Sequence Analysis of a Novel Insertion Site of Transposon IS10

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Acta Genetica Sinica, November

ISSN 0379-4172

2006, 33 (11)：1047–1052

Sequence Analysis of a Novel Insertion Site of Transposon IS10 XIANG Tai-He①, WANG Li-Lin, WANG Hui-Zhong School of Life Sciences, Hangzhou Normal College, Hangzhou 310036, China Abstract: A sacB mutant was obtained by transposon IS10 inactivation of a plasmid pXT3sacB carrying the sacB gene. Sequencing of this mutant plasmid DNA (GenBank accession No. AY580883.1) showed that the IS10 flanking the 22 bp inverted repeats were 5′-CTGAGAGATCCCCTCATAATTT-3′ and 5′-AAATCATTAGGGGATTCATCAG-3′, which were similar to those published in reports previously. However, the target sequence adjacent to IS10 was 5′-TGCTTGGTT-3′ instead of the previously reported 5′-NGCTNAGCN-3′. To our knowledge, this is the first report on the novel insertion site of IS10. In addition, Southern blot hybridization confirmed that the mobile IS10 originated from the chromosomal DNA of the host strain Escherichia coli DH5α and that there were two copies in the DH5α genome. Key words: transposon; IS10; sacB gene; insertion site; sequence

Transposable element (transposon) is widespread among organisms and plays an important role in gene recombination and genome evolution[1,2]. Recently, it has been described that a transposon can induce RNA interference and is functionally related to the regulation of gene expression[3]. It is known

1 1. 1

Materials and Methods Plasmid, bacterial strain, and growth condition Plasmid pXT3sacB with the sacB gene encoding

levansucrase was constructed previously[14]. It was

that a short target sequence is replicated to the flank of the transposon after transposon insertion[4,5]. IS10 is a type of bacterial transposon. It can either function as an individual insertion sequence (IS) or mediate transposition of the whole Tn10 element[6-8]. Accord-

maintained in the host strain Escherichia coli DH5α.

ing to the published reports, IS10 inserts into the receptor at a specific site that contains the sequence

sucrose at 37℃ for 12-48 h. Rotational speed was

5′-NGCTNAGCN-3′[5,9 13]. Among the nine base -

pairs in the target sequence adjacent to IS10, six base pairs are identical. In this article, an inactivating mutation of the sacB gene by IS10 was obtained using plasmid pXT3sacB carrying the sacB gene[14]. The sequence of this mutant plasmid DNA revealed a novel insertion site of IS10.

Host strain with pXT3sacB plasmid is lethal in the presence of 5% sucrose in the agar medium. Bacteria were grown in LB medium with 50 mg/L kanamycin (Km) or LB medium with 50 mg/L Km and 50 g/L adjusted to 180-200 r/min for suspension culture. Agar (10 g/L) was used for solid medium. 1. 2

Selection and analysis of sacB inactivation mutants

Bacterial culture grown overnight in LB medium with 50 mg/L Km was diluted to about 0.1 OD and spread onto LB plates with 50 mg/L Km and 50 g/L

Received: 2006-02-07; Accepted:2006-02-27 This work was supported by the Natural Science Foundation of Zhejiang Province (No.Y304083), the Science Foundation of Department of Education at Zhejiang Province (No.20040069) and the Science Foundation for the talents of Hangzhou Normal College (No.87). ① Corresponding author. E-mail: [email protected]; Tel & Fax: + 86-571-2886 5327

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sucrose. Mutant clones were obtained after 48 h of

the study of Kempter B and Grossbadern K[16]. DNA

culture at 37℃. Single mutant colonies were picked

samples were digested by BamHⅠ, fractionated by

and inoculated into LB liquid medium with 50 mg/L Km and 50 g/L sucrose for propagation. Mutation frequency was investigated according to the method of Sheng Zu-Jia[15]. Plasmid DNA was isolated by the

1% agarose gel electrophoresis, and transferred onto Hybond-Nylon membranes. Based on the IS10 sequence, a pair of primers 5′-TAGAACCCATGGCTCCTTTG-3′ and 5′-TTACCGAACTTGGCCGTAAC3′ were synthesized for PCR amplification of a 680 bp fragment from the mutant plasmid. The PCR product was purified to generate a probe, which was labeled with DIG-High Prime DNA Labeling kit (Roche Ltd) and hybridized to the membrane. The signal was detected by using Detection Starter Kit I (Roche Ltd), following the manufacturer’s instructions.

