Characterization of a novel plasmid from extremely halophilic Archaea: nucleotide sequence and function analysis

FEMS Microbiology Letters 221 (2003) 53^57

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Characterization of a novel plasmid from extremely halophilic Archaea: nucleotide sequence and function analysis Xuecheng Ye, Jianhong Ou, Lina Ni, Wanliang Shi, Ping Shen




Department of Microbiology, Wuhan University, Wuhan 430072, PR China Received 2 December 2002; received in revised form 13 February 2003; accepted 16 February 2003 First published online 21 March 2003

Abstract We determined the complete nucleotide sequence of the 16 341 bp plasmid pHH205 of the extremely halophilic archaeon Halobacterium salinarum J7. The plasmid has a G+C content of 61.1%. A number of direct and inverted repeat sequences were found in pHH205, while no insertion sequences were found. Thirty-eight large open reading frames (ORFs) were identified in both strands, and most of them had no significant similarities to known proteins. A putative protein encoded by ORF31 showed 20^41% homology to some hypothetical proteins, which are annotated in several archaeal genome databases as predicted nucleic acid-binding proteins containing PIN domain. Sequence analysis using the GC skew procedure predicted a possible origin of replication. A 4.8 kb PvuII^SnaBI fragment containing both this region and ORF31 was shown to be able to restore replicate of pWL102, a replicon-deficient plasmid in Haloferax volcanii and in H. salinarum R1. Several methods failed to completely cure H. salinarum J7 of pHH205, suggesting that the plasmid probably played an important role in the growth and metabolism of the host. Our work describes a novel haloarchaeal replicon, which may be useful in the construction of cloning and shuttle vectors. A 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Plasmid pHH205; Sequence and function analysis ; Curing; Halobacterium salinarum

1. Introduction Since the recognition that Archaea are the third form of life [1,2], extensive research has focused on the genetics and molecular biology of these organisms [3^5]. Plasmids and extrachromosomal elements (ECE) are the most important genetic tools for studying the Archaea. The plasmids and ECE of Archaea have been studied intensively in recent years, however, only a few have been completely sequenced. Many distinct features of traditional plasmids have been discovered during the analysis of those sequences. For example, plasmids of Archaea encode genes necessary for survival of the host [6], and plasmid sequences have a high similarity to the host chromosome in nucleotide sequence [7]. These studies have expanded our understanding of the biological signi¢cance of plasmids. Thus, sequencing and analysis of archaeal plasmids provide material and data for further study, allowing new insights in

the functioning of life, and give new information about the essence and origin of plasmids. Halobacteria are a group of extremely halophilic Archaea that require high concentrations of salts for growth. They can easily be maintained and manipulated in the laboratory, which makes them excellent experimental organisms among the Archaea. Halobacterium salinarum J7, isolated from a salt mine in Hubei province, China, was found to contain a plasmid designated pHH205. We previously reported the size, structure, copy number and a simple restriction map of the plasmid [8]. In this study, we sequenced and analyzed the complete nucleotide sequence of pHH205, and identi¢ed the replication region of the plasmid.

2. Materials and methods 2.1. Strains and growth conditions

* Corresponding author. Tel. : +86 (27) 87648533; Fax : +86 (27) 87883833. E-mail addresses : [email protected] (P. Shen), [email protected] (P. Shen).

H. salinarum J7 harboring plasmid pHH205 was grown in a complex medium [8]. Haloferax volcanii WFD11 was kindly provided by W.F. Doolittle. Rich media, minimal

0378-1097 / 03 / $22.00 A 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00175-7

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media and solutions used for transformation were prepared as previously described [9]. Solid minimal medium used to grow H. volcanii transformants contained 15 WM mevinolin. Escherichia coli JM109 was used for cloning and was routinely cultured in Luria broth (LB) supplemented with 50 g ml31 ampicillin when harboring pBluescript II-SKþ or recombinant plasmid.

liquid cultures inoculated from resulting colonies, then examined by agarose gel electrophoresis and hybridization with a DIG-labeled pHH205 as probe. Cell concentration of cultures, dosages of these factors and other operations were performed as described [15].

