A new insertion sequence, IS14999, from Corynebacterium glutamicum

Yota Tsuge1,2, Kana Ninomiya1, Nobuaki Suzuki1, Masayuki Inui1 and Hideaki Yukawa1,2

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A new insertion sequence from Corynebacterium glutamicum ATCC 14999 was isolated and characterized. This IS element, designated IS14999, comprised a 1149 bp nucleotide sequence with 22 bp imperfect terminal inverted repeats. IS14999 carries a single open reading frame of 345 amino acids encoding a putative transposase that appears to have partial homology to IS642, an IS630/Tc1 superfamily element, at the C-terminal region in the amino acid sequence. This indicated that IS14999 belonged to the IS630/Tc1 superfamily, which was first identified in C. glutamicum. IS14999 has a unique distance of 38 amino acid residues between the second and third amino acids in the DDE motif, which is well known as the catalytic centre of transposase. This suggested that IS14999 constituted a new subfamily of the IS630/Tc1 superfamily. A phylogenetic tree constructed on the basis of amino acid sequences of transposases revealed that this new transposable element was more similar to eukaryotic Tc1/mariner family elements than to prokaryotic IS630 family elements. Added to the fact that IS14999 was present in only a few C. glutamicum strains, this implies that IS14999 was probably acquired by a recent lateral transfer event from eukaryotic cells. Analysis of the insertion site in C. glutamicum R revealed that IS14999 appeared to transpose at random and always caused a target duplication of a 5'-TA-3' dinucleotide upon insertion, like the other IS630/Tc1 family elements. These findings indicated that IS14999 could be a powerful tool for genetic manipulation of corynebacteria and related species.


Abbreviations: IR, inverted repeat

IS14999 has been registered with the ISfinder database at http://www-is.biotoul.fr/.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Corynebacterium glutamicum is a Gram-positive bacterium with a high G+C content and a characteristic envelope structure, with an abundant mycolic acid layer outside its cell membrane (Puech et al., 2001). This bacterium has been widely used in industry as a producer of several amino acids (Kinoshita et al., 1957; Terasawa et al., 1990; Sahm et al., 1996; de Graaf et al., 2001). To date, numerous mutants that can produce amino acids at high efficiency have been created. The worldwide demand for amino acids such as L-glutamate, L-lysine and L-threonine for use as animal feed and food additives has continued to increase (Hermann, 2003; Ohnishi et al., 2003). Therefore, it is desirable to develop C. glutamicum strains with improved productivity. However, this is unlikely to be feasible using random mutations.

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and culture conditions.
Bacterial strains and plasmids used in this study are listed in Table 1. C. glutamicum strains were grown in minimal medium containing 10 % (w/v) sucrose, or in A medium, at 33 °C with aeration. Minimal medium contained, per litre, 40 g glucose, 2 g urea, 7 g (NH4)2SO4, 0·5 g K2HPO4, 0·5 g KH2PO4, 0·5 g MgSO4.7H2O, 6 mg FeSO4.7H2O, 6 mg MnSO4.7H2O, 200 µg biotin and 200 µg thiamin.HCl. A medium contained, per litre, 2 g yeast extract and 7 g Casamino acids in addition to the components of minimal medium. Escherichia coli strains were grown in Luria–Bertani (LB) medium (Sambrook & Russell, 2001) at 37 °C with aeration. Where necessary, spectinomycin was added to a final concentration of 200 µg l–1, and kanamycin to 50 µg l–1. Chloramphenicol was used at 50 µg l–1 for E. coli strains, and at 5 µg l–1 for C. glutamicum strains.


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Table 1. Strains, plasmids and primers used in this study

 
DNA techniques and PCR methodology.
E. coli plasmid DNA was extracted using a QIAprep spin Miniprep Kit (Qiagen) according to the manufacturer's instructions. PCR reactions were performed using TAKARA LA Taq DNA polymerase (Takara) in a GeneAmp PCR System 9700 (Applied Biosystems). PCR products were recovered by using a QIAquick Gel Extraction Kit (Qiagen). DNA termini were modified using a TAKARA Blunting Kit (Takara).

