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A Novel Nickel Resistance Determinant Found in Sewage-Associated BacteriaAuthor(s): R. W. Pickup, H. E. H. Mallinson, G. Rhodes, L. K. ChatfieldSource: Microbial Ecology, Vol. 33, No. 3 (May - Jun., 1997), pp. 230-239Published by: SpringerStable URL: http://www.jstor.org/stable/4251493

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MICROBIA

lECOLOGMicrob Ecol (1997) 33:230-239

C 1997 Springer-VerlagNew York Inc.

A Novel Nickel Resistance Determinant Found in Sewage-

Associated Bacteria

R.W. Pickup,' H.E.H. Mallinson,' G. Rhodes,' L.K. Chatfield2

1 Institute of FreshwaterEcology,WindermereLaboratory,FarSawrey,Ambleside, Cumbria,LA22OLP,UK2 Department of Applied Biology, Univesityof Central Lancashire,Preston,LancashirePRI 2TQ, UK

Received:30 January1996;Revised:8 May 1996

IA B S T R A C T

Nickel-resistantbacteria were isolated from effluent discharged rom a sewagetreatment outfall

over an 18-month period. One of these strains,EnterobacterloacaeFBA30,was found to harbor

a narrow host range conjugativeplasmid, designatedpFBA30,which confers nickel resistanceon

its host. A 10.2-kb SstI restrictionfragmentwas cloned from pFBA30and was shown to specify

inducible nickel resistance,both in its originalhost and in laboratorystrainsof Escherichia oli.

ThisDNA fragment,and a 1.75-kbSmaIfragmentderivedfromit, were used as probesto examine

other bacterial trains solated duringthe study. Homologous nickel resistancegenesweredetected

in enteric bacteriaemerging directly from the treatmentplant, but not in strains isolated from

sediments downstreamof the outfall,or in strainsharboringwell-characterized eterminants uch

as ncc/nre, cnr,or Klebsiellaoxytocatype. Thus, this element constitutes a new nickel resistance

determinantand was named nrf

Nickel-resistant trainswere sortedinto two groups, basedon the samplingdate and the size of

restriction ragmentshomologous to probes developedfrom pFBA30.GroupA isolatesall carried

a homologous 6.5-kb PvuIl restrictionfragment and expressednickel resistanceconstitutively.

GroupB strains, solatedindependently rom the sameoutfall,were different rom groupA in that

the nickel resistancewas associatedwith a 4.7-kb PvuIJ ragment,and group B strains displayeda

slowergrowthrateon nickel saltsmedia. Eachgroupcontaineda rangeof entericbacterial pecies,

includingK.oxytoca,Citrobacterfreundii,nd Enterobacterpp. Localizedgeneticexchangeprobably

occurs within wastewater reatmenteffluents, and, once effluents aredischarged nto the environ-

ment, entericbacteriacarryingpFBA30-likedeterminantsdo not persist downstream.

Correspondenceo:R.WPickup.E-mail:[email protected].

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Nickel Resistance n Sewage-AssociatedBacteria 231

Introduction

Plasmid-encodednickelresistance n bacteriahas been char-

acterizedmost extensively in AlcaligeneseutrophusCH34

[21]. This strain harborsthe conjugativeplasmid pMOL28

which confersresistanceo arangeof metals ncludingnickel

and cobalt. Resistance s mediatedby an inducibleenergy-

dependent specific efflux system [11, 25, 34, 35, 36, 41].

Extensivemolecularcharacterization f the nickel effluxsys-

tem was achievedby cloning the resistancegene (cnr)from

pMOL28 [15, 37]. Most related studies have used nickel-

resistant strains isolated from anthropogenicallypolluted

sites, particularlyhoseassociatedwiththemetal-reclamation

industries [20, 39], and those where nickelpercolatesnatu-

rally throughan ecosystem [39]. Resistanceevels conferred

by these plasmid- or chromosomallylocated genes range

from 1 to 40 mm in Tris-gluconatemedia [31, 33].

There is some diversityto be found among nickel resis-

tance determinants.Some nickel resistancegenesshow ahigh

degree of homology with cnr of pMOL28 and ncc of A.

xylosoxidans 1A [32, 33], while others havebeen found to

carrya second resistantdeterminant nre)that has only lim-

itedhomologywithpMOL28.The nredeterminant xpresses

in E. coli and is thought to be widespreadamong nickel-

resistantbacteria[32]. Comparativeanalysisof a large num-

ber of nickel-resistantsolates enabledStoppeland Schlegel[39] to distinguishfour groups of nickel resistance.These

comprised (1) thoseshowing homologywith cnrof pMOL28

[17] and ncc from pTOM (33); (2) a combination of cnr,

ncc,and nre[32];and (3) a determinant ound in K.oxytoca,

showingweakhomology with nre [40]. These loci were not

ubiquitous,however,as indicatedby the existenceof afourth

group of resistantstrainsexhibitingno sequencehomology

with either cnr/nccor nre [32].

