ApnI, a Transmembrane Protein Responsible for Subtilomycin Immunity, Unveils a Novel Model for

13 Pages·2014·2.97 MB·English

ApnI, a Transmembrane Protein Responsible for Subtilomycin
Immunity, Unveils a Novel Model for Lantibiotic Immunity
YunDeng,Cong-ZhiLi,Yi-GuangZhu,Peng-XiaWang,Qing-DongQi,Jing-JingFu,Dong-HaiPeng,Li-FangRuan,MingSun
StateKeyLaboratoryofAgriculturalMicrobiology,CollegeofLifeScienceandTechnology,HuazhongAgriculturalUniversity,Wuhan,People’sRepublicofChina
SubtilomycinwasdetectedfromtheplantendophyticstrainBacillussubtilisBSn5andwasfirstreportedfromB.subtilis
strainMMA7.Inthisstudy,ageneclusterthathasbeenproposedtoberelatedtosubtilomycinbiosynthesiswasisolatedfrom
theBSn5genomeandwasexperimentallyvalidatedbygeneinactivationandheterologousexpression.Comparisonofthe
subtilomycingeneclusterwithotherverifiedrelatedlantibioticgeneclustersrevealedaparticularorganizationofthegenesapnI
andapnTdownstreamofapnAPBC,whichmaybeinvolvedinsubtilomycinimmunity.Throughanalysisofexpressionofthe
D
apnIand/orapnTgenesinthesubtilomycin-sensitivestrainCU1065andinactivationofapnIandapnTintheproducerstrain
o
BSn5,weshowedthatthesinglegeneapnI,encodingaputativetransmembraneprotein,wasresponsibleforsubtilomycinim- w
munity.Toourknowledge,evidenceforlantibioticimmunitythatissolelydependentonatransmembraneproteinisquiterare. n
lo
FurtherbioinformaticanalysisrevealedtheabundantpresenceofApnI-likeproteinsthatmayberesponsibleforlantibioticim- a
munityinBacillusandPaenibacillus.WeclonedthepaeIgene,encodingonesuchApnI-likeprotein,intoCU1065andshowed d
e
thatitconfersresistancetopaenibacillin.However,nocross-resistancewasdetectedbetweenApnIandPaeI,eventhoughsubti- d

lomycinandpaenibacillinsharesimilarstructures,suggestingthattheprotectionprovidedbyApnI/ApnI-likeproteinsinvolves fr
o
aspecific-sequencerecognitionmechanism.Peptiderelease/bindingassaysindicatedthattherecombinantB.subtilis m
expressingapnIinteractedwithsubtilomycin.Thus,ApnIrepresentsanovelmodelforlantibioticimmunitythatappearstobe h
common. tt
p
:
/
/
a
BacillussubtilisisaGram-positive,endospore-formingmodel Alantibioticproducermustpossessoneortwosynergisticim- e
m
bacterium. In general, B. subtilis grows in association with munity mechanisms to protect itself against its own produced .
a
plantsandpromotesplantgrowth(1).However,arecentreport lantibiotics.Thereportedimmunitymechanismsusuallyinvolve s
m
revealed that B. subtilis is also a saprophytic organism in other immunityproteins,includingABCtransporters(LanFE[G]),li-
.
environments, such as in an animal’s gastrointestinal tract (2). poproteinsormembrane-associatedpeptides(LanI),andtrans- o
r
DozensofantibioticsandenzymesproducedbyvariousB.subtilis membraneproteins(LanH)(8).Forlantibioticimmunity,ABC g
/

isolatesareusedinthefoodindustryandagriculturalfields.Be- transportersexportlantibioticstotheextracellularspacebyATP o
n
causeofitsabilitytotakeupDNAandintegrateitintoitsgenome, hydrolysis,andinsomecases,thetransmembraneproteinLanH
A
i.e.,naturalcompetence(3),itsgeneticsequenceshavebeenwell actsasanaccessoryproteinfortheABCtransportersinvolvedin p
characterized,includingthoserelatedtothebiosynthesisandreg- substraterecognition(9,10).Inaddition,lipoproteinsormem- ril
ulationmodeofmostoftheantibioticsdiscoveredinthisspecies brane-associatedproteins(LanI)canworkeithersolelyorsyner- 4
,
todate. gistically with ABC transporters by interacting with lantibiotics 2
B.subtilisisalsoknowntoproducelantibiotics(4),whichare (11,12).MostoftheLanIproteinsthusfarreportedarelocatedon 0
1
lanthipeptideswithantimicrobialactivity(5).Lanthipeptidesare theexternalsurfaceofthemembrane,suchasNisI,SpaI,andPepI. 9

