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Institute of Molecular Medicine and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, ROC
Correspondence
Thy-Hou Lin
thlin{at}life.nthu.edu.tw
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Rousseau et al., 2002). To date, only a few members of the IS3 family have been characterized in lactobacilli, e.g. IS1223 (Walker & Klaenhammer, 1994) in Lactobacillus johnsoni and IS1163 (Skaugen & Nes, 1994) and IS1520 (Skaugen & Nes, 2000) in Lactobacillus sakei. Members of this family are characterized by containing two overlapping open reading frames, namely orfA and orfB, leading to the fusion protein OrfAB produced by programmed translational frameshift between orfA and orfB. A DD(35)E catalytic motif has been identified at the C-terminal region of OrfAB (Fayet et al., 1990). The IS3 family elements are produced by transposition in a simple cut-and-paste manner involving several steps. In the first step of transposition, the transposase OrfAB cleaves a single DNA strand at one IS end to generate a free 3'-OH group and then transfers this end to an open end of the same strand which is several nucleotides distal from the other IS end (Haas & Rak, 2002; Sekine et al., 1999). A ‘figure of eight’ is formed by the IS sequence following repair by the host repair machinery and replication by the IS itself at the junction region (Turlan & Chandler, 1995). In the second step of transposition, the figure of eight is processed into a transposon circle. However, the left inverted repeat (IRL) and right inverted repeat (IRR) junction is created by either intra-IS or occasionally inter-IS recombination to form a head-to-tail IS tandem dimer (Szeverenyi et al., 2003; Ton-Hoang et al., 1998).
The expression of transposase is controlled by an original promoter PIRL upstream of orfA. However, in several cases of the IS3 family, an inwardly directed –10 hexamer has been detected in the IRL (Galas & Chandler, 1989). New promoters, such as Pjun, may be generated by a combination of the –10 hexamer in the IRL with a –35 hexamer in the neighbouring IRR when two ends from an IS are juxtaposed as head-to-tail dimers or circles. Compared with the original PIRL, this new promoter is a relatively strong one in some members of IS3 family, e.g. IS21 (Reimmann, et al., 1989), IS30 (Dalrymple, 1987) and IS911 (Ton-Hoang et al., 1997), which leads to high transposase expression and consequently an increase in the transposition activity. Moreover, Duval-Valentin et al. (2001) demonstrated that inactivation of Pjun strongly decreases IS911 transposition when the corresponding transposase is produced by its natural configuration PIRL. Transpositionally active junctions have been reported experimentally for several other IS families, e.g. IS21 (Reimmann & Haas, 1990), IS30 (Kiss & Olasz, 1999; Olasz et al., 1993) and IS1 (Shiga et al., 1999; Ton-Hoang et al., 1997).
In previous studies, we have shown that ISLC3 (GenBank accession no. AF445084) is inserted into frame orf14 of the temperate bacteriophage 
AT3 of Lactobacillus casei ATCC 393, resulting in the insertion of a premature stop codon (Lo et al., 2005). This insertion causes a deletion of 36 amino acids in Orf14 from the C-terminus of the original protein. Sequence analysis of ISLC3 has revealed that this DNA element belongs to the IS3 family. A high level of sequence similarity is also found between ISLC3 and the putative mobile elements of Lactobacillus casei ATCC 334 (accession no. CP000423, 99 % identity) (Makarova et al., 2006), Lactobacillus coryniformis DSM 20001T (accession no. AJ605769, 99 % identity), Pediococcus pentosaceus ATCC 25745 (accession no. CP000422, 98 % identity) and IS153 of Lactobacillus sanfranciscensis (accession no. AJ239042, 82.1 % identity) (Ehrmann & Vogel, 2001). The total length of ISLC3 has been determined as 1351 bp, and 37 bp IRs are found at each end. Moreover, the flanking sequence of ISLC3 has been determined as a 3 bp (5'-ACC-3') direct repeat (DR) on its target sequence. There are two open reading frames, orfA (from 82 to 333 bp) and orfB (from 387 to 1229 bp) identified on the ISLC3 sequence, which are in phase 0 and –1, respectively. A conserved DD(35)E triad has also been identified in the middle of the OrfB sequence, suggesting the existence of a catalytic domain on the sequence (Rousseau et al., 2002).