The occurrence of metal resistantbacteria n anthropo-

genicallypollutedsites is well documented [10, 38]. Studiesof bacteriaemergingfrom a sewagetreatmentplant in the

EnglishLakeDistrictshowed thepresenceof arangeof metal

resistances,which included nickel [26] and copper [27]. No

selection for resistancedue to metal contaminationwas ap-

parentat this site, since this treatmentsystemreceivedonly

domesticinputs [26]. It has been notedpreviously hat resis-

tance to heavymetalsoccurs at a surprisinglyhigh frequency

in the absence of sourcesof directselection [38].

Sewage effluent, wastewater,and water treatmentshave

been shown to have several effects on bacteria.Wastewater

treatment increases the proportion of bacteria that carry

either single or multiple antibioticresistances;between 0.1

and 1% of total coliforms in fecal materialwere resistant,

and this reachedfrequenciesof 10%in urban wastewater,

50%in riverwater,and 80%in potablewater [16]. Bacteria

that carryresistanceto multiple antibioticshave also beenfound in both drinkingwaterand wastewater 2, 3]. Further-

more, chlorination of water has been shown to select for

multiple antibioticresistance[23]. Resistance o heavymet-

als, linked to that of antibiotics,has been found not only in

bacteriasolated romsewageeffluent[41 butalso inbacteria

from hospitals [19], metal-contaminated stuaries [1], and

soils [18]. Bacteriaaffectedby either water or wastewater

treatmentare eventuallyreleased nto aquaticenvironments.

Almost all nvestigations f antibioticresistancen theaquatic

habitathave concentratedon its incidence n bacteriaof fecalorigin [5, 12-14]. However,a positivecorrelationwasnoted

between he occurrenceofheavymetalandmultipleantibiotic

resistance n a rangeof bacteria solatedfromwater distribu-

tion and treatmentsystems [7]. The aim of this studywas to

developnucleic acid probesto examine the nickel-resistant

bacterial ommunitybeing dispersed romawastewater reat-

mentworks,and to assesswhether metal resistance ransfers

to the bacterial ommunitydownstreamof the outfallpipe.

Materials and Methods

Bacterial trains ndPlasmids

The strainsused in this study are described n Table 1. All nickel-

resistanttrainsweremaintainednnormal-strengthutrient gar

(Oxoid,Basingstoke K) supplementedith5.0mmnickelchlo-

ride. Escherichia oli HB101 and JM103containing recombinant

plasmidswere maintainedon nutrient agarsupplementedwith 50

Figml-' ampicillin.All other strains were maintainedon nutrient

agar.

Sampling nd Isolation f Bacteria

ChurchBeck s a freshwater tream n the ConistonFells, Cumbria,

UK and has been describedpreviously [26, 27]. The sample site

was the sewageoutfall in the lower section of Church Beck which

feedsdirectly nto Coniston (National Grid ReferenceSD 308971).

Bacteriawere isolated from 11 samples of water, as describedby

Pickup [26], except that filterswere placed on nutrientagarsupple-

mentedwith 5 mmNiCl26H2O(pH 7.6). Sedimentswerecollected

downstreamof theoutfallpipein glasscontainers, igorously haken

in the laboratory,and allowed to settle. The supernatantwas then

filtered and strains were isolated in the same way as the water

samples. Plates were incubatedat 20'C until single colonies ap-

8/4/2019 4251493


232 R.W. Pickupet al.

Table 1. Bacterial trainsand plasmids

Straindesignationused in this study Speciesname and strain number Plasmidname/(content) Descriptiona Origin

FBA30 Enterobacterloacae pFBA30/pFBA312) inducibleNir This studyFBA30-1 EscherichiaoliHB101 pFBA30(1) inducibleNir This study

FBA30-2 EnterobacterloacaeNCIB 8151 pFBA30(1) inducibleNir This studyFBA30-3 Enterobactererogenes pFBA30(1) inducibleNir This studyFBA30-4 Serratiarubidae pFBA30(1) inducibleNir This studyFBA13 Klebsiella xytoca group A) 0 constitutiveNir This studyFBA17B Enterobacterakazakii group A) not named (2) constitutiveNir This studyFBA28 Enterobacterloacae group A) not named (2) constitutiveNir This studyFBA1.3.21 Citrobacterfreundiigroup B) not named (1) inducibleNir This studyFBA1.4.2 ndb(group B) not named (1) inducibleNir This studyFBA1.5.43 Enterobacter gglomeransgroup B) not named (1) inducibleNir This studyFBA30-10 EscherichiaoliJM103 pFBA30-10(1) inducibleNir This studyFBA30-11 Escherichia oli JM103 pFBA30-11 (1) inducibleNir This studyFBA30-12 Escherichiaoli JM103 pFBA30-12 (1) constitutiveNir This studyNCIMBC8151 Enterobacterloacae (0) Ni' NCIMB

