Laboratory of Microbial Gene Technology, Agricultural University of Norway, PO Box 5051, N-1432 s-NLH, Norway1
Author for correspondence: Morten Skaugen. Tel: +47 64949466. Fax: +47 64941465. e-mail: morten.skaugen{at}ikb.nlh.no
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ABSTRACT |
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Keywords: transposition, insertion sequence, lantibiotic
The EMBL accession number for the IS1520 nucleotide sequence is AJ250958.
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INTRODUCTION |
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METHODS |
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Plasmids were isolated using a standard alkaline lysis method (Birnboim & Doly, 1979 ), with elevated lysozyme concentrations (final concentration 5 mg ml-1) when isolating plasmids from L. sakei. Total DNA was isolated from cultures of L. sakei as described previously (Skaugen et al., 1994
).
Restriction analysis and molecular cloning.
Enzymes used in restriction analyses and cloning experiments were purchased from New England Biolabs, Promega or Boehringer Mannheim.
E. coli DH5 was transformed by electroporation (Dower et al., 1988
) using the Bio-Rad Gene Pulser. DNA molecular size markers (1 kb ladder) were purchased from Gibco-BRL.
Amplification and nucleotide sequence determination.
DNA (0·110 ng template) was amplified using AmpliTaq polymerase and the Perkin Elmer GeneAmp 9600 or MJ Research PTC-100 thermocycler. Amplification typically proceeded through 35 cycles after a 3 min hot-start (97 °C). The polymerizing time (72 °C) was set to 1 min 30 s in all reactions, except when amplifying from ligation mixes, where the polymerizing time was set to 3 min. The annealing temperature was set at the Tm value for the oligonucleotide having the lowest calculated melting point. Annealing time and denaturing time (94 °C) were set to 1 min in all reactions.
For RT-PCR analysis, total RNA from L. sakei was isolated as described previously (Skaugen et al., 1997 ) and treated with RQ1 DNase (Promega) according to the manufacturers instructions. The first-strand cDNA synthesis was carried out in 20 µl 1x AMV (avian myeloblastosis virus) reaction buffer containing 1 µg total RNA, 100 pmol oligonucleotide primer (antisense of the 5' end of the downstream gene), 40 U RNasin (Promega), 2 mM each dNTP and 5 U AMV RT (Promega) at 42 °C for 1 h. After this time, 80 µl amplification mix [dNTP (0·2 mM final concn), forward primer (100 pmol), Taq buffer (1x final) and Taq polymerase (2·5 U)] was added and the complete reaction was subjected to a standard amplification program [97 °C for 5 min (hot-start), 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min] and analysed on agarose gels. A reaction where AMV RT had been omitted was included as a control in each experiment.
Nucleotide sequences were determined using the cycle sequencing protocol of Perkin Elmer and the ABI Prism 310 or 377 automatic sequencer, with purified PCR product or plasmid DNA as template.
DNADNA hybridization.
Restricted DNA was separated on agarose gels and blotted to GeneScreen Plus membranes (DuPont) using the Vacugene blotting apparatus (LKB Pharmacia). Hybridization probes were labelled using the Boehringer Mannheim Random Primed DNA Labelling kit and [-32P]dCTP (3000 Ci mmol-1; 111 TBq mmol-1) (Amersham). Prehybridization, hybridization and washing conditions were as described previously (Church & Gilbert, 1984
; Skaugen & Nes, 1994
). Autoradiography was carried out using Cronex film (DuPont) and intensifying screen at -85 °C.
Nucleotide sequence analysis.
Nucleotide sequence data were assembled using the ABI/Perkin Elmer Autoassembler software, and analysed using the UWGCG program package (version 8.0). Database searches were performed using BLAST (Altschul et al., 1990 , 1997
) with default settings at the NCBI WWW server.
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RESULTS |
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The GC content of IS1520 is 37·6 mol%, which is similar to IS1163 (39 mol%; Skaugen & Nes, 1994 ), but significantly lower than the genomic value of 4244 mol% for L. sakei (Kandler & Weiss, 1986
). The GC content of the approximately 11 kb las cluster is 30·2 mol%.