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).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isolation of IS elements from C. glutamicum
The Bacillus subtilis sacB gene carrying plasmid pMV5 (Vertès et al., 1994a) was used to isolate new transposable elements in C. glutamicum. C. glutamicum and several other Gram-positive bacteria show sucrose sensitivity in the presence of the SacB product because they contain a mycolic acid layer in their cell envelope (Jager et al., 1992; Pelicic et al., 1996). C. glutamicum cells harbouring plasmid pMV5 were grown overnight in A medium supplemented with spectinomycin. A total of 100 µl of the overnight culture was plated on minimal medium containing sucrose supplemented with spectinomycin. From several sucrose-tolerant colonies that arose after 48 h incubation at 33 °C, sacB-disruption strains carrying an IS element in their sacB genes were obtained. The resistant colonies were cultured in liquid minimal sucrose medium and plasmid DNA was extracted. Then plasmid DNA was used to transform E. coli, and plasmid DNA was extracted again. Restriction analysis of extracted plasmid DNA using SmaI and XbaI revealed that the 1·9 kb sacB-containing fragment was altered when a transposable element was transposed into the sacB gene. After screening of C. glutamicum ATCC strains, one plasmid, named pCRB512, was obtained from C. glutamicum ATCC 14999. Restriction analysis showed that this plasmid contained an insertion of approximately 1·1 kb within the SmaI–XbaI fragment including the sacB gene (data not shown).

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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Table 2. IS630/Tc1 superfamily of transposable elements

 


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Fig. 1. An alignment of segments of transposases encoded by IS630/Tc1 superfamily elements. The D, D and E of the DDE motif are shown on a black background. A grey background indicates similar amino acids. The numbers in parentheses show the distance between amino acid sequences of the DDE motif.

 


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Fig. 2. Phylogenetic tree analysis of IS630/Tc1 superfamily elements. The tree was constructed by the neighbour-joining method (Saitou & Nei, 1987) based on amino acid sequences of transposases. The numbers indicate bootstrap values for 1000 replicates. The scale bar equals a distance of 0·1.

 
Distribution of IS elements in C. glutamicum strains
As mentioned above, a BLAST search revealed that C. efficiens had two IS14999-like elements in its genome, which had 84·6 % and 84·7 % identity with IS14999 at the amino acid level. On the other hand, C. glutamicum R and ATCC 13032 did not have any other IS14999-like element. This indicated that IS14999 could be used as a tool for genomic engineering, especially in C. glutamicum R and ATCC 13032. To investigate whether IS14999 and IS31831 are conventional transposable elements in C. glutamicum, dot-blot analysis was carried out. We selected 50 C. glutamicum strains, including C. glutamicum R, ATCC 13032 and ATCC 14999, in this experiment. Genomic DNA was extracted and digested with PvuII, which does not digest the nucleotide sequence of either of these IS elements. Extracted and denatured genomic DNA was used for the following hybridization. PCR fragments of the nucleotide sequences of IS14999 and IS31831 were used as probes. The result showed that of 50 selected strains, 41 possessed high copy numbers of IS31831. On the other hand, IS14999 was present in only 12 C. glutamicum strains (data not shown). IS14999 and IS31831 were present in three and six copies of the strains from which they were derived, C. glutamicum ATCC 14999 and ATCC 31831, respectively, as verified by Southern blot analysis (data not shown).

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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Fig. 3. Physical map of insertion sites of Tn14999 in the C. glutamicum R genome. Numbers represent the position of the first nucleotide of the 5'-TA-3' duplicated sequences in the C. glutamicum R genome.

 


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Fig. 4. Target preference of IS14999. Numbers represent the percentage occurrence of the preferred base at the positions indicated. The analysis is based on 61 independent insertions (including insertion into sacB). Black backgrounds show duplicated bases. Grey backgrounds indicate preferred bases. Bases below the numbers show the most preferred base at positions – to +3.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In the present study, we isolated and characterized a new IS element from C. glutamicum, IS14999, which encodes a protein with homology to IS630/Tc1 superfamily transposases in the region containing a DDE motif. In the course of the isolation of the IS element, many sucrose-resistant colonies were obtained. This indicated that besides the sucrose resistance caused by mutation of the sacB gene via insertion of the IS element, an as yet unknown resistance mechanism must exist.

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.


   ACKNOWLEDGEMENTS
 
We thank Roy H. Doi (University of California, Davis, USA) and Crispinus Omumasaba for critical reading of the manuscript. This study was carried out as a part of The Project for Development of a Technological Infrastructure for Industrial Bioprocesses on R&D of New Industrial Science and Technology Frontiers by Ministry of Economy, Trade & Industry (METI), and entrusted by the New Energy and Industrial Technology Development Organization (NEDO).


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ABSTRACT
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METHODS
RESULTS
DISCUSSION
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Received 19 August 2004; revised 26 October 2004; accepted 26 October 2004.



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