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Sequence analysis of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (P25X) reveals a novel replication region
FEMS Microbiology Letters 158 (1998) 159^165
Sequence analysis of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (P25X) reveals a novel replication region Stephen M. Kwong, Chew Chieng Yeo, Damian Chuah, Chit Laa Poh * Department of Microbiology, Faculty of Medicine, National University of Singapore, Lower Kent Ridge Road, Singapore 119260, Singapore Received 3 November 1997 ; accepted 6 November 1997
Abstract The replication region of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (strain P25X) was localized within a 5.9kbp DNA fragment and its sequence was determined. An interesting feature of the sequence is the presence of a 1.3-kbp region containing seven, highly conserved, direct repeats of 72 bp in length. The pRA2 replication region has two open reading frames (ORFs). ORF1 appeared to be essential for replication and had the potential to encode a novel 30-kDa protein with a predicted helix-turn-helix motif located at the C-terminal end. ORF2 was not essential for replication and may encode for a 37-kDa protein which shares 41% and 27% amino acid sequence identity to the KfrA proteins from plasmids RK2 and R751, respectively. The essential region of replication was narrowed down to 2819 nucleotides and included four of the seven 72-bp direct repeats, a potential DnaA-binding site and ORF1. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Plasmid replication; Direct repeat; Replication initiation protein; kfrA
1. Introduction Pseudomonads have promising biotechnological potentials in agriculture, the chemical industry and environmental bioremediation due to their vast metabolic versatility. Plasmids are important in the investigation of degradative processes since they often encode catabolic genes and provide a means of genetic transfer between genera. Pseudomonas alcaligenes NCIB 9867 (P25X) is able to degrade 2,5-xylenol, 3,5-xylenol and m-cresol via the gentisate pathway [1]. The native plasmid of P25X, pRA2 * Corresponding author. Tel.: +65 (87) 43674; Fax: +65 (77) 66872; E-mail: [email protected]
(33 kbp), has a narrow-host-range and is self-transmissible but attempts at curing it have proven unsuccessful [2,3]. The plasmid of P. alcaligenes C-0 [4] and more recently those of P. alcaligenes type strain [5] have also been reported to be incurable. This suggests that these plasmids not only provide a selective advantage for the host cell, as with the majority of plasmids, but may actually play a critical role in survival even under optimal growth conditions. Autonomous replication is a fundamental plasmid characteristic. A plasmid replicon usually contains a speci¢c protein that is necessary for initiation of its own replication, while the majority of the requirements for replication such as initiation factors, heli-
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cases, primases and polymerases are provided by the host [6]. By encoding their own initiation proteins, plasmids are able to replicate independently of the chromosome and thus de¢ne their own copy number in order to maintain stable inheritance. Plasmid-encoded initiation proteins, such as RepA from pPS10 [7] and TrfA of RK2 [8], bind to speci¢c repetitive sequences (iterons) which are located within the vegetative origin of replication, oriV. Iterons generally range from 17 to 22 nucleotides in length, exist in multiple copies and occur in the replicons of many Gram-negative bacteria [9]. In this study, we report the novel regions associated with the replication of plasmid pRA2, provide a comparison of regions sharing similarity to previously reported plasmids and de¢ne an essential region for autonomous replication.
2. Materials and methods 2.1. Bacterial strains and media P. putida RA713 [10] was used in the isolation of the mini-replicon while E. coli strain JM109 [11] was used in the generation of recombinant clones. E. coli strains were cultivated at 37³C and P. putida at 32³C in Luria-bertani medium. Antibiotics were used in the following concentrations: ampicillin, 100 Wg ml31 ; kanamycin, 50 Wg ml31 ; streptomycin, 100 Wg ml31 ; and chloramphenicol, 15 Wg ml31 . 2.2. Sequencing and analysis Both strands of the pRA2 mini-replicon were sequenced. Nested deletion clones of the mini-replicon were obtained using a combination of the Deletion Factory1 System (Gibco/BRL) and the double stranded Nested Deletion Kit (Pharmacia Biotech). DNA sequencing and oligonucleotide synthesis were performed using an ABI 373 DNA sequencer and an ABI 392 DNA synthesizer (Applied Biosystems/Perkin-Elmer), respectively. DNA for sequencing was prepared using the mini alkaline lysis method for DNA extraction [12] or the Wizard1 Plus Minipreps (Promega). DNA sequencing was carried out using the ABI Taq Dye Deoxy Cycle Sequencing Kit (Applied Biosystems/Perkin-Elmer) according to
the manufacturers instructions. DNA sequences were compiled and analyzed using DNASIS (Hitachi Software Engineering) and the BLAST server [13]. 2.3. Molecular techniques DNA cloning, agarose gel electrophoresis, preparation of competent cells and plasmid transformations were performed as previously described [12]. Restriction enzymes were obtained from Boehringer Mannheim or New England Biolabs.
