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Mercury resistance (rner) operons in enterobacteria J. L. Hobman', A. M. M. Essa and N. L. Brown School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B I 5 2TT, U.K.
clinically important isolates (reviewed in [3]). T h e first report of Hg' in bacteria was among Staphylococcus aureus in 1960, when Moore [4] found that mercuric salts used to disinfect catgut used in sutures would not inhibit growth of S . aureus isolated from surgical wounds, but would inhibit the growth of S. aureus from other sources. T h e co-transduction of both Hg' and penicillin resistance to sensitive S. aureus was reported 4 years later [5]. Hg'was first detected amongst antibioticresistant clinical enterobacteria in 1967 [6]. Subsequent research by other groups (reviewed in [3,7]) showed that the genetic linkage between antibiotic resistance and mercury resistance in enterobacteria had occurred prior to the late 1950s in Japan. A clinical strain of S h i g e h Jexneri, originally isolated at this time on the basis of its multiple antibiotic-resistance phenotype, was subsequently shown to contain a multi-resistance plasmid, N R l (R100). RlOO carries, amongst other genetic elements, transposon T n 2 1 (reviewed in [7]). T n 2 1 belongs to the T n 3 subgroup of the Class I1 transposable elements. I t is bounded by 38 bp inverted repeat DNA sequences, and encodes a mercury-resistance (rner) operon (merR T P C A D E ) , transposition functions (res, tnpR and t n p A ) ,two other open reading frames (ORFs), urf2 and t n p M , and resistances to streptomycin1 spectinomycin and sulphonamides [6]. These resistances and other ORFs are carried on an 11.O kb integron, a DNA element with the capacity to acquire antibiotic-resistance cassettes (Figure 1 ; reviewed in [3,7]). T n 2 1 and related elements are highly successful and are pandemic. They are found in clinical, environmental and commensal bacteria (reviewed in [7,8]). This ubiquity and variety of T n 2 1-related elements has in part been attributed by Grinsted et al. [8] to the acquisition of the integrase system by an ancestor of Tn21, enabling the rapid development of new types of transposon by gene acquisition. T h e most obvious advantage conferred to T n 2 1 and related transposons by the presence of the integron is the ability to acquire multiple antibiotic resistances. This capability, particularly in medically important bacteria, would have been a
Abstract Mercury resistance is found in many genera of bacteria. Common amongst enterobacteria are transposons related to Tn21, which is both mercuric ion- and streptomycin-/spectinomycinand sulphonamide-resistant. Other TnZ1-related transposons often have different antibiotic resistances compared with T n Z l , but share many non-antibiotic-resistance genes with it. In this article we discuss possible mechanisms for the evolution of T n 2 1 and related genetic elements.
Introduction Mercury and mercury-containing compounds, such as mercuric chloride (HgCl,), mercurous chloride (Hg,Cl,) and mercuric sulphide (HgS), have been used as medicines for the treatment of many human ailments, ranging from constipation to skin disease, for many hundreds of years [l]. Mercury was perhaps most famously (though not necessarily efficaciously) used for the treatment of syphilis prior to the synthesis and use of the first selective-toxicity chemotherapeutic agent, arsphenamine (' Salvarsan '), which was discovered by Paul Ehrlich and Sahachiro Hata in 1907. T h e production of penicillin in large quantities during the 1940s and its highly effective treatment of syphilis superceded both mercurials and ' Salvarsan '. However, mercury-containing compounds such as mercuric chloride, methylmercury, phenylmercury, merbromin and thimerosal were still in widespread use in hospitals until at least the early 1970s in the U.K., U.S.A., Germany and Japan [2], and later in other countries. Indeed, mercurials are still in use today as preservatives in vaccines and some healthcare products and in dental amalgams.
