Analysis for the presence of determinants involved in the transport of mercury across bacterial membrane from polluted water bodies of India

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ht t p : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Analysis

for

the

presence

of

determinants

involved

in

the

transport

of

mercury

across

bacterial

membrane

from

polluted

water

bodies

of

India

Arif

Tasleem

Jan

a,∗

_,

_Mudsser

_Azam

b

_,

_Inho

_Choi

a

_,

_Arif

_Ali

b

_,

_Qazi

_Mohd.

_Rizwanul

_Haq

b

a_School_of_{Biotechnology,}_Yeungnam_University,_Gyeongsan_712-749,_Republic_of_Korea b_Department_of_Biosciences,_Jamia_Millia_Islamia,_New_Delhi_110025,_India

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Articlehistory:

Received7January2015 Accepted14June2015 AssociateEditor:MiguelJuan Beltran-Garcia Keywords: Bioremediation Mercury Resistancedeterminants Mercurytransport

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Mercury,whichisubiquitousandrecalcitranttobiodegradationprocesses,threatenshuman healthbyescapingtotheenvironmentviavariousnaturalandanthropogenicactivities. Non-biodegradabilityofmercurypollutantshasnecessitatedthedevelopmentand imple-mentationofeconomicalternativeswithpromisingpotentialtoremovemetalsfromthe environment.Enhancementofmicrobialbasedremediationstrategiesthroughgenetic engi-neeringapproachesprovidesonesuchalternativewithapromisingfuture.Inthisstudy, bacterialisolatesinhabitingpollutedsiteswerescreenedfortolerancetovarying concen-trationsofmercuricchloride.Followingidentiﬁcation,severalPseudomonasandKlebsiella

specieswerefoundtoexhibitthehighesttolerancetobothorganicandinorganic mer-cury.Screenedbacterialisolateswereexaminedfortheirgeneticmake-upintermsofthe presenceofgenes(merPandmerT)involvedinthetransportofmercuryacrossthe mem-braneeitheraloneorincombinationtodealwiththetoxicmercury.Genesequenceanalysis revealedthatthemerPgeneshowed86–99%homology,whilethemerTgeneshowed>98% homologywithpreviouslyreportedsequences.Byexploringthegenesinvolvedin impart-ingmetalresistancetobacteria,thisstudywillservetohighlightthecredentialsthatare particularlyadvantageousfortheirpracticalapplicationtoremediationofmercuryfromthe environment.

∗ _{Corresponding}_author_at:_School_of_{Biotechnology,}_Yeungnam_University,_Gyeongsan_712-749,_Republic_of_Korea.

E-mail:[email protected](A.T.Jan).

http://dx.doi.org/10.1016/j.bjm.2015.11.023

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Introduction

Pollutionwithtoxicmetalshasaccelerateddramaticallysince the beginning of the industrial age. Mercury is the sixth mostabundanttoxicelementamong6millionknowntoxic substances. Beingrecalcitrant tobiodegradation, it persists in the environment though bioaccumulation, thereby pre-sentingagreatthreattohumanhealth.Soonafteritsrelease into the environment in metal or ionic form, mercury is able to become methylated to highly toxic organomercu-rialcompounds.1,2 _Mercury_{contamination}_presents_a_major

healthproblemowingtoitsabilitytocrosstheplacentaland blood-brainbarrier.3,4 _Intentional_or_{unintentional}_exposure

to mercury resultsin acquisition of resistancein bacteria, enablingthemtothriveinenvironmentswithconcentrations farabovenormallevels.Mercuryresistancedeterminantsthat occurgloballyinbacteriafromnaturalenvironments facili-tatetheirtransformationtoovercometheirdeleteriouseffects onhumanhealth.5–7 _The_most_studied_mechanism_involves

enzymatic transformation based on clustering ofdifferent determinantsinan operon(mer operon).The meroperons, which show somegenetic variation instructure, are com-posedofgenes encodingfunctional proteinsfor regulation (merR,merD),transportgenes(merT,merP)andgenesinvolved inreduction(merB,merA).8,9_{Additionally,}_genes_such_as_merC,

merE,merHandmerF(allmembraneproteins)arebelievedto assistintransportfunctions,10–12_and_merG_confers_resistance

tophenylmercury.13,14

Environmentaldecontaminationofpollutedsitesremains oneofthemainchallengesforsustainabledevelopment.In our previous study, we showed that, among the screened bacterialisolates,onlythree(Pseudomonasaeruginosa(ARY1),

