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Journal of Clinical Microbiology, August 2005, p. 4298-4300, Vol. 43, No. 8
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.8.4298-4300.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Correctly Identifying the Streptothricin Resistance Gene Cassette

Top Letter References

 
If analysis of the gene cassettes located in class 1 or class 2 integrons found in surveys of antibiotic resistance genes is to be useful in tracking the prevalence and movement of resistance genes, the cassettes must be accurately identified. This can be simply achieved by citing the GenBank accession numbers for the relevant reference cassettes listed in available compilations (e.g., references 6 and 9). However, confusion is arising around the single cassette known to include a streptothricin acetyltransferase gene (GenBank accession no. X15995), which was first identified in transposons Tn1825 and Tn1826 isolated from bacteria resistant to streptothricin (4, 12). These transposons are close relatives of Tn7 (14), and the sat cassette was subsequently identified in Tn7 (3, 11, 13), where it confers only modest levels of resistance to the antibiotic (11, 13). Thus, Tn7, Tn1825, and Tn1826 are all class 2 integrons that appear to differ only in the composition of the cassette array (see reference 9) (Fig. 1A).

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FIG. 1. Maps of integrons containing the sat2 or estX cassette. Thick black lines represent the 5' CS (intI region), and the attI1 site is represented by an open box with a number indicating the integron class. Each cassette is shown as a narrow open or hatched box with an adjacent filled box representing the 59-be (59-base element). Labeled arrows indicate the positions of the genes. The positions of the TAG stop codon at the end of estX and the mutant version (TAT) in Tn1825 are indicated. The maps of Tn7, Tn1825, and Tn1826 were drawn using sequences from GenBank accession nos. AP002527 (Tn7), X56185 (Tn1825), and X15995 plus additional map information from reference 14 for Tn1826. Maps for the remaining cassette arrays are from the GenBank accession numbers shown. The estX gene/cassette is annotated as sat in AB121039 and AB161461-3 but has been corrected in AY090896; sat2 is named sat1 in reference 1a and AB161461-3.

The confusion has arisen because proteins of different sizes were produced by the cloned streptothricin resistance determinants of Tn1825 and Tn1826, and the genes were named sat1 and sat2 (15). Tn1826, in which the sat cassette is first in the cassette array, produces the shorter Sat-2 protein, which is sufficient to confer streptothricin resistance. In Tn1825 the sat cassette is preceded by another previously unnamed cassette (estX in Fig. 1), and the protein produced by this cassette is fused to Sat-2, forming the longer Sat-1 (15). Further sequences of cassette arrays equivalent to that of Tn1825 are now available (GenBank accession nos. AB161461 to AB161463[1]), and it appears that the protein fusion, which may increase the level of resistance, results from a mutation in the termination codon of the estX gene of Tn1825, leading to translational readthrough (Fig. 1A). Thus, sat2 is the only known cassette-associated streptothricin resistance gene, and sat1 is an estX-sat2 fusion.

Recently, the first cassette of Tn1825 has been found in class 1 integrons in GenBank accession nos. AY090896 (2) and AB121039 (1) (see Fig. 1B) but has been recorded as a sat cassette. In fact, the predicted 280-amino-acid polypeptide is not an acetyltransferase but is over 40% identical to several predicted proteins annotated as putative esterases or hydrolases of the /ß fold superfamily (8). These are encoded in the genomes of Mesorhizobium loti, Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis and in a Sinorhizobium meliloti plasmid (Fig. 2). We have therefore named the cassette gene estX for esterase X. EstX is 42% identical to a protein encoded in multidrug resistance plasmids from Escherichia coli (7) and over 30% identical to RdmC, DauP, and DnrP from Streptomyces species. The latter are methylesterases that catalyze one of the final "tailoring" reactions in the biosynthesis of rhodomycin, daunomycin, and daunorubicin, respectively (see reference 5). Whether EstX can inactivate any known antibiotic remains to be established.

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FIG. 2. Alignment of proteins related to estX. Residues conserved in all sequences are shown as black on white. The residues that constitute the catalytic triad of RdmC (Ser102, Asp248, and His276) are indicated by asterisks in the top line, and Ser102 lies at the center of the G-X-S-X-G motif typical of a serine hydrolase active site (5). The proteins are translated from the following sources: RdmC (GenBank accession no. U10405); DauP (L35154); SMa, product of SMa1327 from Sinorhizobium meliloti plasmid pSymA (AE007259); mll, product of mll3776 from Mesorhizobium loti (AP003002); BA, product of BA2738 from Bacillus anthracis (AE017032), representing all of the homologous (>88% identical) Bacillus proteins; pTZ, "RdmC homologue" from pTZ3721 (AB020531) and pTZ3723 (AB038654); EstX, cassette-encoded protein (AY090896). The completely conserved residues shown are also conserved in all additional homologues (not shown): DnrP (L40425), 94.6% identical to DauP; products of BCE2771 (AE017273) and BC2784 (AE017007) from B. cereus; EstA (AE017355) from B. thuringiensis; and all other variants of EstX (AB161461 to AB161463 and AB121039).

Reports of the presence of a sat cassette should be treated with caution, unless the standard sat2 cassette (GenBank accession no. X15995) is cited (e.g., in reference 10) or an accompanying database entry is available for confirmation (e.g., AB186119). An approximately 0.65-kb cassette array PCR product obtained with standard 5'-conserved segment (CS) and 3'-CS primers (e.g., references 16 and 17) is, however, consistent with the presence of the sat2 cassette.

