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Representational difference analysis uncovers a novel IS10-like insertion element unique to pathogenic strains of Yersinia enterocolitica
FEMS Microbiology Letters 210 (2002) 251^255
www.fems-microbiology.org
Representational di¡erence analysis uncovers a novel IS10-like insertion element unique to pathogenic strains of Yersinia enterocolitica A. Iwobi a , A. Rakin a
a;
, E. Garcia b , J. Heesemann
a
Max-von-Pettenkofer Institut fu«r Hygiene und Medizinische Mikrobiologie, Pettenkoferstrasse 9a, 80336 Munich, Germany Lawrence Livermore National Laboratory, Human Genome Center L-452, 7000 East Ave., Livermore, CA 94550, USA
b
Received 11 February 2002; accepted 13 March 2002 First published online 25 April 2002
Abstract The method of suppressive subtractive hybridization was employed to map out genomic differences between the highly pathogenic Yersinia enterocolitica (Ye) biogroup 1B, serotype O:8 strain (WA-314) and the closely related apathogenic Y. enterocolitica biogroup 1A, serotype O:5 strain (NF-O). A novel IS10-like element, IS1330, uncovered by this technique was found to be uniquely present in high copy numbers among the highly pathogenic Y. enterocolitica 1B strains, while a single copy of the element was found in the low pathogenic Ye biogroup 4 serotype O:3 strain. The 1321-bp repetitive element has 19-bp imperfect inverted terminal repeats and is bracketed by a 10-bp duplication of the target sequence. The predicted transposase shares high homology with the IS10 open reading frame of the large virulence plasmid pWR501, of Shigella flexneri, with IS10 transposase of Salmonella typhi, and with IS1999 (tnpA) of Pseudomonas aeruginosa. The IS1330 tnp gene is transcribed in vitro and in vivo in HeLa cells. At least one copy of IS1330 flanks the recently described chromosomal type III secretion cluster in Y. enterocolitica WA-314, O:8, and future studies should shed light on whether this novel transposase mediates transposition events in highly pathogenic Y. enterocolitica strains, thus enhancing the genetic plasticity of this species. : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Subtractive hybridization ; IS1330; Yersinia enterocolitica
1. Introduction The Yersinia species are an important genus among the pathogenic Enterobacteriaceae, with Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica representing the major strains of medical interest. Y. pseudotuberculosis and Y. enterocolitica are human enteropathogenic species and cause food-borne diseases (with a broad spectrum of intestinal and extraintestinal symptoms) ranging from acute enteritis, enterocolitis, terminal ileitis, and mesenteric lymphadenitis to reactive arthritis. Y. pestis on the other hand is the etiological agent of bubonic plague, a systemic and more fatal form of yersiniosis [1^3].
* Corresponding author. E-mail address : [email protected] (A. Rakin).
Central to the pathogenicity of the Yersinia is the possession of a 70-kb pYV plasmid while the chromosomally encoded Ail (Attachment invasion locus) and Inv (Invasin) proteins may be important enteropathogenic virulence markers [1]. While Y. pestis and Y. pseudotuberculosis share greater than 90% DNA homology [4], Y. enterocolitica di¡ers signi¢cantly in terms of genomic content and organization. Pathogenic Y. enterocolitica species are differentiated into two groups: the highly pathogenic, New World or American serotypes, which are mouse lethal, and the low pathogenic, Old World or European species, which are not lethal for mice [2,3]. Although having almost all virulence factors in common, the high pathogenic serotypes possess in addition a chromosomal locus designated the HPI (high pathogenicity island) which is involved in iron sequestration from the host [5^7]. Transposons are mobile, discrete genetic elements that insert into DNA molecules through non-homologous recombination, sometimes with crucial consequences for the recipient DNA, for example, gene inactivation/activation,
0378-1097 / 02 / $22.00 : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 6 3 0 - 4
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or enhancement of the genetic plasticity of the recipient strain through introduction of portable regions of homology. Recently the genome sequence of Y. pestis revealed four IS elements distributed in 140 copies within the genome [8,9]. In this work, we report the discovery of a new IS10-like element, IS1330, with apparent exclusivity to the high-pathogenic Y. enterocolitica species, and which £anks on one side the chromosomal type III protein secretion cluster [10] in Y. enterocolitica WA-314, serotype (ST) O:8.
manufacturer’s instructions. Analysis of sequences was carried out with the Blast program from NCBI and the TIGR-CMR program. Reverse transcription analysis was performed using bacterial RNA harvested under normal growth conditions and RNA harvested from an infected HeLa cell line, a method previously described as SCOTS (selective capture of transcribed sequences) [15]. 2.2.1. Nucleotide sequence accession number The sequence of IS1330 was deposited at the EMBL/ GenBank under accession No. AJ344215.
