- Email: [email protected]
Identification and sequence analysis of IS1297, an ISS1-like insertion sequence in a Leuconostoc strain
GENE A N I N T E R N A T I O N A L ,,JOURNAL ON
GENES AND GENOME5
ELSEVIER
Gene 174 (1996) 259-263
Identification and sequence analysis of IS/297, an ISSl-like insertion sequence in a Leuconostoc strain Lawrence J.H. Ward *, Julie C.S. Brown, Graham P. Davey New Zealand Dairy Research Institute, Private Bag 11029, Palmerston North, New Zealand
Received 18 October 1995; revised 30 November 1995; accepted 18 January 1996
Abstract The insertion sequence (IS) ISS1 from Lactococcus lactis was amplified from lactococcal genomic DNA using a primer to the 18-bp inverted repeat sequence. The amplified product hybridized to a single EcoRI fragment in a total genomic DNA digest of Leuconostoc mesenteroides ssp. dextranicum NZDRI 2218. The DNA sequence of this ISSl-like element (IS1297) and the Le. mesenteroides sequences flanking the IS were determined and compared with other iso-ISS1 elements. No direct repeats were found immediately flanking IS1297; however, direct repeats were present approximately 60 bp on either side of the insertion site. IS1297 contained a major open reading frame (ORF) of 681 bp, encoding a putative 226-amino-acid protein with 96.5% homology to the presumed transposase of ISS1. An overlapping ORF of 174 bp in the same orientation was also present. A putative ORF in the opposite orientation to the transposase ORF, which has been shown in some iso-ISS1 elements, was not present in IS1297. IS1297 was shown to hybridize with other dairy Leuconostoc strains. This is the first sequence of an ISSl-like element from a genus other than Lactococcus; however, IS1297 has close similarity to the lactococcal iso-ISS1 elements, especially the iso-ISS1 element from the lactose plasmid, pTD1. Keywords: Lactococcus; Transposase; Polymerase chain reaction; DNA sequencing
1. Introduction A number of insertion sequence (IS) elements have been reported and characterized in the lactic acid bacteria, especially Lactococcus, where IS elements are associated with m a n y industrially important characteristics (Romero and Klaenhammer, 1993). The IS element ISS1 is present in multiple copies in most lactococcal strains. It is characterized by 18 base inverted repeats and a single open reading frame (ORF) encoding a 226 amino acid (aa) transposase. Other IS's similar to ISS1 (isoISS1 elements) have been characterized and sequenced, particularly from lactococcal plasmids associated with lactose metabolism (Polzin and Shimizu-Kadota, 1987; Sch~ifer et al., 1991), proteinase activity ( H a a n d r i k m a n et al., 1990) or phage resistance (Romero and * Corresponding author. Tel. + 64 6 3504649; Fax + 64 6 3504740; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pairs(s); IS, insertion sequence(s) (Fiandt et al., 1972); La., Lactococcus; Le., Leuconostoc; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction. 0378-1119/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S 0 3 7 8 - 1 1 1 9 ( 9 6 ) 0 0 0 9 1 - 1
Klaenhammer, 1990; Cluzel et al., 1991). ISS1 has also been reported to hybridize to some Pediococcus and Leuconostoc strains (Polzin et al., 1993). This paper presents data demonstrating the presence of an ISS1like element in a Leuconostoc strain and compares the nt and putative transposase sequences to other iso-ISS1 sequences.
2. Experimental and discussion 2.1. Identification of an ISSl-like element in Leuconostoc
Primer I S S I I R (Table 1) complementary to the 18 base inverted repeat of ISS1 was synthesized and the ISS1 sequence was amplified from lactococcal D N A by polymerase chain reaction (PCR). The 808 base pair (bp) product was used as a probe to Southern blots of genomic D N A from strains ofLa. lactis and Le. mesenteroides ssp. dextranicum N Z D R I 2218 (Fig. 1). The L'euconostoc strain showed one band of hybridization to the probe compared to multiple bands of hybridization observed in the lactococcal strains, which suggested that
260
L.Z H. Wardet al./Gene 174 (1996) 259 263
Table 1 Primers used for amplification and/or DNA sequencing Name
Size
Sequence
1SS1 IR CR CL ISS1R ISS/L IS/297EXTL IS1297EXTR
18-mer 20-mer 20-mer 20-mer 20-mer 20-mer 20-mer
5
1234
56
5 5 5 5 5 5
ggt caa gct cat gca ctg gtc
Position in ISS1 Sequence tct act taa cga ggt ttg aag
789
m
G ,wlwm I
gtt gtt cct cct agt ctc cca
gca tta tag att aac cag aac
aag tgg ata aaa caa cat act
ttt agt tct cga cag aat tta
3' cg gg cg cg ac ag
3 3 3 3 3 3
1-18, 808-791 446 428 363 382 581-600 140 121
like element (Fig. 2). The sequence of these amplification products was determined using the P r o m e g a f-mol e sequencing system with direct incorporation of 35S-dATP. The sequencing strategy is described in Fig. 2 and the complete sequence presented in Fig. 3. The IS has been assigned the n u m b e r IS1297 in accordance with the Plasmid Reference Center Registry allocations (Campbell et al., 1979).
