ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2003, p. 1115–1119 0066-4804/03/$08.00ϩ0 DOI: 10.1128/AAC.47.3.1115–1119.2003 Copyright 2003, American Society for Microbiology. All Rights Reserved.
Mercury Resistance Determinants Related to Tn21, Tn1696, and Tn5053 in Enterobacteria from the Preantibiotic Era Ashraf M. M. Essa, Daniel J. Julian, Stephen P. Kidd, Nigel L. Brown, and Jon L. Hobman* School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom Received 9 July 2002/Returned for modification 4 October 2002/Accepted 2 December 2002 Three mer transposons from the Murray collection of preantibiotic enterobacteria show >99% sequence
identity to current isolates. Tn5073 is most closely related to Tn5036 and Tn1696, and Tn5074 is most closely
related to Tn5053. Tn5075 is most closely related to Tn21 but lacks integron In2 and is flanked by insertion
Tn21 encodes mercuric ion resistance (Hgr) and contains the PCR was performed with part of the mer operon from each class I integron In2, encoding resistance to sulfonamides (sul) plasmid as described elsewhere (3). PCR products purified by and streptomycin-spectinomycin (aadA) (9, 18). It is carried by using a QIAquick PCR purification kit (Qiagen, Ltd., Crawley, the conjugative plasmid NR1 (R100), which was isolated in United Kingdom) were sequenced with the Big Dye terminator Japan in the 1950s (21). More recently, Tn21 (18) and other cycle sequencing kit (PE Applied Biosystems, Warrington, Tn21-like transposons carrying integron-associated antibiotic United Kingdom) and an Applied Biosystems 3700 sequencer, resistance (7) have been detected in Escherichia coli from ag- according to the manufacturer’s protocols. Further sequence ricultural (1, 28) and nonclinical sources (19), as well as from analysis was performed by using primers designed from the mercury amalgam-exposed, gram-negative gut bacteria (16, 17, sequences so obtained and from merA gene primers (5) (the 33), clinical bacterial isolates (13, 31, 36), and intercontinental primers used are described at http://www.biosciences.bham.ac plasmids carried by clinical isolates (10, 22).
.uk/labs/brown/mer_primers.htm). The transposon terminal in- It is now thought that Tn21 evolved by the insertion of an verted repeat DNA and flanking sequences were amplified by In2 ancestor (lacking IS1353) into the urf2M gene of a hypo- inverse PCR (26). Genetic maps of the sequenced mer operons thetical mercury resistance transposon, Tn21⌬ (18) (also called are shown in Fig. 1A. DNA alignments and analysis were TnX [25]), probably catalyzed by transposition proteins en- performed with the University of Wisconsin Genetics Com- coded in trans (2, 18). A similar event led to the formation of puter Group version 9.0 suite of programs at the University of Tn1696 from plasmid R1033, where In4 inserted at the res site of a Tn5036-like mer transposon (25).
The three mer operons that we sequenced represent differ- Hughes and Datta identified three Hgr bacterial strains, ent lineages and are not closely related to each other, but they M426, M567, and M634, from a total of 433 strains from the are closely related to mer transposons isolated since the 1950s.
Murray collection of preantibiotic era enterobacteria (11). In Table 2 shows the percent identities between the sequences of this study, we sequenced the mer operons from M426, M567, the genes from Tn5073 (Klebsiella pneumoniae M426), Tn5074 and M643 in order to investigate the relationships between Hgr (Morganella morganii M567), Tn5075 (E. coli M634) and pub- sequences from clinical bacteria that had been isolated before antibiotics came into widespread use and present-day Hgr se- The sequenced merRTPCAD genes (3,788 bp) of the Tn5073 mer operon (Fig. 1A) had the highest identity at the DNA level The plasmids, bacterial strains, antimicrobial resistance of to those from Tn5036 (35), a Tn5036-like mer transposon from these strains, and the 16S ribosomal DNA sequence identifi- Salmonella enterica serovar Typhi CT18 plasmid pHCM1 (24), cations of the three Hgr strains from this study (determined as and to the sequenced merAD genes of Tn1696, which carries previously described [32]) are shown in Table 1. All bacteria In4 (25) (Table 2). In comparison to the sequence encoded by were grown at 37°C in Luria broth (LB) or on LB agar (27).