3. Results 3.1. Isolation of a mini-replicon from pRA2 A mini-replicon of the P. alcaligenes P25X plasmid pRA2 was constructed by partial digestion of the 33kbp plasmid with Sau3AI and ligation of the resulting fragments to a 2.2-kbp kanamycin resistance gene (KmR ), isolated from a mini-Tn5 transposon [14]. The ligation mixture was transformed into P. putida RA713, a plasmid-free strain of Pseudomonas putida NCIB 9869 (strain P35X) [10], and transformants were selected on LB plates supplemented with kanamycin. A single KmR transformant was isolated and analysis of its plasmid showed that it contained approximately 6 kbp of the pRA2 plasmid. 3.2. DNA sequence of the replication region Restriction analysis of the mini-replicon allowed us to localize the position of the replication region with respect to the pRA2 restriction map (Fig. 1). Sequence analysis of the mini-replicon showed that it consisted of a continuous 5990-bp fragment derived from pRA2 (GenBank accession no. AF020268). The DNA sequence revealed a highly repetitive region of approximately 1.3 kbp (nt 879^2185). Within this region are seven well conserved 72-bp direct repeats which vary from the deduced consensus sequence by only one or two bases (Fig. 2). The repeats are arranged into two groups, being separated by 302 bp of non-repetitive DNA. The ¢ve spacer regions between the repeats of each group also share considerable sequence homology to one
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Fig. 1. Schematic representation of the pRA2 replication region with reference to the pRA2 restriction map. The single BglII restriction site on pRA2 was arbitrarily taken as coordinate 0 [2]. Sequence analysis revealed a highly repetitive region (dark arrows), two signi¢cant ORFs (open arrows), a potential DnaA-binding site (shaded diamond) and an A+T-rich region (dark box). The lines below indicate the relative size of the replication region covered by the deletion clones. Heavy lines indicate those that retained replicative ability in P. putida RA713 while thin lines represent deletion clones that lost the ability to replicate in P. putida RA713.
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another and range from 86 to 108 nucleotides in length. The replication region has an 88% A+T-rich region of 59 bp (nt 4424^4482) adjacent to a 87% G+C-rich region of 45 bp (nt 4369^4413) and a possible DnaA-binding site, 5P-TTATCCACA-3P (nt 3713^3721), matching the consensus sequence [TT(T/A)TNCACA] of the binding site for DnaA in E. coli [15]. The replication region sequence had an overall G+C content of 60%. 3.3. Potential open reading frames Two signi¢cant ORFs were identi¢ed in the replication region of pRA2. ORF1 (nt 2745^3575) is located 559 bp downstream from the repetitive region and encodes a putative 276-aa protein with a molecular weight of 30 kDa. A BLAST search failed to ¢nd any sequence similarity to proteins listed in the databases but the predicted secondary structure of ORF1 indicates a helix-turn-helix motif located at the C-terminal end. ORF2 (nt 4493^5518) has the potential to encode a 37-kDa protein that shares amino acid sequence homology to the KfrA protein from plasmid RK2 (41% identity, 59% similarity) as well as the KfrA protein of plasmid R751 (27% identity, 45% similarity). 3.4. Deletion analysis Deletion clones of the pRA2 mini-replicon were used to de¢ne a minimal region for autonomous replication. Two of the clones (7.4SS and 8.15SS) which
had deletions in the same direction were found to retain 4059 and 3673 bp of the replication region, respectively (Fig. 1). The larger subclone 7.4SS could be transformed and maintained in P. putida RA713 but repeated attempts to transform clone 8.15SS into RA713 were unsuccessful. Both clones 7.4SS and 8.15SS had deletions which removed the A+T-rich region as well as the ORF2. Clone 7.4SS was found to retain the DnaA-binding site which had been deleted in 8.15SS (Fig. 1). Several clones (H2K8, 4509 bp; H2K25, 5449 bp; H2K4, 5845 bp) which were larger than clone 7.4SS could also be transformed and maintained in RA713 while the smaller clone 11.11SS (2515 bp) could not be maintained in RA713. Only one of the deletion clones from the opposite direction (clone 3.4KC) could be transformed and maintained in RA713 and this clone was the result of the removal of 1240 bp from the 5P end which included two entire direct repeats as well as the ¢rst 7 bp of a third repeat (Fig. 1).
