Exploiting gene regulation as an approach to identify, analyze and utilize the biosynthetic pathways of the glycopeptide ristomycin A and the zincophore [S,S]-EDDS in Amycolatopsis japonicum

DISSERTATION der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)

vorgelegt von Marius Steffen Spohn aus Tettnang

Tübingen 2015 Exploiting gene regulation as an approach to identify, analyze and utilize the biosynthetic pathways of the glycopeptide ristomycin A and the zincophore [S,S]-EDDS in Amycolatopsis japonicum

DISSERTATION der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)

vorgelegt von Marius Steffen Spohn aus Tettnang

Tübingen 2015

Erklärung

Ich erkläre hiermit, dass ich die zur Promotion eingereichte Arbeit selbständig verfasst, nur die

angegebenen Quellen und Hilfsmittel benutzt und Stellen, die wörtlich oder inhaltlich nach den

Werken anderer Autoren entnommen sind, als solche gekennzeichnet habe. Eine detaillierte

Abgrenzung meiner eigenen Leistungen von den Beiträgen meiner Kooperationspartner habe Tag der mündlichen Qualifikation: 27.01.2016 ich im Anhang vorgenommen. Dekan: Prof. Dr. Wolfgang Rosenstiel

1. Berichterstatter: PD Dr. Evi Stegmann ...... 2. Berichterstatter: Prof. Dr. Karl Forchhammer Unterschrift 3. Berichterstatter: Prof. Dr. Christian Hertweck Tübingen, den 30.11.2015

Table of content Table of content Table of content Table of content

Table of content 2.2 Identification of the [S,S]-EDDS biosynthetic genes by exploiting knowledge of zinc Zusammenfassung i dependent transcriptional regulation 16 Abstract iii 2.2.1 The zinc responsive zinc uptake regulator Zur controls the [S,S]-EDDS List of publications v biosynthesis 16 Contributions vi 2.2.2 Zur controls the high affinity zinc uptake system ZnuABC 17 Abbreviations vii 2.2.3 The rational screening of the A. japonicum Zur regulon revealed putative [S,S]- EDDS biosynthetic genes 17 1. Introduction 1 2.2.4 The transcription of the aesA-D operon is zinc responsively regulated by Zur 18 1.1 The genus Amycolatopsis: a valuable source of secondary metabolites 1 2.2.5 The aes genes are essential for [S,S]-EDDS production 19 1.2 Glycopeptide antibiotics 2 2.2.6 The [S,S]-EDDS biosynthesis as response to zinc deficiency is phylogenetically 1.2.1 Structural categorization of glycopeptides 3 clustered 19 1.2.2 Glycopeptide antibiotics biosynthesis: precursor supply 3 1.2.3 Glycopeptide antibiotics biosynthesis: assembly of the heptapeptide 4 3. Discussion 21 1.2.4 Glycopeptide antibiotics biosynthesis: modification of the heptapeptide 5 3.1 A. japonicum genome contains a silent gene cluster directing the biosynthesis of 1.2.5 Transcriptional regulation of glycopeptide biosynthesis 5 ristomycin A 21 1.3 Ionophores: naturally occurring chelating agents 6 3.2 The [S,S]-EDDS biosynthetic genes were identified by a novel approach exploiting 1.3.1 Siderophore biosynthesis 6 knowledge in the field of zinc responsive gene regulation 25 1.3.2 Metal responsive regulation of ionophore biosynthesis 6 3.2.1 [S,S]-EDDS exemplifies the rare functionally class of zinc responsive ionophores 25 1.3.2.1 The ferric uptake regulator (Fur) 7 3.2.2 Elucidation of the Zur mediated zinc regulation enabled the identification of the 1.3.2.2 The iron dependent DtxR protein family 7 [S,S]-EDDS biosynthetic genes 28 1.3.2.3 The zinc uptake regulator (Zur) 8 3.2.3 The biogenesis of [S,S]-EDDS 30 1.4 Ethylenediamine-disuccinate (EDDS) 8 3.2.4 Biotechnological [S,S]-EDDS production 31 1.5 Genome mining: an empirical strategy to discover new natural products 9 1.6 Aim of the work 12 4. References 33

2. Results 13 5. Publications 43 2.1 Activation of a silent glycopeptide gene cluster in A. japonicum 13 5.1 Publication 1 43 2.1.1 A. japonicum encodes a silent glycopeptide gene cluster 13 5.2 Publication 2 81 2.1.2 Genome sequencing and Genome mining revealed a glycopeptide gene cluster 13 5.3 Publication 3 84 2.1.3 The cluster encoded transcriptional activator AjrR is functional 14 2.1.4 Structure elucidation revealed ristomycin A as product of A. japonicum 15 2.1.5 A. japonicum ristomycin A induces in vitro platelet aggregation 15

Zusammenfassung Zusammenfassung Zusammenfassung usammenassung

Zusammenfassung einen inksensitiven egulator gewhrleistet wird ie akterielle inkomostase wird meistens durch den gloalen inkspeziische ranskriptionsregulator ur reguliert as ur rotein von A. Der mikrobielle Sekundärmetabolismus ist eine reichhaltige Quelle für Naturstoffe, von denen viele japonicum wurde identiiziert und detailliert charakterisiert s konnten gezeigt werden, dass urA klinische beziehungsweise industrielle Anwendung gefunden haben. Die Gattung Amycolatopsis ist die ranskription des hoch ainen inkaunahmesstems nuAA durch seine inkahngige für die Synthese vieler Naturstoffe bekannt. Beispielsweise werden viele Glykopeptid-Antibiotika, wie indung an speziische A indeseuenzen reguliert iese urindeseuenzen wurden das klinisch relevante Vancomycin oder das Balhimycin, von Stämmen dieser Gattung produziert. Im verwendet, um das A. japonicum Genom nach weiteren, urA regulierten, Genen zu durchsuchen Gegensatz dazu wurde der Stamm Amycolatopsis japonicum nie als Produzent einer biologisch ies hrte zur Auindung des aesA-D perons mangreiche ranskriptionsntersuchungen aktiven Substanz beschrieben. Dieser Stamm produziert jedoch unter Zinkmangelbedingungen das ergaen, dass aesA-D inkahngig von urA reguliert wird ie eteiligung von aesA-D an der S,S EDTA-Isomer Ethylendiamindisuccinat ([S,S]-EDDS). Diese zinkabhängige [S,S]-EDDS Produktion lässt konnte durch naktivierungsversuche nachgewiesen werden ustzlich hrte die eletion des darauf schließen, dass [S,S]-EDDS ein Zinkophor ist, das an der Zinkaufnahme beteiligt ist. inkregulators urA A. japonicum Δzur dazu, dass auch in Gegenwart von hohen ink [S,S]-EDDS weist Komplexbildungseigenschaften auf, die mit denen von EDTA vergleichbar sind. Im onzentrationen S,S in hohen engen produziert wird Gegensatz zu EDTA ist [S,S]-EDDS jedoch biologisch abbaubar. Die weitverbreitete industrielle A. japonicum Δzur ist eine erolgversprechende Ausgangsasis, um einen nachhaltigen und Anwendung von EDTA in Kombination mit dessen Unzugänglichkeit für biologische Abbauprozesse wirtschatlich verwertaren S,S roduktionsprozess zu entwickeln, der keiner imitierung führt zu einer umweltgefährdenden EDTA-Persistenz in aquatischen Lebensräumen. Der Naturstoff durch negative inlsse von ink unterliegt [S,S]-EDDS ist deshalb ein nachhaltiger EDTA Ersatz mit einem verbesserten ökologischen ie trategie, ein vorhergesagtes, stilles Gencluster durch die xpression eines speziischen Fingerabdruck. egulators zu aktivieren, sowie auch die trategie, neue iosntheseGene durch die In dieser Arbeit wurden zwei molekulargenetische Strategien entwickelt, um die Biosynthese des harakterisierung eines gloalen egulators, der speziische mweltsignale wahrnimmt, zu Glykopeptid-Antibiotikums Ristomycin A zu aktivieren und um die [S,S]-EDDS-Biosynthese-Gene in identiizieren, ermglichte die harakterisierung neuer aturstosnthesewege in A. japonicum A. japonicum zu identifizieren. eide Anstze nutzen rkenntnisse er regulatorische echanismen und esitzen das otenzial Untersuchungen des genetischen Potenzials der Gattung Amycolatopsis ließen vermuten, dass auch zukntig angewendet zu werden, um neue aturstoe und neue nthesewege zu identiizieren A. japonicum die Fähigkeit besitzt, ein Glykopeptid-Antibiotikum zu synthetisieren. Um dieses nicht exprimierte, sogenannte „stille Gencluster“ zu aktivieren, wurde ein molekulargenetischer Ansatz verwendet, bei dem der Biosynthese-spezifische Aktivator Bbr heterolog in A. japonicum exprimiert wurde. Bbr reguliert die Balhimycin-Biosynthese in Amycolatopsis balhimycina. In A. japonicum induzierte dessen Expression die Produktion von Ristomycin A, was durch HPLC-DAD, MS, MS/MS, HR-MS, und NMR-Analysen bestätigt werden konnte. Ristomycin A ist ein vielfach glykosyliertes Heptapeptid, das als Hauptwirkstoff in Diagnoseverfahren zur Bestimmung von angeborenen und weitverbreiteten Blutgerinnungsstörungen verwendet wird. Die Sequenzierung des A. japonicum Genoms und dessen computergestützte Auswertung führten zur Identifizierung des Biosynthese- Genclusters, das für die Synthese von Ristomycin A verantwortlich ist. Solche computergestützten Genomanalysen mittels verschiedenster bioinformatischen Plattformen werden heutzutage standardmäßig zur Identifizierung von Sekundärmetabolit-Gencluster angewandt, die bekannten Synthesemechanismen zugeordnet werden können. Allerdings konnten die [S,S]-EDDS-Biosynthese-Gene mit diesen Tools nicht entdeckt werden, was auf einen bislang nicht bekannten Biosynthesemechanismus hindeutet. Um diesen zu identifizieren, wurde ein neuer Ansatz entwickelt, der auf der Annahme beruht, dass die Zink-reprimierte [S,S]-EDDS-Biosynthese durch

i ii i ii Abstract Abstract Astract Astract

Abstract the identiication o the operon aesA-D xtensive transcriptional analses and and shit assas aesA-D S,S he microial secondar metaolism is a rich source or valuale products that have ound their wa revealed that is zinc responsivel regulated ur and involved in iosnthesis, as S,S into various clinical and industrial applications A particularl productive acterial genus or the shown inactivation studies he iosnthesis was uncoupled rom zinc repression zur S,S discover o natural products is Amycolatopsis he most reuentl reported tpe o secondar deleting his mutant sets the stage to estalish a sustainale production process metaolites produced this genus are glcopeptide antiiotics like alhimcin or the medicall without limits ormerl imposed zinc repression relevant vancomcin n contrast to most other memers o the Amycolatopsis genus, Amycolatopsis he strateg to awake predicted silent gene clusters using a characterized regulator as well as the japonicum was never descried to produce an product with antiacterial activit his strain strateg to identi new iosnthetic genes characterizing an environmental signalsensing A. japonicum however is known to snthesize the chelating agent ethlenediaminedisuccinate S,S, a regulator enaled the isolation o novel iosnthetic pathwas in oth approaches iodegradale A isomer in response to zinc deicienc his zinc responsive repression o S,S ollow the oint concept to exploit knowledge o regulator pathwas and have the prospect to e production indicates a possile contriution o S,S to zinc uptake and that it might generall applicale in order to guide uture detection o new natural products elong to the rarel descried phsiological group o zincophores omining excellent chelating properties with the accessiilit to iodegradation, S,S is considered as a sustainale chelating agent, possessing the potential to replace A and other environmentall threatening chelating agents in various applications n this stud, two distinct molecular genetic strategies were developed and implemented to activate S,S the iosnthesis o the glcopeptide antiiotic ristomcin A and to identi the Amycolatopsis japonicum iosnthetic genes in Amycolatopsis A. japonicum Genetic evaluation o the antiiotic iosnthetic potential indicated that might has the capailit to produce a glcopeptide antiiotic ince the iosnthesis o the predicted glcopeptide was not detected altering the culture conditions, a molecular genetic approach was emploed to activate its production eterologous expression o the characterized pathwa speciic activator r, naturall inducing the alhimcin iosnthesis in A. balhimycina, induced the snthesis A. japonicum o a ioactive sustance in he ioactivit could e assigned to the production o ristomcin A, a highl glcoslated peptide antiiotic which is used in diagnostic kits to detect widespread hereditar coagulation disorders ull seuencing o the A. japonicum genome and its computational analsis led to the identiication o the corresponding iosnthetic gene cluster which is directing the iosnthesis o ristomcin A uch computational genome analses various ioinormatic tools are nowadas standardized applied strategies to identi secondar metaolite gene clusters hese approaches however ailed to detect the S,S iosnthetic genes his reuired the development o a new approach which relies on the assumption that the zinc repressed iosnthesis o S,S is regulated a zinc responsive regulator element hereore, the maor zinc responsive transcriptional regulator o A. japonicum ur was characterized in detail ur regulates the expression o the high ainit zinc uptake sstem nuA inding to a speciic A inding seuence he screening o the A. japonicum genome or urther ur regulated genes using this deduced ur inding seuence led to

iii iv iii iv List of publications Contributions ist o pulications ontriutions

List of publications Contributions

n pulication pohn et al., , overexpressed the proteins, perormed transcriptional analsis Publication 1 and the ermentative production process Also the analtical analses A, and Spohn, M, irchner, , ulik, A, ochim, A, ol, , nzer, , orst, , Gross, , ohlleen, were perormed me urthermore, designed most o the experiments, analzed and interpreted , and tegmann, verproduction o ristomcin A activation o a silent gene cluster in the data and wrote most o the manuscript or the ermentation and the analtic was assisted Amycolatopsis japonicum G Antimicrobial Agents and Chemotherapy 58, A ulik supervised ol during his initial attempt to overexpress bbr and during the computational analsis o the gene cluster A ochim cloned ajrR and overexpressed it under m

supervision irchner and Gross isolated ristomcin A rom the culture roth and determined its Publication 2 structure , and spectroscop Gross also wrote the corresponding paragraph or tegmann, , Alersmeier, A, Spohn, M., Gert, , eer, , ohlleen, , alinowski, , and the manuscript nzer and orst perormed the platelet aggregation assa and wrote the ckert, omplete genome seuence o the actinoacterium Amycolatopsis japonica corresponding paragraph or the manuscript tegmann and ohlleen supervised the proect, G producing ,,ethlenediaminedisuccinic acid Journal of took part in designing the experiments and revised the manuscript Biotechnology 189,

n pulication tegmann et al., , contriuted to manual gene annotation and perormed the Publication 3 computational analsis o the A. japonicum potential to produce secondar metaolites also took Spohn, M., ohlleen, , and tegmann, lucidation o the zinc dependent regulation in part in editing the manuscript Amycolatopsis japonicum enaled the identiication o the ethlenediaminedisuccinate S,S genes Environmental Microbiology 18, n pulication pohn et al., , all the experiments were designed and perormed me analzed and interpreted the data and wrote the manuscript tegmann and ohlleen supervised the proect, took part in designing the experiments and revised the manuscript

v vi v vi Abbreviations Areviations Introduction ntroduction

Abbreviations 1. Introduction

A Adenine 1.1 The genus Amycolatopsis: a valuable source of secondary metabolites A. Amycolatopsis econdar metaolites are natural products which are considered to e not essential or vegetative Ala Alanine growth, development or reproduction o an organism ut rather unction in increasing the B. Bacillus evolutionar itness and survivailit under certain specialized growth conditions an microial p ase pair secondar metaolites either unction as growth inhiitors, having antiacterial, antiungal or C. Corynebacterium antiviral activit or, as growth promoters eg enhancing the uptake o scarce nutrient sources ae ,diaminoethane atural compounds have the potential to e developed into drug sustances which can e used as ap ,diaminopropionic acid antiinective or anticancer therapeutic agents or clinical application or or agricultural use as pg ,dihdroxphenlglcine ungicides, insecticides, hericides or ertilizers E. Escherichia A known acterial genus producing valuale compounds which alread ound medical application is G Guanine the genus Amycolatopsis he medicall most relevant antiacterial agents are riamcin, the lead G βglucuronidase drug o riampicin, and vancomcin, a glcopeptide antiiotic emers o this genus however are pg phdroxphenlglcine descried to produce man urther secondar metaolites o various structural classes, relecting its M. Mycobacterium particularl high potential as sources or valuale compounds onriosomal peptidesnthetase idependent siderophore he polketide riamcin was irst identiied as a product o A. mediterranei in ensi et al., onriosomal peptidesnthetase ts derivatives are important antiiotics or the treatment o inectious disease caused olmerase chain reaction Mycobacterium tuberculosis ther Amycolatopsis polketide snthase derived antiiotics are olketide snthase chelocardin, produced A. sulphurea, a roadspectrum tetracclic antiiotic exhiiting potent S. Streptomyces acterioltic activit ukezic et al., and , a polene which was identiied in A. S. aureus Staphylococcus aureus orientalis A anskota et al., possesses strong activit against gram S. typhimurium Salmonella typhimurium positive acteria, including methicillinresistant Staphylococcus aureus A and vancomcin taphloerrin resistant enterococci hmine he most reuentl reported tpe o secondar metaolites produced the genus Amycolatopsis, ltraviolet however, are glcopeptide antiiotics n addition to the actuall known glcopeptide producing βt βhdroxtrosine Amycolatopsis species, screening indicated the genetic potential o several urther strains to also

produce glcopeptides verest and eers, his iosnthetic potential however is not a common eature o the entire Amycolatopsis genus ut rather o a certain phlogenetic clade ancomcin, which was isolated rom A. orientalis righam and ittenger, inds clinical application as antiiotic to treat severe inections with enterococci and A strains esides vancomcin, several other glcopeptides were identiied as Amycolatopsis products alhimcin and chloroeremomcin, isolated rom A. balhimycina adkarni et al., and an A. orientalis strain van ageningen et al., , respectivel share an identical heptapeptide ackone with vancomcin and dier onl in the glcoslation pattern A. orientalis produces norvancomcin, whose chemical structure is almost the same as that o vancomcin, except or an vii vii 1 Introduction Introduction ntroduction ntroduction asent methl group at the terminus ei et al., Avoparcin is a product o A. coloradensis other to orm a rigid cup shaped structure ischo et al., reuired or the interaction with unstmann et al., and has een used as growthpromoting eed additive in agricultural their molecular target, the alanl–alanine AlaAla terminus o acterial cell wall precursors applications aeda, his glcopeptide slightl diers rom the vancomcinlike glcopeptides enolds, his inding intereres with the acterial cell wall iosnthesis and thus leads to in the composition o the heptapeptide ackone istomcin A also called ristocetin A, which is cell death produced A. lurida Grund et al., causes thromoctopenia and platelet agglutination and is thereore no longer used or the treatment o human staphlococcal inections hese tructural categorization o glcopeptides therapeuticall unavorale unctions however are nowadas exploited to detect widespread he structural diversit o glcopeptides arises rom the variation o incorporated amino acids and hereditar genetic disorders in coagulation such as the von illerand disease and the ernard rom the variale decoration o the heptapeptide ackone glcoslic moieties, methl groups, oulier sndrome utilizing ristomcin A as an in vitro diagnosis compound ari et al., chlorine atoms and att acid residues, respectivel ased on the incorporated amino acids, the level o compete with other soildwelling microorganisms, Amycolatopsis strains do not onl produce o side chain crosslinking and the decoration with a att acid residue, glcopeptides can e compounds with antimicroial activit ut also chelating compounds ionophores to monopolize categorized into distinct classes he tpe glcopeptides are characterized two aliphatic amino scarce metal ion resources o soluilize and to acilitate the uptake o iron under iron deicient acids at position and o the heptapeptide ackone his class comprises eg vancomcin, growth conditions microes secrete siderophores iderophores are small molecular weight norvancomcin, alhimcin and chloroeremomcin Avoparcin is exempliing the tpe compounds which exhiit high iron chelating ainit uch secreted chelating agents are descried glcopeptides with incorporated aromatic amino acids also at position and hese amino acids rom Amycolatopsis sp AA amchelin eedsaamdost et al., and A. alba alachelin however do not undergo an oxidative crosslinking in contrast to the tpe glcopeptides, odani et al., oth structurall related molecules are composed o six siderophore characterized a ull crosslinked heptapeptide ackone he model tpe glcopeptide is the characteristic amino acids, while amchelin is additionall decorated with a hdroxenzol group highl glcoslated ristomcin A ther iron chelating agents are the siderochelins produced Amycolatopsis sp iu et al., he glcopeptide iosnthesis pathwa ollows three distinct steps the precursor suppl speciic he siderochelins which show antimcoacterial activities were isolated a ioassaguided iosnthesis pathwas, the asseml o this speciic uilding locks and the inal modiication o the ractionation his activities however are rather due to their ion chelating properties than due to a heptapeptide ackone various tailoring reactions tegmann et al., direct target interaction in means o classical antimicroial chemotherap iu et al., u et al.,

A dierent kind o chelating agent is ethlenediaminedisuccinate , which is Glcopeptide antiiotics iosnthesis precursor suppl snthesized the strain Amycolatopsis japonicum G in the S,Sconiguration ishikiori he nonproteinogenic aromatic amino acids which are incorporated into glcopetides have to e et al., S,S production does not occur in response to iron deicienc ut in response to speciicall supplied All genes reuired or the snthesis o pg, pg and βt are located within the zinc deicienc eulla, wicker et al., his indicates that the ionophore corresponding gene clusters onadio et al., contriutes to zinc uptake and that it elongs to the rarel descried phsiological group o the or the snthesis o pg and pg seven genes are reuired in total pg derives rom zincophores antke, a hao et al., hdroxphenlpruvate, the direct precursor o trosine droxphenlpruvate is converted into he road variet o Amycolatopsis secondar metaolites with respect to phsiological unction as the pg precursor phdroxphenlgloxlate the enzmatic activities o a phdroxmandelate active deense or in competition or resources and with respect to iosnthesis and iochemistr snthase and a phdroxmandelate oxidase uard et al., astner et al., pg is not descried so ar indicates the road potential o this genus to snthesize valuale sustances derived rom a proteinogenic amino acid ut rom our malonloA suunits and snthesized a

polketide snthase mechanism eier et al., he concerted enzmatic catalsis o pgA 1.2 Glycopeptide antibiotics leads to the pg precursor ,dihdroxphenlgloxlate he transamination o oth, p he glcopeptide heptapeptide ackone is predominantl assemled the nonproteinogenic hdroxphenlgloxlate and ,dihdroxphenlgloxlate to ield the end products pg and pg aromatic amino acids ,dihdroxphenlglcine pg, phdroxphenlglcine pg and β is catalzed the same trosine dependent aminotranserase eier et al., hdroxtrosine βt hese aromatic amino acid side chains are oxidativel crosslinked with each

2 3 Introduction Introduction

β β by loading tyrosine onto a peptide synthase module and its hydroxylation at the β et al. et al. β β et al. oxy a β et al. et al. β et al.

et al. et al. et al. et al.

et al. Streptomyces griseus Streptomyces glausescens et al. et al. et al. et al.

4 5 Introduction Introduction

1.3 Ionophores: naturally occurring chelating agents

Streptomyces coelicolor et al. et al. et al. et al. et al. et al. Escherichia coli et al. Salmonella typhimurium S. typhimurium fur et al. E. coli et al.

Corynebacterium et al. Mycobacterium et al. Streptomyces 6 7 Introduction Introduction

S,S Mycobacterium smegmatis et al. et al. Streptomyces et al.

Bacillus subtilis E. coli A. japonicum et al. S,S et al. A. japonicum S,S today’s S,S znuABC A. japonicum S. coelicolor S,S et al. S,S et al. S. coelicolor et al. 1.5 Genome mining: an empirical strategy to discover new natural products

1.4 Ethylenediamine-disuccinate (EDDS)

et al. S,S R,R R,S S,R S,S et al. et al. S,S et al.