HB1I1 EscherichiaoliHB101 (0) Smr Ni' 6

JM103 Escherichia oli JM103 (0) Ni' 43PaW340 Pseudomonas utidaPaW340 (0) Smr trp- 42M695 EscherichiaoliJM109 pSK-::KOHI4-1 Nir K. oxytoca 40

M696 Escherichia oli JM109 pSK-::TBA9a Nir nre/ncc 32,33

M697 Escherichiaoli JM109 pHLI4 Nir cnr 17

" rp, Tryptophan;Nir,nickel resistance;Sm',streptomycinresistance

bnd, Not determinedcNCIMB, National Collection of Industrialand Marine Bacteria,Aberdeen

peared.All colonies were purifiedand retained.API identification

strips (BiomerieuxLtd., Basingstoke,UK) were used, as directed

by the manufacturer.

BacterialConjugation

ExogenousPlasmid Isolation.Exogenous plasmid isolation was

modified fromthe method of Bale et al. [4]. Water/effluent amples

of 50, 100, and 500 ml were mixed with 1 ml of recipient strain

culture(PaW340or HBI01) and filtered hrougha 0.22-jim Milli-

pore HC filter under vacuum. Filterswere incubated on nutrient

agar at 30?Covernight. The accumulatedgrowthwas resuspended

in sterile distilledwater,seriallydiluted,and spreadonto nutrient

agarplatessupplementedwith NiCl2 5 mM)andstreptomycin 100

jig ml-1). Plateswereexaminedfor singlecolonies after ncubation

at 30?C.Appropriatedonor (water/effluent ample) and recipient

controlswere performedsimultaneously.

Endogenous lasmidTransfer.Conjugationexperimentswere per-

formed using a plate mating method with purified donor strains

isolated from the sewageoutfall,as describedby Pickup[28], using

both PaW340 and HB101 as recipients. To facilitatescreening a

large number of isolates, streakmatingswere used. The recipient

was streakedverticallydown the left side of the selection plate.

Donors were then streakedhorizontally, n one movement, from

the left to the right side through the recipientstreak. The donor

streak o the left of the recipient servedas a control.Nutrientagar

platessupplementedwith NiCl2(5 mM)and streptomycin 100 jg

ml-') wereuse astheselectivemedium.Singlecolonieswere solated,

purified,and examined for the presence of plasmids.

DNAManipulations

Plasmids from environmental isolates and transconjugantswere

extractedusingthe method of WheatcroftandWilliams [42]. Total

genomic DNA was extractedusing the method of Pitcher et al.

[29], and recombinantplasmidswere preparedby the method of

Close and Rodriguez[8]. Plasmidsused for genetic manipulation

were purifiedby SDSlysis/CsClcentrifugation 30].

Restriction ndonucleaseswereobtainedfromGibcoBRL Pais-

ley, UK). Agarosegel electrophoresiswascarriedout by established

procedures. Reactionconditions for DNA ligation with T4 DNA

ligasewere asrecommendedby the supplier GibcoBRL).Transfor-mation into E. coli JM103was achievedby the procedureof Cohen

et al. [9].DNAprobes(either rompurifiedDNAor fromrestriction

fragmentsexcisedfrom low melting temperatures els, as directed

by the manufacturer)were abeledwith [32p] dCTPusing a Random

Primed DNA LabellingKit (Boehringer,LewesUK). pUC19 gene

banks were screenedby the method of Close and Rodrigues[9].

DNA hybridizationwas carriedout as describedpreviously[22].

Inductiontudies

Bacteriawereculturedovernight n 50 ml normal-strength utrient

broth and in 50 ml nutrient broth supplementedwith 0.5 mm

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12 3 4 5 6

(kb)

21.2 ___ .

5.1 5.0 !

4.3-

2.0

1.9/

Fig. 1. HindIll digestion of plasmidsfound in FBA30and trans-

conjugants.Lanes:1, A (HindIll); 2, FBA30(pFBA30:pFBA31);,

E. coli JM103(pFBA30); , E. cloacaeNCIMB8151(pFBA30);5, E.

aerogenes(pFBA30);, Serratiarubidae(pFBA30).

