1 Research Institute of Innovative Technology for the Earth (RITE), 9-2 Kizugawadai, Kizu-cho Soraku-gun, Kyoto 619-0292, Japan
2 Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Ikoma, Nara 630-0101, Japan
Correspondence
Hideaki Yukawa
mmg-lab{at}rite.or.jp
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ABSTRACT |
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IS14999 has been registered with the ISfinder database at http://www-is.biotoul.fr/.
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INTRODUCTION |
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Our knowledge of C. glutamicum has advanced greatly due to the completion of the genome sequence of C. glutamicum R (unpublished data), C. glutamicum ATCC 13032 (Kalinowski et al., 2003; Ikeda & Nakagawa, 2003
) and the closely related Corynebacterium efficiens (Nishio et al., 2003
). One approach to developing strains of higher productivity using genome information has been reported (Ohnishi et al., 2003
). But it does not yet deviate from the range of a single gene manipulation, and there still remain many unknown functions of genes in C. glutamicum. The easiest way to analyse genes of unknown function is to disrupt them and analyse the resultant strains. Insertion sequences are widely used as efficient tools for genome-wide mutagenesis to disrupt a single gene at a time (Hutchison et al., 1999
).
IS elements are the simplest form of transposable elements, and they have been found on chromosomes and plasmids of numerous bacteria (Mahillon & Chandler, 1998). However, the majority of IS elements have been discovered by analysis of genome sequences. Therefore their transposition activity was not experimentally confirmed. IS elements generally have a gene encoding a transposase that usually has a triad DDE motif as a catalytic domain and terminal inverted repeat sequences (IRs) (Mahillon & Chandler, 1998
). Upon insertion, they generate duplication of a sequence of specific base pairs at the target site. They are classified into several families based on the homology of amino acid sequence of the transposase, the IRs, and the length of the target site sequence (Mahillon & Chandler, 1998
).
To date, hundreds of IS elements have been identified in many bacteria, but not many IS elements are known from C. glutamicum and Brevibacterium lactofermentum. Moreover, only a few IS elements have been verified to possess transposition activity (Vertès et al., 1994a; Bonamy et al., 1994
, 2003
; Jager et al., 1995
). Two previous studies have described transposon mutagenesis of the C. glutamicum genome using IS31831 and IS1207 (Vertès et al., 1994b
; Bonamy et al., 2003
). These two elements belong to the same ISL3 family and share 99 % identity. Our unpublished data suggest that although IS31831 is a useful tool for random mutagenesis, it still has a tendency to transpose into specific sites. We initiated studies aimed at isolation of new functional IS elements from C. glutamicum. As a result, a new IS element, named IS14999, was successfully isolated from C. glutamicum ATCC 14999. It had transposition activity, and belonged to a new family in C. glutamicum, the IS630/Tc1 superfamily. Phylogenetic analysis showed that IS14999 was more similar to eukaryotic Tc1/mariner family elements than prokaryotic IS630 family elements. This new IS element will provide us with another useful tool for genetic or genomic engineering in C. glutamicum and related species.
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METHODS |
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Detection of transposition events.
Transposition was detected on minimal medium containing 10 % sucrose and spectinomycin by disruption of the sacB gene in plasmid pMV5.
Construction of transposon vector.
Plasmid pCRB512 was digested with HpaI and DraI, and subcloned into the HindIII site of plasmid pHSG398, which cannot replicate in C. glutamicum, to yield plasmid pCRB201. A kanamycin-resistance cassette was amplified using PCR with primers P1 and P2 from template pUC4K DNA. The PCR product was subcloned into HindIII-digested and blunt-ended pCRB201 to construct plasmid pCRB203.
Transformation of C. glutamicum.
All plasmid DNA used in the transformation of C. glutamicum was extracted from E. coli JM110 (dam dcm). Plasmid DNA extracted from a dam+ dcm+ E. coli strain cannot efficiently transform C. glutamicum because of the presence of a methyl-specific restriction system in C. glutamicum (Vertès et al., 1993). One microgram of unmethylated plasmid was used to transform C. glutamicum cells using a GenePulser II (Bio-Rad) as previously described (Kurusu et al., 1990
). One millilitre of A medium was added to electroporated cells, and the mixture was incubated for 2 h at 33 °C. An appropriate volume of culture was plated on A medium containing appropriate antibiotic to select transformants.
DNA sequencing and sequence analysis.
Nucleotide sequence determination was performed by the dideoxy chain-termination method (Sanger et al., 1977) using an ABI PRISM 3100 genetic analyser (Applied Biosystems). For sequence determination of IS14999, plasmid pCRB512 was sequenced using primer walking with synthetic oligonucleotides. Nucleotide sequences were determined on both strands independently. DNA sequence data were analysed with the GENETYX program (Software Development). Comparison searches of DNA and deduced protein sequences were performed with IS FINDER (http://www-is.biotoul.fr/is.html) and with the BLAST search program (Altschul et al., 1997
) provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast). Multiple alignment was done using CLUSTAL W version 1.83. Based on the amino acid sequence of the transposase, a phylogenetic tree was generated using TreeView version 1.6.0.
