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

FEMSLE 05923

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

Gareth Lloyd-Jones *'u, A. Mark osborn u, Donald A. Ritchie u, peter strikeJon L. Hobman u,b, Nigel L. Brown b and Duncan A. Rouch "o Department of Genetics and Microbiology, Donnan Laboratoies, The Uniuersity of Liuerpool, Liuerpool L6g 3BX, IJK and" School of Biological Sciences, The Uniuersity of Birmingham, Birmingham Bis 2TT, 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. Specificmembrane-proximal reduction is proposed as the mechanism of resistance to tellurite coded by all three determinants, despite theirlack of sequence homology.

Key words: Tellurite resistance; pMER610; RK2; pHHl508a; Escherichia coli

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Introduction

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

--or.oponaingauthor.presentaddress:LandcareResearchLimited, Private Bag 3123, Hamilton, New Zealand.

ssDl 03 78- 1 097(94)00068 _3

mination [4-6] and their properties have beenreviewed [7]. The sequence data have shown thethree determinants to be non-homologous, withno statistically significant regions of sequencesimilarity.

During growth of tellurite-resistant Es-cherichia coll strains, tellurite concentrations havebeen shown to decrease in culture supernatants[5,7-10]. Tellurite uptake in E. coli appears tooccur at least partly via the cellular phosphate(Pi) transport systems [11]. Following uptake andaccumulation, reduced tellurite, in the form ofcrystalline deposits of elemental tellurium, hasbeen observed in Te' strains 18,9,121. These de-

tL4

posits, however, are also seen in Tet strains of .E

coli l9l thus casting doubt on a specific role forTe crystallization in resistance.

In this paper we compare the properties ofstrains harbouring the three sequenced Te' de-terminants from plasmids pMER610, pHH1508aand RK2. Studies of tellurite transport and thedistribution of tellurium crystals in these strainshave allowed us to propose a model in which Tecrystallization is essential to the mechanism ofresistance in the three different determinants.

Materials and Methods

Bacterial strains and gowth mediumEscherichin coli strain AB1157 (Sm', F-) was

used as the host strain for the Te' plasmidsdetailed in Table 1. Cells were grown at 37'C inK medium (M9 medium U.3l containing 0.25%w/v casamino acids and 0.8Vo w/v glucose). an-tibiotics were used at the following concentra-tions: 200 pg ml-1 streptomycin; 25 p,g ml-rchloramphenicol, 50 pg ml-1 ampicillin.

Electron microscopyWashed cell pellets of each strain, challenged

with 50 p,M tellurite for 30 min, were fixed usinggluteraldehyde and osmium tetroxide. Sampleswere then dehydrated successively in 30, 50, 70,

90, 100, 100, and l00Vo ethanol, followed byembedding in epory resin. Sectioned material wasleft unstained and examined by transmission elec-tron microscopy.

Telluite transportRadioactive tellurium dioxide, t"T"Or, *us

obtained from the Universities Research Reactor,Risley, Warrington, UK (maximum activity 15

p,Ci mg-1; half life 9.4 h), and was dissolved in100 g.M NaOH to give a stock solution of127reo!-7reO.2-. Cells were harvested by cen-trifugation at 10000 Xg for 10 min, washed, andresuspended in M9 medium at 100 mg wet weightml-1. CeU densities during transport assays weretypically 0.25-0.5 mg dry weight ml-1. Evertedvesicles were prepared as previously described

[14]. Transport assays were conducted at 37'Cwith aeration by shaking in M9 medium supple-mented with 0.8Vo w/v glucose; 0.2-ml sampleswere withdrawn at timed intervals; the cells werecollected by filtration and washed with M9medium, and the incorporated radioactivity wasdetermined. For direct efflux studies, cells werepreincubated with 50 g.M labelled tellurite for 30min at 4'C, washed and resuspended in M9medium. After taking the zero time sample, cellswere 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

t Plasmids used in this study

Plasmid ' Resistancemarkers

Source MIC Copy No. Reference(pM)

pUCSpLV59pMER610RK2pHH1508apMJ600pDT1364pDT1558

Ap'CmHg, TeAp, Km, Tc, TeSm, Su, Tp, TeCm, TeAp, TeAp, Te

Te' pLV59 clone from pMER610 "Te' pUC8 clone from pHH1508aTe' pUC8 clone from RK2Te8ur

NDND1 00020002000I 000

5001 000

500-70020- 30

low4-7

low20- 30

500-700500-700

lztlt22la4lt3lI23lt4lI23ll23l

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

b Last LA plate, supplemented with increasing concentrations of tellurite, showing growth. ND: not determined.