NiCl2 previouslydetermined o be a nonlethalconcentration),with

shaking (100 rpm) at 30?C.The optical densityat 600 nm was then

measured(Cecil InstrumentsCE292Digital UV Spectrophotome-

ter), with nutrient broth as a blank. Each culture was diluted in

nutrient broth to an optical densityof 1.0 at 600 nm. Afteradjust-

ment, 1 ml of each culture was inoculated into duplicate flasks

containing50 ml nutrient both supplementedwith 2.5 mm NiCl2.

These were incubatedat 30?Cwith shaking (100 rpm). The optical

densityof each was measuredhourlyfor a total of 8 h after inocu-

lation.

Results

Isolation f Nickel-Resistanttrains

The sewage outfall was sampled three times (samples 1, 2,

and 3) at intervals of 6 months. Bacteria resistant to 5 mm

NiCl2 were readily isolated on each occasion at a concentra-

tion of approximately 1,000 cells 1-1. In the first instance,

over 100 isolates were retained from sample 1. A total of 100

isolates from samples 2 and 3 were also retained for further

analysis. All were confirmed as oxidase negative. A further

100 isolates, in total, were retained from sediments down-

streamof the outfall duringsampletrips2 and 3. Theconcen-

tration of nickel-resistant solates in the sediments was not

determined.Of the sediment isolates, 60% were confirmed

as oxidase negative.

Conjugationxperiments

An attemptwas made to isolate conjugativenickel-resistant

plasmidsfrom environmentalsamples by transfer o labora-

tory strains of Pseudomonasputida (PaW340) and E. coli

(HB101). No transconjugantswere isolated by this method

when filteredbacteria rom sewage sample 1 or eitherof the

sedimentsampleswere usedas donorpopulations,so purified

strains isolated from sample 1 were examined further by

individual streak matings. Again, no transconjugantswere

observedwhen PaW340was used as a recipient,but a single

isolate regularlyyielded nickel-resistant ransconjugants n

matingswith HB101. This strainwas designatedFBA30.API

identificationand analysis of plasmid DNA confirmed that

the colonies were true transconjugants.The donor isolate

was identified as Enterobacter loacae(probability>99%)

using API test strips. Plasmid analysisof the donor strain

(FBA30) revealed two plasmids (designated pFBA30 and

pFBA31;Fig. 1). Comparison of the plasmid content of

FBA30and the transconjugantsobtained by plate matings

revealedthat one plasmid (pFBA30)had transferredwith a

frequencyof 1.9 X 10-3 transconjugantsper donor cell, and

was stably maintained (Fig. 1). FBA30-1 was retained as

representativeof the transconjugants.Plate matings with

FBA30andFBA30- showedthatplasmidpFBA30was trans-

missiblebetween enteric bacteria (E. cloacaeNCIB8151,E.

aerogenes, erratia ubidae),but not to nonentericstrains(P.

putida PaW340,P. fluorescens).Transconjugantsharboring

pFBA30were isolatedandpurified rom thesematings.These

strains were designatedFBA30-2, FBA30-3, and FBA30-4,

respectively(Table 1). Restriction endonuclease digestion

with a range of enzymes revealed pFBA30to be approxi-mately55 kb in size (datanot presented).

Cloning f NickelResistanceDeterminantrompFBA30

PlasmidDNA from pFBA30was isolated and digestedwith

SstI. Resultant DNA fragmentswere ligated into pUC19,

and recombinantplasmidswere transformed nto JM103.All

clones were tested for nickel resistance,and a single,nickel-

resistant colony was obtained.The recombinantplasmid in

this strainwas found to carryan inserted 10.2-kb SstIrestric-

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pFBA30-10 S,MCS a

aC~Sm Sm HSM p Sm DSD

Vji a | P ID ID G PD p

1 kb

pFBA30-1 b

B P D

P Sm ~ BD

H Sm D | Ori D

Probe2 Probe 1 AmpR

I kb

pFBA30-12 c

B PD

SmD

D,2 Sm H Ori B D

_... .i... ......---.----. .----.....

Probe 1 Probe 2 AmpR

1 kb

Fig.2. Restrictionmapof the DNA insert n pUC19associated

withnickel esistance:a) representsFBA30-10itha 10.2-kb stl

fragment loned ntothe multiple loning ite (MCS)of pUC19;

the shaded rea ndicateshe 2.3-kbPvuII ragmentubclonedn

pUC19; b)and c)represent FBA30-1andpFBA30-12ubclones

of pFBA30-10arrying 2.6-kbPvuIIragmenthat s stillable o

confernickel esistance. , DraI;H, HindIII; , PvuIJ;S, Sall;Sm,

SmaI;S, SstI.

tion fragment,which conferredresistance o NiC12 n its E.