aclassofribosomallysynthesizedandposttranslationallymodi- VeryfewLanIproteins,especiallythosesolelyprovidingimmu- b
y
fiedpeptides(6),whicharealsoreferredtoaslanthionine(Lan)or nity,arelocatedwithinthecellmembrane.
g
3-methyl-lanthionin (MeLan)-containing peptides. The ring- SubtilomycinisarecentlyidentifiedlantibioticproducedbyB. u
e
formingLanandMeLanpeptidesareusuallygeneratedviaatwo- subtilisstrainMMA7isolatedfromthemarinespongeHaliclona s
t
stepmodificationprocess,involvingdehydrationofthethreonine simulans,anditshowsabroadspectrumofactivityagainstGram-
(Thr) and/or serine (Ser) in the precursor peptide to form the positive bacteria, including drug-resistant pathogens (13). The
doublebonds,followedbycyclizationmediatedbyaMichaelad- relationshipbetweensubtilomycinanditsgeneclusterwasestab-
ditionwiththethiolbondofcysteine(Cys)inthepropersite.This lishedbasedontheclosematchbetweentheN-terminalamino
processinvolvesthedehydrataseandcyclase,whichare,respec-
tively,encodedbyeithertwodependentgenes,lanBandlanC,ora
combined gene, lanM. Lanthipeptides are subdivided into four Received9July2014 Accepted29July2014
classes (I to IV) depending on the specific biosynthetic gene(s) Publishedaheadofprint1August2014
involved.Structurallyestablishedlantibioticscanbefurthersub- Editor:H.L.Drake
dividedbasedonsequencesimilarity,suchasthePep5group(7). AddresscorrespondencetoMingSun,[email protected].
ClassIlantibioticsaresynthesizedastheprecursorpeptideLanA Supplementalmaterialforthisarticlemaybefoundathttp://dx.doi.org/10.1128
andaremodifiedbytwodifferentenzymes,LanBenzyme(dehy- /AEM.02280-14.
dratase)andLanCenzyme(cyclase),andareexportedbyLanT, Copyright©2014,AmericanSocietyforMicrobiology.AllRightsReserved.
and then the leading peptides are removed by the extracellular doi:10.1128/AEM.02280-14
serineproteaseLanP.
October2014 Volume80 Number20 AppliedandEnvironmentalMicrobiology p.6303–6315 aem.asm.org 6303
Dengetal.
acidsequenceandthe5=-endsequenceofthestructuralgenesubA. TABLE1Bacterialstrainsandplasmidsusedinthisstudy
PaenibacillinisalantibioticproducedbythestrainPaenibacillus
Sourceor
polymyxaOSY-DF(14),anditsstructurehasbeenelucidated(15); Strainorplasmid Description reference
however, the paenibacillin biosynthetic gene cluster has not yet E.coliDH5(cid:3) Cloninghost 42
beenreported.Sinceitshowshighsequencesimilarity(54%)to
Paenibacillus Wildtype;paenibacillinproducerstrain 28
subtilomycin, the structure of subtilomycin has been proposed ATCC842
usingthestructureofpaenibacillinasatemplate(13).However, B.subtilis
no molecular biological evidence has been provided to validate BSn5 Wildtype;subtilomycinproducer 16
thisproposedbiosyntheticgenecluster. B1201((cid:6)apnB) StrainBSn5derivative;(cid:6)apnB::spc Thisstudy
Inthisreport,weprovidedirectevidencetoconfirmthepres- B1202((cid:6)apnT) StrainBSn5derivative;(cid:6)apnT::kan Thisstudy
B1203((cid:6)ybcL) StrainBSn5derivative;(cid:6)ybcL::kan Thisstudy
enceofasubtilomycingenecluster,termedtheapncluster,inthe
B1204((cid:6)ybdG) StrainBSn5derivative;(cid:6)ybdG::kan Thisstudy
completegenomeofB.subtilisstrainBSn5,whichwaspreviously B1205((cid:6)apnI) StrainBSn5derivative;(cid:6)apnI::kan Thisstudy
isolated from the plant Amorphophallus konjac (16). We com- CU1065 Strain168derivative;trpC2attSP(cid:2) 17
paredthegeneorganizationoftheapnclusterwiththoseofother B1301 CU1065amyE::(spcsubtilomycingene Thisstudy
related gene clusters to determine its specificity and potential cluster) D
function.DNAfragmentscontainingdifferentregionsofthegene B1302 CU1065amyE::(spcfragmentapnI) Thisstudy o
w
clusterwereexpressedintheheterologoushostB.subtilisstrain B1303 CU1065amyE::(spcfragmentapnT) Thisstudy n
CU1065(W168attSP(cid:2)trpC2)(17),andgeneinactivationanalysis B1304 CU1065amyE::(spcfragmentapnI-apnT) Thisstudy lo
B1305 CU1065amyE::(spcfragmentpro apnI) Thisstudy a
wasperformedfortheproducerBSn5straintoidentifythegene(s) apnA d
involvedinconferringimmunity.Ourstudythusprovidesvalu- B1306 CU1065amyE::(spcfragmentproapnAapnT) Thisstudy e
B1307 CU1065amyE::(spcfragmentpro Thisstudy d
ableinformationofararemolecularmodelunderlyinglantibiotic apnA
apnI-apnT) fr
immunity. o
B1308 CU1065amyE::(spcfragmentproapnApaeI) Thisstudy m
B1309 CU1065amyE::(spcfragmentpropaeApaeI) Thisstudy h
MATERIALSANDMETHODS t
Plasmids tp
Bacterialstrains,plasmids,andgeneralmethods.Thestrainsandplas- pDG780 pBluescriptKS(cid:7)harboringkan(ampkan) 43 :/
midsusedinthisstudyarelistedinTable1.B.subtilisstrainsBSn5and pMD18T ClonevectorforE.coli(amp) TaKaRa /a
CU1065,alongwiththeirderivatives,wereculturedinLuria-Bertani(LB) e
simple m
mediumat28°C,andEscherichiacoliDH5(cid:3),whichwasusedasahostfor pIC333 Mini-Tn10transposonsdeliveryvector 44 .
a
clones,wasroutinelyculturedinLBmedium(Invitrogen,Karlsruhe,Ger- harboringspcanderm s
many) at 37°C. For solid culture, LB medium was supplemented with pDG1730 IntegrationvectorharboringamyE::spc 45 m
1.5%agar.Forstockpreparation,theculturewascultivatedovernightat (amperm) .o
28°CinLBmediummixedwithsterileglycerol(finalconcentrationof pB1201 pMD18Tharboringmutationof(cid:6)apnB::spc Thisstudy rg
17%)andstoredat(cid:4)80°C.Forselectivemedia,thefollowingantibiotics pB1202 pDG780harboringmutationof(cid:6)apnT::kan Thisstudy /
wereadded:ampicillin,100(cid:5)g/ml(E.coli);spectinomycin,100(cid:5)g/ml(B. pB1203 pDG780harboringmutationof(cid:6)ybcL::kan Thisstudy on
subtilis);andkanamycin,20(cid:5)g/ml(B.subtilis).Isolationofplasmidsand pB1204 pDG780harboringmutationof(cid:6)ybdG::kan Thisstudy A
chromosomalDNA,restrictionendonucleasedigestion,agarosegelelec- pB1205 pDG780harboringmutationof(cid:6)apnI::kan Thisstudy p
r
trophoresis,PCR,andtransformationofE.coliwereperformedbyfollow- pB1301 pDG1730harboringamyE::(spcapngene Thisstudy il
ingestablishedprotocolsformolecularbiologytechniquesdescribedby cluster) 4
,
SambrookandRussell(18). pB1302 pDG1730harboringamyE::(spcapnI) Thisstudy 2
PlasmidconstructionandmanipulationofB.subtilisstrains.Gene pB1303 pDG1730harboringamyE::(spcapnT) Thisstudy 0
1
disruptionplasmidswereconstructedusingtwoapproaches.Thefirstwas pB1304 pDG1730harboringamyE::(spcapnI-apnT) Thisstudy 9
establishmentoftheapnBmutantallele(apnB::spc)bysubcloningofthe pB1305 pDG1730harboringamyE::(spcproapnA Thisstudy b
spectinomycin resistance gene (spc) from pIC333 into the PCR-intro- apnI) y