Determination of insertion sites and target sites.
Genomic DNA of recombinants was extracted using GenomicPrep Cells and a Tissue DNA isolation Kit (Amersham Bioscience). After digestion with PvuII, it was circularized by self-ligation using TAKARA ligation kit version 2.1 (Takara). The flanking region of insertion sites was amplified by inverse PCR with primers P3 and P4. The PCR product was sequenced with the same primers to determine insertion sites.
Dot-blot hybridization.
Genomic DNA of C. glutamicum strains was extracted and digested with PvuII. After denaturing at 100 °C for 5 min, genomic DNA was transferred to positively charged Hybond-N+ nylon membrane (Amersham Biosciences). We used the full nucleotide sequences of IS14999 and IS31831 amplified by PCR as a probe. DNA probes were prepared using the Gene Image Random Prime Labelling Module (Amersham Biosciences). Other DNA manipulations were performed essentially as described by Sambrook & Russell (2001).
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RESULTS |
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Characterization of IS14999, a new IS element of C. glutamicum
The nucleotide sequence of the 1·1 kb DNA fragment in plasmid pCRB512 was determined. The 1·1 kb DNA fragment comprised 1149 bp, with 22 bp IRs at either end (see GenBank accession no. AB186419). Computer analysis of the DNA fragment indicated the presence of one potential ORF. The ORF begins with an ATG at position 81 and ends at position 1115, and consists of 1035 nucleotides, corresponding to a product of 345 amino acids with a predicted mass of 39·3 kDa. The deduced amino acid sequence of the ORF has significant homology with two putative transposases annotated in C. efficiens YS-314, a species related to C. glutamicum, and also has partial homology in the C-terminal region with the transposase of IS642, which belongs to the IS630 family of Bacillus halodurans C-125. This new IS element was named IS14999. The overall G+C content of IS14999 is 55·1 mol%, which is almost the same as that of C. glutamicum R and ATCC 13032. 5'-TA-3' dinucleotides flanking the element were duplicated upon insertion of IS14999 as a direct repeat. The TA dinucleotide flanking the IS14999 sequence is replicated at position 1222 in the ORF of the sacB gene. IS630 family elements were verified to also duplicate the 5'-TA-3' dinucleotide (Tenzen et al., 1990). These facts indicated that IS14999 belonged to the IS630 family. This is believed to be the first report of a IS630 family transposable element in corynebacteria or mycobacteria.
Phylogenetic relationship between IS14999 and IS630/Tc1 superfamily elements
Transposases exhibit a highly conserved triad DDE motif as a catalytic domain at the C-terminus (Mahillon & Chandler, 1998) and this motif has proved to play a crucial role in transposition (Lohe et al., 1997
). The IS630 family comprises part of the IS630/Tc1 superfamily along with the eukaryotic Tc1/mariner family because of overall sequence similarity and a specific TA dinucleotide insertion target (Doak et al., 1994
; Shao & Tu, 2001
). Multiple alignments based on the transposase of IS14999 and IS630/Tc1 superfamily elements were conducted. The results showed that the transposase of IS14999 has a DDE motif at its C-terminal region and that flanking amino acids of this motif were partially conserved (Table 2
, Fig. 1
). These facts clearly showed that IS14999 belonged to the IS630/Tc1 superfamily. A phylogenetic tree was generated for 18 IS elements belonging to the IS630 family and 6 Tc1/mariner family transposable elements based on the amino acid sequences of their transposases (Fig. 2
). The phylogenetic tree showed that IS14999 is closer to eukaryotic Tc1/mariner family elements than to the prokaryotic IS630 family elements. Moreover, the distance between the last two residues in the DDE catalytic triad of IS14999 was 38 amino acids a unique distance compared to other IS630/Tc1 superfamily elements (Fig. 1
). These facts indicated that IS14999 might be transposed from a Tc1/mariner family element and could form a new subfamily of the IS630/Tc1 superfamily.