alkaline lysis method and digested by ClaⅠto distinguish between point mutation and insertion mutation. 1. 3

Sequence analysis of sacB gene insertion mutation Primers P1 and PR1 were synthesized for se-

quencing according to the sequence of sacB gene (GenBank accession No. X02730). Then, P2-P5, PR2, and PR3 were synthesized for sequence walking (Table 1 and Fig. 1). DNA sequencing was performed by

2

Shanghai Genecore Biotechnologies Co. Ltd using an

2. 1 Analysis of mutant plasmids by restriction endonuclease digestion

automated DNA sequencer, Model ABI3700.

P1

CCTAAAAGACCAAACACAAGA

P2

TGAAACCGCTGATTGCAT

Overnight suspension culture of bacteria carrying pXT3sacB was spread onto LB plates supplemented with 50 mg/L Km and 50 g/L sucrose. Single colonies were present only after 48 h, whereas no clones were present in 24 h. Ten single colonies (No.

P3

TGCTAGAACCCATGGCTC

1-10), of which No. 1-3, No. 4-6, and No. 7-10 were

P4

GGGGATTCATCAGTGCTTGGT

P5

CTGCGTATATCGCTTGCTGA

obtained from three separate lines, were selected to extract plasmid DNA. Digestion of the plasmid DNA

Table 1 tants

Primers used in sequencing sacB insertion mu-

Primers

1. 4

Results

Sequences (5′→3′)

PR1

TGTGCAGTTGTCAGTGTTTGT

PR2

CCAGCAGTGCGGTAGTAA

PR3

CGAAGTGTGAGTAAGTAAAGGT

Southern blot hybridization

with ClaⅠ revealed a distinction between point mutation and insertion mutation (Fig. 2). Three bands of about 10 kb, 2 200 bp, and 350 bp were produced when pXT3sacB was cut with ClaⅠ (Fig. 2, lane 11).

Preparation of DH5α chromosomal DNA was

Three bands of about 10 kb, 3 500 bp, and 350 bp were produced when the insertion mutant was cut

carried out according to the procedure described in

with ClaⅠ (Fig. 2, lanes 1, 4, 6, 8, 9 and 10).

Fig. 1 Diagrammatic sketch of primers for sequencing and AY5800883.1 sequence RI: replicated sequence of insertion site.

XIANG Tai-He et al.: Sequence Analysis of a Novel Insertion Site of Transposon IS10

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The result revealed that the insertion sequence was the same as the transposable element IS10 (GenBank accession No. AF162223.1, AF542545.1, AY533233.1, AY319289.1, J01829.1, AP005147.1, and BX664015.1). The sequences flanking IS10 were 5′-CTGAGAGATCCCCTCATAATTT-3′ and 5′-AAATCATTAGGGGATTCATCAG-3′ and were Fig. 2

Agarose gel electrophoresis of plasmid DNA of

similar to those published in previous reports. Both

sacB gene inactivated mutants digested by ClaⅠ

ends of the insertion sequence were 22 bp long and

M: λDNA/EcoRⅠ+Hind Ⅲ DNA marker; 1, 4, 6, 8, 9, 10:

included a 17 bp reverse complement sequence.

pXT3sacB insertion mutants digested by ClaⅠ; 2, 3, 5, 7:

However, the target sequence adjacent to IS10 was

pXT3sacB spot mutants digested by ClaⅠ; 11: pXT3sacB

5′-TGCTTGGTT-3′ (Figs. 1 and 3）, which was dif-

digested by ClaⅠ.

ferent from the 5′-NGCTNAGCN-3′ that was

However, ClaⅠ digestion did not distinguish between pXT3sacB and plasmids with point mutations (Fig. 2, lanes 2, 3, 5 and 7). Moreover, the average frequency of insertion mutation and point mutation was 1.48 × 10−7% and

published in reports previously[5,9-13]. The 6th and 8th nucleotides in this insert site were G and T instead of A and C. Moreover, sequencing No.4 and No.8 mutants by primers P2 and PR3 consistently showed the target sequence to be 5′-TGCTTGGTT-3′ (data not

1.13 × 10−7%, respectively. The frequency of insertion mutation was a little higher than that of point mutation (Table 2).

shown).

2. 2

verse complementary sequence at the insertion site.