In this study, we investigated the excision process of the ISLC3 transposition reaction. We found that an in vivo genetic rearrangement has occurred at the IRL and IRR of ISLC3 which results in the generation of circles or head-to-tail tandem dimers containing a 3 bp spacer or a deletion of 25 bp at the IRL between the abutted IS ends of the junction region. We also determined the transcription start sites for Pjun and PIRL in several lactobacilli by using a primer extension assay. An extra transcription start site was detected by the same assay as well, indicating that a potential promoter is located in the proximity of Pjun or PIRL. In other primer extension experiments where the complete ISLC3 sequence was cloned into Escherichia coli using plasmid pSA1, we found that this potential promoter was formed by the 25 bp deleted IRL (IRL
25) of the inverted repeat junction sequence, namely, Pdjun. We also found via an in vitro transposition assay that the transposase OrfAB of ISLC3 cleaved a single DNA strand at the IRR but not IRL end in the first step of the transposition process. Moreover, OrfAB activity was also found to be strongly enhanced in the presence of Mn2+ and Mg2+.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. All the lactobacilli used in the study were obtained from the Culture Collection and Research Center (CCRC) of Taiwan and were maintained at –80 °C and subcultured on MRS (Difco) agar plates or in MRS broth anaerobically at 37 °C. The E. coli host used in the cloning procedures was DH5
, which was grown in Luria–Bertani (LB) broth at 37 °C with agitation. Antibiotics were added at the following concentrations: kanamycin (Km), 50 µg ml–1; chloramphenicol (Cm), 50 µg ml–1; and ampicillin (Amp), 50 µg ml–1. The concentration of X-Gal used in all the cloning procedures was 20 µg ml–1. Unless otherwise stated, all the chemicals used were purchased from Sigma. Klenow DNA polymerase, T4 DNA ligase and all restriction enzymes used were purchased from New England Biolabs (NEB) and the manufacturer's instructions were followed.
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and were plated on LB agar containing appropriate antibiotics. All the DNA sequences were determined by Mission Biotech (Taiwan) using ABI377-96 automated sequencers (Applied Biosystems). The BLAST network service of the National Center for Biotechnology Information (NCBI) was used to compare the homologous sequences searched from the GenBank/EMBL/DDBJ and SWISS-PROT databases. The ISLC3 DNA fragment was isolated from the temperate bacteriophage AT3 (GenBank accession no. AY 605066) of L. casei ATCC 393 and was subcloned into vector pBluescriptSK+ (pSK+) (Stratagene) to generate plasmid p2END. Plasmid pSA1 was constructed as follows: a HindIII fragment carrying the intact ISLC3 sequence was amplified by PCR from plasmid p2END using primer Hin-R (5'-GCAAGCTTTGTGGCTAATACCAAAACTTATTTCAA-3'; 15054–15080 bp of phage AT3 genome) and Hin-L (5'-GCAAGCTTGATGGCATGAGCTGAATGATG-3'; 16525–16529 bp of phage AT3 genome). The artificial HindIII site on both 5' ends of the primers is shown in italics. The PCR fragment obtained was cloned into the HindIII site of vector pACYC184 or directly into vector pGEM-T-Easy (Promega) for sequencing. The selection markers were the chloramphenicol (Cmr) or ampicillin (Ampr) resistance genes in E. coli DH5 and the resultant plasmids were designated pSA1 or pGIS, respectively. The alkaline lysis method described by Sambrook et al. (1989) was used for the extraction of plasmid DNA. The QIAquick Gel Extraction kit (Qiagen) was used to extract and purify DNA following separation by the agarose gel electrophoresis. Two ISLC3 complementary primers, 149out (5'-GCTGATTCGCCTTGCTTATATAGG-3'; 149–126 bp of ISLC3) and 999out (5'-GCATGCTTTTTACAATAGGAAGAG-3'; 1130–1153 bp of ISLC3), were used in inverse PCR (IPCR) to amplify the IS inserts from cell lysates of L. casei ATCC 393, L. casei ATCC 334, L. casei ATCC 27139 and E. coli DH5(pSA1), and the PCR products were cloned into vector pGEM-T-Easy for sequencing. The uncut chromosomal DNA of lactobacilli or plasmids was separated by agarose gel electrophoresis and purified using the QIAquick Gel Extraction kit. DNA molecules between 1000 and 4000 bp were designated part A and those smaller than 1000 bp were designated part B. Both parts of DNA were then utilized in IPCR or Southern blot assay.