2.3. Comparison ofiS1297 with other IS elements
III
The sequence of IS1297 exhibited from 79% to 98% sequence identity with published iso-ISSl sequences. The greatest similarity (98%) was to ISS1/pTD1, an isoISS1 element downstream of the P-fl-galactosidase gene in the lactose plasmid pTD1 (Schfifer et al., 1991). The 18 bp inverted repeat sequence at the 3' end differed by one base to that of most iso-ISS1 elements but was identical to the p T D 1 inverted repeat.
2.4. Putative coding regions ofiS1297 and comparison with other iso-ISS1 elements
Fig. 1. Southern blot of total genomic DNA from Le. mesenteroides ssp. dextranicum strain NZDRI 2218 digested with EcoRI (lanes 1 and 2) and HindIII (lanes 8 and 9) and five La. lactis strains digested with EcoRI (lanes 3-7). The filter was probed with the ISSI sequence amplified as described in Section 2.1. The PCR product was labelled and hybridized using the Amersham ECL system in accordance with the manufacturers instructions. the Leuconostoc strain contained one copy of a sequence similar to ISS1. The presence of an ISSl-like element in the Leuconostoc strain was confirmed by the amplification of a P C R p r o d u c t of similar size to ISS1 using the ISS1 inverted repeat primer. 2.2. Sequencing of the 1SSl-like element from Leuconostoc Primers C L and C R (Table 1) were designed to allow amplification of the left and right regions of the ISS1-
IS1297, like all other iso-ISS1 elements examined, contained one major O R F . This O R F consisted of 681 bp encoding a putative protein of 226 aa. In addition, a second overlapping O R F of 174 bp existed in the same orientation but in a different reading frame which potentially encoded a 57-aa protein. It has been noted for m a n y IS elements from Gram-negative bacteria that short overlapping O R F s exist on the opposite strand to the m a j o r O R F (Galas and Chandler, 1989). The presence of such an O R F has been noted for ISS1RS by H u a n g et al. (1992). This O R F was not conserved in IS1297 or in the iso-ISS1 sequence from pTD1 (Cluzel et al., 1991) or for ISS1N and I S S I W ( H a a n d r i k m a n et al., 1990). In IS1297, ISSI/pTD1, I S S 1 N and ISS1W, a single base change at position 290 (G to A) disrupts the reverse direction O R F which is present in IS946, ISS1RS, 1SS1CH, ISS1S, I S S I T and iso-ISSl. Interestingly, this base change does not alter either of the O R F s on the other strand.
L.J.H. Ward et al./Gene 174 (1996) 259-263
261
IS 1297 IS 1297EXTL
ISSIlR ISS1L
CL
CR
•
ISS1R
IS 1297EXTR
A[
• All
ISSIlR
4
,
•
4
---
ID
Fig. 2. Amplification and sequencing strategy used. The D N A sequence was determined from each of the oligonucleotide primers as indicated by the arrows. To obtain the sequence of the Leuconostoc D N A flanking the IS, total genomic D N A from strain 2218 was sequenced using primers ISS1L to obtain sequence to the left of the IS and ISS1R to obtain the right junction sequence. This sequence data was used to design primers IS1297EXTL and IS1297EXTR, complementary to the Leuconostoc sequence flanking the IS to amplify the IS and the adjoining Leuconostoc DNA. This enabled double stranded sequence data to be obtained for the ends of the IS and the site of insertion into the Leuconostoc DNA.
2.5. Direct repeats of host DNA Small direct repeat duplications of the target DNA at the point of insertion of an IS are characteristic of some IS elements (Galas and Chandler, 1989) and are believed to be caused by the staggered cleavage of target DNA by the transposition enzymes. The ability of an IS element to induce transposition, co-integration and deletion is not dependent on the presence of the small flanking duplication; however, these repeats may be required for precise excision of the IS element (Iida et al., 1983). No direct repeats of host DNA were observed at the site of IS1297 insertion. This is similar to observations for ISS1W and ISS1N (Haandrikman et al., 1990), isoISS1 (Cluzel et al., 1991) and IS946 inserted in pTK6 and pTRK18 (Romero and Klaenhammer, 1990). Sequencing of newly transposed IS946 and ISSIS elements, however, showed the presence of 8 bp direct repeats of host DNA at the insertion site (Romero and Klaenhammer, 1990; Polzin and Shimizu-Kadota, 1987). Iida et al. (1983) have noted that some natural isolates lack a duplicated target DNA sequence. This was postulated to indicate restructuring following the initial transposition event. It is likely that the lack of direct repeats flanking IS1297 is due to such a restructuring event rather than any difference in the mechanism of IS1297 transposition compared with other ISS1 elements. 2.6. Analysis of the sequences flanking iso-ISS1 element insertion sites The DNA sequence of the regions flanking IS1297 in the Leuconostoc strain were compared with the regions flanking ISSIN (Haandrikman et al., 1990) and iso-ISS1 (Cluzel et al., 1991). ISS1W is immediately preceded by a partial ISSI copy (Haandrikman et al., 1990) and hence was not included in this analysis. Insufficient data
on the sequences flanking IS946, ISS1R, S, T and ISS1/pTD1 was available to permit analysis. The most striking feature of all the flanking regions analysed was the presence of an 8 bp repeat 60-70 bp on either side of the site of insertion. These repeats did not exhibit any sequence homology between strains. Whether these repeats have any function in insertion is unknown. 2.7. Distribution oflS1297 in dairy Leuconostoc strains The PCR product containing IS1297 was used as a hybridization probe to Southern blots of genomic DNA from a number of dairy Leuconostoc strains. Of the 27 strains examined, 16 contained between 1 and 6 copies of IS1297. Examples are shown in Fig. 4. Thus, ISSIlike elements are present in low copy number in some but not all dairy Leuconostoc strains. This contrasts with the presence of multiple copies of ISSl-like elements in most lactococcal strains.