Analysis of insertion mutants by sequencing

Sequencing mutant clone No.1 using eight primers listed in Table 1 resulted in a 4 425 bp DNA, the sequence of which has been submitted to GenBank (Accession No. AY580883.1). The result indicated that the sacB gene was inactivated by the insertion sequence (IS) at the 1599th nucleotide. The entire length of sacB was 2 007 bp, according to Fouet A et al. (GenBank accession No. X02730)[17], and the entire insertion sequence was 1 329 bp. The insertion sequence was compared with available DNA sequences in the GenBank database by Blast. Table 2

In Fig. 3, the chromatogram at the top showed a partial sequence at the insertion site, whereas the chromatogram at the bottom indicated the partial reThe continuous 9 bp starting from the 10th nucleotide was the sequence of the insertion site (insertion site sequences are underlined in Fig. 3). 2. 3

Southern blot analysis To determine whether IS10 originated from

chromosome of host strain DH5α, chromosomal DNA was extracted and digested by BamHⅠ. Southern blot analysis showed that the two chromosomal DNA fragments hybridized with the probe, whereas no bands were detected from pXT3sacB DNA (Fig. 4). It

Mutation frequency of sacB inactivation

Test lines

Number of mutations in the analysis

1

16

2

18

9

1.18×10−7

9

1.18×10−7

3

16

10

1.70×10−7

6

1.02×10−7

Average

−

−

1.48×10−7

−

1.13×10−7

Insertion mutation Number of insertion Frequency of insertion mutation mutation (%) 9 1.55×10−7

Point mutation Number of point Frequency of point mutation mutation (%) 7 1.20×10−7

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Fig. 3 Chromatogram of sequence of IS10 insertion site Top: partial sequence via primer P2; Bottom: partial sequence via primer PR3.

confirmed that IS10 that jumped into sacB gene in-

published reports of IS10 insertion site consensus

deed originated from the chromosomal DNA of the

sequence 5′-NGCTNAGCN-3′[5,9 13]. IS10 transposi-

host strain and that there were two copies in the ge-

tion is known to increase the fitness of Escherichia

nome.

coli and may have been recruited under the SOS re-

-

sponse to increase cell survival during hostile envi-

3

Discussion

Results from the three lines consistently showed that the specificity of the target sequence in this study was 5′-TGCTTGGTT-3′, which had not been found previously. To our knowledge, this is the first report of this novel insertion site of IS10, in addition to the

ronmental conditions[17,18]. The mutants that resulted from IS10 insertion in this study were produced after bacteria had been grown on LB medium supplemented with 50 g/L sucrose for 48 h, i.e., the bacteria suffered high osmotic pressure and malnutrition. It may be suggested that the stress condition in this

XIANG Tai-He et al.: Sequence Analysis of a Novel Insertion Site of Transposon IS10

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jing: Higher Education Press, 2002, 54-63 (in Chinese). [6] Halling S M, Simons R W, Way J C, Walsh R B, Kleckner N. DNA sequence organization of IS10-right of Tn10 and comparison with IS10-left. Proc Natl Acad Sci USA, 1982, 79(8): 2608-2612. [7] Chalmers R, Sewitz S, Lipkow K, Crellin P. Complete nucleotide sequence of Tn10. J Bacteriol, 2000, 182(10): 2970-2972. [8] Allingham J S, Haniford D B. Mechanisms of metal ion action in Tn10 transposition. J Mol Biol, 2002, 319(1): 53-65. Fig. 4 Host strain DH5α chromosomal DNA hybridization with probe 1: DH5α chromosomal DNA/BamHⅠ; 2: plasmid DNA of insertion mutant; 3: plasmid DNA of pXT3sacB.

study could, to some extent, have a bearing on the change of IS10 insertion site, but further studies need to be conducted. On the other hand, sacB gene from Bacillus subtilis encodes a levansucrase, which catalyzes transfructorylation from sucrose to various acceptors. The production of levansucrase induced by sucrose is lethal to gram-negative bacteria[19], so an inactivated sacB gene can be positively selected on the medium with sucrose. In this study, sacB mutants caused by IS10 insertion were easily obtained following this procedure. Such a system could be valuable for the entrapment of other insertion sequence elements. References: [1] Chao L C, Vargas B, Spear B, Cox E C. Transposable elements as mutator genes in evolution. Nature, 1983, 303(5918): 633-635. [2] Kidwell M G, Lisch D. Transposable elements as sources of variation in animals and plants. Proc Natl Acad Sci USA, 1997, 94(15): 7704-7711. [3] Puig M, Caceres M, Ruiz A. Silencing of a gene adjacent to the breakpoint of a widespread Drosophila inversion by a transposon-induced antisense RNA. Proc Natl Acad Sci USA, 2004, 101(24): 9013-9018. [4] Foster T J, Lundblad V, Hanley-Way S, Halling S M, Kleckner N. Three Tn10-associated excision events: relationship to transposition and role of direct and inverted repeats. Cell, 1981, 23(1): 215-227. [5] Zhu Y X, Li Y. Modern molecular biology. 2nd ed. Bei-