colihost.A comparisonof restriction ndonuclease ragments

fromboth plasmids,andhybridizationo pFBA30DNA (data

not presented)confirmed that this plasmidoriginated rom

pFBA30.The fragmentwas examinedfor internalrestriction

sites,andarestrictionmap compiledforDral,HindIII,PvuII,

Sail, and SmaI (Fig. 2a). The plasmid was designated

pFBA30- 0. Nickelresistancewas retainedby subclonesde-

rived from pFBA30-10by digestion with PvuII and subse-

quent religation. Transformationof re-ligated DNA into

JM103yielded two such clones, here designated pFBA30-

11 and pFBA30-12.Each carries a 2.3-kb PvuII restriction

fragmentwhich occupies oppositeorientations n eachplas-mid. The insert in pFBA3O-1 (Fig. 2b) has the sameorienta-

tion as that of pFBA30- 0,while that of pFBA30-12 Fig. 2c)

has the reverseconfiguration.

When tested on nickel-supplemented media, FBA30,

JM103(pFBA30), nd JM103(pFBA30-10)were resistant to

7.5 mMNi in nutrientagar.JM103(pFBA30-1 1) and JM103

(pFBA30-12)were resistant o only a lowerconcentration 5mM).When Tris-buffered luconateminimal media(TMM)

was used [21], FBA30retained its resistanceprofile while

those plasmidswith E.coliHB101 or JM103 as a host showed

proportionallyreduced levels of resistance:pFBA30- and

pFBA30-10 at 2.5 mM and the subclones (pFBA30-11and

pFBA30-12)at 1 mm.This is becausethe nutrientagar may

act as a complexing agent enhancingnickelavailabilityand

subsequent nhibitoryeffects n TMM[31, 33]. No resistance

to antibiotics, CoCl2,or other metalswas observed.

Development f Probe or Nickel-Resistanceenes

A total of 500 isolates from treated sewage and sediment

were probed in hybridization xperimentswith the complete

pFBA30-10plasmidbearing he 10.2-kb SstIrestriction rag-

ment. Of the 400 sewage solates,95 showed somehomology

to pFBA30-10 under conditions of low stringency,but of

these, only 32 isolatesshowedhomologyunderhighlystrin-

gent conditions.The latterwere retained.None of the isolates

from sediment showed any significant evel of hybridization

to pFBA30-10,even under conditionsof low stringency.The

subclone pFBA30-11was developedas a probe againstthe

32 isolatesby digestionwith PvuIIand SmaI.This yieldeda

1.75-kb SmaI fragmentand a 0.5-kb PvuII-SmaI ragment.

Each was used as a nucleic acidprobe.The 0.5-kb fragment

did not hybridizewith any of the 32 isolatesshowing homol-

ogy to pFBA30-10.The larger probe of 1.75 kb hybridized

stronglywith a numberof isolates under conditions of high

stringency.However,reconfirmationof their status revealed

that only 6 isolates consistently hybridizedto the 1.75-kb

fragment.The fragmentsshowing homology to the 1.75-kb

SmaIfragmentwere sized from a PvuIIdigestof total DNA

from each isolate and were shown to be divisibleinto two

classes,distinguishableby the samplingdate. GroupA com-

prisedisolatesFBA13,FBA17B,and FBA28,eachcontaining

a homologous fragment of approximately6.5 kb. Each of

the group B isolates (FBA1.3.21,FBA1.4.2,and FBA1.5.43)

possessed a PvuIIrestrictionfragmentof 4.7 kb that also

showed significant homology with the 1.75-kbprobe (Fig.

3). Of the groupA and B strains,only FBA13contained no

observable plasmids (Table 1). Hybridization experimentswith plasmids rom these strains uggest hat the homologous

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1 2 3 4 5 6 7 8 9

a

23.1 ____

9.46.6

4.4 __

2.3 _

2.0

(kb)

b

Fig.3. Southernblot analysisof nickel-resistantsolates ndicatinghomology with the nickel resistancelocus of pFBA30. (a) PvuII

digestof total DNA fromFBA30and environmental solates.Lanes:

1, A (HindIII); 2, FBA13;3, FBA17B;4, FBA28;5, FBA1.3.21;6,

FBA1.4.2;7, FBA1.5.43;8, FBA30;9, FBA30-10.(b) Southern blot

analysis using 1.75-kb PvuII fragment (probe 1) of pFBA30-11

showing homology with 6.5-kb fragmentin group A isolates and

4.7-kb fragment n group B isolates, respectively.Lanes:as in Fig.

