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Acta Biochim Biophys Sin 2006, 38: 620-624

doi:10.1111/j.1745-7270.2006.00207.X

Characterization of Nocardia Plasmid pXT107

 

Hai-Yang XIA1,2#, Yong-Qiang TIAN2#, Ran ZHANG2, Kai-Chun LIN1*, and Zhong-Jun QIN2*

 

1 College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China;

2 Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China

 

Received: May 21, 2006

Accepted: June 13, 2006

This work was supported by the grants from the National Natural Science Foundation of China (No. 30170019, 30270030 and 30325003), the National Natural Science Foundation of Shanghai (No. 0202ZA14096) and the Hi-tech Research and Development Program of china (No. 2005AA227020)

# These authors contributed equally to this work

*Corresponding authors:

Kai-Chun LIN: Tel, 86-27-87287657; Fax, 86-27-87287633; E-mail, [email protected]

Zhong-Jun QIN: Tel/Fax, 86-21-54924171; E-mail, [email protected]

 

Abstract������� Nocardia, Rhodococcus and Streptomyces, all members of the actinomycetes family, are Gram-positive eubacteria with high G+C content and able to form mycelium. We report here a newly identified plasmid pXT107 of Nocardia sp. 107, one of the smallest circular plasmids found in Nocardia. The complete nucleotide sequence of pXT107 consisted of 4335 bp with 65% G+C content, and encoded one replication extragenic palindromic (Rep) and six hypothetical proteins. The Rep, double-strand origin and single-strand origin of pXT107 resembled those of typical rolling-circle-replication plasmids, such as pNI100 of Nocardia, pRE8424 of Rhodococcus and pIJ101 of Streptomyces. The Escherichia coli-Nocardia shuttle plasmid pHAQ22, containing the rep gene of pXT107, is able to propagate in Nocardia but not in Streptomyces.

 

Key words������� Nocardia; plasmid; complete nucleotide sequence; replication

 

The genera of Nocardia and its close relative Rhodococcus, belonging to nocardioform actinomycetes, are high G+C Gram-positive eubacteria with fragmentation of mycelium. Many Nocardia species, even clinical isolates, can produce bioactive molecules such as antibiotics [1,2] and enzymes of industrial importance [3], whereas some species cause human and animal diseases of lung and brain [4]. Indigenous circular plasmids have been detected among Nocardia species [5-7], but few are characterized. The replication extragenic palindromic (Rep) protein of plasmid pNI100 of Nocardia italica resembles that of the rolling-circle-replication (RCR) plasmid pSG5 of Streptomyces, and a shuttle vector of pNI100 derivative can propagate in Streptomyces [8].

Nocardia sp. 107 was identified from a soil sample isolated in Sichuan province, China [9]. We report here the identification, sequencing and characterization of the indigenous plasmid pXT107 from Nocardia sp. 107. An Escherichia coli-Nocardia shuttle plasmid, containing the rep gene, can propagate in Nocardia but not in Streptomyces.

 

 

Materials and Methods

 

Bacterial strains and plasmids

 

The list of plasmids and strains used in this work is given in Table 1. Nocardia corallina 4.1037 was purchased from the Chinese General Microbiological Culture Collection Center (Beijing, China).

 

Growth conditions, transformation procedures and DNA manipulations

 

Nocardia strain was grown at 28 �C in Tryptone Soy Broth media. Plasmid DNA was isolated using both non-alkaline denatured [10] and denatured/renatured procedures [11]. Electroporation of N. corallina 4.1037 was done by the method of Yao et al. [12]. Streptomyces culture, protoplast preparation and transformation were carried out by the method of Kieser et al. [11]. The protocol of Sambrook et al. [13] was used in E. coli DNA manipulations. The 16S rDNA fragment amplification was done by using the actinomycetes-specific 16S rDNA primer pair (16S-F, 5'-AGAGTTTGATCCTGGCTCAG-3'; 16S-R, 5'-TACGGCTACCTTGTTACGACTT-3') and high fidelity thermostable DNA polymerase.

 

DNA sequencing and analysis

 

Plasmid sequence was determined using "primer walking" method from both strands on the ABI 3730 automated DNA sequencer (Applied Biosystems, Foster, USA) at the Chinese Human Genome Center (Shanghai, China). Sequence analysis was carried out with FramePlot 3.0beta software (http://watson.nih.go.jp/~jun/cgi-bin/frameplot-3.0b.pl) [14]. Sequence comparisons were done with software from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/). DNA secondary structure was predicted using "mfold" software (http://www.bioinfo.rpi.edu/applications/mfold/old/dna/forml.cgi) [15]. The Genbank accession No. of pXT107 sequence is DQ399903.

