MicrobiologyOpen. 2017;e499.  | 1 of 12https://doi.org/10.1002/mbo3.499

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Received:2February2017  |  Revised:27April2017  |  Accepted:28April2017DOI: 10.1002/mbo3.499

O R I G I N A L R E S E A R C H

Coregulation of the cyclic lipopeptides orfamide and sessilin in the biocontrol strain Pseudomonas sp. CMR12a

Feyisara E. Olorunleke1,* | Nam P. Kieu1,* | Evelien De Waele1 | Marc Timmerman1 |  Marc Ongena2 | Monica Höfte1

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,provided the original work is properly cited.©2017TheAuthors.MicrobiologyOpenpublishedbyJohnWiley&SonsLtd.

1LaboratoryofPhytopathology,FacultyofBioscienceEngineering,GhentUniversity,Ghent,Belgium2GemblouxAgro-BioTech,UniversityofLiège,Gembloux,Belgium

CorrespondenceMonicaHöfte,LaboratoryofPhytopathology,FacultyofBioscienceEngineering,GhentUniversity,Ghent,Belgium.Email: monica.hofte@ugent.be

Funding informationNationalFundsforScientificResearch,Belgium; Schlumberger Foundation; Research FoundationFlanders,Grant/AwardNumber:3G.002.10N

AbstractCyclic lipopeptides (CLPs) are synthesized by nonribosomal peptide synthetases(NRPS),whichareoftenflankedbyLuxR-typetranscriptionalregulators.Pseudomonas sp.CMR12a,aneffectivebiocontrol strain,produces twodifferentclassesofCLPsnamelysessilinsandorfamides.Theorfamidebiosynthesisgeneclusterisflankedup-anddownstreambyLuxR-typeregulatorygenesdesignatedofaR1 and ofaR2,respec-tively,whereas the sessilinbiosynthesisgeneclusterhasoneLuxR-type regulatorygene which is situated upstream of the cluster and is designated sesR. Our study inves-tigated the role of these three regulators in the biosynthesis of orfamides and sessilins. PhylogeneticanalysespositionedOfaR1andOfaR2withLuxRregulatoryproteinsofsimilarorfamide-producingPseudomonas strains and the SesR with that of the tolaasin producer,Pseudomonas tolaasii.LC-ESI-MSanalysesrevealedthatsessilinsandorfa-mides are coproduced and that production starts in the late exponential phase.However,sessilinsaresecretedearlierandinlargeamounts,whileorfamidesarepre-dominantly retained in the cell. Deletion mutants in ofaR1 and ofaR2 lost the capacity toproducebothorfamidesandsessilins,whereasthesesR mutant showed no clear phenotype.Additionally,RT-PCRanalysisshowedthatinthesessilincluster,amuta-tion in either ofaR1 or ofaR2ledtoweakertranscriptsofthebiosynthesisgenes,ses-ABC, andputative transporter genes,macA1B1. In theorfamidecluster,mainly thebiosynthesis genes ofaBCwereaffected,while the firstbiosynthesisgeneofaA and putative macA2B2transportgeneswerestilltranscribed.AmutationineitherofaR1,ofaR2,orsesR genes did not abolish the transcription of any of the other two.

K E Y W O R D S

cycliclipopeptides,LuxR,Pseudomonas,transcriptionalregulators

1  | INTRODUCTION

Cyclic lipopeptides (CLPs) are bacterial metabolites with biosurfac-tant activity composed of a cyclic oligopeptide lactone ring coupled

toafattyacidtail.ThebiosynthesisofCLPsisdrivenbynonribosomalpeptidesynthetases(NRPS),whichareencodedbylargegeneclusters(Raaijmakers,deBruijn,&deKock,2006).CLPshavedrawnincreasinginterest for their versatile functions in plant beneficial Pseudomonas,whichincludeinvolvementinbiofilmformation,motility,andantimi-crobialactivityagainstawiderangeofmicroorganismsincludingfungi,*Joint first author.

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bacteria, viruses, and oomycetes (reviewed by Olorunleke, Kieu, &Höfte,2015).WithinthedifferentCLPfamilies,severalCLPbiosyn-thesisgeneclustershavebeenfullycharacterizedincludingorfamide,viscosin,massetolide,putisolvin,xantholysin,entolysin,andpoaeam-ide, tolaasin, syringomycin, and syringopeptin, WLIP, arthrofactin,bananamide, thanapeptin, nunamycin, and nunapeptin (D’aes etal.,2014;DeBruijnetal.,2007;DeBruijn,deKock,deWaard,vanBeek,& Raaijmakers, 2008; Dubern, Coppoolse, Stiekema, & Bloemberg,2008; Li etal., 2013; Vallet-Gely etal., 2010; Zachow etal., 2015;Scherlachetal.,2013;Wang,Lu,Records,&Gross,2006;Rokni-Zadehetal.,2012;Washio,Lim,Roongsawang,&Morikawa,2010;Nguyenetal.,2016;VanDerVoortetal.,2015;Michelsenetal.,2015).

The LuxR superfamily consists of transcriptional regulators thatcontainaDNA-bindinghelix–turn–helix(HTH)motifintheC-terminalregion(Fuqua,Winans,&Greenberg,1996). Inthissuperfamily,foursubfamilies can be distinguished based on domain architecture and the mechanismofregulatoryactivation.LuxR-likeproteinsSalA,SyrF,andSyrGareapartofthefourthsubfamily,whichischaracterizedbythelackofanydefinedN-terminaldomain.Theseproteinshavebeenasso-ciatedwiththeregulationoftheCLPssyringomycinandsyringopeptinin Pseudomonas syringae pv. syringae, a plant pathogenic bacterium(Vaughn &Gross, 2016). In various other Pseudomonas species and strains,regulatorygenesencodingsimilarLuxR-likeproteinsareposi-tionedup-anddownstreamoftheCLPbiosynthesisgenes(DeBruijn&Raaijmakers,2009a).WithinseveralCLPfamilies,theregulationofCLPbiosynthesis has been attributed to LuxR-type regulators includingPsoR(putisolvin) inP. putida (Dubernetal.,2008),ViscAandViscBC(viscosin) inP. fluorescens SBW25 (DeBruijn&Raaijmakers, 2009a),MassAandMassBC(massetolide)inP. fluorescens SS101 (De Bruijn & Raaijmakers,2009b),ArfF(arthrofactin)inP. fluorescensMIS38(Washioetal.,2010),EtlR(entolysin)inP. entomophiliaL48T(Vallet-Gelyetal.,2010),WlpR (WLIP) inP. putidaRW10S2 (Rokni-Zadehetal.,2012),XtlR(xantholysin)inP. putidaBW11M1(Lietal.,2013),andPcoRandRfiA(corpeptin)inP. corrugataCFBP5454(Stranoetal.,2015).

