Nucleotide sequence and organization of plasmid pMVSCS1 from Mannheimia varigena: identification of a multiresistance gene cluster

Corinna Kehrenberg and Stefan Schwarz,*

Institut für Tierzucht und Tierverhalten der Bundesforschungsanstalt für Landwirtschaft (FAL), Dörnbergstraße 25–27, 29223 Celle, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Objectives: A small resistance plasmid of Mannheimia varigena was analysed with regard to its gene organization and the development of a multiresistance gene cluster.

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.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Bacteria of the family Pasteurellaceae are involved in a variety of economically important diseases in food-producing animals.1 Increasing rates of resistance to various antimicrobial agents among bovine and porcine isolates of the genera Pasteurella and Mannheimia have been recorded during recent years, with resistances to sulphonamides and streptomycin being observed in up to 100% of isolates tested.1 Despite the high frequency of resistance, comparatively little is known about the resistance genes involved and their mechanisms of spreading.1 During the course of a study on transferable tetracycline resistance in bovine Pasteurella and Mannheimia isolates,2 a small plasmid mediating resistance to sulphonamides, chloramphenicol and streptomycin was detected in a M. varigena isolate. This plasmid was analysed with particular reference to its resistance genes and their physical linkage.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The M. varigena isolate S131 was obtained from the respiratory tract of a pig suffering from bronchopneumonia. Its antimicrobial resistance patterns were examined by MIC determination using the broth macrodilution method.3 Plasmids were prepared by alkaline lysis and further purified by affinity chromatography on Qiagen columns (Qiagen, Hilden, Germany).4 Transformation into Escherichia coli JM107 was performed by the CaCl2 method.4 Mapping of plasmid pMVSCS1 using various restriction endonucleases and cloning of HindIII fragments into pBluescript II SK+ (Stratagene, Amsterdam, The Netherlands) followed previously described protocols.4 Complete sequencing of the HindIII fragments was achieved by primer walking on both strands starting with the M13 universal and reverse primers. The sequence of pMVSCS1 has been deposited with the GenBank database under accession number AJ319822. Potential open reading frames were identified by using the ORF finder system (http://www.ncbi.nlm.nih.gov/gorf/gorf.html; last accessed 29 October 2001) while comparisons of the nucleotide sequences or the deduced amino acid sequences were obtained using the BLAST system (http://www.ncbi.nlm.nih.gov/BLAST/; last accessed 29 October 2001).


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Properties of pMVSCS1

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 FigureGo.



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Figure. Comparative analysis of restriction map and structural organization of the plasmids pLS88 from H. ducreyi,8 pYFC1 from P. haemolytica,7 pIG1 from P. multocida, RSF1010 from E. coli5,6 and pMVSCS1 from M. varigena. Restriction endonucleases are abbreviated as follows: B (BclI), C (ClaI), D (DraI), E (EcoRI), EV (EcoRV), H (HindIII), Hp (HpaI), P (PstI), Pv (PvuI), S (SacI), Sm (SmaI), X (XbaI) and Xh (XhoI). A distance scale in kilobases is presented below each map. The reading frames for the genes in plasmid replication (repA, repB, repC, rep), plasmid mobilization (mobA, mobB, mobC), sulphonamide resistance (sulII), streptomycin resistance (strA, strB), chloramphenicol resistance (catAIII) and kanamycin resistance [aph(3')-I] are shown as arrows with the direction of transcription indicated. The prefix {triangleup} indicates functionally inactive truncated genes.