3. Conclusions
The IS element IS1297 has been sequenced from Le. mesenteroides ssp. dextranicum N Z D R I 2218. Although direct evidence for transposition has not been demonstrated, IS1297 has substantial homology to the ISS1 group of lactococcal IS's and shares many of the features of this group of transposable elements. ISSl-like elements are widespread and present in multiple copies in both the plasmid and chromosomal DNA of most lactococci. We have demonstrated the presence of.similar ISSl-like elements in several dairy Leuconostoc strains. The close similarities of the ISS1like elements in Leuconostoc and Lactococcus raise the possibility of genetic exchange between these genera. Additional studies are needed to establish whether this
262
L.J.H. Ward et aL/Gene 174 (1996) 259 263 I
CTGTTGCTCCAGCATAATACAAATTCAACGCAACATTGTGAAAAGTGGGAATCAGTAACA
60
61 TTAGAATTGCTATTATTCCCTTGTATCAGAACCTATATTCAAAAAGGTTCTGTTGCAAAG
120
121 TTTTCCAAAAAATCTATTTTTGTGTAAAATTGAGAAAAAAGACAG~GAGGACAGAGTAAT "-~ M
180
181 GAATCATTTTAAAGGCAAACAATTCAAAAAAGACGTCATTATTGTCG CTGTTGGTTACTA N H F K G K Q F K K D V I I V A V G Y Y
240
241 CCTGCGTTACAATCTAAGCTATCGTGAAGTTCAGGAATTGTTATATGATCGTGGAATAAA 300 L R Y N L S Y R E V Q E L L Y D R G I N M 301 TGTTTGTCATACTACGATTTATCGTTGGGTGCAAGAGTACAGCAAAGTCCTCTATTATCT V C H T T I Y R W V Q E Y S K V L Y Y L F V I L R F I V G C K S T A K S S I I F
360
361 TTGGAAGAAGAAAAATAGACAATCCTTCTATTCATGGAAAATGGACGAAACCTATATCAA W K K K N R Q S F Y S W K M D E T Y I K G R R K I D N P S I H G K W T K P I S K
420
421
AATTAAGGGACGTTGGCATTATCTTTATCGTGCAATTGATGCGGACGGTTTAACCTTAGA 480 I L
481
K G R W H Y L Y R A I D A D G L T R D V G I I F I V Q L M R T V *
L
D
TATCTGGTTACGAAAGAAACGGGATACGCAAGCAGCCTATGCTTTCTTAAAACGACTCCA I W L R K K R D T Q A A Y A F L K R L H
540
541 TAAACAGTTTGGTGAGCCGAAATCAATTGTGACCGATAAAGCACCTTCTCTTGGCTCCGC K Q F G E P K S I V T D K A P S L G S A
600
601 CTTTAGAAAGTTACAGAGTGTGGGTTTATATACTAAGACAGAGCACCGAACTGTGAAGTA F R K L Q S V G L Y T K T E H R T V K Y
660
661
TCTTAACAATTTAATAGAACAAGACCATCGACCTATTAAACGACGGAATAAATTTTATCA L N N L I E Q D H R P I K R R N K F Y Q
720
721 AAGTCTCCGTACAGCCTCTTCCACGATTAAGGGCATGGAGACCCTTCGAGGAATATATAA S L R T A S S T I K G M E T L R G I Y K
780
781 AAAGAACCGAAGAAATGGAACGCTCTTCGGCTTTTCGGTGTCTACTGAAATCAAGGTATT K N R R N G T L F G F S V S T E I K V L
840
841
AATGGGAATAACAGCCTAAGATATTTGGAGTTCATAGAGGGCGCGTTTGATTTTCAAACT M G I T A *
900
901
TCGCAACAGAACCGCGATTTGGATCTTCCGAGTACATAGGTAACTACCCCCTACACGTCA
960
961
TATATACGCATACCCCTTAAAGTGTTTGGCTTCACTCTTTTTGAATCACCCACATTTACA 1020
1021 ACACAAGGTTATGTTATAT 1039
Fig.3.DNAsequence•f•s1297andthe•ankingregi•ns•fh•stDNA.Putativetrans•ati•ns•f•RFsdescribedinSecti•n2.4arepr•vided.The invertedrepeats•secti•n2.3)areindicatedbyarr•ws.Thentsequence•f•S12•7hasbeendep•sitedinGenBank(accessi•nN•.U59•••). is t h e c a s e a n d t o e x a m i n e t h e p o s s i b l e r o l e of I S ' s in this process.