ducedClaIandXbaIsitesoftheapnBgeneinpMD18T-apnB,resultingin pB1306 pDG1730harboringamyE::(spcpro Thisstudy g
apnA u
plasmidpB1201(Table1).Thesecondapproachwastheestablishmentof apnT) e
apnT,ybcL,ybdG,andapnImutantalleles(apnT::kan,ybcL::kan,ybdG:: pB1307 pDG1730harboringamyE::(spcpro Thisstudy s
apnA t
kan,andapnI::kan,respectively)byamplifyingtheupstreamanddown- apnI-T)
streamarmsofeachgenefragmentintotheupstreamanddownstream pB1308 pDG1730harboringamyE::(spcpro paeI) Thisstudy
apnA
restrictionsitesoftheintegrationvectorpDG780,respectively,resulting pB1309 pDG1730harboringamyE::(spcpro paeI) Thisstudy
paeA
inplasmidspB1202,pB1203,pB1204,andpB1205(Table1).TheB.sub-
tiliscompetentcellpreparationwasperformedbyfollowingtheclassical
nutritionaldownshiftmethoddescribedbyAnagnostopoulosandSpiz-
izenin1960(3),withmodificationsdescribedbyYasbinetal.(19).A resistanceevaluation.ProperallelicreplacementwasconfirmedbyPCR
seriesofatleastfive10-minintervalgradesweresettoensurethatthet0 andsequencing.
point(i.e.,thepointatwhichthecultureleavesthelogarithmicgrowth Differentfragmentscontainingthewholesubtilomycingenecluster,
phase)couldbecapturedateverycycleconsideringthelowertransforma- apnI,apnT,orapnI-apnTwereamplifiedusingtheprimerslistedinTable
tionefficiencyofwild-typestrainBSn5thanofB.subtilis168.Chromo- 2.Then,thesefragmentswerecutusingproperrestrictionendonuclease
somalgenereplacementofwild-typeapnB,apnT,ybcL,andybdGwiththe andclonedintoanotherintegrationvector,pDG1730,resultinginthe
correspondingconstructedmutationalleleswascarriedoutbytransform- heterologous expression alleles amyE::(spc subtilomycin gene cluster),
ingBSn5withtheconstructedplasmids,resultinginthemutantsB1201, amyE::(spcfragmentapnI),amyE::(spcfragmentapnT),andamyE::(spc
B1202,B1203,B1204,andB1205.B1201wasselectedforspectinomycin fragmentapnI-apnT)inplasmidspB1301,pB1302,pB1303,andpB1304,
resistanceevaluation,andtheothermutantswereselectedforkanamycin respectively.Thefragmentcontainingthepredictedpromoterregionof
6304 aem.asm.org AppliedandEnvironmentalMicrobiology
SubtilomycinGeneandLantibioticImmunity
TABLE2Primersequencesusedinthisstudy
Primer Sequence(5=¡3=)a Use
ybcLUpF CTAACTAGTCAGAGATCAGTAAGTCC ybcLgenedisruption
ybcLUpR TGTCTGCAGAGGTTCCAACTAAAAAGC ybcLgenedisruption
ybcLDownF GGACTCGAGAGTTTGGTTGGATCAAAG ybcLgenedisruption
ybcLDownR TGAGGTACCCTCAGTAATCTTATTAGCC ybcLgenedisruption
ApnBUpF TTTGCATGCTTAGTATTCTCGAGCAG apnBgenedisruption
ApnBDownR TGCGGATCCATTAAATAGGGATAGTG apnBgenedisruption
ApnBUpR CCTTCGTATCCTATTACTCGCATTT apnBgenedisruption
ApnBDownF AGAATCTAGAGCAATCACCTATTCTAAGAGC apnBgenedisruption
SpcF AGTATCGATGCGGTGCTACAGAGTTCTTG Amplificationofspectinomycinresistancegene
SpcR GGGTCTAGAGTAAACGCTGAATATCGTGTT Amplificationofspectinomycinresistancegene
ApnTUpF TTTGGATCCGGTTGTTGAAGACGCAG apnTgenedisruption
ApnTDownR TCCTTTCCCATACTCTTC apnTgenedisruption
ApnTDownF CAACTCGAGCGAGTTCTCTAAGTTA apnTgenedisruption
ApnTDownR TTTGGTACCTGGTCAGAGACCGCATCT apnTgenedisruption D
YbdGUpF GGGACTAGTTCGAGATAG ybdGgenedisruption o
w
YbdGUpR CACCTGCAGATTATATACAGCGGAAG ybdGgenedisruption n
YbdGDownF TATCTCGAGGGTTCGCTAGGATGAAGG ybdGgenedisruption lo
YbdGDownR TGAGGTACCGGTTTAAACATGCTGTC ybdGgenedisruption a
d
ApnIUpF GAAACTAGTTCTAGAGGCGGAACAAGAT apnIgenedisruption e
ApnIDownR GGGGAATTCATGAAAAGAACTGCGAACC apnIgenedisruption d