Klebsiellasp. (ND3)and Klebsiellapneumonia sp.(ND6)) con-tained the broad spectrum mercury resistance operon.15

Theseresultsindicatedthatresistanceinmostofourisolates

is mediated byother genes ofmer operons. Although this bacterialresistancesystemrepresentsamodelforbiological detoxiﬁcationoforganicmercury,theseﬁndingsindicatethat studiesofdeterminantsinvolvedinthetransportofmercury across thebacterialmembraneisessential beforetheycan beemployedtoachievemercuryremediationfrompolluted sites.Incontinuationofourpreviousstudy,thepresent inves-tigationwascarriedouttoexaminethegeneticmake-upof mercuryresistantbacteriaintermsofthepresenceof differ-entgenesofthemeroperoneithersinglyorincombination to dealwith toxicmercury. Despite the fact that mercury-reducingbacteriarepresentanimportanttoolforremediation ofcontaminatedsites,itisstillnecessarytoinvestigatethe genesinvolvedinthetransportofmercury(Hg2+₎_into_the_cell

forreductiontothevolatileelementalformtoenabledesign of strategiestocombat its removalfrom the environment. As microbebaseddetoxiﬁcationofmercury ison forefront ofremediationstrategies,studiesbasedoncharacterization ofmercuryresistantdeterminantsinvolvedinthetransport wouldprovideagoodfoundationforunderstandingthe com-pletestructureoftypicalmercuryresistancemodulesamong screenedbacteriaisolatestofacilitatetheirmanipulationfor bioremediationofcontaminatedsites.

Materials

and

methods

Screeningofbacteriaandgrowthinhibitionassay

Followingcoldvaporatomicabsorptionspectroscopy(CVAAS) ofcollectedwatersamplesforthedeterminationofmercury load,screenedbacterialisolateswere checkedfortheir tol-erance tovariedconcentrationofmercuric chloride(10␮M, 100␮M,1000␮M),byinoculatingtheminluriabroth,followed byincubationat37◦Cfor16–18honarotatorplatform incu-batorshaker(SCIGENICS)operatingat250rpm.Pseudomonas

Table1–Growthofbacterialisolatesinpresenceofvaryingconcentrationsofmercuricchloride.

Conc Pseudomonas aeruginosa (ATCC 9027) ARY1 (FJ613642) ARY4 (FJ613643) ARY2 (FJ613644) ARY7 (HM149547) ARY3 (FJ613645) ARTK3 (HM149545) ARH4 (HM149546) ARFA (HM149549) ARFB (HM149550) 0.1␮M ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ 1␮M ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ 10␮M ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ 100␮M + ++ ++ + ++ + ++ + ++ ++ 1000␮M – ++ ++ – ++ – ++ – – – 10,000␮M – – – – – – – – – – Conc ARKK (HM149548) ARSA3 (HM149552) ARSA4 (HM149551) ARR4 (HM149544) ND1 (JF927778) ND2 (JF927779) ND3 (JF927780) ND5 (JF927781) ND6 (JF927782) ND7 (JF927783) 0.1␮M ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ 1␮M ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ 10␮M ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ 100␮M ++ + + ++ ++ ++ ++ ++ ++ – 1000␮M – – – ++ – – ++ ++ ++ – 10,000␮M – – – – – – – – – –

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aeruginosaATCC9027wasusedaspositivecontrolinallsets ofexperiment.

Identiﬁcationofbacteriabasedon16SrRNAgeneanalysis

PCR ampliﬁcation of the 16S rRNA gene from different isolates was achieved using two primer sets: Primer set 1 [Pf1 5 GCAGTGGGGAATATTGGACAATCC 3 and

PR1 5 ATGAGGACTTGACGTCATCCCCA 3] and Primer set 2 [PF2 5 AAGGCGACGATCCGTAACTGG 3 and PR2 5 AACCACATGCTCCACCGCTTG 3]. The following ampli-fication profile was used: initial denaturation at 94◦C for 5min, followed by 35 cycles of denaturation at 94◦C for 1min, annealing at 56◦C for 1min and extension at 72◦C for 2min and then a final extension at 72◦C for 5min.

Table2–PhylogeneticafﬁliationandGenBankaccessionnumbersofmerPandmerTgenesequencesofbacterialisolates investigatedinthisstudy.