Plasmid pTL, containing a truncated IRL end, used in the in vitro activity assay for OrfAB, was made by IPCR using the following primers: 5'-GGGTATAATGAATAAATCGTCTC-3' (38–60 bp of ISLC3) and 5'-TGCCTAATGTATGCAAAGGGCGATC-3' (15115–15091 bp of AT3), on plasmid pGIS as a template. Similarly, the truncated-IRR plasmid pTR was made by IPCR from plasmid pGIS using the primers 5'-CATTCAGAACCGCGTTTACCTTCC-3' (1314–1291 bp of ISLC3) and 5'-GTGGGACAAGTTTCTTTTGCCACCC-3' (16474–16498 bp of AT3). The site-directed mutagenesis kit Phusion (Finnzymes) was used in these experiments.
Southern blotting.
Total cellular DNA was extracted from the lactobacilli by treating them with SDS/proteinase K and the DNA was phenol/chloroform treated and then precipitated with ethanol as described by Forsman & Alatossavam (1994). For Southern hybridization analysis, equal amounts of chromosomal DNA from each strain were digested with EcoRV and subjected to electrophoresis on a 1 % agarose gel. The DNA fragments were transferred to a Hybond-N+ membrane (Amersham) and cross-linked by a UV cross-linker (Spectronics). The membrane was pre-hybridized with 0.5 ml of a 1 mg ml–1 solution of sonicated salmon sperm DNA at 42 °C for 1 h. The DNA fragments amplified by PCR from plasmid p2END using primers ISR50 and ISL50 were used as the probe (labelled with [-32P]dATP). The membrane was hybridized with the labelled probe at 42 °C for 12 h. After hybridization, the membrane was washed under high-stringency conditions: 30 min with 2x SSC/0.1 % SDS (1x SSC is 150 mM NaCl plus 15 mM sodium citrate) followed by another 30 min with 0.5x SSC/0.1 % SDS. Both washes were at room temperature and were followed by a final 30 min wash at 55 °C with 0.1x SSC/0.1 % SDS.
RNA extraction and primer extension.
The lactobacilli were cultivated in 40 ml MRS broth at 37 °C. Bacteria were collected at the mid-exponential phase and rapidly pulverized in liquid nitrogen. The total RNA was isolated by using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The RNA pellets obtained were suspended in diethyl pyrocarbonate (DEPC) water treated with RQ1 RNase-free DNase (Promega). The RNA was recovered by extraction with TRIzol reagent again. The quantity of RNA was determined by spectrophotometry (A260 and A280). Analysis of the 5' ends of the OrfA mRNA transcripts was performed by primer extension using 6-FAM-labelled primers (5'-TTGAATTCTTTGTCGTAACGA-3'; 110–190 bp of ISLC3) (Merighi et al., 2006). A total of 100 pmol primer was annealed to 20 µg total RNA. Synthesis of cDNA was performed at 50 °C using the SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. These first-strand cDNAs were analysed in an ABI 3730 capillary electrophoresis sequencer and the corresponding fluorescence intensity was quantified by the GeneMapper V3.7 software (Applied Biosystems).