ApnIDownF TAACTCGAGAGGTTATACATATGGGC apnIgenedisruption fr
o
ApnIDownR GCAACAAAGGTACCTGGTGAAATAG apnIgenedisruption m
apnF CCTGGATCCGCTCTGTTTCCGTTGTCG Cloneofapncluster
h
apnR TGAGGATCCTACCCAAGTGGTCTCATTATTT Cloneofapncluster t
t
ApnIF TTCGGATCCGGTCAAGATTCGTGGGAC CloneoffragmentapnI-apnT(I) p
:
ApnIR GGCGAATTCGTTATTGCCCATATGTATAAC CloneoffragmentapnI //
a
ApnTF AGTGGATCCTAGCGAAAGTGTTCATTC CloneoffragmentapnT e
ApnTR GACGAATTCAAGCAAAACCAAAGCAGCC CloneoffragmentapnI-apnT(T) m
ProapnAF TTTGGATCCGCGAGCTGAAGTACAGTACG CloneoftheapnApromoterregion .a
ProapnAR CTTGGATCCTTCCATTTCCTCCTTTT CloneoftheapnApromoterregion s
m
ProapnAF2 TTTGGATCCGCGAGCTGAAGTACAGTACG CloneoftheapnApromoterregion
.
ProapnAR2 CTTGAATTCCTTAGCTCGAGTTCCATTTCCTCCTTTT CloneoftheapnApromoterregion o
r
PropaeAF AACGGATCCATCGGGACCAAAGTGATTCGACTCA CloneofthepaeApromoterregion g
/
PropaeAR CTTGAATTCTGCCTCTCGAGTTTCCTCTTCCTCTCTGAATTAAAT CloneofthepaeApromoterregion
o
PaeIF TTTGGATCCCTTAGCTCGAGCAGATGCTGGTATTTAGCT CloneofthepaeIgene n

PaeIR CTCCGAATTCGAATGGGCCGTTTGTTTT CloneofthepaeIgene A
p
aRestrictionsitesareunderlined. r
il
4
,

apnA(pro )wasamplifiedwithprimerscontainingBamHIrestriction intoa500-mlflaskcontaining100mlofLBmediumattheratioof1/100 2
apnA 0
sitesandclonedintoalloftheconstructedvectors,exceptforthatcon- (vol/vol).Theflaskwasincubatedat28°Cfor12hinarotaryshakerwith 1
tainingthewholeapncluster.PCRwasusedtoidentifytheinserteddirec- agitationat200rpm.Aftercentrifugationat10,000(cid:8)gfor10minat4°C 9

