FEMS Microbiology Letters 118 (1994) 113-120 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier

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FEMSLE 05923

Accumulation and intracellular fate o f tellurite in tellurite-resistant Escherichia coli: A model for the mechanism of resistance

Gare th Lloyd-Jones .,a, A. Mark Osborn a, Donald A. Ritchie a, Peter Strike ~, Jon L. Hobman a,b, Nigel L. Brown b and Duncan A. Rouch c

a Department of Genetics and Microbiology, Donnan Laboratories, The University of Liverpoo~ Liverpool L69 3BX, UK, and b School of Biological Sciences, The University of Birmingham, Birmingham B15 2T'I~, UK

(Received 27 January 1994; accepted 15 February 1994)

Abstract: The tellurite accumulation properties of three Escherichia coli strains containing different tellurium-resistance determi- nants of Gram-negative origin, from plasmids pMER610, pHH1508a and RK2, were compared. In all three cases membrane-associ- ated tellurium crystallization was observed, and neither reduced uptake nor increased export contributed to the resistance. Specific membrane-proximal reduction is proposed as the mechanism of resistance to teUurite coded by all three determinants, despite their lack of sequence homology.

Key words: Tellurite resistance; pMER610; RK2; pHH1508a; Escherichia coli

Introduction

The toxicity of tellurite to microorganisms is well established, and is believed to be a conse- quence of its properties as a strong oxidant [1]. Plasmid-mediated tellurite resistance (Te r) deter- minants are relatively easily isolated [1-3], and three of these of Gram-negative origin, from the plasmids pMER610, pHH1508a and RK2, have been characterized by nucleotide sequence deter-

* Corresponding author.Present address: Landcare Research Limited, Private Bag 3123, Hamilton, New Zealand.

mination [4-6] and their properties have been reviewed [7]. The sequence data have shown the three determinants to be non-homologous, with no statistically significant regions of sequence similarity.

During growth of tellurite-resistant Es- cherichia coli strains, tellurite concentrations have been shown to decrease in culture supernatants [5,7-10]. Tellufite uptake in E. coli appears to occur at least partly via the cellular phosphate (Pi) transport systems [11]. Following uptake and accumulation, reduced tellurite, in the form of crystalline deposits of elemental tellurium, has been observed in Te r strains [8,9,12]. These de-

SSDI 0378-1097(94)00068-3

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posits, however, are also seen in Te s strains of E. coli [9] thus casting doubt on a specific role for Te crystallization in resistance.

In this paper we compare the properties of strains harbouring the three sequenced Te r de- terminants from plasmids pMER610, pHH1508a and RK2. Studies of tellurite transport and the distribution of tellurium crystals in these strains have allowed us to propose a model in which Te crystallization is essential to the mechanism of resistance in the three different determinants.

Materials and Methods

Bacterial strains and growth medium Escherichia coli strain ABl157 ( S m r, F - ) was

used as the host strain for the Te r plasmids detailed in Table 1. Cells were grown at 37°C in K medium (M9 medium [13] containing 0.25% w/v casamino acids and 0.8% w/v glucose). An- tibiotics were used at the following concentra- tions: 200 /~g ml -~ streptomycin; 25 /~g ml -~ chloramphenicol, 50/~g ml- t ampicillin.

Electron microscopy Washed cell pellets of each strain, challenged

with 50/~M tellurite for 30 min, were fixed using gluteraldehyde and osmium tetroxide. Samples were then dehydrated successively in 30, 50, 70,

90, 100, 100, and 100% ethanol, followed by embedding in epoxy resin. Sectioned material was left unstained and examined by transmission elec- tron microscopy.

Tellurite transport Radioactive tellurium dioxide, 127TEO2, w a s

obtained from the Universities Research Reactor, Risley, Warrington, UK (maximum activity 15 /zCi mg-1; half life 9.4 h), and was dissolved in 100 /~M NaOH to give a stock solution of 127TeO32-/TeO 2-. Cells were harvested by cen- trifugation at 10 000 × g for 10 min, washed, and resuspended in M9 medium at 100 mg wet weight ml-t. Cell densities during transport assays were typically 0.25-0.5 mg dry weight m1-1. Everted vesicles were prepared as previously described [14]. Transport assays were conducted at 37°C with aeration by shaking in M9 medium supple- mented with 0.8% w/ v glucose; 0.2-ml samples were withdrawn at timed intervals; the cells were collected by filtration and washed with M9 medium, and the incorporated radioactivity was determined. For direct efflux studies, cells were preincubated with 50/~M labelled tellurite for 30 min at 4°C, washed and resuspended in M9 medium. After taking the zero time sample, cells were transferred to 37°C, and samples were with- drawn at timed intervals. Uptake of labelled tel- lurite into everted vesicles was carried out as