2. Materials and methods 3. Results and discussion 2.1. Bacterial strains and culture techniques Y. enterocolitica biogroup (BG) 1B strains were: WA314, ST O:8, 8081 (ST O:8), Ye 1209-79 (ST O:13); Ye 1223-75-1 (ST O:20), and Ye 737 (ST O:21). Also employed were Ye MRS40 (BG 2, ST O:9), Ye NF-O (BG 1A, ST O:5), and Y-108 (BG 4, ST O:3). All were from the strain collection of the Max von Pettenkofer Institute. Y. pseudotuberculosis (pstbc) strains employed were Y. pstbc H141/84 (ST O:1a) and Y. pstbc H457/86 (ST O:2a) from S. Aleksic (Hamburg, Germany), Y. pstbc IP3295 (ST O:1) from E. Carniel (Paris, France), and Y. pstbc 346 (ST O:3) from the Max von Pettenkofer Institute strain collection. Y. pestis strains were KIM BG A, KUMA (BG M) and TS (BG O) from R. Brubaker (Michigan, USA). Salmonella serotypes employed in the colony blot reactions were representative strains of groups I^VII of the SARC collection [11]: Salmonella typhimurium, S. typhi, Salmonella arizonae, and Salmonella bongori. The Escherichia coli strains were from the well characterized ECOR collection [12] : ECOR7-11, ECOR31, ECOR35, ECO36, ECOR41, ECOR46, ECOR50, ECOR52, ECOR54, ECOR57, ECOR59, ECOR63, ECOR64, and ECOR65. All other strains were from the strain collection of the Max von Pettenkofer Institute. Typically, bacterial strains were cultured on Luria^Bertani (LB) medium at 27‡C for Yersinia strains, and at 37‡C for all others. 2.2. Molecular biology techniques Bacterial DNA was isolated as previously described [13]. The Y. enterocolitica gene bank was constructed using the Supercos1 cosmid vector as a cloning vehicle (Stratagene). Subtractive hybridization [14] was performed using the modi¢ed protocol from Clontech Laboratories. Generation of the subtracted library was achieved by transformation of the highly e⁄cient MOS cells with pMOSBlue vectors carrying various subtracted fragments. Southern and colony blot hybridizations were performed as previously described [14]. Sequencing was performed on an ABI 373A Sequencer (Applied Biosystems, Inc.) according to the
Apathogenic Y. enterocolitica 1A species typically share a high level of homology with their highly pathogenic, mouse lethal 1B counterparts. In this study, we focused on identifying novel genetic loci that could account for the enhanced virulence of the 1B serotypes alongside the apathogenic 1A strains. In this regard we used chromosomal DNA from the apathogenic Y. enterocolitica (NF-O) biogroup 1A strain as driver DNA and chromosomal DNA from the highly pathogenic Y. enterocolitica (WA-314) biogroup 1B strain as tester DNA in the subtractive hybridization. This technique uncovered a variety of sequences unique to highly pathogenic Y. enterocolitica 1B serotypes. Generally the subtracted fragments fell under three major categories: (1) sequences with homology to known Y. enterocolitica genes and genes from related Enterobacteriaceae, (2) sequences with homology to known phage genes, and mobile genetic elements, and (3) novel sequences without homology to any published genes. Of the 200 subtracted fragments analyzed through sequencing, a fragment (n41) showing signi¢cant homology to genes of previously described transposases was identi¢ed and the cosmid carrying this subtracted fragment was isolated from the cosmid gene library. Further sequencing uncovered a putative insertion element, IS1330, unique to pathogenic Y. enterocolitica strains. IS1330 comprises two imperfect 19-bp inverted repeats (IR) and is £anked by a 10-bp duplication of the target sequence (Fig. 1). At least one GATC methylation site was present in the IS1330 sequence following the transcriptional start site (another one was found 36 nucleotides before the ATG start codon), which could thus in£uence its expression [16]. The putative polypeptide which is predicted to be a 46-kDa protein in size, exhibited a highly basic pI of 10.6, common to transposases [17]. Reverse transcription analysis was performed in order to determine whether IS1330 transposase is transcribed in Y. enterocolitica WA314. The result was positive, also when RNA harvested under in vivo conditions was used as template. IS1330 transposase demonstrated the highest homology (68%) to the IS10 open reading frame of the large virulence plasmid pWR501 of S. £exneri [18], to IS1999 (tnpA)
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253
Fig. 1. Partial nucleotide sequence of IS1330. The positions of direct repeats (DR) and imperfect 19-bp inverted terminal repeats (IR) are indicated in bold letters and with arrows. Sites recognized by deoxyadenine methylase (GATC) are indicated in italics. Putative initiation (ATG) and termination codons (TAA) are indicated by asterisks and in bold letters and the potential ribosome-binding site (S.D) is underlined.