In several Pseudomonasstrains,theprincipalregulatorofCLPbio-synthesisistheGacA/GacStwo-componentsystemsinceamutationinoneofbothencodinggenesleadstoalossinCLPproduction(DeBruijn & Raaijmakers, 2009a). The GacA/GacS system is known toactivatesmallRNAsthatbindtoandsequestertranslational repres-sorproteins,whichblocktheribosomalbindingsitesinthemRNAofGac-regulatedgenes.TwosmallRNAs(sRNAs)andtworepressorpro-teins,RsmAandRsmE,havebeenlinkedtotheregulationofentolysin(Vallet-Gelyetal.,2010)andmassetolideAbiosynthesis(Song,Voort,etal.,2015).InthemassetolideproducerP. fluorescensSS101,theserepressorproteinsmostlikelyblocktranslationoftheLuxR-typetran-scriptionalregulator,MassAR(Song,Voort,etal.,2015),bybindingtoaspecificsitecalledtheGacAbox.ThissitecomprisesanontranslatedleadersequenceupstreamoftheAUGcodononthemessengerRNA.In several CLP-producingPseudomonas strains, a GacA box is pres-entupstreamtheLuxRregulatorsflankingtheCLPbiosynthesisgeneclustersuggestingthatotherCLP-producingPseudomonas strains may show a similar regulation of lipopeptide biosynthesis (Song, Voort,etal.,2015).

Besides the GacA/GacS regulatory system, N-acylhomoserinelactone (N-AHL)-mediated quorum sensing was shown to be re-quiredforviscosinandputisolvinbiosynthesis (Cui,Harling,Mutch,&Darling,2005;Dubern,Lugtenberg,&Bloemberg,2006)inP. fluo-rescensstrain5064andP. putidastrainPCL1445,althoughthisisnotthe case in certain other Pseudomonasstrains(DeBruijnetal.,2008;Dumenyo,Mukherjee,Chun,&Chatterjee,1998;Kinscherf&Willis,1999).InP. putidastrainPCL1445,twoheatshockproteinsDnaKandDnaJlocateddownstreamoftheGacsystemwereshowntoregulateputisolvinbiosynthesis(Dubern,Lagendijk,Lugtenberg,&Bloemberg,2005).RecentstudiesonthegeneticregulationofmassetolideAbio-synthesis in P. fluorescens SS101 revealed that the serine protease ClpP togetherwith the chaperone ClpA regulates the biosynthesisofmassetolidesviaaspecificpathway involvingtheLuxRregulator(MassABC),theheatshockproteinsDnaKandDnaJ,andproteinsofthe tricarboxylicacid (TCA)cycle (DeBruijn&Raaijmakers,2009b;Song,Aundy,vandeMortel,&Raaijmakers,2014;Song,Sundqvist,etal.,2015).

Pseudomonas sp. CMR12a is a biocontrol strain isolated from the cocoyam rhizosphere inCameroon (Perneeletal., 2007).This strainproducestwoclassesofCLPsnamelyorfamidesandsessilinstogetherwith two types of phenazines, phenazine-1-carboxylate (PCA) andphenazine-1-carboxamide (PCN) (D’aes etal., 2014; Perneel etal.,2007).Orfamidesarealsoproducedbybiocontrol agentsbelongingto the P. protegens group (Gross etal., 2007; Jang etal., 2013;Ma,Geudens,etal.,2016;Takeuchi,Noda,&Someya,2014),whilesessi-lins are structurally related to the tolaasins produced by the mushroom pathogen, P. tolaasii. Sessilins are important for biofilm formation,while orfamides are crucial for the swarming motility of CMR12a (D’aesetal.,2014)andbothCLPsareimportantforbiocontrol(D’aesetal.,2011;Hua&Höfte,2015;Ma,Hua,Ongena,&Höfte,2016;Olorunleke,Hua,Kieu,Ma,&Höfte,2015).

In CMR12a, sessilin biosynthesis is governed by three linkedNRPS genes namely sesA, sesB, and sesC (Figure1a) (D’aes etal.,2014).ThesegenesareflankedupstreambyanodT-likegenedesig-nated sesT,anddownstreambymacA1 and macB1genes,whichareprobably involved in sessilin secretion.MacA andMacB are part ofa tripartite secretion system involving an inner membrane protein (MacB),aperiplasmicadaptorprotein(MacA),andanoutermembraneprotein(NodT).Similartosessilin,orfamidebiosynthesisisgovernedby three linkedNRPSgenesnamelyofaA,ofaB, andofaC (Figure1b)(D’aesetal.,2014).MacA- and macB-like genes putatively involved in orfamide secretion are located downstream of ofaC.Intriguingly,thereis no nodT-likegeneintheorfamidegeneclusterofPseudomonas sp. CMR12a,whilethisgeneispresentintheorfamidegeneclustersofP. protegensisolates(Ma,Geudens,etal.,2016).Inaddition,aLuxR-typeregulatorygene,ofaR1, is locatedupstreamof theorfamidebiosyn-thesisclusterandasecondone,ofaR2,issituateddownstreamofthemacA2B2genes,whereasasingleLuxR-typeregulatorygene,sesR,islocatedupstreamofthesessilinbiosynthesisclusternexttothesesT gene(D’aesetal.,2014).

Inthisstudy,wehypothesizedthatinPseudomonassp.CMR12a,OfaR1 and OfaR2 regulate the biosynthesis of orfamides, whereas

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SesR is vital for sessilin biosynthesis. To test our hypothesis, site-directed mutagenesis of the corresponding genes was conducted fol-lowed by biochemical and transcriptional analyses.

