Original Paper
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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
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.
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