Circle junction promoter activity assay.
Sequences for promoters PIRL, Pdjun (the 25 bp truncated junction promoter) and Pjun (the complete junction promoter) were obtained by the following respective PCR reactions: (i) primer ISL (5'-TGGATGTAAGTTGATTCCTGAGA-3'; 1–23 bp of ISLC3) and HiaR (5'-AAAAATTCCTTCCTTTTGAG-3'; 79–60 bp of ISLC3) on plasmid pSA1; (ii) primer 999out and HiaR on the genomic DNA of L. casei ATCC 393; and (iii) primer 999out and HiaR on plasmid pRDRL2 as the template DNA. Plasmid pRDRL2 carrying the complete 5'-IRR-DR(ACC)-IRL-3' fragment was obtained from one of the subclones of the IPCR assay on the E. coli DH5(pSA1) cell lysates. These PCR fragments were cloned into a TA vector, the pBlue-TOPO vector (Invitrogen), and then fused with the lacZ gene to generate plasmids pIRLz, pDSLz and pACLz, respectively. The lacUV5 promoter on plasmid pUV5 was made by using the oligonucleotides UV5+ (5'-ACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGAA-3') and UV5– (5'-TCCACACATTATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTA-3') via annealing and then direct TA cloning on vector pBlue-TOPO. The PUV5 –35 and –10 hexamers are shown in bold in the sequence of primer UV5+. Each of these plasmids was used to transform E. coli DH5 (RecA–) and the transformed cells were grown at 37 °C overnight in LB medium supplemented with appropriate antibiotic(s). The overnight culture was diluted 1 : 50 with fresh medium and grown until the mid-exponential phase (OD600 0.4–0.8) was reached. The cells were collected by centrifugation and lysed; then the β-galactosidase activity was measured as A420, as described by Miller (1992). The β-galactosidase activity measured was converted to Miller units using the following equation: β-galactosidase activity (Miller units)=(A420x1000)/(OD600xtxv), where t is the incubation time (min) and v is the volume of culture (ml).
Protein expression and purification.
The site-directed mutagenesis for the –1 frameshift region upstream of the orfA stop codon was performed by IPCR on the entire orfA and orfB genes on plasmid pGIS with the 5' primer 5'-CTTTCTTGCGCAGAAGTAACCAATA-3' (315–339 bp of ISLC3) and 3' primer 5'-CCTAGGGCTTTTTTTTAGGATTTCGTT-3' (289–314 bp of ISLC3), where the latter was complementary to the 3' end of the orfA gene including an extra T base (bold) of the orfA gene. The site-directed mutagenesis kit Phusion (Finnzymes) was used for this experiment. The mutant plasmid product was purified and used as the PCR template for testing the expression of OrfAB using the Champion pET101 directional TOPO Expression kits (Invitrogen). Overexpression of OrfAB (1–382 aa) was achieved by IPTG (Boehringer Mannheim) induction of an exponentially growing culture of E. coli BL21(DE3) transformed with plasmid pET101/D-TOPO(OrfAB). The OrfAB derivatives were partially purified using the C-terminal histidine tag as indicated by the manufacturer. Induction was achieved by the addition of IPTG to a final concentration of 1 mM and the cells were incubated for a further 90 min before being harvested and then lysed by sonication. The protein purification was performed at 4 °C as follows: the crude extracts (the total protein concentration was about 10 mg ml–1) were subjected to centrifugation at 8000 g for 10 min and then the overexpressed OrfAB protein was purified by a Ni-NTA resin column.
In vitro activity assay for OrfAB.