of P. aeruginosa (36%) [19], and to IS10 transposase of S. typhi (36%) [20]. Southern blot analysis (Fig. 2) using the original n41 subtracted fragment (with homology to the putative IS element) as probe against various representatives of Yersinia isolates showed a preponderance of this putative transposase gene among highly pathogenic Y. enterocolitica strains: O:13 (six copies), O:20 (six copies), and O:21 (11 copies). Only a single copy of the IS1330 gene was present in the low pathogenic isolate, Y. enterocolitica 108 BG 4 ST O:3, while Y. enterocolitica NF-O BG 1A ST O:5 (apathogen), and Y. pestis and Y. pseudotuberculosis strains of di¡erent serotypes were negative for this probe. Interestingly, among the two tested Y. enterocolitica O:8 strains WA-314 and Ye 8081, di¡erences existed in both the copy number of the transposase fragment and their chromosomal locations. For example, WA-314 harbors four copies of this IS element with at least one copy 345 bp downstream from the chromosomal type III cluster described by Haller et al. [10]. Ye 8081 on the other hand carries six copies of the IS1330 sequence and no copy appears to be associated with this chromosomally encoded type III cluster, shedding interesting light on intraspecies di¡erences that could exist between very closely related Y. enterocolitica strains in terms of genomic organization. The result of colony blot hybridizations to identify sequences homologous to IS1330 among various members of the Enterobacteriaceae family is depicted in Table 1. They con¢rm the ¢ndings of our previous Southern blot analysis (Fig. 2), indicating the unique distribution of IS1330 among showing apparent exclusivity of IS1330 among highly pathogenic Y. enterocolitica strains. The Y. enterocolitica O :3 serotypes also moderately reacted with the probe but as evident from the Southern blot may harbor only one or two copies of IS1330 in contrast
to the highly pathogenic serotypes that harbored 4^11 copies. The Yersinia chromosome is subjected to di¡erent types of genetic rearrangements, including deletions and insertions promoted by mobile genetic elements. In Y. pestis for example, IS100 and IS245 are present in multiple copies and are responsible for the inactivation of certain virulence factors [8,21]. In Y. enterocolitica, IS1328 has been implicated in possible deletions [22] of the fyuA/irp2 gene cluster that constitute part of the iron acquisition system in the Yersinia spp. On the other hand, IS elements are involved in genetic mobility of chromosomal fragments, as is exempli¢ed by antibiotic resistance cassettes harbored by a variety of members of the Enterobacteriaceae. Be-
Fig. 2. Southern blot hybridization of EcoRI-digested bacterial chromosomal DNA with DIG-labeled IS1330 probe. Lanes: 1^3, Y. pseudotuberculosis spp. (Y. pstbc H141/84 (ST O:1a), Y. pstbc H457/86 (ST O:2a) and Y. pstbc 346 (ST O:3); 4^6, Y. pestis spp. (KIM ; KUMA ; TS); 7, Y. enterocolitica (Ye) MRS40; 8, Ye WA-314; 9, Ye 8081; 10, Ye 737; 11, Ye 1223-75-1; 12, Ye 1209-79; 13, Ye 108.