Mercury resistance (Hg') in bacteria, and Tn2 I Hg' is found in many genera of bacteria, and has been found in environmental, commensal and Key words: composite transposon, pre-antibiotic era, Tn2 I , Abbreviations used: Hg', mercury resistance; ORF, open reading frame. 'To whom correspondence should be addressed (e-mail J.L.Hobman(a'bham.ac.uk).
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major factor in the success of this transposable element. Although the use of mercury-containing antimicrobials has been largely discontinued, except for specific limited uses noted earlier in this review, there is evidence to show that mercuryresistant bacteria were still being found in hospital bacterial populations, albeit at lower levels than before, several years after the discontinuation of mercurial usage in the hospitals [2]. More recent surveys of bacterial populations have shown that mer operons, particularly those related to Tn21, are present in enterobacteria from both humans and animals, and are associated with integrons and
multiple antibiotic resistances [7,9]. Zuhlsdorf and Weidemann [lo] found that 8 04 of 807 unselected Gram-negative clinical isolates collected in 1986 from hospitals in Austria, Switzerland and Germany hybridized to five Tn21 -related probes, including the I n t l l gene from the Tn21 integron In2 (see Figure 1 ) . A further 11 O:, of the isolates hybridized to between one and four Tn21-specific probes. Bass et al. [9] examined the incidence of integrons in avian pathogenic Escherichia coli isolated from diseased poultry, and found that 6344 of these isolates contained the class Iintegron-associated i n t l l and qacAEl genes, and SOo/, of the isolates contained the genes i n t l l ,
Figure I Genetic maps of T n 2 l [6] and Tn5075 (A. M. M. Essa, D. J. Julian, S. P. Kidd, N. L. Brown and J. L. Hobman, unpublished work) The black boxes represent the Tn2 I 38 bp inverted repeats The gene functions for rnerRJPCADE are reviewed in [3,7],the transposition gene functions f o r m , tnpR and tnpA are reviewed in [7,8] The possiblerole oftnpM is also discussed in [7] Tn5075 IS flanked by the insertion sequence elements lS5075L and IS5075R, which are 9953% identical to each other lntegron genes are rntll, the integrase gene, attl, insertion site aadA I, aminoglycoside adenylyltransferase gene, qocEA I, resistance to quaternary ammonium compounds and sull, sulphonamide resistance The integron contains an ORF of unknown function (orf5) and two IS elements, IS I326 and IS 1353, as well as integron transposition genes tniA and the partially deleted tnr6 [7] IR, inverted repeat
In2 IR
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aadA1, qacAEl and merA, from a Tn21-like
insertion sequence elements (IS5075L and IS5075R; 99.53O; identical to each other) that were 91°/, identical to IS4321L and IS4321R that flank the 'cryptic' transposable element Tn4321 from plasmid R751. However, the highest identity to the Tn5075 mer genes, transposition functions, IS5075L and IS5075R was the mercury-resistance transposon identified in the plasmid p H C M l .This plasmid D N A sequence comes from the recently published genome sequence of the multiply drug-resistant S. enterica serovar Typhi strain CT18 [14], which had been isolated in the Mekong Delta region of Vietnam in 1993 from a juvenile human female. T h e T n 2 1 subgroup of transposable elements is successful because they are often carried on conjugative plasmids, can transpose and have the ability to acquire antibiotic resistances. Our D N A sequence data (A. M. M. Essa, D. J. Julian, S. P. Kidd, N. L. Brown and J. L. Hobman, unpublished work) and that of Parkhill et al. [14] suggest that T n 2 1-related elements may be components of composite transposons (Figure l ) , where the T n 2 1 transposon is flanked at both the mer and transposon ends by virtually identical IS elements. As yet it is not known how widespread these elements are amongst Tn21-type mer resistances, but there is some evidence to suggest that they are not uncommon ( A . M . M . Essa, D. J. Julian, S. P. Kidd, N . L. Brown and J . L. Hobman, unpublished work).
transposon (Figure 1). During investigations on the incidence of phenotypically expressed antibiotic resistance in E . coli from the gut flora of healthy swine, Sunde and S ~ r u m[l 11 found that they could identify a TnZ1-related transposon from one of these isolates by PCR amplification using primers designed for T n 2 I - or integronspecific genes. T h e transposon was carried on a self-transmissible plasmid, pRtl7, which could transfer to pathogenic E . coli, Salmonella enterica serovar Typhimurium and normal pig gut flora E . coli under laboratory conditions.