Typical activity assays for OrfAB (50 µl) contained 500 ng transposase OrfAB and 200–500 ng plasmids and were performed in the following cocktail: 20 mM HEPES pH 7.5, 5 mM DTT, 50 mM MgCl2, 200 mM KCl and 10 % (v/v) glycerol. Assays were incubated at 37 °C. The concentrations of metal ions used are given in the Results. The products of all the in vitro assays were treated with 0.5 mg proteinase K ml–1 for 30 min at 50 °C before being purified with Qiaquick purification minicolumns (Qiagen).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), was conducted to examine whether the free circular form or head-to-tail tandem copies were present extrachromosomally in L. casei ATCC 393, where the presence of ISLC3 was confirmed by IPCR analysis. A small fragment of nearly 350 bp was detected by IPCR (Fig. 1a), suggesting that ISLC3 may circularize itself as observed in other IS elements identified from several Gram-positive bacteria, such as the flanking IS256 copies of Tn4001 tandem dimer in Staphylococcus aureus (Prudhomme et al., 2002). In fact, a fragment of the same size was also detected by IPCR using the same primer set on the extracted chromosomes of L. casei ATCC 27139, L. sanfranciscensis ATCC 27651 and L. delbrueckii subsp. bulgaricus HER1308. The binding sites of the 149out and 999out primers were 98 bp or 161 bp distal from the ends of ISLC3 (Fig. 2). Through sequencing on the subcloned fragment on plasmid pGEM-T-Easy, we obtained some IPCR products that varied around 342 bp in the lactobacilli studied. Furthermore, the difference in such sequences detected for the complete 370 bp circles in L. sanfranciscensis ATCC 27651 or L. delbrueckii subsp. bulgaricus HER1308 was 2 or 5 bp respectively. The sequence of the suspected complete circle formed by ISLC3 was detected as a fragment of 370 bp as shown in Fig. 2. The truncated circle of the 342 bp fragment is also shown.
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AT3 genome. The 3 bp is one of the two copies of the 3 bp DR(5'-ACC-3') target sequence produced in the initial insertion event. However, the complete 5'-IRR-DR-IRL-3' junction sequence characterized by a DR at the insertion site by ISLC3 such as that in the bacteriophage AT3 genome was not detected from the IPCR products of the L. casei ATCC 393 genome or other DR-separated IRs of the junction regions of L. casei ATCC 27139, L. sanfranciscensis ATCC 27651 and L. delbrueckii subsp. bulgaricus HER1308. Instead, the IPCR product was detected as circles or head-to-tail tandem dimers where 25 bp from the IRL junction region was found to be deleted (5'-IRR-IRL25-3').
Detection of ISLC3 circles in E. coli
It is difficult to detect the original circular forms in lactobacilli studied using the Southern hybridization technique due to low copy number. Therefore, we employed a technique which was used by Perkins-Balding et al. (1999) to detect the IS492 circle. The size of digested DNA between 1000 to 4000 bp (part A) was separated from that below 1000 bp (part B) in an agarose gel and each part of DNA was purified using the QIAquick Gel Extraction kit. The DNA sample extracted from each gel slice (parts A and B) was utilized in an IPCR assay (Fig. 3a). Plasmid pSA1 was constructed from pACYC184 to carry a complete wild-type ISLC3 sequence flanked respectively by 50 bp of IRs of orf14 target sequence on the left and right ends (Fig. 3a). This constructed vector was used to transform E. coli DH5 (RecA–) and the corresponding plasmid DNA was extracted using the alkaline lysis method. The DNA samples were separated into two parts by electrophoresis and purified using the QIAquick Gel Extraction kit as described (Fig. 3a, parts A and B). To confirm the formation of the circular form, plasmid DNA was digested with PstI, since the restriction site was located right in the middle of the ISLC3 sequence. Without the presence of intact plasmid pSA1, the amount of circular forms may be enriched by the separation and collection procedures described in Fig. 3(a). The PstI-digested DNA sample of part A (Fig. 3a) was analysed by Southern hybridization using the ISLC3-specific probe. Detection of a signal of 1.3 kb (lane 1 of Fig. 3b) confirms the presence of the circular form of ISLC3 in E. coli. The 5.5 kb signal was the intact plasmid pSA1 leaked out from part A during the electrophoresis process (lane 1 of Fig. 3b). However, both signals (the ISLC3 circles and intact pSA1) were much weaker than those of the undigested pSA1 fraction (lane 2 of Fig. 3b) or L. casei ATCC 393 chromosomal DNA (lane 3 of Fig. 3b). The formation of ISLC3 circles was further examined at the circle junction region using IPCR with primer 149out and 999out for DNA samples collected in part A and B (Fig. 3a), respectively. The presence of circle junction in parts B (lane 4 of Fig. 3b) and A (lane 5 of Fig. 3b) was evident and this further confirmed the presence of ISLC3 circles in E. coli. The IPCR products collected from both parts were subcloned and sequenced. It was found that nearly 10 % (2 out of 21 independent clones) of DNA samples sequenced has the complete 5'-IRR-DR-IRL-3' junction region, and a deletion of 25 bp including the DR of the junction region (IRR-IRL25) was also found.