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Table 1 Result of colony blots to determine the presence of IS1330 among various representatives of Enterobacteriaceae Organism Yersinia spp. Y. enterocolitica spp. Ye O:8 (WA-314) Ye O:8 (8081) Ye O:13 Ye O:20 Ye O:21 Ye O:3 Ye O:1,2a,3 Y. frederiksenii Y. intermedia Y. mollaretti Y. pseudotuberculosis spp. Y. pstbc O:1a Y.pstbc O:2a Y. pstbc IP3295 (ST O:1) Y. pstbc 346 (ST O:3) Y. pestis spp. KIM (BG A) KUMA (BG M) TS (BG O) Salmonella spp. S. typhimurium S. typhi S. arizonae S. bongori Shigella spp. S. sonnei S. £exneri S. boydii Proteus spp. P. vulgaris P. mirabilis Klebsiella spp. K. pneumoniae K. oxytoca E. coli (ECOR) strains
IS1330 probe
+ + + + + + + 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
The E. coli strains are representatives of the ECOR collection listed in Section 2.
cause IS1330 shares some common features with previously described IS10 elements including molecular mass in the 46-kDa range and possibly a related mechanism of regulation by Dam methylase, future studies will shed light on whether IS1330 similarly mediates gene rearrangements and IS10-like transposition related events in the Yersinia chromosome, thus enhancing the genetic plasticity of pathogenic Y. enterocolitica strains.
References [1] Boyd, A.P. and Cornelis, G.Y. (2001) Yersinia. In: Principles of Bacterial Pathogenesis (Groisman, E.A., Ed.). [2] Perry, R.D. (1997) Yersinia pestis ^ Etiologic agent of pest. Clin. Microbiol. Rev. 10, 35^66.
[3] Carter, P.B. (1975) Pathogenicity of Yersinia enterocolitica for mice. Infect. Immun. 11, 164^170. [4] Bercovier, H., Mollaret, H.H., Alonso, J.M., Brqult, J., Fanning, G.R., Steigerwalt, A.G. and Brenner, D.J. (1980) Intra- and interpecies relatedness of Yersinia pestis by DNA hybridization and its relationship to Yersinia pseudotuberculosis. Curr. Microbiol. 4, 25^ 229. [5] Pelludat, C., Rakin, A., Jacobi, C.A., Schubert, S. and Heesemann, J. (1998) The Yersiniabactin biosynthetic gene cluster of Yersinia enterocolitica: organization and siderophore-dependent regulation. J. Bacteriol. 180, 538^546. [6] Rakin, A. and Noelting, C. (2001) Schropp, P. and Heesemann, J. (2000) Integrative module of the high-pathogenicity island of Yersinia. Mol. Microbiol. 39, 407^415. [7] Brem, D., Pelludat, C., Rakin, A., Jacobi, C.A. and Heesemann, J. (2001) Functional analysis of yersiniabactin transport genes of Yersinia enterocolitica. Microbiology 147, 1115^1127. [8] Parkhill, J., Wren, B.W., Thomson, N.R., Titball, R.W., Holden, M.T., Prentice, M.B., Sebaihia, M., James, K.D., Churcher, C., Mungall, K.L., Baker, S., Basham, D., Bentley, S.D., Brooks, K., Cerdeno-Tarraga, A.M., Chillingworth, T., Cronin, A., Davies, R.M., Davis, P., Dougan, G., Feltwell, T., Hamlin, N., Holroyd, S., Jagels, K., Karlyshev, A.V., Leather, S., Moule, S., Oyston, P.C., Quail, M., Rutherford, K., Simmonds, M., Skelton, J., Stevens, K., Whitehead, S. and Barrell, B.G. (2001) Genome sequence of Yersinia pestis, the causative agent of plague. Nature 413, 523^527. [9] Kleckner, N. (1989) Transposon Tn10. In: Mobile DNA (Berg, D.E. and Howe, M.M., Eds.), pp. 227^268. American Society for Microbiology, Washington, DC. [10] Haller, J.C., Carlson, S., Pederson, K.J. and Pierson, D.E. (2000) A chromosomally encoded type III secretion pathway in Yersinia enterocolitica is important in virulence. Mol. Microbiol. 