Sequence analysis of IS1520
Comparisons of nucleic acid and protein sequences suggest that IS1520 belongs to the IS3 subgroup (Mahillon & Chandler, 1998 ) of the IS3 family, which is presently the largest and most widespread group of bacterial insertion sequences. IS1520 is 1302 bp long, has relatively short (10 bp), perfect terminal inverted repeats (Fig. 2
), and the plasmid insertions are flanked by (different) 3 bp direct repeats (Fig. 1
). Although IS1520 undoubtedly belongs to the IS3 family of insertion sequences, one conserved feature is absent: whilst nearly all elements in this group conform to the so-called 5'TG(/A) . . . . CA3' rule (Mahillon & Chandler, 1998
), IS1520 has G (5') and C (3') as its terminal residues (Fig. 2
).
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As is the case for many of its relatives (Mahillon & Chandler, 1998 ), the IS1520 OrfA contains a relatively strong helixturnhelix motif in the N-terminal half of the protein, and a leucine zipper motif towards the C-terminus (Fig. 2
). The function of the former motif is probably the specific recognition of the elements end(s) by OrfAB as well as OrfA (Hu et al., 1996
; Lei & Hu, 1997
; Ton-Hoang et al., 1998
), while the leucine zipper is likely to promote the transposase oligomerization required for transposition (Haren et al., 1998
; Lei & Hu, 1997
). A modulatory role for OrfA in transposition has been demonstrated for at least two members of the IS3 family [IS911 (Polard et al., 1992
) and IS3 (Sekine et al., 1994
)], and the IS2 OrfA has been shown to affect orfA/B/AB transcription by binding to the promoter overlapping the left inverted repeat of this element (Hu et al., 1996
).
orfB encodes the C-terminal two-thirds of the putative IS1520 OrfAB transposase, which constitutes the catalytic domain of the protein with the highly conserved DD(35)E triad (Doak et al., 1994 ). There are no combinations of a proper ribosome-binding site and a standard initiation codon associated with the IS1520 orfB, suggesting that OrfB may not be produced as a separate protein. However, as in IS1163, there is an AUU codon, in reading frame 0, 8 nt downstream of a putative ribosome-binding site and overlapping the proposed heptanucleotide frameshifting site (Fig. 2
). For the related element IS911, it has been demonstrated that a similarly situated AUU codon, in reading frame -1, is utilized as the OrfB initiation codon (Polard et al., 1991
).
The presence of a perfect consensus -35 box hexanucleotide (nt 12891294; Fig. 2) partially overlapping the right inverted terminal repeat suggests that IS1520 could participate in the generation of a hybrid promoter upon insertion, although analysis of the junction sequences created by the insertions failed to identify any such promoters. For at least some of the IS3 family members (Mahillon & Chandler, 1998
) it has been demonstrated that the element can excise as a circle in which the left and right ends are joined via a short spacer, which for IS911 corresponds to one of the direct repeats flanking the element in the donor location (Polard et al., 1992
). In the case of IS911, it has been shown that this circularization assembles a strong promoter that could ensure the high levels of transposase required for the circles to function efficiently as substrates for the subsequent insertion reaction (Ton-Hoang et al., 1997
). Although it is uncertain whether the circularization and hybrid promoter assembly is a general feature of the IS3 family, it is interesting to note that the circularization of IS1520 with a 3 bp spacer between the repeats would place the IRR -35 element at a distance of 17 bp from the near perfect -10 hexanucleotide TATGAT in the IRL, thus creating an at least theoretically strong promoter of orfA and B transcription.
Genomic copy number of IS1520 in L45
To determine the genomic copy number of IS1520, the element was amplified from pCIM157, labelled and used to probe a Southern blot of HindIII- and EcoRI-digested total DNA from various isolates of L45 as well as non-lactocin-S-producing strains from our laboratory collection. The result of one such hybridization is shown in Fig. 3 and indicates a minimum genomic copy number of 9 in L45. All L. sakei strains tested had fragments hybridizing to the IS1520 probe, with a mean (minimum) copy number of 23 (results not shown).