References Campbell, A., Berg, D.E., Botstein, D., Lederberg, E.M, Novick, R.P., Starlinger, P. and Szybalski, W. (1979) Nomenclature of transposable elements in prokaryotes. Gene 5, 197 206. Cluzel, P.-J., Chopin, A., Dusko-Ehrlich, S. and Chopin M.-C. (1991) Phage abortive infection mechanism from Laetocoeeus lactis subsp
lactis, expression which is mediated by an iso-ISSl element. Appl. Environ. Microbiol. 57, 3574 3551. Fiandt, M., Szybalski, W. and Malamy, M.H. (1972) Polar mutations in lac, gal and Phage2 consist of a few IS-DNA sequences inserted with either orientation. Mol. Gen. Genet. 119, 223 231. Galas, D.J. and Chandler, M. (1989) Bacterial insertion sequences. In: Berg D.E. and Howe, M.M. (Eds.), Mobile DNA. American Society for Microbiology, Washington DC, pp. 109 162. Haandrikman, A.J., Van Leeuwen, C., Kok, J., Vos, P., De Vos, W.M. and Venema, G. (1990) Insertion elements on lactococcal proteinase plasmids. Appt. Environ. Microbiol. 56, 1890-1896. Huang, D.C., Novel, M., Huang, X.F. and Novel, G. (1992) Non-iden-
L.J.H. Ward et aL/Gene 174 (1996) 259 263
2 3 4 5 67
8 9 1 0 1112 1314151617
A
I 2 3 4
263
5 6 7 8 9 1011121314151617
B
Fig. 4. (A) Agarose gel of HindIII digests of Leuconostoc genomic DNA. Lanes: 1, 1 kilobase DNA ladder (BRL); 2, Le. mesenteroides ssp. cremoris NZDRI 253; 3, Le. mesenteroides ssp. cremoris NZDRI 502; 4, Le. mesenteroides ssp. cremoris NCDO 523; 5, Le. mesenteroides ssp. dextranicum NZDO 529; 6, Le. mesenteroides ssp. cremoris NCDO 543; 7, Le. paramesenteroides 653; 8, Le. paramesenteroides NCDO 803; 9, Le. mesenteroides ssp. dextranicum NZDO 864; 10, Le. lactis NCDO 956; 11, Le. mesenteroides ssp. cremoris NCDO 1226; lane 12: Le. mesenteroides ssp. cremoris NCDO 2035; 13, Le. mesenteroides ssp. dextranicum ATCC 8082; 14. Le. mesenteroides ssp. cremoris NZDRI 60; 15, Empty; 16, Le. mesenteroides ssp. cremoris NZDRI 83; 17, 2 HindIII molecular weight markers. (B) Southern blot of (A) hybridized with the PCR product containing IS1297. tity between plasmid and chromosomal copies of ISSl-like sequences in Lactococcus lactis subsp lactis CNRZ270 and their possible role in chromosomal integration of plasmid genes. Gene 118, 39-46. Iida, S., Meyer, J. and Arber, W. (1983) Prokaryotic IS elements. In: Shapiro, J.A. (Ed.), Mobile Genetic Elements. Academic Press, New York, pp. 159-221. Polzin, K.M. and Shimizu-Kadota, M. (1987) Identification of a new insertion element, similar to Gram-negative IS26, on the lactose plasmid of Streptococcus lactis ML3. J. Bacteriol. 169, 5481-5488. Polzin, K.M., Romero, D., Shimizu-Kadota, M., Klaenhammer, T.R. and McKay, L.L. (1993) Copy number and location of insertion
sequences ISS1 and IS981 in lactococci and several other lactic acid bacteria. J. Dairy Sci. 76, 1243-1252. Romero, D.A. and Klaenhammer, T.R. (1990) Characterization of insertion sequence IS946, and iso-ISS1 element, isolated from the conjugative lactococcal plasmid pTR2030. J. Bacteriol. 175, 4151-4160. Romero, D.A. and Klaenhammer, T.R. (1993) Transposable elements in lactococci: a review. J. Dairy Sci. 76, 1-19. Sch~ifer, A., Johns, A. Geis, A. and Teuber, M. (1991) Distribution of the IS elements ISSI and IS904 in lactococci. FEMS Microbiol. Lett. 80, 311 318.