tionofthepro fragment.Thesameprocedureasdescribedabovewas usingthecentrifugeSorvallST16R(ThermoFisherScientific;USA),the b
usedtopreparaepnthAecompetentcellsofB.subtilisCU1065.Theheterolo- cell-free supernatant was filtered through a 0.22-(cid:5)m membrane filter. y
g
gousexpressionrecombinantswereselectedforspectinomycinresistance Ammoniumsulfatewasaddedintermittentlytothecell-freesupernatant u
andwereconfirmedbyPCRandsequencing. to30%saturationduringstirringusingamagneticstirrer.Themixture e
s
To express the gene paeI for putative paenibacillin immunity in wassetat4°Cfor2h.Theresultingprecipitatewascollectedbycentrifu- t
CU1065,thefragmentscontainingpaeIandthepredictedpromoterre- gationat12,000(cid:8)gfor30minat4°C,followedbydialyzationdesalina-
gionsofapnAandpaeA(pro andpro ,respectively)wereamplified tion.Theextractwasusedastheactiveammoniumsulfatecrudeextract
apnA paeA
usingtheprimerslistedinTable2andwereT/AclonedintothepMD18T (ASCE)ofthesubtilomycinpreparationforfurtherbioassays,high-per-
Simplevector.Then,thepaeIfragmentswerecutandinsertedintothe formanceliquidchromatography(HPLC),andmatrix-assistedlaserde-
downstreamregionsofthepro andpro fragments,resultingin sorptionionization–timeofflight(MALDI-TOF)massspectrum(MS)
apnA paeA
insertion of pro paeI and pro -paeI, respectively, in the vector analysis.ThesubtilomycinextractsfromBSn5mutantswerepreparedby
apnA- paeA
pMD18TSimple.Finally,thetwofragmentswerecutusingBamHIand followingthesameprotocol.
EcoRIandclonedintotheintegrationvectorpDG1730,resultinginthe Crudesubtilomycinextractswerecondensedbyfilteringthrougha
heterologous expression alleles amyE::(spc fragment pro paeI) and MilliporeAmiconUltra-153Kdeviceandadjustedto25%acetonitrile
apnA-
amyE::(spcfragmentpro -paeI)inplasmidspB1308andpB1309(Ta- (0.1%trifluoroaceticacid[TFA]),followedbycentrifugationat12,000(cid:8)
paeA
ble1).TheB.subtilisCU1065transformationandtheconfirmationof gfor5mintoremoveinsolublematerial.Subtilomycinwaspurifiedby
recombinants were performed by following the procedures described preparativeHPLCusingaWaters1528binaryHPLCpump,Waters2489
above. UV/visibledetector,andWaters2707auto-samplesystemequippedwith
Isolationandpurificationofsubtilomycin.Topreparesubtilomycin, anAgilentEclipseXDB-C18PrepHTcolumn(5(cid:5)m,21.2by150mm).
asinglecolonyofBSn5wasinoculatedfromthecultureplateinto5mlof Themobilephaseconsistedofchromatography-gradewatercontaining
LBmediumwithagitation,andthentheovernightculturewastransferred 0.1%trifluoroaceticacid(TFA)inpumpAand80%chromatography-
October2014 Volume80 Number20 aem.asm.org 6305
Dengetal.
gradeacetonitrilecontaining0.086%TFAinpumpB.Theelutionstep Basedonapreviouslyreportedisolationanddetectionstrategy(14),we
wasperformedunderanacetonitrilegradientof30to70%over20minat aimed to detect and prepare crude extracts of paenibacillin. Briefly, a
aflowrateof9.0ml/min.Subtilomycinwaselutedwithin14.3min(see singlecolonyofATCC842wasactivatedin5mloftrypticsoybrothyeast
Fig.S4inthesupplementalmaterial).Thepurifiedpeptidewaslyophi- extractandincubatedat28°Cfor24h.Theresultingculturewasinocu-
lizedandusedforsubtilomycinquantificationbyHPLC. lated into a 500-ml flask containing 100 ml of tryptic soy broth yeast
Bioassayfordetectionofsubtilomycin.Agarwelldiffusionassays extract.Theflaskwasincubatedat28°Cfor24hinarotaryshakerwith
wereusedtodetecttheantimicrobialactivitiesoftheisolatesaspreviously agitationat200rpm.Aftercentrifugationat9000(cid:8)gfor10min,the
described(20).ThirtymillilitersofLBagar(1.0%,wt/vol)mediumwas cell-freesupernatantwasmixedwithAmberliteXAD-7resin(Sigma)ata
pouredinto100-by100-mm2plasticdishesandinoculated(1%,vol/vol) 10%(wt/vol)ratio,andthemixturewassetforstaticadsorptionovernight
withtheindicatorsuspension.Afterdryingfor30min,nine6-mmwells at4°C.Theabsorbedpaenibacillinresinwaswashedsequentiallywith1
wereboredineachplate.Approximately60(cid:5)lofcrudeextractsamples literofdistilledwaterand500mlof30%(vol/vol)ethanol.Finally,the
fromthesupernatantsofB.subtilisstrainswaspipettedintoeachwell,and resin was eluted with 250 ml of 75% (vol/vol) ethanol (pH 2.0). The
theplatesweremaintainedat4°Cfor4hfordiffusionandthenincubated fractionwascondensedwitharotaryevaporatorat35°Cundervacuum,
overnightat28°C.Thediameteroftheinhibitionzoneswasmeasuredto andtheconcentratewasadjustedtopH7.0withphosphatebufferand
determineantimicrobialactivity. thenwasfreeze-dried.Thepowderwasresolvedindistilledwater,fol-
Detection of subtilomycin by HPLC and liquid chromatography lowedbycentrifugationat12,000(cid:8)gfor5min.Theresultingsupernatant
D
(LC)-MS.ThesameHPLCsystemasdescribedaboveforpurificationwas wasconsideredtobethepaenibacillincrudeextractandwasusedtodetect o
usedforsubtilomycindetection.Thedataweredisplayedandanalyzedby paenibacillinunderLC-MSbyfollowingthesameprocedureasdescribed w
usingaBreezesystem(WatersCorporation,USA).AnAgilentHC-C18 aboveforsubtilomycindetection. n
reverse-phasecolumn(250by4.6mm;particlesizeof5(cid:5)m)wasem- Subtilomycinsensitivityassay.Thesubtilomycinsensitivityassayof lo
a
ployed(Agilent,USA).Themobilephaseconsistedofchromatography- B.subtilisCU1065andrecombinantswasperformedusingagardiffusion d
testsaccordingtoamodificationofapreviouslydescribedprotocol(21). e
gradewatercontaining0.1%TFAinpumpAandchromatography-grade d
acetonitrileinpumpB.Thecrudelantibioticpreparationwasdissolvedin Stationary-phase-grownCU1065anditsrecombinantcultureswithan f
20%acetonitrile(TFA,0.1%),and5to20(cid:5)lwasloaded.Theelutionstep opticaldensityat600nm(OD600)of0.8wereeachinoculatedat1/1,000 ro
intodilutedLBagar(1.0%,wt/vol)at40°Candthenpouredinto90-mm m
wasperformedunderanacetonitrilegradientof20to80%over15minat
petridishes(20ml)anddriedfor30min.Seven6-mmwellswerebored
aflowrateof1ml/min.Thepeaksweredetectedbymeasuringtheabsor- h
intoeachplate.Seriallydiluted(0.125to200(cid:5)M)subtilomycinextracts t
banceat235nmand254nm. (60(cid:5)l)wereloadedintothewellsoftestplates.Theplatesweremain- tp
Liquidchromatographyquadrupole-timeofflight(LC-Q-TOF)MS :/
tained at 4°C fo r 4 h toallow sufficient diffusion and then incubated /
was carried out using an Agilent 1260 LC device equipped with a C a
reverse-phasecolumn(100by1.8mm;particlesizeof3.5(cid:5)m)unde18r overnightat28°C.Theresponsewascalculatedasthemeanareaofthe em
inhibitionhalos.Eachtestwasindependentlyreplicatedthreetimes.The
chromatographydetectionconditionsastheflowratewasdecreasedby .
0.3ml/min;theinjectionvolumewas1(cid:5)l,andthediodearraydetection sameprocedurewasemployedforvancomycin,nisin,andpaenibacillin as
was performed at 254 nm. MS was performed using the Q-TOF MS sensitivitytestsofB.subtilisCU1065andtheapnI-expressingrecombi- m
G6540Asystem(Agilent)equippedwithadual-sourceelectrosprayion- nants(proapnAapnI). .o
ization(ESI)ionsourceandwasoperatedinpositive-ionmode.Calibra- Peptide-bindingassayforsubtilomycin.Thepeptide-bindingassay rg
for subtilomycin was performed according to a previously described
tion was carried out with standard references of mass 121.0509 and /
method,withsomemodifications(21).B.subtilisCU1065anditsrecom- o
922.0098. The source parameters were as follows: gas temperature of n
binantexpressingapnIwereculturedovernight.Thecellswereharvested
350°C, gas flow rate of 9 liters/min, and nebulizer stress of 40 lb/in2 A
andwashedtwicewith50mMsodiumphosphatebuffer(pH7.0)con-
(gauge). The capillary, fragmenter, skimmer, and octopole radio fre- p
quency(RF)peakvoltageweresetat4,000V,250V,65V,and750V, wtaiitnhin1gm1%lofgliunccousbea.tTiohnebceulflfceorncocenntatriantiinogn5w0amsaMdjusosdteidumtopanhoOspDh6a0t0eo,f11%0 ril
respectively,forthescansource.Thequadrupolewassettopassionsfrom 4
glucose,and1MNaCl.Thecellsuspensionwasincubatedwithsubtilo- ,
m/z200to2,000.TheMSscanratewassetto1.5spectra/s. mycinatconcentrationsof15(cid:5)g/mland25(cid:5)g/mlwithshaking(150 2
Afterdeterminationoftheionsofsubtilomycinatm/z1,079.1666by 0
rpm)for30minat30°C.Afterincubation,thecellswerecentrifugedat 1
MS,targettandemMS(MS/MS)modewaschosenforacquisitionofthe 17,000(cid:8)gfor10min.ThesupernatantsweretransferredtotheHPLC 9
fragmentalionsofsubtilomycin.TheMSandMS/MSscanrateswereset systemwiththesameparametersasdescribedabove.Theharvestedcell b
to1.5spectra/sand0.8spectrum/s,respectively.TheMSandMS/MSscan y
pelletsweregentlymixedwith1mlof20%acetonitrileinwatercontain-
g
rangesweresettom/z200to2,000andm/z100to2,000,respectively.The
ing0.1%trifluoroaceticacidandincubatedwithshaking(150rpm)for5 u
targetionswereisolatedandfragmentedinthecollisioncellbyadding minat30°C.Thecellswereremovedbycentrifugationat17,000(cid:8)gfor10 e
s
optimizedcollisionenergyat34eV.DatawereanalyzedusingAgilent min,andtheremainingsupernatantswereloadedintotheHPLCsystem. t
MassHunterqualitativeanalysissoftware.
Subtilomycinamountsincell-freesupernatantsandincellextractswere
DetectionofsubtilomycinbyMALDI-TOFMS.TheMSanalysisof quantitativelydetermined.
thepeptidepreparationswasperformedbyusingthe4800PlusMALDI
TOF/TOF analyzer (Applied Biosystems, USA). Aliquots of 0.5-(cid:5)l ex- RESULTS
tractsweremixedwitha0.5-(cid:5)lsampleofmatrix((cid:3)-cyano-4-hydroxycin- ProductionofsubtilomycininB.subtilisstrainBSn5.Inaccor-
namicacidinacetonitrileand0.1%TFAinwater,1:3).Thesampleswere dancewithapreviouslyreportedmethodforpurifyingsubti-
spottedontotheMALDItargetanddriedinair.Massspectraweremea- lomycin(13),wepreparedASCEstodetecttheproductionof
suredinpositiveion-modeintherangeofm/z800to4,000forsearching
subtilomycinfromthe12-hsupernatantofB.subtilisBSn5.LC-
and2,000to5,000DaforcomparisonandanalyzedusingProteinPilot
electrospray ionization MS of the crude extracts revealed that
software.
BSn5canproduceacompoundwiththesamecharacteristicsas
Preparationofpeptideantibioticsforsensitivityassay.Vancomycin
subtilomycin. The corresponding main peak was detected at a
and nisin were purchased from Sigma-Aldrich, USA. By searching the
retentiontimeof7.9minundertheUVmonitorat254nmusing
NationalCenterforBiotechnologyInformation(NCBI)databaseusing
thepaenibacillinstructuralsequence,wefoundthataBacillusGenetic similarmobile-phaseconditions(Fig.1A),andtheMSextracted
StockCenter(BGSC)preservedisolate,PaenibacillusstrainATCC842, fromthispeakshowedthatthemonoisotopicmassofthequadru-
withcompletegenomesequenceinformation,mayproducepaenibacillin. plycharged[M(cid:7)4H]4(cid:7),triplycharged[M(cid:7)3H]3(cid:7),anddoubly
6306 aem.asm.org AppliedandEnvironmentalMicrobiology
SubtilomycinGeneandLantibioticImmunity
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
a
e
m
.
a
s
m
.
o
r
g
/

