A step forward to empower global microbiome research through local leadership.

Science

&

Society

A

Step

Forward

to

Empower

Global

Microbiome

Research

Through

Local

Leadership

Victor

S.

Pylro,

*

Tsai

S.

Mui,

Jorge

L.M.

Rodrigues,

Fernando

D.

Andreote,

Luiz

F.W.

Roesch,

*

and

the

Working

Group

Supporting

the

INCT

Microbiome

Obtaining the full microbial potential to benefit local communities and citizens, as wellas ongoing conservation efforts, isamajorchallenge forBraziland other developing countries. We propose poli-cies and priorities for organizing micro-biome studies locally and worldwide, aimingforacomprehensivecatalogueof microbiomes,asrecentlyurged.

Microorganismsarerecognizedasa fun-damentalresourceforcreatingafastand efficientstrategyforecosystem manage-ment and scientific and technological development. Microbial community assemblyandfunctionsaretightlylinked to local geographic and environmental features [1]. Developing countries, such as Brazil, house biomes evolving under specific environmental conditions that likely harbor unique microbiomes. Chal-lenges in profiling microbial diversity include a lack of standardized methods and metadata collection that precludes robust interstudy comparisons, limiting thevalueofthesestudies[2–4].Advances in sequencing technologies and associ-ated bioinformatics approaches should nowenablecomparison of thediversity, abundance, and function of microbial communities at much greater resolution thanwaspreviouslypossible[5].Recently, threepapershavepointedtotheneedfor microbiomeresearchcoordination[4,6,7]. Theseproposalsenvisionedguidelinesfor

intellectual-propertyrights,research prior-ities for funding (including a long-term interagency funding strategy),the devel-opment of new analytical tools, training programs, data integration through an in-countrydistributeddatacenterand pol-iciesondatasharing.Weargueherethat thereisaneedfordevelopinglocal lead-ership in microbiome research [8] and proposeestablishinganinitiativetofoster international collaborations in order to marshal microbiome research in Brazil. Recently,theUSgovernment,alongwith theprivatesector,has announceda sig-nificant budget to launch the National MicrobiomeInitiative(NMI)[9]. Consider-inggeographyasanimportantpartofthe microbiome puzzle, the development of local initiatives will strengthen the NMI and provide a basis for a global micro-biomeeffort[6].

In2008,theBrazilianMinistryofScience andTechnologyconsolidatedan impor-tantstrategytorestructurepoliciesof sci-entific development, implementing the NationalInstitutesofScienceand Tech-nology (INCT in Portuguese). Themain objectivesoftheINCTaretostrengthen nationalresearch,increasetraining, inter-nationalize groups, and transfer knowl-edge to society, businesssectors, and government when appropriate. Despite a direct relationship with the strategic themesestablishedbypublicpoliciesof theBraziliangovernment,thesystematic studyofmicrobiomesandtheir biotech-nological potential remain limited. We haveproposedasystematicstudyof Bra-zil's microbial resources as a valuable new focus area for the INCT efforts. Although pressed by the economic slump, political crisis, and their conse-quences for science and technology

[10],Brazilismovingforwardon micro-biomeresearch.TheNationalCouncilof Technological and Scientific Develop-ment(CNPq)hasrecommendedthe cre-ation of a National Institute of Science and Technology to advance microbial research–the INCT Microbiomei. During thenextmonths,acombinedevaluation

bytheCNPqandotherBrazilianagencies supporting research, such as CAPES, FINEPandFAPESP(SãoPauloResearch Foundation),willforgeadefinitive agree-mentforassemblingthisinstitute, poten-tially awarding it a budget of up to 10 million Brazilian Reais(US$ 2.8million). TheINCTMicrobiomeaimstobeacenter of international excellence seeking to coordinate and provide guidance to microbiome researchers, besides bal-ancinginequality ofresearch opportuni-ties in Brazil. Low- and middle-income countriesareusuallypreventedfrom per-forming state-of-the-art microbiome researchduetoalackofstructureorto alackofwell-trainedpeople.Local initia-tives might benefit from a core center housing all the necessary infrastructure and trained scientists, to provide high-qualityprojectdesign,cutting-edge tech-nology(sampleprocessing,sequencing, etc.) and training on data analyses to deliverthebestsolutionstocollaborators. The benefits brought by such an endeavorwillbeessentialtoconsolidate thelocalinitiativeenvisionedtoadvance Brazilianmicrobial studies[8]. Wehope thatthisinitiativecanserveasatemplate forleadershipofmicrobiomeresearchin otherdevelopingcountries.

