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1 Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands
2 Laboratory of Microbial Ecology, Centre for Ecological and Evolutionary Studies (CEES), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands
Correspondence
Lubbert Dijkhuizen
L.Dijkhuizen{at}rug.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). However, to date, only a single phenotype has been found to be associated with some of the actinomycete plasmids and integrative elements: when plasmid-carrying donor cells are grown on plates together with an excess of plasmid-lacking recipient cells, inhibition zones are formed after plasmid transfer, and these are known as pocks (Bibb et al., 1977).
The small circular actinomycete plasmids and integrative elements for which the replication mechanism has been identified have all been found to replicate via the rolling circle replication (RCR) mechanism (Hagege et al., 1993). RCR plasmids are divided into five families: pT181, pC194, pMV158 and pSN2 (del Solar et al., 1998), and a recently described family of RCR replicons of Corynebacterium spp. (Nesvera et al., 1997; Osborn et al., 2000). Actinomycete RCR plasmids and integrative elements belong either to the pC194 plasmid family (Hagege et al., 1993; Muth et al., 1995; Servin-Gonzalez, 1993; Suzuki et al., 1997), which is widely distributed in Gram-positive and Gram-negative bacteria, or to the fifth family (RCR replicons of Corynebacterium spp.). RCR is initiated when the replication initiator (Rep) protein binds to DNA cognate sites, and nicks the plus strand of the double-stranded origin (DSO). The Rep protein is covalently attached to the 5' phosphate at the nick site, while leading strand replication is initiated from the 3' OH end. After the leading strand has been fully displaced, the Rep protein cleaves the displaced ssDNA at the regenerated nick site. A series of cleavage/joining reactions generates a double-stranded plasmid and a circular single-stranded plasmid. The latter is converted into dsDNA using the single-stranded origin and the host replication machinery.
The actinomycete Amycolatopsis methanolica harbours a 13.3 kb integrative element, pMEA300. The complete sequence of pMEA300 has been determined previously (GenBank accession no. L36679), revealing 20 putative ORFs. Construction and characterization of deletion derivatives has allowed the identification of genes required for replication, regulation, integration and conjugation (Vrijbloed et al., 1994, 1995a, b, c). Previous experiments have suggested the presence of a system for high-frequency spontaneous mutagenesis on pMEA300 (Vrijbloed, 1996). Unfortunately, additional experiments have not been able to substantiate this mutator phenotype.
Based on structural and functional similarity, pMEA300 groups into a class that consists of integrative and conjugative elements (ICEs) (Burrus et al., 2002) of several actinomycetes (Raynal et al., 1998): SLP1 from Streptomyces coelicolor A3(2) (Bibb et al., 1981), pSAM2 from Streptomyces ambofaciens (Pernodet et al., 1984), pIJ110 from Streptomyces parvulus (Hopwood et al., 1984), pIJ408 from Streptomyces glaucescens (Hopwood et al., 1984; Sosio et al., 1989), pSG1 from Streptomyces griseus (Cohen et al., 1985), pSE101 (Brown et al., 1988) and pSE211 (Brown et al., 1990) from Saccharopolyspora erythraea, pMEA100 from Amycolatopsis mediterranei (Moretti et al., 1985), and probably pMR2, a plasmid from Micromonospora rosaria that has recently been sequenced (Hosted Jr et al., 2005).
pMEA300 is present mostly as an integrated form within a chromosomal gene encoding isoleucine tRNA (Vrijbloed et al., 1994), but it can also replicate autonomously. The region required for replication of pMEA300 has been previously minimized to two unlinked DNA fragments encoding OrfA and OrfB, and KorA (Vrijbloed et al., 1995a). In this paper, we show that OrfB is the Rep protein of pMEA300, and that it has unique DNA-binding properties.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), and the plasmid-pMEA300-deficient strain WV1 (Vrijbloed et al., 1994, 1995b), were used. The procedures followed for cultivation in batch cultures, harvesting of cells, and growth measurements, have all been described previously (de Boer et al., 1990). Escherichia coli strains JM109 and BL21(DE3) were grown in Luria–Bertani (LB) medium at 37 and 30 °C, respectively.
