Characterization of DIM-1, an Integron-Encoded Metallo-ß- Netherlands

Transcription

1 AAC Accepts, published online ahead of print on March 0 Antimicrob. Agents Chemother. doi:./aac.01-0 Copyright 0, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Revised AAC01-0 Version Characterization of DIM-1, an Integron-Encoded Metallo-ß- Lactamase from a Pseudomonas stutzeri Clinical Isolate in the Netherlands Laurent Poirel, 1 Jose-Manuel Rodríguez-Martínez, 1 Nashwan Al Naiemi, Yvette J. Debets-Ossenkopp, and Patrice Nordmann 1 * 1 Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI, K.-Bicêtre, France, 1 and Dept of Microbiology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands Keywords : Pseudomonas stutzeri, metallo-ß-lactamase, DIM, integron Running title : Metallo-ß-lactamase DIM-1 in Pseudomonas stutzeri L.P. and J.M.R.M. contributed equally to this work * Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, rue du Général Leclerc, Le Kremlin-Bicêtre cedex, France. Phone: Fax: nordmann.patrice@bct.aphp.fr 1

2 A carbapenem-resistant Pseudomonas stutzeri strain isolated from a Dutch patient was analyzed in detail. This isolate produced a metallo-ß-lactamase (MBL) whose gene, of.% GC content, was cloned and expressed in Escherichia coli. ß- Lactamase DIM-1 (for Dutch IMipenemase) was weakly related to other Ambler class B ß-lactamases, sharing less than % amino acid identity with the most closely- related MBL GIM-1, and % with IMP-type MBLs. ß-Lactamase DIM-1 hydrolyzed significantly broad-spectrum cephalosporins and carbapenems, and spared aztreonam. This MBL gene was embedded in a class 1 integron containing two other gene cassettes encoding resistance to aminoglycosides and disinfectants, that was located on a 0-kb plasmid.

3 Metallo-ß-lactamases (MBLs) represent one of the most challenging antibiotic resistance threat in gram negatives (). Those enzymes hydrolyze very efficiently all ß- lactams including carbapenems (with the exception of aztreonam) and are located most often onto transferable genetic platforms (). Different MBL-type enzymes have been described, with IMP- and VIM-derivatives being the most widespread (). The bla IMP-like and bla VIM-like genes have been identified in most clinically-relevant bacteria belonging to the Enterobacteriacae family, in Pseudomonas spp., and in Acinetobacter spp. (). Several other MBLs have been identified in specific geographical locations, being SIM-1 from Acinetobacter baumannii in Korea (1), KHM-1 from Citrobacter freundii in Japan (), and NDM-1 from Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae in India and the UK (, M. Doumith, M. Warner, M. Weinbren, J. Clayton, D.M. 1 Livermore, N. Woodford, Abstract C1-0, presented at the th ICAAC, San Francisco, 1 CA, 1 to 1 September 00), although SPM-1 in Brazil (1, 0, 0), GIM-1 in Germany 1 (), and AIM-1 in Australia (D. Yong, T.R. Walsh, J. Bell, B. Ritchie, R. Pratt, and M.A. 1 Toleman, presented at the th ICAAC, Chicago, DC, 1 to 0 September 00) were all 1 identified from Pseudomonas aeruginosa.

4 The genetic vehicles that carry MBL genes vary, but most of those genes are found as a form of gene cassettes (bla IMP-like, bla VIM-like, bla SIM and bla GIM-1 ) embedded into class 1 integron structures (). In addition, a peculiar insertion sequence named ISCR belonging to the IS1 family and likely moving by rolling-circle transposition has been identified at the origin of mobilization of bla SPM-1 (0, ). This study characterized a novel and clinically-significant MBL which gene was identified in a class 1 integron. MATERIALS AND METHODS Bacterial strains. Pseudomonas stutzeri clinical isolate 1 was identified with the API-0 NE system (biomérieux, Marcy l'etoile, France), and confirmed by rdna sequencing. E. coli TOP was the host for cloning experiments (1). 1 Susceptibility testing. Antibiotic-containing disks were used for routine 1 antibiograms by the disk diffusion assay (Sanofi-Diagnostic Pasteur, Marnes-la-Coquette, 1 France) as recommended (). The ESBL double-disk synergy test was performed with 1 disks containing ceftazidime or cefepime and ticarcillin-clavulanic acid on Mueller-Hinton