3. Results 2.2. DNA preparation and manipulation 3.1. Cloning and sequencing Plasmid pHH205 and total DNA were prepared from H. salinarum J7 as previously described [8]. Plasmids were extracted from H. volcanii as previously described [9]. E. coli plasmid preparation, restriction endonuclease digesting, DNA ligating, E. coli competent cells preparation and transformation were performed as described [10]. 2.3. Cloning and sequencing Overlapping fragments that cover the whole plasmid were de¢ned according to the restriction map of pHH205 [8]. After purifying these fragments by agarose gel electrophoresis, we inserted them into the multiple cloning sites of plasmid pBluescript II-SKþ . The dideoxynucleotide chain termination procedure was applied for DNA sequencing. Each fragment was sequenced separately using the universal primers M13-47 and RV-M with a 377XL DNA sequencer at ¢rst, and then the primer walking strategy was used to complete the rest of the sequences. 2.4. Analysis of the sequence Computer analysis of the sequence was performed using the DNASTAR software package (DNAstar Inc., UK). Databank searches for homology of nucleotide sequence and putative proteins were carried out through NCBI online using BLAST 2.0 [11]. 2.5. Determination of the replication region

According to the restriction map of pHH205, seven overlapping fragments representing the whole plasmid were de¢ned and cloned into pBluescript II-SKþ . Both strands of all these fragments were sequenced. The complete nucleotide sequence of pHH205 was then obtained by connecting the sequences of these fragments. The sequence has been deposited in the GenBank database under accession number AY048850. 3.2. Analysis of the sequence The plasmid was found to be 16 341 bp in size with a G+C content of 61.1%. Databank searches using BLAST revealed that the plasmid had no similarity to the Halobacterium sp. NRC-1 genome or to any other known nucleotide sequence [6]. A number of direct and inverted repeated sequences were found dispersedly in pHH205, which is in concordance with what was previously reported. Thus the F II fraction of the Halobacterium genome contains many repeated sequences. Because so many repeated sequences exist in the plasmid, some regions may form palindrome or hairpin structures. Distinct from other plasmids of halobacteria, there was not any region likely to be an insertion sequence [16,17]. Thirty-eight ORFs were identi¢ed in both strands of pHH205, which putatively encode polypeptides larger than 10 kDa (Table 1). All putative proteins encoded by these ORFs were compared with the sequence databases. None of them shows signi¢cant homology to proteins with known function, but several have some similarity to hypo-

The GC skew procedure was used to predict the origin of replication of pHH205 [12,13]. Fragments containing this region were obtained by endonuclease digestion. After blunting with Klenow fragment of DNA polymerase I, these fragments were inserted into the plasmid pWL102, from which the replication region of pHV2 was deleted [14]. Recombinant plasmids were transformed into H. volcanii WFD11 and the transformants were screened on solid medium containing 15 WM mevinolin. 2.6. Tests of curing H. salinarum J7 of pHH205 Liquid culture was treated with ethidium bromide, acridine orange or ultraviolet or cotreated with all three factors before plating. Total DNA was extracted from 1.5 ml

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Fig. 1. GC skew analysis of pHH205.