DNA manipulations.
A. methanolica plasmid DNA was isolated using the Qiagen Plasmid Midi kit, with the following modifications. Cells were harvested from 50 ml YEME (Vrijbloed et al., 1995a), and incubated at 37 °C for 30 min in a cell resuspension buffer (P1), which was supplemented with 4 mg lysozyme ml–1 (Sigma). After addition of lysis buffer (P2), the mixture was incubated for 15 min at room temperature, and then, after mixing with the protein precipitation solution (P3), it was placed on ice for 30 min. Total DNA was isolated with the Wizard Genomic DNA Purification kit (Promega) from 2 ml of overnight YEME cultures.
Plasmid DNA from E. coli was isolated using the QIAprep Spin Miniprep kit (Qiagen). All other DNA manipulations were done according to standard protocols (Sambrook et al., 1989).
Construction of pMEA300 deletion derivatives.
pMEA300 deletion derivatives used in this study were constructed from the E. coli–A. methanolica shuttle vector pWV136, which is devoid of integrative and conjugational functions (Vrijbloed et al., 1995a). pHK315 was obtained by removing the remaining transfer genes (ClaI–ScaI fragment) from pWV136. pHK313 is a SacI–ScaI deletion construct of pWV136, containing a disruption of the orfA gene that was created by digestion of the BstEII(2) site in orfA, and a subsequent Klenow DNA polymerase fill-in reaction, resulting in a frameshift starting at amino acid residue 66 (the complete length of OrfA is 170 aa).
Transformation of A. methanolica WV1.
A simplified version of a previously described method (Vrijbloed et al., 1995b) was used to transform A. methanolica WV1. This improved method, which omits the soft agar overlay step, is much less laborious, and yields similar transformation frequencies, as reported previously (between 3x104 and 5x104 transformants, using saturating concentrations of >1.0 µg plasmid DNA; Vrijbloed et al., 1995b). Additionally, the new procedure significantly reduces background growth on agar plates. In order to prepare competent cells, overnight cultures of A. methanolica WV1 on 50 ml trypticase soy medium (BBL) were grown to an OD430 of 5.0. After centrifugation (5 min, 3600 g), the cells were washed in 25 ml T10E1 (10 mM Tris/HCl, pH 7.5; 1 mM EDTA) and resuspended in T10E1 to an OD430 of 160. To 100 µl of the cell suspension, 10 µl 0.2 M MgCl2, 60 µl 4.17 M CsCl2, target DNA, and T10E1 to a total volume of 20 µl, were added, and mixed by pipetting. Subsequently, 200 µl 65 % (w/v) PEG-1000 (Koch Light) was added, and gently mixed by pipetting. The transformation mixture was incubated for 40 min at 37 °C. Following incubation, 1 ml T27M [3 % (w/v) trypticase soy broth and 7.3 % (w/v) mannitol] (37 °C) was added, gently mixed, and centrifuged (1 min, 10 000 g). After a second wash step with 1 ml T27M, cells were resuspended in 500 µl T27M, and incubated for 5–7 h at 37 °C in a shaking incubator. Finally, an appropriate amount of cell suspension was transferred to T27M agar plates containing kanamycin (15 or 20 µg ml–1). Transformants appeared after approximately 3 days of incubation at 37 °C.
Analysis of autonomous replication.
Autonomous replication of the plasmid deletion derivatives in A. methanolica WV1 was checked by Southern hybridization of untreated total DNA and plasmid preparations, and on total DNA and plasmid preparations that had been digested with appropriate restriction enzymes. Southern hybridization was performed with a DIG DNA Labelling and Detection kit (Roche Diagnostics) using the orfB (repAM) gene of pMEA300 as a probe.
Construction of RepAM overexpression plasmids.