 

 

Results and Discussion

 

Identification and complete nucleotide sequencing of plasmid pXT107

 

The 16s rDNA fragment of strain 107 was amplified by polymerase chain reaction and sequenced. The sequence was searched for similarity in the National Center for Biotechnology Information database by BLASTN and displayed high homology with that of many Nocardia strains, such as Nocardia sp. DSM 43576 (identity 99%) and Nocardia flavorosea JCM 3342 (99%). Circular plasmid DNA was isolated from Nocardia sp. 107 using both non-alkaline denatured [10] and denatured/renatured procedures [11], and electrophoresed in an agarose gel. A 4.5 kb DNA band (designated pXT107), resistant to alkaline treatment, was detected (data not shown). Treatment with restriction endonucleases showed that pXT107 contained unique sites of BamHI, PstI, XbaI and XhoI (Fig. 1). The BamHI-digested DNA was cloned into E. coli plasmid pBluescript II KS (+) to yield pTQ104. Polymerase chain reaction sequencing of pTQ104 was carried out using "primer walking" (see "Materials and methods"). The complete nucleotide sequence of pXT107 on pTQ104 consisted of 4335 bp. The G+C content was 65%, resembling that of typical Nocardia plasmids (e.g., 67% for pNF1 and 68% for pNF2) [16]. The putative open reading frame analysis with FramePlot 3.0beta predicted seven protein-encoding regions, including one Rep and six hypothetical proteins.

 

Rep, double-strand origin (dso) and single-strand origin (sso) of pXT107 resemble those of typical RCR plasmids

 

All RCR plasmids contain three elements: a gene encoding the initiator protein (Rep), the dso, and the sso [17]. The predicted Rep of pXT107 contained conserved protein motifs I-III [18,19], resembling several RCR plasmids, such as pRE8424 of Rhodococcus erythropolis, pIJ101 of Streptomyces lividans, pNI100 of N. italica and pBL1 of Brevibacterium lactofermentum [Fig. 2(A)]. The experimentally identified dso sequences of Streptomyces RCR plasmids pIJ101, pJV1, pSNA1, pBL1 and pSN22 were conserved, especially the GG dinucleotide at the nick site [Fig. 2(B)] [20-23]. A similar sequence was also found within the rep of pXT107. The sso is required for initiation of lagging strand synthesis [17]. Alignment of the pXT107 sequence to the characterized sso sequences of pIJ101, pSN22, pBL1 and pRE8424 [21-25] displayed high similarity� [Fig. 2(C)]. In addition, like pNI100 [8], the sso of pXT107 could form a structure of stem-loops (Fig. 3). These results indicated a typical RCR mechanism of pXT107.

 

E. coli-Nocardia shuttle plasmid pHAQ22 propagates in Nocardia but not in Streptomyces

 

The PstI fragment of pXT107, containing the intact rep gene (Fig. 1), was cloned into E. coli plasmid pQC156, which contained actinomycetes selection markers tsr and melC [26], to yield pHAQ22. Introduced by transformation into plasmid-free hosts including N. corallina 4.1037 and S. lividans ZX7, thiostrepton-resistant transformants were obtained in strains N. corallina 4.1037 with a transformation efficiency of 3�102 per microgram plasmid DNA, but not in S. lividans ZX7 (plasmid pIJ702 as positive control). Another constructed plasmid pHAQ20, which cloned the BamHI fragment of pXT107 (disrupting the rep) into pQC156, could not propagate in 4.1037 or ZX7. These results indicated the essential function of the rep gene of pXT107 for replication.

 

 

References

 

 1�� Shigemori H, Komaki H, Yazawa K, Mikami Y, Nemoto A, Tanaka Y, Sasaki T et al. Brasilicardin A. a novel tricyclic metabolite with potent immunosuppressive activity from actinomycete Nocardia brasiliensis. J Org Chem 1998, 63: 6900-6904

 2�� Tanaka Y, Grafe U, Yazawa K, Mikami Y, Ritzau M. Nocardicyclins A and B: new anthracycline antibiotics produced by Nocardia pseudobrasiliensis. J Antibiot 1997, 50: 822-827

 3�� Coco WM, Levinson WE, Crist MJ, Hektor HJ, Darzins A, Pienkos PT, Squires CH et al. DNA shuffling method for generating highly recombined genes and evolved enzymes. Nat Biotechnol 2001, 19: 354-359