2. Pleasenote that FBA 17B and FBA1.5.43gave atypical signals;

subsequentSouthernblots reconfirmedthe presenceof the 6.5-kb

and 4.7-kb fragments,respectively data not presented).GroupA

and B isolates refer o the size of fragmenthomologous to the 1.75-

kb probe, which are 6.5 kb and 4.7 kb, respectively.

sequences appearedto be located on the chromosome in

each case as hybridization could not be matched to any of

the plasmidbands (data not presented).

No homology was detected between pFBA30-11 and

pMOL154 which contains the structural cnr genes from

pMOL28(Fig. 1). Similarly,no homology was detected with

plasmidscarryingncc/nre,cnr, or the determinantfrom K.

oxytoca[32, 33, 40] (data not presented).

Identificationf IsolatesShowingHomologyo pFBA30

All nickel-resistant isolates that showed homology with

pFBA30were found to be oxidase negative,but presented

different profiles when tested with the API identification

system. Group A strains were tentativelyidentified as K.

oxytoca strain13;probability85%),E. sakazalai strain17B;

probability97%),and E. cloacae strain28; probability99%).

Group B included E. agglomeransstrain 1.5.43; probability

85%), C.freundii(strain 1.3.2; probability85%),and strain

1.4.2which, althoughnot identifiableby the API test strips,

presented a different profile from the other strains. These

strains hus representa diversepopulationof entericbacteria,

but no one strainpredominatedeithergroupA or B. Growth

on nickel-supplementedmedia showed that all strainswere

resistant o 7.5 mMNi in nutrientbroth. None demonstrated

resistance o low concentrationsup to 1 mm CoCl,.

Inducibilityf Nir

FBA30and laboratoryE. coli strains haboring pFBA30or a

derivativeplasmid were examined for the inducibility of

nickel resistanceby preculturingstrainsin the presenceof a

nonlethalconcentrationof Ni (0.5 mM)priorto inoculation

into nutrient broth supplementedwith 2.5 mm Ni. Control

strains,E. cloacaeNCIB8151and E. coli HB1O1, howed no

growthafter 8 h.

The growth profile of FBA30 indicated that the nickel

resistanceof this strainwas inducible,sincepreculture n 0.5

mMNi had asignificanteffecton the lagtime whencompared

to that of FBA30 n the absenceof preculture Fig. 4a). JM103

(pFBA30-10)showed identical growth characteristics Fig.

4b). This confirmsthat the 10.2-kb SstI ragmentof pFBA30

carriedby pFBA30-10contains the entire nickel resistance

locus, and is still inducible. Both pFBA30-11(Fig. 4c) and

pFBA30-12(Fig. 4d) differed from pFBA30-10 n that each

showed a slower growthrate in the presenceof 2.5 mm Ni.

pFBA30-12displayeda reducedlevel of resistance hat was

not inducible:this presumablyreflects a lower rate of tran-

scription of the nickel resistance ocus from the vector pro-

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(e) KJebsioltaoxytoca (FBA13) (h) Citrobacterfreundii(FBAI.3.21)

0350.7 0.2 TE E~~~~~~~~~~~~~0.6 0.15 1

C" 0.50 01

*0.4 I.

c 0.3 --c

Lvim (h(Vim h

0.2..~ ~ ~ ~ ~ ~ ~ ~ ~~~~C 0.45.

0.1 T0

N --a

0 -.- I I I I I I ~0 I I I I I I I

0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 8

Time (h) Time(h)

(f) EnterobactersakazakiiFBA17B) Unidentified (FBAI.4.2)

0O.702

0.6 C

* 0.4~~~~~~~~~~~~~~~~~~~~~~~~001

c 0.3--e .2- E0.

2 0.1 0

. 0 .0

0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8

Time(h) Time (h)

(g) Enterobacterloacae (FBA 8) U)EnterobacteragglomeransFBA1.5.43)

0.2

0.4~~~~~~~~~~~~~ 0.1

C0.3C

e0.2 I-0 05 -

0.1 ~~~~~~~~~~~~~~~~~~~~~~00 1 I. i i I i I 0 I I I I I I

0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7

Time(h) Time(h)

Fig. 4 continued

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patternsof metal-tolerantbacteria isolated from an estuary.