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). It is also known that a promoter generated by the joining reaction will control the expression of the transposase. Some promoter-like sequences resembling the typical E. coli 
70-dependent promoter sequences, namely TTGACA of the –35 element, and TATAAT spaced by 16–18 bp of the –10 element (Harley & Reynolds, 1987), were found by analysing the transcription regions of the complete 5'-IRR-DR(ACC)-IRL-3' junction region of ISLC3 (Fig. 4a) (Burr et al., 2000). Some primer extension experiments were conducted to analyse the 5' ends of the OrfA mRNA transcripts prepared from L. casei ATCC 393, ATCC 334, ATCC 27139 and E. coli(pDSLz), respectively. The method was used to map the transcription start sites for the OrfA mRNA extracted from these bacteria. Using 6-FAM end-labelled oligonucleotide primers and the total RNA extracted for these lactobacilli grown at 37 °C, three reverse-transcribed DNA fragments, of 55 bp, 80 bp and 90 bp, were detected for each. We found that the transcription start site of the 90 bp fragment of the complete 5'-IRR-DR-IRL-3' junction region of ISLC3 was at the guanine base 61 bp upstream of the OrfA translation start codon (Fig. 4a). This finding confirmed our conjecture that a putative 70 promoter (TTGACT-N17-TAAGCT) including an untypical extended TGn –10 motif (5'-TGTAAGCT-3', where an additional nucleotide ‘n’ is presumably missing) was generated at the 5'-IRR-DR-IRL-3' junction region of ISLC3 (Fig. 4a) (Keilty & Rosenberg, 1987; Ponnambalam et al., 1986; Sabelnikov et al., 1995). In other words, the transcripts made from this promoter started at the guanine base which was eight bases downstream from the –10 hexamer (5'-TAAGCT-3') of the predicted junction promoter Pjun while the –35 hexamer (5'-TTGACT-3') was initiated within the IRR. The transcription start site for the 55 bp reverse-transcribed DNA fragment was 10 bases downstream from a potential –10 hexamer (5'-TATAAT-3') at the guanine base of the indigenous OrfA promoter PIRL (Fig. 4a). The corresponding fluorescence intensity of the 90 bp fragment of Pjun was about 2.5-fold stronger than that of the 55 bp fragment of PIRL (Fig. 4a). However, the transcription start site for the 80 bp reverse-transcribed DNA fragment could not be unambiguously defined. We suspected that this transcript could be made from an alternative transcription start site of promoter Pjun or it could be produced by a new promoter formed by 25 bp deletion of IRL (IRL25) at the junction region, namely Pdjun. Therefore, we performed another primer extension experiment using the same 6-FAM end-labelled oligonucleotide primers on the total RNA extracted from E. coli transformed with plasmid pDSLz, where the 5'-IRR-IRL25-3' fragment was ligated with the lacZ gene on a pBlue-TOPO vector. As shown in Fig. 4(a), a cDNA fragment of nearly 80 bp was generated in the E. coli-based system. However, the fluorescence intensity of transcripts made in E. coli for the 80 bp fragment was only about one-tenth that of the 90 bp reverse-transcribed DNA fragment made in lactobacilli (Fig. 4a). These E. coli transcripts detected confirmed the existence of promoter Pdjun. This putative promoter Pdjun (TGTCTC-N17-TATTGA) made at the 5'-IRR-IRL25-3' region of ISLC3 is a non-typical one since it lacks a recognizable –35 region and its –10 hexamer is far from complete since it relies on an extended and TGn –10 motif. The transcription start site of this unusual promoter was determined to be at the thymine base 16 bases downstream from the –10 hexamer of the promoter (Fig. 4b).