5 agar plates, and the results were interpreted as described previously (). The MBL detection was performed by using Etest MBL strips (AB Biodisk, Solna, Sweden). MICs were determined by an agar dilution technique with Mueller-Hinton agar (Sanofi-Diagnostic Pasteur) with an inoculum of CFU per spot, as described previously (1). All plates were incubated at C for 1 h at ambient atmosphere. MICs of ß-lactams were determined alone or in combination with a fixed concentration of clavulanic acid ( µg/ml) and tazobactam ( µg/ml). MIC results were interpreted according to the guidelines of the CLSI (). PCR and hybridization experiments. Total DNA of P. stutzeri 1 was extracted as described previously (). This DNA was used as a template in standard PCR conditions () with a series of primers designed for the detection of class B ß-lactamase genes 1 bla IMP, bla VIM, bla SPM, and bla SIM (1, ). Southern hybridizations were performed as 1 described by Sambrook et al. () using the ECL nonradioactive labeling and detection kit 1 (GE Healthcare, Orsay, France). 1 Cloning experiments, recombinant plasmid analysis, and DNA sequencing. 1 Total DNA of P. stutzeri 1 isolate was digested by XbaI restriction enzyme, ligated into

6 the XbaI site of plasmid pbk-cmv and transformed in E. coli TOP reference strain, as described (1). Recombinant plasmids were selected onto Trypticase soy (TS) agar plates containing amoxicillin (0 µg/ml) and kanamycin (0 µg/ml). The cloned DNA fragments of several recombinant plasmids were sequenced on both strands with an Applied Biosystems sequencer (ABI 0) (Applied Biosystems, Foster City, Ca). The entire sequence provided in this study was made of sequences of several plasmids that contained overlapping cloned fragments. The nucleotide and deduced amino acid sequences were analyzed and compared to sequences available over the Internet at the National Center for Biotechnology Information website ( Genetic support. Transformation experiments were performed with P. stutzeri 1 DNA into P. aeruginosa PU1 recipient strain, as described (1). Plasmid DNA extraction 1 from P. stutzeri 1 was attempted with the Qiagen plasmid DNA maxi kit (Qiagen, 1 Courtaboeuf, France) and with the Kieser method (1) and visualized and sized as 1 described (1). Hybridization was performed with a -bp probe specific for bla DIM-1 1 gene generated with internal primers DIM-1A ( -TCTATTCAGCTTGTCTTCGC- ) and 1 DIM-1B ( -TGTTAGAGGCTGTCTCAGCC- ).

7 ß-Lactamase purification and isoelectric focusing (IEF) analysis. Cultures of E. coli TOP(pXD-1) were grown overnight at C in four liters of TS broth containing amoxicillin (0 µg/ml) and kanamycin (0 µg/ml). ß-Lactamase was purified by ion- exchange chromatography. Briefly, the ß-lactamase extract obtained by sonication of the cells, resuspended in 0 mm sodium phosphate buffer (ph ), was cleared by ultracentrifugation, treated with DNAse, and dialyzed against 0 mm diethanolamine buffer (ph.). This extract was loaded on the Q-Sepharose column, and the ß-lactamase- containing fractions were eluted with a linear 0 to 0. M NaCl gradient. The fractions containing the highest ß-lactamase activity were again dialyzed against the same buffer mentioned above, and the same procedure repeated by eluting more slowly with a linear 0 to 0. M NaCl gradient. The purity of the enzyme was estimated by sodium dodecyl sulfate 1 (SDS)-polyacrylamide gel electrophoresis analysis. The protein content was measured by 1 the Bio-Rad DC protein assay. 1 IEF analysis was performed with an ampholine polyacrylamide gel (ph. to.), 1 as described previously (1), using a purified ß-lactamase extract from a culture of E. coli