GENES AND GENOME5
ELSEVIER
Gene 174 (1996) 259-263
Identification and sequence analysis of IS/297, an ISSl-like insertion sequence in a Leuconostoc strain Lawrence J.H. Ward *, Julie C.S. Brown, Graham P. Davey New Zealand Dairy Research Institute, Private Bag 11029, Palmerston North, New Zealand
Received 18 October 1995; revised 30 November 1995; accepted 18 January 1996
Abstract The insertion sequence (IS) ISS1 from Lactococcus lactis was amplified from lactococcal genomic DNA using a primer to the 18-bp inverted repeat sequence. The amplified product hybridized to a single EcoRI fragment in a total genomic DNA digest of Leuconostoc mesenteroides ssp. dextranicum NZDRI 2218. The DNA sequence of this ISSl-like element (IS1297) and the Le. mesenteroides sequences flanking the IS were determined and compared with other iso-ISS1 elements. No direct repeats were found immediately flanking IS1297; however, direct repeats were present approximately 60 bp on either side of the insertion site. IS1297 contained a major open reading frame (ORF) of 681 bp, encoding a putative 226-amino-acid protein with 96.5% homology to the presumed transposase of ISS1. An overlapping ORF of 174 bp in the same orientation was also present. A putative ORF in the opposite orientation to the transposase ORF, which has been shown in some iso-ISS1 elements, was not present in IS1297. IS1297 was shown to hybridize with other dairy Leuconostoc strains. This is the first sequence of an ISSl-like element from a genus other than Lactococcus; however, IS1297 has close similarity to the lactococcal iso-ISS1 elements, especially the iso-ISS1 element from the lactose plasmid, pTD1. Keywords: Lactococcus; Transposase; Polymerase chain reaction; DNA sequencing
1. Introduction A number of insertion sequence (IS) elements have been reported and characterized in the lactic acid bacteria, especially Lactococcus, where IS elements are associated with m a n y industrially important characteristics (Romero and Klaenhammer, 1993). The IS element ISS1 is present in multiple copies in most lactococcal strains. It is characterized by 18 base inverted repeats and a single open reading frame (ORF) encoding a 226 amino acid (aa) transposase. Other IS's similar to ISS1 (isoISS1 elements) have been characterized and sequenced, particularly from lactococcal plasmids associated with lactose metabolism (Polzin and Shimizu-Kadota, 1987; Sch~ifer et al., 1991), proteinase activity ( H a a n d r i k m a n et al., 1990) or phage resistance (Romero and * Corresponding author. Tel. + 64 6 3504649; Fax + 64 6 3504740; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pairs(s); IS, insertion sequence(s) (Fiandt et al., 1972); La., Lactococcus; Le., Leuconostoc; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction. 0378-1119/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S 0 3 7 8 - 1 1 1 9 ( 9 6 ) 0 0 0 9 1 - 1
Klaenhammer, 1990; Cluzel et al., 1991). ISS1 has also been reported to hybridize to some Pediococcus and Leuconostoc strains (Polzin et al., 1993). This paper presents data demonstrating the presence of an ISS1like element in a Leuconostoc strain and compares the nt and putative transposase sequences to other iso-ISS1 sequences.
2. Experimental and discussion 2.1. Identification of an ISSl-like element in Leuconostoc
Primer I S S I I R (Table 1) complementary to the 18 base inverted repeat of ISS1 was synthesized and the ISS1 sequence was amplified from lactococcal D N A by polymerase chain reaction (PCR). The 808 base pair (bp) product was used as a probe to Southern blots of genomic D N A from strains ofLa. lactis and Le. mesenteroides ssp. dextranicum N Z D R I 2218 (Fig. 1). The L'euconostoc strain showed one band of hybridization to the probe compared to multiple bands of hybridization observed in the lactococcal strains, which suggested that
260
L.Z H. Wardet al./Gene 174 (1996) 259 263
Table 1 Primers used for amplification and/or DNA sequencing Name
Size
Sequence
1SS1 IR CR CL ISS1R ISS/L IS/297EXTL IS1297EXTR
18-mer 20-mer 20-mer 20-mer 20-mer 20-mer 20-mer
5
1234
56
5 5 5 5 5 5
ggt caa gct cat gca ctg gtc
Position in ISS1 Sequence tct act taa cga ggt ttg aag
789
m
G ,wlwm I
gtt gtt cct cct agt ctc cca
gca tta tag att aac cag aac
aag tgg ata aaa caa cat act
ttt agt tct cga cag aat tta
3' cg gg cg cg ac ag
3 3 3 3 3 3
1-18, 808-791 446 428 363 382 581-600 140 121
like element (Fig. 2). The sequence of these amplification products was determined using the P r o m e g a f-mol e sequencing system with direct incorporation of 35S-dATP. The sequencing strategy is described in Fig. 2 and the complete sequence presented in Fig. 3. The IS has been assigned the n u m b e r IS1297 in accordance with the Plasmid Reference Center Registry allocations (Campbell et al., 1979).