36, 1436^1446. [11] Boyd, E.F., Wang, F., Whittam, T.S. and Selander, R.K. (1996) Molecular genetic relationships of the Salmonellae. Appl. Environ. Microbiol. 62, 804^808. [12] Ochman, H. and Selander, R.K. (1984) Standard reference strains of Escherichia coli from natural populations. J. Bacteriol. 157, 690^ 693. [13] Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (Eds.) (2000) Current Protocols in Molecular Biology. John Wiley and Sons, New York. [14] Diatchenko, L., Lau, Y.-F.C., Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D. and Siebert, P.D. (1996) Suppression subtractive hybridization: a method for generating di¡erentially regulated or tissuespeci¢c cDNA probes and libraries. Proc. Natl. Acad. Sci. USA 93, 6025^6030. [15] Morrow, B.J., Graham, J.E. and Curtiss III, R. (1999) Genomic subtractive hybridization and selective capture of transcribed sequences identify a novel Salmonella typhimurium ¢mbrial operon and putative transcriptional regulator that are absent from the Salmonella typhi genome. Infect. Immun. 67, 5106^5116. [16] Rasmussen, L.J., Marinus, M.G. and Lobner-Oelsen, A. (1994) Novel growth rate of dam gene expression in Escherichia coli. Mol. Microbiol. 12, 631^638. [17] Galas, D.J. and Chandler, M. (1989) Bacterial insertion sequences. In: Mobile DNA (Berg, D.F. and Howe, M.M., Eds.), pp. 109^162. American Society for Microbiology, Washington, DC. [18] Venkatesan, M.M., Goldberg, M.B., Rose, D.J., Grotbeck, E.J., Burland, V. and Blattner, F.R. (2001) Complete DNA sequence and analysis of the large virulence plasmid of Shigella £exneri. Infect. Immun. 69, 3271^3285. [19] Stokes, H.W. and Hall, R.M. (1991) Sequence analysis of the inducible chloramphenicol resistance determinant in the Tn1696 integron suggests regulation by translational attenuation. Plasmid 26, 10^19. [20] Lawley, T.D., Burland, V. and Taylor, D.E. (2000) Analysis of the
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A. Iwobi et al. / FEMS Microbiology Letters 210 (2002) 251^255 complete nucleotide sequence of the tetracycline-resistance transposon Tn10. Plasmid 43, 235^239. [21] Torosian, S.D. and Zsigray, R.M. (1995) A portion of IS100 regulates gene expression in Yersinia pseudotuberculosis and shares essentially identical sequence homology with a repetitive sequence isolated from Yersinia pestis. In: Yersiniosis : Present and Future, Contrib.
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Microbiol. Immunol. (Ravagnan, G. and Chiesa, C., Eds.), pp 314^ 317. Karger, Basel. [22] Rakin, A., Schubert, S., Guilvout, I., Carniel, E. and Heesemann, J. (2000) Local hopping of IS3 elements into the A+T-rich part of the high-pathogenicity island in Yersinia enterocolitica 1B, O:8. FEMS Microbiol. Lett. 182, 225^229.
