Abstract
The multiresistance IncP-1β plasmid pB10 conferring resistance to ampicillin, streptomycin, sulfonamides, tetracycline and mercury ions was previously obtained from activated sludge bacteria by applying the exogenous isolation method with Pseudomonas sp. strain GFP2 as recipient. A pB10 derivative, designated pB10-1, occurred spontaneously and displays an extended NotI restriction fragment. From the pB10 nucleotide sequence, it is known that the corresponding NotI fragment of this plasmid contains a complete class 1 integron with an oxa2 and an orfE-like gene cassette. Sequencing of the integron-specific variable region present on pB10-1 revealed that a second copy of the oxa2 gene cassette has inserted downstream of the orfE-like cassette. Sequences flanking the second oxa2 cassette indicate that this cassette was excised from pB10 and reinserted at a new site in an integrase-catalyzed manner. Duplication of the oxa2 cassette is associated with a higher level of ampicillin resistance. Another pB10 derivative, designated pB10-2, conferring higher resistance to ampicillin, was shown to carry an IS10 insertion upstream of the oxa2 cassette. Since IS10 possesses a promoter-out activity, it can be assumed that the elevated ampicillin resistance level is due to enhanced transcription of the β-lactamase gene.
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References
Bennett PM (1999) Integrons and gene cassettes: a genetic construction kit for bacteria. J Antimicrob Chemother 43:1–4
Blazey DL, Burns RO (1982) Transcriptional activity of the transposable element Tn10 in the Salmonella typhimurium ilvGEDA operon. Proc Natl Acad Sci USA 79:5011–5015
Bunny KL, Hall RM, Stokes HW (1995) New mobile gene cassettes containing an aminoglycoside resistance gene, aacA7, and a chloramphenicol resistance gene, catB3, in an integron in pBWH301. Antimicrob Agents Chemother 39:686–693
Case CC, Roels SM, Gonzalez JE, Simons EL, Simons RW (1988) Analysis of the promoters and transcripts involved in IS10 anti-sense RNA control. Gene 72:219–236
Ciampi MS, Schmid MB, Roth JR (1982) Transposon Tn10 provides a promoter for transcription of adjacent sequences. Proc Natl Acad Sci USA 79:5016–5020
Collis CM, Hall RM (1992) Site-specific deletion and rearrangement of integron insert genes catalysed by the integron DNA integrase. J Bacteriol 174:1574–1585
Collis CM, Hall RM (1995) Expression of antibiotic resistance genes in the integrated cassettes of integrons. Antimicrob Agents Chemother 39:155–162
Collis CM, Recchia GD, Kim MJ, Stokes HW, Hall RM (2001) Efficiency of recombination reactions catalyzed by class 1 integron integrase IntI1. J Bacteriol 183:2535–2542
Davies J (1994) Inactivation of antibiotics and the dissemination of resistance genes. Science 264:375–382
Davison J (1999) Genetic exchange between bacteria in the environment. Plasmid 42:73–91
Dröge M, Pühler A, Selbitschka W (2000) Phenotypic and molecular characterization of conjugative antibiotic resistance plasmids isolated from bacterial communities of activated sludge. Mol Gen Genet 263:471–482
Grant SG, Jessee J, Bloom FR, Hanahan D (1990) Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci USA 87:4645–4649
Heuer H, Krögerrecklenfort E, Wellington EMH, Egan S, van Elsas JD, van Overbeek L, Collard J-M, Guillaume G, Karagouni AD, Nikolakopoulou TL, Smalla K (2002) Gentamicin resistance genes in environmental bacteria: prevalence and transfer. FEMS Microbiol Ecol 42:289–302
Holland S, Dale JW (1984) Improved purification and characterization of the Oxa-2 beta-lactamase. Biochem J 224:1009–1013
Hynes MF, Simon R, Pühler A (1985) The development of plasmid-free strains of Agrobacterium tumefaciens by using incompatibility with a Rhizobium meliloti plasmid to eliminate pAtC58. Plasmid 13:99–105
Kratz J, Schmidt F, Wiedemann B (1983) Transposition of a gene encoding Oxa-2 beta-lactamase. J Gen Microbiol 129:2951–2957
Lévesque C, Brassard S, Lapointe J, Roy PH (1994) Diversity and relative strength of tandem promoters for the antibiotic-resistance genes of several integrons. Gene 142:49–54
Lévesque C, Piche L, Larose C, Roy PH (1995) PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob Agents Chemother 39:185–191
Mahillon J, Chandler M (1998) Insertion sequences. Microbiol Mol Biol Rev 62:725–774
Mazel D, Davies J (1999) Antibiotic resistance in microbes. Cell Mol Life Sci 56:742–754
