Institut für Tierzucht und Tierverhalten der Bundesforschungsanstalt für Landwirtschaft (FAL), Dörnbergstraße 2527, 29223 Celle, Germany
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Abstract |
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Materials and methods: The 5621 bp plasmid pMVSCS1 was transformed into Escherichia coli JM107, cloned and sequenced completely.
Results: Three intact resistance genes, sulII, catAIII and strA, a truncated strB gene and a novel replication gene were identified. A potential recombination site for the integration of catAIII in the spacer region between sulII and strA was identified.
Conclusion: The physical linkage of the plasmid-borne resistance genes organized in a cluster would facilitate the spread of the different resistance genes by co-selection, even in the absence of a direct selective pressure.
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Introduction |
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Materials and methods |
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Results and discussion |
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M. varigena S131 harboured two plasmids of c. 2.7 and 5.6 kb. The 5.6 kb plasmid, designated pMVSCS1, mediated resistance to sulphonamides, chloramphenicol and streptomycin, as confirmed by transformation into E. coli JM107. The MICs of sulphonamides, chloramphenicol and streptomycin of M. varigena S131 were 2048, 64 and 128 mg/L, respectively, while those of the E. coli JM107:pMVSCS1 transformants were even higher, at >2048, 256 and >256 mg/L. Sequence analysis confirmed the size of plasmid pMVSCS1 to be 5621 bp.
Analysis of the reading frames of pMVSCS1
Five reading frames were identified: four coding for proteins involved in antimicrobial resistance and one for a replication protein. The restriction map and the structural organization of plasmid pMVSCS1 in comparison with other completely sequenced small plasmids mediating sulphonamide and streptomycin resistance are shown in the Figure.
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The catAIII gene codes for the 213 amino acid monomer of a type III chloramphenicol acetyltransferase (CAT, EC 2.3.1.28). This CAT variant was indistinguishable in its amino acid sequence from the respective enzyme of an uncultured eubacterium (accession no. AJ271879), but differed by a single conservative amino acid exchange (202N compared with 202S in pMVSCS1) from the corresponding enzyme of plasmid R387 (accession no. X07848) from Shigella flexneri.9
The strA gene specifies a streptomycin phosphotransferase [APH(3"), EC 2.7.1.] of 263 amino acids. It differs in its 3' end from the other 267 amino acid StrA enzymes so far known from pIG1 of P. multocida (accession no. U57647) and pLS88 of H. ducreyi.8 The insertion of an additional C residue immediately after codon 255 caused a switch of the reading frame, resulting in different codons 256263 and a premature translational stop codon at position 264 in strA from pMVSCS1. In comparison with StrA from RSF1010, two more differences in the amino acid sequence (166-LH-167 compared with 166-QL-167 in pMVSCS1) were observed.6 The same two amino acid differences plus another six amino acid differences (115-YRLINV-120 compared with 115-LSVDQC-120 in pMVSCS1) were noted in strA of pYFC1.7
The strB gene, which is located immediately downstream of the strA gene, is deleted in pMVSCS1. While the intact StrB protein as known from RSF1010 consists of 278 amino acids,6 the StrB reading frame of pMVSCS1 consists of only 69 amino acids, the amino terminal 41 amino acids of which correspond almost exactly to the StrB amino acid sequence.6 Analysis of the nucleotide sequence downstream of this homologous region exhibited homology to an internal part of the mobilization gene mobA from M. haemolytica (accession no. Z21724), and a recombinational event between strB and mobA might be considered to be the cause of the truncation of the strB gene in pMVSCS1. The high MICs of streptomycin observed in the original strain as well as in the E. coli JM107:pMVSCS1 transformants suggest that the observed amino acid differences in the C-terminus of the StrA protein did not reduce the activity of the enzyme, and that expression of strA is sufficient to render the host bacterium resistant to clinically achievable levels of streptomycin. Further support for this latter hypothesis comes from other plasmids, such as pLS88, pIG1 and pYFC1 (Figure), known to mediate streptomycin resistance and also to harbour an intact strA gene but a truncated strB gene.7,8
The fifth reading frame detected on the pMVSCS1 sequence codes for a 322 amino acid protein. Database comparisons revealed homology to a wide range of proteins involved in replication of plasmids, mainly from Gram-negative bacteria. This protein from M. varigena was most closely related to the 328 amino acid Rep protein of the Neisseria gonorrhoeae plasmid pFA3 (accession no. M31727) with 66% identity and 81% homology in the amino acid sequence. The analysis of the nucleotide sequences up- and downstream of the presumed rep gene of pMVSCS1 did not show homology to sequences deposited in the databases.
Development of the multiresistance gene cluster on pMVSCS1
Sequence analysis strongly suggested that a catAIII gene similar to that of R3879 has integrated into the non-coding region between sulII and strA to give rise to the resistance gene cluster observed in pMVSCS1 (Figure). Although sulII and strA genes are found in various members of Enterobacteriaceae and Pasteurellaceae,58,10 their intergenic spacer regions vary distinctly. Detailed comparative analysis of these spacer sequences, but also of the sequences up- and downstream of catAIII in R387,9 indicated that a sulIIstrA spacer region similar to that of RSF10105,6 most likely served as a target for catAIII insertion. In this spacer a 17 bp region (5'-CGCGCTTCATCAGAAAA-3') was detected, which in part showed similarity to a 13 bp region (5'-GGTTCTTAGTAAA-3') upstream of catAIII (71.4%), but also to a 13 bp region (5'-CCCTGTTTTATTA-3') downstream of catAIII (61.5%), from R387. Illegitimate recombination may occur at sequences that exhibit limited similarity, as observed between these R387 and RSF1010 sequences. As a result of the integration of catAIII using the sequences described above, closely related 17 bp sequences are found in pMVSCS1 upstream (5'-CGCGCTTCATCAGAAAA-3') and downstream (5'-CCCTGTTCATCAGAAAA-3') of the catAIII gene.
Analysis of the sulIIstrA spacer regions in RSF1010, pLS88 and pYFC1 showed that there are no specific promoter sequences for strA, suggesting that sulII and strA are transcribed from the same promoter upstream of sulII.58 The catAIII upstream region in pMVSCS1 contained only the catAIII-associated ribosome binding site, but not the corresponding promoter sequences available in R387.9 Since no other pMVSCS1 sequence was detectable in this area that could functionally replace the missing catAIII promoter, it is likely that all three genes, sulII, catAIII and strA, are co-transcribed from the sulII promotor.
In summary, this is the first report of a complete sequence of a resistance plasmid from M. varigena. The analysis of this plasmid revealed a novel arrangement of three resistance genes that are widespread among Gram-negative bacteria. Their physical linkage and organization in a resistance gene cluster gives the opportunity for persistence, but also for co-selection and co-transfer, of all three resistance genes by selective pressure as imposed by the use of either sulphonamides, chloramphenicol or streptomycin.
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Acknowledgements |
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Notes |
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References |
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Sundin, G. W. (2000). Examination of base pair variants of the strAstrB streptomycin resistance genes from bacterial pathogens of humans, animals and plants. Journal of Antimicrobial Chemotherapy 46, 8489.
Received 7 August 2001; returned 1 November 2001; accepted 2 November 2001