The repAM gene was PCR-amplified from pMEA300 plasmid DNA using the following primers: 5'-GCGCATATGACCGCCAACCCCGGAGC-3' and 5'-CGCGGATCCTCAGGCGTTGTTGCCGACGAC-3'. The first primer introduces an NdeI restriction site (underlined) at the start codon, and the second primer introduces a BamHI site (underlined) at the termination codon. Reactions were performed with Vent DNA polymerase (New England Biolabs). Restriction-digested PCR fragments were cloned into the NdeI–BamHI sites of pET15B (Novagen), introducing an N-terminal His6 tag in the protein, yielding the expression plasmid pHisRepAM. Sequencing of the pHisRepAM construct (GATC Biotech) showed that no amplification errors had been introduced.
Overexpression and purification of RepAM protein.
A. methanolica pMEA300 RepAM protein was overexpressed in E. coli BL21(DE3)-pHisRepAM. Cells were grown at 30 °C in 50 ml LB medium containing 1 M sorbitol, 2.5 mM betaine, and 50 µg ampicillin ml–1. Protein expression was induced by addition of 0.5 mM IPTG at an OD660 of 0.3, and subsequent incubation at room temperature for 24 h. Cells were harvested by centrifugation, and resuspended in 1 ml 25 mM Tris/HCl, pH 8.5. Cell-free extracts were obtained by sonication and subsequent centrifugation (1 min, 10 000 g). His-tagged RepAM protein was purified with nickel nitrilotriacetic acid (Ni-NTA) resin (Qiagen), using the protocol of the manufacturer.
RepAM-binding assays.
Purified His-tagged RepAM protein was mixed with DNA fragments, and incubated for 30 min at 37 °C in 15 µl reaction buffer (10 mM Tris/HCl, pH 8, 5 mM MgCl2, 100 mM NaCl, 1 mM 2-mercaptoethanol, 5 mM DTT, and 50 µg BSA ml–1). Binding of DNA by RepAM was studied by analysing DNA mobility in 1.2 % (w/v) agarose gels. Following electrophoresis, gels were stained with ethidium bromide and photographed under UV illumination. The oligonucleotide probes used in the assay to determine the binding specificity of RepAM to the conserved 8 bp repeat were as follows: 5'-GGGCTGCTATCCTCTAGCGCCGTACCTATGAACGTGTCCCAGGC-3', and 5'-GGGCTGCTATCCTCTAGCTTGAAGTGTATGAACGTGTCCCAGGC-3'. dsDNA probes were obtained by mixing equivalent amounts of the 43-mer oligonucleotides with their complementary strand, heating to 95 °C for 10 min, and then slowly cooling to room temperature.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Deletion of the korA gene resulted in loss of replication in A. methanolica. The korA gene product probably has a regulatory function in replication, as its amino acid sequence shows extensive similarity to proteins of the GntR family of transcriptional repressors (Vrijbloed et al., 1995a). Deletion or disruption of orfB completely abolishes replication in A. methanolica (Vrijbloed et al., 1995a). Although orfA is located in an operon directly upstream of orfB, disruption of orfA (construct pHK313) did not lead to loss of autonomous replication, since free circular pHK313 DNA could still be isolated from the transformants (this study, data not shown). This leaves orfB as the sole candidate to encode the Rep protein. Similar to the position of the gene encoding the Rep protein (repSA) on pSAM2 (Hagege et al., 1994), orfB is located directly upstream of the genes encoding the excisionase (xis), the integrase (int), and the chromosome attachment site (attP). This conserved arrangement of rep, xis, int and attP seems to be a general feature of the actinomycete ICE class. Although no sequence similarity has been found between OrfB and RepSA, the deletion studies and the conserved location substantiate our conclusion that OrfB is the Rep protein of pMEA300. Accordingly, orfB and its product are referred to as repAM and RepAM, respectively. Interestingly, RepAM of pMEA300 shows no similarity to any known bacterial Rep protein. However, RepAM is highly similar to uncharacterized ORFs of two integrative elements: pSE211 (18.1 kb) of Sac. erythraea (Brown et al., 1990), and pMEA100 (23.7 kb) of A. mediterranei (Madon et al., 1987; unpublished data), which is currently being sequenced by our group. These two elements also belong to the actinomycete ICE class, and their repAM homologues are also located directly upstream of xis, int and attP. This suggests that these proteins also function as Rep proteins.