8 9 Introduction Introduction todto todto

o s ots ay ad t t ass o ts osato oday t aatst ooos assoatd t oy ts sssy d to t soato taot od ata s o t od atoytas so a t o saata day et al. adata o ts aoa s tat t aato o otta to aat sa os tat ds t osd sty y a s t od ata s ot dd o o ass tat dto o ysoa osatos td tat a a ooto o sttay o ata odts ad to ots ddd o t t st oato aot do t st aay ad soatd ad t as assd tat t a oods a sad dtto d od o aoas a to ad staty s o t atato o ass s oas ssta osytt s ad t sst oaat aayss o t d ty ad t s ot dttd oods a atod to to dstt os dtta tat taoos od to dty a ood os sytss s dtd y t atatd os a odd y t ooass ad sad t assa s st s staty o s a stasd t aato syst o t od aoas d to ts aayts o o aato o t data ad to t o o sta a a t ato o t st as ts staty as sssy ata odts a ot odd d assa ad tato odtos ad a ad as t dtato o t S. coelicolor sdoo o at et al. ts aaytay t dtta osod sts o t odd t ot o as dtd at t atato o t od cchH ad t dttd taots a d to as yt o oa sts t sts ssty od oaat tao o a y ooy ssd o ot ssd at a a d to as st sts s aoas o a o oo tato ty a ot sta to yd s data ds d to t dot o atat tods to dsoy ata oods o ot ssd st sts s a aoas tat ot od oods todooy s o t aato o t t oato as t as t d o tastoa ato dod to aa ts st soday o atoa t o taots oad et al. staty to t ass to ts taot sts od to a t osod odt ass o dtto ad oos t otta y ot ts yt o st sts s t o soato t aay aad atoy asads o soday taot osytss aoa a ots o o a t odta aato o t t ato t st assoatd atoy s do o t ost doo et al. oato t sst atato ad a otato o ts yt sts o atd stats dty atta ts o atay s atos ad y o d to t oa toty o stts t s o o ys ad t t o o t ata atoy syst ad to at a sytt dssd at o t soday taot sts ad d odts t dtato atoy stat sso o ost atay s atos ay as o atoato ad ttato o t oos oato as ty a o ds t tasto o stta s ad ts s a ooy sd staty o t t a da o t sytt otta o a ta sta a ady ad ay o ooata o odto yd o o aa st sts doo et al. too at as dod et al. at s t st os a atay s atato tat aoa as ad Streptomyces ambofaciens to aa o dty ad ato osytt o o t o a o d t sso o a st st at et al. osttt sso o dsd soday taot ood asss a st assoatd ost ato td t sso o t osod o tay s ts aaa t oatos to t ass to tat aa oods osytt s ad d to t dtato o t staoys yosyatd aods t sa dstt o ts dod ad sssy td ta os atto atty ass t ooata aayss a ad to a odta dto o aatst stta o ato o a st y a tastoa sso dos sstat ts dto ats o t odt ad ts to a dto o t ysoa ots y ad stad o ts osso to d st sso s staty as ood to atoy as s dtd ots a t t tatd y adatd aayta o t adoy st o Streptomyces venezuelae a et al. tods atat t dtato o t osod tao odt dtato o atato o a st adat ss d to osttt odto o adoy saata s y ts staty day et al. ayss o a oytd st tot t t o a stss tatt od ssta o to odto t d t a atoyt Salinispora tropica sstd tat t ods o a o ysd ty sta s s o o t st sssy ad aoas o dt a ss atoy oy aoata oytd atoa aato o a tat ot o oods

10 11 Introduction Results todto sts

t a yt st to a t odto o atd soday taots ad 2. Results ts a t dtta 2.1 Activation of a silent glycopeptide gene cluster in A. japonicum a aoa to aat atoy s as t ass to a t ad d tas o t s Amycolatopsis a o yotd atot ods to A. o soday taot dsoy atoa otato o od ato o japonicum as tsy stdd o ay yas o odt t atata atty od soday taot osytss o a ot oy to ass a o to a o dtd so a o os os t as so tat A. japonicum ods st t aso t to ass o s to t osytss o a o o yotd sstat a sos et al. ad tat t o otas a oo t aato ad t osdato o ass o osytt st ato oxyB od a oooyas s ssta o t odto o yotds d so ts ad atoy asads a t ott to t data st ad ys o ts sts t as assd tat A. japonicum as t otta dstad o t ysooa tos o ata odts ad aso to addss t sto to od a yotd o tst t A. japonicum o otas s a st yotd at o do ta ooass ossss s a d too o o ata odts st t od o ts tastoa ato as otd

1.6 Aim of the work A. japonicum ods a st yotd st o ts stdy as to do ad to t a atoy aoas to say o yotd sts a tastoay otod y a atays t stat std asts o A. japonicum soday taos ato oado et al. ad a st atato staty y oss

t od t aatd atays tastoa ato o t ay o t oo stos ad to asd st bbrAba ay et al. A. japonicum t t a to aa t sstd st  os A. japonicum ossss t t otta to od a yotd a yotd st oat sta oss bbrAba ad t A. japonicum d say atatd ty sta o a d sta o yotd odto ad t satats  a a od t d o ato ad ot t to dty t S,S sd o odto o a atotay at odt y a ot to assay od osytt s t oaat tao o as t satat o A. japonicum d ty dd ot

ota ay ooa at ood t t satat o t oat sta

stoy td ot o a dato sta ato oaat tao

o aoa s ad a a t satat o t oat A.

japonicum sta oss bbrAba as ast t oatoa o t A. japonicum d ty satat ato dttd a ad s sta t saty to sta o ot dsd yotds

o s ad o ad a yotd st

od to dty t st o t osytss o t yotd t o of A. japonicum as sd o ossts o to os t ooso ad t asd ya od a tota o ot od s a ato o aayss ad t s o a ty yd st sod saty to aady dsd yotd sts s st ossts o dstt o ad as ato t a tota s o aost t s dtd

12 13 sts Results Results sts to od ys sos o assy ad ot o t yotd sssta ad tt dato ad stoy as odt o A. japonicum

ato t ast to dads osda oss as ad dstad t od to dt t a stt o t yotd A. japonicum oss t ts ad osty o yotd osytss o t tat tos o a atay s ato a as o a t o to ato od odts od ddd y oaat ao ad s aayss t ooos yotd odto as dttd at o ot ad ad a a aot to o o yotd sts a ato ood od o t dda − ato tty dd soato as od a a adsot s ad ys atat yotd osytss as t oss to aost oty dt sst sd as aat d ood as aayd y aos t stt o t a odt in silico data stoy sstd tat t a odt o t aayta tods ad stosoy to dtd st s a sod yosyatd t tyatd oaoatd ad y oss dt ts a stt stt dato ad tat t dtd st d yotd t oaato ad doa oosto o t ss a s dt t odto o stoy stoy s a ty yotd t a o t tatd t t ao ad s βt βt odaty ossd ao ad ao osst o βtβt s dydoyyy pydoyyy βt βydoytyos doatd y s yosd sds ad to tyatos ato ooato o aoat ao ads at osto ad tot t t dtd ot yd tatd s tat ts st dts t sytss o a ty yotd A. japonicum stoy ds in vitro att aato ato stoy ass tooytoa ad att atato s t s ad to assay tos

tatay aoa tos in vitro as a daost ood to dtt dsad st odd tastoa atato s toa dtay t dsods s as o ad dsas ad ado sydo a et y dtd st aos a os ddd odt ts al. tty o t o ad ato s asa y t stoyattdd saty to t t tastoa atato o t ay st a o atato tod a att aato assay t as dostatd tat stoy soatd a tats t tasto o t yotd st A. balhimycina t A. o A. japonicum as t sa in vitro to as t oa stoy japonicum st s o st at d dta ot odtos o aat ts in vivo ato toaty as ssd d t oto o a osttt oot A. japonicum satat o t oat A. japonicum sta osttty ss ajrR td ot o a dato sta as so o t tooos sso o bbra ato

aayss od t osytss o t yotd o stat t o t t tastoa atatos o sso o t dtd yotd st t tastoa att o stat st s dtd t A. japonicum d ty ad t to oat stas ss bbra ad ajrR sty o tast o ajrR ad t o ay ot aayd stta as dttd A. japonicum d ty

ato o osso o ot bbra ad ajrR dd t tasto o a

statd stta s otast tasto o t vanHAX yotd ssta asstt as dttd A. japonicum d ty tot t t o a s tastoa tato y ajrR

14 15 Results Results sts sts

2.2 Identification of the [S,S]-EDDS biosynthetic genes by exploiting knowledge of zinc osytt s a d ts sos oto addto to t ddt dependent transcriptional regulation S,S odto t dto o zur d to a saty asd tota S,S yd

A. japonicum as dsd as t od o S,S so et al. o oad to t d ty sta t ys d o osytss a ot dtd d t o a t assa aoas s as tooos sso o a A. japonicum osd ay a otos t aty ta syst ood sta oaat otos o s o t s ad t as o os aatd so a say os s t dt tos ot atd o s o dtd osytt ys o atoy ts t dat to oostass ata t aty ta syst as dtd as s a ad sssy od to dty t S,S osytt s atd ay ata ad s dy dsttd toot t ata do at ta o as ass to t A. japonicum o s ad at aoy et al. a et al. et al. a et al. aod t in silico data aayss to aat t osytt otta o A. japonicum s in silico ast aayss s t osy dsd syst o S. coelicolor et al. aayss ad t s o aos tat sts od t sytss o ata ad t s o to tat tasots os ddd ot ss so odts A. japonicum a ato s o saty to o S. coelicolor ato o dat o t aatst stta ats o S,S od ot assd to ay o t tasot syst s ysooay otd to ta A. japonicum t tastoa dtd assy ass odd y t dtd sts o t dot o atts statd t st to t s o aos dat ta os a atat staty o t dtato o t S,S osytt s as d tasto o o ta syst as say ssd y aas t sod y assto o t stasd aoa s tat t ssd osytss o S,S o as say ssd y ato o t ysooa tos o s datd y a sos atoy t oa t aayss o t aty ad aas ta assd to t osod ta systs ato ad t sst otatoa s o ssd s odtd to o aat t atoy to o o znuABC tasto znuB tasto as stat S,S osytss aayd A. japonicum Δzur otast to t d ty znuB tasto s asoty ssd y inc concentrations ≥ ts tasto od ddty A. sos ta ato otos t S,S osytss japonicum Δzur t dtta sso at y atd otatos znu ao sos tastoa ato s at t sso o s ato o aay t ds to t oot o a ddt a od ta ad oato tos oayots s t ta ato toot oty st assays od s ad a ddt ad znu at ast aayss o t A. japonicum o s t S. coelicolor ot et s d o d ot to t oot o ato al. as y ad a os oosd odt ts saty sot A. japonicum S,S o ts otta o as ao ato otatoa ao ad s aayss sod atoa s o t o ad tat tat t ao ads dsd to od d a y osd osytt s ato od o o A. japonicum zur as dtd A. japonicum Δzur to stat ddt d o to t znu oot o sstd t s o a s ts o as ato o t S,S osytss od to stdy t t o t dto t d s o dty ts ot a otatoa at o t znu oot o to tat sta ad t A. japonicum d ty sta o sytt d s o osy dsd d ss o ot aost ata as od as otatos t otatos ≥ 2 µM S,S odto as td s at ad a ado s t t znu oot o t t A. japonicum d ty ato otast ddt ad ostat S,S saty to t osy dsd ots y dtd s as sd to dd osytss as osd A. japonicum Δzur o t tota a o t ad a o s A. japonicum d s ato adt ato odto o S,S ts tat s o s ot as t sd to s t A. japonicum o to dty t atd s sstd tat t dtd ods t ss ato dd ad tat t S,S ata t S,S osytt s o oa ooa aato s s 16 17 Results Results sts esuts

ad a tat d ot t to s tasd ds dtos aesE ad data ere generated in spetrophotometri assas suseuent nder suinhiitor in aesA ad ato aesE ods a ot o to t adas sat onentrations the strain representing A. japonicum id tpe reeaed atiit ees ay aesA s t st o a oo aesA-D oosd odts o aesA aesB signifiant aoe those of the negatie ontros ig puiation inear dereasing ad aesC sa sat saty to ys od t sytss o stayo atiit as measured in the range of partia inhiitor in onentrations o atiit as ato a dd sdoo o S. aureus et al. otd detetae in this strain hen gron at in onentrations higher than n ontrast yat atts o t t s oooos o S. aureus ad to t ato o a ta atiities in A. japonicum ∆zur arring the reporter onstrut ere measurae in presene of daota a oty st ato so t S,S stt s eeated in onentrations at the same ee as in asene of in his atiit as inreased aatd y s a ta a oty ody t ato o t ta a oty fod ompared to the atiit in A. japonicum id tpe arring the reporter onstrut after S,S osytss s sstd to o a sa ay as osytss o a groth in asene of in ddt sts assy o t s d os as to dd o S,S osytss s o syttas s odd t A. japonicum o at a 5 he aes genes are essentia for S,S prodution

o proe hether the in reguated aesA-D operon is inoed in S,S iosnthesis the entire tasto o t aesA-D oo s sosy atd y genomi region ontaining aesA-C as deeted he mutant strain A. japonicum ∆aesA-C as not ae S,S s oy odd d t odtos t tasto o t dtd to produe S,S ig puiation o erif that the oss of S,S prodution in A. addat s aesA-D as aaysd to ossy dtt ay oaty t toy japonicum ∆aesA-C is on due to the aesABC deetion a geneti ompementation as performed ts o S,S odtty ad tasto tasto att o aesA as integrating a pasmid into the A. japonicum ∆aesA-C genome hih harours the ompete aesA-D dtd at ot o A. japonicum s o aos otatos s operon donstream of its natie in repressed promoter his genetia ompement mutant oatd tasto o aesA to asd otato as osd y o shoed restored S,S prodution ig puiation ue to the transriptiona ontro of tast as dttd o otatos ao ato toy the integrated aesA-D operon the natie promoter prodution of S,S ourred soe in t as aso s o t s aesB-D ato A. japonicum ∆zur o in depeted medium he amount of S,S produed this reominant strain as inreased aesAD sso as ddt ato aesA as sd as o to to three times ompared to A. japonicum id tpe. st t t oo td a osttt tasto o t o a o he genes aesF aesG and aesH are oated adaent to aesE ig puiation and are predited ad adt t ∆zur aod ato to enode a s fami transriptiona reguator a steine diogenase and an aettransferase o t y t datd ddt sso o aesAD st assays s respetie o further narro don the S,S iosntheti gene uster and to inestigate the d and the 5’ upstream region of aesA od d od say to inoement of these genes in the iosnthesis of S,S the genomi region haroring aesE-H t o s o o d od t as o as deeted his mutant as not ae to produe S,S ig 5 puiation oeer the ato aesA-D operon as sti in dependent transried in this mutant onfirming that the promoter o attaty dt t tastoa o aesA-D a ddt a a region of aesA as not affeted this mutation hese resuts demonstrated that at east one of the tastoa so o t aesA oot o t t ot gusA od a β aesE-H genes is reuired for the prodution of S,S in addition to aesA-C odas as osttd ot ostts tatd to A. japonicum d ty ad A. japonicum ∆zur os oat stas o s o he S,S iosnthesis as response to in defiien is phogenetia aos otatos o t aayss o atts tay atts ustered assayd asd oo assays A. japonicum d ty ota t ot ostt he genera miroia potentia to produe S,S as assessed eauating the phogeneti soy td atty o at o otatos otast atty as aundane of the aes genes omputationa anases his reeaed that the ourrene of the aso s A. japonicum ∆zur at atd otatos ato attat aesA-H genes is restrited to ertain strains of the genus Amycolatopsis f a anaed genomes 18 19 Results Discussion esuts suss soe these of Amycolatopsis sp 5 Amycolatopsis orientalis and Amycolatopsis 3. Discussion lurida haror the eight ustered genes aesA-H entire ith identia gene arrangement 3.1 A. japonicum genome contains a silent gene cluster directing the biosynthesis of and ith high seuene simiarit ompared to A. japonicum ae puiation Amycolatopsis ristomycin A decaplanina 5 and Amycolatopsis alba genomes miss the termina aesH peptes are a mar tpe sear metates prue te eus Amycolatopsis homoog nteresting the uro is high onsered in a disoered aesA 5’ upstream regions tras pssess te at t prue peptes te t uster teter peeta ae puiation eidening the ommon ur mediated and in dependent transriptiona t mst ut t a te esre pruers e t te Amycolatopsis peet ae repression hese identified strains are a ose reated to A. japonicum and are assified into the erest a eers s te stra A. japonicum as t e te pruer Amycolatopsis phogenti ade n eeption ithin this phogenti ade hoeer is te eat aet S,S es e t ts ae Amycolatopsis azurea 5 hih does not sho a onsered S,S uster n ontrast preus esre pepte pru Amycolatopsis stras ae ee ete as to the tota aes gene uster homoogs of the aesA-D suuster are uite aundant in arious pruers att ue sree appraes ese stras epress te rresp genera of atinoateria and proteoateria att ee uster uer erta uture ts trast A. japonicum es t prue o eentua orreate this geneti information to the atua apait to produe S,S the a ate mpu uer uture ts usua sutae r pepte prut phogeneti ade strains A. lurida A. decaplaina A. alba and A. azurea ut aso Amycolatopsis eer te appat a meuar eet appra eae te atat pepte balhimycina 5 Amycolatopsis nigrescens and Amycolatopsis nigrescens prut te prete pruer stra A. japonicum s as aee eterus as representaties of other phogeneti ades ere gron under S,S prodution epress te aratere pata spe atatr r a et al. te am onditions A. lurida A. decaplaina and A. alba gron ithout the suppementation of in produed ee uster A. balhimycina r ue te trasrpt te strutura ees reure r S,S hie no prodution as oserae after groth of these strains in presene of in ig pepte stess A. japonicum puiation o S,S prodution oud e deteted after utiation of A. azurea A. e atat a rata prete sear metate ee uster erepress a balhimycina 5 A. mediterranei nor A. nigrescens empra se trasrpta atatr s a prms strate u e eera appe t hese resuts sho that S,S prodution as an eoutionar response to in defiien is ie purpseu atate arus s sear metate asses atera e truput a uniue feature of the Amycolatopsis phogeneti ade appat ts strate atat erta sear metate usters e atera stra

ets eer ma e mte te reuremet atera aesst t eet mapuats trast t usua eme m appraes use te aesse eet rmat as ase prr t atate set ee usters ts appra es t eesstate aaat eet rmat a ts eauat eer te as strate t erepress a trasrpta atatr rer t atate a set ee usters s sare t arus suessu ute assa eme m appraes e aass te S. ambofaciens eme r ts stet pteta e reeae te presee a set ee uster a te tere ee pata spe atatr te u prte am prr t spe uster atat ts mus erepress auret et al. e etae uersta pepte stess maes t psse t rea eue te struture te a prut rm te eet rmat t a erta eree e in silico aass te ete ee uster A. japonicum sueste te stess a s sate a te metate eptapepte ae sst see armat am as se se 20 21 Discussion Discussion suss suss

as are u rsse ese rmats rreate t te ema struture te tpe e uster rer a tree esre rstm ee uster are emte te vanHAX pepte rstm reus te struture rstm eer et al. resstae assette e vanHAX resstae assette s t a atr eature pete ee ams et al. ut t te rresp stet ee uster as ee reprte e usters vanHAX ees are e preset te ee usters ret te stess a tere s eter a ter tpe pepte ee uster esre te ete A. japonicum tepa e t te am a reremm ee usters a et al. 5 ee uster s te rst reprte ee uster eemp ts tpe sat te pepte e trasrpt ts resstat assette s usua reuate te t mpet sstem a a suseuet struture euat a reeae tat te A. japonicum tpe pepte ses te presee peptes A. balhimycina eer te vanHAX reuat s ee uster s ee ret te stess rstm e ute reae in silico pret epeet rm te vanRSe t mpet sstem s u aaet t te am te ema struture rm te eet rmat ee te ee uster ser te ee uster ere et al. e tat ees e te a ere ete pru ee pete stess ss te per aas aaae aere te Amycolatopsis sp 5 a A. lurida emes ruma et al. r te rmats ts A. japonicum eme s mpes tat vanHAX epress s a epeet as tese e apat t prue rstm eer s t a uue eature A. japonicum se ts stras rasrpta aass vanH A. japonicum tpe reeae ee ts sttute sate pepte as ta ete as a prut A. lurida ru et al. 5 epress epeet rstm prut a et t te serats tat te A. a reet as as a prut Amycolatopsis sp 5 ruma et al. t stras japonicum tpe stra s pru a pepte resstat e a ereas pepte are se reate t A. japonicum e t stras A. lurida a Amycolatopsis sp 5 prut u e etete ere et al. as rstm pruer ere ete a assa att ue sree uture param att resstae s tat resstat etermats are epresse eart t superatat rats ase Amycolatopsis sp 5 a tarete tstep assa sstem att prut t aapt te pru stra t te presee te mpu erere spee t etet te presee pepte atts as appe s sree appra vanH epress a stess resstat e as te tpe A. japonicum ma reet tat rees te reprter stra S. coelicolor ∆femX se eet aru eesstates te presee te uster s rater er pr epresse ta asute set mea tat tere s a asa a pepte r at ruma et al. s ustrates eret as a ea t te rstm prut s aata t etetae ut eerteess ea t te same a a trast t te r a resure sum att ue sree reuremet resstae ts ase te erepress te pata spe tre reuatrs preures te suessu appe meuar eet atat rstm prut A. u rater e a e ptmat appra ta a eme m appra ea t atat japonicum str atate te r ea t te sat te ate mpu a set ee uster s epete mputata aass te reet puse eme seuees A. lurida u e tre epeet vanH trasrpt stas trast t te essetat tre prte a a Amycolatopsis sp 5 reeae te presee ee usters tat et r te epress te strutura rstm ees s s areemet t te serat eta eet raat a smart uete ee t ea ter ruma et al. tat epress a streptm stet ees s trepeet eses te epress a as t te uster A. japonicum te resstat ee m et al. 5 tre epeet ee epress te resstae tese ee usters ta smar tre trasrpta atatrs eer assette A. japonicum ma reet a eutar aapt te stra t te presee ter trast t te A. japonicum ee uster s set uer aratr ts te prut mpet se atera t te pteta t prue peptes rater ta a rstm urs tut spe atat A. lurida a Amycolatopsis sp 5 e umesa prtet measm rreat t te prut te pepte set state te A. japonicum rstm uster s t ue t te ajrR re tse se ts att in vivo utat u e s ut rater ue t te at tat ajrR s t trasre uer urret A. lurida s ute r mmera prut rstm s eee r ts staare utat ts A. japonicum erere e assume tat tere s a st appat as ast mpu ate tere are esre trasrmat prts u superr trer reure r ajrR atat s eter tta mss A. japonicum aaae t eeta mapuate A. lurida a Amycolatopsis sp 5 s seems t mt te r mre prae aset uer te estate uture ts e ptmat rstm prut tese stras t assa stra eepmet rete eut a t assa ermetate ptmat trast seera eret

22 23 Discussion Discussion suss suss preures t eeta mapuate A. japonicum are estase tema et al. ese 3.2 The [S,S]-EDDS biosynthetic genes were identified by a novel approach exploiting preures are eer at east t appae r A. lurida e atat rstm knowledge in the field of zinc responsive gene regulation prut te eeta eas aesse stra A. japonicum ers te psst t S,S eempes te rare uta ass respse pres ptme te prut a rata maer meta eeer appraes a t rease A. japonicum prues te eat aet S,S as respse t ee trast t te urret tae rstm e m sat utat su appraes ter meta s t s a represse eet S,S stess erere t s u e s te ase am prut ere prutt A. balhimycina as sere tat S,S s a eutar aaptat t ee trutes t reasae ret meta ues t rease te aaat spe preursrs aer et uptae aa t serpresmeate r aust te term pre as true al. e traser ts ee t A. japonicum mt ea t a ptme prut r S,S eua 5 ate a stra t rease rstm e t utmpete te aas ustra ute A. lurida eera mra eat aets r pres are assae t uta asses

ar t ter respse meta t respet t ter trasrpta reuat a t

spe reassat te meta pre mpe t te e

ate r aur serpres are te ma rup esre eat mpus prue

mres e eutar suess serpres sueste tat mrrasms as emp

aaus sstems t seuester pr aesse s ter ta r rer t eae ter

assmat ampes esr su eat aets eer ae t ee e mu erae

terature tu

ser te essetat r t es a te premat urree r as

stae a sue err e rate mpees aer ermets at e

assmate mrrasms as 5 maes t er e tat te ast mart mra

pres are ee r aur serpres te ter a ts suests tat prut

pres respse t meta s ter ta r ma e a prprta rare aapte

measm erta speae mres tat eter ae t aee seere ee erta

s ter atura ermet r reure a mparate amut erta s ue t ter

metasm eerteess ma pres are tut t e serpres ae eer ee

epermeta ere t u te rtera t e asse as su at e atates

etete ee usters autmata as serpre ee usters tut ser

pretae rmats r respse trasrpta reuat ta mputata

sree r meta reuatr stes t te rresp ee usters prete

t ret te stess pres u e urter st t ter psa ut a

u trute t ue sat mre pres rare r ee e asses rete

rmat aases ue uete seuee ata sets m te uersta

pre stess a meta respse ee reuat ma ea t etat

urter pres ut assmat erta meta s ter ta r a rae

te uersta atera sura stratees

5 24 25 Discussion Discussion suss suss

e e eampes rpre sstems are eter r pper respse serpre mpe ete atera te ma rute r serpremeate r aust sstem sst a serete atr ra spea reassates uptae s represete te mprt err serpre mpees t te tpasm e etr t te e surae a epresse trasprter prte rt s ute te mar uma te err serpre mpees passe us s eer preete te tpasm ua pate Candida albicans t assmate te esseta utret rm ts st tu et al. memrae ease a ers us uptae urs te mpe t a ter ass pres are te metaats prue erta metatrp memrae assate prte prr t ts ate trasprt arss te tpasm memrae atera m et al. e metaats are serete t aure pper rm te a assette trasprter sstem tt et al. rau rau a ermet s esseta r tese atera t sere as eme atr r te meta ate u su uptae sstems are usua epresse t te rresp pata metae at r t te ree r r pper as a aa t serpre stet ees arame et al. trasprt sstem s u serpres te metaats are reerre t as apres ee a see aaet t te S,S stet ees trast t serpres se stess s ma rete r patas te C. e eetua rem te r rm ts eatr s te same as semes at te euar albicans ra a te metaats are rsma stese peptes ereas ra s surae r te tpasm e r reease s eter atate te mpett r serete as a uatere pepte metaats ere rm a prepepte emrau et al. rat t a prtat pepeet reease te rss te serpre s etese psttrasata me ee a see ese rsma ae rt reease r te reut te err eter reute reease ete stese eat prtes eer are assa meuar et sear metates e mart r rema presses are estrute assmatr reut presses ut rater aaus t atera empres esau et al. serete prtes aptur ree err r mpees e e assumpt ts press s tat serpres ae at eme r etrat eme rm em r te err at a mu er at r te errus at err reut s respse sstem s prete t ret te stess a mpu t suseuet e te sptaeus reease r mpette seuestrat te reue strutura eatures araterst r eat aets s esre r S. coelicolor ete et al. spees ete a arae e ee epress ts ee uster s respse tre ur aas e reut err t errus r s ma emata atae reutases e te et al. u prut as asse t ts ee uster et rmata eauat rat utat prte u Vibrio cholerae uttert a aer te te eet rmat sueste te stess a saate ta mpu terme etraeuar ermet ts reute rema s te ret upe t at uptae eat ete et al. ue t te respse repress te uster ees t s te reease errus r spees st e su a reute reease as t e eue r te sueste tat eat seres as pre r S. coelicolor a et al. S,S atate uptae se te emstr s mate ts at state e meuar et S,S es te mst eret pture as a eampe r a pre s reert sstems ree et al. e prue respse t meta s ter ta r eer tu te strt represse e estrute rt reease s ease r a mrt serpres ue t strutura prut suests a ut S,S spe uptae epermeta pr r ts reuremets su as te trut rater ustae ester s t te serpre sa ptess s st a ate eter reassat S,S mpe t te A. ete Such ferric siderophore hydrolyzing enzymes generally belong to the α/βrases japonicum e surae r atua S,S meate uptae t te tpasm as s superam et esterase att E. coli utes a set tree serpre esterases sst rma a e te rresp pre me a apture meta ets aesse te ts r a es a te perpasmat r t re err eterat a ts r spe euar uptae t eetua mere t te euar meta p erere te sate erates et al. 5 u et al. 5 e S,S es t arr a ester me meta as t e reease rm ts pre a a traserre t te tpasm e bond such a α/βrases superam prte atae eraat te ae mpe meuar measms ts uptae ae ee etese estate r serpre meate as t e rue ut r uptae aret measms are te upe t at uptae ae ee aueus sut te rmat r serpre mpees s aete te p e ree t atate te rema r rm ter serpre as e r reease a eter ur at prts a te r mpete r te ree serpre as s rat emstr s te euar surae assat t ree r uptae r ater euar uptae te err epte te r rema press Saccharomyces cerevisiae upes a emat err