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Representational di¡erence analysis uncovers a novel IS10-like insertion element unique to pathogenic strains of Yersinia enterocolitica A. Iwobi a , A. Rakin a
a;
, E. Garcia b , J. Heesemann
a
Max-von-Pettenkofer Institut fu«r Hygiene und Medizinische Mikrobiologie, Pettenkoferstrasse 9a, 80336 Munich, Germany Lawrence Livermore National Laboratory, Human Genome Center L-452, 7000 East Ave., Livermore, CA 94550, USA
b
Received 11 February 2002; accepted 13 March 2002 First published online 25 April 2002
Abstract The method of suppressive subtractive hybridization was employed to map out genomic differences between the highly pathogenic Yersinia enterocolitica (Ye) biogroup 1B, serotype O:8 strain (WA-314) and the closely related apathogenic Y. enterocolitica biogroup 1A, serotype O:5 strain (NF-O). A novel IS10-like element, IS1330, uncovered by this technique was found to be uniquely present in high copy numbers among the highly pathogenic Y. enterocolitica 1B strains, while a single copy of the element was found in the low pathogenic Ye biogroup 4 serotype O:3 strain. The 1321-bp repetitive element has 19-bp imperfect inverted terminal repeats and is bracketed by a 10-bp duplication of the target sequence. The predicted transposase shares high homology with the IS10 open reading frame of the large virulence plasmid pWR501, of Shigella flexneri, with IS10 transposase of Salmonella typhi, and with IS1999 (tnpA) of Pseudomonas aeruginosa. The IS1330 tnp gene is transcribed in vitro and in vivo in HeLa cells. At least one copy of IS1330 flanks the recently described chromosomal type III secretion cluster in Y. enterocolitica WA-314, O:8, and future studies should shed light on whether this novel transposase mediates transposition events in highly pathogenic Y. enterocolitica strains, thus enhancing the genetic plasticity of this species. : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Subtractive hybridization ; IS1330; Yersinia enterocolitica
1. Introduction The Yersinia species are an important genus among the pathogenic Enterobacteriaceae, with Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica representing the major strains of medical interest. Y. pseudotuberculosis and Y. enterocolitica are human enteropathogenic species and cause food-borne diseases (with a broad spectrum of intestinal and extraintestinal symptoms) ranging from acute enteritis, enterocolitis, terminal ileitis, and mesenteric lymphadenitis to reactive arthritis. Y. pestis on the other hand is the etiological agent of bubonic plague, a systemic and more fatal form of yersiniosis [1^3].
* Corresponding author. E-mail address : [email protected] (A. Rakin).
Central to the pathogenicity of the Yersinia is the possession of a 70-kb pYV plasmid while the chromosomally encoded Ail (Attachment invasion locus) and Inv (Invasin) proteins may be important enteropathogenic virulence markers [1]. While Y. pestis and Y. pseudotuberculosis share greater than 90% DNA homology [4], Y. enterocolitica di¡ers signi¢cantly in terms of genomic content and organization. Pathogenic Y. enterocolitica species are differentiated into two groups: the highly pathogenic, New World or American serotypes, which are mouse lethal, and the low pathogenic, Old World or European species, which are not lethal for mice [2,3]. Although having almost all virulence factors in common, the high pathogenic serotypes possess in addition a chromosomal locus designated the HPI (high pathogenicity island) which is involved in iron sequestration from the host [5^7]. Transposons are mobile, discrete genetic elements that insert into DNA molecules through non-homologous recombination, sometimes with crucial consequences for the recipient DNA, for example, gene inactivation/activation,
0378-1097 / 02 / $22.00 : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 6 3 0 - 4
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or enhancement of the genetic plasticity of the recipient strain through introduction of portable regions of homology. Recently the genome sequence of Y. pestis revealed four IS elements distributed in 140 copies within the genome [8,9]. In this work, we report the discovery of a new IS10-like element, IS1330, with apparent exclusivity to the high-pathogenic Y. enterocolitica species, and which £anks on one side the chromosomal type III protein secretion cluster [10] in Y. enterocolitica WA-314, serotype (ST) O:8.
manufacturer’s instructions. Analysis of sequences was carried out with the Blast program from NCBI and the TIGR-CMR program. Reverse transcription analysis was performed using bacterial RNA harvested under normal growth conditions and RNA harvested from an infected HeLa cell line, a method previously described as SCOTS (selective capture of transcribed sequences) [15]. 2.2.1. Nucleotide sequence accession number The sequence of IS1330 was deposited at the EMBL/ GenBank under accession No. AJ344215.