[9] Bogosian G, Bilyeu K, O'Neil J P. Genome rearrangements by residual IS10 elements in strains of Escherichia coli K-12 which had undergone Tn10 mutagenesis and fusaric acid selection. Gene, 1993, 133(1): 17-22. [10] Huisman O, Errada P R, Signon L, Kleckner N. Mutational analysis of IS10's outside end. EMBO J, 1989, 8(7): 2101-2109. [11] Bender J, Kleckner N. IS10 transposase mutations that specifically alter target site recognition. EMBO J, 1992, 11(2): 741-750. [12] Bender J, Kleckner N. Tn10 insertion specificity is strongly dependent upon sequences immediately adjacent to the target-site consensus sequence. Proc Natl Acad Sci USA, 1992, 89(17): 7996-8000. [13] Eichenbaum Z, Livneh Z. Intermolecular transposition of IS10 causes coupled homologous recombination at the transposition site. Genetics, 1995, 140(3): 861-874. [14] Xiang T H, Yang J B, Somers D A. Disarming of wild type Agrobacterium rhyzogenes K599. Hereditas (Beijing), 2001, 23(4): 336-340 (in Chinese with an English abstract). [15] Sheng Z J. Microbiology. Beijing: Higher Education Press, 1994, 76-80 (in Chinese). [16] Kempter B, Grossbadern K. Quick preparation of high molecular weight DNA by freezing. Trends Genet, 1992, 8(7): 226. [17] Skaliter R, Eichenbaum Z, Shwartz H, Ascarelli-Goell R, Livneh Z. Spontaneous transposition in the bacteriophage λ cro gene residing on a plasmid. Mutat Res, 1992, 267(2): 139-151. [18] Eichenbaum Z, Livneh Z. UV light induces IS10 transposition in Escherichia coli. Genetics, 1998, 149(7): 1173-1181. [19] Fouet A, Arnaud M, Klier A, Rapoport G. Characteriza-

tion of the precursor form of the exocellular levansucrase from Bacillus subtilis. Biochem Biophys Res Commun, 1984, 119(2): 795-800.

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转座因子 IS10 一个新的插入靶位点的序列分析 向太和，王利琳，王慧中 杭州师范学院生命科学学院，杭州 310036 摘 要: 转座因子在生物体内广泛存在，它在研究基因的重组机理以及生物染色体的进化方面有着重要意义。IS10 是细 菌中的一种转座因子，它既能单独作为插入序列，也能作为Tn10 的一部分进行转座。利用含sacB基因的质粒pXT3sacB， 获得了由转座因子IS10 插入而导致sacB基因失活的突变体。通过对插入突变体质粒DNA的序列测定(GenBank登记号为 AY580883.1) ， 结 果 表 明 IS10 两 端 分 别 包 括 22 bp 倒 置 重 复 区 CTGAGAGATCCCCTCATAATTT 和 AAATCATTAGGGGATTCATCAG，这与前人的报道一致；而IS10 两端的插入靶位点序列为TGCTTGGTT，该 9 bp靶位点序列与前人 报道的序列NGCTNAGCN不同。根据文献资料，本研究中的靶位点序列是首次报道。此外，通过Southern blot杂交分析， 插入sacB基因中的IS10 来源于宿主大肠杆菌DH5α染色体DNA，并且IS10 在DH5α 染色体中为两个拷贝。此外，本研究利 用sacB基因捕获到转座因子IS10，该方法为研究其他插入序列提供了一个有益的体系。 关键词: 转座因子；IS10；sacB 基因；插入位点；序列 作者简介: 向太和（1965-），男，安徽青阳人，教授，博士，研究方向：分子遗传学。E-mail: [email protected]