Table 1

Plasmids used in this study

Plasmid a Resistance Source b MIC Copy No. Reference markers (~M)

pUC8 Ap r - N'D 500-700 [21] pLV59 Cm - N D 20- 30 [22] pMER610 Hg, Te - 1000 low [4] RK2 Ap, Kin, Tc, Te - 2000 4 - 7 [3] pHH1508a Sin, Su, Tp, Te - 2000 low [23] pMJ600 Cm, Te Te r pLV59 clone from pMER610 ¢ 1000 20 - 30 [4] pDT1364 Ap, Te Te r pUC8 clone from pHH1508a 500 500-700 [23] pDT1558 Ap, Te Te r pUC8 clone from RK2Te8ur 1000 500-700 [23]

a Resistance determinants: Ap = ampicillin, Cm = chloramphenicol, Hg = mercury, Km ffi kanamycin, Sm = streptomycin, Su = sulphonamide, Tc = tetracycline, Te ffi tellurite.

b Last LA plate, supplemented with increasing concentrations of tellurite, showing growth. ND ~ not determined. c The Te r determinant f rom pMER610 cannot be subcloned into pUC vectors due to instability of resulting subclones.

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above, but in the presence of 5 mM NADH, 5 mM NADPH or 5 mM ATP(Mg2+), or in the absence of any co-factors.

Results

Localization of tellurium deposition Although tellurium deposition in strains con-

taining the three determinants has been exam- ined previously, this was performed under differ- ent conditions in each case [8,9,12] thus denying proper comparison. Here the conditions were standardized, with the three determinants, as

present on pMER610, pHH1508a and RK2, be- ing carried by the same host strain, ABl157, grown under the same conditions and prepared in similar fashion for electron microscopy. The re- suiting electron micrographs showed that for all three Te r strains the presence of a number of black crystals was regularly observed in cells (Fig. la,b,c). Such electron-dense crystals have been shown previously to consist of elemental tel- lurium [8, 9, 12]. Although some tellurium crys- tals appeared elsewhere in the cytoplasm of each strain, there was a marked tendency towards local deposition of crystals near the cytoplasmic mem- brane. A similar result was observed for the plas-

Fig. 1. Scanning transmission electron micmgraphs (upper magnification) of strains challenged with 50 #M tellurite (a) ABl157(pMER610), (b) AB1157(RK2), (e) ABl157(pHH1508a), and (d) ABl157. Scale bar = 0.5/zrn.

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midless host Te ~ strain ABl157 (Fig. ld), al- though in this case the cells are killed by this concentration of tellurite.

Tellurite uptake in M9 medium supplemented with glucose

In further work subclones of the three Te r determinants were used (Table 1); pMJ600 (from pMER610), pDT1364 (from pHH1508a) and pDT1558 (from RK2). Cellular uptake of tellurite in M9 medium in the absence of glucose occurred at low rates, with rates increasing in the presence of glucose. All assays were therefore carried out in M9 medium supplemented with 0.8% w/v glucose. Tellurite was taken up by all Te r plas- mid-containing strains at rates equal to or greater than the isogenic plasmidless host strain E. coli ABl157 (Fig. 2).

Tellurite uptake at varied concentrations of teUurite When the uptake of tellurite by the Te s strain

ABl157, and its Te r plasmid-containing deriva- tives, was measured in the presence of 10, 100, 500 and 1000 /tM tellurite (Fig. 3), the uptake

35000

30000 / /

25000 = ._~

20000

E 15000

{3.

o 10000

5000

O I I I I I I

0 30 60 90 120 150

t(min)

Fig. 2. Accumulation with time of teilurite from 100 /~M t27TeO2-/TeO2- by: ABl157(pMJ600) (e), ABl157(pDT 1367) (v ) , ABl157(pDT1558) ( v ) and the Te s host strain E. coli ABl157 (o), in M9 medium, supplemented with glucose, at 37°C with aeration. Each point is the mean of three uptake

assays.

30000 i i i i i

v

25000

5000

Ol l l l l 1 0 200 4-00 600 800 1000

[ T e l l u r i t e ] u M

Fig. 3. Accumulation of teilurite as a function of initial tellurite concentration by: AB1157(pMJ600) (e), ABl157 (pDT1364) (v ) , AB1157(pDT1558) ( v ) and the Te' host strain E. coil ABl157 (O), in M9 medium supplemented with glucose, at 37°C with aeration. Each point is the mean of

three uptake assays.

E 20000

15000

E ~- 10000

rates in cells containing pDT1558 were three-fold higher than those in cells containing pMJ600 or pDT1364 and than those in the Te s strain ABl157, which did not differ significantly from one another.

No decreased uptake of tellurite due to any of the three Te' determinants could be detected relative to the Te s host strain (Fig. 3). In addi- tion, no efflux could be detected from the strains preloaded with tellurite; measurements of la- belled tellurite retention after 30 min showed values of 97% to 102% retention in the resistant and sensitive strains. Uptake of tellurite was simi- lar in everted vesicles prepared from each of the resistant strains and from the sensitive parental strain, thus providing no evidence for a mecha- nism involving increased efflux of tellurite in the resistant strains (data not shown).