Hg' in 'pre-antibiotic era' bacteria Logically, one would expect that Hg' is a genotype that bacteria have carried for a considerable period of time, given that mercury has been liberated into the biosphere by geological processes for millennia, and has been used as an anti-microbial or medicine for many hundreds of years. T h e Murray collection of enterobacteria collected from largely clinical sources by E. D. G. Murray between 1917 and 1954 contains Hg' bacteria. When the sealed bacterial cultures that formed the Murray collection were opened in 1980, they were tested for resistance to a range of antibiotics and heavy metals. There were very few bacteria from the collection that were antibiotic-resistant (2/433 were penicillin-resistant, 9/43 3 were tetracyclineresistant), larger numbers that were resistant to copper, tellurite and arsenic, and three strains were identified that were Hg' [12]. We recently confirmed that none of the three Hg' bacteria were antibiotic-resistant, and determined the D N A sequences of the mer operons from all three of them, and flanking D N A from one of them (A. M. M . Essa, D. J. Julian, S. P. Kidd, N . L. Brown and J. L. Hobman, unpublished work). D N A sequence analysis has shown that two of the Murray collection isolates contained mer operons that were 99 O 0 identical to mer transposons that have been previously isolated from environmental samples (Tn5053 and Tn5036; reviewed in [13]). T h e third isolate (from Murray strain E . coli M634, which was originally isolated in or before 1931) was 9900 identical to Tn21, across the mer genes and transposition functions, but no integron was present. Instead of urf2 and tnpM, a single O R F , urf 2M, was present (Figure 1). T h e other major difference between T n 2 1 and Tn5075 was that Tn5075 was flanked by
References I
Goldwater, L. J. ( I 972) Mercury: a History of Quicksilver, York Press, Baltimore 2 Porter, F. D., Silver, S., Ong. C. and Nakahara, H. (1982) Antimicrob. Agents Chernother. 22, 852-858 3 Hobman, J. L. and Brown, N. L. ( I 997) in Metal Ions in Biological Systems (Sigel, A. and Sigel, H., eds), pp. 527-568, Marcel Dekker, New York 4 Moore, B. ( 1960) Lancet 2,453458 5 Richmond, M. H. and John,M. ( I 964) Nature (London) 202, I 360- I 36 I 6 Smith, D. H. (1967) Science 156, I I 14-1 I I6 7 Liebert, C. A., Hall, R. M. and Summers, A. 0. (1999) Microbiol. Mol. Biol. Rev. 63,507-522 8 Grinsted, J., de la Cruz, F. and Schmitt, R. ( 1990) Plasmid
26, 163- I89 9
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Bass, L., Liebert, C. A., Lee, M. D.. Summers, A. O., White, D. G., Thayer, S. G. and Maurer, J, G. ( I 999) Antirnicrob. Agents Chernother. 43,2925-2929 Zuhlsdotf M. T. and Weidernann, B. ( 1992) Antimicrob. Agents Chernother. 36,I9 15- I92 I Sunde, M. and S0rurn, H. (200 I ) Microb. Drug Resist. 7,
191-196
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I2 Hughes, V. M. and Datta, N. ( 1983) Nature (London) 302, I3
14 Parkhill,J., Dougan, G.,James,K. D.,Thornson, N. R., Pickard, D.,Wain, J., Churcher, C., Mungall, K. L., Bentley, S.D., Holden, M. T. G.et al. (200 I ) Nature (London) 4 13,
725-726 Nikiforov, V. G., Bass, I. A,, Bogdanova, E. S., Gorlenko, Zh. M., Kalyaeva, E. S., Kopteva, A. V., Lomovskaya, 0 .L., Minakhin, L S , Minakhina, S V , Mindlin, S Z et al. ( I 999) Molekulyama Biologiya 33, 44-50
848-852 Received I March 2002
Molecular evidence for the role of a ferric reductase in iron transport A. T. McKie*', G. 0.Latunde-Dada*, S. Miret*, J. A. McGregor", G. J. Anderson+, C. D. Vulpef, J. M. Wrigglesworth* and R. J. Simpson" +Division of Life Sciences, King's College London, Franklin-Wilkins Building, I50 Stamford Street, London SE I 9NN, U.K., tQueensland lnstitue of Medical Research, P.O. Royal Brisbane Hospital, Hereston, Queensland 4029, Australia, and $Department of Nutritional Sciences, University of California, Berkeley, CA, U.S.A.