Tn5036, there were two amino acid differences in the sequence Hgr plasmids from M426, M567, and M634 were mated with E. encoded by Tn5073: MerR A173V and MerA V2503A. The coli TG2 (20), and Hgr transconjugants were grown overnight Tn5073 merT gene carries five GTCTGAACCACAAAA du- on LB agar plates containing tetracycline (15 ␮g/ml) and plications at the 5Ј end (Fig. 1A). Multiple repeats of this sequence have also been observed in enterobacterial mercury The E. coli TG2 Hgr transconjugants from each of the three resistance determinants from primates (16) and in Tn5036, Murray strains contained an ϳ60-kb plasmid that conferred Hgr. Plasmid DNA was isolated by standard methods (27), and The sequenced merRTPFADE genes (3,647 bp) from the Tn5074 mer operon (Fig. 1A) had the highest identity at the DNA level to those from Tn5053 (14) and pMER327/419 (8) * Corresponding author. Mailing address: School of Biosciences, (Table 2). In comparison to the sequence encoded by Tn5053, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. Phone: 44 (0)121 414 6558. Fax: 44 (0)121 414 5925.
there were four amino acid differences in the sequence en- coded by Tn5074: MerR A1193S and K1213Q and MerA TABLE 1. Bacterial strains and plasmids, antibiotic resistance phenotypes, 16S rRNA sequences, and sources of the strains used in this study K-12 lac-prosrl-recA 306::Tn10 proAϩ Bϩ laclq lacZ⌬M15] a Tcr, tetracycline resistance; Rifr, rifampicin resistance; Apr, ampicillin resistance; Kmr, kanamycin resistance.
b NCTC, National Collection of Type Cultures, Colindale, London, United Kingdom.
V2323A and S2893G. The Tn5053 type mer operon was first Tn21, resulting in a frameshift and a 314-amino-acid protein, isolated in environmental bacteria (8, 14) and has also been rather than the 328-amino-acid protein predicted previously found in the fecal flora of primates and humans exposed to The insertion (IS) elements IS5075L and IS5075R (which In total, 11,298 bp of Tn5075 were sequenced. The 3,962-bp are 1,351 bp and Ͼ99.5% identical to each other) flank mer operon from Tn5075 carrying merRTPCADE has the high- Tn5075, forming a composite transposon (Fig. 1A). IS5075L est identity at the DNA level to the equivalent regions of Tn21 and IS5075R belong to the IS110 family and are Ͼ99.6% (18) and the Tn21-like mer transposon from S. enterica serovar identical to the IS elements flanking the Tn21-like mer trans- Typhi CT18 plasmid pHCM1 (24) (Fig. 1A) (Table 2). The poson from plasmid pHCM1 (Fig. 1A). IS5075L and IS5075R Tn5075 res site and the transposition genes (3,529 bp) tnpR and are between 91 and 93% identical to IS element sequences tnpA again had the highest identity to those of Tn21. In com- flanking other mercury resistance-encoding genes from gram- parison to the sequence encoded by Tn21, there were three amino acid differences in the sequence encoded by Tn5075: MerA, Q5583H; TnpR, T1653A; and TnpA R4553A. Most Transposition of the Hgr phenotype was determined by a importantly, Tn5075 did not carry the integron In2, which in mate-out assay (15) with E. coli TG2 carrying plasmid RK2, Tn21 is located between urf2 and the putative transposition conjugated with Hgr plasmids from M426, M567, or M634, into gene tnpM (12) (Fig. 1A). Instead, in Tn5075, there is a single E. coli KH802. Transconjugants were selected on LB agar 945-bp open reading frame (urf2M) of unknown function plates containing HgCl2 (20 ␮g/ml) and carbenicillin (200 ␮g/ rather than the predicted 987-bp hypothetical urf2M from ml), and the donor strain was counterselected with rifampin Tn21⌬ proposed by Liebert et al. (18). Tn5075 urf2M is 97.1% (50 ␮g/ml). Purified plasmid RK2 DNA from the transconju- identical at the DNA level to the hypothetical urf2M that gants was analyzed for transposon insertion by PstI digestion Liebert et al. (18) postulated to exist in the ancestor of Tn21.
and gel electrophoresis (27). Tn5073 transposed into plasmid The reason for this lower percent identity is a 23-bp deletion in RK2 at a frequency of 6.3 ϫ 10Ϫ5 per donor cell. Tn5074 the Tn5075 urf2M sequence compared to the tnpM sequence in transposed into RK2 at a frequency of 2.9 ϫ 10Ϫ4 per donor FIG. 1. (A) Genetic maps of the sequenced regions of Tn5073, Tn5074, Tn5075, Tn21 (18), and the Tn21-like transposon from pHCM1 (24).