 
Present address: Centre for Infectious Diseases and Microbiology, Westmead Hospital, Westmead, NSW 2145, Australia.

Top Letter References

 

1
1. Ahmed, A. M., and T. Shimamoto. 2004. A plasmid-encoded class 1 integron carrying sat, a putative phosphoserine phosphatase genes and aadA2 from enterotoxigenic Esherichia coli O159 isolated in Japan. FEMS Microbiol. Lett. 235:243-248.[CrossRef][Medline]
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2. Ahmed, A. M., H. Nakano, and T. Shimamoto. 2005. Molecular characterization of integrons in non-typhoid Salmonella serovars isolated in Japan: description of an unusual class 2 integron. J. Antimicrob. Chemother. 55:371-374.[Abstract/Free Full Text]
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3. DeLappe, N., F. O'Halloran, S. Fanning, G. Corbett-Feeney, T. Cheasty, and M. Cormican. 2003. Antimicrobial resistance and genetic diversity of Shigella sonnei isolates from western Ireland, an area of low incidence of infection. J. Clin. Microbiol. 41:1919-1924.[Abstract/Free Full Text]
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4. Hall, R. M., D. E. Brookes, and H. W. Stokes. 1991. Site-specific insertion of genes into integrons: role of the 59-base element and determination of the recombination cross-over point. Mol. Microbiol. 5:1941-1959.[Medline]
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5. Heim, U., E. Tietze, W. Weschke, H. Tschäpe, and U. Wobus. 1989. Nucleotide sequence of a plasmid born streptothricin-acetyl-transferase gene (sat-1). Nucleic Acids Res. 17:7103.[Free Full Text]
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6. Jansson, A., J. Niemi, P. Mäntsälä, and G. Schneider. 2003. Crystal structure of aclacinomycin methylesterase with bound product analogues: implications for anthracycline recognition and mechanism. J. Biol. Chem. 278:39006-39013.[Abstract/Free Full Text]
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8. Noguchi, N., J. Katayama, and M. Sasatsu. 2000. A transposon carrying the gene mphB for macrolide 2'-phosphotransferase II. FEMS Microbiol. Lett. 192:175-178.[Medline]
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9. Ollis, D. L., E. Cheah, M. Cygler, B. Dijkstra, F. Frolow, S. M. Franken, M. Harel, S. J. Remington, I. Silman, and J. Schrag. 1992. The /ß hydrolase fold. Protein Eng. 5:197-211.[Abstract/Free Full Text]
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10. Recchia, G. D., and R. M. Hall. 1995. Gene cassettes: a new class of mobile element. Microbiology 141:3015-3027.[Free Full Text]
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11. Sandvang, D., and F. M. Aarestrup. 2000. Characterization of aminoglycoside resistance genes and class 1 integrons in porcine and bovine gentamicin-resistant Escherichia coli. Microb. Drug Resist. 6:19-27.[Medline]
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12. Sundström, L., P. H. Roy, and O. Sköld. 1991. Site-specific insertion of three structural gene cassettes in transposon Tn7. J. Bacteriol. 173:3025-3028.[Abstract/Free Full Text]
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13. Tietze, E., and J. Brevet. 1990. Nucleotide sequence of the streptothricin-acetyl-transferase gene sat-2. Nucleic Acids Res. 18:1283.[Free Full Text]
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14. Tietze, E., and J. Brevet. 1991. The trimethoprim resistance transposon Tn7 contains a cryptic streptothricin resistance gene. Plasmid 25:217-220.[CrossRef][Medline]
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15. Tietze, E., J. Brevet, and H. Tschäpe. 1987. Relationships among the streptothricin resistance transposons Tn1825 and Tn1826 and the trimethoprim resistance transposon Tn7. Plasmid. 18:246-249.[CrossRef][Medline]
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16. Tietze, E., J. Brevet, H. Tschäpe, and W. Voigt. 1988. Cloning and preliminary characterization of the streptothricin resistance determinants of the transposons Tn1825 and Tn1826. J. Basic Microbiol. 28:129-136.[Medline]
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17. White, D. G., A. Datta, P. McDermott, S. Friedman, S. Qaiyumi, S. Ayers, L. English, S. McDermott, D. D. Wagner, and S. Zhao. 2003. Antimicrobial susceptibility and genetic relatedness of Salmonella serovars isolated from animal-derived dog treats in the USA J. Antimicrob. Chemother. 52:860-863.
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18. Zhao, S., S. Qaiyumi, S. Friedman, R. Singh, S. L. Foley, D. G. White, P. F. McDermott, T. Donkar, C. Bolin, S. Munro, E. J. Baron, and R. D. Walker. 2003. Characterization of Salmonella enterica serotype Newport isolated from humans and food animals. J. Clin. Microbiol. 41:5366-5371.[Abstract/Free Full Text]

						Sally R. Partridge Department of Biological Sciences Macquarie University Sydney, NSW 2109, Australia Ruth M. Hall* Department of Molecular and Microbial Biosciences The University of Sydney NSW 2006, Australia
						* Phone: (612) 9351 6014, Fax: (612) 9351 4571, E-mail: Ruth.Hall{at}mmb.usyd.edu.au

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Journal of Clinical Microbiology, August 2005, p. 4298-4300, Vol. 43, No. 8
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.8.4298-4300.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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