2  | MATERIALS AND METHODS

2.1 | Bacterial strains and culture conditions

Bacterial strains, plasmids, andprimersused in this studyare listedin Table 1. Pseudomonassp.CMR12awasculturedonLuria–Bertani(LB)agarplatesorinliquidLBbrothat28°C.Allmoleculartechniqueswere performed using standard protocols (Sambrook, Frithsch, &Maniatis,1989).Escherichia colistrainsweregrownonLBagarplatesor LB broth amended with appropriate antibiotics. Saccharomyces cerevisiae InvSc1wascultivatedonyeastextract–peptone–dextrose(YPD)(Shanks,Caiazza,Hinsa,Toutain,&O’Toole,2006).Escherichia colistrainWM3064wasusedasahostfortheplasmidsusedinsite-directed mutagenesis.

2.2 | Analysis of CLP production

ForLC-ESI-MSanalyses,bacterialstrainsweregrownat28°Cinsix-wellplateswith2.5mlLBbrothperwell.Culturesweremaintainedfor variable time periods after which 1 ml of each was centrifuged at 18,900g for 4min. Filter-sterilized supernatantswere subjected toreverse-phaseLC-ESI-MSasdescribedbyD’aesetal. (2014).Cellsobtained after the centrifugation step were washed once with ster-iledistilledwaterresuspendedin1mlofacetonitrilesolution(50%)after which sonication was carried out for 30 s. Following centrifuga-tion, thecell supernatantwas filter sterilizedand subjected toLC-ESI-MSanalysis.Datageneratedfromsupernatantandcellanalyses

wereprocessedtoeitherextractchromatogramsorobtaintherela-tiveproductionofsessilinsandorfamidesusingtheMassLynxV4.1software.

2.3 | Site- directed mutagenesis

Site-directedmutagenesis of theofaR1,ofaR2, and sesR genes was performed based on methods described previously (D’aes etal.,2014). To construct eachmutant, a fragment of the correspondingLuxRbiosynthesisgenewasdeletedbyallelicreplacementwithvectorpMQ30(Shanksetal.,2006).Primersusedforpolymerasechainreac-tion(PCR)andplasmidsaredescribedinTable1.Toobtainadeletionplasmid,twocodingregionsofeachLuxRgenewereamplifiedbyPCRandtheseproductswereclonednexttoeachotherbyhomologousrecombination in S. cerevisiaeInvSc1.ThisplasmidwasmobilizedintoCMR12a by conjugation with E. coliWM3064andselectionongenta-mycin.Subsequently,transconjugantsthathadlosttheplasmidduringthesecondcrossovereventwereselectedonLBwith10%sucroseafter which gene deletion was confirmed by PCR and sequencing(LGCGenomics,Germany).

2.4 | Construction of pME6032- based vectors for complementation

AfragmentcontainingtheluxRgenewasobtainedbyPCRwithspe-cificprimers(Table1).ThesePCRproductsweresubsequentlyclonedin the expression vector pME6032 comprising the pTac promoter.The plasmids obtained, pME6032-OfaR1, pME6032-OfaR2, andpME6032-SesRweretransformedintoE. coliWM3064viaheatshockafterwhichtransformedcolonieswereselectedonLBagarplatessup-plementedwithtetracycline50μg/ml. Correct integration of fragments

F IGURE  1 Schematicrepresentationofsessilin(a)andorfamide(b)geneclustersofPseudomonassp.CMR12a.Onbothclusters,RT-PCRampliconpositionsareletteredAtoX.SesT(NodT-likeoutermembranelipoprotein);SesR:LuxR-typetranscriptionalregulator;SesD:SyrD-likeABCtransporterprotein;OfaR1:LuxR-typetranscriptionalregulatorupstreamoftheorfamidegenecluster;OfaR2:LuxR-typetranscriptionalregulatordownstreamoftheorfamidegenecluster;MacA:periplasmicmembraneprotein;MacB:innermembraneprotein.MacA1andMacB1:associatedwiththesessilingenecluster;MacA2andMacB2:associatedwiththeorfamidegenecluster.MacA1/MacA2andMacB1/MacB2share78%and80%identity,respectively

SesA SesCSesB MacA1SesRSesT SesD MacB1

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wasverifiedbyPCRanalysis,restrictionanalysisofisolatedplasmids,andsequencing.ThesethreepME6032-basedE. coliWM3064vectorswere transformed into the corresponding Pseudomonas sp. CMR12a LuxR mutants by conjugation. Transformed cells were selected onLB supplementedwith 100μg/ml tetracycline and the presence of pME6032-OfaR1, pME6032-OfaR2, or pME6032-SesR was con-firmedbyPCRanalysisusingprimersspecific forpME6032andthecorresponding luxR gene.

2.5 | White line- in- agar and swarming motility assays

Thewhiteline-in-agartest(Rokni-Zadeh,Li,Yilma,Sanchez-Rodriguez,&DeMot,2013)wasperformedintriplicateonKings’B(KB)medium.BacterialstrainswereculturedinLBbrothfor16hrandwashedtwicewithsalinesolution (0.85%).The lineofbacterial indicatorstrain (P. protegensPf-5)inthemiddleoftheplateswasmadefromthreedrops

TABLE  1 Strains,plasmids,andsite-directedmutagenesisprimersusedinthisstudya

Strains, plasmids, and primers Relevant characteristics Reference/Source

Pseudomonas

CMR12a PHZ+,CLP1+,CLP2+,wildtype(Cameroon) Perneeletal.(2007)

CMR12a-ΔsesR Mutant with deletion of luxR gene in sessilin cluster This study

CMR12a-ΔofaR1 Mutant with deletion of luxRgene,upstreamorfamidecluster This study

CMR12a-ΔofaR2 Mutant with deletion of luxRgene,downstreamorfamidecluster This study

P. protegensPf-5 Orfamide,wildtype LoperandGross(2007)

Escherichia coli

DH5α Host for cloning Hanahan(1983)

WM3064 Strain for conjugation; λpir,DAPauxotroph Saltikov and Newman(2003)

