Westfälische Wilhelms-Universität Münster, Institut für Mikrobiologie, Corrensstraße 3, 48149 Münster, Germany1
Author for correspondence: Friedhelm Meinhardt. Tel: +49 251 83 39825. Fax: +49 251 83 38388. e-mail: meinhar{at}uni-muenster.de
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ABSTRACT |
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Keywords: extrachromosomal element, cryptic plasmid, cloning, pP811, repA
Abbreviations: ds, double-stranded;; DSO, double-strand origin;; IR, imperfect repeat;; KmR, kanamycin resistance;; RC, rolling circle;; RCR, rolling circle replicating;; S/D, ShineDalgarno;; SSB, single-strand DNA-binding protein;; ss, single-stranded;; SSO, single-strand origin.
The EMBL and GenBank accession number for the nucleotide sequence of pPP81 is AJ289784.
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INTRODUCTION |
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The phenol-degrading Gram-negative bacterium Pseudomonas putida P8 has attracted considerable interest as a system for studying the degradation of harmful compounds such as phenol (Bettmann & Rehm, 1984 ; Zache & Rehm, 1989
), particularly for elucidating the molecular mechanism by which bacteria adapt their membranes to such substances prior to degradation. Recently, we isolated and functionally characterized the chromosomal cti gene (Holtwick et al., 1997
) encoding a cis/trans isomerase for unsaturated fatty acids a cytochrome-c-type polypeptide (Holtwick et al., 1999
) enabling P. putida P8 to change membrane fluidity.
Here, we report on the occurrence of three differently sized plasmids in P. putida P8, the cloning, complete sequencing and functional characterization of the smallest of these extrachromosomal elements, and its use as a cloning vector. On the basis of sequence comparisons with known elements and demonstration of the characteristic ssDNA intermediate, this extrachromosomal element has emerged as the first RC-type plasmid found in a representative of this physiologically extremely diverse bacterial group.
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METHODS |
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Solid (1·5% agar) and liquid media were supplemented, when required, with ampicillin (50 µg ml-1) and kanamycin (2550 µg ml-1).
The commercially available pUCBM20 cloning plasmid (Boehringer Mannheim) was used for cloning pPP8-1 and for the construction of subclones.
Recombinant DNA techniques.
P. putida DNA was prepared according to the protocol of Rodriguez & Tait (1983) . Plasmid DNA was isolated from E. coli as described by Birnboim & Doly (1979)
. pUT-mini-Tn5-Km (de Lorenzo et al., 1990
) served as the source of the kanamycin-resistance marker.
Restriction endonuclease digestions, ligation, agarose electrophoresis and additional recombinant DNA techniques were carried out as described by Sambrook et al. (1989) unless otherwise specified.
Southern blot hybridization for the verification of ssDNA replication intermediates was performed essentially as described by te Riele et al. (1986) , using plasmid pPP8-1 as the probe. Label was added by random priming using a non-radioactive labelling kit (Boehringer Mannheim) according to the instructions of the supplier.
Transformation procedures.
Plasmids were introduced into E. coli by the transformation of CaCl2-treated cells with purified plasmid DNA (Sambrook et al., 1989 ).
P. putida was transformed by electroporation, as described by Taghavi et al. (1994) but with the following modifications. P. putida strains were grown at 30 °C in complete medium to an OD600 of 0·8. Cells were collected by centrifugation (10 min, 7000 g) and washed twice with 1 vol. ice-cold buffer [10% (v/v) glycerol, 90% (v/v) H2O]. Subsequently, the cells were concentrated 100-fold. To 40 µl of the suspension, 4 µl plasmid DNA (total 1 µg) was added; the preparation was then transferred into a 2 mm electroporation cuvette (Elektroporator 2510; Eppendorf). The best results were obtained with the following settings: voltage, 2400 V; capacitance, 10 µF; resistance, 600
. After electroporation, cells were incubated in 1 ml complete medium for 1 h at 30 °C before being plated onto selective medium.
Sequencing.
Small fragments (0·51 kb) of pPP8-1 were subcloned in pUCBM20 and sequenced with IRD41-labelled universal and reverse primers by using a Thermo Sequenase fluorescent primer cycle sequencing kit with 7deaza-dGTP (Amersham Buchler) and an automatic LI-COR sequencer.