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Table 1 Putative ORFs in pHH205 Putative ORF

Length (amino acids)

Position (nucleotides)

Molecular weight (kDa)

ORF1 ORF2 ORF3 ORF4 ORF5 ORF6 ORF7 ORF8 ORF9 ORF10 ORF11 ORF12 ORF13 ORF14 ORF15 ORF16 ORF17 ORF18 ORF19 ORF20 ORF21 ORF22 ORF23 ORF24 ORF25 ORF26 ORF27 ORF28 ORF29 ORF30 ORF31 ORF32 ORF33 ORF34 ORF35 ORF36 ORF37 ORF38

343 225 100 107 110 327 186 314 148 154 109 369 197 201 156 197 118 275 158 113 239 166 138 137 134 190 170 121 151 223 140 121 117 154 267 178 134 177

141^1170 1172^1847 2134^2434 2696^3017 3699^4029 4284^5265 5170^5728 6401^7343 6856^7300 8201^8663 8373^8700 8662^9769 9200^9791 9768^10371 10377^10845 10844^11435 11268^11622 11624^12449 12973^13447 13130^13469 13455^14172 14458^14956 15463^15887 16069^16341^142 1565^1163 3777^3207 3964^3454 4576^4213 6896^6443 7041^6372 7698^7278 10260^9897 11066^10715 13075^12613 13930^13129 14821^14287 15471^15069 15923^15392

36884.44 24459.82 10594.33 11913.49 12058.58 37235.58 20575.01 35235.18 16668.01 17341.70 11862.51 41573.98 21842.24 22285.09 16520.67 22207.73 13285.13 30426.25 17430.49 12356.90 26543.69 16117.23 12953.65 15365.09 14176.19 19740.86 18909.03 13047.37 16125.11 25489.29 16051.07 12402.08 12643.66 17572.95 30016.43 18413.73 14390.82 19072.22

thetical proteins. It is interesting that a smaller predicted protein, encoded by ORF31, shows 20^41% similarity to some hypothetical proteins of Halobacterium sp. NRC-1 and other archaeal species (Table 2), and is annotated as a

Predicted pI 3.975 4.565 8.005 8.005 8.005 5.335 4.035 6.145 11.845 7.385 12.645 3.915 12.825 3.545 3.185 4.285 11.235 4.525 4.505 12.895 4.675 2.575 3.605 3.905 11.425 12.395 11.415 11.855 13.125 4.295 5.355 8.915 12.485 5.725 10.265 7.405 11.395 6.725

‘predicted nucleic acid-binding protein, containing PIN domain’. In addition, there are a number of smaller ORFs for which statistical veri¢cation becomes increasingly di⁄cult. Most of them overlap with the larger ORFs.

Table 2 Hypothetical proteins of archaeal genomes that have similarity to a predicted protein encoded by ORF31 Archaea genomes

Hypothetical proteins

Database no.

Identity (%)

Archaeoglobus fulgidus Halobacterium species NRC-1

hypothetical protein AF1683 hypothetical protein Vng6166h hypothetical protein Vng0072h hypothetical protein Vng6396h hypothetical protein PAB1599 hypothetical protein PAB1672 hypothetical protein hypothetical protein conserved hypothetical protein 144 amino acids long conserved hypothetical protein 134 amino acids long conserved hypothetical protein 128 amino acids long conserved hypothetical protein

O28590 AAG20837 AAG18709 AAG21007 H75096 E75082 AAL62561 AAK41864 AAK43178 BAB66769 BAB66723 BAB64987

32 41 27 20 23 22 30 27 34 26 29 21

Pyrococcus abyssi Pyrobaculum aerophilum Sulfolobus solfataricus Sulfolobus tokodaii

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the control. After treatment, liquid cultures were plated on solid complex medium and incubated for 7 days at 37‡C. Only a few colonies were found on the test plates, whereas the control plate was almost ¢lled with colonies. DNA extracted from those colonies showed obvious plasmid bands upon agarose gel electrophoresis. This indicated that these treatments killed most of the cells, while only cells still carrying pHH205 could survive. Removal of pHH205 from the host by curing resulted in the loss of growth, suggesting that pHH205 is essential for the growth and metabolism of the host. On the other hand, treatments with these factors failed to completely cure H. salinarum J7 of pHH205. Thus, the plasmid is very stable in the host. Fig. 2. Recombinant plasmid pHSP1.