Saccharomyces cerevisiae InvSc1

Yeast strain for in vivo recombination (ura3-52/ura3-52mutation) Invitrogen

Plasmids

pMQ30 GenereplacementvectorforPseudomonas species; sacB,URA3,GmR Shanksetal.(2006)

pMQ30-ΔsesR VectorforsitespecificmutagenesisofluxR gene in sessilin cluster This study

pMQ30-ΔofaR1 VectorforsitespecificmutagenesisofluxRgene,upstreamorfamidecluster This study

pMQ30-ΔofaR2 VectorforsitespecificmutagenesisofluxRgene,downstreamorfamidecluster This study

pME6032 Expressionvectorwithtac promoter; TcR Heeb,Blumer,andHaas(2002)

pME6032-SesR ExpressionvectorwithsesR of CMR12a under tac promoter; TcR This study

pME6032-OfaR1 ExpressionvectorwithofaR1 of CMR12a under tac promoter; TcR This study

pME6032-OfaR2 ExpressionvectorwithofaR2 of CMR12a under tac promoter; TcR This study

Primers(5′→3′)

OfaR1-Up-F GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTGGAAGTCGTGAAAGGCCAGT This study

OfaR1-Up-R GCTGTTCTTGACGCTCAGGGAGGTTGCTGCTCAGACTCA(911bp) This study

OfaR1-Down-F TGAGTCTGAGCAGCAACCTCCCTGAGCGTCAAGAACAGC This study

OfaR1-Down-R CCAGGCAAATTCTGTTTTATCAGACCGCTTCTGCGTTCTGATTTCAGTGTGCGACTCAATCC(885bp) This study

OfaR2-Up-F GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTGGCTGCCTTCACCTTCTATGC This study

OfaR2-Up-R CTCACTCAGGTTTGCTGCTGATGACCTTGCCAATGTGAGG(883bp) This study

OfaR2-Down-F CCTCACATTGGCAAGGTCATCAGCAGCAAACCTGAGTGAG This study

OfaR2-Down-R CCAGGCAAATTCTGTTTTATCAGACCGCTTCTGCGTTCTGATCGTCAGCCACCTGTACTTCA(896bp) This study

SesR-Up-F GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTGCTTGAGGCCAAAGACCAGAC This study

SesR-Up-R CACTTGGTCAATCCATGTCGTGAATGCTGCTCGTCATTTC(953bp) This study

SesR-Down-F GAAATGACGAGCAGCATTCACGACATGGATTGACCAAGTG This study

SesR-Down-R CCAGGCAAATTCTGTTTTATCAGACCGCTTCTGCGTTCTGATAACCAGCAACGTCAGGCTAT(863bp)

This study

aPHZ,phenazines;CLP1,sessilins;CLP2,orfamides;GmR,TcR,AmpR,KmR,resistanttogentamycin,tetracyclin,ampicillin,kanamycin,respectively.

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(5μl per drop) of the suspension. Subsequently, 5μl suspension of each test bacterial strain was spotted at both sides of the line within a 1-cmdistance.Whiteprecipitateformationintheagarwasevaluatedafter3daysofgrowthat28°C.

Forswarmingmotilityassays,5μl suspension of each test strain wasspotted inthecenterofLBplatescomprising0.6%agar, lefttodry briefly and incubated at 28°C for 24hr (D’aes etal., 2014). Atleast tworeplicatesperstrainwere included,andexperimentswererepeated at least twice.

2.6 | RNA extraction and reverse transcription- PCR (RT- PCR)

Bacterialcellsweregrowninstillculturesusingasix-wellplatecon-taining2.5mlLBbrothperwellat28°C.At24hr,growthofstrainswas determined by measuring optical density OD620 of 100 μl in a 96-well plate using a Bio-Rad 680 microplate reader after which1mlofcellculturewascollectedandspundown.Cellswerefrozenin liquid N2andstoredat−80°C.FortheRNAextractionandcom-plementaryDNA (cDNA) synthesis, twobiological replicateswereused.RNAwas isolated from frozenbacterial cellswith theTrizolreagent(Sigma),followedbygenomicDNAremovalusingtheTurboDNA-freekit(Ambion/AppliedBiosystems).cDNAwassynthesizedby using the GoScript Reverse Transcription System (Promega).cDNAwithRNAequivalentof100–200ngwas subjected toPCRwith specific primers listed in Table S1. The thermal profile used consistedofaninitialdenaturationstepat95°Cfor2min,followedby30cyclesof94°C for30s,54°C for30s, and72°C for1min.Theprimerpairswereused to amplify cDNAobtained from tran-scripts corresponding to genes of the sessilin and orfamide biosyn-thesis gene clusters and their flanking genes including the sesT,sesR,ofaR1, ofaR2, ofaABC, sesABC, and themacAB genes. Transcripts covering adjacent gene pairs of the aforementioned genes were also amplified.

2.7 | Bioinformatic analyses

LuxR-like protein sequences for Pseudomonas sp. CMR12a were obtained from the nucleotide sequences of the sessilin and orfa-mide biosynthesis gene clusters with GenBank accession num-bers JQ309920 and JQ309921, respectively. Other amino acidsequences used for phylogenetic analyses were collected from the National Centre for Biotechnology Information (NCBI) web-site. Characteristics of strains and protein sequences used in the phylogeneticanalysesofLuxRproteinsarepresented inTableS2.Sequence alignments were made using Muscle (Edgar, 2004) viathesoftwarepackageMEGA6(Tamura,Stecher,Peterson,Filipski,&Kumar,2013).Phylogenetictreewas inferredbymaximumlike-lihood (ML) using 1000 bootstrap replicates andwas rootedwiththeLuxR (quorumsensingprotein) fromVibrio fischeri.ProteinsofN-acyl-L-homoserine lactones (acyl-HSLs)-binding regulators ofCMR12a,CmrRandPhzR (DeMaeyer,D’aes,Hua,Nam,&Höfte,2013),wereincludedinthisanalysis.

Furthermore,bioinformatictoolswereemployedtocheckforthepresence of Rsm binding sites upstream of the ofaR1,ofaR2,andsesR genes. The query search was conducted using the conserved motif 5′-A/UCANGGANGU/A-3′,whereNdenotes any nucleotide (Song,Voort, etal., 2015). Subsequently, similar nontranslated leader se-quencesflankingtheLuxRtranscriptional regulatorsofseveralCLP-producing Pseudomonas strains were aligned with the three LuxRregulators of Pseudomonas sp. CMR12a.