The Brazilian Microbiome Project(BMP)ii

[11]wassetupin2013,andthiswillform thebasisfortheINCTMicrobiome.Now, Brazilisexpectedtoadvanceonit,uniting microbiome investigatorsto support the developmentofanunprecedented knowl-edge base of microbial resources. In 2012,Brazilmovedforwardinthe assess-mentanduseofbiodiversitybyjoiningthe Global Biodiversity Information Facility (GBIF) and by creating the Biodiversity Portaliiiin2015.However,microbial infor-mation has been ignored, as has hap-pened in previous attempts to explore biologicalresources inBrazil[12].Anew databaseisrequiredtocombineBrazilian microbialdatawithotherbiodiversitydata, includingGBIF,andinagreementwiththe Brazilianlawsofbiodiversitydatasharing (normative instruction 02/2015 of the

Chico Mendes Institute for Biodiversity Conservation–ICMBio).

Four key issues have been identified as prioritiesforscientificresearch:(i) standard-ization of methods and integration with existinggroupsdevelopingprotocolsand standards,suchastheEarthMicrobiome Project(EMP)iv,TerraGenomevandQIITAvi, toensurethatstudiesaresystematicand comparable,allowingthedevelopmentofa dynamicand constantlyexpanding data-basethatcanprovidereal-timeinformation forallusers;(ii)characterizingmicrobiomes under natural conditions, which is of fundamental importance for establishing

comparativemodels;(iii)developing micro-bial interventions that allow the mainte-nance of the quality and productivity of the environment or host; and (iv) develop/expand partnership among aca-demic studies,agriculture,medicine,and industry. This well-defined approach is expected to increase the understanding ofBrazil'smicrobialresourceswiththegoal ofdevelopingstrategiesto:mitigate envi-ronmentalpollution;increasetheactivityof beneficialmicroorganismsfromsoils; sup-presspathogenicmicroorganismsinplants andhumans;andcreateanefficient strat-egyforscientificandtechnological biopro-specting.Toachievethesegoals,theINCT

Microbiomeneedstobeinherently collab-orative. We propose an organizational model that represents specific scientific researchdomains,andstrategic commit-tees focused on training and transfer of knowledge and technology (Figure 1). Researchdomaincommitteeswillbe the-matic, addressing microbial interactions withplants,animals,soils,aquatic environ-ments,andhumansaswellason biopro-specting. Each theme will consider research drivers, scan the horizon, and translateresearchintosocioeconomic rel-evance. Promising translational areas include the effects of pollution and land use change, improving agricultural

Bioinformacs knowledge transfer Plant microbiome Animal microbiome Soil microbiome Water microbiome Human microbiome Bioprospecng Standardizaon of methods and integraon with exisng efforts Characterizing microbiomes and developing microbial intervenons Linking academic studies to industry and society

Others...

Figure1.OrganizationalandFunctionalStructureoftheINCTMicrobiome.BrokenlinesrepresentpossiblefutureinteractionsoftheINCTDatabasewithotherinitiatives underway.

Box1.PrimaryHabitatsandHostsCurrentlyBeingAddressedbytheINCTMicrobiomeCollaborators

Amicrobiomeisdefinedas‘theentirehabitat,includingthemicrobes(bacteria,archaea,lowerandhighereukaryotes,andviruses),theirgenomes(i.e.,genes),andthe surroundingenvironmentalconditions’[14].Megadiversecountriesrepresentpromisingscenariosforbiotechnologicaladvances,whichcanbeenhancedbythelocal researcher'smicrobiomeconsortia.Acatalogueoftargetsformicrobialprospection,permeatingtheentireBrazilianterritory,rangingfromagriculturalimprovementsto humanhealthpromotion,isprovidedbelowasanexample:

(i) Microbiomesassociatedwithplants

 Themicrobiomeofwheatcultivarsandtheirancestors:manymicroorganismsassociatedwithrootscanbenefitplantsonnutritionandtolerancetobioticand abioticstressfactors.Foroveracentury,breedershaveexploredplantgenestoimprovetheirperformanceandproductivity,buttherhizospheremicrobiome hasbeenneglected.Exploringthemicrobiomeofwheatcultivarsandtheirancestorswillallowforthedevelopmentofnewapproachesapplyingmicroorganisms tosustainablyimproveplantdevelopmentandhealth.