Sample collectionsite Bestmatch (GenBank Acc.no.) Similarity (%) Microbialgroup afﬁliation GenBank Acc.no.of 16SrRNA GenBank Acc.no.of merPgene GenBank Acc.no.of merTgene Rajkotdrain (Gujrat),India Aeromonasveronii (AB472977) 99.7 Aeromonasveronii HM149544 JN188345 JN188348 Hooglyriver (Kolkata),India Pseudomonassp. (HQ105014) 99.8 Pseudomonasstutzeri HM149545 JN188343 JN188351 Hooglyriver (Kolkata),India Unculturedbacteria (HM328775)or Acinetobactersp. (FN377701) 98.8 Acinetobactersp. HM149546 – JN188352 Yamunariver (Agra),India Pseudomonassp. (HM234002) 99.4 Pseudomonas aeruginosa HM149547 JN188338 JN188356 Kodaikanallake (Tamilnadu), India Pseudomonassp. (HM566026) 99.3 Pseudomonasstutzeri HM149548 – – Kodaikanallake (Tamilnadu), India Citrobacterfreundii (HM756481) 98.9 Citrobacterfreundii HM149549 JN188335 JN188346 Kodaikanallake (Tamilnadu), India Citrobacterfreundii (HM756481) 99.7 Citrobacterfreundii HM149550 JN188340 – Hindonriver (Ghaziabad), India Uncultured ␥-proteobacteria (AB234527)or Enterobactersp. (FJ668827) 98.9 Enterobactersp. HM149551 JN188337 JN188350 Hindonriver (Ghaziabad), India Pantoeaagglomerans (EF429005) 98.6 Pantoeaagglomerans HM149552 JN188336 JN188354 Yamunariver (Okhla),India Pseudomonassp. (HM234002) 99.8 Pseudomonas aeruginosa FJ613642 JN188334 JN188355 Yamunariver (Okhla),India Unculturedbacteria (HQ008634) 99.4 E.coli FJ613643 JNN188342 JN188349 Yamunariver (Agra),India Unculturedbacteria (HM335010) 99.2 Citrobacterfreundii FJ613644 JN188344 JN188347 Yamunariver (Faridabad), India Citrobacterfreundii (HM756481) 99.5 Citrobacterfreundii FJ613645 – – Najafgarhdrain (Delhi),India

Aeromonasjandaei 99.4 Aeromonassp. JF927778 JN188332 –

Najafgarhdrain (Delhi),India Uncultures Pseudomonassp. 99.5 Pseudomonassp. JF927779 – – Najafgarhdrain (Delhi),India Klebsiellavariicola strainJDM-14 99.2 Klebsiellasp. JF927780 JN188341 JN188353 Najafgarhdrain (Delhi),India Acinetobactersp. F71019 99.7 Acinetobactersp. JF927781 – – Najafgarhdrain (Delhi),India Klebsiellavariicola strainJDM-14 99.6 Klebsiellapneumonia sp. JF927782 JN188333 – Najafgarhdrain (Delhi),India Acinetobactersp. F71019 99.4 Acinetobactersp. JF927783 – –

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DNAextractionandPCRampliﬁcationofmercury resistantdeterminants

Toinvestigatethediversityofmercuryresistantdeterminants (merPandmerTgenes),primersfortheCDSregionofmerPand

merTgenesweredesignedsothattheywerenotself compli-mentarytopreventtheformationofprimerdimers.Following analysisofthesequencesretrievedfromGenBankusingthe CLUSTAL W option in the BioEdit 5.0.9 sequence analysis software,thefollowingrespectiveprimersweredesignedfor ampliﬁcationofDNAfragmentscorrespondingtomerPand

merTgenes:Pf5 ATGAAGAAACTGTTTGCCTCC3 andPR5 TCACTGCTTGACGCTGGACG 3 and Tf 5 ATGTCTGAACCA-CAAAACGGG 3 and TR 5 TTAATAGAAAAATGGAACGAC3. PCRampliﬁcationformerPandmerTgenesfromdifferent iso-lateswascarriedout ina50␮lreactionvolume containing 2␮l DNA (110ng/␮l), 15␮l10× Taq DNA Polymerase buffer

(with1.0–2.5mMMgCl2),2␮lprimer(10picomolarforwardand

reverse),5␮lof10×dNTPmix,2unitsofTaqDNApolymerase (Fermentas,USA)and22␮lsterilewaterinanautomated ther-mocycler (Techne Tc-312) with the following amplification profile:initialdenaturationat92◦Cfor5min,followedby35 cyclesofdenaturationat92◦Cfor1min,annealingat58.5◦C (merP) or55.5◦C (merT)for1minandextensionat72◦Cfor 1min,followedbyfinalextensionat72◦Cfor10min.