3  | RESULTS

3.1 | Growth and production of sessilins and orfamides by CMR12a in shaken and still LB broth cultures

To quantify the production of sessilins and orfamides by CMR12a in shaking(150rpm)andstillLBbrothcultureconditions,filter-sterilizedsupernatantsandcellswerecollectedatvarioustimepoints,preparedandsubjectedtoLC-ESI-MSanalysis.Timepointschosen—17,20,24,and41hr—correspondedtothelateexponentialgrowthphase,earlystationarygrowthphase, stationarygrowthphase,anddeathphaseofCMR12a.AnalysesofrelativeCLPproduction(relativepeakarea/OD620)showedthatinbothcultureconditions,coproductionofsessil-insandorfamidesstartedat17hr(Figure2).Mostofthesessilinspro-ducedwere immediatelysecreted intothesupernatant,while loweramountswere kept inside (Figure2a and b). In contrast, orfamidesweremainlyretainedinthecells(Figure2c).Unlikesessilinsecretion,thesecretionoforfamidesintotheLBbrothoccurred7hrafterthestartofCLPproductioninbothcultureconditions(Figure2d).Aeratedcultures reached a higher biomass than still cultures (Figure2e). Ingeneral, aerationhadnostrongeffectonCLPproduction,althoughat24hritseemedthatmoreCLPswereretainedinsidethecellinstillconditions.

3.2 | Functional analysis of luxR- type regulatory genes in sessilins and orfamides biosynthesis

LC-ESI-MSanalysis revealed the completeabolishmentoforfamideand sessilin production in the ofaR1 and ofaR2mutants (Figure3a).However,themutantinthesesRgene,locatedupstreamofthesessilinbiosynthesiscluster,stillproducedsessilinsandorfamides.

Additionally, quantitative measurements (relative peak area/OD620) of the two CLPs did not reveal any difference betweenCMR12aandCMR12a-∆sesR(datanotshown).Restoredsessilinandorfamide production was observed in the complemented ofaR1 mu-tant,butnotinthecomplementedofaR2mutant(Figure3a).

Previous results showed that orfamides are important in theswarming motility of CMR12a and that sessilins and orfamides inter-act togiveawhite lineonKBmedium (D’aesetal.,2014). Inorderto ascertain the cessation of sessilin and orfamide production by the LuxRmutantsofCMR12a,swarmingmotilityandwhitelinetestswereconducted. Similar to CMR12a, the sesR mutant swarmed on 0.6%LBagar.However,ofaR1 and ofaR2mutantsdidnotexhibitswarming

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motility(Figure3b).Complementationofthemutantswitheachofthecorresponding target genes cloned into the stablevectorpME6032restored swarming motility in the ofaR1mutant,butnotintheofaR2

mutant.Thewhiteline-in-agarformationistypicalforCMR12awhenit interacts with an orfamide producer such as P. protegensPf-5andis indicative for sessilin production. In our study, CMR12a-∆ofaR1,

F IGURE  2 Quantificationofsessilinsandorfamidesproducedandsecretedbywild-typestrain,Pseudomonassp.CMR12a,instillandshakengrowthconditions.(a)Sessilinsincells,(b)sessilinsinsupernatants,(c)orfamidesincells,(d)orfamidesinsupernatants,(e)growthcurveofPseudomonassp.CMR12aovertimepoints.Bacteriaculturesweregrowninstillandshaking(150rpm)LBbrothconditionsat28°C.Ateachtimepoint,celldensitywasmeasuredspectrophotometrically(OD620)andmeanvaluesfromthreereplicateswererecorded.Timepointswererepresentative of different growth phases of Pseudomonassp.CMR12a.17h:lateexponentialgrowthphase;20h:earlystationarygrowthphase;24h:stationarygrowthphase;41h:deathphase.Forallgraphs,differentscaleswereusedtorepresentpeakarea/OD.Valuesaremeans± standard error (n=3)

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(c)

(d)

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CMR12a-∆ofaR2, and the complemented ofaR2 mutants no longer secrete sessilins, since they did not give the white line-in-agar in-teractionwhen challengedwith the orfamide producer,P. protegens

Pf-5.Thewhite line-in-agar phenotypewas observed, however, forCMR12a, CMR12a-∆sesR, and the complemented ofaR1 mutant strains(Figure3b).

F IGURE  3 CLPcharacterizationofCMR12a,LuxRmutantsandcomplementedstrains.(a)LC-ESI-MSchromatogramsofcell-freeculturesupernatantsofwild-typePseudomonassp.CMR12a,LuxRmutants,andcomplementedstrainsafter24hrofincubation.Wild-typeproducessessilins(analogs—A,B,andC)andorfamides(analogs—B,D,andE).CMR12a:wild-typePseudomonas sp. CMR12a; OfaR1: OfaR1 biosynthesis mutant; OfaR1+c: complement of OfaR1 biosynthesis mutant; OfaR2: OfaR2 biosynthesis mutant; OfaR2+c: complement of OfaR2 biosynthesis mutant;SesR:SesRbiosynthesismutant;SesR+c:complementofSesRbiosynthesismutant.(b)SwarmingabilityofCMR12aandLuxRmutantson0.6%LBagarandwhiteline-in-agartestsonKBagarmedium.Bacterialculturesweregrownfor17hrinLBbrothandwashedtwicewithsalinesolution(0.85%).Fivemicroliterofthesuspensionswasspottedinthecenteroftheplatesandincubatedat28°Cfor24hr.Forthewhitelinetest,thepicturewastaken3daysafterincubationat28°C.(1)CMR12a,wild-typePseudomonas sp.CMR12a;(2)CMR12a-ΔOfaR1,OfaR1biosynthesismutant;(3)CMR12a-ΔOfaR2,OfaR2biosynthesismutant;(4)CMR12a-ΔSesR,SesRbiosynthesismutant;(5)CMR12a-ΔOfaR1+c,ofaR1genecomplementofOfaR1biosynthesismutant;(6)CMR12a-ΔOfaR2+c,ofaR2genecomplementofOfaR2biosynthesismutant;(7)CMR12a-ΔSesR+c,sesR gene complement of SesR biosynthesis mutant

(a)

(b)

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3.3 | Transcriptional analysis of flanking and CLP biosynthesis genes in CMR12a and LuxR mutants

Figure1aandbshowprimerpositionsforRT-PCRonthesessilinandorfamidegeneclusters,respectively.Inordertoinvestigatethetran-scriptional analysis for ofaABC,sesABC,andtheirflankinggenes,RT-PCRwas conducted forCMR12aandLuxRmutantsusingbacterialcell cultureswhichweregrown in still LB cultures for24hr in tworeplicates(Figure4a–c).