 Themicrobiomeofsugarcane:plantsseekthehelpofmicrobesEvidencesuggeststhatplantsunderattack‘askforhelp’fromthemicrobiomeandactively recruitbeneficialmicrobesfortheirhealth.Asaresult,soilbecomessuppressivetodiseases.Characterizingthemicrobiomeofsugarcanewithcontrastinglevels ofresistancemightcontributetoempoweringbiologicalcontrolthroughspecificmicrobeactivities.

(ii) Microbiomesassociatedwithanimals

 Thecattle'sintestinalandskinmicrobiome:ruminants’microbiomesarenowbeingexploredassourcesofbiomass-degradingenzymestobeappliedinthe industrialproductionofbiofuels.Also,ticksusceptibilityincattlehasnowbeenrelatedtovolatilecompoundsproducedbyspecificskinmicrobiomeprofiles, makingitsmodulationahopefulstrategyforpromotingresistancetoectoparasites.Thecomparisonsofmicrobiomesofdifferentcattlebreeds,indifferent geographicalregionsandsubjecttodifferentmanagementpractices,willallowverificationofthedegreeofcontributionofgeneticmakeupandtheenvironment. Theknowledgegeneratedwillenablethedevelopmentofinnovativeandsustainabletechnologiesfortickcontrol,suchasprobioticsand/orrepellents.

 Intestinalmicrobiotaassociatedwithproductivityperformanceinbroilerchickens:theuseofantibioticsinanimalfeedasgrowthpromotershasresultedin increasedsusceptibilitytoentericdiseasesanddysbiosis,andcausedanegativeeffectontheproductivityofbroilerchickens.Researchhasshownvarious beneficialeffectsofusingprobiotics,prebiotics,symbiotic,organicacidsandplantextractsinthedietsofbirds.Thesumoftheresultsofthesestudieswillallow thenutritionisttoadoptoptimizedfeedingstrategies,inordertomodulatetheintestinalmicrobiotaofbroilerchickensforhigherperformance.

(iii) Microbiomesassociatedwithsoils

 Microbiomesofmangrovesoils:mangrovesarehighlyproductiveandhavearichassociatedbiodiversity.Theyaresubjecttoconstantanthropicimpacts,which arethemaincauseofdeteriorationoftheseecosystems.ThislineofresearchseekstoaccessthemicrobiomeofmangrovesoilsoftheNorth,Northeast, Southeast,andSouthernregionsofBrazil,correlatingthemwithenvironmentalvariablestobetterunderstandingthemicrobialtaxonomyandfunctioningin mangroves.

 MicrobiomeofCaatingabiomesoils(aridsoils):theCaatingabiomesuffersconstantlimitationsrelatedtowaterdeficit.Inrecentyears,aridityoftheCaatingahas beenincreasingduetohumanactivities,resultinginincreasederosionofsoils,microclimatechanges,anddesertification.Obtaininginformationregardingthe effectsofthesehabitatsonthediversityandfunctionofthemicrobialcommunityisessentialtoguidesustainablerecoverypracticesforthisbiome.Atthesame time,thisregionisapromisingtargettounravelnovelstrategiesofplantdroughtresistancemediatedbymicrobes.

 Microbiomeofagriculturalsoils:definingtheorganizingprinciplesthatcontroltheassemblyofsoilmicrobialcommunitiesunderland-usechangewillhelpto providethebasistounderstandhowindividualmicrobesandmicrobialcommunitiesfunctioninresponsetochangingenvironmentalparameters(atamolecular scale)andviceversa.Microbial-community-mediatedbiologicalprocesseswillbelinkedtoenvironmentalparameters,allowingustobuildpredictivemodelsof metabolicandregulatoryprocessesrelatedtothechangescausedbyagricultureandadoptingstrategiesformitigatingglobalheating.

(iv) Microbiomesassociatedwithaquaticenvironments

 Characterizingthemicrobiomeofwaterandsedimentsoftheriversandwaterreservoirs:environmentalpollutionisalsoanimportantissue,andinitiativesto mitigateitarehighlyimportanttopreserveriversandwaterreservoirs.Increasinganthropogenicactivitycanleadtoecologicalimbalanceofwaterbodiesandthe extinctionofkeymicrobialspecies.Acomprehensivemetagenomicstudycorrelatingthemicrobiomeandthetrophicconditionsallowforproposingsolutionsfor improvingthequalityofwateravailableinthereservoirs.