Database searches were performed with the BLAST program provided by EMBL/Heidelberg, using the default parameter settings (Altschul et al., 1997 ). Multiple sequence alignments were carried out using the CLUSTAL-W programs (Thompson et al., 1994
). Further analysis of the sequence was performed by using the HUSAR Genius net service package of EMBL/Heidelberg.
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RESULTS |
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Since pPP8-1 was found to harbour a single HindIII site (see Fig. 1), the entire plasmid was inserted into the corresponding restriction enzyme site of the E. coli cloning vector pUCBM20, resulting in plasmid pUCPP81, which was subsequently used to construct subclones useful for sequencing.
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Demonstration of single-strand replication intermediates
When DNA is isolated from strains carrying RCR plasmids and subjected to gel electrophoresis, ssDNA can be detected by immediate blotting and UV cross-linking to nylon membranes without denaturation, since a plasmid-specific probe hybridizes to ssDNA rather than dsDNA. Digestion of ss intermediates by single-strand-specific nucleases, such as S1 nuclease or mungbean nuclease, prior to gel electrophoresis results in the disappearance of hybridization signals. The results of such experiments are shown in Fig. 5, clearly proving the existence of pPP8-1 ssDNA intermediates in P. putida P8.
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The putative promoter of repA is obviously located at position 13741402 and the repA terminator is located immediately 3' downstream of the coding region at position 22722288, with a calculated free energy (G) of -47·6 kJ mol-1. Apparently, a transcriptional terminator sequence with a calculated
G of -124·6 kJ mol-1 is also located on the opposite strand at position 12761329. No ORF preceding this terminator could be identified; however, a potential promoter is situated at position 15421523. Accordingly, the repA mRNA and its antisense transcript overlap one another by 104 nt, comprising 28 nt 5' upstream of the repA translational start and 76 nt of the repA coding sequence.
Vector construction
In our initial attempts to obtain a hybrid vector on the basis of pPP8-1, we tried to integrate the kanamycin-resistance (KmR) gene of Tn5 into the single HindIII site of pPP8-1. After Ca2+-mediated transformation, direct selection did not give rise to antibiotic-resistant E. coli clones (data not shown). Since the HindIII site is located within ORF B, disruption of the gene by integration of the antibiotic-resistance marker might have interfered with plasmid replication and maintainance, eventually resulting in the failure to obtain transformants. However, when the KmR gene was ligated into the SmaI site located in the intergenic region between ORF D and ORF B, E. coli transformants carrying the desired hybrid plasmid (pPP8-1KmR in Fig. 1) were easily obtained. As for the wild-type pPP8-1 in P. putida P8 (see Fig. 5
), single-strand intermediates of the hybrid vector were detected in E. coli transformants. Plasmid DNA isolated from those transformants was characterized by restriction-enzyme analysis (see Fig. 1
, pPP8-1KmR) and used in electroporation transformation experiments with P. putida P8; the number of transformants obtained was up to 3·6x104 (µg DNA)-1.
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DISCUSSION |
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Plasmid pKB740 from Pseudomonas sp. (Altenschmidt et al., 1992 ) may also replicate by an RC replication mechanism, on the basis of homology between the putative protein encoded by ORF 5 of this plasmid and that of the gene II protein, involved in RC replication of the filamentous bacteriophage I2-2 (Stassen et al., 1992
).
By analogy with pC194 and X174, the strictly conserved sequence CTT
GATA carrying the indicated nick site (
) within the putative DSO was identified in pPP8-1, along with highly conserved amino acid residues (Tyr and Glu) in the catalytic domain of the predicted RepA polypeptide (Fig. 3
, IV). Both amino acid residues were previously shown to be instrumental in the nicking process and in circularization of single-strand intermediates (Noriot-Gros et al., 1994
). Though the predicted RepA protein of pPP8-1 is only 2530% similar to the corresponding proteins of the pC194-plasmid family, it harbours the five functional domains identified in this type (Fig. 3
). It is presumed that the strikingly conserved cysteines, as well as aromatic and charged amino acids (see Fig. 3
), are instrumental in the recognition of, and binding to, the target sequence (Noriot-Gros et al., 1994
).