8 TOP(pXD-1). The focused ß-lactamases were detected by overlaying the gel with 1 mm nitrocefin (Oxoid, Dardilly, France) in 0 mm phosphate buffer (ph.0). Kinetic measurements. Purified ß-lactamase was used for kinetic measurements performed at 0 C with 0 mm Hepes buffer (ph.) supplemented with 0 µm ZnSO with an ULTROSPEC 000 UV spectrophotometer (Amersham Pharmacia Biotech) as described (). The specific activity of the purified ß-lactamase from E. coli TOP(pXD-1) was obtained as described previously, with imipenem as substrate (). One unit of enzyme activity was defined as the activity which hydrolyzed 1 µmol of imipenem per min per mg of protein. The total protein content was measured with the DC Protein assay kit (Bio-Rad, Ivry-sur-Seine, France). In order to evaluate whether the zinc ion concentration might have an impact on DIM-1 hydrolytic activity, specific activities were measured using the 1 purified enzyme and variable concentrations of ZnSO (0, 0, 0, 0, 00, 00 µm and 1 1mM) and using imipenem as substrate. 1 Fifty percent inhibitory concentration (IC 0 ) was determined for DIM-1 as the 1 concentration of EDTA that reduced the hydrolysis rate of 0 µm benzylpenicillin by

9 0%, under conditions in which DIM-1 was preincubated with various concentrations of EDTA for min at 0 C, before adding the substrate. Nucleotide sequence accession number. The nucleotide sequence data reported in this work have been deposited in the GenBank nucleotide database under accession no. GU01. RESULTS and DISCUSSION Properties of P. stutzeri isolate 1. This strain was isolated in June 00 at the VU Medical Center, Amsterdam (The Netherlands) from purulent exudate obtained from tibial osteomyelitis in a -year old patient who did not have any history of recent travel or hospitalization elsewhere. P. stutzeri 1 was resistant to ticarcillin, piperacillin, piperacillin-tazobactam, and imipenem, had reduced susceptibility to ceftazidime, 1 cefepime, cefpirome and remained fully susceptible to aztreonam. Double-disk synergy 1 test was negative with clavulanate-ceftazidime and clavulanate-imipenem (data not 1 shown), but was positive with the MBL E-test (MIC of IMP at µg/ml vs MIC of 1 IMP/EDTA at µg/ml). This P. stutzeri isolate was also resistant to gentamicin,

10 tobramycin, fluoroquinolones, rifampin, chloramphenicol and tetracycline, and remained susceptible to amikacin, netilmicin, and colistin. Cloning and sequencing of the ß-lactamase gene. Preliminary attempts to detect MBL encoding genes by PCR failed (data not shown). Using total DNA of P. stutzeri 1 as a template in cloning experiments, several E. coli strains harboring recombinant plasmids including pxd-1 were obtained. Sequence analysis of a ca. -kb cloned fragment of pxd-1 revealed a -bp long open reading frame (ORF) encoding a 1-amino-acid preprotein corresponding to an Ambler class B ß-lactamase designated DIM-1 (for Dutch IMipenemase) (1). It possessed the conserved motifs characteristic of MBL enzymes (Fig. 1), including the consensus zinc binding motif HXHXD (residues to ), together with the critical residues for MBL activity, such as His, His, Asp, His1, 1 Cys1, and His according to the BBL nomenclature (, 1-) (Fig. 1). According to 1 its amino acid sequence, it can be classified as a member of subclass 1 MBL, together with 1 IMP- and VIM-types ß-lactamases (). DIM-1 was distantly related to other Ambler class 1 B ß-lactamases. Indeed, the highest percentages of amino acid identity were % with 1 GIM-1 (), % with a putative MBL identified in-silico in the genome of Shewanella

11 denitrificans (Genbank NC_00), and % with KHM-1 (). ß-Lactamase DIM-1 shared % identity with the widespread IMP-type enzymes, and only 0% with the VIM- type enzymes. The G+C content of the bla DIM-1 gene was.%, a value which fits with that of many gram negative species but differs significantly from the G+C content of the P. stutzeri genome being % according to the Genbank database (n NC_00), thus suggesting an horizontal acquisition of bla DIM-1. ß-Lactam susceptibility. MICs of ß-lactams for E. coli TOP(pXD-1) indicated the expression of an MBL that hydrolysed expanded-spectrum cephalosporins (including cephamycins) together with carbapenems, that conferred reduced susceptibility to imipenem and meropenem, and that paradoxally spared aztreonam (Table 1). Addition of ß-lactamase inhibitors such as clavulanic acid or tazobactam did not restore the 1 susceptibility to ß-lactams. 1 Biochemical properties of DIM-1. IEF analysis showed that P. stutzeri 1 and 1 E. coli TOP(pXD-1) had ß-lactamase activities with a pi value of., corresponding to 1 that of DIM-1 (data not shown). The specific activity of the purified ß-lactamase DIM-1 1 was 1 U.mg of protein -1 with benzylpenicillin as substrate. Its overall recovery was 0%