4. Discussion 3.3. Determination of the replication region The nucleotide sequence of plasmid pHH205 was analyzed using the GC skew procedure. The switch in C-G deviation of the replication region was statistically signi¢cant at about 9000 nucleotides (Fig. 1). Several fragments containing such a region were inserted into the plasmid pWL102 from which the replication region was deleted, and then transformed into H. volcanii WFD11. The transformants were selected on solid medium containing 15 WM mevinolin. A recombinant plasmid (named pHSP1, restriction map shown in Fig. 2) was isolated from these transformants, which contained a 4.8 kb PvuII^SnaBI fragment from pHH205. This fragment includes, in addition to a possible origin of replication at 9000 nucleotides, ORF31 that is annotated as a ‘predicted nucleic acid-binding protein, containing PIN domain’ and ORF12, the largest ORF on pHH205 (Fig. 3). A retransformation experiment in which recombinant plasmid pHSP1 (Fig. 2) was transformed into H. salinarum R1 further proved the replication function of the 4.8 kb fragment. Moreover, further deletion of the fragment led to loss of its replication capability. Taken together, these results indicate that the PvuII^SnaBI fragment (Fig. 3) is the minimal functional replication region on pHH205. 3.4. Test of curing H. salinarum J7 of pHH205 Treatment with ethidium bromide, acridine orange and ultraviolet was used to cure H. salinarum J7 of pHH205. A culture without treatment with these factors was used as

We formerly reported the size, structure, copy number and a simple restriction map of plasmid pHH205 [8]. We here performed sequence and function analysis for the plasmid, revealing the replication region and some unusual characteristics. We summarize several conclusions from our work about pHH205: 1. It is a high copy plasmid (about 50 per cell), which is rare in most natural plasmids. It is unusual stable in the host, although it contains a large number of repeated sequences. It seems that the plasmid includes essential genes or functional regions for host survival, which is di¡erent from common natural plasmid. 2. The putative ORF31 protein (predicted DNA-binding protein) has high homology to some archaeal hypothetical proteins. The other 37 putative proteins of pHH205 show no signi¢cant similarity to any known proteins. They may have some unknown functions related to the unique features of pHH205, which are worth further analysis. 3. The G+C content of the plasmid is higher than the average G+C content of the F II fraction of the halobacterial genome. There is no obvious insertion sequence found in the plasmid. These features may contribute to maintaining pHH205 with stable structure and function. Our work reveals that pHH205 is a novel haloarchaeal plasmid, which may be useful in construction of cloning and shuttle vectors. The plasmid vector systems available for use in Archaea have not been developed to a satisfactory degree, limiting their genetical manipulation. Major advantages of pHH205 plasmid, the possible use as a vector, are that its nucleotide sequence, restriction map and

Fig. 3. Minimum replication function region (PvuII^SnaBI fragment) on pHH205.

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replication origin are known, and that there are higher copy number and nature of stabilizing their maintenance in the host. Another advantage of the plasmid is that it is possible to delete unnecessary repeated sequences and insert polycloning sites for conveniencing manipulation and cloning of foreign DNA. Further research for pHH205 plasmid as an excellent vector in halophilic archaeon is in progress.

[7]

[8]

Acknowledgements [9]

We thank Dr. W.F. Doolittle for kindly providing the pWL102 plasmid. We are grateful to Dr. Merck and Dr. Sharp and Dohme Research Lab. for providing mevinolin. This work was supported by the National Science Foundation of China (Nos. 39070009, 39770009).