o
n

A
p
r
il
4
,

2
0
1
9

b
y

g
u
e
s
t
FIG1Detectionoftheproductionofsubtilomycinbyliquidchromatography-electrosprayionization-massspectrometry(LC-ESI-MS).(A)Chromatography
profileofactiveammoniumsulfatecrudeextractsoftheBacillussubtilisBSn5culturesupernatant.Thetargetcompoundisindicatedbyanarrow.(B)ESI-MS
ofthecompoundindicatedbythearrowinpanelA.(C)Magnificationofthedoublychargedions[M(cid:7)2H]2(cid:7)inpanelB;themonoisotopicmasspeakis
indicatedbyanarrow.(D)Fragmentalionsobtainedfromtargettandemmassspectrometryofthepeptidewithamassof3,234.48Da.Ionsyandbindicatethe
fragmentedpeptidesextendedfromthecarboxylterminusandaminoterminus,respectively.(E)Predictedstructureofsubtilomycinandestablishedstructure
ofpaenibacillin(15).Thepredictedstructuremotifsareshownindifferentcolors.Oburefersto2-oxobutyryl.
October2014 Volume80 Number20 aem.asm.org 6307
Dengetal.
charged [M (cid:7) 2H]2(cid:7) ions present in the sample were m/z observedwithrespecttotheproductionofsubtilomycin(Fig.2B).
809.6273,m/z1,079.1666,andm/z1,618.2471,respectively,which Thedetectionofsubtilomycinproductionwasalsoevaluatedus-
areallequivalenttothemassof3,234.48Da,whichcorresponds ingMALDI-TOFMS,andtheresultswereconsistentwiththose
extremely well to the theoretical mass of subtilomycin of fromthebioassayandHPLCanalyses(seeFig.S1inthesupple-
3,234.4806Da(Fig.1BandC). mentalmaterial).
BasedontheMS/MSresults,wefurtherverifiedthepredicted Inaddition,theB.subtilisCU1065recombinantharboringthe
N-terminal modification of 2-oxobutyrate. Similar to the re- wholeapnclusterwasconstructed.Subtilomycinproductionwas
portedN-terminalmodificationofPep5(22),theN-terminalDhb evaluatedinthe12-to16-hculturedsupernatantoftherecombi-
(C H NO—) is spontaneously deaminated into 2-oxobutyrate nant by LC-MS (Fig. 2C; see also Fig. S2 in the supplemental
4 6
(C H O —)insubtilomycin,resultinginanaccuratemassgainof material),whichfurtherverifiedthattheminimalapnclusterwas
4 5 2
0.9843 Da. All the measured b ions (extending from the amino responsibleforsubtilomycinbiosynthesis.
terminus)areconsistentwiththemassofpeptideswithanN-ter- The putative transmembrane protein-encoding gene apnI
minal 2-oxobutyrate modification (see Table S1 in the supple- confers subtilomycin immunity. A structurally elucidated lan-
mentalmaterial).Previously,Lietal.usedtandemmassspectro- tibiotic, paenibacillin, produced by Paenibacillus polymyxa
metricanalysistodeterminetheringtopologyoflanthipeptides OSY-DFwasidentifiedwiththehighestidentity(54%)tosubti- D
thatdonotcontainoverlappingrings(23).Astheproposedsub- lomycin;however,itsbiosyntheticgeneclusterhasnotbeenpub- o
w
tilomycinstructuredoescontainoverlappingrings,wecouldnot lished.Inaddition,otherlantibioticsfromthePep5groupshowed
n
directly determine the subtilomycin structure based on our homologywithsubtilomycin,includingepilancin15X,epilancin lo
MS/MS results. However, significantly increased fragmentation K7,Pep5,andepicidin280.Wecomparedthesubtilomycinbio- a
d
was observed at the N-terminal region, which contains no pre- syntheticclusterwithfourreportedandrelatedlantibioticgene e
d
dictedthioetherrings,therebyprovidingsubstantialsupportfor clustersfromthePep5group(Fig.3).Fourgeneshomologousto