Sequencingandphylogeneticanalysisofmercuryresistant determinants

Forphylogeneticstudies,PCRproductscorrespondingtothe expected size of the merP and merT gene sequence were puriﬁedusingaQIAquickspincolumn(QiagenInc.)under themanufacturerspeciﬁcations.Sequencingreactions corre-sponding tomerP and merTgenes from the isolates under

Pseudomonas stutzeri ARTK3

E.coli ARY4

Klebsiella sp.ND3

Pseudomonas aeruginosa ARY7

Pseudomonas aeruginosa ATCC 9027

Citrobacter freundii ARFB

TRICHOTOMY 561 442 732 938 987 709 Aeromonas sp. ND1 1000 981 1000 1000 1000 Salmonella enterica

Citrobacter freundii ARY2

973

0.1

Aeromonas veronii ARR4

Aeromonas salmonicida A449

972

Pantoea agglomerans ARSA3

E. coli plasmid NR1

Plasmid R100 Enterobacter sp. ARSA4 Klebsiella pneumoniae ND6

Citrobacter freundii ARFA

713 ₉₅₇

Fig.1–Phylogramdrawnusingtheneighbornetmethod(bootstrapanalysiswith1000replicates)illustratingphylogenetic relationshipsbasedonmultiplealignmentsofmerPnucleotidesequencesfromstudiedisolateswithotherknown

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studyweredeterminedusingdeﬁnedprimerswithan auto-mated sequencer (ABI 1377) at Xcelris Laboratory, Gujarat (India).NucleotidesequencescorrespondingtomerPandmerT

genes were aligned with the CLUSTAL W algorithm using theBioEdit program.Thetopologyofthe phylogenetictree constructedwithnucleotidesequenceswasassessedbythe neighbor-joining(NJ)methodwith1000bootstrapreplications.

Nucleotidesequenceaccessionnumber

CompletegenesequencesofmerPandmerTgeneshavebeen deposited inthe GenBankdatabase under accession num-bersJN188332–JN188356,whileJF927784correspondstomerT

ofATCC9027.

Results

and

discussion

Following cold vapor atomic absorption spectroscopy (CV-AAS) for the determination of mercury load in samples collected from different polluted sites, samples were sub-jected to screeningformercury resistant bacteria on Luria Agarsupplementedwith0.1␮Mofmercuric chloride.After initialscreeningon Luria agar,the abilityofscreened bac-terialisolatestotoleratevariousconcentrationsofmercuric chloride(10␮M,100␮M,1000␮M)wasinvestigated.Following initialscreening,18of80bacterialisolatesfoundtobe tol-erant tovariousconcentrations ofmercuric chloride, along withonesensitiveisolatefromNajafgarhdrain,wereselected for furtherscreening (Table1). Isolates ARY1,ARY4,ARY7,

E.coli ARY4 150 480 576 542 975 867 879 595 1000 940 1000 Salmonella enterica

Pseudomonas aeruginosa ARY7

394

902 0.1

Pseudomonas stutzeri ARTK3

Plasmid R100

Aeromonas salmonicida A449

TRICHOTOMY

E.coli plasmid NR1 Pantoea agglomerans ARSA3 Enterobacter sp. ARSA4 Citrobacter freundii ARFA

955 552

Aeromonas sp. ND1

Klebsiella pneumoniae ND6 Klebsiella sp. ND3

Citrobacter freundii ARFB

Fig.2–Phylogramdrawnusingtheneighbornetmethod(bootstrapanalysiswith1000replicates)illustratingphylogenetic relationshipbasedonmultiplealignmentsofmerTnucleotidesequencesfromstudiedisolateswithotherknown

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ARTK3,ARR4,ND1,ND3andND6werefoundtogrowon mer-curicchlorideandtolerategreaterconcentrations(1000␮M) thantherestoftheisolates,whichcouldonlytolerate100␮M. Theseresultsclearlydemonstratethatthecollectedisolates show variable tolerance to mercuric chloride. Speciﬁcally, growthwasobviouslysuppressedinpresenceof1000␮M mer-curicchloride,withdelayedexponentialphasesaccomplished uponconversion ofthetoxicformofmercurytoless toxic formsbyenzymesencodedbydifferentmeroperongenes.