12 with a -fold purification. The purity of the enzyme was estimated to be more than % according to SDS-gel electrophoresis analysis (data not shown). Kinetic parameters of DIM-1 showed its broad-spectrum activity against most ß-lactams, including oxyimino- cephalosporins, cephamycins, and carbapenems but excluding aztreonam (Table ). Analysis of the relative hydrolysis rates of DIM-1 showed that cefotaxime was hydrolyzed at a similar level to that of benzylpenicillin, and cefoxitin was also a good substrate. Ceftazidime was significantly hydrolyzed, with a K m value of 0 µm reflecting a relatively good affinity of DIM-1 for that substrate, as commonly observed for many MBLs. On the opposite, the monobactam aztreonam was not hydrolyzed by DIM-1, as for all MBLs (, ). IC 0 determinations performed with benzylpenicillin as a substrate showed that DIM-1 activity was inhibited by EDTA (1 µm). 1 In addition, we observed that the DIM-1 hydrolytic activity was correlated with the 1 zinc ion concentration. Whereas, the specific activity of DIM-1 for imipenem was U.mg of protein -1 in absence on ZnSO, it was respectively 0.1, 0.1, 0.1, 0., 0., and 1 0. U.mg of protein -1 in presence of increased concentrations of ZnSO (ranging from 0, 1 0, 0, 0, 00, 00 µm and 1mM), thus indicating a significant correlation. 1

13 Genetic environment and support of the bla DIM-1 gene. Sequence analysis of recombinant plasmid pxd-1 harboring the bla DIM-1 gene revealed that it was as a form of a gene cassette, which was inserted at the atti1 recombination site (), similarly to the other acquired MBL encoding genes identified in gram negatives such as bla IMP, bla VIM, and bla SIM genes. Analysis of the -end sequence of the integron showed that the P C promoter sequences were located in the structural integrase gene but no secondary promoter P was identified (1). Thus, the gene cassettes located in that integron are under the control of weak promoter sequences. The dim-1 gene cassette possessed imperfect core (GTTAGAG) and inverse core (CGCTAAC) sites, that latter being located inside the bla DIM-1 coding sequence ( bp from the -end of the gene). The length of its -be sequence was only 1 bp, in which the 1 usually conserved R and L regions of -bes were not detected, indicating that this - 1 be has been likely truncated and therefore suggesting that the dim-1 cassette may not be 1 functional in recombination anymore. A second gene cassette was identified, containing 1 the aadb gene encoding resistance to aminoglycosides (Fig. ). The third gene cassette 1 contained the qach gene encoding resistance to disinfectants. Inside the qach gene 1

14 cassette, the ISKpn insertion sequence was identified that had targeted the -be (), as previously noticed with other members of the IS family that target preferentially the gene cassette -bes (1, ). Analysis of the right extremity of this integron showed that the -conserved segment made of the qace 1 and sul1 genes usually identified were absent, but the tnic gene of transposon Tn00 encoding a 0 amino acid long resolvase was identified. The tnpa gene of transposon Tn was identified (a Tn-like transposon), as well as its IRR extremity (Fig. ). The left-hand extremity of class 1 integrons defined by an inverted repeat structure was identified downstream of the int1 gene, truncating a tnpa gene (subsequently lacking bp at its -extremity) that encodes the transposase of transposon Tn. The IRR of 1 Tn was also identified, that was preceeded by a novel insertion sequence element 1 named ISPst (Fig. ). ISPst is 1, bp long, belongs to the IS0 family, and its 1 transposase shares 0% amino acid identity with that of ISPst1 also identified from a P. 1 stutzeri isolate ( Its transposition had generated a -bp 1 duplication. 1