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). All these plasmids plus plasmid pUV5, where a lacUV5 constitutive promoter was used to drive the expression of β-galactosidase, were transformed into E. coli DH5 (RecA–). The transformed bacteria were incubated in LB with the addition of X-Gal so that the activity of β-galactosidase could be measured as A420. The β-galactosidase activities measured for promoters Pjun, PIRL and Pdjun were 3.5-, 0.7- and 0.5-fold of that of promoter lacUV5 (data not shown). This result indicated that the promoter activity of Pjun was much stronger than that of both PIRL and Pdjun in E. coli.
OrfAB activity in vitro
To examine the activity of OrfAB in vitro, plasmid pGIS carrying the wild-type ISLC3 and plasmid pTL or pTR carrying the truncated IRL or IRR ends, respectively, were incubated with a highly enriched fusion protein OrfAB expressed by plasmid pEAB. The protein was made as an ISLC3 derivative by inserting 1 bp in the sequence of the A7G frameshifting site between orfA and orfB, which rendered the transposase gene orfAB in-frame. The in vitro experiments performed by incubating OrfAB and plasmid pGIS together in the presence of various divalent metal ions revealed that the protein was able to linearize pGIS to give a 4.5 kb product (Fig. 5a and b). Some sparsely present high-molecular-mass species were recognized as the autointegation products (Fig. 5a, b) (Ton-Hoang et al., 1998). As shown in Fig. 5(c) by a densitometer tracing of the gel images, about 85 % of pGIS was influenced by OrfAB while the autointegration products were gradually increased to nearly 20 % within the 30 min incubation with OrfAB. In contrast, no linearization product was found for the case where plasmid pTR, carrying a 37 bp truncation at the IRR end, was incubated with OrfAB (Fig. 5d). However, OrfAB was able to linearize pTL, where the 37 bp truncation was at the IRL end (Fig. 5d) and no corresponding high-molecular-mass autointegration product was detected in this case (Fig. 5d). These results showed that IRR was the first target chosen by OrfAB when the transposase cleaved a single DNA strand at one IS end to generate a free 3'-OH group in the first step of transposition. The effect of divalent metal ions on the OrfAB activity was examined at pH 7.5. The results showed that OrfAB was almost inactive in the presence of EDTA or in the absence of metal ions. The OrfAB activity was strongly enhanced in the presence of Mn2+ and Mg2+ in vitro but was severely impaired if these metal ions were replaced by Ca2+ (Fig. 5b). We also observed that the transposase preferred Mg2+ over Mn2+ in vitro since the concentration of the former and latter required by the enzyme to achieve the optimal activity was 10 and 50 mM, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Szeverenyi et al., 1996; Lewis et al., 2004). It has been shown for IS911, IS2 or other IS elements of Gram-negative or positive bacteria that formation of the IRL-IRR circle junction will generate a promoter whose activity may be stronger than that of the indigenous one in driving the transcription of transposase (Polard et al., 1992; Turlan & Chandler, 1995). However, no intact IRR-DR-IRL junction has been found in the lactobacilli studied here, although we did observe the presence of an intact IRR-DR-IRL junction in E. coli transformed by a plasmid carrying a wild-type ISLC3 copy. We also found that the copy number of intact IRR-DR-IRL junction in the lactobacilli studied was much lower than that of the 25 bp deletion junction (IRR-IRL25), the major product detected in an IPCR assay.
