 and pKFW141 were maintained in strains J53-2 and J53-1 respectively. Bacterial cultures were grown on tryptone soya broth (Oxoid). Minimal medium was prepared by standard methods (Sambrook et al., 1989 ). Antibacterial agents were used at the following concentrations: ampicillin, 100 µg ml-1; chloramphenicol, 50 µg ml-1; trimethoprim, 20 µg ml-1; nalidixic acid, 30 µg ml-1; rifampicin, 50 µg ml-1; kanamycin, 100 µg ml-1.


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Table 1. E. coli strains and plasmids used in this study

 
Plasmid preparation and DNA manipulations.
All plasmid DNA was isolated according to the method of Birnboim & Doly (1979) followed by caesium chloride/ethidium bromide density gradient ultracentrifugation. Restriction enzymes were supplied by Roche Molecular Biochemicals or Promega. T4 DNA ligase was obtained from Promega. All restriction digests and DNA ligations were performed as recommended by the manufacturers. DNA manipulations, transformations and agarose gel electrophoresis were carried out by standard procedures (Sambrook et al., 1989 ).

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{alpha} 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{alpha} 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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Subclones and miniplasmids of R478
An origin of replication and a HI2-specific incompatibility region were previously reported within the 51 kb SalI-B fragment of R478 (Whelan & Colleran, 1992 ). A series of transfer-defective (Tra-) Tn7 transposon insertion mutants of R478 have also been described where the Tn7 insertion was mapped within the SalI-B fragment (Page et al., 1999 ). Miniplasmids of these Tra- mutants, including pDTP5, were generated by restricting with SalI, religating and selecting on media containing trimethoprim. Further derivatives of these miniplasmids, including pDTP25, were similarly generated using EcoRI (Fig. 1). Autoreplicative regions were identified by ligation to the chloramphenicol (Cm) resistance cassette of the interposon plasmid, pHP{Omega}-Cm, transformation and selection of E. coli DH5{alpha} cells on media containing Cm.



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Fig. 1. Generation of miniplasmids of the transfer-defective Tn7 insertion mutant of R478, pKFW141. RepHI2A (Rep) is represented by a black dot. Tn7, indicated by a hatched box, contains a single EcoRI recognition site which divides the transposon into two fragments, Tn7L (5·1 kb) and Tn7R (8·9 kb) (Gosti-Testu et al., 1983 ). pDTP5 contains many unmapped EcoRI sites. *EcoRI within pDTP5 denotes the known EcoRI site within Tn7. Tn7L encodes the determinant conferring Tp resistance. E denotes EcoRI recognition sites within pDTP25. The EcoRI site (E) indicated by an arrow occurs within RepHI2A. The size (kb) of each EcoRI restriction fragment of pDTP25 is indicated by the numbers inside the circle map (not drawn to scale).

 
RepHI2A
A total of 2889 bp of DNA sequence was obtained from the 12·8 kb, EcoRI-derived, miniplasmid of R478, pDTP25 (Fig. 1). This region was found to contain genetic structures consistent with their being an iteron-controlled plasmid replicon region. These structures include: (i) a large ORF (1·1 kb) that shows significant amino acid homology with replication initiation proteins (Rep) of many different iteron-controlled plasmid replicons; (ii) a series of short, tandemly repeated oligonucleotide sequences (iterons) which flank the large ORF; (iii) characteristic E. coli origin elements upstream of the ORF including multiple recognition sites for host DNA adenine methylase (Geiger & Modrich, 1979 ) and a consensus recognition site for the binding of the host replication-associated DnaA protein (Fuller & Kornberg, 1983 ). This region of R478 was designated RepHI2A.

Eight different 18 bp iterons flank the rep homologue of RepHI2A (four upstream iterons, A–D, and four downstream iterons, E–H). Nine different 76 bp iterons, I–Q, 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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Fig. 2. Sequences and alignments of the small (A–H and R) and the large (I–Q) iterons of RepHI2A. Sequences are shown 5'->3'. The consensus iteron sequence of RepHI2A is shown underneath. N, random nucleotide; R purine nucleotide; Y, pyrimidine nucleotide.

 
RepHI1A(R478)
A total of 2998 bp of DNA sequence was generated from the construct pKFW50, which is a 15 kb EcoRI subclone of the SalI-B fragment of R478 (Whelan & Colleran, 1992 ). As with RepHI2A, this DNA sequence was found to contain genetic structures associated with iteron-controlled replicons, including a large ORF, flanking iteron sequences and consensus recognition sites for host E. coli replication-associated elements. The sequence of this replicon-associated region of R478 shows very similar homology to that of the RepHI1A replicon of the IncHI1 plasmid R27 (Newnham & Taylor, 1994 ), including 73·5% nucleotide identity and 85·9% amino acid identity (Fig. 4) between the large ORFs (which are each 873 bp in length) and very similar iteron consensus sequences (Fig. 3). Nucleotide sequence comparisons of RepHI1A with RepHI1A(R478) show that the ORFs, iterons, methylation sites and DnaA recognition sites are perfectly aligned (Fig. 5). No other significant homologies or alignments were observed between RepHI1A and this region of R478 (mapped to within coordinates 27 and 30), which was designated RepHI1A(R478). Four of the iterons occurring downstream of the rep gene of RepHI1A(R478) were direct repeats of the upstream iteron sequences, whereas the remaining downstream iterons were inverted repeats.