2. Materials and methods 3. Results and discussion 2.1. Bacterial strains and culture techniques Y. enterocolitica biogroup (BG) 1B strains were: WA314, ST O:8, 8081 (ST O:8), Ye 1209-79 (ST O:13); Ye 1223-75-1 (ST O:20), and Ye 737 (ST O:21). Also employed were Ye MRS40 (BG 2, ST O:9), Ye NF-O (BG 1A, ST O:5), and Y-108 (BG 4, ST O:3). All were from the strain collection of the Max von Pettenkofer Institute. Y. pseudotuberculosis (pstbc) strains employed were Y. pstbc H141/84 (ST O:1a) and Y. pstbc H457/86 (ST O:2a) from S. Aleksic (Hamburg, Germany), Y. pstbc IP3295 (ST O:1) from E. Carniel (Paris, France), and Y. pstbc 346 (ST O:3) from the Max von Pettenkofer Institute strain collection. Y. pestis strains were KIM BG A, KUMA (BG M) and TS (BG O) from R. Brubaker (Michigan, USA). Salmonella serotypes employed in the colony blot reactions were representative strains of groups I^VII of the SARC collection [11]: Salmonella typhimurium, S. typhi, Salmonella arizonae, and Salmonella bongori. The Escherichia coli strains were from the well characterized ECOR collection [12] : ECOR7-11, ECOR31, ECOR35, ECO36, ECOR41, ECOR46, ECOR50, ECOR52, ECOR54, ECOR57, ECOR59, ECOR63, ECOR64, and ECOR65. All other strains were from the strain collection of the Max von Pettenkofer Institute. Typically, bacterial strains were cultured on Luria^Bertani (LB) medium at 27‡C for Yersinia strains, and at 37‡C for all others. 2.2. Molecular biology techniques Bacterial DNA was isolated as previously described [13]. The Y. enterocolitica gene bank was constructed using the Supercos1 cosmid vector as a cloning vehicle (Stratagene). Subtractive hybridization [14] was performed using the modi¢ed protocol from Clontech Laboratories. Generation of the subtracted library was achieved by transformation of the highly e⁄cient MOS cells with pMOSBlue vectors carrying various subtracted fragments. Southern and colony blot hybridizations were performed as previously described [14]. Sequencing was performed on an ABI 373A Sequencer (Applied Biosystems, Inc.) according to the
Apathogenic Y. enterocolitica 1A species typically share a high level of homology with their highly pathogenic, mouse lethal 1B counterparts. In this study, we focused on identifying novel genetic loci that could account for the enhanced virulence of the 1B serotypes alongside the apathogenic 1A strains. In this regard we used chromosomal DNA from the apathogenic Y. enterocolitica (NF-O) biogroup 1A strain as driver DNA and chromosomal DNA from the highly pathogenic Y. enterocolitica (WA-314) biogroup 1B strain as tester DNA in the subtractive hybridization. This technique uncovered a variety of sequences unique to highly pathogenic Y. enterocolitica 1B serotypes. Generally the subtracted fragments fell under three major categories: (1) sequences with homology to known Y. enterocolitica genes and genes from related Enterobacteriaceae, (2) sequences with homology to known phage genes, and mobile genetic elements, and (3) novel sequences without homology to any published genes. Of the 200 subtracted fragments analyzed through sequencing, a fragment (n41) showing signi¢cant homology to genes of previously described transposases was identi¢ed and the cosmid carrying this subtracted fragment was isolated from the cosmid gene library. Further sequencing uncovered a putative insertion element, IS1330, unique to pathogenic Y. enterocolitica strains. IS1330 comprises two imperfect 19-bp inverted repeats (IR) and is £anked by a 10-bp duplication of the target sequence (Fig. 1). At least one GATC methylation site was present in the IS1330 sequence following the transcriptional start site (another one was found 36 nucleotides before the ATG start codon), which could thus in£uence its expression [16]. The putative polypeptide which is predicted to be a 46-kDa protein in size, exhibited a highly basic pI of 10.6, common to transposases [17]. Reverse transcription analysis was performed in order to determine whether IS1330 transposase is transcribed in Y. enterocolitica WA314. The result was positive, also when RNA harvested under in vivo conditions was used as template. IS1330 transposase demonstrated the highest homology (68%) to the IS10 open reading frame of the large virulence plasmid pWR501 of S. £exneri [18], to IS1999 (tnpA)
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Fig. 1. Partial nucleotide sequence of IS1330. The positions of direct repeats (DR) and imperfect 19-bp inverted terminal repeats (IR) are indicated in bold letters and with arrows. Sites recognized by deoxyadenine methylase (GATC) are indicated in italics. Putative initiation (ATG) and termination codons (TAA) are indicated by asterisks and in bold letters and the potential ribosome-binding site (S.D) is underlined.