" The Te' determinant from pMER61.0 cannot be subcloned into pUC vectors due to instability of resulting subclones.

above, but in the presence of 5 mM NADH, 5

mM NADPH or 5 mM ATP(Mg2*), or in theabsence of any co-factors.

Results

Localization of tellurium depositionAlthough 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 denyingproper comparison. Here the conditions werestandardized, with the' three determinants, as

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present on pMER6L0, pHH1508a and RK2, be-ing carried by the same host strain, A81157,grown under the same conditions and prepared insimilar fashion for electron microscopy. The re-sulting electron micrographs showed that for allthree Ter strains the presence of a number ofblack crystals was regularly observed in cells (Fig.1a,b,c). Such electron-dense crystals have beenshown previously to consist of elemental tel-lurium 18, 9, l2l. Although some tellurium crys-tals appeared elsewhere in the cytoplasm of eachstrain, there was a marked tendency towards localdeposition of crystals near the cytoplasmic mem-brane. A similar result was observed for the plas-

Scanning transmission electron micrographs (upper magnification) of strains challenged with 50 g,M tellurite (a)A8115(pMER610), (b) A81157(RK2), (c) AB1157(pHH1508a), and (d) ,481157. Scale bar: 0.5 rr.m.

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midless host Te' strain AB1157 (Fig. 1d), al-though in this case the cells are killed by thisconcentration of tellurite.

Tellurite uptake in M9 medium supplemented withglucose

In further work subclones of the three Te'determinants were used (Table 1); pMJ600 (frompMER610), pDT1364 (from pHH1508a) andpDT1558 (from RK2). Cellular uptake of telluritein M9 medium in the absence of glucose occurredat low rates, with rates increasing in the presenceof glucose. All assays were therefore carried outin M9 medium supplemented with 0.8Vo w /vglucose. Tellurite was taken up by all Te' plas-mid-containing strains at rates equal to or greaterthan the isogenic plasmidless host strain E. coliAB\157 (Fie. 2).

Tellurite uptake at uaried concentrations of telluriteWhen the uptake of tellurite by the Te' strain

AB1157, and its Te' plasmid-containing deriva-tives, was measured in the presence of 10, L00,500 and 1000 pM tellurite (Fig. 3), the uptake

20000

1 5000

1 0000

5000

00 30 60 90 120 150

t(min)

Fig. 2. Accumulation with time of tellurite from 100 pM127TeOl- 7'teor2- by: AB1151pMJ600) (o), A81157(pDT1367) (r), AB1151pDTl558) (v) and the Te" host strain E.coli ABll57 (o), in Mg medium, supplemented with glucose,

at 37'C with aeration. Each point is the mean of three uptakeassays.

c'E

co'6

=-!oE

o-O

30000

25000

20000

1 5000

1 0000

5000

0

E.9O

=

!oE

LO

0 200 400 600 800 1 000

[Telluriie] uM

Fig. 3. Accumulation of tellurite as a function of initialtellurite concentration by: A81157(pMJ600) (o), antfSZ(pDT136a) (v), A81157(pDT1558) (v) and the Tes hoststrain E. coli AB1,l57 (o), in M9 medium supplemented withglucose, at 37"C with aeration. Each point is the mean of

three uptake assays.

rates in cells containing pDT1558 were three-foldhigher than those in cells containing pMJ600 orpDT1364 and than those in the Tes strainABII57, which did not differ significantly fromone another.

No decreased uptake of tellurite due to any ofthe three Te' determinants could be detectedrelative to the Te' host strain (Fig. 3). In addi-tion, no efflux could be detected from the strainspreloaded with tellurite; measurements of la-belled tellurite retention after 30 min showedvalues of 97Vo to l02Vo retention in the resistantand sensitive strains. Uptake of tellurite was simi-lar in everted vesicles prepared from each of theresistant strains and from the sensitive parentalstrain, thus providing no evidence for a mecha-nism involving increased efflux of tellurite in theresistant strains (data not shown).