For thesessilinbiosyntheticgenecluster,RT-PCRanalysisof theWT strain revealed the transcription of sesA,sesB,andflankinggenes,sesT, sesR, macA1, and macB1, whereas sesC was not transcribed. Additionally,thecoexpressionofsesT-sesR,sesA-sesB,sesB-sesC,sesC-macA1,andmacA1-macB1 gene combinations indicate that the sesT-sesR genes on one hand and the sesABC together with macA1B1 genes on theotherhandaretranscribedfromapolycistronicoperon(Figure4a).

In contrast, analysis of theCMR12a-∆ofaR1 mutant mainly revealed the transcription of sesT-sesR and macA1B1 genes.Furthermore, thismutantwascharacterizedbythepresenceofweaksesAB transcripts. For the CMR12a-∆ofaR2 mutant, similar transcription results as theCMR12a-∆ofaR1mutantwereobtained.Additionally,RT-PCRanalysesoftheCMR12a-∆sesRmutantrevealedsimilarresultsastheWTexceptfortheexpectedabsenceofsesT-sesR and sesRexpression.

Transcriptional analyses of the orfamide biosynthesis gene clus-ter were also conducted after growing bacterial cultures for 24 hr. In theWTstrain,ofaR1,ofaA,ofaB,ofaC,ofaR2,andthegenecombina-tions of ofaB-ofaCwereclearlytranscribed,whereasofaA-ofaB gave a weak transcript (Figure4b).Additionally, the transcriptionofmacA2 and macB2 and gene combinations of ofaC-macA2 and macA2-macB2 show that ofaABC and macA2B2 are also transcribed from a poly-cistronic operon. For the sesRmutant, expression and coexpressionanalyses of all genes and gene combinations showed similar results withCMR12a. Incontrast,theCMR12a-∆ofaR1 mutant showed the transcription of ofaA,macA2B2 and ofaR2 coupled with weak ofaB,andofaB–ofaCtranscripts.Moreso,CMR12a-∆ofaR2 mutant only showed ofaR1,ofaA,macA2B2,andweakofaBCtranscripts(Figure4b).

Furthermore, amutation in eitherof the threeLuxR-typegenesof CMR12a did not appear to abolish the transcription of the other (Figure4A and B). OfaR1 and ofaR2 mutants appeared to show a weaker transcription of the sesD (syrD-like) gene,whereas the sesR mutantshowedsimilarresultswithCMR12a(Figure4c).

3.4 | Phylogenetic analyses of LuxR- type regulatory proteins associated with CLP gene clusters

Phylogeneticanalysesof theCLPcluster-associatedLuxR-typepro-teins of CMR12a together with that of other Pseudomonas strains,showedseveraldistinctclusters(Figure5)asfollows:OfaR1andSesRproteinsclusteredtogetherwithotherLuxR-typeregulators locatedupstreamofCLPbiosynthesisgenes.Similarly,OfaR2clusteredwithLuxR-type regulators located downstream of the CLP biosynthesisgenes. Specifically, SesR clusteredwith other LuxR-type regulatorswithin the tolaasin group, while OfaR1 and OfaR2 clustered withregulatorswhichflankorfamide-codinggenes inotherPseudomonas strains including P. protegensPf-5 (Loper&Gross,2007).TheAHL-binding regulators of CMR12a, CmrR and PhzR, formed a separateclustertogetherwiththeLuxRofV. fischeri indicating that they belong toaseparatesubfamilyofregulators(Figure5).

3.5 | Presence of Rsm binding sites upstream of LuxR transcriptional regulators

Genomic search for putative Rsm binding sites was conductedwithin the sequences upstream of the three luxR regulatory genes of CMR12a.ConservedGGAmotifsupstreamof theATGstart codoncould be identified. Sequence alignment of these sequences with their homologsinCLP-producingPseudomonas strains showed the similar-ity of these regions upstream of sessilins and orfamide biosynthetic geneclusterswiththoseofpreviouslydescribedCLPs(Figure6).

F IGURE  4 RT-PCRanalysesforthesessilin(a)andorfamide(b)biosynthesisgeneclustersandflankinggenesinCMR12aandLuxRmutants,(c)sesD (syrD-like)geneassociatedwiththesessilinsgenecluster.Bacterialcellsanalyzedwerecollectedfrom24hrcultureofPseudomonassp.CMR12aanditsLuxRmutants.Foreachgenewithinthesessilinandorfamidegeneclusters,thesamebacterialculturewasanalyzedinduplicateandrepresentativeresultsareshownforoneexperiment.Agarosegelresultsareshownforanalysisofsinglegenestogetherwithgenecoexpressiontodistinguishmonocistronicandpolycistronictranscription.PrimersusedarelistedinTableS1and the amplicon positions are as indicated in Figures 1a and b

CMR12a

OfaR1

OfaR2

SesR

CMR12a

OfaR1

OfaR2

SesR

ofaR1 ofaAofaA-ofaB ofaB

ofaB-ofaC ofaC

ofaC-macA macA

macA-macB macB ofaR2

sesT sesT-sesR sesR sesA

sesA-sesB sesB

sesB-sesC sesC

sesC-macA macA

macA-macB macB

SesDCMR12a OfaR1 OfaR2 SesR

(a)

(b)

(c)

     |  9 of 12OLORUNLEKE Et aL.