4. Discussion The majority of plasmids found in Gram-negative bacteria are known to possess one or more clusters of direct repeats, designated as iterons, within their replication origin region. Studies so far have shown that iterons are usually comprised of 17^22 nucleotides and serve as speci¢c binding sites for the replication initiation protein [7,8,16], thus being essential for plasmid replication. The repetitive region of the pRA2 replicon is shown to contain direct repeats that are far larger than any of the previously re-
Fig. 2. Nucleotide sequences of the seven highly conserved 72-bp direct repeats found in the replication region of pRA2. Each repeat is consecutively numbered 1^7 in the order in which they occur. Boxed regions indicate perfect nucleotide sequence conservation while the deduced consensus sequence is shown below.
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ported iterons [9]. While these direct repeats could have a probable role analogous to iterons in the replication of other plasmids, further investigations are clearly needed to elucidate the signi¢cance of the relatively large and highly conserved sequences of the pRA2 iterons. We believe that the putative protein encoded by ORF1 is the pRA2 replication initiation protein based on the following observations:
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(i) ORF1 is the only open reading frame located within the minimal region of replication which could potentially encode a protein that falls within the size range of most plasmid-encoded replication initiation proteins (25^49 kDa); (ii) the predicted secondary structure of the ORF1-encoded protein shows a probable helix-turn-helix DNA-binding motif at the C-terminal end; and (iii) no transformants were iso-
Fig. 3. A: The aligned amino acid sequences of the KrfA proteins from plasmids pRA2 of Pseudomonas alcaligenes NCIB 9867, RK2 of Pseudomonas auroginosa, and R751 of Enterobacter aerogenes. Identical amino acids are in bold letters and marked with an asterisk. Dashes represent gaps which have been inserted to maximize homology. B : DNA sequences of the promoter regions found upstream of the respective kfrA start codons. Arrows represent inverted repeats present in the promoter regions of kfrAR751 and kfrApRA2 . Also shown is the DNA sequence homology between the promoter regions of kfrApRA2 and kfrARK2 .