Meyer F, Goesmann A, McHardy AC, Bartels D, Bekel T, Clausen J, Kalinowski J, Linke B, Rupp O, Giegerich R, Pühler A (2003) GenDB—an open source genome annotation system for prokaryote genomes. Nucleic Acids Res 31:2187–2195
Nücken EJ, Henschke RB, Schmidt FR (1989) Nucleotide sequence of an OXA-2 beta-lactamase gene from the R-plasmid R1767 derived plasmid pBP11 and comparison to closely related resistance determinants found in R46 and Tn2603. J Gen Microbiol 135 (Pt 4):761–765
van Overbeek LS, Wellington EMH, Egan S, Smalla K, Heuer H, Collard J-M, Guillaume G, Karagouni AD, Nikolakopoulou TL, van Elsas JD (2002) Prevalence of streptomycin-resistance genes in bacterial populations in European habitats. FEMS Microbiol Ecol 42:277–288
Partridge SR, Recchia GD, Scaramuzzi C, Collis CM, Stokes HW, Hall RM (2000) Definition of the attI1 site of class 1 integrons. Microbiology 146 (Pt 11):2855–2864
Radström P, Sköld O, Swedberg G, Flensburg J, Roy PH, Sundström L (1994) Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements. J Bacteriol 176:3257–3268
Recchia GD, Hall RM (1995) Gene cassettes: a new class of mobile element. Microbiology 141 (Pt 12):3015–3027
Rowe-Magnus DA, Mazel D (1999) Resistance gene capture. Curr Opin Microbiol 2:483–488
Rowe-Magnus DA, Mazel D (2001) Integrons: natural tools for bacterial genome evolution. Curr Opin Microbiol 4:565–569
Rowe-Magnus DA, Guerout AM, Mazel D (2002) Bacterial resistance evolution by recruitment of super-integron gene cassettes. Mol Microbiol 43:1657–1669
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Labolatory, Cold Spring Harbor
Schlüter A, Heuer H, Szczepanowski R, Forney LJ, Thomas CM, Pühler A, Top EM (2003) The 64 508 bp IncP-1beta antibiotic multiresistance plasmid pB10 isolated from a waste-water treatment plant provides evidence for recombination between members of different branches of the IncP-1beta group. Microbiology 149:3139–3153
Simons RW, Hoopes BC, McClure WR, Kleckner N (1983) Three promoters near the termini of IS10: pIN, pOUT, and pIII. Cell 34:673–682
Smalla K, Sobecky PA (2002) The prevalence and diversity of mobile genetic elements in bacterial communities of different environmental habitats: insights gained from different methodological approaches. FEMS Microbiol Ecol 42:165–175
Smith CA, Thomas CM (1987) Comparison of the organisation of the genomes of phenotypically diverse plasmids of incompatibility group P: members of the IncP beta sub-group are closely related. Mol Gen Genet 206:419–427
Staden R (1996) The Staden sequence analysis package. Mol Biotechnol 5:233–241
Stokes HW, Hall RM (1989) A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol Microbiol 3:1669–1683
Stokes HW, Hall RM (1992) The integron In1 in plasmid R46 includes two copies of the oxa2 gene cassette. Plasmid 28:225–234
Tauch A, Schlüter A, Bischoff N, Goesmann A, Meyer F, Pühler A (2003) The 79,370-bp conjugative plasmid pB4 consists of an IncP-1beta backbone loaded with a chromate resistance transposon, the strA–strB streptomycin resistance gene pair, the oxacillinase gene bla(NPS-1), and a tripartite antibiotic efflux system of the resistance-nodulation-division family. Mol Genet Genomics 268:570–584
Tennstedt T, Szczepanowski R, Braun S, Pühler A, Schlüter A (2003) Occurrence of integron-associated resistance gene cassettes located on antibiotic resistance plasmids isolated from a wastewater treatment plant. FEMS Microbiol Ecol 45:239–252
Thorsted PB, Macartney DP, Akhtar P, Haines AS, Ali N, Davidson P, Stafford T, Pocklington MJ, Pansegrau W, Wilkins BM, Lanka E, Thomas CM (1998) Complete sequence of the IncPbeta plasmid R751: implications for evolution and organisation of the IncP backbone. J Mol Biol 282:969–990
Tschäpe H (1994) The spread of plasmids as a function of bacterial adaptability. FEMS Microbiol Ecol 15:23–31
Acknowledgements
We thank the Bioinformatics group at the Lehrstuhl für Genetik (Bielefeld) in particular for their support concerning bioinformatics. This work was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF), Förderkennzeichen: 0312384.
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Szczepanowski, R., Krahn, I., Pühler, A. et al. Different molecular rearrangements in the integron of the IncP-1β resistance plasmid pB10 isolated from a wastewater treatment plant result in elevated β-lactam resistance levels. Arch Microbiol 182, 429–435 (2004). https://doi.org/10.1007/s00203-004-0726-9
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DOI: https://doi.org/10.1007/s00203-004-0726-9