15 Electro-transformation experiments did not result into the transfer of bla DIM-1 either to P. aeruginosa PU1 or E. coli TOP recipients strains. However, analysis of plasmid content of P. stutzeri 1 identified a single 0-kb plasmid that harbored the bla DIM-1 gene, as confirmed by Southern hybridization (data not shown). However, the negative mating- out result prevented to know which other antibiotic resistance markers were plasmid- associated with the bla DIM-1 gene. As a conclusion, we identified here a novel MBL sharing weak amino acid identity with other MBLs but sharing similar biochemical properties. The dissemination of the bla DIM-1 gene among other gram negative isolates, and especially in Enterobacteriaceae, remains to be evaluated. Indeed, several pieces of evidence had indicated that environmental species such as Pseudomonas sp. may act as intermediate reservoirs for 1 capturing antibiotic resistance genes from other environmental species and then 1 exchanging those genes with Enterobacteriaceae. 1 1 ACKNOWLEDGMENTS This work was mostly funded by the INSERM, France, and by grants from the Ministère 1 de l'education Nationale et de la Recherche (UPRES-EA), Université Paris XI, France 1 and from the European Community (DRESP, LSHM-CT-00-0 and TROCAR, 1

16 HEALTH-F-00-01). J.M. R.M. was funded by a postdoctoral grant from the Ministerio de Educación y Ciencia from Spain (00/0). REFERENCES 1. Ambler, R. P., A. F. Coulson, J.-M. Frère, J. M. Ghuysen, B. Joris, M. Forsman, R. C. Levesque, G. Tiraby, and S. G. Waley. 11. A standard numbering scheme for the class A ß-lactamases. Biochem. J. :-0.. Bellais, S., D. Aubert, T. Naas, and P. Nordmann Molecular and biochemical heterogeneity of class B carbapenem-hydrolyzing ß-lactamases in Chryseobacterium meningosepticum. Antimicrob. Agents Chemother. :1-1.. Bellais, S., D. Girlich, A. Karim, and P. Nordmann. 00. EBR-1, a novel 1 Ambler subclass B1 ß-lactamase from Empedobacter brevis. Antimicrob. Agents 1 Chemother. :-. 1. Bush, K. 1. Metallo-β-lactamases: a class apart. Clin. Infect. Dis. (Suppl. 1 1):S-S. 1

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24 FIGURE LEGENDS Figure 1. Panel A. Comparison of the amino acid sequence of ß-lactamase DIM-1 to those of other acquired MBLs (GIM-1, SIM-1, IMP-1, VIM-1, NDM-1 and SPM-1), and several naturally-occurring MBLs sharing some degree of similarities (IND-1 from Chryseobacterium indologenes, JOHN-1 from Flavobacterium johnsoniae, SLB-1 from Shewanella livingstonensis, and SFB-1 from Shewanella frigidimarina). Shaded amino acids are those conserved with DIM-1. Numbering of ß-lactamases is according to BBL (). Stars indicate conserved amino acid residues. Dashes correspond to the sequence gaps. Panel B. Secondary structure of DIM-1 as compared to that of VIM-. The beta strands and alpha helix are indicated above the DIM-1 sequence. The conserved residues are 1 indicated in black. The conservative amino acid substitutions are boxed. The figure was 1 obtained with ESPript software (). 1 1 Figure. Schematic map representing the In integron structure containing the bla DIM-1 1 gene, together with its flanking sequences. The -bes are indicated by white circles. The 1 horizontal arrows indicate the transcription orientations. The insertion sequences ISPst

25 and ISKpn are represented by rectangles. The right inverted repeats (IRR) of transposons Tn and Tn are represented by black triangles.

26 TABLE 1. MICs of β-lactams for P. stutzeri 1 clinical isolate, E. coli TOP harboring recombinant plasmid pxd-1 expressing DIM-1, and E. coli TOP reference strain. β-lactam (s) a P. stutzeri E. coli E. coli 1 TOP TOP (pxd-1) Amoxicillin >1 >1 Amoxicillin+ CLA >1 >1 Ticarcillin >1 >1 Ticarcillin + CLA >1 >1 Piperacillin >1 1 1 Piperacillin + TZB Cefoxitin 1 1 Ceftazidime 0.0 Cefotaxime Cefepime Aztreonam Imipenem 0.0 a CLA, clavulanic acid at a fixed concentration of µg/ml; TZB, tazobactam at a fixed concentration of µg/ml.