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lated when ORF1 was disrupted by insertional mutagenesis. While the amino acid sequence of ORF1 does not show any similarity to amino acid sequences deposited in the databases, short nucleotide sequences within the pRA2 repetitive region were found to share similarity with the pECB2 plasmid of P. alcaligenes type strain [5]. The sequence 5PGCCGGAATCTCTCCCTGAT-3P that is present ¢ve times in the iteron region of pRA2 is also found in the replication region of pECB2, albeit only once. However, a shorter subsequence of this, 5PCCGGAATC-3P, is found to occur eleven times in pRA2 and three times in plasmid pECB2. Despite these similarities it appears that the two plasmids may have acquired di¡erent modes of replication since the putative initiation protein of pECB2 does not share any amino acid sequence homology with the ORF1-encoded protein of pRA2, but rather, shares 71% amino acid sequence identity to the RepA initiation protein of pPS10 isolated from Pseudomonas syringae pv. savastanoi [5,17]. Analysis of deletion clones derived from the pRA2 mini-replicon demonstrated that two of the 72-bp direct repeats (proximal to the 5P region), the A+Trich region and ORF2 are not essential for pRA2 replication (Fig. 1). However, the potential DnaAbinding site may be vital for pRA2 replication as the di¡erence in clone 7.4SS, which could replicate in strain RA713, and clone 8.15SS, which could not, lies in the presence of the DnaA-binding site in clone 7.4SS and the absence of the site in the latter. The DnaA-binding sites found in the replication regions of both pPS10 and RK2 were reportedly required for e¤cient plasmid replication [8,17,18] while the DnaA-binding site in plasmid pSC101 was found to be essential for its replication [19]. The similarity in amino acid sequence between the ORF2-encoded protein (KfrApRA2 ), the KfrARK2 and KfrAR751 proteins (Fig. 3A) strongly suggests that they have a similar function and evolutionary origin. KfrARK2 has been proposed to be involved in plasmid partition [20,21]. Sequence conservation between the three KfrA-like proteins is highest at the C-terminal end, implying functional importance in this region. The N-terminal regions of KfrARK2 and KfrAR751 , suspected to contain DNA-binding motifs, have diverged considerably as have their respective promoter regions which are the potential KfrA-bind-
ing sites and points of autorepression [20]. Investigations of the KfrARK2 protein revealed that its transcription is autoregulated by the KfrARK2 protein itself in addition to being suppressed by the korABF operon [21]. The promoter region of kfrApRA2 shows some DNA sequence similarity to the kfrARK2 promoter and, like the kfrAR751 promoter, contains an inverted repeat capable of forming a palindrome (Fig. 3B). Sequences downstream of the pRA2 replication region suggest further similarity to plasmids of the IncP group with additional open reading frames encoding proteins similar to KorA as well as other proteins involved in conjugation in both RK2 and R751 (data not shown). Therefore plasmid pRA2 may provide further insight into our understanding of plasmid functions since it appears to share similar systems of plasmid partition and conjugal transfer to plasmids of IncP while the mode of replication seems to be quite di¡erent to those studied to date.
Acknowledgments This work was supported by the National University of Singapore academic research Grant no. RP95-0383 to C.L. Poh.
References [1] Hopper, D.J. and Chapman, P.J. (1971) Gentisic acid and its 3- and 4-methyl substituted homologues as intermediates in the bacterial degradation of m-cresol, 3,5-xylenol and 2,5-xylenol. Biochem. J. 122, 19^28. [2] Yeo, C.C. (1997) Mobile genetic elements of Pseuodomonas alcaligenes NCIB 9867. PhD. thesis, National University of Singapore. [3] Poh, C.L. and Tham, J.M.L. (1993) Genetic system in Pseudomonas alcaligenes NCIB 9867. FEMS Microbiol. Lett. 106, 253^258. [4] Kroëckel, L. and Focht, D.D. (1987) Construction of chlorobenzene-utilizing recombinants by progenitive manifestation of a rare event. Appl. Environ. Microbiol. 53, 2470^2475. [5] Charnock, C. (1997) Characterization of the cryptic plasmids of the Pseudomonas alcaligenes type strain. Plasmid 37, 189^ 198. [6] Kuëes, U. and Stahl, U. (1989) Replication of plasmids in gram-negative bacteria. Microbiol. Rev. 53, 491^516. [7] Garc|èa de Viedma, D., Giraldo, R., Ruiz-Echevarr|èa, M.J., Lurz, R. and D|èaz-Orejas, R. (1995) Transcription of repA,
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[8]
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the gene of the initiation protein of the Pseudomonas plasmid pPS10, is autoregulated by interactions of the RepA protein at a symmetrical operator. J. Mol. Biol. 247, 211^223. Pinkney, M., Diaz, R., Lanka, E. and Thomas, C.M. (1988) Replication of mini RK2 plasmid in extracts of Escherichia coli requires plasmid-encoded protein TrfA and host-encoded proteins DnaA, B, G, DNA gyrase and DNA polymerase III. J. Mol. Biol. 203, 927^938. Helinski, D.R., Toukdarian, A.E. and Novick, R.P. (1996) Replication control and other stable maintenance mechanisms of plasmids. In: Escherichia coli and Salmonella: Cellular and Molecular Biology, Vol. 2, 2nd edn., pp. 2295^2324. American Society for Microbiology, ASM Press, Washington, DC. Jain, R.K., Bayly, R.C. and Skurray, R.A. (1984) Characterization and physical analysis of a 3,5-xylenol degradative plasmid in Pseudomonas putida. J. Gen. Microbiol. 130, 3019^ 3028. Yannisch-Peron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains : nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103^119. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403^410. de Lorenzo, V., Eltis, L., Kessler, B. and Timmis, K.N. (1993)