Antimicrob Agents Chemother12:545-547

2. Armstrong JL, Shigeno DS, Calomiris JJ,Seidler RJ (1981)

Antibiotic-resistantbacteria n drinkingwater. Appl Environ

Microbiol 42:277-283

3. Armstrong JL, Calomiris JJ, Seidler RJ (1982) Selection of

antibiotic resistantstandardplate count bacteriaduring water

treatment.Appl Environ Microbiol44:30-316

4. Bale JM, Fry JC, Day MJ (1988) Transferand occurrence

of large mercuryresistanceplasmids in riverepilithon. Appl

Environ Microbiol 54:972-9785. Bayne S, Blankson M, Thirkell D (1983) Enumerationand

speciation of group D streptococci from above and below a

sewage outfall, their susceptibilityto six antibiotics, and a

comparison with clinical isolates. Anton Van Leewen 49:299-310

6. Boyer HW, Roulland-DussoixD (1969) A complementation

analysisof the restrictionand modificationof DNA in Esche-

richiacoli.JMol Biol 41:459-472

7. Calomiris JJ,ArmstrongJL, SeidlerRJ(1981) Association of

metal tolerancewith multiple antibioticresistanceof bacteria

isolated from drinking water. Appl Environ Microbiol 47:

1212-1238

8. Close TJ,RodriguezRL(1982) Constructionand characteriza-

tion of the chloramphenicol-resistance ene cartridge:a new

approach o the transcriptionalmappingof extrachromosomal

elements. Gene 20:305-3169. Cohen SN, Chang ACY, Hsu CL (1972) Nonchromosomal

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(a) Enterobacterloacae FBA30(pFBA30IpFBA31) (b) E. coi JM103 FBA30-10(pFBA30-10)

0.30.3

0.25 E 0.25

o 0

0.20 0.* ~~~~~~~~~~~~~~~~~~~~~~0

E 1. 25 0.18o 10.

U) 0

.00.05 .00.05.

0 ,0

0 1 2 3 4 5 6 7 80 1 2 3 4 5 6 7 8

Time (h) Time (h)

(c) E. coil JMIO FBA3O0-1t(pFBA30-1) (d) E. coil JM103FBA30-12(pFBA30-12)

duplicate pinsx ii gotwh )ad iht-)ruuen. Nov7t8hei.rh(d0.2

eo E

co0.15c 0.

0~~~~~~~~~~~~~~~~~~~~~~(0~~~~~~~~~~~~~~~0.15- 0.15

0 0

0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8

Time (h) Time (h)

Fig. 4. Comparisonof growthof strainscarrying he pFBA30-typenickel determinant n the presenceof NiCl2.Eachgraphrepresents

duplicate experiments examining growthwith (um)and without (As) preculture n 0.5 mmiNi over a 7- to 8-h period. Graphs(a-d)

represent he wild-type and recombinantstrains; e-g), groupA strains;and (h-j), group B strains.

demonstrated constitutivenickel resistance,while those of

group B displayed nduction of resistancewhen precultured

in low levels of NiCl2.Thus, the nickel resistancegenes of

groupsA and B are taken as representatives f two branches

of a divergent gene family,which differ in their regulation

and flankingrestriction endonuclease sites. Furtherstudies

are underway o confirm the relationshipbetween the group

A and group B nickel resistance oci.

No homology with pFBA30was detected in nickel-resis-

tant bacteria isolated from sediments downstreamof the

outfall pipe. The time scale of the sampling indicatedthat

resistantbacteriaareconstantlydischarged rom the sewage

treatmentplant into the river. The differentnickel-resistant

populations found in eachsample further ndicatethatthese

resistantbacteriado not constitute a stablepopulation, but

rather one that changes with time in terms of its species

and genetic constitution. These bacteriarepresentpotentialcolonists of the sediments downstreamof the effluent pipe,

but isolates from these sediments did not possesshomology

with the nickel resistancedeterminantof pFBA30.Thispre-

sumably reflects he narrowhost rangeof this plasmid,and

a low freqencyof conjugative ransferbetween entericbacte-

ria and indigenous speciesin this ecosystem.

Acknowledgments

Support rom the Instituteof Freshwater cologyand Natural

EnvironmentResearchCouncil is gratefullyacknowledged.

Iwish to thankProf.Dr. H.G.Schlegel orsupplyingplasmids

carryingncc/nre,cnr, and Klebsiella xytoca-type esistances.

In addition,I would like to thankAnnickWilmotte (VITO,

Belgium)for supplyingpMOL154, and PamelaBroadbent,

Nick Whalley,and FrancisO'Neil, who have all contributedto this manuscript.

8/4/2019 4251493


236 R.W.Pickupet al.

moter. pFBA30- 1,with its insert in the same orientation as

that of pFBA30-10,displayeda slow growthrate and a re-

duced form of inducibility.

Environmentalsolates were also examined for inducibil-

ity of Nir.GroupA strains13, 17B,and 28 demonstrateda

common growth profileindicativeof a noninducibleconsti-tutive resistance Fig.4e,f,g). In contrast, groupB strainsall

showed a clear inducibleresponse(Fig. 4h,i,j). Growthrates

for these strainsin the presenceof nickelwere consistently

slowerthan that for FBA30.