2.3. Comparison ofiS1297 with other IS elements
III
The sequence of IS1297 exhibited from 79% to 98% sequence identity with published iso-ISSl sequences. The greatest similarity (98%) was to ISS1/pTD1, an isoISS1 element downstream of the P-fl-galactosidase gene in the lactose plasmid pTD1 (Schfifer et al., 1991). The 18 bp inverted repeat sequence at the 3' end differed by one base to that of most iso-ISS1 elements but was identical to the p T D 1 inverted repeat.
2.4. Putative coding regions ofiS1297 and comparison with other iso-ISS1 elements
Fig. 1. Southern blot of total genomic DNA from Le. mesenteroides ssp. dextranicum strain NZDRI 2218 digested with EcoRI (lanes 1 and 2) and HindIII (lanes 8 and 9) and five La. lactis strains digested with EcoRI (lanes 3-7). The filter was probed with the ISSI sequence amplified as described in Section 2.1. The PCR product was labelled and hybridized using the Amersham ECL system in accordance with the manufacturers instructions. the Leuconostoc strain contained one copy of a sequence similar to ISS1. The presence of an ISSl-like element in the Leuconostoc strain was confirmed by the amplification of a P C R p r o d u c t of similar size to ISS1 using the ISS1 inverted repeat primer. 2.2. Sequencing of the 1SSl-like element from Leuconostoc Primers C L and C R (Table 1) were designed to allow amplification of the left and right regions of the ISS1-
IS1297, like all other iso-ISS1 elements examined, contained one major O R F . This O R F consisted of 681 bp encoding a putative protein of 226 aa. In addition, a second overlapping O R F of 174 bp existed in the same orientation but in a different reading frame which potentially encoded a 57-aa protein. It has been noted for m a n y IS elements from Gram-negative bacteria that short overlapping O R F s exist on the opposite strand to the m a j o r O R F (Galas and Chandler, 1989). The presence of such an O R F has been noted for ISS1RS by H u a n g et al. (1992). This O R F was not conserved in IS1297 or in the iso-ISS1 sequence from pTD1 (Cluzel et al., 1991) or for ISS1N and I S S I W ( H a a n d r i k m a n et al., 1990). In IS1297, ISSI/pTD1, I S S 1 N and ISS1W, a single base change at position 290 (G to A) disrupts the reverse direction O R F which is present in IS946, ISS1RS, 1SS1CH, ISS1S, I S S I T and iso-ISSl. Interestingly, this base change does not alter either of the O R F s on the other strand.
L.J.H. Ward et al./Gene 174 (1996) 259-263
261
IS 1297 IS 1297EXTL
ISSIlR ISS1L
CL
CR
•
ISS1R
IS 1297EXTR
A[
• All
ISSIlR
4
,
•
4
---
ID
Fig. 2. Amplification and sequencing strategy used. The D N A sequence was determined from each of the oligonucleotide primers as indicated by the arrows. To obtain the sequence of the Leuconostoc D N A flanking the IS, total genomic D N A from strain 2218 was sequenced using primers ISS1L to obtain sequence to the left of the IS and ISS1R to obtain the right junction sequence. This sequence data was used to design primers IS1297EXTL and IS1297EXTR, complementary to the Leuconostoc sequence flanking the IS to amplify the IS and the adjoining Leuconostoc DNA. This enabled double stranded sequence data to be obtained for the ends of the IS and the site of insertion into the Leuconostoc DNA.
2.5. Direct repeats of host DNA Small direct repeat duplications of the target DNA at the point of insertion of an IS are characteristic of some IS elements (Galas and Chandler, 1989) and are believed to be caused by the staggered cleavage of target DNA by the transposition enzymes. The ability of an IS element to induce transposition, co-integration and deletion is not dependent on the presence of the small flanking duplication; however, these repeats may be required for precise excision of the IS element (Iida et al., 1983). No direct repeats of host DNA were observed at the site of IS1297 insertion. This is similar to observations for ISS1W and ISS1N (Haandrikman et al., 1990), isoISS1 (Cluzel et al., 1991) and IS946 inserted in pTK6 and pTRK18 (Romero and Klaenhammer, 1990). Sequencing of newly transposed IS946 and ISSIS elements, however, showed the presence of 8 bp direct repeats of host DNA at the insertion site (Romero and Klaenhammer, 1990; Polzin and Shimizu-Kadota, 1987). Iida et al. (1983) have noted that some natural isolates lack a duplicated target DNA sequence. This was postulated to indicate restructuring following the initial transposition event. It is likely that the lack of direct repeats flanking IS1297 is due to such a restructuring event rather than any difference in the mechanism of IS1297 transposition compared with other ISS1 elements. 2.6. Analysis of the sequences flanking iso-ISS1 element insertion sites The DNA sequence of the regions flanking IS1297 in the Leuconostoc strain were compared with the regions flanking ISSIN (Haandrikman et al., 1990) and iso-ISS1 (Cluzel et al., 1991). ISS1W is immediately preceded by a partial ISSI copy (Haandrikman et al., 1990) and hence was not included in this analysis. Insufficient data
on the sequences flanking IS946, ISS1R, S, T and ISS1/pTD1 was available to permit analysis. The most striking feature of all the flanking regions analysed was the presence of an 8 bp repeat 60-70 bp on either side of the site of insertion. These repeats did not exhibit any sequence homology between strains. Whether these repeats have any function in insertion is unknown. 2.7. Distribution oflS1297 in dairy Leuconostoc strains The PCR product containing IS1297 was used as a hybridization probe to Southern blots of genomic DNA from a number of dairy Leuconostoc strains. Of the 27 strains examined, 16 contained between 1 and 6 copies of IS1297. Examples are shown in Fig. 4. Thus, ISSIlike elements are present in low copy number in some but not all dairy Leuconostoc strains. This contrasts with the presence of multiple copies of ISSl-like elements in most lactococcal strains.