[15]
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Analysis of Pseudomonas gene products using lacIq /Ptrpÿlac plasmids and transposons that confer conditional phenotypes. Gene 123, 17^24. Schaper, S. and Messer, W. (1995) Interaction of the initiator protein DnaA of Escherichia coli with its DNA target. J. Biol. Chem. 270, 17622^17626. Perri, S. and Helinski, D.R. (1993) DNA sequence requirements for interaction of the RK2 replication initiation protein with plasmid origin repeats. J. Biol. Chem. 268, 3662^3669. Nieto, C., Giraldo, R., Fernaèndez-Tresguerres, E. and Diaz, R. (1992) Genetic and functional analysis of the basic replicon of pPS10, a plasmid speci¢c for Pseudomonas isolated from Pseudomonas syringae patovar savastanoi. J. Mol. Biol. 223, 415^426. Sugiura, S., Ohkubo, S. and Yamaguchi, K. (1993) Minimal essential origin of plasmid pSC101 replication: requirement of a region downstream of iterons. J. Bacteriol. 175, 5993^6001. Felton, J. and Wright, A. (1979) Plasmid pSC101 replication in integratively suppressed cells requires DnaA function. Mol. Gen. Genet. 175, 231^233. Macartney, D.P., Williams, D.R., Sta¡ord, T. and Thomas, C.M. (1997) Divergence and conservation of the partitioning and global regulation functions in the central control region of the IncP plasmids RK2 and R751. Microbiology 143, 2167^ 2177. Jagura-Burdzy, G. and Thomas, C.M. (1992) kfrA gene of broad host range plasmid RK2 encodes a novel DNA-binding protein. Mol. Biol. 225, 651^660.
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Sequence analysis of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (P25X) reveals a novel replication region Stephen M. Kwong, Chew Chieng Yeo, Damian Chuah, Chit Laa Poh * Department of Microbiology, Faculty of Medicine, National University of Singapore, Lower Kent Ridge Road, Singapore 119260, Singapore Received 3 November 1997 ; accepted 6 November 1997
Abstract The replication region of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (strain P25X) was localized within a 5.9kbp DNA fragment and its sequence was determined. An interesting feature of the sequence is the presence of a 1.3-kbp region containing seven, highly conserved, direct repeats of 72 bp in length. The pRA2 replication region has two open reading frames (ORFs). ORF1 appeared to be essential for replication and had the potential to encode a novel 30-kDa protein with a predicted helix-turn-helix motif located at the C-terminal end. ORF2 was not essential for replication and may encode for a 37-kDa protein which shares 41% and 27% amino acid sequence identity to the KfrA proteins from plasmids RK2 and R751, respectively. The essential region of replication was narrowed down to 2819 nucleotides and included four of the seven 72-bp direct repeats, a potential DnaA-binding site and ORF1. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Plasmid replication; Direct repeat; Replication initiation protein; kfrA
1. Introduction Pseudomonads have promising biotechnological potentials in agriculture, the chemical industry and environmental bioremediation due to their vast metabolic versatility. Plasmids are important in the investigation of degradative processes since they often encode catabolic genes and provide a means of genetic transfer between genera. Pseudomonas alcaligenes NCIB 9867 (P25X) is able to degrade 2,5-xylenol, 3,5-xylenol and m-cresol via the gentisate pathway [1]. The native plasmid of P25X, pRA2 * Corresponding author. Tel.: +65 (87) 43674; Fax: +65 (77) 66872; E-mail: [email protected]
(33 kbp), has a narrow-host-range and is self-transmissible but attempts at curing it have proven unsuccessful [2,3]. The plasmid of P. alcaligenes C-0 [4] and more recently those of P. alcaligenes type strain [5] have also been reported to be incurable. This suggests that these plasmids not only provide a selective advantage for the host cell, as with the majority of plasmids, but may actually play a critical role in survival even under optimal growth conditions. Autonomous replication is a fundamental plasmid characteristic. A plasmid replicon usually contains a speci¢c protein that is necessary for initiation of its own replication, while the majority of the requirements for replication such as initiation factors, heli-
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cases, primases and polymerases are provided by the host [6]. By encoding their own initiation proteins, plasmids are able to replicate independently of the chromosome and thus de¢ne their own copy number in order to maintain stable inheritance. Plasmid-encoded initiation proteins, such as RepA from pPS10 [7] and TrfA of RK2 [8], bind to speci¢c repetitive sequences (iterons) which are located within the vegetative origin of replication, oriV. Iterons generally range from 17 to 22 nucleotides in length, exist in multiple copies and occur in the replicons of many Gram-negative bacteria [9]. In this study, we report the novel regions associated with the replication of plasmid pRA2, provide a comparison of regions sharing similarity to previously reported plasmids and de¢ne an essential region for autonomous replication.
