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1 Department of Bioscience, Tokyo University of Agriculture, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
2 Department of Life Science, College of Science, Rikkyo (St Paul's) University, Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan
Correspondence
Hirofumi Yoshikawa
hiyoshik{at}nodai.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-glutamate (-PGA)-deficient mutants of Bacillus subtilis (natto) appear to have resulted from the insertion of IS4Bsu1 exclusively into the comP gene. However, complete genomic analysis of B. subtilis 168, a close relative of B. subtilis (natto), revealed no IS4Bsu1 insertion. Preliminary experiments using a transformable ‘natto’ strain indicated that the frequency of transposition of IS4Bsu1 was exceptionally high under competence-developing conditions. On the other hand, such high-frequency transposition was not observed when cells were grown in a rich medium, such as LB medium, suggesting that there must be suitable environmental conditions that give rise to the transposition of IS4Bsu1. To assess the behaviour of IS4Bsu1 and explore any host factors playing roles in IS transposition, an intermolecular transposition assay system was constructed using a modified IS4Bsu1 element in B. subtilis 168. Here, the details of the intermolecular transposition assay system are given, and the increase in transposition frequency observed under high-temperature and competence-inducing conditions is described.
-PGA, poly--glutamate |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Insertion sequences (ISs) are small and simple transposons in bacteria; they are components of chromosomes and plasmids. Many IS elements have been identified in various bacteria and classified on the basis of transposase homology, IRs and the length of the target sequence. It is widely believed that transposition of IS elements plays an important role as a motive force in genomic evolution (Craig et al., 2002). The transposition activity of transposons is mediated by various host factors that are specific for each element. Histone-like proteins (or DNA chaperones) such as HU and integration host factor (IHF) function in the transposition reaction of some bacterial transposons (Kleckner et al., 1996; Lavoie & Chaconas, 1996). While a nucleoid-associated protein, H-NS, is required for IS1 transposition, other histone-like proteins are not (Shiga et al., 2001). Therefore, there is no particular host factor essential for the transposition of all transposons. Other known host factors include the replication initiator DnaA, the protein chaperones/proteases ClpX, ClpP and ClpA, the SOS control protein LexA and the Dam methylase (Mahillon & Chandler, 1998).
‘Natto’ is a traditional fermented food derived from soybeans; it is widely consumed by the Japanese. A ‘string-like’ component contributes to its characteristic sticky texture; it has been found to be a fermentation product, poly--glutamate (-PGA), of a variant strain of Bacillus subtilis (natto). B. subtilis (natto) is closely related to B. subtilis Marburg 168, the best-characterized Gram-positive bacterium, whose entire genome has been sequenced (Kunst et al., 1997). Among B. subtilis strains, Marburg 168 can be transformed at a high frequency with its own natural genetic competence; other B. subtilis strains, including B. subtilis (natto), yield transformants at a frequency as low as that of spontaneous mutation.
In the B. subtilis 168 strain, cell-density-dependent phenotypes are regulated by a quorum-sensing mechanism involving the ComP–ComA two-component regulatory system (Hahn & Dubnau, 1991; Nakano et al., 1991; Roggiani & Dubnau, 1993; Solomon et al., 1995; Weinrauch et al., 1990). The synthesis of -PGA in B. subtilis (natto) is also controlled by this system. The ability to produce -PGA is occasionally lost through serial cultivation, and this phenomenon is associated with the transposition of an IS into the comP gene (Nagai et al., 2000). The IS found in natto strains is a member of the IS4 family, designated IS4Bsu1, which is 1406 bp in length and has imperfect 18 bp terminal IRs. It also contains an ORF that encodes a 374 aa transposase and generates a 9 bp duplication of the target site during insertion (Nagai et al., 2000).
Transposition studies have focused on analysing the detailed mechanisms of transposition; however, the cellular conditions that induce transposition remain to be elucidated. Therefore, we constructed a transposition assay system using B. subtilis 168 and modified IS4Bsu1. Our results revealed an increase in transposition frequency under high-temperature and competence-developing conditions.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was obtained from F. Kawamura (Rikkyo University). These strains were grown in Luria–Bertani (LB) or CI medium (Anagnostopoulos & Spizizen, 1961) at 37 or 49 °C. Escherichia coli DH10B [F– mcrA 
(mrr-hsdRMS-mcrBC) 
80dlacZ M15 lacX74 deoR recA1 araD139 (ara leu)7697 galU galK 
– rpsL endA1 nupG] was grown in LB medium at 37 °C. The shuttle vector pDG148 [ampicillin resistant (AmpR) for E. coli and kanamycin resistant (KmR) for B. subtilis] (Stragier et al., 1988) was used for the intermolecular transposition assay. When necessary, biotin was added at a final concentration of 0.1 µg ml–1. Antibiotics were used at the following concentrations: chloramphenicol, 5 µg ml–1 (B. subtilis) or 20 µg ml–1 (E. coli); ampicillin, 50 µg ml–1; kanamycin, 5 µg ml–1; and spectinomycin, 50 µg ml–1.