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Fig. 4. CLUSTAL W alignment of the amino acid sequences encoded by the Rep proteins of RepHI1A(R478) and RepHI1A(R27). * indicates identical amino acids.

 


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Fig. 3. Sequences and alignments of the fourteen iterons of RepHI1A(R478). Sequences are shown 5'->3'. The consensus iteron sequence is also shown and is compared with the consensus iteron sequence of RepHI1A(R27).

 


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Fig. 5. CLUSTAL W alignment of the DNA sequences of RepHI1A(R478) with RepHI1A(R27). Shown are the upstream iterons as well as the consensus recognition sites for host E. coli DNA adenine methylase (underlined) and DnaA (italics). Significant homology was not observed further upstream or downstream of the regions shown. * indicates identical nucleotides. Start and stop codons are indicated in bold. The consensus iteron sequences of RepHI1A(R478) are boxed and are indicated in numerical sequence above each box. An arrow indicates the orientation of each iteron. The upstream iteron sequences of the RepHI1A(R27) replicon are also indicated within boxes.

 
Incompatibility assays
RepHI2A. The construct pDTP60 consists of a 2·4 kb StuI–SacI fragment of pDTP25 containing RepHI2A cloned into pUC119 (Fig. 6a). pDTP60 contains all of the upstream iterons, and 10 of the downstream iterons (E–N) of RepHI2A. This construct did not show incompatibility with representative plasmids from each of the IncH groups and subgroups: R27 (IncHI1), R478 (IncHI2), MIP233 (IncHI3) and pHH1508a (IncHII). In a control experiment for the incompatibility assay, pDTP60 showed strong incompatibility with pDTP61, which consists of the 2·4 kb RepHI2A fragment ligated to the Cmr cassette of pHP{Omega}-Cm (Fig. 6b). pDTP62 was generated from pDTP60 by deleting the 819 bp EcoRI–SacI fragment. Only a single downstream iteron, iteron E, occurs on pDTP62 (Fig. 6c), and this was found to have similar incompatibility characteristics to pDTP60.



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Fig. 6. Linear maps of constructs containing replicon regions cloned from R478 (not drawn to scale). Construct names and replicons are indicated above each map, with the plasmid sizes (bp) indicated below the map. The ORF of each replicon region is indicated by a hatched box, with the start codon shown by an internal arrow. Iteron sequences are indicated by arrows with the orientation of each iteron following the arrow direction. Recognition sites for endonucleases StuI, EcoRI, SacI, ClaI and HindIII are indicated. (a, b, c) The shorter, 18 bp iterons of RepHI2A are indicated by solid arrowheads; the longer, 76-base pair iterons are indicated by open arrows. pDTP60 was generated by ligating the 2389-base pair StuI–SacI fragment (containing RepHI2A) of R478 to pUC119 double-digested with SmaI and SacI. The other constructs were generated as described in the text. Ampr indicates the ampicillin resistance determinant of the vector plasmid, pUC119. Cmr refers to the 3·1 kb chloramphenicol resistance cassette from the interposon plasmid pHP{Omega}-Cm.

 
RepHI1A(R478). pDTP70 was obtained by ligating a 2·497 kb ClaI–HindIII fragment of pKFW50, containing RepHI1A(R478), to the phagemid vector pUC119 (Fig. 6d). This fragment was also ligated to the Cmr cassette of pHP{Omega}-Cm to generate pDTP71 (Fig. 6e). Incompatibility tests were carried out between pDTP70 and the test IncH plasmids as listed above. pDTP70 was strongly incompatible with both R478 and R27 and was compatible with pHH1508a and MIP233. Strong incompatibility was also observed in a control incompatibility test between pDTP70 and pDTP71.

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{alpha} 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{alpha} 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.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This work describes the cloning, DNA sequence and analysis of two autoreplicative determinants, RepHI2A and RepHI1A(R478), of the IncHI2 plasmid R478. These are the first such elements from IncHI2 subgroup plasmids to be described.

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.


   ACKNOWLEDGEMENTS
 
The interposon plasmid pHP{Omega}-Cm was a generous gift from Professor Dr Joachim Frey, Institute of Veterinary Bacteriology, University of Berne, Laenggassstrasse 122, 3012 Bern, Switzerland. This work was supported by a grant from Forbairt (grant no. Sc/95/132). D.T.P. was the recipient of a Forbairt studentship. K.F.W. was the recipient of an Eolas studentship.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 4 December 2000; revised 15 February 2001; accepted 19 February 2001.



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