Following identiﬁcation based onbiochemical tests, 16S rRNAgeneanalysiswasperformed.Thefeasibilityofusing 16SrRNAsequencesfortheidentiﬁcationofscreenedbacterial isolateshasbeenreportedpreviously.15_Sequences_of_the_16S

rRNAgenefromallisolateswerealignedagainsttheavailable sequenceswithwhichtheyshowedaclosematchinthe Gen-BankdatabaseandanalyzedusingtheCLUSTALWoptionin theBioEdit5.0.9sequenceanalysisprogram(Table2).Species identiﬁcationwasdeterminedfromthebest-scoringreference sequence in the databases,with ≥98% homology withthe querysequencebeingtakentoindicateaperfectmatch.

Afterscreening,allbacterialisolateswereanalyzedforthe presenceofmercuryresistancedeterminantsthatarebelieved

toplayanimportantroleinimpartingresistancetodifferent formsofmercurybyPCR.Byusingthedesignedgenespeciﬁc primers,segmentsofDNAsequencescorrespondingtomerP

andmerTgenesencodingmercurytransportingproteinswere ampliﬁed.Ofthe18screenedisolates,only14(inthecaseof

merPgene)and12(inthecaseofmerTgene)generatedpositive ampliﬁcationproductsof∼276bpand∼351bpwithprimers speciﬁcforthemerPandmerTgenes,respectively.Moreover,

merPandmerT,whichareinvolvedinthetransportofHg2+

acrossbacterialmembrane,werefoundtobemoreprevalent thanothermercuryresistantdeterminantssuchasthemerB

gene.

Ampliﬁedproductscorresponding tothe merPandmerT

genes were sequenced directly using the deﬁned primers. Sequence homology analysis of the transporter proteins encodedbythemerPand merTgenethatform characteris-ticfeaturesofthemeroperonwereperformedtoinvestigate the variabilityofthesegenesinthebacterialisolatesbeing investigated (S.Figs. 1and 2). DNAsequencing ofthe PCR productsfollowedbysequencesimilaritysearchesusingthe advancedBLASTsearchprogramoftheNCBIdatabaseagainst the retrievedsequences revealedthatthe screenedisolates

Enterobacter sp. ARSA4

103 89

Citrobacter veronii ARY2

TRICHOTOMY

Klebsiella sp. ND3 425 6 520 871 E.coli E.coli ARY4

Pseudomonas aeruginosa ARY7 958

0.01 245

Pantoea agglomerans ARSA3

955

937 948

Enterobacter cloacae

Pseudomonas aeruginosa plasmid R1033

Salmonella enterica Pseudomonas aeruginosa ARY1

464

417

Acinetobacter sp. ARH4

Fig.3–Phylogramdrawnusingtheneighbornetmethod(bootstrapanalysiswith1000replicates)illustratingphylogenetic relationshipsbasedonmultiplealignmentsofMerPsequencesfromstudiedisolateswithotherknownsequences.

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Klebsiella sp. ND3

33 52 25 10 36 659 666 915 913 841 870 Enterobacter cloacae

Pseudomonas aeruginosa ATCC 9027 669

0.01

Pseudomonas aeruginosa plasmid R1033

Salmonella enterica Pantoea agglomerans ARSA3

E.coli E.coli ARY4

Acinetobacter sp. ARH4

25

TRICHOTOMY

Enterobacter sp. ARSA4 Aeromonas veronii ARR4

Fig.4–Phylogramdrawnusingtheneighbornetmethod(bootstrapanalysiswith1000replicates)illustratingphylogenetic relationshipsbasedonmultiplealignmentsofMerTsequencesfromstudiedisolateswithotherknownsequences.