[10]

[11]

References [12] [1] Woese, C.R., Kandler, O. and Wheelis, M.L. (1990) Towards a natural system of organisms : proposal for the domains Archaea, Bacteria, and Eucarya. Proc. Natl. Acad. Sci. USA 87, 4576^4579. [2] Gray, M.W. (1996) The third form of life. Nature 383, 299^300. [3] Bell, S.D. and Jackson, S.P. (1998) Transcription and translation in Archaea : a mosaic of eukaryal and bacterial features. Trends Microbiol. 6, 225^228. [4] Bernander, R. (2000) Chromosome replication, nucleoid segregation and cell division in Archaea. Trends Microbiol. 8, 278^283. [5] Sowers, K.R. and Schreier, H.J. (1999) Gene transfer systems for the Archaea. Trends Microbiol. 7, 212^219. [6] Ng, W.L., Kennedy, S.P., Mahairas, G.G., Berquist, B., Pan, M., Shukla, H.D., Lasky, S.R., Baliga, N., Thorsson, V., Sbrogna, J., Swartzell, S., Weir, D., Hall, J., Dahl, T.A., Welti, R., Goo, Y.A., Leithauser, B., Keller, K., Cruz, R., Danson, M.J., Hough, D.W., Maddocks, D.G., Jablonski, P.E., Krebs, M.P., Angevine, C.M., Dale, H., Isenbarger, T.A., Peck, R.F., Pohlschrod, M., Spudich, J.L., Jung, K.H., Alam, M., Freitas, T., Hou, S., Daniels, C.J., Den-

[13]

[14]

[15]

[16] [17]

57

nis, P.P., Omer, A.D., Ebhardt, H., Lowe, T.M., Liang, P., Riley, M., Hood, L. and DasSarma, S. (2000) Genome sequence of Halobacterium species NRC-1. Proc. Natl. Acad. Sci. USA 97, 12176^ 12181. Bult, C.J., White, O., Olsen, G.J., Zhou, L., Fleischmann, R.D., Sutton, G.G., Blake, J.A., FitzGerald, L.M., Clayton, R.A., Gocayne, J.D., Kerlavage, A.R., Dougherty, B.A., Tomb, J.F., Adams, M.D., Reich, C.I., Overbeek, R., Kirkness, E.F., Weinstock, K.G., Merrick, J.M., Glodek, A., Scott, J.L., Geoghagen, N.S. and Venter, J.C. (1996) Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science 273, 1058^1073. Shen, P. and Chen, Y. (1994) Plasmid from Halobacterium halobium and its restriction map. Chinese J. Genet. 21, 409^416. Cline, S.W., Lam, W.L., Charlebois, R.L., Schalkwyk, L.C. and Doolittle, W.F. (1989) Transformation methods for halophilic archaebacteria. Can. J. Microbiol. 35, 148^152. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn., pp. 17^57. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Altschul, S.F., Madden, T.L., Scha¡er, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSIBLAST: A new generation of protein database search programs. Nucleic Acids Res. 25, 3389^3403. Lobry, J.R. (1996) Asymmetric substitution patterns in the two DNA strands of bacteria. Mol. Biol. Evol. 13, 660^665. Picardeau, M., Lobry, J.R. and Hinnebusch, B.J. (2000) Analyzing DNA strand compositional asymmetry to identify candidate replication origins of Borrelia burgdorferi linear and circular plasmids. Genome Res. 10, 1594^1604. Charlebois, R.L., Lam, W.L., Cline, S.W. and Doolittle, W.F. (1987) Characterization of pHV2 from Halobacterium volcanii and its use in demonstrating transformation of an archaebacterium. Proc. Natl. Acad. Sci. USA 84, 8530^8534. Crosa, J.H., Tolmasky, M.E., Actis, L.A. and Falkow, S. (1994) Methods for General and Molecular Bacteriology (Gerhardt, P., Murray, R.G.E., Wood, W.A. and Krieg, N.R., Eds.), pp. 365^386. American Society for Microbiology, Washington, DC. Petrov, D.A. (2001) Evolution of genome size: new approaches to an old problem. Trends Genet. 17, 23^25. Pfeifer, F. and Blaseio, U. (1989) Insertion elements and deletion formation in a halophilic archaebacterium. J. Bacteriol. 171, 5135^ 5140.

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