(v) Microbiomesassociatedwithhumanhealth

 Biocontrolofhumanvector-bornediseases:inthetropics,severalemergingmaladiesandvector-borneviraldiseaseshavethepotentialtocauseepidemics, suchasmalaria,denguefever,chikungunya,andmorerecentlytheZikavirus.Combatingthesevectorsandpathogenicagentsissomehowineffectiveby traditionalapproaches,butnovelinterventionssuchastheintroductionofaWolbachiastraininAedesaegyptihasreducedtheinfectionwithdengue, chikungunya,andZikavirusesaswellasPlasmodiumparasites.Biologicalcontrolanditsecologicalimplicationsarepromisingforsolvingpublichealthissues.

 Associationamongfetalmicrobiota,prematurity,andpretermmorbidities:thebiologicalcausesforpretermdeliveriesremainpoorlydefined.Multiplelinesof evidenceindicatethatthemicrobiomeinexpectantmotherscantriggerprematuredelivery.Identifyingsuchamechanismwouldhaveimportantimplicationsfor designingnutritionalinterventionsinpregnantmothersaimedatestablishingamicrobiotawithlowerriskforpretermdelivery.

(vi) Bioprospecting

 Oneofthemostpromisingapproachesforthediscoveryofnewbioactivecompoundsistheuseofmetagenomicsinthesearchfornewbiosyntheticgenesfor cloningandexpressioninaheterologousorhomologoushost.Bioprospectingallowsforthedevelopmentofnewbiotechnologicalsustainableprocesses relatedtobioremediation,biorefining,miningbioleaching,oilrecovery,orcleaningofoilstoragetanks,basedontheisolationofmicroorganismscapableof performingthesefunctions.

(vii) Bioinformatics

 Dataanalysisandpipelines:computationalresourcescanbedifficulttohandle,andtheeffectiveexecutionandreproducibilityofanalysesisachallengegiventhe amountandnatureofthedata.MetagenomicanalyseshavebeendescribedasoneoftheleastreproducibleNGSapplications,mainlyduetothelackof integratedandstandardizedsolutions.Therecentdevelopmentofstandardmethodsandprotocolsallowedproperdataanalysisandinterpretation.Uptonow, thisinitiativeprovidedthecommunitywithawebplatformfordataanalysis,aLinux-basedOperationalSystemdedicatedtodataanalysesandstandard methods,anddataanalysespipelinesii.Morethan10000usersfrom112countries(mostfromBrazil,theUSA,andtheUK)havealreadybenefited.

practices, water treatment and manage-ment, animal breeding, and microbial effectsonhumanhealth(Box1givesfor adetailedlistofprimarytargetsinBrazil). Expanding capacity in bioinformatics is critical because the current bottleneck forbiosciencesishowtoanalyze‘bigdata’

[4,13]. In-depth analysis of the growing numberofcompletelysequenced micro-bialgenomesinpublicdatabasesis pro-viding fascinating contributions to our understandingofhowmicrobesare genet-icallytailoredtotheirlifestyles.Thequality of this information critically depends on high-qualitygenomeannotation.Accurate annotation is challenging, and perhaps evenmoreimportanttonew understand-ingofmicrobes thanthedevelopmentof newsequencingtechnologies.For exam-ple,mobilegenomicelements(MGEs)are importantforpathogenicityand antibiotic resistance, but are often poorly under-stood because they are not well anno-tated. To address the annotation and otherrelatedissues,aneffective Bioinfor-maticsCommitteeshouldbeassembledto support satellite bioinformatics laborato-riesfor trainingcollaborative teams. This effortisparamounttoimprovedata inter-pretationandreproducibility,andtofoster thedevelopmentofnewprotocolstomeet thedemandsofadiversegroupof collab-orators.Studiesofmicrobiomesin partic-ularareunder-fundedinBrazil,andtiesto internationaleffortsandtoindustrywillbe importantfordevelopingappropriate bio-informaticstechniques.