Discussion

The rates of tellurite accumulation due to thethree Te' determinants, relative to the isogenic

/

/

"/,/|

host strain, show that a decreased uptake oftellurite does not contribute to the resistancemechanisms encoded by any of these plasmids.Reduced uptake has been found to contribute toother metal oryanion resistance mechanisms, e.g.chromate resistance in Enterobacter cloacae [1,5f.Similarly the lack of any detectable export mech-anisms may rule out direct export of tellurite aspart of the resistance mechanisms. Again, othermetal resistances have export mechanisms whichhave been readily demonstrated using the proto-cols employed here e.g. cadmium, zinc and cobaltextrusion in Alcaligenes eutrophus CH34 [16] orarsenate, 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-genic host E. coli AB1l57 did not differ signifi-cantly; however, the rates were three-fold greaterfor cells containing pDT1558 (Figs. 2 and 3). Thisdifference may be due to greater uptake occur-ring in a strain with the highest degree of protec-tion from tellurite-induced cellular damage, thusmaintaining total cellular viability over a longerperiod (pDT1558-containing cells have a higherMIC than those containing pDTl364, andpDT1558 has a higher copy number than pMJ600(Table 1)). The striking 200-fold difference in theremoval (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 surprisingly notsupported by these tellurite accumulation assays.These results appear to rule out both reduceduptake and export of tellurite as components ofthe resistance mechanisms encoded by the threeTe' determinants.

Tellurium crystallization upon exposure to 50pM tellurite occurred in a similar pattern instrains containing the three Te' determinants.Crystal deposition was also observed in the Teshost strain, confirming a previous observation[L,9], and appearing to rule out a role in resis-tance for the reduction of tellurite to non-toxictellurium. However, upon incubation in mediacontaining 50 pcM tellurite resistant strains retainfull growth potential, while the sensitive strainfails to grow and loses viability (not shown). These

tl1

data, together with the crystallization data, sug-gest that the resistant and sensitive cells handletellurite differently. Tellurite is strongly oxidising,and its toxicity in the sensitive strain would beexpected to occur through non-specific oxidationof cellular materials following its uptake into thecytoplasm. Such oxidation would result in con-comitant reduction of the tellurite, and the insol-uble tellurium produced by complete reductionwould form crystals. Consistent with this notion isthe observed pattern of Te deposition in .{81157cells. The proximity of Te crystals to the cytoplas-mic membrane can be explained on the basis thatthe cell constituents closest to the sites of tellu-rite uptake in the membrane have the highestprobability of reacting with the tellurite.

A resistance mechanism must protect the cellcytoplasm against non-specific oxidation. Thesimplest way this protection could be afforded,consistent with the data, would be through spe-cific high-affinity reduction of tellurite by theresistance mechanism, thereby protecting essen-tial cell constituents from oxidative damage, butstill resulting in reduction of tellurite to non-toxictellurium. The mechanism of resistance specifiedby all three determinants is therefore proposed toinvolve specific reduction of tellurite to tellurium.

An alternative mechanism to prevent toxicity iscytoplasmic sequestration of tellurite in a non-crystalline form. The transport data presentedhere 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 significantTe concentrations in regions of the cell otherthan the tellurium crystals (not shown). Thus,such a cytoplasmic sequestration mechanism isunlikely 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 fourelectrons to tellurite for reduction to tellurium,or which, may transiently bind the tellurite priorto reduction [19]. In the RK2 determinant therysteine pair (Cys125, Qrs132) 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 wouldaccount for the failure to detect specific reduc-tion in cell extracts [9], as electron transportwould be disrupted. Moreover, a membrane-basedreductase would account for the observed mem-brane association of Te crystals in resistant cells;this being similar to the location of crystals insensitive cells, but for a different reason. InRhodobacter spheroi.des an intact electron trans-port system is essential for resistance to telluriteand other rare earth oxyanions [20]. Further studywith 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 morecytoplasmic polypeptides (D.A. Rouch, unpub-lished, from data in [4-6]), which, by extension ofthe reduction model, may act to bind and channeltellurite to the proposed membrane based reduc-tase. Mutagenesis (including deletion) studiesshould 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 [127TeO?-];O.p^ Taylor(Department of Medical Microbiology and Infec-tious Diseases, University of Alberta, Edmonton,Canada) for supplying the Te' determinantspDT1364 and pDT1558; J. Smith for assistancewith the electron micrographs; the SERC forresearch grant GR/F2376.7 (G.L.-J., D.A.Ritchie and P.S.); and the MRC for research

erant G.909025236C8 (N.L.B. and D.A. Rouch).

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