26 27 Discussion Discussion suss suss

reut t a etraeuar aat esusse et al. 5 e ere p eas t a assumpt usterer s tat ee u stet pata asses are er eret parta estaat te err serpre mpe ue t a rease a prtat rm es te ute te same ra eme ames r te atass e reats e a am S,S a mpe a rae meta s er a ra p rae erere usterer prets putate usters etet erta mas are t mama eat eets urr apprmate at eutra r st aae p Kołodyńska, ate utse a mpreese set stet ee uster tpes eer et al. e apprmate p rae s sutae r eat aet s S,S s 5 ese stet ee ustere res are sueste t putate ret te stess etee p 5 a t p er ta S,S s preset as ree t u ap rm a sear metate a e pata ass eer as ser ts aae etet rama et al. artms e te etete rpa stet ee usters A. japonicum u e e serat serpre meate r uptae measms psema rmata asse t te araterst strutura eatures S,S e eetua prpertes as ert a A. japonicum eets a ue urter estats putate S,S ete S,S stet ees esape ee etet tese appraes s suests meate uptae e asee a uptae sstem te S,S uster ma a e a uue S,S assem measm tat s eret rm te tpa r ate a reease rater at te euar surae assat t spe uptae patas r pre stess a as eates sat rm a ter preus aate uptae sstem mat S,S a traser t te tpasm esre sear metate patas mt e u e ut a ate memrae assate prte rerut te e aure et te S,S stet ees us rma appe ema S,S mpe mt e perrme te prete pprte ete te u a rmata stratees r te sat sear metate ee usters eesstate sstem A. japonicum ets a terma sa pepte t sere p te eepmet a e appra rees te eptat ee te e terest as te u sstems ter Amycolatopsis spees ere s t prue trasrpta reuat r te etat te S,S stet ees ts appra S,S are etee ts pprte e t s t u pru Amycolatopsis ree te araterat te ur meate trasrpta reuat A. japonicum a spees e eetua reease es pra t ur er te mpe stat te suseuet mputata sree te eme seuee r represse ees us te reut a eter r S,S esterases eer S,S eue ur ste as uer eraate emes ea te etee e su resue a te etra ae ere e A. japonicum ur ste as eue estat te ur meate reuat area sate tse a esa et al. ese ar tre ases are u eta e A. japonicum eme tas t uptae sstems et smar t te arsuate ase r a a a a eme ata te ast step smart t te u sstem S. coelicolor eer trast t S. coelicolor eter are stess e A. japonicum eme ees ust e smar prte se eet tem s ae aaet t te zur re et al. at t te at suests ts ut are stess rater ta S,S rss respse u A. japonicum pssesses as a maaese respse trasprter s sstem s prete t ae maaese uptae ut smar t te t sstem B. subtilis ue a ema euat ts sstem s pra meate a maaese uat te ur meate reuat eae te etat te S,S respse tam prte e t B. subtilis ase et al. a r Treponema stet ees pallidum se et al. A. japonicum pssess t tam prtes a r respse e e eauat te eera A. japonicum stet pteta rmat ts e reuat e serpre stess a a se e et er am a seuee at er a mpreese ppee apae et stet er te smart t t B. suts ta t reatera r respse t prtes e rae sear metate mpu asses et al. eer et al. 5 pe ur t te znuABC a te aesA prmter re A. japonicum as s ue te etat te atmra mpus rstm a 5 a a r s u ur as ae t reas etrats a se ur st as sere respse stese ere serpre trast S. coelicolor ur rms mutpe mpees t ts taret s epeee eer a e mtat tese prete ts s te euse etat stet ee rease ur etrat et al. aas et al. t s tut tat tese mutpe usters area tpes erme ts mtat te mst reet ers at sts represet epeet eets seera ur mers t te eer r E. coli eer et al. 5 ues te usterer artm merma et al. e e 28 29 Discussion Discussion suss suss

ur su termeate spees prte s ere sere ere t ur mers aprtee am a ap a urtermre aaet a s as a prete preursr t te a perate a reea se sts epermets st et S,S al. mparae t te sere se ur st r A. japonicum a eue putate S,S stess pata puat te t preursrs ap terest te prete ur ste t te aesA prmter re s ate urter a aaet a are asseme a et u measm t rm a me termeate upstream te aesA start mpare t S. coelicolor ur stes a te prete suseuet earat te ap met ea t te ae met s sueste t e ur ste t te A. japonicum znu prmter trast erap te prete atae es ar t te reat stess eu et al. a 5 prmter eemets et al. eer epeet A. japonicum ur ar reat te aetermeate a a se aaet a meue upe t a t te aesA prmter re es eerteess t trasrpta epress aesA suseuet reut te t ue s u ea t te uta S,S ut te tree prete S,S u s are assume t e suppe te reat e eess S,S es a es e supp e meue aaet a ts reat pss reees te e e prpse ee pruts aesA aesB a aesC sare sat smart t a u te e s t suppse t e represse ee ts mateae a tus respete emes e te stess te ere serpre S. aureus aeuate eer supp memraeu resprat seems t ae spea sae r S,S eu et al. e araterst strutura eature a S,S s a etra ae stess se ts prut urs euse epeta rt pase met s met s rare atura mpus a t as sere tat t s restrte t smutaeus t mass rmat er et al. rt epeet prut s as stet mpus euse uetse a esre r serpres e te atmetes eserrame ees et al. s S. aureus stess starts t te rmat amprp a ap te reets tat meta supp pres s rua r prerat es t mata te emat prut te amat pspsere t te amr utam a ar et al. att arus metaemes are reure a aret prmar metasm patas s reat s rate atae a e urter assem te se s s trast t ast mart sear metates prue atmetes se u s (2x Dap, citric acid and αetutar a urs a tree stt stess s eera assate t spe rt rates 5 ae et al. stetases a ta s reure t atae te earat e et S,S prut t epeta rt pase seems t t re te a aptermeate eas t te ae met eu et al. puat trasrpta tr te aesA-D per aesA-D epress eet ts s as araa t te reuremet te e termeates citric acid and αetutar a as etetae statar rt pase ppse t S,S prut stp urtermre tu preursrs te serpre S. aureus emps a rspar respse asee r aesA-D transcription is constitutive in the Δzur aru S,S prut s st upe t eas t repress te e a t a a meta reraat tat aurs e prerat ts eet mutat s mt ate tat respse S,S eer prut te ss e emat att a eer ree prut s uaratee ur reuate preursr supp e rt epeet prut s stess rm ts meta mtats mpse te rspar respse s reat ur epeet e psttrasrpta measms r reuat te et u eers t αetutar a a ap rm ssere metates ar et al. assem maer seuet a aapte S,S prut t te assate psa e et al. ts eesstat ts presee ame ee a aa patas e trast t r stress starat s t suppse t ue e repress a s metasm seems t e uaratee a mutatra reuatr etr tus eter ea t mtat e termeates u supp te u

ap r S,S stess A. japonicum s sueste t ur a smar a as S. aureus tea S,S prut e ee stues t stpe aee aspart a sueste ts trasamat t aaet a prr t ts rprat t S,S eua 5 t s e t assume tat ese erse parameters ue te S,S stess ae t e sere r te es a es use aspart a as am r r te ers pspsere t ap eptua es a tea S,S prut press e rt epeet stea utam a s prete es a es atae reat u tus eer te mtat S,S prut as area assesse preus e eepmet a tre

30 31 Discussion References suss eerees

e at ermetat press t pre epeta rt pase e t prut e 4. References

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5 euat sear metasm streptmetes Curr Opin Microbiol 8 5

s ster erta eer tema s eer eer ee a ssmut 5 e stess amtpe pepte atts a me r ate sea rss eases upe t te at pepte stetases Chembiochem 6

eema aempur sa ret aa a eer at a ersate patrm r eme m sear metate pruers Nucleic Acids Res 41

e ete s a ee eets rm sma aes psse as t epre atures ema erst Chembiochem 3

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as 5 e strute atera pata r serpre stess epeet ere eu ra are are ata a 5 rsma pepte stetases Chembiochem 6 tatera ser a eepmet rm ee t prut a a Biomed Research International e u a a eta mpeat a eraat a a est uta ter stu J Phys Chem A 114 5 eer e are a uts truture rstet Proc Natl Acad Sci U S A 69 eu ease u ae a ers euar araterat staperr stess Staphylococcus aureus Mol Microbiol 74 5 at e err uptae reuatr superam erst a ersatt e trasrpta reuatrs Arch Biochem Biophys 546 5 merma eema aese urta r armmats at re erse ar rre aa a t a ras ase ur stese Amycolatopsis japonicum sa sts t sear metasm rm a a aass prart University of Tübingen pma ess stet ee usters Cell 158 aaa a ema etat a spe metareuatr prte ur tu ase ram rue pe r ue a tr trasprt pers Bacillus subtilis J Bacteriol 180 555 s Candida albicans saees st a ra ur etea as Plos Pathogens 8 er r a as esttut a araterat te Escherichia coli eterat stetase rm t t a t Biochemistry 37 5 e a am ateatesaate eterpas emstrat a ateate t saate rat ae Inorganic Chemistry 39 st a arus aaerae e ue ra a ara trutura a east ass euat rss te E. coli esa aer amuss a eer stereseete artre ur eu PLoS Biol 12 e ase rm Ralstonia sp eaes t tree smers msuate Biodegradation 15 ase ue ema a rea truture te maaeseu maaese trasprt reuatr Bacillus subtilis Nature Structural Biology 10 55 rsa a as eets a assem e em serpre stess atera Microbiology and Molecular Biology Reviews 66 ru ar eraut ste er arre r er a ester 5 stet mr prpertes Antibiot Annu 5 34 35 References References eerees eerees

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aats ut meerer teue ee ssmut a tema ree a a aret tate re emstr metate a te e stess tepatpe pepte atts assmet 5 m tr utuats e sa Arch Biochem Biophys 463 eases t se a ats pepte Chem Biol 14 ustma tser rter a a are a e a a a araterat a ueteseuee aass te att ermetat sat a araterat Antimicrob Agents Chemother 8 5 argH ee rm Campylobacter jejuni e arsuate ase J Bacteriol 176 5 u a rat eme seuee Amycolatopsis lurida pruer te pepte am att rstet Genome Announc 2 ate euat err r trasprt Escherichia coli sat a sttute mutat Mol Gen Genet 182 aea 5 Amycolatopsis coloradensis sp te apar pru stra Int J Syst Bacteriol 45 ate a atera trasprters a reuatrs Biometals 14 auret ua rre e as a e etat ate r a meta reuat atera Curr Opin Microbiol 4 a ate 5memere mare mpe atat a set pete stase Streptomyces ambofaciens Proc Natl Acad Sci U S A 108 5 u ase res e ase r tte s a ereea resut 5 rsta struture psppase rm Bacillus autru eet ae a as 5 ser a e pepte atura cereus Nature 338 5 prut Streptomyces coelicolor eme m Nat Chem Biol 1 5

uar mas a as stess prpee a e ua a a 5 rat eme seuee prtee am a sttuet pepte atts Chem Biol 7 rampru stra Amycolatopsis orientalis Genome Announc 3

aas ase e traamere a aet e esusse asterassm a ae 5 errreutase att Saccharomyces respse reuatr ur trs epress te eat ee uster Streptomyces cerevisiae a ter u rmetr assas aar pates Anal Biochem 226 5 coelicolor J Bacteriol 192 sa u a as 5 In vitro araterat same a aster er atesa ute a ett rpee eterat trate rases r r a es J Am Chem Soc 127 5 stess Herpetosiphon aurantiacus a ase ee upat a atat eree Archives of Microbiology 194 555 u ser es r a rpe re er uuts ept a es ere a e errus eat aet prue ee a see eme m r metaats Journal of Biological Nocardia J Antibiot (Tokyo) 34 Inorganic Chemistry 19 u e a e 5 eres t atmatera att rm eera a amse sauts ames a usaturate as atet Amycolatopsis sp Chin J Nat Med 13 55 uare uss arma a eeraue a rsta m raam prt terma aea are suss a struture a ut te uptae reuatr ur rm Mycobacterium tuberculosis J Biol er etaat a pperaust mpu rm metae Chem 282 atera Science 305 5 ue es rse rat a aa e aspr err ar r aaama a ers a urp uter a et etat te ear stet ee uster rm tess amprp a a serpre a att preursr Chem Biol 21 Amycolatopsis sulphurea a patrm r pru e tetrae atts Microbiology 159 55

a ma uu emm a sameama 5 sat a aa raua u a eresstae measm Streptomyces struture etermat e serpre aae rm Amycolatopsis alba Biometals 28 peucetius erepress drrA drrB a drrC r ru eaemet Microbiol Res 165 5

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ees eer er etsrapp a u rut ater a ate e u at uptae sstem a ts reuatr ur eserrame a e aas rete ermetat a ee ermetat Appl Escherichia coli Mol Microbiol 28 Microbiol Biotechnol 32 555 a uert sep aus e a e e respse ete euar stratees mra r assmat rm at mpees reu Neisseria meningitidis mprses ees uer tr a ur eemet J Bacteriol 194 t atr assem sstems Metallomics 5 5 5

ete a arae erprease r aust a pate tr eer s es etea eer a rer Microbiology and Molecular Biology Reviews 71 5 ee a eer pete stase pepte stess e stess te prtee am a 5rpee Journal of tersuu er stese Amycolatopsis japonicum University of Biological Chemistry 276 Tübingen pma ess mes a a rsta struture a atatate ptera t ar r a s a a ser e ate repressr mpe reeas a meta e ma J Mol Biol 292 5 meues rm mra sures Microb Biotechnol 7 mes a rsta struture te repeet reuatr rta ataa ta usma a sua r Vibrio e rm Mycobacterium tuberculosis ss t meta stes u upe J Mol Biol parahaemolyticus s a are mutru eu pump J Bacteriol 182 285 55

rta ama ta e ataa usma a sua r a a eas terat a r trasprt mpu rm Salmonella a putate mutru eu prte Vibrio parahaemolyticus a ts m Escherichia coli typhimurium Biochem Biophys Res Commun 38 Antimicrob Agents Chemother 42 se aram rrs a erar araterat a maaese ua e tte ura ee ssmut a a ee epeet reuatr prte r rm Treponema pallidum Proc Natl Acad Sci U S A 96 e testerase p s e te rmat etartrse ur am stess Amycolatopsis balhimycina Chembiochem 11 u s tte eer est tema ssmut a ee aar ate atteree aaumar esa uma au stess retartrse a prtee am a te pept a mert am a e pepte att prue Amycolatopsis ae pepte atts J Bacteriol 186 sp am prut sat a a att J Antibiot (Tokyo) 47 u uer s etea u ssmut a ee ee a eer pepte stess Amycolatopsis mediterranei 5 ea a se terespe as a ter mpees at mpe ut a aease a a aperaseperrase Chem Biol 9 55 eteeamesu a Inorganic Chemistry 7 5 ama a ra eme s a eetr meue r repeet sr uama aaaa ata amaa aeu a a eraat te atera r respse reuatr rr prte Proc Natl Acad Sci U S A 96 5 meaa rut atmetes eteeamesu a a tr psppase J Antibiot (Tokyo) 37 ue a ema aaese mestass Bacillus subtilis s reuate t a re a s truture etere a reate r uta reuatr reate t te ptera t repressr am prtes Mol Microbiol 35 esere uates rm Escherichia coli Biochim Biophys Acta 215 5

rama e aare a sea mpeat a me eta a ster 5 e reuatr streptm ee epress tr steresmers eteeamesu a t e u a s Streptomyces griseus s a atatr prte t mutpe ret stes Mol Microbiol aueus sut Journal of the Chemical Society-Dalton Transactions 18 5

esstaes a a as e ere em a es truture emstr a measm at pepte atts rsma pepte stetaseepeet pata r rueeerr serpre Eur J Clin Microbiol Infect Dis 8 5 stess Chem Commun (Camb) 55

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ae ae rer arauate mer ae a ae tema ersmeer p ert eer ee as a as umarampe rueaa ae a u ar sure ert mpete eme seuee te ataterum Amycolatopsis japonica reuat att prut Journal of Antibiotics 63 5 pru eteeamesu a J Biotechnol 189C ar tratt aer a rus ature era atr a e assa a a spe tr Proc Natl Acad Sci U S A 71 tema ras a ee pepte stess te tet as euar uts Curr Opin Microbiol 13 55 ere mer ras tte u tte aer ee a tema eresstae a e a mpst te pepte tema eer s u tert ut ere s smut pruer Amycolatopsis balhimycina Antimicrob Agents Chemother 55 a ee eet aass te am amtpe ease ees J Biotechnol 124 5 ae ete ers artma eere a ars eraat a me steresmers etee ame su a a tema eer e a ee eepmet tree eret ee trast meta eatr Chemosphere 34 5 sstems r eet estat te e spees Amycolatopsis japonicum te eteeamesu a pruer J Biotechnol 92 5 emrau aaea prt aa aa erma reemeer ara a re at ueumer a urre t arra aar er rett s a a etaat a m r ert t at as te pperst metatrps Environ erate te pepte prue atat te etarase ee Microbiol 15 Nonomuraea sp FEMS Microbiol Lett 256 5

es arat a ma 5 m a e att premar reprt tt ares u a am ra r trasprt a a serpre Farmaco Sci 14 sutte memrae trasprt param Proc Natl Acad Sci U S A 97

eesaamst raer e ter a ar truture a aaas umt uu a eraates eteeame stess ame a uusua mea serpre rm Amycolatopsis sp J Am su a a ter eat aets Biosci Biotechnol Biochem 61 55 Chem Soc 133 aer ese ee eer utet at a tema a u etrre eer aa ee a tema rease pepte prut ater erepress smate pata ees e part e rer seuee te am stess ee uster rm Amycolatopsis balhimycina te am stet ee uster Metabolic Engineering 12 55 tas bbr e a tre pataspe reuatr J Mol Microbiol Biotechnol 13 m sa amaa s a ru 5 rasrpta tr e ara a ers e att Staphylococcus aureus s atr strR te pataspe trasrpta atatr r streptm stess esseta r rreuate stess staperr ut t staperr te eet a Streptomyces griseus J Bacteriol 187 5555 se trate stase Mol Microbiol 92 ruma u e a u a tt resstae m a e e respse reuatr ur trs a measms rm ser etat a araterat a e Amycolatopsis stra uptae sstem a sme rsma prtes Streptomyces coelicolor J Bacteriol 189 pru rstet Antimicrob Agents Chemother 58 555 ua arrer aaa ue a art rasrpta reuat p rer u m er rst rss ee te eserrame ee uster Streptomyces coelicolor s meate m t a tema erprut rstm atat a set ee uster a r te prmter te desA ee Febs Journal 274 Amycolatopsis japonicum Antimicrob Agents Chemother 58 5 ar eer sar a apus ea ese a re p tema ee a eer steseee u eme seue reeas mpe sear metame te mare atmete prtee u erare ur stese erma atet ppat Salinispora tropica Proc Natl Acad Sci U S A 104 5 a aee rpatr ams arrs ersa ear p ee a tema uat te epeet reuat es es a eer eue a aass ees e te Amycolatopsis japonicum eae te etat te eteeamesuate S,S stess a am rup att Chem Biol 5 55 ees Environ Microbiol 18 eer uea ru m ruer ee sa uer tema euareetse u emse tersuue er rut ee ret aa a eema 5 at a Amycolatopsis japonicum University of Tübingen tra ess 40 41 References Publicaton 1 eerees

mpreese resure r te eme m stet ee usters Nucleic Acids Res 43 5. Publications 5.1 Publication 1 ams aaaa a ama te struture a me at te att rstet Journal of the Chemical Society-Perkin Transactions 1 Spohn, M tse a urat a araterat a ase rm te era atera stra tat ataes te sptt eteeamesuate a Amycolatopsis japonicum Antimicrobial Agents and Chemotherapy 58 strutura smer p Biodegradation 9

a a a 5 euat am prut Streptomyces venezuelae 5 emet a repressr ee jadR2 J Bacteriol 177

er a as tratees r te ser e atura pruts eme m Chembiochem 10 5

a er e e aterma a am trutura aass trme 5 5 e te stess te pre eat Int J Mol Sci 13 55

u aeet ema a ate 5 uts te serpre esterases r a r rsame utat Microbiology-Sgm 151

er ea er a eer ptmat ermetat ts r te prut eteeamesu a Amycolatopsis orientalis J Ind Microbiol Biotechnol 19 –5