 
The sulII gene of pMVSCS1 specifying a type II dihydropteroate synthase (DHPS, EC 2.5.1.15) of 271 amino acids was closely related to the sulII genes known so far; its gene product differed by one amino acid (246I compared with 246S in pMVSCS1) from those of the enterobacterial plasmids RSF1010 (accession no. A34950)5,6 and pGS05 (accession no. M36657),5 as well as that of pIE1115 (accession no. AJ293027) from an uncultured eubacterium. The type II DHPS variant of plasmid pYFC1 from Pasteurella haemolytica (accession no. M83717)7 showed this amino acid difference, but due to a mutation in the translational stop codon also has an extension of 12 amino acids at the C-terminus. Two amino acid differences (112T and 246I compared with 112A and 246S in pMVSCS1) were noted between the type II DHPS from pMVSCS1 and the corresponding enzyme from the Pasteurella multocida plasmid pIG1 (accession no. U57647). The type II DHPS from the multiresistant Salmonella enterica serovar Typhi strain CT18 (accession no. AL513383) also exhibited two amino acid differences (66E and 246I compared with 66A and 246S in pMVSCS1). The 263 amino acid type II DHPS from pLS88 of Haemophilus ducreyi (accession no. L23118)8 differed from the DHPS of pMVSCS1 by two amino acids in the N-terminus (39S and 94S compared with 39A and 94Y in pMVSCS1). Owing to single base pair insertions downstream of codon 225, the C-terminus of the type II DHPS from pLS88 differed completely from all known type II DHPS variants, including that of pMVSCS1. The high sulphonamide MICs associated with pMVSCS1 indicate that the amino acid substitutions seen in the type II DHPS of pMVSCS1 do not seem to adversely affect the biological activity of this enzyme.

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 256–263 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 (FigureGo), 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 (FigureGo). Although sulII and strA genes are found in various members of Enterobacteriaceae and Pasteurellaceae,5–8,10 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 sulII–strA 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 sulII–strA 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.5–8 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
We wish to thank Erika Nußbeck, Vera Nöding and Gisela Niemann for expert technical assistance.


    Notes
 
* Corresponding author. Tel: +49-5141-384673; Fax: +49-5141-381849; E-mail: stefan.schwarz{at}fal.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
1 . Kehrenberg, C., Schulze-Tanzil, G., Martel, J.-L., ChaslusDancla, E. & Schwarz, S. (2001). Antimicrobial resistance in Pasteurella and Mannheimia: epidemiology and genetic basis. Veterinary Research 32, 323–39.[ISI][Medline]

2 . Kehrenberg, C. (2000). Molecular basis of tetracycline resistance among isolates of the genera Pasteurella and Mannheimia: identification and characterization of novel plasmids and transposons. PhD thesis. Hannover School of Veterinary Medicine, Hannover, Germany.

3 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals: Approved Standard M31-A. NCCLS, Wayne, PA.

4 . Kehrenberg, C. & Schwarz, S. (2000). Identification of a truncated, but functionally active tet(H) tetracycline resistance gene in Pasteurella aerogenes and Pasteurella multocida. FEMS Microbiology Letters 188, 191–5.[ISI][Medline]

5 . Radstrom, P. & Swedberg, G. (1988). RSF1010 and a conjugative plasmid contain sulII, one of two known genes for plasmid-borne sulfonamide resistance dihydropteroate synthase. Antimicrobial Agents and Chemotherapy 32, 1684–92.[ISI][Medline]

6 . Scholz, P., Haring, V., Wittmann-Liebold, B., Ashman, K., Bagdasarian, M. & Scherzinger, E. (1989). Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010. Gene 75, 271–88.[ISI][Medline]

7 . Chang, Y. F., Ma, D. P., Bai, H. Q., Young, R., Struck, D. K., Shin, S. J. et al. (1992). Characterization of plasmids with antimicrobial resistant genes in Pasteurella haemolytica. DNA Sequence 3, 89–97.[Medline]

8 . Dixon, L. G., Albritton, W. L. & Willson, P. J. (1994). An analysis of the complete nucleotide sequence of the Haemophilus ducreyi broad-host-range plasmid pLS88. Plasmid 32, 228–32.[ISI][Medline]

9 . Murray, I. A., Hawkins, A. R., Keyte, J. W. & Shaw, W. V. (1988). Nucleotide sequence analysis and overexpression of the gene encoding a type III chloramphenicol acetyltransferase. Biochemical Journal 252, 173–9.[ISI][Medline]

10 . Sundin, G. W. (2000). Examination of base pair variants of the strA–strB streptomycin resistance genes from bacterial pathogens of humans, animals and plants. Journal of Antimicrobial Chemotherapy 46, 848–9.[Free Full Text]

Received 7 August 2001; returned 1 November 2001; accepted 2 November 2001