It is currently unknown why the intra-molecular recombination by the transposition of ISLC3 in lactobacilli will cause a frequent deletion of 25 bp at the IRL region. While the head-to-tail tandem dimer carrying the truncated IRR-IRL25 junction predominantly existed as a stable copy (or copies) on the chromosome of lactobacilli studied here, the formation of intact IRR-DR-IRL circular form could be an extrachromosomal intermediate occasionally generated during the transposition of the IS. This is evidenced by significant difference either in the amount of the two types of structures detected or in the corresponding promoter activities measured here or reported elsewhere by others (Olasz et al., 1993; Szeverenyi et al., 2003) using the same type of promoter activity assays.
The corresponding promoter activity of Pjun was found to be much stronger than that of Pdjun, which is a putative promoter formed by the truncated IRR-IRL25. Moreover, Pjun was the strongest promoter among the three promoters studied. Although some variations in the –10 region (TAAGCT) of Pjun with the consensus –10 hexamer box were also found (Fig. 4a
), the presence of TGn motifs directly upstream of the –10 region of Pjun may enhance its binding with the RNA polymerase, which renders Pjun a stronger promoter than PIRL or Pdjun (Burr et al., 2000). The sequence of the –10 region (TATAAT) of PIRL appears to be the most conserved one among the three promoters studied, but the –35 region (CTGAGA) of PIRL was not conserved with the consensus TTGACA box. On the other hand, the –35 hexamer (TGTCTC) of Pdjun was less conserved, since only 1 bp matched the consensus TTGACA box (McCracken et al., 2000). However, a number of promoters have now been reported where the specific –35 regions are not required for transcription initiation (Bown et al., 1997; Chan et al., 1990). Although no TGn motif was detected in Pdjun, the sequence of the –10 region (TATAAT) of Pdjun appears to be the most conserved one, which gave rise to its transcription activity (Fig. 4b).
It is known that in the initial step of circle formation by IS3 family members one IS end acts as the donor and the other one as the target. The donor DNA is cleaved and then transferred to the DNA adjacent to the other end. However, both ends of most of the IS3-related elements act equally well as a donor or target (Lewis & Grindley, 1997). The intra-molecular recombination between two ends of an IS element is the key for formation of circles or tandem repeats (Szeverenyi et al., 2003). In the transposition by IS911, an initial single-strand cleavage at the donor end resulting from intra-molecular recombination creates a free 3' OH group which will attack the target end on the same strand of DNA.
The first step in the transposition reaction by the IS3 family is an asymmetric single-stranded cleavage at the active donor end, followed by the DNA strand transfer process. This was confirmed here through an in vitro activity assay for the overexpressed transposase OrfAB, where the cleavage of IRR by the enzyme was found to be prior to that of IRL. Furthermore, the donor activity of the initial asymmetric DNA strand transfer by the transposase virtually vanished if the IRR was deleted. For the transposition of IS911 studied by others (Turlan et al., 2000), both IRR and IRL ends are found to be used in the intermolecular transposition, while in some minor cases only a single end is used. It is known that an IR-damaged IS can not transpose alone and will eventually lead to elimination of the transposon if the DNA sequence at the recipient end is mutated before the strand transfer process (Mahillon & Chandler, 1998; Wagner, 2006). On the other hand, each IS can either transpose alone or it can transpose together with the other IS in a composite transposon made by two identical ISs. This composite transposon is evolutionarily stable and the corresponding ISs will act in concert in the transposition process to nearby genomic locations. The single IRL25 structure detected here in the head-to-tail tandem dimer could be evolutionarily unstable since it was unable to form a composite transposon.
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ACKNOWLEDGEMENTS |
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Edited by: M. Kleerebezem
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 17 September 2007;
revised 20 December 2007;
accepted 12 January 2008.
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