42 43 Publicaton 1 Publicaton 1

Overproduction of Ristomycin A by Activation of a Silent Gene Spohn et al. Cluster in Amycolatopsis japonicum MG417-CF17 tide-resistant pathogens, like glycopeptide-intermediate Staphy- liquid chromatography (HPLC) coupled with a diode array detector Downloaded from lococcus aureus and vancomycin-resistant enterococci (VRE). A (DAD). Five microliters of each sample was analyzed by HPLC with gra- Marius Spohn,a Norbert Kirchner,b,c Andreas Kulik,a Angelika Jochim,a Felix Wolf,a Patrick Muenzer,d Oliver Borst,e Harald Gross,b,c particular function is described for the highly glycosylated glyco- dient elution. For gradient elution, solvent A was 0.1% phosphoric acid Wolfgang Wohlleben,a,b Evi Stegmanna,b peptide ristomycin A (also called ristocetin A), previously identi- and solvent B was acetonitrile, and the gradient elution was performed as Interfaculty Institute of Microbiology and Infection Medicine Tuebingen, Microbiology/Biotechnology, University of Tuebingen, Tuebingen, Germanya; German Centre for follows: t0 ϭ 0% solvent B, t7 ϭ 30% solvent B, t8 to t10 ϭ 100% solvent B. fied in Amycolatopsis lurida (11). Since ristomycin A causes Ϫ1 Infection Research (DZIF), Partner Site Tuebingen, Tuebingen, Germanyb; Pharmaceutical Institute, Department of Pharmaceutical Biology, University of Tuebingen, thrombocytopenia and platelet agglutination, it is no longer used The flow rate was 0.85 ml min . Gradient elution was conducted using a Downloaded from c d Nucleosil 100-C column (5 ␮m; 125 by 3 mm) (precolumn, 20 by 3 Tuebingen, Germany ; Department of Physiology I, University of Tuebingen, Tuebingen, Germany ; Department of Cardiology and Cardiovascular Medicine, University of for the treatment of human staphylococcal infections but solely 18 Tuebingen, Tuebingen, Germanye mm) (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). Detection applied to assay those therapeutically unfavorable functions in was carried out at 210, 230, 260, 280, 310, 360, 400, 435, and 500 nm (1260 vitro as a diagnosis compound to detect widespread hereditary The emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new anti- http://aac.asm.org/ Infinity diode array detector; Agilent Technologies, Waldbronn, Ger- genetic disorders such as von Willebrand disease and Bernard- many). bacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary me- Soulier syndrome (12). Analytical instrumentation. Nuclear magnetic resonance (NMR) tabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize In this study, we were able to identify and activate a new type III spectra were recorded on a Bruker Avance III 500 HD spectrometer, medically important products is Amycolatopsis. However, Amycolatopsis japonicum does not produce an antibiotic under stan- glycopeptide gene cluster in A. japonicum coding for ristomycin A equipped with a BBFO cryo probe head. Spectra were referenced to resid- dard laboratory conditions. In contrast to most Amycolatopsis strains, A. japonicum is genetically tractable with different meth- production. Although ristomycin A has been in use for many years, ual protonated solvent signals with resonances at ␦H/C 2.50/39.5 (deuter- http://aac.asm.org/ ods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of no type III glycopeptide gene cluster has been published so far. Het- ated dimethyl sulfoxide [d6-DMSO]). Circular dichroism (CD) spectra were measured on a Jasco J-720 spectropolarimeter. High-resolution balhimycin biosynthesis, the bbr gene from Amycolatopsis balhimycina (bbrAba), into A. japonicum. This resulted in the produc- erologous expression of the balhimycin pathway-specific regulator (HR) electrospray ionization-time of flight mass spectrometry (ESI-TOF tion of an antibiotically active compound. Following whole-genome sequencing of A. japonicum, 29 cryptic gene clusters were gene bbrAba (bbr gene from A. balhimycina)(13) in A. japonicum identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic on December 25, 2016 by UNIVERSITAETSBIBLIOTHEK TUEBINGEN enabled us to activate the cryptic ristomycin gene cluster. MS) data were recorded using a Bruker Daltonic maXis 4G instrument. Semipreparative HPLC was conducted using a Waters system consisting tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of The activation of ristomycin A production in A. japonicum of a 600 pump, a 996 photodiode array detector, a 7725i rheodyne injec- von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses now offers the possibility of optimizing production of ristomycin tor, and a PerkinElmer vacuum degasser series 200. Ristomycin monosul- by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and A in a genetically accessible strain. fate A standard was purchased from Aldrich and used without further nuclear magnetic resonance (NMR) spectroscopy confirmed the in silico prediction that the recombinant A. japonicum/pRM4- purification for MS analyses. For NMR and CD analyses, the standard was on December 25, 2016 by UNIVERSITAETSBIBLIOTHEK TUEBINGEN bbrAba synthesizes ristomycin A. MATERIALS AND METHODS desalted. HPLC–ESI-MS and HPLC-MS/MS. Culture broths were prepared by Bacterial strains and plasmids. Escherichia coli XL1-Blue (14) was used centrifugation or filtration and partially purified via adsorption chroma- for cloning purposes, and the methylation-deficient strain E. coli ET12567 tography with Amberlite XAD16 and subsequent ethyl acetate extraction. he nocardioform actinomycete Amycolatopsis japonicum MG417- secondary metabolite biosynthesis and the high number of puta- (15) was used to obtain unmethylated DNA for Amycolatopsis japonicum transformations. A. japonicum MG417-CF17 (1) is the (S,S)-EDDS-pro- Portions (2.5 ␮l) of the different fractions were analyzed by means of TCF17 (DSM 44213) is known as the producer of the hexaden- tive gene clusters, the identification, categorization, and interpre- HPLC–ESI-MS and HPLC-MS/MS using a Nucleosil 100-C column (3 18 tate chelating agent (S,S)-ethylenediaminedisuccinic acid [(S,S)- tation of the information encoded within the genomes required ducing wild type and was used to generate bbrAba and ajrR overexpression strains (this study). The overexpression plasmids pRM4-bbr and ␮m, 100 by 2 mm) (precolumn, 10 by 2 mm) (Dr. Maisch GmbH, Am- EDDS] (1), which is a biodegradable EDTA isomer (2) with automation. Bioinformatic tools such as antiSMASH 2.0 (7) offer Aba pRM4-ajrR derive from pRM4 (16), a pSET152-derived nonreplicative, merbuch-Entringen, Germany) coupled to an ESI mass spectrometer. similar properties. Though members of the genus Amycolatopsis a comprehensive pipeline capable of discovering and characteriz- ⌽C31 integration vector with an integrated constitutive ermEp* pro- LC-MS measurements were obtained from a LC/MSD Ultra Trap system are major producers of various antimicrobial compounds, such as ing biosynthetic loci covering the whole range of described sec- moter, an artificial ribosomal binding site, and an apramycin resistance XCT 6330 (Agilent Technologies, Waldbronn, Germany). Detection of the medically relevant rifamycin and vancomycin, no bioactive ondary metabolite compound classes. cassette. m/z values was conducted with Agilent DataAnalysis for 6300 series Ion secondary metabolite has been isolated from A. japonicum so far. Trap LC/MS 6.1 version 3.4 software (Bruker-Daltonik GmbH). Analysis In order to use this information, various strategies to activate Media and culture conditions. E. coli strains were grown in Luria Ϫ1 Ϫ1 was carried out at a flow rate of 0.4 ml min with gradient elution. Genome sequencing projects have revealed that the potential cryptic secondary metabolite gene clusters have been applied. In broth medium (17) at 37°C and were supplemented with 100 ␮gml Solvent A was 0.1% formic acid in acetonitrile, and solvent B was 0.06% of actinomycetes for the production of valuable secondary metab- many cases, stress conditions or variations of culture conditions apramycin when necessary to maintain plasmids. Liquid cultures of A. japonicum were cultivated in 100 ml of R5 medium (18) in an orbital formic acid in acetonitrile. Gradient elution was performed as follows: olites is much larger than previously expected. In certain cases, led to the production of new metabolites. More-targeted ap- shaker (220 rpm) in 500-ml baffled Erlenmeyer flasks with steel springs at t0 ϭ 0% solvent B, t7 ϭ 30% solvent B, t8 to t10 ϭ 100% solvent B. The flow more than 30 gene clusters encoding components of pathways for Ϫ1 proaches are the exchange of promoters or heterologous expres- Ϫ1 rate was 0.4 ml min , and the temperature was 40°C. Electrospray ion- secondary metabolite biosynthesis have been found per actinomy- 27°C. Liquid/solid media were supplemented with 100 ␮g ml apramy- sion of all relevant genes in a suitable host (8, 9). cin to select for strains carrying integrated antibiotic resistance genes. To ization (alternating positive and negative ionization) in Ultra Scan mode cete genome. For example, Streptomyces coelicolor, Streptomyces Previously we elucidated the biosynthesis of the glycopeptide detect glycopeptide production, wild-type A. japonicum or A. japonicum with a capillary voltage of 3.5 kV and a drying gas temperature of 350°C avermitilis, Streptomyces griseus, and Saccharopolyspora erythraea was used for LC-MS analysis. For tandem MS experiments, the analysis balhimycin in detail in Amycolatopsis balhimycina (10). The genus carrying the pRM4-bbrAba or pRM4-ajrR plasmid, respectively, were in- are each known to produce three to five secondary metabolites but Amycolatopsis is known to produce various glycopeptides that are cubated for 5 days in R5 medium without apramycin. was carried out either in negative mode with an Agilent LC/MSD Ultra actually possess more than 20 gene clusters that are predicted to Trap system XCT 6330 or in positive ionization mode employing an AB important clinical emergency antibiotics. New glycopeptides Construction of the integrative expression vector pRM4-bbrAba/ encode components of biosynthetic pathways for secondary me- SCIEX QT3200 instrument. might be key compounds to treat currently spreading glycopep- ajrR. For the overexpression of bbrAba and ajrR, the bbrAba and ajrR cod- tabolites (3, 4, 5, 6). This exemplifies that a large number of these ing regions were amplified by using the primer pair bbrAba-FP (FP stands Scaled-up cultivation, extraction, and purification of ristomycin A. pathways are cryptic, meaning that they are expressed poorly or for forward primer) and bbrAba-RP (RP stands for reverse primer) and A. japonicum/pRM4-bbrAba was cultivated in a 20-liter fermentor (b20; not at all under standardized laboratory conditions. One strategy the primer pair ajrR-FP and ajrR-RP (see Table S1 in the supplemental Giovanola). The fermentor was inoculated with 2% (vol) of shaking cul- Received 30 May 2014 Returned for modification 15 June 2014 tures grown for 48 h in 500-ml Erlenmeyer flasks with one baffle and steel to obtain access to this enormous genetic potential is the genome material), respectively. The 968-bp (bbrAba) and the 981-bp (ajrR) PCR Accepted 27 July 2014 products were integrated into pRM4 via the primer-attached NdeI and spring in tryptic soy broth (TSB). The fermentation was carried out at mining approach. The crucial point of genome mining is in learn- Published ahead of print 11 August 2014 XbaI sites for bbrAba and NdeI and HindIII sites for ajrR, downstream of 27°C with an agitation rate of 1,000 rpm and an aeration rate of 0.5 vol/ ing how to identify, subsequently activate, and finally exploit these Address correspondence to Evi Stegmann, the ermEp* promoter. vol/min. After 50 h of fermentation, 1 liter of fermentation broth was gene clusters, making their product accessible for evaluation as [email protected]. Direct transformation of A. japonicum. For transformation of A. taken, and bacterial cells were removed by centrifugation. Diaion HP-20 drug leads and for other biotechnological applications. The avail- Supplemental material for this article may be found at http://dx.doi.org/10.1128 japonicum, the direct transformation method of Stegmann et al. (19) was adsorbent resin (50 g literϪ1) was added to a portion of the supernatant ability of hundreds of actinomycete genome sequences and the /AAC.03512-14. modified. Mycelia were grown in 100 ml of TSB-D (17 g Bacto tryptone, 3 (900 ml), and the mixture was agitated in a 1.5-liter Erlenmeyer flask at Copyright © 2014, American Society for Microbiology. All Rights Reserved. rapidly decreasing costs of genome sequencing have made ge- g peptone 110, 5 g NaCl, 2.5 g K2HPO4, and 2.5 g glucose per liter Milli- 120 rpm for 2 h. The HP-20 resin was then passed through a fritted funnel nome mining the most promising tool to generate raw data for doi:10.1128/AAC.03512-14 pore H2O) for 24 h in a 1-liter Erlenmeyer flask with 4 bottom baffles at and washed with water, and the bound metabolites were eluted using a drug discovery. However, due to the biochemical heterogeneity in 30°C and 220 rpm; 5-ml portions from these precultures were used to stepwise gradient of isopropanol-H2O (acidified with 1% acetic acid inoculate 100 ml of TSB-D, which were incubated for 36 h under the same [HOAc]) to produce five fractions (A to E). Fractions B, C, and D, eluting conditions as the preculture. with 5, 10, and 15% isopropanol in acidified water, respectively, were Detection of ristomycin biosynthesis by HPLC-DAD. Glycopeptide found to potently inhibit the growth of B. subtilis in an agar diffusion October 2014 Volume 58 Number 10 Antimicrobial Agents and Chemotherapy p. 6185–6196 aac.asm.org 6185 production was determined by bioassays with Bacillus subtilis ATCC 6633 assay. Each bioactive fraction was rechromatographed with reversed- as the test organism after growth in R5 medium or by high-performance phase HPLC (RP-HPLC). For RP-HPLC separation, a Phenomenex Syn-

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Activation of a Silent Ristomycin A Cluster Spohn et al. Downloaded from Downloaded from

FIG 1 Bioassay after cluster activation by expression of bbrAba and ajrR, respectively. Wild-type A. japonicum (1), A. japonicum/pRM4-bbrAba (2), and A. japonicum/pRM4-ajrR (3) were grown for 5 days in R5 medium, and 20 ␮l of culture supernatant was assayed for bioactivity against B. subtilis.

ergi Hydro-RP 80A column (10 by 250 mm; 4 ␮m) in combination with a tial for the production of secondary metabolites. They are major pro- http://aac.asm.org/ http://aac.asm.org/ Phenomenex SecurityGuard AQ C18 precolumn (10 by 10 mm) was used. ducers of various glycopeptides like balhimycin (A. balhimycina), the Ϫ1 The flow rate was 2.0 ml min . UV monitoring at 210 and 254 nm was medically relevant vancomycin (Amycolatopsis orientalis NRRL performed. Elution was performed as follows: (i) isocratic elution at 20:80 2452), and rifamycin (Amycolatopsis mediterranei), which is one acetonitrile (MeCN)-H2O (0.1% trifluoroacetic acid [TFA]) over a period component in the drug cocktail for the treatment of tuberculosis and of 10 min, (ii), gradient elution from 20:80 to 40:60 MeCN-H O (0.1% 2 inactive meningitis. However, these species are difficult to manipu- TFA) over 10 min, (iii) gradient elution from 40:60 to 100:0 MeCN-H2O (0.1% TFA) over 20 min, and (iv) isocratic elution at 100% MeCN for an late genetically because of the lack of efficient transformation systems. additional 10 min. This procedure yielded collectively 33 mg of ristomy- In contrast to these species, we were able to establish a DNA trans- cin A. fer protocol for A. japonicum (19). Although we have been work- on December 25, 2016 by UNIVERSITAETSBIBLIOTHEK TUEBINGEN on December 25, 2016 by UNIVERSITAETSBIBLIOTHEK TUEBINGEN Platelet aggregation. For preparation of platelet-rich plasma (PRP), ing with this species in our laboratory for many years, no antibi- citrate-anticoagulated human blood was centrifuged at 200 ϫ g for 10 otically active product could be identified so far. In previous work, min. After centrifugation, the PRP was collected in a fresh tube, and the it was shown that A. japonicum produces glycopeptide-resistant remaining blood was centrifuged at 2,500 ϫ g to obtain platelet-poor cell wall precursors (20) and that the genome contains an oxyB plasma (PPP). Afterwards the platelet count in the PRP was estimated gene, which is involved in the production of glycopeptides (21). with a KX21-N automatic hematology analyzer (Sysmex, Norderstedt, Germany). After adjusting the PRP to a platelet concentration of 200 ϫ From these results, we assumed that A. japonicum may have the 105 ␮lϪ1 with the obtained PPP, aggregation was estimated from light potential to produce a glycopeptide. transmission measurements determined with a luminoaggregometer To test whether A. japonicum has this potential, we made use of (model 700; Chrono-Log Corp., Havertown, PA, USA). Following cali- the observation that all known glycopeptide clusters are con- bration, agonists were added at the indicated concentrations, and aggre- trolled by a pathway-specific StrR-like regulator (22). Hence, we gation was measured for 10 min with a stir speed of 1,000 rpm at 37°C. The applied a new cluster activation strategy, overexpressing the gene extent of aggregation was quantified as percentage of light transmission. encoding the characterized pathway-specific transcriptional reg- Data analysis was performed with the aggrolink8 software (Chrono-Log). ulator of the balhimycin gene cluster, bbr (13), in A. japonicum The adjusted PRP was either treated with agonists at the indicated con- Aba centrations or not treated with an agonist. aiming to awake the cryptic glycopeptide gene cluster. Therefore, Gene expression analysis by RT-PCR. For reverse transcription-PCR bbrAba was cloned into the integrative vector pRM4 under the

(RT-PCR) experiments, the wild-type A. japonicum and the bbrAba and control of the constitutive promoter ermEp* and transferred into ajrR overexpression strains were grown in R5 medium. After 25 h, the cells A. japonicum by direct transformation. The recombinant species were harvested and disrupted using glass beads and a Precellys homoge- A. japonicum carrying the pRM4-bbrAba plasmid was grown in R5 nizer (Peqlab). RNA preparations were treated twice with DNase I (Fer- medium for 5 days, and the culture supernatant was analyzed in a mentas). To exclude DNA contamination, negative controls were carried growth inhibition assay. Whereas the supernatant from wild-type FIG 2 HPLC chromatograms of wild-type A. japonicum and A. japonicum/pRM4-bbrAba after growth for 5 days in R5 medium and ristomycin standard. The out by using total RNA as the template for a PCR using the primer pair A. japonicum did not contain any biologically active compound, boxed regions show the corresponding DAD spectra. Rt, retention time; mAU, milliabsorbance units. sigB-RT-FP and sigB-RT-RP (see Table S1 in the supplemental material). the culture supernatant from the recombinant A. japonicum- cDNA from 3 mg RNA was generated with random hexameric primers, reverse transcriptase, and cofactors (Fermentas). PCRs were performed (pRM4-bbrAba) strongly inhibited growth of the indicator species, with the primers listed in Table S1. PCRs were carried out under the B. subtilis (Fig. 1). The metabolic profiles of the supernatants were open reading frames (ORFs) (45). Extensive genome analysis us- (Table S2) which showed high similarity to described glycopeptide following conditions: (i) an initial denaturation step (94°C for 2 min); (ii) determined by HPLC-DAD (Fig. 2). The chromatogram of the A. ing antiSMASH 2.0 (7) revealed the presence of 29 putative sec- gene clusters, such as the balhimycin (25), teicoplanin (26), and 27 cycles of PCR, with 1 cycle consisting of denaturation (95°C for 30 s), japonicum/pRM4-bbrAba supernatant revealed two peaks with gly- ondary metabolite gene clusters (see Table S2 in the supplemental A47934 (27) gene clusters. Cluster 24 consists of 39 distinct ORFs annealing (59°C for 30 s), and polymerization (72°C for 30 s); and (iii) an copeptide-specific DAD spectra. These peaks were absent in the material). One of the gene clusters (cluster 4) encodes the synthe- (AJAP_31985 to AJAP_32175) (Fig. 3) with a total size of almost 69 additional polymerization step (72°C for 1 min). Each PCR mixture (25 chromatogram of the wild-type A. japonicum supernatant (Fig. 2). sis of the polyketide synthase (PKS) compound ECO-0501, which kb. The putative functions of all 39 gene products were deduced by ␮l) contained a 1-␮l aliquot of RT reaction product. As a positive control, From these results, we concluded that A. japonicum does indeed was already identified in the vancomycin producer Amycolatopsis comparative amino acid sequence analysis with homologues from cDNA was amplified from the major vegetative sigma factor (sigB) tran- have the genetic potential to produce a glycopeptide antibiotic, script, which is produced constitutively. The PCR products were analyzed orientalis ATCC 43491 by a genome-scanning technology (23). known glycopeptide clusters (Table 1). Cluster 24 is predicted to en- by agarose gel electrophoresis (2.0%). which is synthesized only after transcriptional activation. ECO-0501 defines a new structural class of the polyketide antibi- code all the enzymes required for biosynthesis of a glycopeptide, Nucleotide sequence accession number. The GenBank accession Genome mining in A. japonicum. In order to identify the gene otic octanoic acid glucuronide and possesses activity against comprising enzymes responsible for assembly and export of the gly- number of the genome sequence of A. japonicum is CP008953 (45). cluster for biosynthesis of the new glycopeptide, its regulation, Gram-positive bacteria, including methicillin-resistant Staphylo- copeptide, self-resistance, and gene regulation. By considering these and the self-resistance mechanism and to evaluate the potential to coccus aureus (MRSA) and vancomycin-resistant enterococci (VRE) deduced functions, the boundaries of the glycopeptide gene cluster 24 RESULTS AND DISCUSSION synthesize additional secondary metabolites, the genome of A. (24). However, since this cluster and the corresponding product were were predicted. The left border is probably delimited by orf1,adahp Activation of a cryptic glycopeptide gene cluster. Members of the japonicum was sequenced. The genome sequence of A. japonicum already known, we focused our attention on the type III PKS/nonri- homologue, involved in tyrosine precursor supply for the synthesis of genus Amycolatopsis are known to possess a particularly high poten- is approximately 8.96 Mb in size and contains 8,464 putative bosomal peptide synthetase (NRPS) hybrid gene cluster (cluster 24) the aproteinogenic amino acids (28), and the right border is most

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Activation of a Silent Ristomycin A Cluster Spohn et al. ␤ - e formation Ht formation (aa 5 to 7) (aa 4 to 6) (aa 1 to 3) (aa 2 to 4) deacetylation Downloaded from Downloaded from Hpg formation Cross-linking Cross-linking Cross-linking Cross-linking Peptide synthesis/ Mannosyl transfer Sugar/ristosamine Dpg formation Biosynthetic role ␤ -Ht formation Peptide methylation Methylation Mannosyl transfer Dpg/Hpg formation d http://aac.asm.org/ http://aac.asm.org/ N -acetylglucosamine deacetylase Sugar addition/ -acetylglucosamine transferase activity (type III PKS) 5-epimerase activity dihydroxyphenylglycine aminotansferase synthetase/adenylation domain Nonribosomal peptide synthetase Nonribosomal peptide synthetase Nonribosomal peptide synthetase Peptide synthesis p -Hydroxymandelate oxidase Mannosyltransferase p -Hydroxymandelate synthase Methyltransferase Methyltransferase p -Hydroxy- and 3,5- on December 25, 2016 by UNIVERSITAETSBIBLIOTHEK TUEBINGEN on December 25, 2016 by UNIVERSITAETSBIBLIOTHEK TUEBINGEN 94809291 C-3 aminotransferase NAD-dependent epimerase/dehydratase NDP-hexose 2,3-dehydratase 97 3,5-Dihydroxyphenylacetyl-CoA synthase 91 dTDP-4-keto-6-deoxy-glucose- 889085 P450 monooxygenase 86 P450 monooxygenase 94 P450 monooxygenase P450 monooxygenase Adenylation, loading of NRPS 9083 3,5-Dihydroxyphenylacetyl-CoA oxidase Enoyl-CoA hydratase/enhances DpgA 8790 3-Deoxy-7-phosphoheptulonate synthase Precursor supply Enoyl-CoA hydratase/enhances DpgA Amino acid similarity (%) Proposed function 81 92 P450 monooxygenase 73 UDP- N 7081 UDP- N -acetylglucosamine transferase UDP- N -acetylglucosamine transferase Sugar addition 7873 Inactive UDP- N -acetylglucosamine transferase 79 Mannosyltransferase 889194 Nonribosomal peptide Hydrolase TeiB (Dpg) TEI [-] [-] [-] Hmo DpgA [-] [-] OxyC* OxyB OxyE OxyA* MbtH DpgC DpgB [-] DpgD HmaS [-] [-] [-] GtfB Orf2/Dbv21* GtfA* [-] [-] Orf15* [-] [-] HpgT a

FIG 3 Genetic organization of the ristomycin A (ris) cluster identified in A. japonicum. Predicted ORFs are represented by an arrow drawn to scale and are numbered as in Table 1. Gene names are indicated underneath the corresponding ORFs. Predicted functions of genes are listed. TE, thioesterase. StaC (Hpg–Hpg– ␤ -Ht) TeiC (Hpg–Hpg– ␤ -Ht) STA [-] [-] [-] Hmo DpgA [-] [-] StaJ StaH StaG* StaF StaE DpgC DpgB [-] DpgD HmaS [-] [-] [-] [-] [-] [-] [-] [-] [-] [-] [-] HpgT c likely delimited by orf39,avanH homologue, involved in self-resis- peptide gene cluster contains four NRPS genes (orf33 to orf30) that tance (20)(Table 1). are predicted to encode the enzymes catalyzing the assembly of the Proposed glycopeptide biosynthesis in A. japonicum. In the heptapeptide backbone. The genetic organization and domain last 2 decades, considerable progress has been made in under- composition of these NRPS genes and the predicted specificity BAL standing the genetics and biochemistry of glycopeptide biosynthe- of the A domains indicate that the ORF33 protein, including Glycopeptide cluster sis (10, 22, 29). The profound knowledge of the individual en- module 1 and module 2, incorporates Hpg and ␤-Ht; ORF32, zymes participating in glycopeptide biosynthesis makes it possible including module 3, incorporates Dpg; ORF31, including 1050 BspB (Hpg–Hpg– ␤ -Ht) StaB (Dpg) 369325469 DvaB* DvaE* 356 DvaA* Hmo* No. of amino acids 366 DpgA* 575205 [-] DvaD* 413398 OxyC 385 OxyB* 391 [-] 69 OxyA MbtH* 404220 DpgC* DpgB* 358263 Dahp* DpgD* 350396 HmaS* OxyD* 385 BgtfA* 404405 BgtfC* BgtfB* 281374 Bal2 270 [-] [-] 346605 [-] [-] to almost completely predict the structure of the final product. modules 4, 5, and 6, incorporates Hpg, Hpg, and ␤-Ht; and 577274 BpsB* 453 Bhp* PgaT* (i) Synthesis of the nonproteinogenic amino acids. Glyco- ORF30, including module 7, incorporates Dpg. This amino peptides consist of a heptapeptide backbone constituted mainly acid composition was confirmed by all three amino acid pre-

by aprotenogenic aromatic amino acids such as 3,5-dihydroxy- diction tools used by antiSMASH 2.0 (7, 36). Hence, the assem- cluster ORFs with ORFs from published glycopeptide clusters 1 phenylglycine (Dpg), p-hydroxyphenylglycine (Hpg), and ␤-hy- bled heptapeptide has the predicted amino acid sequence Hpg – ris 2 3 4 5 6 7

droxytyrosine (␤-Ht). Since detailed information on their biosyn- ␤-Ht –Dpg –Hpg –Hpg -␤-Ht –Dpg . The organization of the (Dpg-TE) 1853 BspC (Dpg-TE) StaD (Dpg-TE) TeiD (Dpg-TE) (Hpg-Hpg- ␤ -Ht) 3994 (Dpg) thesis is available (30, 31, 32, 33, 34), it is possible to conclude that epimerization domains within the modules predicts a stereochemis- hmaS oxyD rpsB riaC riaB riaA hmo gtfA gtfC gtfB orf2 gtfD mtfA mtfB gtfE rpsE rhp pgat dpgA gtfF riaD oxyB oxyE oxyA mbtH rpsD rpsC dpgC dpgB dahp dpgD most likely, orf5 to orf2 and orf16 are responsible for the synthesis try of L-D-L-D-D-L-L, which is consistent with the stereochemistry of oxyC of Dpg, orf11, orf12, and orf16 are responsible for Hpg synthesis, teicoplanin (26), but inconsistent with L-D-D-D-D-L-D of A47934 (27) and orf15 to orf13 are responsible for synthesis of ␤Ht (Fig. 3 and and D-D-L-D-D-L-L of balhimycin (35), respectively. orf29, located

Table 1). downstream of the NRPS genes encodes a 69-amino-acid small Comparison of the AJAP_32105 (ii) Synthesis of the aglycon. (a) Synthesis of the linear back- MbtH-like polypeptide. MbtH-like peptides are common features b

bone. The amino acids are assembled by nonribosomal peptide of NRPS biosynthesis gene clusters acting as facilitators of the ORF 910 AJAP_32025 AJAP_32030 11 AJAP_32035 7 AJAP_32015 4 AJAP_32000 2 AJAP_31990 12 AJAP_32040 13 AJAP_32045 32 AJAP_32140 8 AJAP_32020 24 AJAP_32100 2223 AJAP_32090 AJAP_32095 1819 AJAP_32070 AJAP_32075 2021 AJAP_32080 AJAP_32085 17 AJAP_32065 14 AJAP_32050 15 AJAP_32055 16 AJAP_32060 31 AJAP_32135 5 AJAP_32005 6 AJAP_32010 26 AJAP_32110 27 AJAP_32115 28 AJAP_32120 29 AJAP_32125 30 AJAP_32130 1 AJAP_31985 TABLE 1 ris ORF no. Locus tag Gene 3 AJAP_31995 synthetases to form a heptapeptide (35). The A. japonicum glyco- peptide-assembling machineries by stimulating adenylation reac- 25

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tions (37, 38, 39, 40). The predicted incorporation of aromatic amino acids into the heptapeptide revealed that the A. japonicum glycopeptide belongs to either the type II, type III, or type IV glycopeptides but not to the type I (vancomycin type) glycopep- tides characterized by aliphatic amino acids at positions 1 and 3 Downloaded from Downloaded from Precursor supply Regulation Excretion Resistance (Fig. 4). (b) Cross-linking of the aromatic amino acid residues. Dur- ing peptide formation, the aromatic residues are connected due to