3. Conclusions
The IS element IS1297 has been sequenced from Le. mesenteroides ssp. dextranicum N Z D R I 2218. Although direct evidence for transposition has not been demonstrated, IS1297 has substantial homology to the ISS1 group of lactococcal IS's and shares many of the features of this group of transposable elements. ISSl-like elements are widespread and present in multiple copies in both the plasmid and chromosomal DNA of most lactococci. We have demonstrated the presence of.similar ISSl-like elements in several dairy Leuconostoc strains. The close similarities of the ISS1like elements in Leuconostoc and Lactococcus raise the possibility of genetic exchange between these genera. Additional studies are needed to establish whether this
262
L.J.H. Ward et aL/Gene 174 (1996) 259 263 I
CTGTTGCTCCAGCATAATACAAATTCAACGCAACATTGTGAAAAGTGGGAATCAGTAACA
60
61 TTAGAATTGCTATTATTCCCTTGTATCAGAACCTATATTCAAAAAGGTTCTGTTGCAAAG
120
121 TTTTCCAAAAAATCTATTTTTGTGTAAAATTGAGAAAAAAGACAG~GAGGACAGAGTAAT "-~ M
180
181 GAATCATTTTAAAGGCAAACAATTCAAAAAAGACGTCATTATTGTCG CTGTTGGTTACTA N H F K G K Q F K K D V I I V A V G Y Y
240
241 CCTGCGTTACAATCTAAGCTATCGTGAAGTTCAGGAATTGTTATATGATCGTGGAATAAA 300 L R Y N L S Y R E V Q E L L Y D R G I N M 301 TGTTTGTCATACTACGATTTATCGTTGGGTGCAAGAGTACAGCAAAGTCCTCTATTATCT V C H T T I Y R W V Q E Y S K V L Y Y L F V I L R F I V G C K S T A K S S I I F
360
361 TTGGAAGAAGAAAAATAGACAATCCTTCTATTCATGGAAAATGGACGAAACCTATATCAA W K K K N R Q S F Y S W K M D E T Y I K G R R K I D N P S I H G K W T K P I S K
420
421
AATTAAGGGACGTTGGCATTATCTTTATCGTGCAATTGATGCGGACGGTTTAACCTTAGA 480 I L
481
K G R W H Y L Y R A I D A D G L T R D V G I I F I V Q L M R T V *
L
D
TATCTGGTTACGAAAGAAACGGGATACGCAAGCAGCCTATGCTTTCTTAAAACGACTCCA I W L R K K R D T Q A A Y A F L K R L H
540
541 TAAACAGTTTGGTGAGCCGAAATCAATTGTGACCGATAAAGCACCTTCTCTTGGCTCCGC K Q F G E P K S I V T D K A P S L G S A
600
601 CTTTAGAAAGTTACAGAGTGTGGGTTTATATACTAAGACAGAGCACCGAACTGTGAAGTA F R K L Q S V G L Y T K T E H R T V K Y
660
661
TCTTAACAATTTAATAGAACAAGACCATCGACCTATTAAACGACGGAATAAATTTTATCA L N N L I E Q D H R P I K R R N K F Y Q
720
721 AAGTCTCCGTACAGCCTCTTCCACGATTAAGGGCATGGAGACCCTTCGAGGAATATATAA S L R T A S S T I K G M E T L R G I Y K
780
781 AAAGAACCGAAGAAATGGAACGCTCTTCGGCTTTTCGGTGTCTACTGAAATCAAGGTATT K N R R N G T L F G F S V S T E I K V L
840
841
AATGGGAATAACAGCCTAAGATATTTGGAGTTCATAGAGGGCGCGTTTGATTTTCAAACT M G I T A *
900
901
TCGCAACAGAACCGCGATTTGGATCTTCCGAGTACATAGGTAACTACCCCCTACACGTCA
960
961
TATATACGCATACCCCTTAAAGTGTTTGGCTTCACTCTTTTTGAATCACCCACATTTACA 1020
1021 ACACAAGGTTATGTTATAT 1039
Fig.3.DNAsequence•f•s1297andthe•ankingregi•ns•fh•stDNA.Putativetrans•ati•ns•f•RFsdescribedinSecti•n2.4arepr•vided.The invertedrepeats•secti•n2.3)areindicatedbyarr•ws.Thentsequence•f•S12•7hasbeendep•sitedinGenBank(accessi•nN•.U59•••). is t h e c a s e a n d t o e x a m i n e t h e p o s s i b l e r o l e of I S ' s in this process.