Southern blot analysis.
For genomic Southern blot analysis, 6 µg chromosomal DNA was digested with EcoRV (TaKaRa) and resolved on an 0.8 % agarose gel. The DNA was then transfer-blotted onto a Hybond-N+ nylon membrane (Amersham Bioscience). Hybridization was with a DIG-labelled (DIG High Prime DNA labelling and detection starter kit II; Roche Diagnostics) IS4Bsu1-specific probe using the PCR-generated fragment (primers ISin3' and ISin5', Table 1) according to the manufacturer's instructions.
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) and the C-terminal region (primers P9 and P10), respectively. Fragment 2 included both the IRL and IRR of IS4Bsu1 and a cat gene (primers P3 and P4). Fragment 3 carried PS10 and the Shine–Dalgarno sequence of the rpsJ gene of B. subtilis (primers P5 and P6). Fragment 4 (primers P7 and P8) included the tnp gene derived from IS4Bsu1. Templates for generating these fragments were B. subtilis 168 genomic DNA (fragments 1, 3 and 5), pBEST4C (Itaya et al., 1990) (fragment 2), and B. subtilis (natto) OK2 (Ashikaga et al., 2000) genomic DNA (fragment 4). Primers were designed to have 5' add-on sequences to create overlapping sequences between flanking fragments. To generate the desired secondary PCR constructs, fragment 6 (primers P11 and P12) was amplified by the recombinant PCR method, using the three-piece primary PCR products (fragments 2, 3 and 4) (Higuchi, 1989). Likewise, the tertiary PCR product (primers P1 and P10), generated from fragments 1, 5 and 6, was used to transform B. subtilis 168 to construct the assay strain NBS040 (Fig. 2). As control strains, the IRL-deleted strain NBS041 (using primers P15 and P16 instead of P3 and P2) and the tnp-depleted strain NBS042 (using primers P13 and P14 instead of P4 and P9) were constructed using NBS040 genomic DNA as template (Fig. 2). Pyrobest DNA polymerase (TaKaRa) was used for all PCR assays.
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), and 0.5 µg plasmid DNA was introduced into E. coli DH10B cells via electroporation with a Micro Pulser (Bio-Rad). After appropriate dilution, cells were plated on LB plates containing 50 µg ampicillin ml–1 with or without 20 µg chloramphenicol ml–1. The transposition frequency was estimated as the ratio of the number of AmpR/CmR transformants to the number of total AmpR transformants.
DNA sequencing.
The DNA sequence of the target duplication and the target sites of the mini-IS transposition into the pDG148 plasmid (Fig. 3, Table 4) were analysed using BigDye Terminator version 3.1 cycle sequencing kits and the GeneAmp PCR system 2700 (Applied Biosystems). The primers were out-cat3up and out-cat4down (Table 1); they anneal to the N- and C-terminal regions of the cat gene, respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). While two extra copies of IS4Bsu1 were clearly identified in the genome of cells grown in CI medium, the pattern of the genome of cells grown in LB medium was identical to that of the initial strain, RIK7102, indicating that the transposition frequency of IS4Bsu1 was exceptionally high in competence-inducing medium. We also determined some of the loci of IS4Bsu1 in the genome of RIK7102; the loci that correspond to those in strain 168 are summarized in Table 2. Based on these observations, we posit that the localization of these loci is random and that transposition events are related to culture conditions.
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). Using this assay system and the detection techniques described in Methods, we were able to monitor quantitatively the transposition frequencies of mini-IS from the chromosome in the target plasmid pDG148.
To assess whether the transposition frequency of mini-IS changed under different conditions, we performed an intermolecular transposition assay. First we examined the mini-IS transposition frequency under normal culture conditions (LB medium at 37 °C). We found that the transposition frequency increased 15.7-fold when cells were grown under conditions leading to genetic competence induction, e.g. in CI medium, as compared to normal culture conditions (Table 3). In addition, cultivation in LB medium at 49 °C rather than 37 °C yielded a 4.4-fold higher transposition frequency. With strains NBS041 and NBS042 we obtained no AmpR CmR transformants under any of the tested conditions. Our results indicate that the appearance of CmR transformants depends on the transposase and inverted repeat of IS4Bsu1 and that the phenomena we observed were a consequence of mini-IS transposition events.