ATCC 31121. [-], absence. *, the the activity of P450 monooxygenases (Oxy) to form a rigid cup- shaped structure (41, 42) required for the interaction with their molecular target, the D-alanyl–D-alanine (D-Ala–D-Ala) terminus of bacterial cell wall precursors. The A. japonicum glycopeptide gene cluster contains four oxy genes (orf28 to orf25), putatively http://aac.asm.org/ encoding P450 monooxygenases, as within the teicoplanin, http://aac.asm.org/ A47934, and dalbavancin gene clusters, all characterized by a fully

-Ala dipeptidase -Lac ligase cyclized heptapeptide backbone (22). These cross-linked glyco- D D Actinoplanes teichomyceticus peptides contain three ether bridges (between amino acids 1 and 3, -Ala– -Ala– -Lactate dehydrogenase Nonribosomal peptide synthetase D D D 2 and 4, and 4 and 6) and one C-C link (between amino acids 5 and 7). On the basis of the model proposed for the balhimycin and A47934 oxygenases (42, 43) and using amino acid sequence sim- ilarity as a criterion, it is likely that OxyA is involved in the cross- on December 25, 2016 by UNIVERSITAETSBIBLIOTHEK TUEBINGEN on December 25, 2016 by UNIVERSITAETSBIBLIOTHEK TUEBINGEN 88 88 9091 Prephenate dehydrogenase StrR-like regulator 85 ABC transporter 86 linking of the aromatic residues of amino acids 2 and 4, while OxyB is involved in the cross-linking of amino acids 4 and 6 and OxyC is involved in the cross-linking of amino acids 5 and 7. The NRRL15009; TEI, teicoplanin, fourth encoded oxygenase (OxyE) catalyzes the type III and type IV glycopeptide-specific cross-linking between Hpg at position 1 and Dpg at position 3 (43)(Fig. 4 and Table 1). A backbone consisting of seven aromatic amino acids and a predicted cyclization of all of these residues is a characteristic of VanH* VanX* Pdh StrR-like Orf4 VanA* glycopeptide classes III and IV and excludes the classification to Streptomyces toyocaensis type II where the residues of amino acids 1 and 3 are not linked (Fig. 4). (iii) Tailoring reactions by adding sugars and methyl groups. Structural diversity within the glycopeptide family is generated by different optionally and variably occurring glycosylation, methyl- FIG 4 Classification of the glycopeptides. (A) Type I glycopeptides exemplified by vancomycin. These glycopeptides contain aliphatic chains in amino acids1 ation, acetylation, halogenation, and sulfation patterns. The A. and 3. (B) Type II glycopeptides exemplified by actinoidin A. These glycopeptides contain aromatic aliphatic chains in amino acids 1 and 3. (C) Type III VanH VanX Pdh StaQ StaU VanA DSM5908; STA, A47934, japonicum gene cluster contains six glycosyltransferase genes glycopeptides exemplified by ristomycin A. These glycopeptides are like type II glycopeptides, and they contain an extra F-O-G ring system. Ring abbreviations: (orf24 to orf22, orf20, orf6, and orf18) presumably responsible for A, Me-L-Dpg; B, D-Hpg; C, L-␤-Ht; D, D-Hpg; E, D-␤-Ht; F, Me-L-Dpg; G, L-Hpg. (D) Type IV glycopeptides exemplified by teicoplanin. These glycopeptides the decoration of the aglycon with sugar moieties. No other de- are like type III glycopeptides plus they have aliphatic side chains on sugar. scribed glycopeptide gene cluster contains such an extended amount of glycosyltransferases. For example, the balhimycin gene cluster encodes three glycosyltransferases (BgtfA, BgtfB, and are not directly involved in the synthesis and tailoring of the gly- mize the precursor supply of the aromatic amino acids (28). While BgtfC), while the teicoplanin gene cluster encodes three glycosyl- copeptide, are located at the left (orf1) and right (orf34 to orf39) prephenate dehydrogenase genes are encoded in all described clus-

Amycolatopsis balhimycina transferases (GtfA, GtfB, and one putative mannosyltransferase) borders (Fig. 3). orf39 to orf37 encode the VanHAX-like glycopep- ters, the DAHP synthase-encoding gene is lacking in some gene clus- (Table 1). orf10 to orf7 encode peptides that exhibit high similarity tide resistance cassette. However, neither a vanRS (encoding the ters, like the teicoplanin and A47934 gene clusters (22). to the TDP-L-epivancosamine biosynthesis enzymes EvaA to two-component system) nor a vanY (D,D-carboxypeptidase) Categorization of the A. japonicum glycopeptide by consid- 298 [-] 202 [-] 276265 Pdh* Bbr* 650 Tba* 348 [-] EvaD (Fig. 3 and Table 1) of chloroeremomycin biosynthesis (44), homologue are located within the A. japonicum glycopeptide gene ering the genetic features. The in silico data strongly suggested suggesting that they are involved in the biosynthesis of an uncommon cluster. Previous cell wall precursor analyses (20) demonstrated that the final product of the identified gene cluster is a 6-fold- amino-deoxy sugar, which finally decorates the A. japonicum glyco- that A. japonicum synthesized a glycopeptide-resistant cell wall, glycosylated, twice methylated, and fully cross-bridged glycopep- peptide. Besides the glycosyltransferases, two methyltransferase ho- although no glycopeptide production could be observed, suggest- tide. The genetic organization and domain composition of the 1 2 mologues are encoded within the A. japonicum gene cluster (orf17 ing constitutive expression of the resistance genes. NRPS specifies an amino acid sequence L-Hpg –D-␤-Ht –L- 3 4 5 6 7 and orf21), but the cluster does not encode further tailoring enzymes, The orf34-encoded protein is likely to specify export functions, Dpg –D-Hpg –D-Hpg –L-␤-Ht –L-Dpg . The incorporation of like sulfotransferases, halogenases, or acetyltransferases. The occur- since it is predicted to be an ABC-type transporter exhibiting high aromatic amino acids at positions 1 and 3 together with the pre- vanX pdh ajrR rpsA (Hpg- ␤ -Ht)tri 2062 BspA (Leu– ␤ -Ht–Asn) StaA (Hpg-Tyr) TeiA (Hpg-Tyr) vanA vanH

ORFs with selected genes from previously published glycopeptide clusters. rence of an acetyltransferase is a specific feature of the lipoglycopep- similarity to the balhimycin exporter Tba (15)(Table 1). dicted complete cross-linked heptapeptide by the four oxygenases ris tide class (teicoplanin type or type IV glycopeptides). The absence of The orf36 encodes a StrR-like transcriptional activator with and the absence of any acyltransferase or halogenase imply that an acetyltransferase gene within the identified gene cluster suggests high similarity to the balhimycin pathway-specific transcriptional the A. japonicum gene cluster has the biosynthetic potential to

that the A. japonicum glycopeptide exemplifies a type III glycopeptide activator BbrAba (13) (see below). produce a type III glycopeptide (Fig. 4). Until now, only the struc- (Fig. 4). Two additional genes, presumably encoding a 3-deoxy-7-phos- ture of the type III glycopeptides, exemplified by ristomycin A Resistance, export, regulation, and precursor supply. The phoheptulonate synthase (DAHP synthase) homologue (orf1) and a (produced by A. lurida) was known; no biosynthesis gene cluster CoA, coenzyme A. Comparison of the ris , ristomycin A. aa, amino acids. Homologues in other glycopeptide clusters. BAL, balhimycin, highest score is reported. The pair responsible for the score is indicated by an asterisk. 36 AJAP_32160 37 AJAP_32165 35 AJAP_32155 3334 AJAP_32145 AJAP_32150 a b c d e 3839 AJAP_32170 AJAP_32175 seven genes of the A. japonicum glycopeptide gene cluster, which prephenate dehydrogenase homologue (orf35) are predicted to opti- was known. Therefore, the A. japonicum gene cluster identified

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Activation of a Silent Ristomycin A Cluster Spohn et al.

here is the first type III glycopeptide gene cluster described so far duced from the genome sequence, the chemical structure of the (Fig. 3). The amino acid sequence and stereochemistry of the gly- glycopeptide was analyzed. Initial experiments with A. japonicum/

copeptide encoded by the A. japonicum gene cluster are consistent pRM4-bbrAba growing in shake flasks revealed a glycopeptide pro- with the sequence and stereochemistry of ristomycin A. Further- duction with quantities up to 50 mg literϪ1. The crude extract was more, the number and predicted function of the tailoring enzymes fractionated and analyzed for the presence of the glycopeptide Downloaded from (six glycosyltransferases and two methyltransferases) are in agree- Downloaded from (Fig. 2). A major compound with a quasimolecular ion [M ϩ ment with the ristomycin A decorations (Fig. 4). Considering all 2H]2ϩ ϭ m/z 1,034.8 (Fig. 6) and a minor compound with [M ϩ these congruences of the described ristomycin A structure and the 2H]2ϩ ϭ m/z 887.7 (data not shown) were detected. Since the genetic content within the identified A. japonicum gene cluster, we masses of the major and minor components were in agreement propose that this gene cluster encodes a glycopeptide which is with the masses of ristomycin A and B, the isolated glycopeptides either ristomycin A itself or a highly similar derivative. were compared with a commercially available ristomycin stan- Expression of the A. japonicum StrR-like regulator AjrR. The dard. HPLC-DAD and HPLC–ESI-MS analyses showed that both two closely related species A. japonicum and A. balhimycina each the glycopeptides synthesized by A. japonicum/pRM4-bbrAba and http://aac.asm.org/ harbor a glycopeptide gene cluster encoding the regulators AjrR http://aac.asm.org/ ristomycins A and B eluted at the same time, possessed the same and Bbr , respectively. These StrR-like regulators exhibit 84 and UV profile (Fig. 2), and showed in ESI-MS/MS analyses (positive Aba FIG 7 Batch fermentation of A. japonicum/pRM4-bbr in R5 medium. Bio- Aba and negative mode) an identical fragmentation pattern (see Fig. 91% amino acid identity and similarity, respectively. However, mass (o), ristomycin A (), and partial O pressure (pO )() are depicted. 2 2 S1 to Fig. S3 in the supplemental material). BbrAba initiates the transcription of the glycopeptide gene cluster A. japonicum/pRM4-bbrAba reached its maximal biomass value after 44 h of in A. balhimycina, while the A. japonicum gene cluster is of cryptic incubation, decreasing afterwards slowly during 2 days of further fermenta- To corroborate the findings and to obtain additional informa- nature under the identical laboratory conditions. tion. Metabolizing of different sugars in R5 medium (sucrose [␣-1,2-glyco- tion on the nature of the sugar units, the major glycopeptide syn- sidic linked glucose and fructose] and glucose) is reflected by a diauxic shift thesized by A. japonicum/pRM4-bbr was isolated in a pure form One reason why the glycopeptide cluster in A. japonicum is not (between 24 and 44 h) during cultivation, apparent in growth retardation and Aba and compared with a ristomycin A standard employing NMR, expressed under standard conditions could be that the regulator pO2. on December 25, 2016 by UNIVERSITAETSBIBLIOTHEK TUEBINGEN AjrR is not functional. To evaluate its functionality, we overex- on December 25, 2016 by UNIVERSITAETSBIBLIOTHEK TUEBINGEN CD, and high-resolution mass spectrometry. For this purpose, the pressed ajrR under the control of the constitutive ermEp* in A. initial shake flask cultivation was scaled up to a 20-liter volume japonicum. The supernatant of the recombinant A. japonicum/ suggest that ajrR is not transcribed in the wild-type species. There- using a Giovanola b20 fermentor. Glycopeptide production was pRM4-ajrR inhibited growth of B. subtilis, while the supernatant fore, transcriptional analyses of ajrR and representative biosyn- detected after 24 h and reached a maximum amount up to 200 mg Ϫ1 FIG 5 Transcriptional pattern of representative ristomycin biosynthesis of wild-type A. japonicum did not (Fig. 1). HPLC-DAD analyses thetic genes (riaA, dpgA, bhp, oxyA, hmaS, and vanH) were liter after 50 h and stayed almost constant during 2 more days of genes. The gene names are indicated to the right of the gel. Cultures were performed after growth of wild-type A. japonicum, A. japonicum/ fermentation (Fig. 7). For the isolation of the glycopeptide, 1 liter grown in R5 medium for 25 h. sigB is the major sigma factor of A. japonicum confirmed the biosynthesis of the glycopeptide (data not shown). and was used as a housekeeping gene to normalize the RNA. WT, wild type. These results demonstrated the in vivo functionality of AjrR and pRM4-bbrAba and A. japonicum/pRM4-ajrR in production me- of fermentation broth was taken after 50 h of fermentation and dium R5 (Fig. 5). In wild-type A. japonicum, no ajrR transcription separated by centrifugation into supernatant and mycelium. Sub- was detected, and it is therefore not surprising that no other in- sequent workup of the extract led to the isolation of the major vestigated biosynthetic gene was transcribed. However, in A. ja- glycopeptide in a highly pure form at a yield of 37 mg lϪ1. HR-MS

ponicum/pRM4-ajrR and A. japonicum/pRM4-bbrAba, transcrip- analysis confirmed that the isolated glycopeptide showed the tion of all the investigated genes could be observed. Coincident to same exact mass as that of the ristomycin A standard and there- the observations of Schäberle et al. (20) that A. japonicum pro- fore possessed the same molecular formula of C95H110N8O44 (see duces a glycopeptide-resistant cell wall, whereas no glycopeptide Fig. S4 and S5 in the supplemental material). 1H and the 13C NMR production could be detected, we could observe transcription of spectra of the isolated glycopeptide and the ristomycin A standard the vanH resistance gene in the wild type. It will be interesting to were absolutely superimposable, with the exception of additional

ascertain why bbrAba is transcribed under standard conditions peaks in the commercially available ristomycin standard which whereas ajrR is not. can be attributed to the presence of up to 10% ristomycin deriva- Isolation and spectroscopic characterization of the A. ja- tives in the sample (Fig. S6 to S14). Further analysis of the one- ponicum glycopeptide. In order to confirm the assumption de- dimensional (1D) and two-dimensional (2D) NMR data (Fig. S15

FIG 8 Ristomycin-dependent platelet aggregation. (Left) Representative tracings of aggregometry after stimulation of human platelets with 2.5 mg mlϪ1 commercial (black line) or A. japonicum ristomycin A (light gray line) as well as 10 ␮M ADP (dark gray line) and water control. (Right) Results of aggregometry FIG 6 HPLC–ESI-MS analysis (positive mode) of A. japonicum glycopeptide and ristomycin A. (Top) Ristomycin A standard (0.1 mg mlϪ1). (Bottom) A. after stimulation of human platelets with 2.5 mg mlϪ1 commercial or A. japonicum ristomycin A as well as 10 ␮M ADP. The values are arithmetic means plus 2ϩ japonicum/pRM4-bbrAba glycopeptide. [M ϩ 2H] was observed at m/z 1,034.8 and 1,034.9. The relative intensity is shown on the y axes. standard errors of the means (SEM) (error bars) for four experiments.

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Activation of a Silent Ristomycin A Cluster Spohn et al. to Fig. S20) allowed the complete assignment of all hydrogen and 10. Stegmann E, Frasch HJ, Wohlleben W. 2010. Glycopeptide biosynthesis gene cluster. Metab. Eng. 12:455–461. http://dx.doi.org/10.1016/j.ymben Robinson J, Süssmuth RD, Wohlleben W. 2006. Genetic analysis of the carbon atoms (Table S3), which supported the hypothesis that the in the context of basic cellular functions. Curr. Opin. Microbiol. 13:595– .2010.05.001. balhimycin (vancomycin-type) oxygenase genes. J. Biotechnol. 124:640– isolated compound possessed the same planar structure and the 602. http://dx.doi.org/10.1016/j.mib.2010.08.011. 29. Wohlleben W, Stegmann E, Süssmuth RD. 2009. Molecular genetic 653. http://dx.doi.org/10.1016/j.jbiotec.2006.04.009. 11. Grundy WE, Sinclair AC, Theriault RJ, Goldstein AW, Rickher CJ, approaches to analyze glycopeptide biosynthesis. Methods Enzymol. 458: 38. Boll B, Taubitz T, Heide L. 2011. Role of MbtH-like proteins in the adeny- same relative configuration as that of ristomycin A. Since the CD Warren HB, Jr, Oliver TJ, Sylvester JC. 1956–1957. Ristocetin, micro- 459–486. http://dx.doi.org/10.1016/S0076-6879(09)04818-6. lation of tyrosine during aminocoumarin and vancomycin biosynthesis. J. spectra of the isolated glycopeptide and a commercially available biologic properties. Antibiot. Annu. 1956–1957:687–692. 30. Hubbard BK, Thomas MG, Walsh CT. 2000. Biosynthesis of L-p- Biol. Chem. 286:36281–36290. http://dx.doi.org/10.1074/jbc.M111.288092. Downloaded from ristomycin A standard were consistent (Fig. S21), it was deduced 12. Sarji KE, Stratton RD, Wagner RH, Brinkhous KM. 1974. Nature of von Downloaded from hydroxyphenylglycine, a non-proteinogenic amino acid constituent of 39. Herbst DA, Boll B, Zocher G, Stehle T, Heide L. 2013. Structural basis that the isolated glycopeptide also possessed the same absolute Willebrand factor: a new assay and a specific inhibitor. 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Activation of the production of ristomycin A. gene cluster from Amycolatopsis balhimycina contains bbr, encoding a 557–566. http://dx.doi.org/10.1007/s00203-012-0789-y. pacidamycin PacL adenylation domain by MbtH-like proteins. Biochem- Ristomycin A-dependent platelet aggregation. Measuring StrR-like pathway-specific regulator. J. Mol. Microbiol. Biotechnol. 13: 32. Pfeifer V, Nicholson GJ, Ries J, Recktenwald J, Schefer AB, Shawky RM, istry 49:9946–9947. http://dx.doi.org/10.1021/bi101539b. von Willebrand factor (vWF) activity is essential for the diagnosis 76–88. http://dx.doi.org/10.1159/000103599. Schröder J, Wohlleben W, Pelzer S. 2001. A polyketide synthase in 41. Bischoff D, Bister B, Bertazzo M, Pfeifer V, Stegmann E, Nicholson GJ, 14. Bullock WO, Fernandez JM, Short JM. 1987. Xl1-Blue: a high-efficiency glycopeptide biosynthesis: the biosynthesis of the non-proteinogenic Keller S, Pelzer S, Wohlleben W, Süssmuth RD. 2005. The biosynthesis of of von Willebrand disease (vWD). A common test method is the plasmid transforming recA Escherichia coli strain with ␤-galactosidase se- amino acid (S)-3,5-dihydroxyphenylglycine. J. Biol. Chem. 276:38370– vancomycin-type glycopeptide antibiotics–a model for oxidative side-chain http://aac.asm.org/ ristomycin platelet-induced agglutination method, which allows lection. Biotechniques 5:376–378. http://aac.asm.org/ 38377. http://dx.doi.org/10.1074/jbc.M106580200. cross-linking by oxygenases coupled to the action of peptide synthetases. discrimination among specific subtypes of the vWD. In a platelet 15. MacNeil DJ, Gewain KM, Ruby CL, Dezeny G, Gibbons PH, MacNeil 33. Puk O, Bischoff D, Kittel C, Pelzer S, Weist S, Stegmann E, Süssmuth Chembiochem 6:267–272. http://dx.doi.org/10.1002/cbic.200400328. aggregation assay, we could demonstrate that ristomycin A iso- T. 1992. Analysis of Streptomyces avermitilis genes required for avermectin RD, Wohlleben W. 2004. Biosynthesis of chloro-beta-hydroxytyrosine, a 42. Stegmann E, Rausch C, Stockert S, Burkert D, Wohlleben W. 2006. The biosynthesis utilizing a novel integration vector. Gene 111:61–68. http: nonproteinogenic amino acid of the peptidic backbone of glycopeptide small MbtH-like protein encoded by an internal gene of the balhimycin lated from A. japonicum has the same in vitro function as the //dx.doi.org/10.1016/0378-1119(92)90603-M. antibiotics. J. Bacteriol. 186:6093–6100. http://dx.doi.org/10.1128/JB.186 biosynthetic gene cluster is not required for glycopeptide production. reference ristomycin (Fig. 8). 16. Menges R, Muth G, Wohlleben W, Stegmann E. 2007. The ABC trans- .18.6093-6100.2004. FEMS Microbiol. Lett. 262:85–92. http://dx.doi.org/10.1111/j.1574-6968 porter Tba of Amycolatopsis balhimycina is required for efficient export of 34. Mulyani S, Egel E, Kittel C, Turkanovic S, Wohlleben W, Süssmuth RD, .2006.00368.x. ACKNOWLEDGMENTS the glycopeptide antibiotic balhimycin. Appl. Microbiol. Biotechnol. 77: van Pée KH. 2010. The thioesterase Bhp is involved in the formation of 43. Hadatsch B, Butz D, Schmiederer T, Steudle J, Wohlleben W, Süssmuth We are very grateful to A. Steck, Analytical Services & Applied NMR 125–134. http://dx.doi.org/10.1007/s00253-007-1139-x. beta-hydroxytyrosine during balhimycin biosynthesis in Amycolatopsis RD, Stegmann E. 2007. The biosynthesis of teicoplanin-type glycopeptide Development, at Bruker Biospin GmbH for the generous support and 17. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a labora- balhimycina. Chembiochem 11:266–271. http://dx.doi.org/10.1002/cbic antibiotics: assignment of p450 mono-oxygenases to side chain cycliza- on December 25, 2016 by UNIVERSITAETSBIBLIOTHEK TUEBINGEN NMR measurements. We also thank D. Wistuba and her team at the Mass tory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Har- on December 25, 2016 by UNIVERSITAETSBIBLIOTHEK TUEBINGEN .200900600. tions of glycopeptide A47934. Chem. Biol. 14:1078–1089. http://dx.doi bor, NY. 35. Recktenwald J, Shawky R, Puk O, Pfennig F, Keller U, Wohlleben W, .org/10.1016/j.chembiol.2007.08.014. Spectrometry Department, Institute for Organic Chemistry, University of 18. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA. 2000. Pelzer S. 2002. Nonribosomal biosynthesis of vancomycin-type antibiot- 44. Chen H, Thomas MG, Hubbard BK, Losey HC, Walsh CT, Burkart MD. Tuebingen, for HR-MS measurements. Practical Streptomyces genetics. John Innes Foundation, Norwich, United ics: a heptapeptide backbone and eight peptide synthetase modules. Mi- 2000. Deoxysugars in glycopeptide antibiotics: enzymatic synthesis of TDP- This work was supported by grants from the DFG (SFB 766) to Evi Kingdom. crobiology 148:1105–1118. L-epivancosamine in chloroeremomycin biosynthesis. Proc. Natl. Acad. Sci. Stegmann and from the BMBF (ERA-IB, GenoDrug) to Wolfgang Wohl- 19. Stegmann E, Pelzer S, Wilken K, Wohlleben W. 2001. Development of three 36. 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Table 1 Gordon, D., 2003. Viewing and editing assembled sequences using consed. Curr. Contents lists available at ScienceDirect Genome features of A. japonica MG417-CF17T. Protoc. Bioinform., 12 (Chapter 11, Unit 11). Gordon, D., Abajian, C., Green, P., 1998. Consed: a graphical tool for sequence finish- Features Chromosome Plasmid pAmyja1 ing. Genome Res. 8, 195–202. Journal of Biotechnology Length (bp) 8,961,318 92,539 Jankowitsch, F., Schwarz, J., Rückert, C., Gust, B., Szczepanowski, R., Blom, J., Pelzer, S., Streptomyces DNA-coding regions (bp) 8,156,541 80,232 Kalinowski, J., Mack, M., 2012. Genome sequence of the bacterium davawensis JCM 4913 and heterologous production of the unique antibiotic rose- G + C content (%) 68.89 68.23 oflavin. J. Bacteriol. 194, 6818–6827. CDS 8298 126 journal homepage: www.elsevier.com/locate/jbiotec Meyer, F., Goesmann, A., McHardy, A.C., Bartels, D., Bekel, T., Clausen, J., Kalinowski, rRNA genes (operons) 12 (4) 0 J., Linke, B., Rupp, O., Giegerich, R., Pühler, A., 2003. GenDB – an open source tRNA genes 55 0 genome annotation system for prokaryote genomes. Nucleic Acids Res. 31, Genome Announcement 2187–2195. Myronovskyi, M., Tokovenko, B., Manderscheid, N., Petzke, L., Luzhetskyy, A., 2013. Using the software antiSMASH 2.0 (Blin et al., 2013), in total 30 Complete genome sequence of Streptomyces fulvissimus. J. Biotechnol. 168, Complete genome sequence of the actinobacterium Amycolatopsis gene clusters encoding the biosynthesis of secondary metabolites 117–118. Nishikiori, T., Okuyama, A., Naganawa, H., Takita, T., Hamada, M., Takeuchi, T., T were identified, a number roughly comparable to other Pseudono- japonica MG417-CF17 (=DSM 44213T) producing Aoyagi, T., Umezawa, H., 1984. Production by actinomycetes of (S,S)-N,N - cardiaceae (Strobel et al., 2012). Among these clusters are one novel ethylenediamine-disuccinic acid, an inhibitor of phospholipase C. J. Antibiot. glycopeptide gene cluster (Spohn et al., 2014) as well as several (Tokyo) 37, 426–427. (S,S)-N,N�-ethylenediaminedisuccinic acid Rückert, C., Szczepanowski, R., Albersmeier, A., Goesmann, A., Fischer, N., Steinkam- clusters encoding the synthesis of polyketide, nonribosomal pep- per, A., Pühler, A., Biener, R., Schwartz, D., Kalinowski, J., 2014. Complete genome a, b a a a,c tide antibiotics, and lanthipeptides. sequence of the actinobacterium Actinoplanes friuliensis HAG 010964, producer Evi Stegmann ∗, Andreas Albersmeier , Marius Spohn , Helena Gert , Tilmann Weber , of the lipopeptide antibiotic friulimycin. J. Biotechnol. 178, 41–42. a b b, Wolfgang Wohlleben , Jörn Kalinowski , Christian Rückert ∗∗ Rückert, C., Szczepanowski, R., Albersmeier, A., Goesmann, A., Iftime, D., Musiol, Nucleotide sequence accession numbers E.M., Blin, K., Wohlleben, W., Pühler, A., Kalinowski, J., Weber, T., 2013. Com- a Mikrobiologie und Biotechnologie, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin/Deutsches Zentrum für Infektionsforschung, plete genome sequence of the kirromycin producer Streptomyces collinus Tu 365 Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany The complete genome sequence has been deposited in consisting of a linear chromosome and two linear plasmids. J. Biotechnol. 168, b Technology Platform Genomics, Center for Biotechnology (CeBiTec), Bielefeld University, Sequenz 1, 33615 Bielefeld, Germany 739–740. DDBJ/EMBL/GenBank under accession nos. CP008953 (chromo- c The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Alle 6, 2970 Hørsholm, Denmark Schowanek, D., Feijtel, T.C., Perkins, C.M., Hartman, F.A., Federle, T.W., Larson, R.J., some) and CP008954 (pAmyja1). 1997. Biodegradation of [S,S], [R,R] and mixed stereoisomers of ethylene diamine disuccinic acid (EDDS), a transition metal chelator. Chemosphere 34, 2375– Acknowledgements 2391. a r t i c l e i n f o a b s t r a c t Schwientek, P., Szczepanowski, R., Rückert, C., Kalinowski, J., Klein, A., Selber, K., Wehmeier, U.F., Stoye, J., Pühler, A., 2012. The complete genome sequence of the acarbose producer Actinoplanes sp. SE50/110. BMC Genomics 13, 112. T T This work was supported by grants of the BMBF (GenBioCom- Article history: We report the complete genome sequence of Amycolatopsis japonica MG417-CF17 (=DSM 44213 ) Schwientek, P., Szczepanowski, R., Rückert, C., Stoye, J., Pühler, A., 2011. Sequencing Received 20 August 2014 0315585A, 0315585B, and 0315585J) to Wolfgang Wohlleben, which was identified as the producer of (S,S)-N,N�-ethylenediaminedisuccinic acid during a screening of high G + C microbial genomes using the ultrafast pyrosequencing technology. Accepted 25 August 2014 for phospholipase C inhibitors. The genome of A. japonica MG417-CF17T consists of two replicons: the Tilmann Weber, and Jörn Kalinowski, respectively. J. Biotechnol. 155, 68–77. Available online 3 September 2014 chromosome (8,961,318 bp, 68.89% G + C content) and the plasmid pAmyja1 (92,539 bp, 68.23% G + C con- Spohn, M., Kirchner, N., Kulik, A., Jochim, A., Wolf, F., Muenzer, P., Borst, O., Gross, H., Wohlleben, W., Stegmann, E., 2014. Overproduction of ristomycin A by activa- tent), encoding a total of 8422 protein coding genes. Analysis of the sequence data revealed 30 clusters References tion of a silent gene cluster in Amycolatopsis japonicum MG417-CF17. AAC, pii: Keywords: encoding the biosynthesis of secondary metabolites. Amycolatopsis AAC.03512-14. © 2014 Elsevier B.V. All rights reserved. Anonymous, 1997. Validation of the publication of new names and new combina- Stegmann, E., Pelzer, S., Wilken, K., Wohlleben, W., 2001. Development of three Genome sequence tions previously effectively published outside the IJSB, List No. 63. Int. J. Syst. different gene cloning systems for genetic investigation of the new species Ethylenediaminedisuccinic acid Bacteriol. 47, 1274. Amycolatopsis japonicum MG417-CF17, the ethylenediaminedisuccinic acid pro- Secondary metabolites Blin, K., Medema, M.H., Kazempour, D., Fischbach, M.A., Breitling, R., Takano, E., ducer. J. Biotechnol. 92, 195–204. Weber, T., 2013. antiSMASH 2.0 – a versatile platform for genome mining of Strobel, T., Al-Dilaimi, A., Blom, J., Gessner, A., Kalinowski, J., Luzhetska, M., Püh- secondary metabolite producers. Nucleic Acids Res. 41, W204–W212. ler, A., Szczepanowski, R., Bechthold, A., Rückert, C., 2012. Complete genome Goodfellow, M., Brown, A.B., Cai, J.P., Chun, J.S., Collins, M.D., 1997. sequence of Saccharothrix espanaensis DSM 44229T and comparison to the other Amycolatopsis japonicum sp. nov, an actinomycete producing (S,S)-N,N - completely sequenced Pseudonocardiaceae. BMC Genomics 13, 465. ethylenediaminedisuccinic acid. Syst. Appl. Microbiol. 20, 78–84. Introduction phenotypic properties resulted in the renaming of the strain in “Amycolatopsis japonicum” sp. nov., corrected to A. japonica upon The bacterial order of the Actinomycetales contains a large num- validation (Anonymous, 1997). A. japonica is of great interest ber of genera that are capable of synthesizing a wide variety of because the strain is genetically accessible (Stegmann et al., 2001) secondary metabolites (e.g., Jankowitsch et al., 2012; Myronovskyi and has the potential to produce a wide spectrum of secondary et al., 2013; Rückert et al., 2014; Schwientek et al., 2012). The metabolites (Spohn et al., 2014). nocardioform actinomycete Amycolatopsis japonica MG417-CF17T To obtain the complete genome sequence, data from two (=DSM 44213T) is the producer of [S,S]-ethylenediaminedisuccinic sequencing libraries were combined, a whole genome shotgun acid (EDDS) which is a hexadentate chelating agent. [S,S]-EDDS is library and a 8 k long paired end library. Both libraries were an isomer of ethylenediaminetetraacetic acid (EDTA) with compa- sequenced on a 454 GS-FLX platform using the Titanium chem- rable chelating properties. However, EDDS is in contrast to EDTA istry to avoid problems caused by the expected high G + C content biodegradable (Schowanek et al., 1997). (Schwientek et al., 2011). In total, 732,707 reads (213,725,262 The strain A. japonica MG417-CF17T was discovered in a search bases) were assembled in two scaffolds using Newbler v2.5.3, rep- for specific inhibitors of phospholipases (Nishikiori et al., 1984). resenting the circular chromosome (51 unique contigs) and plasmid Preliminary cultural and morphological studies indicated that the pAmyja1 (1 contig). In total 62 contigs larger than 500 bp were organism belonged to the family Pseudonocardiaceae and it was assembled, the average coverage of the assembled contigs was classified as Amycolatopsis orientalis. However, 16S rRNA anal- 23.6-fold. To obtain the complete sequence, gaps caused by repeats yses performed by Goodfellow et al. (1997) and a number of were closed using CONSED (Gordon, 2003; Gordon et al., 1998). To correct for homopolymer errors, additional data from an Illumina GA IIx single read run of 150 bp were used, similar to the approach for Streptomyces collinus (Rückert et al., 2013). A set of 2,786,834 Corresponding author. Tel.: +49 7071 2978840. ∗ reads was mapped on the sequence, allowing to identify and cor- ∗∗ Corresponding author. Tel.: +49 (0)521 106 12252. E-mail addresses: [email protected] (E. Stegmann), rect 31 indel errors. The polished sequences were annotated using [email protected] (C. Rückert). GenDB (Meyer et al., 2003), the results are listed in Table 1. http://dx.doi.org/10.1016/j.jbiotec.2014.08.034 0168-1656/© 2014 Elsevier B.V. All rights reserved.