Discussion

The rates of tellurite accumulation due to the three Te r determinants, relative to the isogenic

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host strain, show that a decreased uptake of tellurite does not contribute to the resistance mechanisms encoded by any of these plasmids. Reduced uptake has been found to contribute to other metal oxyanion resistance mechanisms, e.g. chromate resistance in Enterobacter cloacae [15]. Similarly the lack of any detectable export mech- anisms may rule out direct export of tellurite as part of the resistance mechanisms. Again, other metal resistances have export mechanisms which have been readily demonstrated using the proto- cols employed here e.g. cadmium, zinc and cobalt extrusion in Alcaligenes eutrophus CH34 [16] or arsenate, arsenite and antimonite export in E. coli or in Staphylococcus aureus [17,18].

Tellurite accumulation rates in strains contain- ing pMJ600 and pDT1364 and that of the iso- genie host E. coli ABl157 did not differ signifi- cantly; however, the rates were three-fold greater for cells containing pDT1558 (Figs. 2 and 3). This difference may be due to greater uptake occur- ring in a strain with the highest degree of protec- tion from tellurite-induced cellular damage, thus maintaining total cellular viability over a longer period (pDT1558-containing cells have a higher MIC than those containing pDT1364, and pDT1558 has a higher copy number than pMJ600 (Table 1)). The striking 200-fold difference in the removal (or decrease in bioavailability) of tellu- rite from growth medium due to pDT1364-con- taining cells over those containing pDT1558 ob- served by Walter et al. [10], was surprisingiy not supported by these tellurite accumulation assays. These results appear to rule out both reduced uptake and export of tellurite as components of the resistance mechanisms encoded by the three Te r determinants.

Tellurium crystallization upon exposure to 50 /xM tellurite occurred in a similar pattern in strains containing the three Te r determinants. Crystal deposition was also observed in the Te S host strain, confirming a previous observation [1,9], and appearing to rule out a role in resis- tance for the reduction of tellurite to non-toxic tellurium. However, upon incubation in media containing 50 t~M tellurite resistant strains retain full growth potential, while the sensitive strain fails to grow and loses viability (not shown). These

data, together with the crystallization data, sug- gest that the resistant and sensitive cells handle tellurite differently. Tellurite is strongly oxidising, and its toxicity in the sensitive strain would be expected to occur through non-specific oxidation of cellular materials following its uptake into the cytoplasm. Such oxidation would result in con- comitant reduction of the tellurite, and the insol- uble tellurium produced by complete reduction would form crystals. Consistent with this notion is the observed pattern of Te deposition in ABl157 cells. The proximity of Te crystals to the cytoplas- mic membrane can be explained on the basis that the cell constituents closest to the sites of tellu- rite uptake in the membrane have the highest probability of reacting with the tellurite.

A resistance mechanism must protect the cell cytoplasm against non-specific oxidation. The simplest way this protection could be afforded, consistent with the data, would be through spe- cific high-affinity reduction of tellurite by the resistance mechanism, thereby protecting essen- tial cell constituents from oxidative damage, but still resulting in reduction of tellurite to non-toxic tellurium. The mechanism of resistance specified by all three determinants is therefore proposed to involve specific reduction of tellurite to tellurium.

An alternative mechanism to prevent toxicity is cytoplasmic sequestration of tellurite in a non- crystalline form. The transport data presented here do not rule out this model, but energy-dis- persive X-ray analysis of different areas of resis- tant cells showed the absence of any significant Te concentrations in regions of the cell other than the tellurium crystals (not shown). Thus, such a cytoplasmic sequestration mechanism is unlikely to be the basis for resistance.

Each of the three resistance determinants en- code a presumptive cytoplasmic membrane pro- tein [4-6], which may act in tellurite reduction. Each of these proteins contains at least two cys- teine residues, which could help channel four electrons to tellurite for reduction to tellurium, or which may transiently bind the tellurite prior to reduction [19]. In the RK2 determinant the cysteine pair (Cys125, Cys132) in the TelB mem- brane protein is essential for resistance [6,19]. Cellular electron transport may provide electrons

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for tellurite reduction. This explanation would account for the failure to detect specific reduc- tion in cell extracts [9], as electron transport would be disrupted. Moreover, a membrane-based reductase would account for the observed mem- brane association of Te crystals in resistant cells; this being similar to the location of crystals in sensitive cells, but for a different reason. In Rhodobacter spheroides an intact electron trans- port system is essential for resistance to tellurite and other rare earth oxyanions [20]. Further study with E. coli mutants defective in electron trans- port is warranted.

In addition to a membrane protein, each de- terminant is predicted to encode one or more cytoplasmic polypeptides (D.A. Rouch, unpub- lished, from data in [4-6]), which, by extension of the reduction model, may act to bind and channel tellurite to the proposed membrane based reduc- tase. Mutagenesis (including deletion) studies should allow testing of this hypothesis.

Acknowledgements

We thank the Staff of the Universities Re- search Reactor, Risley, Warrington, UK for assis- tance in the synthesis of [127TEO32-]; D . E . Taylor (Department of Medical Microbiology and Infec- tious Diseases, University of Alberta, Edmonton, Canada) for supplying the Te r determinants pDT1364 and pDT1558; J. Smith for assistance with the electron micrographs; the SERC for research grant GR/F2376.7 (G.L.-J., D.A. Ritchie and P.S.); and the MRC for research grant G.909025236CB (N.L.B. and D.A. Rouch).

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