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). Although the identification of 9 bp target sequence duplications at the insertion sites was evidence for the occurrence of IS transposition, the 9 bp random target sequences at different sites, usually AT-rich, were not conserved (Table 4). These results further demonstrated that our assay system reflected the ability of IS4Bsu1 to transpose into AT-rich random sequences and to yield 9 bp target sequence duplications. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). We identified 9 bp target sequence duplications on both sides of the mini-IS (Table 4), indicating that our assay system actually detected phenomena resulting from transpositional mechanism(s). We first established that the transposition frequency of IS4Bsu1 in B. subtilis (natto) was remarkably high under competence-developing conditions (Fig. 1), suggesting the existence of some conditions that promote IS4Bsu1 transposition, including conditions that can lead to activation of host factors for transposition. Such environmental conditions have been described elsewhere (Eichenbaum & Livneh, 1998; Nagy & Chandler, 2004; Ohtsubo et al., 2005). Although Nagai et al. (2000) identified six to 11 copies of IS4Bsu1 in the B. subtilis (natto) genome, no transposons were found in the genome of the closely related B. subtilis 168 strain (Kunst et al., 1997; Qiu et al., 2003, 2004). Accordingly, we postulated that an intermolecular transposition assay system using B. subtilis 168 would facilitate simple and detailed transposition analysis.
Our intermolecular transposition assay revealed that under typical culture conditions (37 °C, LB medium; 1.4x10–8 per target plasmid) the frequency of mini-IS transposition was low. Bacterial transposition activity is generally maintained at a low level because high transposition activity and the accompanying mutagenic effects of genomic rearrangement would be detrimental to the host cell (Mahillon & Chandler, 1998). We noted that the transposition frequency of mini-IS increased dramatically (15.7-fold) under our competence-developing (37 °C, CI medium) conditions and moderately (4.4-fold) under the high-temperature (49 °C, LB medium) conditions (Table 3). With respect to transposition frequency at high temperature (42 °C), contrasting results have been reported; it is higher in Burkholderia multivorans ATCC 17616 cells (Ohtsubo et al., 2005) and lower in E. coli cells (Nagy & Chandler, 2004).
Our results provide what is believed to be the first evidence for a high transposition frequency of mini-IS under competence-developing conditions. In B. subtilis, many different regulation pathways constitute the gene regulatory network that controls the development of competence (Hamoen et al., 2003). In particular, the competence transcription factor ComK activates expression of various genes involved in DNA binding, uptake and recombination (van Sinderen et al., 1995). The apparent high transposition frequency under competence-developing conditions in our study raises the possibility that in B. subtilis there are links between the regulatory pathways involved in competence development and transpositional events.
It is of note that these transposition phenomena could be achieved by expressing a PS10-regulated tnp gene on the chromosome. The overexpression of Tn5 transposase in E. coli reportedly results in filamentation, aberrant nucleoid segregation and cell death (Weinreich et al., 1994). Similarly, in B. subtilis we noted filamentation and growth inhibition when we tried to construct a plasmid carrying the PS10-controlled tnp of IS4Bsu1 (data not shown). However, the expression level of the transposase gene in the NBS040 strain seems sufficient not to affect cell growth of B. subtilis while at the same time promoting the transposition of mini-IS.
Some transposons require a host factor(s) for transposition. We showed that in B. subtilis 168, modified IS4Bsu1 isolated from B. subtilis (natto) can transpose, at either high or low frequency, under different conditions (Table 3). These results suggest two possibilities. First, IS4Bsu1 does not require a host factor and depends only on itself. Second, IS4Bsu1 requires a host factor(s) but is able to transpose in B. subtilis 168 because the bacterium supplies this factor(s). There are different conditions that induce mini-IS transposition, and significantly high homology has been shown between the 168 and ‘natto’ strains. We favour the second possibility because, interestingly, no difference or bias was observed in transposition or target sites under the different conditions tested (Fig. 3, Table 4), suggesting strongly that the difference in transposition frequencies under these conditions is not due to a modification of DNA recognition by transposase per se.
The in vivo regulation of transposition is still poorly understood. Our goal was to explore transposition-inducing conditions and we succeeded in transferring an active IS4Bsu1 transposition system into B. subtilis 168. Our transposition assay system may be a powerful tool for a better understanding of the regulation of cellular transposition.
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ACKNOWLEDGEMENTS |
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Edited by: M. Hecker
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REFERENCES |
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Received 16 February 2007;
revised 31 March 2007;
accepted 27 April 2007.
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