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Transposition of IS14999 into C. glutamicum R and its target preference
To assess whether IS14999 could be used as a tool for genetic engineering, a mutagenesis vector of IS14999 was constructed (plasmid pCRB203) and was used to mutagenize the C. glutamicum R genome. Transposition efficiency of Tn14999 was 22 c.f.u. per µg DNA, calculated by the number of kanamycin-resistant clones on the selective plate by averaging five experiments. Among 96 transformants, 91 strains showed kanamycin resistance and chloramphenicol sensitivity, which indicates that transposition had occurred. To determine insertion sites in the C. glutamicum R genome, genomic DNA of transformants was extracted and digested with PvuII, which did not digest the transposition sequence, followed by circularization by self-ligation. The flanking regions of insertion sites were amplified by inverse PCR and were sequenced. Sixty insertion sites were determined and the results showed that IS14999 transposed at random sites in the C. glutamicum R genome (Fig. 3). To investigate whether IS14999 recognized other sequences besides the duplicated 5'-TA-3' dinucleotide, flanking regions of the target sequence were analysed in detail. The results revealed that IS14999 did indeed duplicate the 5'-TA-3' dinucleotide, and moreover, it preferentially recognized the eight-base AGCTAGCT palindrome sequence (Fig. 4
).
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DISCUSSION |
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IS630 family elements are known to preferentially transpose to and consequently duplicate upon insertion a 5'-TA-3' dinucleotide sequence (Ohtsubo & Sekine, 1996; Mahillon & Chandler, 1998
). This feature is recognized also in eukaryotic Tc1/mariner family elements (Plasterk et al., 1999
). Moreover, Tc1/mariner family elements, like IS630 family elements, also have conserved DDE or DDD triad amino acids as an essential part of their catalytic site, and mutations in the triad abolished transposase activity (Lohe et al., 1997
). Consequently, these two families form a mega-family, the IS630/Tc1 superfamily, whose elements share a similar signature sequence or motif in the catalytic domain of their respective transposases (Shao & Tu, 2001
; Urasaki et al., 2002
). The only difference is that Tc1/mariner family elements appear to be excised from their donor molecules, leaving an empty site with an extra sequence, called a footprint (Plasterk, 1996
), like those of other element families.
IRs of IS630 family elements are not as conserved as other IS element families (data not shown). In addition, IRs of Tc1/mariner family elements, each of unique length, show partial conservation only in the first four IR nucleotides (Plasterk 1996). These characteristics are part of the reason for the broad diversity of IS630/Tc1 superfamily elements beyond the frame of prokaryotes and eukaryotes. The phylogenetic tree of IS630/Tc1 superfamily elements showed that some elements were clustered together based on the distance between the second and third amino acids in their DDE motifs. IS14999 is positioned among the eukaryotic Tc1/mariner family elements in spite of its prokaryotic origin. It should be noted that the distance between the second D and third E residues of the transposase of IS14999 in the DDE motif, the catalytic triad, was invariably 38 residues. The distances between the first two Ds are variable while the distances between the last two residues in the DDE motif are mostly invariable for a given IS or transposon family. Most IS630I/Tc1 superfamily elements show distances of 34, 35 or 37 residues between the latter two residues, and form subfamilies depending on these distances (Shao & Tu, 2001
). Surprisingly, IS14999 had a unique distance of 38 residues in IS630/Tc1 superfamily. We suggest that IS14999 may form a new subfamily of the IS630/Tc1 superfamily because of its unique DD38E motif.
Analysis of insertion sites of IS14999 showed that the 5'-TA-3' dinucleotide was duplicated upon insertion, and moreover, it preferably transposed into an 8 bp (AGCTAGCT) palindrome sequence in the C. glutamicum R genome. In this sequence, the A at position 3 and the T at position +3 are the most conserved (55·7 % and 70·5 % respectively). A few detailed analyses of preferred insertion sites of the other IS630/Tc1 superfamily elements have been reported. IS630 has been reported to transpose preferentially to the 5'-CTAG-3' sequence (Tenzen & Ohtsubo, 1991). Tc1 and Tc3, 5'-GAKATATGT-3' (K=G or A) or 5'-AYATATRT-3' (Y=C or T; R=G or A) and 5'-ATATATTT-3' respectively, were preferentially recognized (Mori et al., 1988
; Korswagen et al., 1996
; Preclin et al., 2003
). In Tc1, the A at position 3 and the T at position +3 are the more highly conserved (75 % and 73 % respectively) (Preclin et al., 2003
). This high conservation of the A at position 3 and the T at position +3 is identical to the situation in IS14999. We presume that IS14999 is close to eukaryotic transposable elements because of the similarity of their preferred target sequences and the result of phylogenetic analysis.
Of 60 C. glutamicum R : : Tn14999 strains, 45 strains had the IS inserted in the gene-coding region, but the other 15 strains had the insertion in non-coding regions, including a promoter region (data not shown). IS14999 is thought not to need any host factors in its transposition because it has been reported that IS630/Tc1 superfamily elements do not require any host factors (Craig, 1997; Urasaki et al., 2002
). This would be an advantage for using IS14999 as a tool for genetic manipulation in various bacteria. Study of random chromosomal deletion of C. glutamicum R using IS14999 and IS31831 is in progress.
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ACKNOWLEDGEMENTS |
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Received 19 August 2004;
revised 26 October 2004;
accepted 26 October 2004.
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