A Committee for Knowledge Transfer is essentialtointegrateallothergroupsand tolinkuniversity/researchinstitutionsand society.Inadditiontoprovidingresources forimprovingbasiceducation,local lead-erships will make scientific knowledge accessibleto all, transferring information generated by the different research groups to society. Intellectual property (IP)issuesrepresentamajorconcernfor developing countries, and the improve-mentofalreadyestablishedIP organiza-tions should be addressed. Strong IP

protectionsystems benefitthecountry's economybyattractinginvestorsand fos-teringinnovation.Therestrictedbudgetof such countries reflects the lack of IP expertisehindering patent deposition.In agreement with the recently approved Congressional Law Project (PLC) 77/ 2015,whichregulateslong-term partner-shipsbetweenthepublicandprivate sec-tors inBrazil,localactivities allow better integrationofscientificdatawith entrepre-neurs. Eliminating these bureaucratic obstaclespromotesinnovationand tech-nologicaladvancesforexploringBrazilian biodiversity. The challengehere includes thecreationofa‘one-stopshopping ser-vice’ providing a single point ofcontact betweenpublicandprivatesectors, capa-bleofaddressingindividualchallenges,to offerboth sidesthemostappropriateIP solutions. Protecting the particular inter-ests oftraditional societies(e.g., indige-nous people) and sharing the benefits generated from the use of traditional knowledge in biotechnological develop-mentfairlyisalsoanimportantand ongo-ing discussion, now supported by the Brazilian Biodiversity Law 13.123/2015 anditsrecentregulatorydecree. Weurgesupportofthe integrative solu-tionsproposedhere,throughthecreation oftheINCTMicrobiome.Thislocal micro-biome initiative will not only addressthe Brazilianproblemsofimbalanceof micro-biome studies and data fragmentation, butwillserveasamodelhelpingtoguide futureinteragency funding efforts world-wide,besidesensuringappropriate proj-ect alignment with other international efforts [6,7]. We aim to empower local researchtosolveourproblemsathome, generatingknowledgeandtechnologyto furtheraddressglobalquestions.

Acknowledgments

We thankConselho Nacional de Desenvolvimento CientíficoeTecnológico(CNPq/Brazil),Fundaçãode Amparo à Pesquisa do Estado de São Paulo (FAPESP), Fundação de Amparo à Pesquisa do EstadodoRiodeJaneiro(FAPERJ)andFundação deAmparoàPesquisadoEstadodeMinasGerais (FAPEMIG) for supporting microbiome studies in

Brazil.Fundsforourresearchhavebeensupported bytheNationalScienceFoundation–Dimensionsof Biodiversity (DEB 14422214) and NSF-FAPESP (2014/50320-4).

DisclaimerStatement

Theauthorsdeclare that theyhave nocompeting interests. Resources i http://inct-microbiome.org ii http://brmicrobiome.org iii http://portaldabiodiversidade.icmbio.gov.br iv http://earthmicrobiome.org v http://terragenome.org vi http://qiita.ucsd.edu SupplementalInformation

Supplementalinformationrelatedtothisarticlecanbe foundonlineathttp://dx.doi.org/10.1016/j.tim.2016.

07.007.

1_{BiosystemsInformaticsandGenomicsGroup,René}

RachouResearchCenter–CPqRR,Av.AugustodeLima, 1715,30190-002,BeloHorizonte,MinasGerais,Brazil

2_{CellandMolecularBiologyLaboratory,Centerfor}

NuclearEnergyinAgriculture–CENA,UniversityofSao PauloUSP,Piracicaba,SãoPaulo,Brazil

3_{DepartmentofLand,AirandWaterResources,Collegeof}

AgriculturalandEnvironmentalSciences,Universityof California–Davis,Davis,California,USA

4_{DepartmentofSoilScience,“LuizdeQueiroz”Collegeof}

Agriculture,UniversityofSãoPaulo–ESALQ/USP, Piracicaba,SãoPaulo,Brazil

5_{FederalUniversityofPampa–UNIPAMPA,CampusSão}

Gabriel,SãoGabriel,RS,Brazil

6_{Forafulllistofauthors,pleaseseeTableS1inthe}

supplementalmaterialonline.

*Correspondence: victor.pylro@brmicrobiome.org

(V.S.Pylro)and luizroesch@unipampa.edu.br

(LuizF.W.Roesch).

http://dx.doi.org/10.1016/j.tim.2016.07.007

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Spotlight

Antibiotic

Methylation:

A

New

Mechanism

of

Antimicrobial

Resistance

Kerri

M.

Malone

and

Stephen

V.

Gordon

*

InnewresearchonMycobacterium

tuberculosis, the causative agent

of tuberculosis, Warrier and

col-leagues have discovered a novel

modeofbacterialdrug resistance,

namely antibiotic inactivation via

N-methylation.