References Campbell, A., Berg, D.E., Botstein, D., Lederberg, E.M, Novick, R.P., Starlinger, P. and Szybalski, W. (1979) Nomenclature of transposable elements in prokaryotes. Gene 5, 197 206. Cluzel, P.-J., Chopin, A., Dusko-Ehrlich, S. and Chopin M.-C. (1991) Phage abortive infection mechanism from Laetocoeeus lactis subsp
lactis, expression which is mediated by an iso-ISSl element. Appl. Environ. Microbiol. 57, 3574 3551. Fiandt, M., Szybalski, W. and Malamy, M.H. (1972) Polar mutations in lac, gal and Phage2 consist of a few IS-DNA sequences inserted with either orientation. Mol. Gen. Genet. 119, 223 231. Galas, D.J. and Chandler, M. (1989) Bacterial insertion sequences. In: Berg D.E. and Howe, M.M. (Eds.), Mobile DNA. American Society for Microbiology, Washington DC, pp. 109 162. Haandrikman, A.J., Van Leeuwen, C., Kok, J., Vos, P., De Vos, W.M. and Venema, G. (1990) Insertion elements on lactococcal proteinase plasmids. Appt. Environ. Microbiol. 56, 1890-1896. Huang, D.C., Novel, M., Huang, X.F. and Novel, G. (1992) Non-iden-
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2 3 4 5 67
8 9 1 0 1112 1314151617
A
I 2 3 4
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5 6 7 8 9 1011121314151617
B
Fig. 4. (A) Agarose gel of HindIII digests of Leuconostoc genomic DNA. Lanes: 1, 1 kilobase DNA ladder (BRL); 2, Le. mesenteroides ssp. cremoris NZDRI 253; 3, Le. mesenteroides ssp. cremoris NZDRI 502; 4, Le. mesenteroides ssp. cremoris NCDO 523; 5, Le. mesenteroides ssp. dextranicum NZDO 529; 6, Le. mesenteroides ssp. cremoris NCDO 543; 7, Le. paramesenteroides 653; 8, Le. paramesenteroides NCDO 803; 9, Le. mesenteroides ssp. dextranicum NZDO 864; 10, Le. lactis NCDO 956; 11, Le. mesenteroides ssp. cremoris NCDO 1226; lane 12: Le. mesenteroides ssp. cremoris NCDO 2035; 13, Le. mesenteroides ssp. dextranicum ATCC 8082; 14. Le. mesenteroides ssp. cremoris NZDRI 60; 15, Empty; 16, Le. mesenteroides ssp. cremoris NZDRI 83; 17, 2 HindIII molecular weight markers. (B) Southern blot of (A) hybridized with the PCR product containing IS1297. tity between plasmid and chromosomal copies of ISSl-like sequences in Lactococcus lactis subsp lactis CNRZ270 and their possible role in chromosomal integration of plasmid genes. Gene 118, 39-46. Iida, S., Meyer, J. and Arber, W. (1983) Prokaryotic IS elements. In: Shapiro, J.A. (Ed.), Mobile Genetic Elements. Academic Press, New York, pp. 159-221. Polzin, K.M. and Shimizu-Kadota, M. (1987) Identification of a new insertion element, similar to Gram-negative IS26, on the lactose plasmid of Streptococcus lactis ML3. J. Bacteriol. 169, 5481-5488. Polzin, K.M., Romero, D., Shimizu-Kadota, M., Klaenhammer, T.R. and McKay, L.L. (1993) Copy number and location of insertion
sequences ISS1 and IS981 in lactococci and several other lactic acid bacteria. J. Dairy Sci. 76, 1243-1252. Romero, D.A. and Klaenhammer, T.R. (1990) Characterization of insertion sequence IS946, and iso-ISS1 element, isolated from the conjugative lactococcal plasmid pTR2030. J. Bacteriol. 175, 4151-4160. Romero, D.A. and Klaenhammer, T.R. (1993) Transposable elements in lactococci: a review. J. Dairy Sci. 76, 1-19. Sch~ifer, A., Johns, A. Geis, A. and Teuber, M. (1991) Distribution of the IS elements ISSI and IS904 in lactococci. FEMS Microbiol. Lett. 80, 311 318.