were 86–99% homologous with the reported sequences of the merP gene and >98% homologous with the reported sequencesofthemerTgeneatthenucleotidelevel, respec-tively(Figs.1and2).When comparedtomerP,the deduced aminoacidsequenceforthecorrespondingregionofthemerT

geneshowedhighsimilaritytopreviouslyreportedsequences ofthemerPandmerTgenes(Figs.3and4).Thecurrent inves-tigationdemonstratedthat,inadditiontocarryingthegene encodingorganomercuriallyase,isolatesARY1(Pseudomonas

aeruginosa)fromtheYamunaRiver,okhlaandND3(Klebsiella sp.)fromNajafgarhdraininDelhiwerealsopositiveforthe

merPandmerTgene.Moreover,isolateND6(Klebsiella pneumo-niasp.)fromNajafgarhdrainwasfoundtobepositiveforthe presenceofmerPandmerB,but waslackingthemerT com-ponentofthemeroperon.Theseﬁndings indicate thatthe functionofmerTinthisisolateiseithercompensatedforby someothertransportergeneorperformedbythemerPgene alone.TheresultsofthisstudyalsoindicatedthatARY1,ND3 andND6harboringresistancedeterminantsindifferent com-binations, could tolerate the highestconcentrationof both organic(PMA)andinorganic(HgCl2)formsofmercury.

Com-paredtootherisolates,ARY1andND3,whichcontainedboth mercuryresistancedeterminants(merPandmerT)inaddition tothemerBgene,aresuitablecandidatesthatcanbeutilized

for the remediation of mercury from heavily polluted sites.

Conclusion

Owingtothewidedistributionofmercuryanditspotential deleterious effects on human health, interest in biodegra-dationmechanismshasreceivedincreasingpublicinterest. Whencomparedtophysicalandchemicalmethods,useof bio-logicalagentstoremediatecontaminants,especiallymercury, isofgreatpracticalimportancebecausetheyprovidesimple but effectivesystemsforremediationofpolluted surround-ings.Theabilityofbacteriatowithstandhighconcentrations ofmercurybyintracellularsequestrationfollowed by enzy-matic reductiontolesseror non-toxicformshasincreased interestinisolatingstrainswithhighcapacitytoremediate mercurycompounds.Genesconferringresistancetoorganic andinorganicmercurycompoundsarecommonamong bacte-ria. Among the various resistance systems that bacteria employ to overcome the toxicity of mercury compounds, the most studied mechanism is the enzymatic transfor-mation of organomercurials to Hg2+_, _and _its _subsequent

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ions (Hg2+₎ _is _conferred_by _mercuric _reductase, _which

cat-alyzestheNAD(P)H-dependentreductionofHg2+_to_Hg0_,_that

volatilizesintotheimmediateenvironment.1_The_above

bacte-rialdefensesystemtodetoxifymercuryisbasedonclustering genesthatactinacoordinatedmannertotransfermercury intotheinteriorforreductiontovolatilemetallic(Hg0_).

Resistancetomercurythatisaccreditedtoitsreduction into less toxic Hg0 _by_the _merB _and _merA_genes _encoding

organomercuriallyaseandmercuricreductase,respectively, isdependentonthegenesinvolvedinthetransportof mer-curyintotheinteriorofthecell.16–19_{Investigation}_of_the_genes

thatperformthetransportfunction,merPandmerT,revealed thattheyweremoreprevalentamongthescreenedbacterial isolates. These genesact ina coordinated mannerto gov-ernuptakeofmercury(Hg2+₎_across_the_bacterial_membrane,

afterwhichtheyundergotransformationintolesstoxicforms. Additionally,genessuchasmerC,mere,merFandothergenes knowntoassistinthetransportfunctionarebelievedto facili-tatethetransportofmercuryacrossthemembraneinisolates thatlackthemerPandmerTgenes.Growthinthepresence of1000␮M dilution ofboth C6H5Hg+ and Hg2+ among

iso-latesARY1,ND3 andND6 bearingthemerP, merTandmerB

genessuggestthattheseisolatesarebetteradaptedtosurvive atsiteswithhighlevelsofmercuryandcapableof transfor-mingC6H5Hg+andHg2+,insteadofanybuild-up.Becausenot

everymicrobepossessestheabilitytodegrademercury com-poundsornorpossessingcapacitytotransformingtransform it,exploitingbacteriahavingenzymesforthisprocessto hap-penwouldfacilitategeneticmanipulationoftheseorganisms toachievedecontaminationofpollutedsites.

Conﬂict

of

interest

Theauthorshavenoconﬂictofinteresttodeclare.

Acknowledgement

Arif Tasleem Jan thanks Yeungnam University for provid-ingfacilitiestoperformexperiments.Additionally,Mudsser AzamthankstheCouncilforScientiﬁcandIndustrialResearch (CSIR),India,forﬁnancialassistancethroughanawarded fel-lowship.

Appendix

A. Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2015.11.023.

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