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Spohn, M. Environmental Microbiology (2016)(2015) 18(4), 1249–1263 doi:10.1111/1462-2920.13159doi:10.1111/1462-2920.13159 Amycolatopsis japonicum S,S Environmental Microbiology 18 Elucidation of the zinc-dependent regulation in Amycolatopsis japonicum enabled the identification of the ethylenediamine-disuccinate ([S,S]-EDDS) genes

Marius Spohn,1 Wolfgang Wohlleben1,2 and Introduction Evi Stegmann1,2* Ethylenediamine-disuccinate (EDDS) is an aminopoly- 1Interfaculty Institute of Microbiology and Infection carboxylic acid, which forms typical sixfold coordinated Medicine Tuebingen, Microbiology/Biotechnology, complexes with transient metal ions (Chen et al., 2010). University of Tuebingen, 72076 Tuebingen, Germany. EDDS exhibits two asymmetric C-atoms, allowing the for- 2Partner Site Tuebingen, German Centre for Infection mation of four optical stereoisomers: [S,S]-, [R,R]-, the Research (DZIF), Tuebingen, Germany. meso-isomers [R,S]- and [S,R]-EDDS. The [S,S]- configuration is the only stereoisomer readily biodegrad- Summary able to complete mineralization (Schowanek et al., 1997; Takahashi et al., 1997). The actinomycete Amycolatopsis japonicum pro- The actinomycete strain Amycolatopsis japonicum duces the complexing agent ethylenediamine- MG417-CF17 (formerly described as Amycolatopsis disuccinate ([S,S]-EDDS), which is an isomer of orientalis; Goodfellow et al., 1997) produces [S,S]-EDDS the widely industrially applied ethylenediamine- as a natural compound. [S,S]-EDDS was originally dis- tetraacetate (EDTA). In contrast to EDTA, [S,S]-EDDS covered during a screening for phospholipase C inhibitors is readily biodegradable and is therefore an alterna- (Nishikiori et al., 1984) and exerts its inhibitory effect by tive with a favourable environmental profile. complexing zinc ions, which are essential cofactors of this Biotechnological production of [S,S]-EDDS, however, zinc-metalloenzyme (Hough et al., 1989). [S,S]-EDDS is is not currently possible because its biosynthesis is an ethylenediamine-tetra acetate (EDTA) isomer (Fig. 1). inhibited by low-micromolar zinc concentrations. EDTA is used commercially in large quantities for many Here we illustrate the development of a new strategy applications, such as in cosmetic, food and medical prod- for identifying a biosynthetic pathway that is based ucts as well as in the textile and paper industries or in on the elucidation of transcriptional regulation and laundry detergents. However, due to its low biodegrada- the screening for binding sites of the respective regu- bility, EDTA cannot be removed by conventional waste- lator that controls the [S,S]-EDDS biosynthesis water treatments and remains at high concentrations in genes. To achieve this, we identified the zinc uptake aquatic environments, where it evokes constant environ- regulator Zur in A. japonicum and showed that it mental threats by undesirably mobilizing metal ions. mediates the repression of the zinc uptake system Because [S,S]-EDDS exhibits similar chelating properties ZnuABC . The Zur-binding motif, recognized by the Aj as EDTA, it offers a biodegradable alternative that zinc-bound Zur protein in the upstream region of is chemically produced from non-renewable fossil znuABC , was used to screen the genome, leading to Aj resources. the identification of the aes genes. Transcriptional To date, siderophores are the main group of described analysis and shift assays reveal specific zinc- chelating compounds produced by microbes. They are responsive regulation of the aes genes by Zur, and produced under iron-deficient conditions to solubilize gene inactivation shows their involvement in [S,S]- and facilitate the uptake of iron. Also, strains of the EDDS biosynthesis. Zur-mediated zinc repression of genus Amycolatopsis are known siderophore producers the [S,S]-EDDS biosynthesis genes is abolished in a (Meiwes et al., 1990; Seyedsayamdost et al., 2011). Δzur mutant, which offers now the opportunity to Although there are secondary metabolites that complex develop a biotechnological process. certain metal ions with high affinity, like the zinc-binding zincophorin (Brooks et al., 1984) or the copper-binding Received 7 October, 2015; revised 13 November, 2015; accepted 27 closthioamide (Kloss et al., 2013), they are neither syn- November, 2015. *For correspondence. E-mail evi.stegmann@ biotech.uni-tuebingen.de; Tel. +49 7071 29 78840; Fax +49 7071 29 thesized in response to deficiency of the corresponding 5979. metal ion, nor reported to mediate its uptake.

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12502 M. Spohn,M. Spohn, W. Wohlleben W. Wohlleben and E. and Stegmann E. Stegmann Analysis ofAnalysis zinc regulation of zinc regulation and biosynthesis and biosynthesis of [S,S of]-EDDS [S,S]-EDDS12513

sion of genes encoding zinc uptake and zinc mobilization Results and discussion domain hinge loop (Fillat, 2014). From the crystal struc- functions is Zur (zinc uptake regulator), initially described in tures of Zur from Mycobacterium tuberculosis (Lucarelli The [S,S]-EDDS biosynthetic gene cluster cannot be Bacillus subtilis and Escherichia coli (Gaballa and et al., 2007) and S. coelicolor (Shin et al., 2011), three identified by classical methods and stays hidden to Helmann, 1998; Patzer and Hantke, 1998). Zur belongs to ZBSs per monomer were determined. One ZBS is genome-mining approaches the Fur (Ferric uptake regulator) protein family of transcrip- required to ensure the dimeric structural integrity of the tion regulators. Within the Fur family, there is a huge Amycolatopsis japonicum was described as the producer proteins, whereas the other two ZBSs possess regulatory diversity in metal selectivity and biological function, includ- of [S,S]-EDDS in 1984 (Nishikiori et al., 1984). However, roles to modulate Zur activity (D’Autreaux et al., 2007; ing sensors of not only zinc, iron, manganese and nickel, the enzymes required for [S,S]-EDDS biosynthesis have Shin et al., 2011). In S. coelicolor, it is suggested that the but also of oxidative or acid stress. Fur proteins are typi- not yet been identified. During the pre-genomic era, differ- two regulatory ZBSs serve as an on–off switch to activate cally transcriptional repressors that bind specifically to ent classical approaches, such as heterologous expres- Zur and to mediate graded gene expression as a corresponding palindromic A/T-rich sequences found in sion of an A. japonicum cosmid library in a non-producer response to altered zinc concentration (Shin et al., 2011). the promoters of their DNA targets when bound to their strain, or comparative proteomics of cells growing in the In support of its potential role as a major zinc regulator, cognate metal ion cofactors (Fillat, 2014). Under zinc- presence or absence of zinc, have been applied unsuc- PREDZINC 1.4 (Shu et al., 2008) analysis and deficient conditions, Zur is inactive and has negligible cessfully. In addition, a reverse genetic approach with multisequence alignments revealed the presence of all affinity for the operator sequence. The inactive Zur binds degenerated primers did not result in the identification of three putative ZBSs in the AJAP_29045 gene product

only one zinc ion per monomer within a structural zinc- any genes in the A. japonicum genome encoding NIS (Fig. S1), which is henceforth designated ZurAj.

binding site (ZBS) to maintain its homodimeric state synthetases, commonly involved in ionophore To investigate the role of ZurAj as a regulator of the

(Lucarelli et al., 2007; Ma et al., 2011; Shin et al., 2011). biosynthesis. Access to the A. japonicum genome [S,S]-EDDS biosynthesis in A. japonicum, the entire zurAj- The increased concentration of free zinc ions leads to sequence (GenBank accession number CP008953) coding region was deleted in frame with a markerless Fig. 1. Chemical structures of [S,S]-EDDS and EDTA. further binding of zinc to additional regulatory ZBSs, (Stegmann et al., 2014) allowed data analysis using procedure (A. japonicum Δzur) (the deletion procedure is causing a conformational change, which gives rise to a fully bioinformatic tools such as antiSMASH 3.0 (Weber et al., illustrated in Fig. S2). For this purpose, the flanking regions

Chelating agents (ionophores), which are predicted to active form of the repressor. Zinc-bound Zur exhibits a high 2015) to evaluate the biosynthetic potential of of zurAj were integrated into pGusA21 (Table S1, Fig. S2), function in the acquisition of transition metals other than affinity for its cognate DNA motifs upstream of their target A. japonicum. antiSMASH 3.0 is a bioinformatic tool which contains the gusA reporter gene encoding a iron, however, have been disregarded for a long time. The genes (Choi and Bird, 2014). One of the best-investigated capable of discovering and classifying biosynthetic loci β-glucuronidase (GUS). The gene inactivation plasmid examples described include the zincophores, such as Zur regulators in actinomycetes, to date, is the Zur protein covering a wide range of known secondary metabolite pGusA21Δzur (Table S1) was integrated into the pyridine-2,6-bis(thiocarboxylic acid) from Pseudomonas of S. coelicolor. In S. coelicolor, the Zur regulon includes compound classes. This in silico analysis revealed the A. japonicum genome via a single crossover. This event putida (Leach et al., 2007) and coelibactin from znuABC, encoding an ATP-binding cassette (ABC) uptake presence of various putative gene clusters encoding the was selected by using the plasmid-encoded apramycin Streptomyces coelicolor (Kallifidas et al., 2010), and the system (Shin et al., 2007), an NRPS gene cluster, pre- synthesis of natural products in A. japonicum (Spohn et al., resistance. The integration of the plasmid was verified by copperphore methanobactin from methane-oxidizing bac- dicted to direct synthesis of coelibactin (Kallifidas et al., 2014). Two of the identified gene clusters could be polymerase chain reaction (PCR). To obtain a deletion teria (Kim et al., 2004). Ionophores are either synthesized 2010), and a set of genes encoding alternative, zinc-free assigned to their products: ECO-0501, a polyketide mutant, a second homologous recombination event was by non-ribosomal peptide synthetases (NRPS) (e.g. ribosomal proteins (Owen et al., 2007). synthase-derived polyene (Shen et al., 2012) and provoked by stressing selected colonies using tempera- enterobactin; Pollack et al., 1970; Rusnak et al., 1991) or Because the identification of the EDDS biosynthetic ristomycin A (Spohn et al., 2014), a glycopeptide antibiotic. ture shifts and protoplast formation as described previ- by NRPS-independent siderophore (NIS) synthetases genes was not possible using strategies normally applied However, none of the remaining clusters could be assigned ously (Puk et al., 2002). Protoplast regeneration was (e.g. aerobactin; Gibson and Magrath, 1969; de Lorenzo to the isolation of secondary metabolite gene clusters, we to the [S,S]-EDDS structure, indicating a novel and unique carried out on agar plates containing the GUS substrate and Neilands, 1986). Because [S,S]-EDDS production is developed a new approach to identify natural compound biosynthesis mechanism different from the typical NRPS or 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (X-Gluc). The tightly inhibited by traces of zinc, whereas iron had only a biosynthesis pathways by exploiting knowledge in the NIS pathways known for siderophore synthesis. Therefore, loss of GUS activity (visible as white-coloured colonies) minor effect even at elevated concentrations (Cebulla, field of transcriptional regulation, which we termed the development of an alternative strategy for the identifi- indicated the excision of the plasmid. GUS-negative colo-

1995; Zwicker et al., 1997), it is considered that [S,S]- INBEKT. Using this approach, we could elucidate the cation of the [S,S]-EDDS biosynthesis genes was required. nies were selected, and the deletion of the zurAj gene was

EDDS is an evolutionary response to zinc deficiency, ZurAj-mediated zinc regulation in A. japonicum in detail. confirmed by PCR. serving as a zincophore to contribute to zinc uptake. The constructed A. japonicum ΔzurAj mutant showed To study the effect of the zurAj deletion on [S,S]-EDDS Zur controls the expression of the Zinc is an essential trace element for all living cells, and elevated EDDS production, even in the presence of high production, A. japonicum Δzur and A. japonicum wild [S,S]-EDDS biosynthesis it is estimated that 5–10% of all proteins contain zinc as a zinc concentrations, revealing the EDDS biosynthesis type (WT) were grown in 12-well microtitre plates cofactor (Andreini et al., 2006). Zinc in proteins either genes as a member of the ZurAj regulon. The subsequent Because [S,S]-EDDS production is repressed when containing 3 ml of synthetic medium (SM) per well participates directly in chemical catalysis or is important for screening of the A. japonicum genome with the deduced A. japonicum is grown in zinc-containing medium (>2 μM with various increasing zinc concentrations. [S,S]-EDDS maintaining protein structure and stability. However, highly Zur box enabled the identification of EDDS biosynthesis zinc), we assumed that a zinc-responsive regulator con- production was quantified by high performance liquid concentrated zinc can inhibit physiological functions of genes for the first time. Transcriptional analyses and gel trols the [S,S]-EDDS biosynthesis genes. BLAST analyses chromatography-dioden array detector (HPLC-DAD) proteins by blocking important thiols and by competing with shift experiments confirmed that they are strictly zinc of the A. japonicum genome using the protein sequence analyses after growth for 72 h from three biological repli- other metal ions for binding sites (Kasahara and Anraku, repressed by ZurAj. The analysis of the phylogenetic abun- of ZurSC, the zinc uptake regulator of S. coelicolor (Owen cates to determine the production yield in [S,S]-EDDS/ 1974; Aagaard and Brzezinski, 2001). Hence, all cells have dance of the identified EDDS biosynthesis genes, com- et al., 2007), revealed the presence of AJAP_29045 biomass (mg g−1). A. japonicum WT produced [S,S]-EDDS to maintain zinc homeostasis optimal for cell survival. In bined with the evaluation of the capability of certain (GenBank: AIG78644) with high similarity (67% amino only with zinc concentrations lower than 2 μM (Fig. 2). In prokaryotes, this precise balance is predominantly main- Amycolatopsis strains to produce EDDS, provides insight acid identity). All of the available Zur structures demon- contrast, zinc independent and constant [S,S]-EDDS tained using zinc-responsive transcriptional factors, which into its role as an evolutionary response to zinc deficiency strate a common homodimeric quaternary structure with biosynthesis was observed in A. japonicum Δzur over the sense zinc deficiency and zinc excess (Choi and Bird, and contributes to the determination of the EDDS each monomer consisting of an N-terminal DNA-binding total range of the applied zinc gradient (Fig. 2), an effect 2014). The major prokaryotic factor regulating the expres- biosynthesis gene cluster. domain, a C-terminal dimerization domain and an inter- that was even seen at a zinc concentration of 5 mM.