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Complementation of the insertion mutations by reintroducing the affected gene cloned in a suitable vector is, in practice, prevented by the difficulties with the transformation of L45. Therefore, to test for polarity of the insertions (i.e. premature termination of transcription), RT-PCR experiments were set up using RNA isolated from the mutants (see Methods). Antisense primers for either of the downstream genes (lasT, 5'-ATGCATACTTAGTCGTCTCC-3'; or lasY, 5'-TCCACACTAGATTAGCACAGAAG-3') were used for first-strand cDNA synthesis, and were complemented with either of the two IS1520-specific primers indicated in Fig. 2 for the PCR step. RNA-specific amplification was achieved in all three cases (shown for L45-5.7 in Fig. 4
), indicating that the IS1520 insertions described here do not have polar effects.
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DISCUSSION |
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The simplest explanation for the observed inactivation of the las operon is that insertion results in aborted translation of the target ORF, indicating a role for the affected gene/gene product in lactocin S production. The polarity tests performed support this conclusion, although we cannot at this point exclude the possibility that outward transcription from the element may at least contribute to these results.
As is the case with the related, co-resident element IS1163, the identified IS1520 insertion sites have no obvious common features suggesting any target specificity of insertion. Such specificity could, however, be of a regional kind, which is not easily identified by sequence analysis. It is worth noting in this context that the GC content of the pCIM1 region to which all IS1520 insertions have been mapped is 29·8 mol%. This value is significantly lower than the estimated genomic value of 43 mol% for L. sakei (Kandler & Weiss, 1986 ), thereby indicating a possible preference of transposition to AT-rich regions (Craig, 1997
).
The sequence and genetic organization of IS1520 place the element in the IS3 family of bacterial insertion sequences (Mahillon & Chandler, 1998 ). This group is probably the most widespread of all IS elements, and at least three distinct members of the IS3 family have previously been identified in the genus Lactobacillus (Shimizu-Kadota et al., 1985
; Skaugen & Nes, 1994
; Walker & Klaenhammer, 1994
).
For the IS3-type elements where the issue has been addressed experimentally, it has been shown that a frameshift event fusing the OrfA and OrfB proteins into the transposase, OrfAB, is required for transposition (Polard et al., 1991 ; Sekine et al., 1994
; Vögele et al., 1991
). This so-called programmed frameshifting (Chandler & Fayet, 1993
) occurs at a specific slippery heptanucleotide in the overlap region and its efficacy is, at least in some cases, modulated by an upstream ShineDalgarno-like sequence and/or a downstream secondary structure of varying complexity (Rettberg et al., 1999
; Sekine et al., 1994
; Vögele et al., 1991
). The dependence on frameshifting for the expression of essential genes is a feature the IS3 family shares with retroviruses as well as many bacterial and bacteriophage genes (Atkins et al., 1990
; Fayet et al., 1990
), and the model for ribosomal rephasing was originally proposed to account for the presence of transframe proteins in retroviral systems (Jacks et al., 1988
).
Structures in IS1520 which may be of significance in the expression of the transposase genes are suggested by sequence alignments, and the IS1520 frameshiftheptanucleotide thus indicated has the sequence 5'-UUUAAAA-3', which is identical to that proposed for IS1163 (Skaugen & Nes, 1994 ). Downstream of these tandem slippery codons is a region which could form a stemloop structure masking the stop codon of orfA, and a ShineDalgarno motif is found upstream at a distance indicating a role in frameshifting and possibly in initiating translation of orfB. The AUU codon, which is the initiation codon of the IS911 orfB (Polard et al., 1991
), is present in both IS1163 and IS1520, but in frame 0 rather than -1. The use of this codon in initiating translation of orfB would therefore require a -1 shift of reading phase immediately after initiation, an event which has been shown to be a prerequisite for translation of the second ORF (insB) of IS150 (Vögele et al., 1991
).
The low overall sequence similarity between IS1520 and IS1163 suggests that they have entered their common host as distinct elements. The high similarity between the two elements proposed frameshifting regions may therefore reflect independent adaptations to the host rather than conservation of a common original sequence. Whichever is true, the similarity suggests functional significance of the sequence motifs in the region, thus adding to the diversity of this particular kind of gene expression regulator. It should be noted at this point that the suggested functional regions of IS1520 lack experimental support, and that the significance of the features discussed above with respect to the IS1520 transpositional activities remains to be determined. The results reported here should provide a basis for future experiments addressing these basic issues as well as for the continued exploitation of a natural transposon mutagenesis system in L. sakei.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 16 November 1999;
revised 4 February 2000;
accepted 10 February 2000.