 4�� Brown JM, McNeil MM, Desmond EP. Nocardia, Rhodococcus, Gordonia, Actinomadura, Streptomyces, and other actinomycetes of medical importance. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH eds. Manual of Clinical Microbiology, 7th edn. Washington: American Society for Microbiology Press 1999

 5�� Kirby R, Usdin K. The isolation and restriction mapping of a miniplasmid from the actinomycete Nocardia corallina. FEMS Microbiol Lett 1985, 27: 57-59

 6�� Oh YK, Fare LR, Taylor DP, Widger J, Nisbet LJ. A cryptic plasmid from Nocardia orientalis NRRL 2452, a vancomycin producer. J Antibiot 1986, 39: 694-698

 7�� Provost F, Blanc MV, Beaman BL, Boiron P. Occurrence of plasmids in pathogenic strains of Nocardia. J Med Microbiol 1996, 45: 344-348

 8�� Liu YT, Su CM, Lee CH, Sui MJ, Chang YH, Lin WP, Wu WT et al. Cloning and characterization of the replicon of the Nocardia italica plasmid, pNI100. Plasmid 2000, 43: 223-229

 9�� Tian Y. Biology of genetic elements in the rare actinomycetes. Shanghai: Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences 2005, PhD thesis

10� Qin Z, Cohen SN. Replication at the telomeres of the Streptomyces linear plasmid pSLA2. Mol Microbiol 1998, 28: 893-903

11� Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA. Practical Streptomyces Genetics. Norwich: The John Innes Foundation 2000

12� Yao W, Yang Y, Chiao J. Cloning vector system for Nocardia sp.. Cur Microbiol 1994, 29: 223-227

13� Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press 1989

14 Ishikawa J, Hotta K. FramePlot: a new implementation of the frame analysis for predicting protein-coding regions in bacterial DNA with a high G+C content. FEMS Microbiol Lett 1999, 174: 251-253

15� Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003, 31: 3406-3415

16� Ishikawa J, Yamashita A, Mikami Y, Hoshino Y, Kurita H, Hotta K, Shiba T et al. The complete genomic sequence of Nocardia farcinica IFM 10152. Proc Natl Acad Sci USA 2004, 101: 14925-14930

17� Khan SA. Plasmid rolling-circle replication: highlights of two decades of research. Plasmid 2005, 53: 126-136

18� Gruss A, Ehrlich SD. The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol Rev 1989, 53: 231-241

19� Ilyina TV, Koonin EV. Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria. Nucleic Acids Res 1992, 20: 3279-3285

20� Serv�n-Gonz�lez L. Relationship between the replication functions of Streptomyces plasmids pJV1and pIJ101. Plasmid 1993, 30: 131-140

21� Suzuki I, Seki T, Yoshida T. Nucleotide sequence of a nicking site of the Streptomyces plasmid pSN22 replicating by the rolling circle mechanism. FEMS Microbiol Lett 1997, 150: 283-288

22� Fernandez-Gonzalez C, Cadenas RF, Noirot-Gros MF, Martin JF, Gil A. Characterization of a region of plasmid pBL1 of Brevibacterium lactofermentum involved in replication via the rolling circle model. J Bacteriol 1994, 176: 3154-3161

23� Mendes MV, Aparicio JF, Martin JF. Complete nucleotide sequence and characterization of pSNA1 from pimaricin-producing Streptomyces natalensis that replicates by a rolling circle mechanism. Plasmid 2000, 43: 159-165

24� Deng ZX, Kieser T, Hopwood DA. �Strong incompatibility� between derivatives of the Streptomyces multi-copy plasmid pIJ101. Mol Gen Genet 1988, 214: 286-294

25� Nakashima N, Tamura T. Isolation and characterization of a rolling-circle-type plasmid from Rhodococcus erythropolis and application of the plasmid to multiple-recombinant-protein expression. Appl Environ Microbiol 2004, 70: 5557-5568

26� Qin Z, Shen M, Cohen SN. Identification and characterization of a pSLA2 plasmid locus required for linear DNA replication and circular plasmid stable inheritance in Streptomyces lividans. J Bacteriol 2003, 185: 6575-6582

27� Zhou X, Deng Z, Firmin JL, Hopwood DA, Kieser T. Site-specific degradation of Streptomyces lividans DNA during electrophoresis in buffers contaminated with ferrous iron. Nucleic Acids Res 1988, 16: 4341-4352

28� Katz EC, Thompson CJ, Hopwood DA. Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. J Gen Microbiol 1983, 129: 2703-2714