2. Materials and methods 2.1. Bacterial strains and media P. putida RA713 [10] was used in the isolation of the mini-replicon while E. coli strain JM109 [11] was used in the generation of recombinant clones. E. coli strains were cultivated at 37³C and P. putida at 32³C in Luria-bertani medium. Antibiotics were used in the following concentrations: ampicillin, 100 Wg ml31 ; kanamycin, 50 Wg ml31 ; streptomycin, 100 Wg ml31 ; and chloramphenicol, 15 Wg ml31 . 2.2. Sequencing and analysis Both strands of the pRA2 mini-replicon were sequenced. Nested deletion clones of the mini-replicon were obtained using a combination of the Deletion Factory1 System (Gibco/BRL) and the double stranded Nested Deletion Kit (Pharmacia Biotech). DNA sequencing and oligonucleotide synthesis were performed using an ABI 373 DNA sequencer and an ABI 392 DNA synthesizer (Applied Biosystems/Perkin-Elmer), respectively. DNA for sequencing was prepared using the mini alkaline lysis method for DNA extraction [12] or the Wizard1 Plus Minipreps (Promega). DNA sequencing was carried out using the ABI Taq Dye Deoxy Cycle Sequencing Kit (Applied Biosystems/Perkin-Elmer) according to
the manufacturers instructions. DNA sequences were compiled and analyzed using DNASIS (Hitachi Software Engineering) and the BLAST server [13]. 2.3. Molecular techniques DNA cloning, agarose gel electrophoresis, preparation of competent cells and plasmid transformations were performed as previously described [12]. Restriction enzymes were obtained from Boehringer Mannheim or New England Biolabs.
3. Results 3.1. Isolation of a mini-replicon from pRA2 A mini-replicon of the P. alcaligenes P25X plasmid pRA2 was constructed by partial digestion of the 33kbp plasmid with Sau3AI and ligation of the resulting fragments to a 2.2-kbp kanamycin resistance gene (KmR ), isolated from a mini-Tn5 transposon [14]. The ligation mixture was transformed into P. putida RA713, a plasmid-free strain of Pseudomonas putida NCIB 9869 (strain P35X) [10], and transformants were selected on LB plates supplemented with kanamycin. A single KmR transformant was isolated and analysis of its plasmid showed that it contained approximately 6 kbp of the pRA2 plasmid. 3.2. DNA sequence of the replication region Restriction analysis of the mini-replicon allowed us to localize the position of the replication region with respect to the pRA2 restriction map (Fig. 1). Sequence analysis of the mini-replicon showed that it consisted of a continuous 5990-bp fragment derived from pRA2 (GenBank accession no. AF020268). The DNA sequence revealed a highly repetitive region of approximately 1.3 kbp (nt 879^2185). Within this region are seven well conserved 72-bp direct repeats which vary from the deduced consensus sequence by only one or two bases (Fig. 2). The repeats are arranged into two groups, being separated by 302 bp of non-repetitive DNA. The ¢ve spacer regions between the repeats of each group also share considerable sequence homology to one
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Fig. 1. Schematic representation of the pRA2 replication region with reference to the pRA2 restriction map. The single BglII restriction site on pRA2 was arbitrarily taken as coordinate 0 [2]. Sequence analysis revealed a highly repetitive region (dark arrows), two signi¢cant ORFs (open arrows), a potential DnaA-binding site (shaded diamond) and an A+T-rich region (dark box). The lines below indicate the relative size of the replication region covered by the deletion clones. Heavy lines indicate those that retained replicative ability in P. putida RA713 while thin lines represent deletion clones that lost the ability to replicate in P. putida RA713.