27 Table. Kinetic parameters of purified ß-lactamase DIM-1 a Substrate k cat (s -1 ) K m (µm) k cat /K m (mm -1.s -1 ) Benzylpenicillin 0 0,00 Ampicillin 0 0 Ticarcillin 0 1,00 Cephalothin 0,000 Ceftazidime 0 0 Cefotaxime.,000 Cefepime 0 1 Cefoxitin 0 00 Aztreonam < 0.01 ND ND Imipenem 0 0 Meropenem 0 00 a Data are the means of three independent experiments. Standard deviations were within % of the means. b ND, no detectable hydrolysis (< 0.01 s -1 ).

28 Figure 1 (A) DIM MRTHFTALLLLFSLSSLANDEVPELRIEKVKENIFLHTSYSRVNGFGLVSSNGLVVIDKG-NAFIVDTPWSDRDTETLVHWIRKNG-YELLGS GIM MKNVLVFLILLVALPALAQGHKP-LEVIKIEDGVYLHTSFKNIEGYGLVDSNGLVVLDNN-QAYIIDTPWSEEDTKLLLSWATDRG-YQVMAS SIM MRTLLILCLFGTLNTAFAEEAQPDLKIEKIEEGIYLHTSFQEYKGFGIVKKQGLVVLDNH-KAYLIDTPASAGDTEKLVNWLEKND-FTVNGS IMP MSKLSVFFIFLFCSIATAAESLPDLKIEKLDEGVYVHTSFEEVNGWGVVPKHGLVVLVNA-EAYLIDTPFTAKDTEKLVTWFVERG-YKIKGS SLB MLSLPSYSHEVEP----TSTTIQSVTSSLEGQLSISKLADGVYLHHSYKNVSNFGLVEANGLVVIKDK-QAFIIDTPWTDNDTQKLVDWITQQG-FIPVAS SFB MISAPSFAHENEQQTDQSNTDAVKKPQQQPTELFLSPLVPDVYLHQSYKQVSGFGLVESNGLVVVQNK-QAFIIDTPWTDSDTAKLVDWITQQG-LTVTAS JOHN MRKLASIILFLAAVSNSLGQSKNSPLQISHLTGDFYVYRTFNDYKGT-KISANAMYVVTDK-GVVLFDAPWDKTQFQPLLDSIKAKHNKEVVML IND MKKSIRFFIVSILLSPFASAQVKDFVIEPPIKNNLHIYKTFGVFGGK-EYSANSMYLVTKK-GVVLFDVPWEKIQYQSLMDTIKKRHNLPVVAV VIM MLKVISSLLVYMTASVMAVASPLAHSGEPSGEYPTVNEIPVGEVRLYQIADGVWSHIATQSFDGA-VYPSNGLIVRDGD-ELLLIDTAWGAKNTAALLAEIEKQIGLPVTRA NDM-1 MELPNIMHPVAKLSTALAAALMLSGCMPGEIRPTIGQQMETGDQRFGDLVFRQLAPNVWQHTSYLDMPGFGAVASNGLIVRDGG-RVLVVDTAWTDDQTAQILNWIKQEINLPVALA SPM MNSPKSRALLGFMGAFCLLLVAGAPLSAKSSDHVDLPYNLTATKIDSDVFVVTDRDFYSSNVLVAKMLDGTVVIVSSPFENLGTQTLMDWVAKTMKPKKVVA 1 1 DIM-1 VSTHWHEDRTAGIKWLNDQSISTYATTSTNHLLKENKKEPAKYTLK-GNESTLVDGL IEVFYPGGGHTIDNVVVWLPKSKILFGGCFVRSL GIM-1 ISTHSHEDRTAGIKLLNSKSIPTYTSELTKKLLAREGKPVPTHYFK-DDEFTLGNGL IELYYPGAGHTEDNIVAWLPKSKILFGGCLVRSH