For RCR-type plasmids in general, the identified putative SSO shows no obvious sequence similarities to other RC elements. Also, blocks of nucleotides conserved in hitherto known SSOs, as shown by Kramer et al. (1999) , could not be identified in pPP8-1. Since an SSO generally characterized by extensive secondary structures (five inverted repeats in pPP8-1) must be recognized by host-specific proteins for initiation and completion of synthesis of the double strand, it is probably adjusted to the respective host bacterium, ensuring proper replication (Boe et al., 1989
; Gruss et al., 1987
; del Solar et al., 1987b
).
Besides the repA locus, pPP8-1 harbours three additional ORFs, the predicted polypeptides of which share no significant similarities with known proteins. From BLAST searches, it became evident, however, that ORF C shares similarities with a possible ORF (position 75333) of a recently described RCR-type plasmid of Nitrosomonas sp. (Yamagata et al., 1999 ).
Since it was not possible to obtain hybrid plasmids in which the antibiotic-resistance marker was integrated into the single HindIII site, an intact ORF B seems to be essential. ORFs B and C apparently constitute a transcriptional unit, encoding polypeptides of 105 and 101 aa, respectively. Only short regions of the predicted proteins slightly matched DNA-binding proteins such as DNA polymerases and regulator proteins (data not shown). Because of their small size, the gene products of both ORF B and ORF C cannot be polymerases; they presumably represent DNA-binding proteins of unknown function.
ORF D, though overlapping the putative SSO, was identified as a potential coding region because of a ShineDalgarno (S/D) sequence at an appropriate distance from the start codon, a possible promoter and a corresponding distinct transcriptional terminator. In computer-aided searches, at the DNA level we found a 97 nt region (244367, Fig. 2) with 80% similarity to the functionally uncharacterized ORF 110 of single-strand phage Pf1 (Hill et al., 1991
). At the amino acid level, slight similarities to DNA-binding proteins such as polymerases and recombinases (data not shown) were obtained. In view of the small size of the predicted ORF D polypeptide (73 aa), it might represent a single-strand DNA-binding protein (SSB), as its size corresponds to known SSBs such as that of phage Pf3 (78 aa; Luiten et al., 1985
), phage IF1 (96 aa; Carne et al., 1991
) and Bacillus thuringiensis plasmid pTX14-3 (68 aa; Madsen et al., 1993
). In general, SSBs do not share noticeable structural similarities.
It remains to be determined whether plasmid-encoded proteins are instrumental in copy-number control in pPP8-1. However, regardless of such a possible involvement, copy-number control appears to be influenced by an antisense RNA overlapping the translational start (S/D and ATG) of the repA transcript. The size of the overlap corresponds well with other RCR plasmids, ranging from 40 nt in pJDB21 from Selenomonas ruminantium (Zhang & Brooker, 1993 ), up to 148 nt in pLAB1000 of Lactobacillus hilgardii (Alonso & Tailor, 1987
; Josson et al., 1990
).
A number of RCR plasmids from Gram-positive bacteria, such as pC194, pMV158 and pSA5700, but also pKYM of the Gram-negative Shigella, were found to replicate autonomously in E. coli (Goze & Ehrlich, 1980 ; Barany et al., 1982
; del Solar et al., 1987a
; Yasukawa et al., 1991
). The same holds true for the hybrid plasmid pPP8-1KmR, which can be maintained in E. coli, albeit only under selective pressure. The cultivation of plasmid-containing E. coli cells overnight without kanamycin resulted in segregational loss of the plasmid (data not shown). Nevertheless, the plasmid fulfils several criteria deemed useful for a potential Pseudomonas cloning vector. It is relatively small (2534 bp), it contains restriction-enzyme sites (SmaI) into which foreign DNA can be integrated without disrupting an essential locus, and standard cloning procedures can be performed in E. coli, from which the plasmid can be isolated and transferred to P. putida and possibly other hosts as well.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Altenschmidt, U., Bokranz, M. & Fuchs, G.(1992). Novel aerobic 2-aminobenzoate metabolism. Nucleotide sequence of the plasmid carrying the gene for the flavoprotein 2-aminobenzoyl-CoA monooxygenase/reductase in denitrifying Pseudomonas sp. Eur J Biochem 207, 715-722.[Abstract]
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J.(1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402.