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another and range from 86 to 108 nucleotides in length. The replication region has an 88% A+T-rich region of 59 bp (nt 4424^4482) adjacent to a 87% G+C-rich region of 45 bp (nt 4369^4413) and a possible DnaA-binding site, 5P-TTATCCACA-3P (nt 3713^3721), matching the consensus sequence [TT(T/A)TNCACA] of the binding site for DnaA in E. coli [15]. The replication region sequence had an overall G+C content of 60%. 3.3. Potential open reading frames Two signi¢cant ORFs were identi¢ed in the replication region of pRA2. ORF1 (nt 2745^3575) is located 559 bp downstream from the repetitive region and encodes a putative 276-aa protein with a molecular weight of 30 kDa. A BLAST search failed to ¢nd any sequence similarity to proteins listed in the databases but the predicted secondary structure of ORF1 indicates a helix-turn-helix motif located at the C-terminal end. ORF2 (nt 4493^5518) has the potential to encode a 37-kDa protein that shares amino acid sequence homology to the KfrA protein from plasmid RK2 (41% identity, 59% similarity) as well as the KfrA protein of plasmid R751 (27% identity, 45% similarity). 3.4. Deletion analysis Deletion clones of the pRA2 mini-replicon were used to de¢ne a minimal region for autonomous replication. Two of the clones (7.4SS and 8.15SS) which
had deletions in the same direction were found to retain 4059 and 3673 bp of the replication region, respectively (Fig. 1). The larger subclone 7.4SS could be transformed and maintained in P. putida RA713 but repeated attempts to transform clone 8.15SS into RA713 were unsuccessful. Both clones 7.4SS and 8.15SS had deletions which removed the A+T-rich region as well as the ORF2. Clone 7.4SS was found to retain the DnaA-binding site which had been deleted in 8.15SS (Fig. 1). Several clones (H2K8, 4509 bp; H2K25, 5449 bp; H2K4, 5845 bp) which were larger than clone 7.4SS could also be transformed and maintained in RA713 while the smaller clone 11.11SS (2515 bp) could not be maintained in RA713. Only one of the deletion clones from the opposite direction (clone 3.4KC) could be transformed and maintained in RA713 and this clone was the result of the removal of 1240 bp from the 5P end which included two entire direct repeats as well as the ¢rst 7 bp of a third repeat (Fig. 1).
4. Discussion The majority of plasmids found in Gram-negative bacteria are known to possess one or more clusters of direct repeats, designated as iterons, within their replication origin region. Studies so far have shown that iterons are usually comprised of 17^22 nucleotides and serve as speci¢c binding sites for the replication initiation protein [7,8,16], thus being essential for plasmid replication. The repetitive region of the pRA2 replicon is shown to contain direct repeats that are far larger than any of the previously re-
Fig. 2. Nucleotide sequences of the seven highly conserved 72-bp direct repeats found in the replication region of pRA2. Each repeat is consecutively numbered 1^7 in the order in which they occur. Boxed regions indicate perfect nucleotide sequence conservation while the deduced consensus sequence is shown below.