SIM-1 ISTHFHDDSTAGIEWLNTKSIPTYASKLTNELLNKNGKTQAKHSFD-KESFWLVKNK IEIFYPGPGHTQDNEVVWIPNKKILFGGCFIKPN IMP-1 ISSHFHSDSTGGIEWLNSRSIPTYASELTNELLKKDGKVQATNSFS-GVNYWLVKNK IEVFYPGPGHTPDNVVVWLPERKILFGGCFIKPY SLB-1 ISTHSHQDRAGGIGYLNRQGITTTVSETTQQILTENDKTTAKSTFT-GMQYIMKTDL VEVYDLGAGHTKDNLVVWLPTQQILFGGCLIKSL SFB-1 ISTHSHQDRAGGIGYLNSQGIATWVSDKTQRLLTANKLSTASHTFR-TKQHTLQQQL IEVYDLGAGHTVDNLLVWLPKQQILFGGCLIKSL JOHN-1 FGTHSHEDRAGGFDFYKKKGIKTYSIKLTDDILKKNKEPRAEFIISNDTTFTVGNHT FEVYYPGKGHAPDNIVAWFKKEKILYGGCFVKSA IND-1 FATHSHDDRAGDLSFFNNKGIKTYATAKTNEFLKKDGKATSTEIIKTGKPYRIGGEE FVVDFLGEGHTADNVVVWFPKYNVLDGGCLVKSN VIM-1 VSTHFHDDRVGGVDVLRAAGVATYASPSTRRLAEAEGNEIPTHSLEGLSSSGDAVRFGP VELFYPGAAHSTDNLVVYVPSANVLYGGCAVHEL NDM-1 VVTHAHQDKMGGMDALHAAGIATYANALSNQLAPQEGMVAAQHSLTFAANGWVEPATAPNFGP LKVFYPGPGHTSDNITVGIDGTDIAFGGCLIKDS SPM-1 INTHFHLDGTGGNEIYKKMGAETWSSDLTKQLRLEENKKDRIKAAEFYKNEDLKRRILSSHPVPADNVFDLKQGKVFSFSNELVEVSFPGPAHSPDNVVVYFPKKKLLFGGCMIKPK * * * * * ** *** DIM-1 DSEGLGYTGEAHIDQWSRSAQNALSRYSEAQIVIPGHGKIGDIALLKHTKSLAETASNKSIQPNANASAD GIM-1 EWEGLGYVGDASISSWADSIKNIVSKKYPIQMVVPGHGKVGSSDILDHTIDLAESASNKLMQPTAEASAD SIM-1 ---GLGNLSDANLEAWPGSAKKMISKYSKAKLVIPSHSEIGDASLLKLTWEQAIKGLNESKSKPPLIN-- IMP-1 ---GLGNLGDANIEAWPKSAKLLKSKYGKAKLVVPSHSEVGDASLLKLTLEQAVKGLNESKKPSKPSN-- SLB-1 NSSTLGYTGEADLQQWPLTIAKVQAQFPQVKIVVPGHGQVGDKALLEHTIELLIPK-NETVNSSS SFB-1 SSRTLGYTGEADLEQWPLTVAKVQAQFIQAKIVVPGHGKIGDTSLLSHTIDLLTQ JOHN-1 EALDLGYLGDADVKEWQKSIKKVQAKFKKPDYIISGHDDWTSKESLNHTLKLVDEYLAQKSAGKK----- IND-1 SATDLGYIKEANVEQWPKTINKLKAKYSKATLIIPGHDEWKGGGHVEHTLELLNKK VIM-1 SSTSAGNVADADLAEWPTSVERIQKHYPEAEVVIPGHGLPGGLDLLQHTANVVKAHKNRSVAE NDM-1 KAKSLGNLGDADTEHYAASARAFGAAFPKASMIVMSHSAPDSRAAITHTARMADKLR SPM-1 E---LGYLGDANVKAWPDSAR--RLKKFDAKIVIPGHGEWGGPEMVNKTIKVAEKAVGEMRL * * * *

29 (B)

30 Figure ISPst ΔtnpA In int1 bla DIM-1 aadb qach ISKpn tnic tnpa IRR IRR Tn Tn-like