f
thepredictedsubtilomycinstructure(Fig.1DandE). lanA,lanP,lanB,andlanCwerefoundtoberelativelyconservedin r
o
Identificationandcloningofthesubtilomycinbiosynthetic allofthegeneclustersexamined,includingthesubtilomycinclus- m
genecluster.Throughscanningthewholegenomesequenceof ter.However,thegenesresponsibleforlantibioticexport,immu- h
t
strainBSn5,aputativesubtilomycingenecluster(BSn5_12550 nity,andN-terminalmodificationshowedvariationsintermsof t
p
to BSn5_12575), termed the apn cluster, that spans positions theirarrangement.Thegenesresponsibleforlantibioticexportin :/
/
2428600to2436982wasidentified(16).Althoughtherelation- thePep5grouparelocatedupstreamofthegeneslanAPBCinthe a
e
ship between subtilomycin and its biosynthetic gene cluster direction opposite to that observed in the subtilomycin cluster. m
wasrecentlyestablishedbasedonthesinglematchbetweenthe The transporter gene is missing in the gene cluster for epicidin .a
primaryaminoacidsequenceoftheN-terminalregionofsub- 280,whichwasproposedtobereplacedbyothertransportersin s
m
tilomycinandthe5= endofthestructuralgenesubA(13),no thehost(24).TheimmunitygenespepIandeciIarelocatedclosely
.
o
directmolecularbiologicalevidencehasyetbeenprovidedto upstreamofthestructuralgenespepAandeciA,respectively.The r
g
validate the function of the proposed subtilomycin biosyn- putativeepilancin15XimmunitygeneelxIislocateddownstream /

thetic gene cluster. Therefore, we explored the effect of gene andnexttoelxC. o
n
deletionofapnB,whichencodesaLanB-likedehydratase,and In the apn cluster, the gene products of apnAPBC were pre-
A
apnT, which encodes a putative transporter, to identify the dicted, respectively, as structural peptide ApnA, leader peptide- p
r
functionofthegenecluster(Fig.2A). cleaving protease ApnP, and lanthionine synthetases ApnB and il
BioassaysandHPLCwereusedtodetectthechangeinsubti- ApnC.ThegeneorganizationapnI-apnT,whichislocateddown- 4
,

lomycinproductioninthemutants(Fig.2B).Incontrasttothe streamofapnAPBC,isdifferentfromthatoftheotherreported 2
wild-typestrain,theculturesupernatantfromthe(cid:6)apnBmu- geneclusters(Fig.3).AsdeletionofapnTledtoaconsiderable 01
tants failed to inhibit the target indicator strain B. subtilis loss of subtilomycin production in the supernatant, apnT, 9

CU1065, and no production of subtilomycin was detected by which encodes an ABC transporter, likely plays a key role in b
y
HPLCfromtheASCEsofthe(cid:6)apnBmutant(Fig.2B).Therefore, subtilomycin export. Through protein BLAST using the se- g
apnBisnecessaryforsubtilomycinproduction.Theresultsfrom quenceoftheapnI-encodedprotein,nofunctionallyvalidated u
e
bioassaysandHPLCshowedthatverylittlesubtilomycincouldbe homolog could be found in the NCBI database. Membrane s
detectedfromthesupernatantofthe(cid:6)apnTmutant.TheapnT helixpredictionusingTMHMM(25),HMMTOP(26),andthe t
gene,whichencodesaputativeABCtransporter,waspredictedto DAS-TM filter (27) revealed the presence of five transmem-
beatransporterfortheexportofsubtilomycin.Wepreparedin- branedomainsintheputativeproteinApnI.Immunitygenes
tracellularextractsfromthe10-hculturedcellsofthewild-type areusuallypresentinlantibioticbiosyntheticclustersandare
BSn5andthe(cid:6)apnTmutantbyliquidnitrogengrindingtodetect necessaryforlantibioticproduction.Therefore,thefactthatno
theprecursorpeptideofsubtilomycinbyhigh-resolutionLC-MS. immunity-related gene was found in the subtilomycin gene
However,nopredictedmass(6,250.35Da)ofprecursorpeptideor clusters indicated that apnI and/or apnT is likely involved in
significantaccumulationofmassesfrom5,500to6,500Dacould subtilomycinimmunity.
beobservedinBSn5orthe(cid:6)apnTmutant(datanotshown).In To investigate which genes, apnI and/or apnT, could confer
ordertoconfirmthepresenceoftheminimalsubtilomycingene resistanceonthesubtilomycin-sensitivehostB.subtilisCU1065
cluster,twoflankinggenesoftheclusterweredeletedasdescribed (B.subtilis168background),wefirstpredictedtwoputativepro-
inMaterialsandMethods.TheybcLgene,whichencodesaputa- motersintheapncluster,whicharelocatedontheupstreamgenes
tiveeffluxtransporter,andybdG,whichencodesaputativehydro- apnA and apnT. Different fragments that contained the whole
lase, located upstream and downstream of the cluster, respec- cluster,apnI-apnT,apnT,orapnIwereclonedintotheintegration
tively, were disrupted (Fig. 2A). No significant change was vector pDG1730. The promoter region of apnA (pro ) was
apnA
6308 aem.asm.org AppliedandEnvironmentalMicrobiology
SubtilomycinGeneandLantibioticImmunity
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
a
e
m
.
a
s
m
.
o
r
g
/