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12524 M. Spohn,M. Spohn, W. Wohlleben W. Wohlleben and E. and Stegmann E. Stegmann Analysis ofAnalysis zinc regulation of zinc regulation and biosynthesis and biosynthesis of [S,S of]-EDDS [S,S]-EDDS12535

that of znuABCSc. Moreover, locus 1 is extended by a gene (ajap_18675) that is predicted to encode a lipoprotein containing an N-terminal signal peptide with a conserved lipobox (L-A-A-C). To elucidate which locus is connected to zinc uptake in A. japonicum, we performed qualitative reverse transcrip- tion PCR (RT-PCR). The transcriptional pattern of the two znuABC homologues was investigated with respect to the presence of zinc ions. Hence, cultures were grown in the presence (25 μM) and in the absence of zinc, and RNA was isolated after 10 h (early exponential phase) and 70 h (stationary phase). The presence of ajap_32775 (locus 2) and ajap_18670 (locus 1) transcripts were monitored with specific primer pairs (Table S2). RT-PCR analysis showed

Fig. 2. Zinc-dependent production of [S,S]-EDDS. Strains were that locus 1 expression was repressed in the presence of grown for 72 h in 12-well microtitre plates in synthetic medium (SM) zinc, in contrast to locus 2 whose expression was zinc supplemented with different concentrations of ZnSO4. n = 3. independent (Fig. 3C). From these results we concluded that locus 1 encodes an ABC uptake system sensitive for

Additionally, the deletion of zurAj led to an increased total zinc, and we designated it henceforth znuABCAj. To further [S,S]-EDDS yield (4 to 4.5-fold) compared with the WT specify the function of locus 2, we analysed its transcrip- strain. To genetically complement A. japonicum Δzur,a tional pattern after growth in SM supplemented with 2+ 2+ 2+ 2+ single-coding region of the entire zurAj gene was cloned various metal ions (Fe , Ni , Co and Mn ). ajap_32775 into the integrative vector pRM4 under the control of the expression was specifically repressed in the presence of constitutive promoter ermEp* and transferred into manganese (Fig. 3C). Therefore, locus 2 was considered A. japonicum Δzur. Amycolatopsis japonicum Δzur:pRM4- to encode an ABC transporter with a manganese uptake zur showed the same phenotype as the WT; it produced function in A. japonicum, similar to the mntABCD system [S,S]-EDDS exclusively in the absence of zinc (Fig. S3). of B. subtilis (Que and Helmann, 2000). The transcription [S,S]-EDDS production in A. japonicum Δzur in the pres- of znuB (ajap_18670) is not repressed by any ion other ence of zinc suggested that the ajap_29045-encoded than zinc, revealing the specific function of ZnuABC as a protein is the zinc-sensing regulator ZurAj and that the zinc acquisition system. [S,S]-EDDS biosynthesis genes are under its zinc- To assess the physiological range in which the identi- responsive control. fied zinc uptake system is required to supply the cells with zinc, A. japonicum WT and A. japonicum Δzur were grown Fig. 3. Gene organization and zinc-specific regulation of znuABC homologues loci in A. japonicum. in microtitre plates in a zinc gradient. RNA isolation was A. Comparison of the gene organization pattern of the znuABC locus in S. coelicolor with homologues in A. japonicum. The Zur regulon of A. japonicum contains the performed after 72 h of growth and used for RT-PCR B. Homology analyses using BLAST: identity/similarity of locus 1 and 2 to ZnuABC proteins of S. coelicolor on amino acid level. high-affinity zinc uptake system znuABC C. Trace metal-dependent transcriptional pattern of the putative high-affinity metal uptake systems of A. japonicum. Cultures were grown in analyses. A znuBAj transcript was only detectable in WT SM in the absence of any trace element (−) or supplemented with 25 μM Fe2+, Zn2+, Ni2+, Co2+ or Mn2+, and samples were taken after 10 h Zur regulons characterized thus far usually comprise samples after growth with zinc concentration of ≤2.0 μM (exponential growth phase) and 70 h (stationary growth phase) of incubation to isolate RNA. sigB was used as a housekeeping gene to normalize the RNA. znuB (ajap_18670) and mntA (ajap_32775) were chosen as probes to represent the entire locus. 10–30 genes with different functions, often related to zinc (Fig. 4). This indicates the necessity of ZnuABCAj to homeostasis. In particular, the high-affinity zinc uptake enhance zinc uptake in an environment characterized by system ZnuABC has been identified as Zur regulated in low zinc bioavailability (≤2.0 μM). In contrast, transcrip- labelled DNA with purified ZurAj in binding buffer with or increased Zur concentration (Shin et al., 2007; Kallifidas many bacteria and is widely distributed throughout the tional analysis of znuBAj in the A. japonicum Δzur mutant without 25 μM ZnSO4. Binding of ZurAj occurred only in the et al., 2010). It is thought that these multiple shifts repre- bacterial kingdom (Patzer and Hantke, 1998; Campoy occurred over the whole range of applied zinc concentra- presence of zinc (Fig. 5). Although ZurAj was also added in sent independent binding events of several Zur dimers to et al., 2002; Lucarelli et al., 2007; Shin et al., 2007; Pawlik tions, with detectable expression even at the most various higher concentrations, only a single ZurAj shift was the DNA. However, in E. coli, no such intermediate et al., 2012). We identified two loci in A. japonicum elevated zinc concentrations (100 μM) (Fig. 4). observed. In contrast, S. coelicolor Zur forms multiple species of protein–DNA bindings were observed. There, whose deduced protein sequences show high levels of Because znuCAj and znuBAj are transcribed in reverse complexes with its target DNAs in dependence on two Zur dimers bind to the DNA in a highly cooperative similarity to the previously described ZnuABC system of orientation we assumed the presence of a ZurAj box in this S. coelicolor (Shin et al., 2007) (Fig. 3B). However, in con- intergenic region. To analyse whether ZurAj binds to this Fig. 4. Zinc-dependent gene transcription in trast to S. coelicolor, neither of them is localized closely to region in a zinc-dependent manner, electrophoretic mobil- A. japonicum strains. Strains were grown for zur . The two loci are encoded by ajap_18665-80 (locus 1) ity shift assays (EMSAs) were performed. His-tagged 72 h in 12-well microtitre plates in synthetic Aj medium (SM) supplemented with different and ajap_32775-85 (locus 2), and both encode a putative ZurAj was purified from E. coli BL21(DE3) pLys trans- concentrations of ZnSO4 prior to RNA ATPase, a putative membrane permease and a putative formed with pET-30-zur. Cy5-labelled DNA, covering the isolation. sigB was used as a housekeeping gene to normalize the RNA. znuB metal-binding lipoprotein. Locus 2 exhibits an identical region from −110 to +35 bp with respect to the znuBAj GTG (ajap_18670) and aesA (ajap_08425) were gene arrangement to the znuABCSc genes in S. coelicolor, start codon (iznuCB), was used as a probe in the EMSA. amplified using specific primers (Table S2). whereas the gene arrangement of locus 1 deviates from The binding reaction was carried out by incubating

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12546 M. Spohn,M. Spohn, W. Wohlleben W. Wohlleben and E. and Stegmann E. Stegmann Analysis ofAnalysis zinc regulation of zinc regulation and biosynthesis and biosynthesis of [S,S of]-EDDS [S,S]-EDDS12557 family. Enzymes of this family catalyse the hydrolysis of amide bonds using a highly conserved Ser-Ser-Lys cata- lytic triad, which is also present in the predicted amino acid sequence of AesE (Ser106, Ser130 and Lys31). ajap_08425 is the first gene of an operon consisting of four overlapping genes (ajap_08425-40) (Fig. 7A). The genes were designated aesA, aesB, aesC and aesD respectively. The proposed gene products of aesA, aesB and aesC share significant similarity to SbnB, SbnH and SbnA, respectively, enzymes involved in the synthesis of staphyloferrin B (SB) (Fig. 7B). SB is a NIS-derived siderophore of Staphylococcus aureus (Cheung et al., 2009) containing a central 1,2-diaminoethane (Dae) moiety (Fig. 7C). Central Dae moieties are rare in natural compounds, and it was long considered that this moiety

Fig. 5. Zinc-dependent binding of purified ZurAj to znuCB and aesA promoter region. Purified ZurAj was incubated with Cy5-labelled DNA fragment upstream of znuCB (iznuCB) or aesA (upaesA) (4 nM) in the absence or in the presence of 25 μM ZnSO4 (indicated by – and + respectively). To confirm the specificity of the binding complexes, either an excess amount of non-specific competitors [NS; pRM4 (70 nM)] or of specific competitors [S; unlabelled fragment upstream of znuCB DNA (1,1 μM) or aesA (0.65 μM)] was added to the binding mixture.

way, revealing single shifts in EMSA experiments (Gilston

et al., 2014), comparable with the observed single ZurAj

shift. To identify a conserved ZurAj box we used MEME,a tool for discovering motifs in a group of related DNA (Bailey et al., 2009). The Zur consensus sequences of M. tuberculosis (Maciag et al., 2007), Corynebacterium glutamicum (Schröder et al., 2010) and S. coelicolor (Owen et al., 2007) were aligned to the intergenic region

of znuCBAj to identify a conserved ZurAj box. The align- ment revealed an A/T-rich palindromic sequence consist- ing of a 7-1-7 arrangement in the intergenic region between znuC and znuB (iznuCB), exhibiting high simi- larity to previously described Zur boxes (Fig. 6A). Using

this newly identified motif, a specific A. japonicum ZurAj box was deduced (Fig. 6B).

The ZurAj regulon of A. japonicum contains putative [S,S]-EDDS biosynthetic genes

To identify further ZurAj-regulated genes, in particular the

[S,S]-EDDS biosynthesis genes, the deduced ZurAj box Fig. 6. MEME-FIMO analysis to screen for Zur boxes in A. japonicum was submitted to FIMO, a software tool for scanning DNA genome. sequences with motifs described as position-specific A. MEME alignment of znuCB intergenic region and Zur consensus scoring matrices (Bailey et al., 2009), to screen the sequences of M. tuberculosis (Mtb con), C. glutamicum (Cgl con) and S. coelicolor (Sco con). A. japonicum genome. This revealed a putative binding B. Deduced Zur box used for screening all 5′UTRs of the motif with high similarity to known Zur boxes between A. japonicum genome by MEME-FIMO. Fig. 7. Proposed [S,S]-EDDS biosynthesis. A. Genetic organization of ajap_08400-55. The operon aesA-D (ajap_08425-40) downstream of the Zur-binding site (indicated by an orange ajap_08420 (aesE) and ajap_08425 (aesA) (Fig. 6C), two C. Genetic organization and Zur box locations in the znuCB and aesA (ajap_08425) promoter regions. Nucleotides underlined asterisk) is highlighted in blue and aesE-H in red. The black bar illustrates EMSA shift fragment upaesA (Fig. 5). genes transcribed in opposite directions (Fig. 7A). aesE indicate the start codons. Putative Zur boxes are indicated by grey B. Homology analyses using BLAST: identity/similarity of AesA-D to staphyloferrin (SB) biosynthesis enzymes. encodes a protein belonging to the amidase signature highlighting. C. Comparative alignment of the SB biosynthesis pathway and predicted [S,S]-EDDS pathway. Dae moieties are highlighted in blue.

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12568 M. Spohn,M. Spohn, W. Wohlleben W. Wohlleben and E. and Stegmann E. Stegmann Analysis ofAnalysis zinc regulation of zinc regulation and biosynthesis and biosynthesis of [S,S of]-EDDS [S,S]-EDDS12579

is restricted to synthetic compounds exclusively To quantitatively determine the transcriptional level of (Bucheli-Witschel and Egli, 2001). However, the [S,S]- aesA-D in a zinc-dependent manner, we constructed a EDDS structure also harbours such a moiety. In transcriptional fusion of the aesA promoter region (PaesA) S. aureus, SB biosynthesis starts with the formation of with the gusA gene as a reporter system using the vector diaminopropionate (Dap), the product of the amidation of pGus (Myronovskyi et al., 2011). PaesA was amplified by O-phospho-L-serine with the amino donor L-glutamate PCR and fused to gusA by the primer-attached restriction (Kobylarz et al., 2014). This reaction is coordinately cata- sites, yielding pGusPaesA (Table S2). pGusPaesA was inte- lysed by SbnA and SbnB. The further assembly of the grated into A. japonicum WT and A. japonicum Δzur to single SB building blocks occurs via three distinct NIS generate A. japonicum WT:pGusPaesA and A. japonicum synthetases (SbnE, SbnF and SbnC). Additionally, SbnH Δzur:pGusPaesA respectively. Exconjugants contain- is required to catalyse the decarboxylation of a Dap inter- ing unaltered pGus (A. japonicum WT:pGusP and mediate, which leads to the Dae moiety (Cheung et al., A. japonicum Δzur:pGusP) served as negative controls. The 2009). A NIS-dependent, stepwise assembly can be strains were grown for 72 h in microtitre plates containing SM excluded for [S,S]-EDDS biosynthesis because no NIS with differing zinc concentrations. Initially, GUS activities synthetase is encoded in the A. japonicum genome. were assayed in cell-based chromogenic assays (Fig. 8B). Therefore, an alternative pathway has to be postulated for No X-Gluc turnover was visible in the A. japonicum WT and [S,S]-EDDS: we assumed that the first step, the supply of A. japonicum Δzur strains and their corresponding recombi- the building block Dap, occurs in a similar way and is nant strains carrying the unaltered pGus. Amycolatopsis catalysed by the SbnB and SbnA homologues AesA and japonicum WT:pGusPaesA exhibited GUS activity (visible AesC. A stepwise assembly of Dap with dicarboxylic acids as blue colour) solely when grown at low zinc concentrations, (e.g. oxaloacetic acid) and the decarboxylation of the Dap whereas GUS activity was also visible in A. japonicum Fig. 8. Zinc-dependent GUS activity in A. japonicum WT and A. japonicum Δzur containing aesA promoter-gusA fusions after growth in SM moiety (presumably catalysed by the SbnH homologue Δzur at elevated zinc concentrations (Fig. 8B). Quantitative with various zinc concentrations. AesB) should lead to the functional [S,S]-EDDS with its data were generated using the soluble GUS substrate A. Spectrophotometric assay using p-nitrophenyl-β-D-glucuronide as the substrate. Miller units are shown per milligram of cells. characteristic central Dae moiety (Fig. 7C). [S,S]-EDDS p-nitrophenyl-β-D-glucoronide in spectrophotometric B. Chromogenic assay in SM containing the substrate 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (X-Gluc). can then be transported over the cell membrane by AesD, assays (Fig. 8A). Under sub-inhibitory zinc concentrations a protein similar to the multidrug and toxic compound (0–0.5 μM), A. japonicum WT:pGusPaesA revealed GUS The aes genes are essential for [S,S]-EDDS production analysed by HPLC-DAD over 5 days of growth. Although extrusion protein family (Morita et al., 1998; 2000). This activity levels [1.86-1.64 Miller units (MU)] significantly above the supernatant of A. japonicum WT contained [S,S]- deduced [S,S]-EDDS pathway does not belong to any those of the negative controls (0.28–0.44 MU). Linear To prove whether the zinc-regulated aesA-D operon is EDDS (Fig. 9), no [S,S]-EDDS was detected in the super- previously described secondary metabolite pathway decreasing Gus activity is seen in the range of partial inhibi- involved in [S,S]-EDDS biosynthesis, the in frame deletion natant of the deletion mutant, confirming the involvement class, giving an explanation as to why these genes could tory zinc concentrations of 0.5 to 2 μM, correlating to [S,S]- mutant aesA-C was constructed using pGusA21 aesA-C of at least one of the aesA-C genes in [S,S]-EDDS not be identified using classical bioinformatics tools for EDDS production in A. japonicum WT (Fig. 2). No GUS Δ Δ in analogy to the procedure described for zur deletion. biosynthesis. To verify that the loss of [S,S]-EDDS produc- genome mining. activity was detectable in this strain when grown at zinc Aj A. japonicum ΔaesA-C and A. japonicum WT were grown tion in A. japonicum ΔaesA-C is due to the aesABC dele- concentrations higher than 2 μM. For A. japonicum in the absence of zinc and [S,S]-EDDS production was tion and not due to any polar effects, we genetically Δzur:pGusPaesA, GUS activities were determined after The transcription of the aesA-D operon is zinc regulated growth without ZnSO4 and with ZnSO4 at concentrations of 2 Because [S,S]-EDDS is only produced under zinc- and 100 μM. The GUS activities were measurable in the Fig. 9. Time-dependent [S,S]-EDDS limiting conditions, the transcriptional pattern of the iden- presence of 2 μM (7.53 MU) and 100 μM (8.15 MU) zinc and production by A. japonicum WT and tified candidate genes was determined by RT-PCR. The were comparable to the GUS activity in the absence of zinc A. japonicum ΔaesA-C:pSET-aesA-D. The transcription of aesA was analysed with respect to the (8.24 MU) (Fig. 8). The GUS activity in A. japonicum strains were grown in deionized flasks in zinc-deficient SM. Samples were taken presence of various zinc concentrations. An inverse cor- Δzur:pGusPaesA showed a 4.4 fold increase compared with directly after inoculation of the main culture related transcription level of aesA to increased zinc con- the activity in A. japonicum WT:pGusPaesA after growth in (t = 0 h) and further time points of incubation centration was observed, whereas no transcript was the absence of zinc. This result reflects the significantly and quantified by HPLC-DAD. n = 3. detected for zinc concentrations above 2 μM (Fig. 4). higher expression level of the aesA-D operon in This result is in agreement with the observation that no A. japonicum Δzur, which leads to an increased [S,S]-EDDS [S,S]-EDDS production was detectable in SM with zinc yield in this deletion mutant (Fig. 2). concentration of ≥2 μM (Fig. 2). The zinc inhibitory effect To further verify the zinc-dependent repression of the was also seen for the genes aesB-D (Fig. S4). The pres- operon genes (aesA-D), we performed gel shift assays 2+ 2+ 2+ ence of other divalent metal ions (Fe , Ni , Co and using ZurAj and the Cy5-labelled region upstream of aesA Mn2+) did not affect transcription. In the A. japonicum (upaesA) including −185 to +36 bp with respect to its ATG

Δzur background, however, aesA-D expression was zinc start codon. Purified ZurAj bound specifically to the DNA independent with constitutive transcription over the probe in the presence of zinc, whereas no binding occurred whole range of the applied zinc gradient (Fig. 4). This in the absence of zinc (Fig. 5). This zinc-dependent binding

argues that ZurAj is likely the repressor that controls the of ZurAj to the intergenic region of aesE-A leads to the operon aesA-D. repression of aesA transcription (Fig. 4).

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125810 M.M. Spohn, Spohn, W. Wohlleben W. Wohlleben and E. and Stegmann E. Stegmann AnalysisAnalysis of zinc regulation of zinc regulation and biosynthesis and biosynthesis of [S,S of []-EDDSS,S]-EDDS125911 complemented A. japonicum ΔaesA-C. Hence, we con- Amycolatpsis orientalis HCCB10007 and Amycolatpsis Because A. decaplaina and A. alba also produce Media and culture conditions structed the plasmid pSET152-aesA-D, which harbours lurida DSM 43134 harbour the eight clustered genes [S,S]-EDDS but do not encode an AesH homologue, its E. coli and A. japonicum strains were cultured and manipu- the complete aesA-D operon downstream of its native, entirely with identical gene arrangement and with high- involvement in [S,S]-EDDS biosynthesis can be lated as described previously (Kieser et al., 2000; Stegmann zinc-repressed promoter and applied it to complement the sequence similarity compared with A. japonicum excluded. We therefore consider the cluster border et al., 2001; Kołodyn´ska, 2011; Spohn et al., 2014). mutant. Amycolatopsis japonicum ΔaesA-C:pSET-aesA-D (Table S3). The Amycolatpsis decaplanina DSM 44594 downstream of the aesH coding region. The opposite Liquid cultures of A. japonicum strains in 100 ml volume showed restored [S,S]-EDDS production (Fig. 9). Due to and Amycolatpsis alba DSM 44262 genomes exhibit a border of the [S,S]-EDDS cluster is most probably were grown to detect [S,S]-EDDS production according to the transcriptional control by the native promoter, deviation in that they are both missing the terminal aesH defined by ajap_08445, putatively encoding a function in Zwicker and colleagues (1997). The optimized SM consisted −1 −1 A. japonicum ΔaesA-C:pSET-aesA-D produces [S,S]- homologue. Interestingly, the Zur box is highly conserved sporulation, and the unit ajap_08450-55, encoding a of glycerol (25 g l ), MgSO4 × 7H2O (1.2 g l ), Ferric (III) citrate (60 mg l−1), KH PO (8gl−1), Na HPO 2HO EDDS solely in zinc-depleted medium. The amount of in all discovered aesE-A intergenic regions (Table S4), bifunctional catalase–peroxidase and its transcriptional 2 4 2 4 × 2 (12 g l−1) and sodium glutamate monohydrate (11.3 g l−1), [S,S]-EDDS produced by this recombinant strain was evidencing the common Zur-mediated and zinc-dependent regulator, which are involved in oxidative stress which was used as the nitrogen source. To grow the strains in increased two to three times compared with that by transcriptional repressions. According to Everest and response. The phylogenetic analysis of the abundance a reduced 3 ml volume, 12-well microtitre plates were used. A. japonicum WT, likely due to the presence of a second, Meyers (2009), the members of the genus Amycolatopsis of aes genes, combined with the capability of certain The cultures were grown on a rotary shaker (120 r.p.m.) at plasmid-encoded copy of the exporter gene aesD. can be classified into six phylogentic clades (A–F) Amycolatopsis strains to produce [S,S]-EDDS confirmed 30°C. Precultures were grown in complex culture medium Overexpression of exporter genes is a generally applied (Fig. S6). All strains encoding clustered aes genes are the [S,S]-EDDS biosynthesis via the coordinated action (glycerol (20 g l−1); soybean meal (20 g l−1) at pH 7.5) for 48 h. strategy in metabolic engineering approaches to increase members of the Amycolatopsis phylogentic clade A. An of the enzymes encoded by the aes genes and contrib- A total of 150 μl of this preculture was used to inoculate 3 ml of SM supplemented with ZnSO to reach the final zinc con- production yields. For example, overexpression of theABC exception, however, is Amycolatopsis azurea DSM 43854, uted to assigning the cluster borders. Furthermore, 4 centrations of 0, 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, 5, 10, 25 and transporter DrrABC in Streptomyces peucetius ATCC which also belongs to clade A but does not show a con- these results show that [S,S]-EDDS production, as an 100 μM. The cultures were grown for a further 72 h before 27952 led to a 2.4-fold increase of doxorubicin production served [S,S]-EDDS cluster. In this strain, no aesD evolutionary response to zinc deficiency, seems to be a being analysed by means of [S,S]-EDDS production, RT-PCR (Malla et al., 2010), which is in a similar range as the homologues could be identified and the clustered putative quite common feature of the Amycolatopsis phylogenetic and GUS activity. observed increase in [S,S]-EDDS production due to aesD aesA-C homologues show a significant drop in similarity clade A. overexpression. Putting these results together with the (Table S3). Moreover, in contrast to all other clade A proposed biosynthetic pathway, we conclude that strains, no conserved Zur box is present upstream of the Construction of the inactivation plasmids pGusA21Δzur, the whole aesA-D operon is involved in [S,S]-EDDS aesA-like gene in A. azurea DSM 43854 (Table S4). Inter- Perspective pGusA21ΔaesA-C and pGusA21ΔaesE-H biosynthesis/export. estingly, the aes genes could be identified exclusively in Bioinformatic tools, such as antiSMASH 3.0, are com- The upstream and downstream regions of zurAj, aesA-C and The genes ajap_08415, ajap_08410 and ajap_08405 Amycolatopsis strains belonging to clade A. In contrast to monly used for the identification of gene clusters encod- aesE-H were amplified using the primers listed in Table S2. (aesF, aesG and aesH) are located downstream of aesE the total aes gene cluster, homologues of the aesA-D ing secondary metabolites. However, the identification of The purified PCR products were introduced into pJet1.2 as an and are predicted to encode a LysR family transcrip- subcluster are quite abundant in various genera of clusters is limited to biosynthetic pathways of already intermediate vector and verified by sequencing. By using the primer-attached restriction sites, the upstream and down- tional regulator, a cysteine dioxygenase and an actinobacteria and proteobacteria. However, no aesA-D known mechanisms. In this study, we developed a new stream fragments were consecutively cloned into the plasmid acetyltransferase respectively. To investigate the involve- homologues could be identified, for example, in firmicutes strategy, which is INBEKT. We think that INBEKT is ment of these genes in the biosynthesis of [S,S]-EDDS, or in any Amycolatopsis strain not belonging to clade A. pGusA21, resulting in the inactivation plasmids pGusA21Δzur, generally applicable to search for genes encoding pGusA21ΔaesA-C and pGusA21ΔaesE-H, used for transfor- we constructed the deletion mutant A. japonicum The abundance of the aesA-D subcluster, when related to zincophore, or more generally ionophore, biosynthesis mation of A. japonicum WT. ΔaesE-H. This mutant was not able to produce [S,S]- the biosynthesis of Dae moieties, might indicate the poten- and potentially other pathways consisting of unusual EDDS (Fig. S5). However, the aesA-D operon was still tial of certain strains to produce metabolites containing this or novel biosynthetic steps. Our approach does not zinc dependently transcribed in this mutant, confirming building block. require any information regarding the structure or the Construction of the complementation plasmid pRM4-zur that the promoter region of aesA was not affected by To correlate the genetic information regarding the abun- biosynthesis mechanism of the compound of interest this mutation. These results demonstrate that at least dance of putative biosynthesis genes to the capability in For the complementation of A. japonicum Δzur, the entire and would therefore allow the identification of totally new coding region of the zur gene was amplified using the primer one of the aesE-H genes is required for the production producing [S,S]-EDDS, we cultivated the strains A. lurida Aj secondary metabolite pathways and classes. In addition, pair pzur-F and pzur-R. The 429 bp PCR product was inte- of [S,S]-EDDS in addition to aesA-D. (Lechevalier et al., 1986), A. decaplaina (Wink et al., we illustrate how knowledge-based approaches can be grated into pRM4 (Menges et al., 2007) via the primer- 2004), A. alba (Mertz and Yao, 1993) and A. azurea used to increase productivity, either by deleting nega- attached restriction sites downstream of the ermEp* promoter. (Henssen et al., 1987) belonging to the phylogenetic The [S,S]-EDDS biosynthesis as response to zinc tively acting regulators, or by overexpressing specific clade A in SM in the absence and in the presence of zinc. deficiency is phylogenetically clustered exporters. The ongoing work on the elucidation of the Construction of the complementation plasmid Culture filtrates of A. lurida, A. decaplaina and A. alba [S,S]-EDDS biosynthetic pathway will provide deeper pSET-aesA-D The distribution of the genetic potential to produce [S,S]- grown without the supplementation of zinc showed a peak insights into discrete biosynthetic steps and deliver addi- EDDS in bacteria was assessed by evaluating the whose retention time and UV-DAD spectrum corre- tional possibilities for yield optimization. This will further The cosmid pTWPI1-edds includes a 31 kb insert represent- phylogenetic abundance of the aes genes by EDGAR, sponded to the [S,S]-EDDS standard (Fig. S7). This peak promote the establishment of a biotechnological [S,S]- ing 1 665 131–1 696 415 bp of the A. japonicum genome. a software framework for comparative analysis of was not observable after growth of these strains in the EDDS production process with a favourable environ- This genome fragment includes the entire coding regions prokaryotic genomes (Blom et al., 2009) and a presence of zinc. As expected, no [S,S]-EDDS production mental profile. from ajap_08305 to ajap_08450 and partial sequences of combinatorial BLAST and MULTIGENEBLAST (Medema et al., could be detected after cultivation of A. azurea. Further- ajap_08300 and ajap_08455. A 6545 bp fragment from the 2013) analysis. Computational analysis using aesA-H as a more, representatives of the phylogenetic clades C and D, A. japonicum genome from 1 687 557–1 694 101 bp was isolated from the cosmid using the restriction enzymes query sequence for searching conserved homologues in a namely Amycolatopsis balhimycina DSM 5908 (Nadkarni Experimental procedures EcoRI and BglII. This fragment contains the entire operon GenBank database covering all entries revealed that the et al., 1994; Wink et al., 2003), Amycolatopsis Strains, plasmids and oligonucleotides aesA-D downstream of its native promoter beside partial occurrence of the aesA-H genes is restricted to certain mediterranei DSM 43304 (Margalith and Beretta, 1960) sequences of the genes encoded upstream (ajap_08420) strains of the genus Amycolatopsis. Of all the analysed and Amycolatopsis nigrescens DSM 44992 (Groth et al., The strains and plasmids used in this study are listed in and downstream (ajap_08445). The purified 6545 bp was genomes, solely those of Amycolatpsis sp. MJM2582, 2007), do not produce [S,S]-EDDS (Fig. S7). Table S1. The oligonucleotides are listed in Table S2. cloned into pSET152 (Bierman et al., 1992) via EcoRI and

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121260M.M. Spohn, Spohn, W. Wohlleben W. Wohlleben and E. and Stegmann E. Stegmann AnalysisAnalysis of zinc regulation of zinc regulation and biosynthesis and biosynthesis of [S,S of []-EDDSS,S]-EDDS126113