of P. aeruginosa (36%) [19], and to IS10 transposase of S. typhi (36%) [20]. Southern blot analysis (Fig. 2) using the original n41 subtracted fragment (with homology to the putative IS element) as probe against various representatives of Yersinia isolates showed a preponderance of this putative transposase gene among highly pathogenic Y. enterocolitica strains: O:13 (six copies), O:20 (six copies), and O:21 (11 copies). Only a single copy of the IS1330 gene was present in the low pathogenic isolate, Y. enterocolitica 108 BG 4 ST O:3, while Y. enterocolitica NF-O BG 1A ST O:5 (apathogen), and Y. pestis and Y. pseudotuberculosis strains of di¡erent serotypes were negative for this probe. Interestingly, among the two tested Y. enterocolitica O:8 strains WA-314 and Ye 8081, di¡erences existed in both the copy number of the transposase fragment and their chromosomal locations. For example, WA-314 harbors four copies of this IS element with at least one copy 345 bp downstream from the chromosomal type III cluster described by Haller et al. [10]. Ye 8081 on the other hand carries six copies of the IS1330 sequence and no copy appears to be associated with this chromosomally encoded type III cluster, shedding interesting light on intraspecies di¡erences that could exist between very closely related Y. enterocolitica strains in terms of genomic organization. The result of colony blot hybridizations to identify sequences homologous to IS1330 among various members of the Enterobacteriaceae family is depicted in Table 1. They con¢rm the ¢ndings of our previous Southern blot analysis (Fig. 2), indicating the unique distribution of IS1330 among showing apparent exclusivity of IS1330 among highly pathogenic Y. enterocolitica strains. The Y. enterocolitica O :3 serotypes also moderately reacted with the probe but as evident from the Southern blot may harbor only one or two copies of IS1330 in contrast
to the highly pathogenic serotypes that harbored 4^11 copies. The Yersinia chromosome is subjected to di¡erent types of genetic rearrangements, including deletions and insertions promoted by mobile genetic elements. In Y. pestis for example, IS100 and IS245 are present in multiple copies and are responsible for the inactivation of certain virulence factors [8,21]. In Y. enterocolitica, IS1328 has been implicated in possible deletions [22] of the fyuA/irp2 gene cluster that constitute part of the iron acquisition system in the Yersinia spp. On the other hand, IS elements are involved in genetic mobility of chromosomal fragments, as is exempli¢ed by antibiotic resistance cassettes harbored by a variety of members of the Enterobacteriaceae. Be-
Fig. 2. Southern blot hybridization of EcoRI-digested bacterial chromosomal DNA with DIG-labeled IS1330 probe. Lanes: 1^3, Y. pseudotuberculosis spp. (Y. pstbc H141/84 (ST O:1a), Y. pstbc H457/86 (ST O:2a) and Y. pstbc 346 (ST O:3); 4^6, Y. pestis spp. (KIM ; KUMA ; TS); 7, Y. enterocolitica (Ye) MRS40; 8, Ye WA-314; 9, Ye 8081; 10, Ye 737; 11, Ye 1223-75-1; 12, Ye 1209-79; 13, Ye 108.
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Table 1 Result of colony blots to determine the presence of IS1330 among various representatives of Enterobacteriaceae Organism Yersinia spp. Y. enterocolitica spp. Ye O:8 (WA-314) Ye O:8 (8081) Ye O:13 Ye O:20 Ye O:21 Ye O:3 Ye O:1,2a,3 Y. frederiksenii Y. intermedia Y. mollaretti Y. pseudotuberculosis spp. Y. pstbc O:1a Y.pstbc O:2a Y. pstbc IP3295 (ST O:1) Y. pstbc 346 (ST O:3) Y. pestis spp. KIM (BG A) KUMA (BG M) TS (BG O) Salmonella spp. S. typhimurium S. typhi S. arizonae S. bongori Shigella spp. S. sonnei S. £exneri S. boydii Proteus spp. P. vulgaris P. mirabilis Klebsiella spp. K. pneumoniae K. oxytoca E. coli (ECOR) strains
IS1330 probe
+ + + + + + + 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
The E. coli strains are representatives of the ECOR collection listed in Section 2.
cause IS1330 shares some common features with previously described IS10 elements including molecular mass in the 46-kDa range and possibly a related mechanism of regulation by Dam methylase, future studies will shed light on whether IS1330 similarly mediates gene rearrangements and IS10-like transposition related events in the Yersinia chromosome, thus enhancing the genetic plasticity of pathogenic Y. enterocolitica strains.
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