F IGURE  5 PhylogeneticanalysisoftheLuxR-typeregulatorsflankingtheorfamideandsessilinbiosynthesisgenesofPseudomonas sp. CMR12a(highlightedingray).AlsoincludedinthisanalysisaretheLuxR-typeregulatorsofotherPseudomonasCLPbiosynthesisgenes,andAHL-bindingregulatorsLuxRfromVibrio fischeri,andPhzRandCmrRfromPseudomonas sp. CMR12a. The dendrogram was generated by maximumlikelihoodusing1,000resampleddatasets.Percentagebootstrapvaluesareindicatedatbranchingnodeswhilethebarindicatessequence divergence

Upstream of CLP biosynthesis genes

Downstream of CLP biosynthesis genes

AHL-binding regulators

P. putida BW11M1 XtlRP. entomophilia L48T EtlRP. putida PCL1445 PsoRP. putida RW10S2 WlpR

P. syringae pv. tomato DC3000 SyfRP. fluorescens Pf0-1 LuxRupPseudomonas sp. MIS38 ArfF

Pseudomonas sp. CMR12a OfaR1P. protegens Pf-5 LuxRupP. poae RE*1-1-14 LuxRupP. fluorescens SBW25 ViscARP. fluorescens SS101 MassARP. costantinii DSM 16734 LuxRPseudomonas sp. CMR12a SesR

P. tolaasii PMS117 LuxRP. syringae pv. syringae B301D SyrF

P. syringae pv. tomato DC3000 pspto2833P. putida PCL1445 LuxRdown

P. syringae pv. syringae B301D SalAP. poae RE*1-1-14 LuxRdownP. fluorescens SBW25 ViscBCR

P. fluorescens SS101 MassBCRP. fluorescens Pf0-1 LuxRdownPseudomonas sp. CMR12a OfaR2

P. protegens Pf-5 LuxRdownP. syringae pv. syringae B301D SyrG

Pseudomonas sp. CMR12a PhzRPseudomonas sp. CMR12a CmR

Vibrio fischeri LuxR

97

80

93

92

99

98

93

88

96

94

100

0.5

F IGURE  6 AlignmentoftheregionsupstreamoftheLuxRtranscriptionalregulatorygeneswhichflankdifferentlipopeptidebiosynthesisgene clusters including Pseudomonas fluorescensSS101,Pseudomonassp.MIS38,P. fluorescensPf0-1,P. protegensPf-5,P. putidaPCL1445,P. entomophiliaL48T,P. syringaepv.tomatoDC3000,andourstudystrainPseudomonassp.CMR12a.TheconservedGGAmotifishighlightedinred.ThetranslationinitiationATGcodonisindicatedatthe3′end,while*indicatesthesequencesoftheteststrainusedinthisstudy

10 of 12  |     OLORUNLEKE Et aL.

4  | DISCUSSION

Our study revealed that the LuxR-like transcriptional regulators,OfaR1 and OfaR2, which are associated with the orfamide genecluster not only regulate orfamide biosynthesis but also sessilin bio-synthesis,whilewecouldnotfindaclearfunctionfortheLuxR-likeregulator,SesR,associatedwiththesessilingenecluster.

LC-ESI-MSanalysisrevealedthatorfamideandsessilinproductioncommencesconcurrentlyinthelateexponentialphase,butorfamideis mainly retained inside the cell and secreted much later and in lower amounts than sessilin. We have previously shown that the release of orfamide in the environment is hampered by sessilin and hypothe-sized thatboth compounds compete for the sameoutermembraneefflux transporter,SesT (D’aesetal.,2014).Here,weshowthat thesesTgene,locatedupstreamofthesessilinbiosyntheticcluster,isex-pressed from an operon together with sesR.AmutationinsesR,how-ever, does not seem to affectCLPproductionor secretion.We arecurrently investigating the secretion of orfamides and sessilins in more detail by mutant analysis of putative transport genes including macAB,sesT,andsesD. Incontrast,ofaR1 and ofaR2 mutants completely lost the capacity to produce both sessilins and orfamides as evidenced by theabsenceofswarming,lackofawhiteline-in-agarphenotype,andconfirmedbyLC-ESI-MSanalysis.Also,inthebiocontrolstrainP. flu-orescensSBW25,mutationsintheLuxR-typeregulatorygenesviscAR and viscBCR,locatedup-anddownstreamoftheviscosinbiosynthesiscluster,ledtoalossofviscosinproduction(DeBruijn&Raaijmakers,2009a). Other homologs of ofaR1, located upstream of their NRPSgenes,havebeenshowntobenecessaryfortheproductionofputi-solvin (psoR) (Dubern etal., 2008), arthrofactin (arfF) (Washio etal.,2010),andentolysin(etlR)(Vallet-Gelyetal.,2010).

Sofar,coregulationofdifferentclassesofCLPsinthesamestrainhas only been demonstrated for plant pathogenic Pseudomonas bacte-ria. In the bean pathogen P. syringae pv. syringaeB728a,threeLuxR-likeproteins,SalA,Syrf,andSyrG,wereshowntocontrolthebiosynthe-sis of the CLPs syringopeptin and syringomycin (Vaughn & Gross,2016).SalAcontrolstheexpressionofbothsyrG and syrF(Lu,Scholz-Schroeder,&Gross,2002).Furthermore,qRT-PCRanalysisofdeletionmutants in syrF and syrG showed that both genes require a functional salAgeneforactivation. Inaddition,SyrGappearstofunctionasanupstream transcriptional activator of syrF(Vaughn&Gross,2016).Thesituation in Pseudomonas sp. CMR12a is different since a mutation in either ofaR1,ofaR2, or sesR did not abolish the transcription of the other,althoughthetranscriptofofaR1 may seem weaker in the ofaR2 mutant. Our method does not allow precise transcript quantification andfurtherinvestigationbyquantitativeRT-PCRisneeded.Likewiseintheviscosinproducingstrain—P. fluorescensSBW25,amutationineither viscAR or viscBCR, luxRgenes locatedup-anddownstreamofthe viscABCbiosynthesisgenes,didnotsubstantiallyaffectthetran-scriptionoftheother(DeBruijn&Raaijmakers,2009a)indicatingthatbothLuxRregulatorsdonottranscriptionallyaffecteachother.