Abstract
uptake pathway for iron in the intestine involved a divalent cation transporter [Nramp2, divalentcation transporter 1 ( D C T l ) or divalent-metal transporter 11 [5-71 strongly suggested that ferrous iron was the preferred form of iron absorbed and gave further impetus to the search for a intestinal ferric reductase.
Duodenal cytochrome b (Dcytb) is a haem protein similar to the cytochrome b561 protein family. Dcytb is highly expressed in duodenal brushborder membrane and is implicated in dietary iron absorption by reducing dietary ferric iron to the ferrous form for transport via Nramp2/DCT1 (divalent-cation transporter 1)/D M T 1 (divalent metal-transporter 1). T h e protein is expressed in other tissues and may account for ferric reductase activity at other sites in the body.
Characterization of a duodenal ferric reductase activity T h e presence of such a surface ferric reductase activity in the duodenal mucosa was first described by the Iron Metabolism Group at Kings College London some time before the discovery of D C T l [8,8a]. In that study it was shown that a ferric reductase activity could be measured at the surface of the duodenal mucosa. T h e reductase activity was strongly stimulated by hypoxia and iron deficiency, both of which stimulate increased absorption of dietary iron. In addition it was found that the activity was highest in the duodenum and lowest in the ileum, compatible with the profile of iron absorption along the gut, which is highest in duodenum and lowest in ileum. Attempts to purify the protein responsible for this activity provided evidence that the activity was associated with a b-type haem protein that was immunologically distinct from the NADPH oxidase GP91 -Phox [9]. T h e protein was never successfully purified using biochemical techniques as the haem was lost early in the purification. Given the reasonable homology between GP91 -phox and the yeast and plant ferric reductase proteins one might have expected the duodenal ferric reductase to be a homologous protein. However, degenerate PCR approaches using regions of high homology between the yeast F R E genes and GP91 -phox were
Background Crane and his colleagues [l] suggested the role of a plasma-membrane reductase in iron uptake and provided evidence for diferric transferrin reductase activity in microsomes isolated from liver. Since this time a number of plasma-membrane redox proteins that specifically reduce ferric iron chelates have been identified at the molecular level in yeast as well as plant roots [2-4]. This provided strong evidence for the requirement of a reduction step prior to uptake of iron by yeast and plant cells. T h e ferric reductase proteins described in yeast and plant systems belong to the same family as GP91 -phox, a major component of the respiratory burst oxidase found in mammals. In mammals dietary iron is absorbed in the proximal intestine, mainly in the duodenum and it was known for many years that ferrous iron is better absorbed than ferric. T h e discovery in 1997 that a major
Key words: duodenal cytochrorne b, intestine, neutrophil. Abbreviations used: Dcytb, duodenal cytochrome b ; DCT, divalent-cation transporter. 'To whom correspondence should be addressed (e-mail
[email protected]).
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