Discussion

Transferof nickel resistance rom environmental amplesto

HB101or PaW340was not detectedby the exogenous plas-

mid isolation procedure. Endogenousmatings,againusing

the samerecipients, dentifiedonly a singlestrain out of 100

isolates that could transfernickel resistanceby conjugation.

This may indicate the inabilityof these laboratory trainsto

act as recipientsin matingswith recentlyisolated bacteria

undertheconditions used here. It is significant hatallnickel-

resistantstrains were entericspecies,and this, in itself,may

preclude PaW340 from stablyinheritingnarrow host range

plasmidsfrom thesesources.However,previousstudies have

isolated metal resistant trainsby these methods(silverresis-

tant plasmids, Pickup, unpublished data), and the results

mayinstead indicate a verylow frequencyof plasmid-borne

resistance n these bacterialpopulations.

FBA30 was tentativelyidentified as E. cloacae and was

found to containtwo plasmids.One of thesewas designated

pFBA30and is describedas a conjugative nickel-resistance

plasmid of approximately55 kb. It is considered to have a

narrowhost rangesince it wasreadily ntroduced nto enteric

relativessuch as E. coli, E. aerogenes, nd S. rubidae,but not

intooxidase-positive trains uch as P.putidaor P.fluorescens.TheplasmidpFBA3 found in FBA30s smaller hanpFBA30

and was not transmissible o otherstrains;t wasnot consid-

ered further.

The nickel-resistance ocus was cloned on a 10.2-kb SstI

restrictionfragment of pFBA30 DNA, and localized to an

internal2.3-kb PvuII ragment.Preliminary haracterization

of the nickelresistancedeterminantcarriedby pFBA30 ndi-

cates that it is not relatedto any of the previouslydescribed

nickelresistance amiliessuch as ncc/nre, nr,or the determi-

nant from K. oxytoca [32, 33, 39, 40], and is apparentlyconfined to entericbacteria.Furthermore, nlike othernickel

resistancesystems [21, 24, 32, 37], that of pFBA30does not

provide resistanceto metals such as cobalt, zinc, mercury,

chromate, or cadmium. Therefore, the nickel resistance

gene(s)of pFBA30representsanother,unrelatedgene family

and was designated he nrfdeterminant.

We alsoreportherein hat this nickelresistancewasinduc-ible, both in E.cloacae nd in other entericspecies.Inducibil-

ity was retainedby the 10.2-kb SstI restrictionfragmentof

pFBA30-10,but not by the 2.3-kb PvuIIfragmentderived

fromthis plasmid.pFBA30-11andpFBA30-12, achcarrying

the PvuII ragmentn oppositeorientations,displayed onsti-

tutiveexpressionand slowergrowth n thepresenceof nickel.

This may stem from the loss of regulatory genes located

outside the fragment,but this has yet to be confirmedby

complementationstudies. It is consideredunlikelythat the

effect is due to vector promotersoverridingthe inducibleexpressionof the cloned nickelresistancegene(s), since con-

struction of these plasmidsremoves the regulatedplac pro-

moter of pUC19, and the only otheravailablepromoters ie

in the ori region of the plasmid.

Use of the 10.2-kbSstIrestriction ragmentas ahybridiza-

tion probe againstDNA extracted rom environmental so-

lates of the Church Beck site identified 95 strains(24% of

nickel-resistantsolates) sharing imited homology with the

nickel resistanceocus of pFBA30.Ofthese,32 showedstrong

homology with the probe fragmentunderconditions of high

stringency.These 32 isolates were furtherchallengedby hy-

bridizationexperimentsusinga 1.75-kbSmaI ragment rom

pFBA30-10.Six of these demonstratedextensivehomology

with this probe and were retained for furtherstudy. The

nickel resistanceof these six strainsappeared o be located

on the bacterialchromosome of each,rather han on a plas-

mid. The majority of previously reported nickel resistance

geneshave been shown to reside on plasmids[39], although

chromosomal resistancehas been reported for Alcaligenes

denitrificansa-2 and K. oxytoca[15, 40].

These six strainswere classified nto two groups, desig-

nated A and B, distinguishableby the date on which each

was sampled from the environment.The two groups can

be furtherdistinguishedby the size of chromosomal PvuII

restriction ragments haringhomologywith the nickel resis-

tance gene(s) of pFBA30.GroupA and B strainscarry6.5-

and 4.7-kbPvuII ragments,respectively.which arehomolo-

gous with the 1.75-kb SmaIprobe fragment from pFBA30.

All these strains are enteric species associatedwith sewage

discharge.It is worth noting that one of these species (K.

oxytoca; strain13 of groupA) has already een associatedwith nickel resistance [39, 40]. All three group A strains

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