Baas, P. D. & Jansz, H. S.(1988). Single stranded DNA phage origins. Curr Top Microbiol Immunol 136, 31-70.[Medline]
Barany, F., Boeke, J. D. & Tomasz, A.(1982). Staphylococcal plasmids that replicate and express erythromycin resistance in both Staphylococcus pneumoniae and Escherichia coli. Proc Natl Acad Sci USA 79, 2991-2995.[Abstract]
Bettmann, H. & Rehm, H. J.(1984). Degradation of phenol by polymer entrapped microorganisms. Appl Microbiol Biotechnol 22, 389-393.
Birnboim, H. C. & Doly, J.(1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7, 1513-1522.[Abstract]
Boe, L., Gros, M. F., te Riele, H., Ehrlich, S. D. & Gruss, A.(1989). Replication origins of single-stranded-DNA plasmid pUB110. J Bacteriol 171, 3366-3372.[Medline]
Boronin, A. M.(1992). Diversity and relationship of Pseudomonas plasmids. In Pseudomonas: Molecular Biology and Biotechnology , pp. 329-340. Edited by E. Galli, S. Silver & B. Witholt. Washington, DC:American Society for Microbiology.
Carne, A., Hill, D. F., Stockwell, P. A., Hughes, G. & Petersen, G. B.(1991). The putative single-stranded DNA binding protein of the filamentous bacteriophage, IF1. Amino acid sequence of the protein and structure of the gene. Proc R Soc Lond B Biol Sci 245, 23-30.[Medline]
Goze, A. & Ehrlich, S. D.(1980). Replication of plasmids from Staphylococcus aureus in Escherichia coli. Proc Natl Acad Sci USA 77, 733-737.
Gruss, A., Ross, H. & Novick, R.(1987). Functional analysis of a palindromic sequence required for normal replication of several staphylococcal plasmids. Proc Natl Acad Sci USA 84, 2165-2169.[Abstract]
Hill, D. F., Short, N. J., Perham, R. N. & Petersen, G. B.(1991). DNA sequence of the filamentous bacteriophage Pf1. J Mol Biol 218, 349-364.[Medline]
Holtwick, R., Meinhardt, F. & Keweloh, H.(1997). Cistrans isomerisation of fatty acids: cloning and sequencing of the cti gene from Pseudomonas putida P8. Appl Environ Microbiol 63, 4292-4297.[Abstract]
Holtwick, R., Keweloh, H. & Meinhardt, F.(1999). Cis/trans isomerase of unsaturated fatty acids of Pseudomonas putida P8: evidence for a heme protein of the cytochrome c type. Appl Environ Microbiol 65, 2644-2649.
Horinouchi, S. & Weisblum, B.(1982). Nucleotide sequence and functional map of plasmid pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol 150, 815-825.[Medline]
Josson, K., Soetaert, P., Michiels, F., Joos, H. & Mahillon, J.(1990). Lactobacillus hilgardii plasmid pLAB1000 consists of two functional cassettes commonly found in other gram positive organisms. J Bacteriol 172, 3089-3099.[Medline]
Khan, S. A.(1997). Rolling-circle replication of bacterial plasmids. Microbiol Mol Biol Rev 61, 442-455.[Abstract]
Kramer, M. G., Espinosa, M., Misra, T. K. & Khan, S. A.(1999). Characterization of a single-strand origin, ssoU, required for broad host range replication of rolling-circle plasmids. Mol Microbiol 33, 466-475.[Medline]
de Lorenzo, V., Herrero, M., Jacubzik, U. & Timmis, K. N.(1990). Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol 172, 6568-6572.[Medline]
Luiten, R. G., Putterman, D. G., Schoenmakers, J. G., Kronigs, R. N. & Day, L. A.(1985). Nucleotide sequence of the genome of Pf3 and IncP-1 plasmid specific filamentous bacteriophage of Pseudomonas aeruginosa. J Virol 56, 268-276.[Medline]
Madsen, S. M., Andrup, L. & Boe, L.(1993). Fine mapping and DNA sequence of replication functions of Bacillus thuringiensis plasmid pTX14-3. Plasmid 30, 119-130.[Medline]
Meijer, W. J., Wisman, G. B., Terpstra, P., Thorsted, P. B., Thomas, C. M., Holsappel, S., Venema, G. & Bron, S.(1998). Rolling-circle plasmids from Bacillus subtilis: complete nucleotide sequences and analyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and comparisons with related plasmids from gram-positive bacteria. FEMS Microbiol Rev 21, 337-368.[Medline]
Noriot-Gros, M. F., Bidnenko, V. & Ehrlich, S. D.(1994). Active site of the replication protein of the rolling circle plasmid pC194. EMBO J 13, 4412-4420.[Abstract]
Novick, R. P.(1989). Staphylococcal plasmids and their replication. Annu Rev Microbiol 43, 537-565.[Medline]
te Riele, H., Michel, B. & Ehrlich, S. D.(1986). Single stranded plasmid DNA in Bacillus subtilis and Staphylococcus aureus. Proc Natl Acad Sci USA 83, 2541-2545.[Abstract]
Rodrigues, R. L. & Tait, R. C. (1983). Recombinant DNA Techniques: an Introduction. Stanford: Addison-Wesley.