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ported iterons [9]. While these direct repeats could have a probable role analogous to iterons in the replication of other plasmids, further investigations are clearly needed to elucidate the signi¢cance of the relatively large and highly conserved sequences of the pRA2 iterons. We believe that the putative protein encoded by ORF1 is the pRA2 replication initiation protein based on the following observations:
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(i) ORF1 is the only open reading frame located within the minimal region of replication which could potentially encode a protein that falls within the size range of most plasmid-encoded replication initiation proteins (25^49 kDa); (ii) the predicted secondary structure of the ORF1-encoded protein shows a probable helix-turn-helix DNA-binding motif at the C-terminal end; and (iii) no transformants were iso-
Fig. 3. A: The aligned amino acid sequences of the KrfA proteins from plasmids pRA2 of Pseudomonas alcaligenes NCIB 9867, RK2 of Pseudomonas auroginosa, and R751 of Enterobacter aerogenes. Identical amino acids are in bold letters and marked with an asterisk. Dashes represent gaps which have been inserted to maximize homology. B : DNA sequences of the promoter regions found upstream of the respective kfrA start codons. Arrows represent inverted repeats present in the promoter regions of kfrAR751 and kfrApRA2 . Also shown is the DNA sequence homology between the promoter regions of kfrApRA2 and kfrARK2 .
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lated when ORF1 was disrupted by insertional mutagenesis. While the amino acid sequence of ORF1 does not show any similarity to amino acid sequences deposited in the databases, short nucleotide sequences within the pRA2 repetitive region were found to share similarity with the pECB2 plasmid of P. alcaligenes type strain [5]. The sequence 5PGCCGGAATCTCTCCCTGAT-3P that is present ¢ve times in the iteron region of pRA2 is also found in the replication region of pECB2, albeit only once. However, a shorter subsequence of this, 5PCCGGAATC-3P, is found to occur eleven times in pRA2 and three times in plasmid pECB2. Despite these similarities it appears that the two plasmids may have acquired di¡erent modes of replication since the putative initiation protein of pECB2 does not share any amino acid sequence homology with the ORF1-encoded protein of pRA2, but rather, shares 71% amino acid sequence identity to the RepA initiation protein of pPS10 isolated from Pseudomonas syringae pv. savastanoi [5,17]. Analysis of deletion clones derived from the pRA2 mini-replicon demonstrated that two of the 72-bp direct repeats (proximal to the 5P region), the A+Trich region and ORF2 are not essential for pRA2 replication (Fig. 1). However, the potential DnaAbinding site may be vital for pRA2 replication as the di¡erence in clone 7.4SS, which could replicate in strain RA713, and clone 8.15SS, which could not, lies in the presence of the DnaA-binding site in clone 7.4SS and the absence of the site in the latter. The DnaA-binding sites found in the replication regions of both pPS10 and RK2 were reportedly required for e¤cient plasmid replication [8,17,18] while the DnaA-binding site in plasmid pSC101 was found to be essential for its replication [19]. The similarity in amino acid sequence between the ORF2-encoded protein (KfrApRA2 ), the KfrARK2 and KfrAR751 proteins (Fig. 3A) strongly suggests that they have a similar function and evolutionary origin. KfrARK2 has been proposed to be involved in plasmid partition [20,21]. Sequence conservation between the three KfrA-like proteins is highest at the C-terminal end, implying functional importance in this region. The N-terminal regions of KfrARK2 and KfrAR751 , suspected to contain DNA-binding motifs, have diverged considerably as have their respective promoter regions which are the potential KfrA-bind-
ing sites and points of autorepression [20]. Investigations of the KfrARK2 protein revealed that its transcription is autoregulated by the KfrARK2 protein itself in addition to being suppressed by the korABF operon [21]. The promoter region of kfrApRA2 shows some DNA sequence similarity to the kfrARK2 promoter and, like the kfrAR751 promoter, contains an inverted repeat capable of forming a palindrome (Fig. 3B). Sequences downstream of the pRA2 replication region suggest further similarity to plasmids of the IncP group with additional open reading frames encoding proteins similar to KorA as well as other proteins involved in conjugation in both RK2 and R751 (data not shown). Therefore plasmid pRA2 may provide further insight into our understanding of plasmid functions since it appears to share similar systems of plasmid partition and conjugal transfer to plasmids of IncP while the mode of replication seems to be quite di¡erent to those studied to date.
Acknowledgments This work was supported by the National University of Singapore academic research Grant no. RP95-0383 to C.L. Poh.
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