By 2050, 10 million lives a year and a cumulative US$100 trillion of economic output are at risk due to the rise of drug-resistant infectionsi. Tuberculosis (TB)representsjustone oftheinfectious diseasesthatwemustfindnewways to control if this trend is to be halted and reversed. TB kills someone every 25seconds, a shocking statistic that makes Mycobacterium tuberculosis one ofthedeadliestpathogensontheplanet

[1]. M. tuberculosis was one of the first pathogensto beidentified as the aetio-logicalagentof aninfectiousdiseaseby RobertKochin1882.BacillusCalmette– Guérin(BCG),theTBvaccine,isthemost widelyusedvaccineintheworld,givento

over100millionchildreneveryyear,while cheap, effective drugs are available for treatment of drug-sensitive infections. SowhydoesTBcontinuetoexactsuch aheavytollofmorbidityandmortality? ControlofTBiscomplicatedbytheunique biology of M. tuberculosis, the variable efficacyprovidedbytheBCGvaccineto protectthoseimmunizedagainstdisease, restricted healthcare access for those affected, HIVcoinfection, andprolonged drugtherapythatisdrivingtheemergence ofdrugresistance[1].Itispredictedthat over 2 billion people harbor latent M. tuberculosis infection, providing a vast reservoir of infection [1]. These hurdles have not, however, limited the vision of TB-controlpolicieswiththeWorldHealth Organization (WHO) End TB Strategy seeking to end the global TB epidemic by2035throughachievinga95%decline inTBdeathsanda90%reductionin dis-ease incidence as compared to 2015ii. Delivery ofthisvision will needprogress onmultiple fronts, andwill hingeonthe developmentofnewanti-TBdrugs. The drugs currently available for the treatmentofTBaremainlytheproducts of antibiotic discovery efforts in the 1940s–1960s.Onlysincethedeclaration of TB as a global public health emer-gency bythe WHOin 1993did the TB drugdevelopmentpipeline sputterback to life. However, decades of neglect, increasedregulations,andalackof inter-estfrompharmaceuticalcompanieshas meantthat thepipelinecontainsamere trickle of new compounds; further researchisdesperately neededto iden-tifynoveldrugcandidatesandtounravel drug-resistance mechanisms of the TB bacillus.

Investigations into the antimycobacterial properties of imidazopyridine- and pyr-ido[1,2-/]benzimidazole-containing com-pounds have shown that these compoundsareactiveagainst M. tuber-culosis, and against multidrug-resistant (MDR)- and extensively drug resistant

(XDR)-TB clinical isolates[2–5]. Through the characterization of one such com-poundcontainingapyrido-benzimidazole core designated ‘14’ [4], Warrier et al. have described an entirely novel mode of drug resistance for bacteria, namely inactivation ofanantibioticvia N-methyl-ation[6].

Toelucidatethemode ofaction of‘14’, Warrierandcolleagues isolatedmultiple M. tuberculosis clones resistant to the compound. Whole-genome sequencing oftheseclonesidentified16unique can-didate resistance-conferring mutations, withnineofthesemutationsmappingto the rv2887genealone [6].TheRv2887 protein belongs to the MarR family of transcription factors, with the resis-tance-conferringmutationspredicted to affecttheabilityofRv2887tobindDNA. Binding assays showed that Rv2887 bound directly to the promoter region upstream of rv0560c,a gene encoding a putative methyltransferase. rv0560c wasfoundtobeexpressedat400-fold higherlevelsinresistantM.tuberculosis clones than in sensitive wild-type M. tuberculosis,whileexposureofwild-type M.tuberculosisto‘14’causedconsistent upregulation of rv0560c expression. Basedonthesefindingsitwasdeduced thatRv2887wasanegativeregulatorof rv0560c expression, with resistance-conferring mutationsinrv2887foundto inhibittheabilityoftheRv2887 transcrip-tion factortobind DNA,resulting inthe upregulated expression of rv0560c in resistantclones[6].

TheroleofRv0560cinresistance ofM. tuberculosis to‘14’was nextexplored. As it was reported that Rv0560c was nonessentialtothe survivalofM. tuber-culosis [7],itwasunlikely thatRv0560c wasthetargetof‘14’butratherthatits enzymaticactivitymustrender‘14’ inac-tiveinsomemanner.Characterizationof the methyltransferase activityof recom-binant Rv0560c showed that it directly methylated‘14’attheN-5position, lead-ingtothelossof14'santimycobacterial