Ronald, S., Farinha, M. A., Allan, B. J. & Kropinski, A. M.(1992). Cloning and physical mapping of transcriptional regulatory (sigma) factors from Pseudomonas aeruginosa. In Pseudomonas: Molecular Biology and Biotechnology , pp. 249-257. Edited by E. Galli, S. Silver & B. Witholt. Washington, DC:American Society for Microbiology.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
del Solar, G., Diaz, R. & Espinosa, M.(1987a). Replication of the streptococcal plasmid pMV158 and derivatives in cell-free extracts of Escherichia coli. Mol Gen Genet 206, 428-435.[Medline]
del Solar, G., Puyet, A. & Espinosa, M.(1987b). Initiation signals for the conversion of single stranded to double stranded DNA forms in the streptococcal plasmid pLS1. Nucleic Acids Res 15, 5561-5580.[Abstract]
del Solar, G., Moscoso, M. & Espinosa, M.(1993). Rolling circle-replicating plasmids from Gram-positive and Gram-negative bacteria: a wall falls. Mol Microbiol 8, 789-796.[Medline]
del Solar, G., Giraldo, R., Ruiz-Echevarria, M. J., Espinosa, M. & Diaz-Orejas, R.(1998). Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev 62, 434-464.
Stassen, A. P., Schonmakers, E. F., Yu, M., Schoenmakers, J. G. & Konings, R. N. H.(1992). Nucleotide sequence of the genome of the filamentous bacteriophage I2-2: module evolution of the filamentous phage genome. J Mol Evol 34, 141-152.[Medline]
Taghavi, S., van der Lelie, D. & Mergeay, M.(1994). Electroporation of Alcaligenes eutrophus with (mega) plasmids and genomic DNA fragments. Appl Environ Microbiol 60, 3585-3591.[Abstract]
Thompson, J. D., Higgins, D. G. & Gibson, T. J.(1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignments through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.[Abstract]
Tinoco, I.Jr, Borer, P. N., Dengler, B., Levine, M. D., Uhlenbeck, O. C., Crothers, D. M. & Gralla, J.(1973). Improved estimation of secondary structure in ribonucleic acid. Nature New Biol 246, 40-41.[Medline]
Yamagata, A., Kato, J., Hirota, R., Kuroda, A., Ikeda, T., Takiguchi, N. & Ohtake, H.(1999). Isolation and characterization of two cryptic plasmids in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11. J Bacteriol 181, 3375-3381.
Yanisch-Perron, C., Vieira, J. & Messing, J.(1985). Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33, 103-119.[Medline]
Yasukawa, H., Hase, T., Sakai, A. & Masamune, Y.(1991). Rolling-circle replication of the plasmid pKYM isolated from a gram-negative bacterium. Proc Natl Acad Sci USA 88, 10282-10286.[Abstract]
Wagner, E. G. & Simons, R. W.(1994). Antisense RNA control in bacteria, phages and plasmids. Annu Rev Microbiol 48, 713-742.[Medline]
Zache, G. & Rehm, H. J.(1989). Degradation of phenol by a coimmobilized entrapped mixed culture. Appl Microbiol Biotechnol 30, 426-432.
Zhang, N. & Brooker, J. D.(1993). Characterization, sequence, and replication of a small cryptic plasmid from Selenomonas ruminantium subspecies lactilytica. Plasmid 29, 125-134.[Medline]
Received 10 July 2000;
revised 16 October 2000;
accepted 24 October 2000.
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