BamHI restriction sites resulting in the recombinant pasmid Construction of the gusA promoter probe software framework for the comparative analysis of Hough, E., Hansen, L.K., Birknes, B., Jynge, K., Hansen, S., pSET-aesA-D. plasmid pGusPaesA prokaryotic genomes. BMC Bioinformatics 10: 154. Hordvik, A., et al. (1989) High-resolution (1.5 A) crystal Brooks, H.A., Gardner, D., Poyser, J.P., and King, T.J. (1984) structure of phospholipase C from Bacillus cereus. Nature A 167 bp DNA fragment containing the complete intergenic The structure and absolute stereochemistry of zincophorin 338: 357–360. RT-PCR analysis region from aesE (ajap_08420) to aesA (ajap_08425) was (antibiotic M144255) – a monobasic carboxylic-acid Kallifidas, D., Pascoe, B., Owen, G.A., Strain-Damerell, C.M., PCR amplified using the primer pair PaesA-F and PaesA-R. ionophore having a remarkable specificity for divalent- Hong, H.J., and Paget, M.S. (2010) The zinc-responsive RNA isolation and RT-PCR analyses were performed accord- The PCR fragment was cloned into pJet1.2 and verified by cations. J Antibiot 37: 1501–1504. regulator Zur controls expression of the coelibactin gene ing to Spohn and colleagues (2014) with the primer pairs sequencing before integration into the corresponding sites of Bucheli-Witschel, M., and Egli, T. (2001) Environmental fate cluster in Streptomyces coelicolor. J Bacteriol 192: 608– listed in Table S2. the pGus vector via XbaI and SpeI to generate pGusPaesA. and microbial degradation of aminopolycarboxylic acids. 611. FEMS Microbiol Rev 25: 69–106. Kasahara, M., and Anraku, Y. (1974) Succinate- and NADH Spectrophotometric and chromogenic assay of Campoy, S., Jara, M., Busquets, N., Perez De Rozas, A.M., oxidase systems of Escherichia coli membrane vesicles. Overexpression and purification of A. japonicum Zur in Badiola, I., and Barbe, J. (2002) Role of the high-affinity Mechanism of selective inhibition of the systems by zinc GUS activity E. coli zinc uptake znuABC system in Salmonella enterica serovar ions. J Biochem 76: 967–976. For the quantification of expression levels, the spectro- Typhimurium virulence. Infect Immun 70: 4721–4725. Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F., and The coding region of the zurAj gene was amplified using the photometric method of Myronovskyi and colleagues (2011) Cebulla, I. (1995) Gewinnung komplexbildender Substanzen Hopwood, D.A. (2000) Practical Streptomyces Genetics. primers HIS-pzur-F and HIS-pzur-R. The 441 bp PCR frag- was modified. After cultivation for 72 h, cells were harvested mittels Amycolatopsis orientalis. University of Tübingen, Norwich, UK: John Innes Foundation. ment was cloned into pET30 Ek/LIC (Novagen) via its com- and washed twice with SM before the biomass was adjusted to Doctoral Thesis. Kim, H.J., Graham, D.W., DiSpirito, A.A., Alterman, M.A., plementary overhangs, according to manufacturer’s protocol. 200 mg ml−1 with SM containing glycerol.Atotal of 800 μl of the Chen, L., Liu, T., and Ma, C. (2010) Metal complexation and Galeva, N., Larive, C.K., et al. (2004) Methanobactin, a Escherichia coli BL21(DE3) pLys was transformed with the cell stock solutions were centrifuged and re-suspended in 1 ml biodegradation of EDTA and S,S-EDDS: a density func- copper-acquisition compound from methane-oxidizing bac- resulting plasmid pET-30-zur. The overexpression and puri- of lysis buffer and incubated for 30 min at 37°C. Cell debris tional theory study. J Phys Chem A 114: 443–454. teria. Science 305: 1612–1615. fication of the His-tagged protein was performed as previ- was removed by centrifugation and 100 μl of the cell lysate Cheung, J., Beasley, F.C., Liu, S., Lajoie, G.A., and Kloss, F., Pidot, S., Goerls, H., Friedrich, T., and Hertweck, C. ously reported (Shin et al., 2007). was assayed. Optical density at 405 nm was measured in a Heinrichs, D.E. (2009) Molecular characterization of (2013) Formation of a dinuclear copper(I) complex from the 96-well plate on a Dynamic Microplate MicroTek DS staphyloferrin B biosynthesis in Staphylococcus aureus. Clostridium-derived antibiotic closthioamide. Angew Chem spectrophotometer (Bio-Tek Kontron instuments). Miller units Mol Microbiol 74: 594–608. Int Edit 52: 10745–10748. Kobylarz, M.J., Grigg, J.C., Takayama, S.J., Rai, D.K., Gel mobility shift assays for DNA–Zur binding were calculated as 1000× (OD405 of sample-OD405 of Choi, S., and Bird, A.J. (2014) Zinc’ing sensibly: controlling blank) / (time of reaction in minutes × volume of culture zinc homeostasis at the transcriptional level. Metallomics Heinrichs, D.E., and Murphy, M.E. (2014) Synthesis of DNA fragments containing the analysed intergenic regions assayed). For the chromogenic assay, 50 μl from 6: 1198–1215. L-2,3-diaminopropionic acid, a siderophore and antibiotic were amplified from A. japonicum genomic DNA using the A. japonicum strain stock solutions (200 mg ml−1 in SM- D’Autreaux, B., Pecqueur, L., Gonzalez de Peredo, A., precursor. Chem Biol 21: 379–388. primers listed in Table S2. Fragment labelling was performed glycerol) were mixed with 150 μl of 1.5 mg ml−1 X-Gluc Diederix, R.E., Caux-Thang, C., Tabet, L., et al. (2007) Kołodyn´ska, D. (2011) Chelating agents of a new generation as described by Tiffert and colleagues (2008). Approximately (5-bromo-4-chloro-3-indolyl-β-d-glucuronide) and photo- Reversible redox- and zinc-dependent dimerization of the as an alternative to conventional chelators for heavy metal 4 nM of the Cy5-labelled DNA fragments and purified tagged graphed after 24 h at 30°C. Escherichia coli Fur protein. Biochemistry 46: 1329–1342. ions removal from different waste waters. In Expanding protein were incubated in reaction buffer [20 mM Tris-HCl Everest, G.J., and Meyers, P.R. (2009) The use of gyrB Issues in Desalination. Ning, R.Y. (ed.). Rijeka/Croatia: (pH = 7,8), 50 mM KCl, 1 mM DTT, 0.1 mg of bovine serum sequence analysis in the phylogeny of the genus InTech, pp. 339–370. −1 albumin ml and 5% glycerol] with or without 25 μM ZnSO4 Acknowledgements Amycolatopsis. Antonie Leeuwenhoek 95: 1–11. Leach, L.H., Morris, J.C., and Lewis, T.A. (2007) The role of for 20 min at 30°C. The binding mixture was subjected to the siderophore pyridine-2,6-bis (thiocarboxylic acid) This work was supported by ERA-IB GenoDrug (BMBF, FKZ Fillat, M.F. (2014) The FUR (ferric uptake regulator) super- electrophoresis on a 2% TB (89 mM Trizma base, 89 mM (PDTC) in zinc utilization by Pseudomonas putida DSM 0315930) and the German Center for Infection Research family: diversity and versatility of key transcriptional regu- boric acid) agarose gel in TB buffer. DNA bands were visu- 3601. Biometals 20: 717–726. (DZIF, TTU 09.802 and TTU 09.704). The authors would like lators. Arch Biochem Biophys 546: 41–52. alized by fluorescence imaging using a Typhoon (GE Lechevalier, M.P., Prauser, H., Labeda, D.P., and Ruan, J.-S. to acknowledge C. Hertweck and J. Wink for the provision of Gaballa, A., and Helmann, J.D. (1998) Identification of a Healthcare) fluorescence laser scanner. (1986) Two new genera of nocardioform actinomycetes: Amycolatopsis strains, A. Luzhetskyy for the provision of zinc-specific metalloregulatory protein, Zur, controlling zinc Amycolata gen. nov. and Amycolatopsis gen. nov. Int J pGUS, G. Muth and K. Hantke for helpful discussions, M. transport operons in Bacillus subtilis. J Bacteriol 180: Syst Evol Microbiol 36: 29–37. Schorn for proofreading the manuscript and A. Kulik for assis- 5815–5821. Detection of [S,S]-EDDS biosynthesis using HPLC-DAD de Lorenzo, V., and Neilands, J.B. (1986) Characterization of tance in HPLC analyses. Gibson, F., and Magrath, D.I. (1969) The isolation and char- acterization of a hydroxamic acid (aerobactin) formed by iucA and iucC genes of the aerobactin system of plasmid For the detection of [S,S]-EDDS, the fermentation broth was Aerobacter aerogenes 62-I. Biochim Biophys Acta 192: ColV-K30 in Escherichia coli. J Bacteriol 167: 350– centrifuged, and 1 ml of supernatant was thoroughly mixed References 175–184. 355. with 20 μl of CuSO4 (100 mM) and once again centrifuged Gilston, B.A., Wang, S., Marcus, M.D., Canalizo-Hernandez, Lucarelli, D., Russo, S., Garman, E., Milano, A., before using the supernatant for HPLC analysis. [S,S]-EDDS Aagaard, A., and Brzezinski, P. (2001) Zinc ions inhibit oxi- M.A., Swindell, E.P., Xue, Y., et al. (2014) Structural and Meyer-Klaucke, W., and Pohl, E. (2007) Crystal structure analyses were carried out on a HP1090M liquid chromato- dation of cytochrome c oxidase by oxygen. FEBS Lett 494: mechanistic basis of zinc regulation across the E. coli Zur and function of the zinc uptake regulator FurB from graph equipped with a thermostated autosampler, a diode- 157–160. regulon. PLoS Biol 12: e1001987. Mycobacterium tuberculosis. J Biol Chem 282: 9914– array detector and an HP Kayak XM 600 ChemStation Andreini, C., Banci, L., Bertini, I., and Rosato, A. (2006) Zinc Goodfellow, M., Brown, A.B., Cai, J., Chun, J., and Collins, 9922. (Agilent). A total of 10 μl of samples were injected onto a through the three domains of life. J Proteome Res 5: 3173– D.M. (1997) Amycolatopsis japonicum sp. nov., an Ma, Z., Gabriel, S.E., and Helmann, J.D. (2011) Sequential Hypersil ODS column (125 × 4 mm, 3 μm) fitted with a guard 3178. actinomycete producing (S,S)-N,N’-ethylenediamine- binding and sensing of Zn(II) by Bacillus subtilis Zur. column (10 × 4 mm, 3 μm; Stagroma) and analysed by Bailey, T.L., Boden, M., Buske, F.A., Frith, M., Grant, C.E., disuccinic acid. Syst Appl Microbiol 20: 78–84. Nucleic Acids Res 39: 9130–9138. isocratic elution with solvent A – acetonitrile (96:4, v/v) at a Clementi, L., et al. (2009) MEME SUITE: tools for motif Groth, I., Tan, G.Y.A., Gonzalez, J.M., Laiz, L., Carlsohn, Maciag, A., Dainese, E., Rodriguez, G.M., Milano, A., −1 flow rate of 1 ml min . Solvent A consisted of 20 mM discovery and searching. Nucleic Acids Res 37: W202– M.R., Schütze, B., et al. (2007) Amycolatopsis nigrescens Provvedi, R., Pasca, M.R., et al. (2007) Global analysis of Sorensen’s phosphate buffer (pH 7.2) with 5 mM tetrabutyl- W208. sp nov., an actinomycete isolated from a Roman catacomb. the Mycobacterium tuberculosis Zur (FurB) regulon. J ammoniumhydrogensulfate. UV detection was performed at Bierman, M., Logan, R., O’Brien, K., Seno, E.T., Rao, R.N., Int J Syst Evol Microbiol 57: 513–519. Bacteriol 189: 730–740. 253 nm. For data analysis, Chemstation LC3D software Rev. and Schoner, B.E. (1992) Plasmid cloning vectors for the Henssen, A., Kothe, H.W., and Kroppenstedt, R.M. (1987) Malla, S., Niraula, N.P., Liou, K., and Sohng, J.K. (2010) A.08.03 was used. Commercial [S,S]-EDDS in solution conjugal transfer of DNA from Escherichia coli to Transfer of Pseudonocardia azurea and Pseudonocardia Self-resistance mechanism in Streptomyces peucetius: (Sigma Aldrich) was precipitated to obtain crystalline powder Streptomyces spp. Gene 116: 43–49. fastidiosa to the genus Amycolatopsis, with emended Overexpression of drrA, drrB and drrC for doxorubicin as previously described (Zwicker et al., 1997) and used as a Blom, J., Albaum, S.P., Doppmeier, D., Pühler, A., Vorhölter, species description. Int J Syst Bacteriol 37: 292–295. enhancement. Microbiol Res 165: 259–267. standard. F.J., Zakrzewski, M., and Goesmann, A. (2009) EDGAR: a

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141262M.M. Spohn, Spohn, W. Wohlleben W. Wohlleben and E. and Stegmann E. Stegmann AnalysisAnalysis of zinc regulation of zinc regulation and biosynthesis and biosynthesis of [S,S of []-EDDSS,S]-EDDS126315

Margalith, P., and Beretta, G. (1960) Rifamycin. IX. Taxo- regulator related to the diphtheria toxin repressor family of hensive resource for the genome mining of biosynthetic and pΔzur-US-R (Table S2). Yellow: downstream flanking nomic study on Streptomyces mediterranei nov. sp. proteins. Mol Microbiol 35: 1454–1468. gene clusters. Nucleic Acids Res 43: W237–W243. region of zur amplified by using the primer pair pΔzur-DS-F Mycopathol Mycol Appl 13: 321–330. Rusnak, F., Sakaitani, M., Drueckhammer, D., Reichert, J., Wink, J., Gandhi, J., Kroppenstedt, R.M., Seibert, G., and pΔzur-DS-R (Table S2). Hatched red and yellow bars Medema, M.H., Takano, E., and Breitling, R. (2013) Detecting and Walsh, C.T. (1991) Biosynthesis of the Escherichia coli Sträubler, B., Schumann, P., and Stackebrandt, E. (2004) illustrate the PCR-generated upstream and downstream sequence homology at the gene cluster level with siderophore enterobactin: sequence of the entF gene, Amycolatopsis decaplanina sp. nov., a novel member of fragments, which were integrated into pGusA21. MultiGeneBlast. Mol Biol Evol 30: 1218–1223. expression and purification of EntF, and analysis of cova- the genus with unusual morphology. Int J Syst Evol pGusA21Δzur-zur is depicted in dashed lines to illustrate that Meiwes, J., Fiedler, H.P., Zahner, H., Konetschnyrapp, S., lent phosphopantetheine. Biochemistry 30: 2916–2927. Microbiol 54: 235–239. this plasmid is not replicating in A. japonicum. The proposed and Jung, G. (1990) Production of desferrioxamine E and Schowanek, D., Feijtel, T.C., Perkins, C.M., Hartman, F.A., Wink, J.M., Kroppenstedt, R.M., Ganguli, B.N., Nadkarni, gene products of ajap_2935 – 60 are a conserved putative- new analogs by directed fermentation and feeding fermen- Federle, T.W., and Larson, R.J. (1997) Biodegradation of S.R., Schumann, P., Seibert, G., and Stackebrandt, E. secreted protein, a HTH-type transcriptional repressor, a Fur tation. Appl Microbiol Biotechnol 32: 505–510. [S,S], [R,R] and mixed stereoisomers of ethylene diamine (2003) Three new antibiotic producing species of the genus family transcriptional regulator, a hypothetical protein, a Menges, R., Muth, G., Wohlleben, W., and Stegmann, E. disuccinic acid (EDDS), a transition metal chelator. Amycolatopsis, Amycolatopsis balhimycina sp. nov., A. membrane permease and a second membrane permease (2007) The ABC transporter Tba of Amycolatopsis Chemosphere 34: 2375–2391. tolypomycina sp. nov., A. vancoresmycina sp. nov., and respectively. The deletions of aesA-C and aesH-E were per- balhimycina is required for efficient export of the Schröder, J., Jochmann, N., Rodionov, D.A., and Tauch, A. description of Amycolatopsis keratiniphila subsp. formed accordingly. glycopeptide antibiotic balhimycin. Appl Microbiol (2010) The Zur regulon of Corynebacterium glutamicum keratiniphila subsp. nov. and A. keratiniphila subsp. Fig. S3. HPLC analysis of the zinc-dependent [S,S]-EDDS Biotechnol 77: 125–134. ATCC 13032. BMC Genomics 11: 12. nogabecina subsp. nov. Syst Appl Microbiol 26: 38–46. production. Strains were grown in deionized flasks in SM

Mertz, F.P., and Yao, R.C. (1993) Amycolatopsis alba sp. Seyedsayamdost, M.R., Traxler, M.F., Zheng, S.L., Kolter, R., Zwicker, N., Theobald, U., Zähner, H., and Fiedler, H.P. either with or without supplementation of 6 μM ZnSO4. nov., isolated from soil. Int J Syst Bacteriol 43: 715–720. and Clardy, J. (2011) Structure and biosynthesis of (1997) Optimization of fermentation conditions for the pro- Commercial [S,S]-EDDS from Sigma Aldrich was used as a Morita, Y., Kodama, K., Shiota, S., Mine, T., Kataoka, A., amychelin, an unusual mixed-ligand siderophore from duction of ethylene-diamine-disuccinic acid by standard. Mizushima, T., and Tsuchiya, T. (1998) NorM, a putative Amycolatopsis sp. AA4. J Am Chem Soc 133: 11434– Amycolatopsis orientalis. J Ind Microbiol Biotechnol 19: Fig. S4. Zinc-dependent transcriptional pattern of the puta- multidrug efflux protein, of Vibrio parahaemolyticus and its 11437. 280–285. tive [S,S]-EDDS biosynthesis genes aesA-D of A. japonicum. homolog in Escherichia coli. Antimicrob Agents Chemother Shen, Y., Huang, H., Zhu, L., Luo, M., and Chen, D. (2012) Cultures were grown in SM in absence of any trace element 42: 1778–1782. Type II thioesterase gene (ECO-orf27) from Amycolatopsis (−) or supplemented with (+)6μM Zn2+ respectively and Morita, Y., Kataoka, A., Shiota, S., Mizushima, T., and orientalis influences production of the polyketide antibiotic, Supporting information samples were taken after 25 h of incubation to isolate RNA. Tsuchiya, T. (2000) NorM of Vibrio parahaemolyticus is an ECO-0501 (LW01). Biotechnol Lett 34: 2087–2091. sigB was used as housekeeping gene to normalize the RNA. Additional Supporting Information may be found in the online Na(+)-driven multidrug efflux pump. J Bacteriol 182: 6694– Shin, J.H., Oh, S.Y., Kim, S.J., and Roe, J.H. (2007) The Left panel shows transcription pattern in WT background, version of this article at the publisher’s web-site: 6697. zinc-responsive regulator Zur controls a zinc uptake right panel in the Δzur background. Myronovskyi, M., Welle, E., Fedorenko, V., and Luzhetskyy, system and some ribosomal proteins in Streptomyces Fig. S1. (A) Zinc-binding site prediction by PREDZINC Fig. S5. HPLC chromatograms of culture supernatants of

A. (2011) Beta-glucuronidase as a sensitive and versatile coelicolor A3(2). J Bacteriol 189: 4070–4077. version 1.4 (Shu et al., 2008) of ZurAj (GenBank: AIG78644). A. japonicum strains grown in deionized flasks in SM in reporter in actinomycetes. Appl Environ Microbiol 77: Shin, J.H., Jung, H.J., An, Y.J., Cho, Y.B., Cha, S.S., and Predicted zinc-binding residues are highlighted in red. Cys, absence of zinc. Commercial [S,S]-EDDS from Sigma Aldrich 5370–5383. Roe, J.H. (2011) Graded expression of zinc-responsive His, Asp and Glu are bolded. Residues are predicted as zinc was used as a standard. Nadkarni, S.R., Patel, M.V., Chatterjee, S., Vijayakumar, genes through two regulatory zinc-binding sites in Zur. binding if the score is ≥0.450. (B) Structure-based multiple Fig. S6. 16S rRNA gene phylogenetic tree for 16 members E.K., Desikan, K.R., Blumbach, J., et al. (1994) Balhimycin, Proc Natl Acad Sci USA 108: 5045–5050. sequence alignment of Zur from M. tuberculosis (Lucarelli of the genus Amycolatopsis chosen by criteria of available a new glycopeptide antibiotic produced by Amycolatopsis Shu, N., Zhou, T., and Hovmöller, S. (2008) Prediction et al., 2007) and S. coelicolor (Shin et al., 2011) with its puta- genome sequence data. The tree was constructed using sp. Y-86,21022. Taxonomy, production, isolation and bio- of zinc-binding sites in proteins from sequence. tive orthologue from A. japonicum. Sequences were aligned neighbour joining with CLUSTALW and MEGA4 software. The logical activity. J Antibiot (Tokyo) 47: 334–341. Bioinformatics 24: 775–782. by using CLUSTALW2. Zinc-binding sites: site 1 (structural), classification into the phylogenetic clades A–F occurred Nishikiori, T., Okuyama, A., Naganawa, H., Takita, T., Spohn, M., Kirchner, N., Kulik, A., Jochim, A., Wolf, F., site 2, site 3. Protein domains are indicated on the top. accordingly to Everest and Meyers (2009). The percentage Hamada, M., Takeuchi, T., et al. (1984) Production by Münzer, P., et al. (2014) Overproduction of ristomycin A by Green bar indicates the N-terminal DNA-binding domain, red bootstrap values of 1000 replications are shown at each node actinomycetes of (S,S)-N,N’-ethylenediamine-disuccinic activation of a silent gene cluster in Amycolatopsis bar indicates the amino acids involved in forming the inter- (only values above 40% are shown). The scale bar indicates acid, an inhibitor of phospholipase C. J Antibiot (Tokyo) 37: japonicum MG417-CF17. Antimicrob Agents Chemother domain hinge loop and the blue bar indicates the C-terminal 1 nucleotide substitution per 100 nucleotides. Streptomyces 426–427. 58: 6185–6196. dimerization domain. Amino acid similarity is indicated at scabies was used as an out-group. Owen, G.A., Pascoe, B., Kallifidas, D., and Paget, M.S. Stegmann, E., Pelzer, S., Wilken, K., and Wohlleben, W. the bottom [identical (*), highly similar (:) and similar (.) Fig. S7. HPLC chromatograms of culture supernatants of (2007) Zinc-responsive regulation of alternative ribosomal (2001) Development of three different gene cloning systems respectively]. several Amycolatopsis strains grown in deionized flasks in

protein genes in Streptomyces coelicolor involves Zur and for genetic investigation of the new species Amycolatopsis Fig. S2. The construction of the zur in frame deletion strain SM either with or without supplementation of 25 μM ZnSO4. σR. J Bacteriol 189: 4078–4086. japonicum MG417-CF17, the ethylenediaminedisuccinic A. japonicum Δzur using the plasmid pGusA21Δzur Commercial [S,S]-EDDS from Sigma Aldrich was used as Patzer, S.I., and Hantke, K. (1998) The ZnuABC high-affinity acid producer. J Biotechnol 92: 195–204. (Table S1) via homologous recombination. First and second standard. zinc uptake system and its regulator Zur in Escherichia coli. Stegmann, E., Albersmeier, A., Spohn, M., Gert, H., Weber, homologous recombinations are exemplary illustrated as Table S1. Bacterial strains and plasmids used in this study. Mol Microbiol 28: 1199–1210. T., Wohlleben, W., et al. (2014) Complete genome succession of (first) downstream recombination and Table S2. Oligonucleotides used in this study. Pawlik, M.C., Hubert, K., Joseph, B., Claus, H., Schoen, C., sequence of the actinobacterium Amycolatopsis japonica (second) upstream recombination but could also occur in Table S3. Manual BLAST analysis of AesA-H. Comparison of and Vogel, U. (2012) The zinc-responsive regulon of MG417-CF17 (=DSM 44213T) producing (S,S)-N,N’- opposite succession. aac(3)-IV, apramycin resistance gene; A. japonicum AesA-H to homologues in other Amycolatopsis Neisseria meningitidis comprises 17 genes under control of ethylenediaminedisuccinic acid. J Biotechnol 189C: 46–47. gusA, β-glucuronidase (GUS) gene. Red: upstream flanking species. a Zur element. J Bacteriol 194: 6594–6603. Takahashi, R., Fujimoto, N., Suzuki, M., and Endo, T. (1997) region of zur amplified by using the primer pair pΔzur-US-F Table S4. Zur boxes 5′ upstream of aesA homologues. Pollack, J.R., Ames, B.N., and Neilands, J.B. (1970) Iron Biodegradabilities of ethylenediamine-N,N’-disuccinic acid transport in Salmonella typhimurium: mutants blocked in (EDDS) and other chelating agents. Biosci Biotechnol the biosynthesis of enterobactin. J Bacteriol 104: 635–639. Biochem 61: 1957–1959. Puk, O., Huber, P., Bischoff, D., Recktenwald, J., Jung, G., Tiffert, Y., Supra, P., Wurm, R., Wohlleben, W., Wagner, R., Sussmuth, R.D., et al. (2002) Glycopeptide biosynthesis in and Reuther, J. (2008) The Streptomyces coelicolor GlnR Amycolatopsis mediterranei DSM5908: function of a regulon: identification of new GlnR targets and evidence for halogenase and a haloperoxidase/perhydrolase. Chem a central role of GlnR in nitrogen metabolism in Biol 9: 225–235. actinomycetes. Mol Microbiol 67: 861–880. Que, Q., and Helmann, J.D. (2000) Manganese homeostasis Weber, T., Blin, K., Duddela, S., Krug, D., Kim, H.U., in Bacillus subtilis is regulated by MntR, a bifunctional Bruccoleri, R., et al. (2015) antiSMASH 3.0 – a compre-

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Table S1: Bacterial strains and plasmids used in this study

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Table S2: Oligonucleotides used in this study Table S3: Manual BLAST analysis of AesA-H. Comparison of A. japonicum AesA-H to homologs in other Amycolatopsis species.

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Table S4: Zur-boxes 5‘ upstream of aesA homologues

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