Transcriptional analyses showed that for both the sessilin and or-famide gene clusters, their biosynthesis genes, sesABC and ofaABC,together with putative transport genes, macAB, are most likely

transcribedfromapolycistronicoperon,whichisprobablyregulatedby OfaR1 and OfaR2. The absence of a sesC transcript in CMR12a couldbeduetoprimerspecificityproblemssinceacoexpressionwasobserved for sesB-sesC and sesC-macA1. With respect to the orfamide genecluster,worthyofnotewasthefactthatmutantsinofaR1 and ofaR2 still showed clear transcripts for ofaA and macA2B2 genes. These results indicate that besides the single promoter which enables the transcription of ofaABC and macA2B2genes,separatepromotersfor ofaA and macA2B2maybepresent,whicharenotcontrolledbyOfaR1andOfaR2.Unfortunately,littleinformationisavailableaboutthe gene coexpression of otherCLP gene clusters except forWLIP(Rokni-Zadehetal.,2012),sowecouldnotascertainifthepresenceof multiple promoters as was observed in the orfamide gene cluster is afrequentoccurrence. Inthisrespect, it is interestingtonoticethatin beneficial Pseudomonasspp.,thegenomicregionencodingthefirstCLPbiosynthesisgene isoftenunlinkedwith theother twobiosyn-thesisgenes,whicharecoexpressed.Thisisforinstancethecaseforviscosin, massetolide, WLIP, xantholysin, entolysin, and poaeamide(DeBruijnetal.,2007,2008;Lietal.,2013;Rokni-Zadehetal.,2012;Vallet-Gelyetal.,2010;Zachowetal.,2015).

Duringthisstudy,wewereunabletocomplementtheCMR12a-∆ofaR2 mutant. Considering the fact that the macB2 gene associated with the orfamide gene cluster gave a weaker transcript than macA2 forCMR12a,itispossiblethatofaR2 is transcribed from a longer tran-script which spans across part of the macB2 gene. This would result inanantisenseoverlapthatcouldinfluencetheexpressionofmacB2 by transcription attenuation (Sesto,Wurtzel,Archambaud, Sorek, &Cossart,2012).Thisobviouslyrequiresfurtherinvestigation.

In our study, phylogenetic analysis of LuxR-type proteins, po-sitionedup- anddownstreamof theCLP gene clusters ofCMR12atogetherwithpreviouslydescribedCLP-associatedLuxRregulatorsre-vealedthatOfaR1andSesRclusteredtogetherwithknownLuxR-typeregulatorslocatedupstreamoftheCLPbiosynthesisgenes,whereasOfaR2clusteredwiththoselocateddownstream.LuxRregulatorsfromstrainswhichproducesimilarCLPs,forexample,orfamideproducersP. protegensPf-5andPseudomonassp.CMR12a,clustertogether.Anex-ceptionistheLuxRregulatorforpoaeamide,P. poaeRE*1-1-14whichalthoughsharesastructuralrelationshipwithorfamide(Zachowetal.,2015),clusterswithLuxRregulatorsofCLPsbelongingtotheviscosinfamily.TheLuxRregulator (WipR)oftheWLIPproducer—P. reactans LMG5329,showedahigherhomologywithLuxRregulatorsofthevis-cosinfamilycomparedwiththatofanotherWLIPproducer—P. putida RW10S2 (Rokni-Zadeh etal., 2013). This decreased conservationsuggests that the biosynthetic gene cluster of poaeamide might have evolvedseparately.OurresultsfurtherindicatethatLuxR-typeregu-lators of CMR12a belong to the same subfamily as in other plant ben-eficial Pseudomonas strains including P. protegensPf-5,P. fluorescens SS101, andP. fluorescens SBW25,which produce orfamide,masse-tolide,andviscosin,respectively(DeBruijn&Raaijmakers,2009a;DeBruijnetal.,2008;Loper&Gross,2007).GiventhatLuxRtranscrip-tional regulators of P. syringae pv. syringaeclusterwithallLuxRregula-torsanalyzedduringthisstudy,ourresultsindicatethatsimilartothisplantpathogenicstrain,theseotherLuxRregulators,includingOfaR1,

     |  11 of 12OLORUNLEKE Et aL.

OfaR2,andSesR,belongtothefourthLuxRfamilywhich ischarac-terizedbytheabsenceofanydefinedN-terminaldomain(Vaughn&Gross,2016).

Duringthisstudy,agenomicsearchandsubsequentalignmentofsequences upstream of ofaR1,ofaR2,andsesR with their homologs in other lipopeptide biosynthesis genes of Pseudomonasstrains,showedthat Rsm binding sites were located upstream of all three luxR-likegenesofCMR12a.GiventhefactthatthisRsmbindingsite,alterna-tivelycalledtheGacAbox,wasfoundupstreamofmultipleCLPbio-synthesisgenes(Song,Voort,etal.,2015a)indifferentPseudomonas strains, our results suggest that the Gac/Rsm-mediated regulationof CLPs might be a general phenomenon in most biocontrol CLP-producing Pseudomonas spp.

In conclusion, this study establishes that sessilin and orfamideproduction in CMR12a are coregulated by two of the three luxR-typegenes namely ofaR1 and ofaR2. Our findings show that either OfaR1 orOfaR2canregulatethebiosynthesisofthesetwoCLPs,whilethefunction of SesR remains unclear.

ACKNOWLEDGMENTS

Feyisara Eyiwumi Olorunleke is funded by the Schlumberger Faculty for the Future Fellowship. M. Ongena is a research associate at the National Funds for Scientific Research in Belgium. This work was fundedbytheINTERREGIVprogrammeFrance-Wallonie-Vlaanderen(Phytobio project) and by a grant from the Research FoundationFlanders (FWO project 3G.002.10N). The authors thank LaurentFranzilofGemblouxAgro-BiotechfortheLC-ESI-MSinjections.

CONFLICT OF INTEREST

None declared.

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How to cite this article:OlorunlekeFE,KieuNP,WaeleED,TimmermanM,OngenaM,HöfteM.Coregulationofthecycliclipopeptides orfamide and sessilin in the biocontrol strain Pseudomonas sp. CMR12a. MicrobiologyOpen. 2017;e499. https://doi.org/10.1002/mbo3.499