Vertical black bars represent the 10- to 12-bp inverted repeat sequences flanking the IS elements. Vertical unfilled bars represent the 38bp inverted repeat sequences at the ends of the class II transposition module. The transposition (tnp) regions consist of the transposase gene (tnpA), the resolvase gene (tnpR), the putative transposon regulator (tnpM), and the resolution site, res. urf2M in Tn5075 is of unknown function. The points at which In2 or antibiotic resistance-encoding IS elements have inserted into Tn21 and pHCM1 are marked with black arrows. The mercury resistance-encoding operons consist of the regulatory genes merR and merD, the mercury transport genes merT, merP, and merC or merF, the putative transporter merE, and the gene encoding mercuric reductase, merA. urfI and urf2 are of unknown function. All mer genes are marked in the figure with a single letter. The position of the multiple DNA sequence repeats found in merT from Tn5073 are indicated (). (B) Postulated evolutionary pathway for Tn5075, Tn21, and the Tn21-like transposon in pHCM1. An ancestral mer transposon (a) could acquire either IS5075L and IS5075R to become Tn5075 (b) or an integron related to In2, resulting in the formation of Tn21 (c). Gene insertions and deletions in In2 would lead to Tn21 variants (d), or Tn21 could have acquired IS elements, leading to the formation of a precursor to the Tn21-like transposon in pHCM1 (e). Deletions of transposition genes and partial deletion of In2, followed by insertion of antibiotic resistance gene-carrying IS elements, would result in the formation of the Tn21-like transposon in pHCM1 (f).
TABLE 2. Comparison of DNA sequence identities between Tn5073, Tn5074, Tn5075, and published DNA sequences from other transposons % Identity to mer transposon (genes)a Ͼ99.9 Tn5036-like transposon pHCM1 Ͼ99.9 Tn21-like transposon from pHCM1 (merRTPCADE) Ͼ99.8 Tn21 (res tnpA tnpR) Ͼ97.1 hypothetical urf2m from Tn21⌬ Ͼ99.6 Tn21-like transposon from pHCM1 (IS elements) Ͼ91.0 IS4321L, IS4321R, from R751 cell. We found no transposition of Tn5075 in our assays, which are as follows: Tn5073 (strain M426), AF461013; Tn5074 (strain could detect frequencies of Ͼ10Ϫ7.
M567), AF461012; and Tn5075 (strain M634), AF457211. The In conclusion, the internal genetic structure of Tn5075 is 16S rRNA gene sequence accession number for strain M567 is consistent with the recently proposed structure of Tn21⌬ (Fig. 1B) (2, 18), and Tn5073 is closely related to Tn5036 and Tn1696 (25). The sequence data from Tn5075 support We thank Julian Davies, Didier Mazel, and Barry Holmes for their the hypothesis that Tn21 evolved from an Hgr transposon sim- help. We also thank Chris Thomas and Peter Strike for the gifts of ilar to Tn5075, rather than from Tn2613 (29). Tn1696 and Tn21 plasmids and strains and Gennady Kholodii and anonymous referees for suggested improvements to the paper.
represent independent lineages of mer transposons that have This work was supported by a postgraduate research scholarship to acquired integrons (25). The dates of isolation of Tn5073 A.M.E. from the Egyptian Ministry of Higher Education, and the work (1940) and Tn5075 (1931) and the close relationship of these was partially supported by a BBSRC grant 6/G07943 to N.L.B. and a transposons to other lineages (Tn5073 merAD genes are Ͼ99.9% BBSRC/Wellcome Trust Joint Infrastructure Fund Grant (6/JIF13209).
identical to those of Tn1696; Tn5075 is Ͼ99.6% identical to Support in bioinformatics was from MRC grant G.4600017.
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