RepAM binds to the 3' end of its own coding sequence
Initiation of plasmid replication often involves binding of a plasmid-encoded replication protein to DNA cognate sites in the origin of replication (ori) of the plasmid (Espinosa et al., 2000). To determine whether RepAM is able to bind pMEA300 DNA, binding assays were performed with purified His-tagged RepAM protein, and pMEA300-derived DNA fragments. To obtain RepAM protein, the repAM gene was cloned into the expression vector pET15b, and transformed to E. coli BL21(DE3). Cell extracts from E. coli transformed with pHisRepAM revealed an additional band of approximately 48 kDa on SDS-PAGE gels, which was in agreement with the expected size of RepAM (45.3 kDa). RepAM was purified from the cell extracts using Ni-NTA column chromatography.
BssHII-restricted pMEA300 plasmid DNA was incubated with different concentrations of RepAM protein. Fig. 1 shows that addition of RepAM caused a shift in mobility of the 1007 bp BssHII fragment. This fragment contains the 3' part of repAM, the complete xis gene, and the first part of the int gene. The extent of retardation depended on the concentration of RepAM, since increasing amounts of RepAM gradually decreased the mobility of the bound DNA fragment. This indicates that either the bound DNA fragment contains more than one binding site for RepAM, or RepAM binds to this DNA fragment as a multimer.
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). Addition of purified RepAM protein prevented migration of the repAM PCR fragment, the 454 bp AvaI fragment, and the 1096 bp BssHII fragment of pHK315. The RepAM-bound DNA did not migrate through the agarose gel, but remained in the slots, which could be observed by ethidium bromide DNA staining, and by Coomassie brilliant blue protein staining (not shown). In this way, we were able to narrow down the RepAM-binding site to a 237 bp DNA fragment; therefore, the putative origin of replication locates within the 3' end of the repAM gene. No retardation was observed when eluates obtained from E. coli BL21(DE3) containing the empty vector pET15b were used, showing that the observed retardation was RepAM dependent.
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G of –57 kcal mol–1 (–238.5 kJ mol–1). The hairpin starts with a large 23 bp inverted repeat and includes three identical 8 bp inverted repeats (GCCGTACC). Similar hairpin structures with multiple identical 8 bp inverted repeats were found at the 3' end of the putative replication initiator genes of pSE211 [174 bp; G, –67 kcal mol–1 (–280.3 kJ mol–1)] and pMEA100 [178 bp; G, –74 kcal mol–1 (–309.6 kJ mol–1)] (Fig. 3). Moreover, all three hairpins contain a consensus sequence that is similar to the nicking site (5'-CTTGAT-3') of the pC194 RCR family of plasmids (Gruss & Ehrlich, 1989) (Fig. 4). These putative nicking sites are directly flanked by the conserved 8 bp repeats. Secondary DNA structures predicted from the hairpin sequences, using the online secondary structure prediction tool mfold (Zuker, 2003), confirmed that the putative nicking sites are located within unpaired regions of the hairpins (Fig. 5). The in vivo conformation of the RepAM-binding region, and the conformation of the DNA in our DNA-binding assays, are unknown. The DNA can have a ‘normal’ linear dsDNA conformation, or it can fold into two predicted hairpin structures – one on the leading strand, and one on the lagging strand – resulting in a cruciform structure. Independent of the conformation, the short 8 bp repeats will be paired with either their inverse complement on the lagging strand or their counterpart within the putative hairpin structure, whereas loops are formed only when in the hairpin conformation. If the secondary structure predictions reflect the in vivo conformation upon binding of RepAM, then it would be possible for RepAM to access the nicking site for cleavage; this process is known to occur in plasmids that replicate via the RCR mechanism (Khan, 1997; del Solar et al., 1998).
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). Our results show that the 8 bp inverted repeats play an essential role in RepAM binding, since gel retardation was observed only when the DNA fragments included at least one of the six conserved repeat sequences.
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35 % in RepAM-dependent binding. A residual binding (20 %) of the probe without the conserved 8 bp was most likely to be due to non-specific binding, which is frequently observed for DNA-binding proteins (Lane et al., 1992). These results show that RepAM binds specifically to DNA fragments containing the 8 bp repeat, and thus that the hairpin structure contains multiple RepAM-binding sites.
RepAM belongs to a novel class of Rep proteins
The DSO region of many RCR plasmids have secondary structures, such as hairpins and cruciforms (Gros et al., 1987; Moscoso et al., 1995; Noirot et al., 1990; Wang et al., 1993). The presence of the hairpin structure at the 3'end of the repAM gene, to which the RepAM initiator protein binds, and the presence of a putative nicking site within the hairpin, suggest that pMEA300 also replicates via the rolling circle mechanism. However, the amino acid sequence of RepAM does not share any similarity with known RCR Rep proteins, or with any other Rep protein identified so far. We were also unable to detect any of the consensus sequences characteristic for RCR Rep proteins, such as motifs of the catalytic domain, or those of a putative metal-binding domain (del Solar et al., 1998; Ilyina et al., 1992). Another distinctive feature of pMEA300 is that the putative origin of replication is located within the repAM gene itself, at the 3' end. This functional organization differs markedly from other RCR plasmids, in which the DSO is located either upstream of rep (pC194, pMV158 and pSN2) or in the proximal part of the rep gene, as in the pT181 family (Novick, 1989). A similar atypical distal location of the DSO has been found in the rep gene of plasmid pGA1 of the actinomycete Corynebacterium glutamicum; this plasmid is a member of the fifth family of RCR plasmids (RCR replicons of Corynebacterium spp.) (Abrhamova et al., 2002). However, the Rep proteins of pGA1 and pMEA300 show no significant sequence similarity (<20 %). Furthermore, the sizes of pMEA300 (13.3 kb), pSE211 (18.1 kb) and pMEA100 (23.7 kb) are believed to be too large for a rolling circle mechanism of replication, due to structural instability of large ssDNA intermediates (Helinski et al., 1996). However, ssDNA intermediates can be coated by ssDNA-binding proteins (SSBs), protecting them against nuclease attack and formation of undesired secondary structures (Greipel et al., 1987). Moreover, host-encoded SSBs are known to play a role in replication of plasmids (Helinski et al., 1996; Khan, 1997). Preliminary sequence analysis of pMEA100 has revealed the presence of a unique SSB-encoding gene. Possibly, this additional SSB is required, in addition to chromosomally encoded SSBs, to maintain stability of the 23.7 kb ssDNA intermediate. pMEA300 might be small enough to allow RCR-type ssDNA intermediates to be stabilized by the host SSBs.
In comparison with the Rep proteins of pC194 family of RCR plasmids, RepAM of pMEA300 shares similarity within the putative nicking site only (Fig. 4
), and does not contain any of the other conserved motifs found in pC194, or the four other families of RCR plasmids (del Solar et al., 1998). Replication proteins and structural features, such as iterons, as found in theta-replicating plasmids (del Solar et al., 1998), are also absent on pMEA300. Therefore, we conclude that RepAM of pMEA300, and its homologues of pSE211 and pMEA100, are members of a novel class of Rep proteins that replicate most probably via an RCR-type replication mechanism. Elucidation of the 3D structure of RepAM could provide important information on the DNA–protein interactions, and the mechanism of replication, of this novel class of Rep proteins.
In addition to functioning as the origin of replication, the unique location of the RepAM-binding site might have additional effects on the expression regulation of the gene encoding RepAM, or on that of the gene encoding Xis, which is located directly downstream of repAM, and is co-transcribed with it.
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ACKNOWLEDGEMENTS |
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Received 6 December 2005;
revised 22 June 2006;
accepted 5 July 2006.
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