o
n

A
p
r
il
4
,

2
0
1
FIG2Thesubtilomycingeneclusterandinactivationanalysisofrelatedgenes.(A)Theapnclustergenesareshownindifferentcolors:theprecursorpeptide 9
encodedbyapnAisshowninblack,thegenesencodingmodifyingenzymesareshowninred,thegeneencodingproteaseisshowninyellow,thegeneinvolved
b
inputativeimmunityisshowningray,andthegeneencodingthetransporterisshowninblue.Theflankinggenesareshowninwhiteandarenumbered.The y
blackarrowindicatesrelatedgenesinterruptedbyspectinomycin(Spc)orkanamycin(Kan)resistancegenes.(B)Bioassayandhigh-performanceliquid g
chromatographyresultsfordetectionofsubtilomycinfromactiveammoniumsulfatecrudeextractsofwild-typestrainBSn5anditsmutants.1,wild-typestrain u
BSn5; 2, (cid:6)apnB mutant; 3, (cid:6)ybcL mutant; 4, (cid:6)ybdG mutant; 5, (cid:6)apnT mutant. The black ring refers to the respective target peaks of subtilomycin in e
s
high-performanceliquidchromatography.(C)ESI-MSdetectionofsubtilomycinproductioninthe12-hsupernatantoftherecombinantharboringtheapn t
cluster.Thetriplychargedion[M(cid:7)3H]3(cid:7)isshown.
clonedinfrontofeachoftheconstructedfragmentsapnI-apnT, sensitivitytosubtilomycinaswild-typeCU1065(Fig.4BandC),
apnT, and apnI, resulting in the vectors pro apnI-T, which suggested that apnT does not confer subtilomycin resis-
apnA
pro apnT,andpro apnI,respectively,toensuretheexpres- tanceonthehost.OurresultsindicatedthatthesinglegeneapnI
apnA apnA
sionofeachgene(Fig.4A).Theconstructedvectorsweretrans- couldconferprotectionagainstsubtilomycinonthesensitivehost
formed and integrated into the amyE locus of the B. subtilis strainB.subtilisCU1065.
CU1065chromosome,resultingincorrespondingrecombinants. TodeterminewhethertheapnIgeneplaysaroleinimmunity
Subtilomycinsensitivitytestswereconductedtodetermineif inaproducerstrain,subtilomycinsensitivitytestswereconducted
theclonedfragmentsconferredresistanceondifferentB.subtilis usingwild-typeBSn5andits(cid:6)apnIand(cid:6)apnTmutants.There-
recombinants. The fragments containing apnI with pro re- sultsshowedthatwild-typestrainBSn5andthe(cid:6)apnTmutant
apnA
sulted in the same CU1065 resistance level as that observed for couldtoleratehighsubtilomycinconcentrations(uptoapproxi-
fragmentscontainingthewholecluster(Fig.4BandC).There- mately0.3mM),whereasthe(cid:6)apnImutantwassignificantlysen-
combinantsnormallyexpressingapnTshowedthesamelevelof sitivetosubtilomycin(Fig.4DandE).
October2014 Volume80 Number20 aem.asm.org 6309
Dengetal.
D
o
FIG3ComparisonoftheorganizationofsubtilomycingeneclusterswithbiosyntheticgeneclustersofPep5grouplantibiotics,epilancin15X(39),epilancinK7(40), w
epicidin280(24),andPep5(41).Genesarelabeleddifferentlydependingonthepredictedfunctionoftheirproducts:precursorpeptides(black),proteases(horizontal n
stripes),modificationenzymes(verticalstripes),immunity(white),andtransporters(gray).TheunavailablesequencesinstrainK7areshownasabrokenline. lo
a
d
e
d
Discovery of the ApnI-like proteins for lantibiotic immu- transformedthemintoB.subtilisCU1065.Aconcentrationgra-

f
nity.Assubtilomycinsharesstructuralsimilaritywithpaenibacil- dientofcrudepaenibacillinwasemployedforthesensitivitytestof r
o
lin,wethuswonderedwhatgenedoesplayaroleinpaenibacillin thethreestrains.Theresultsshowedthatthefragmentcontaining m
immunity. Using the reported draft genome sequence (28), we paeIunderthecontrolofpro couldconfercertainresistance h
apnA
t
foundthattheannotatedgenesoftheputativepaenibacillinbio- on CU1065 at the low concentration dilutions of paenibacillin t
p
syntheticgeneclusterarepresentintheorderpaeNAPBCIT.We (the2ndand3rddilutions),comparedwiththeothertwostrains :/
/
thencomparedtheproposedpaenibacillingeneclustertotheapn (Fig. 6). At the increasing paenibacillin concentrations (the 5th a
e
cluster(Fig.5).Interestingly,aputativetransmembraneprotein- and 6th dilutions), no significant difference could be observed m
encoding gene, paeI, is present downstream of the conserved amongthethreestrains(Fig.6A),whichindicatesthatthepaeni- .a
lanAPBC gene organization. Despite the low sequence identity bacillinresistancelevelconferredbyPaeIwasmuchlowerthanthe s
m
(27%)betweenApnIandPaeI,theirsimilartopologicalstructures ApnI-conferredsubtilomycinresistancelevelinthebackground
.
o
andthesamegenearrangementoftheclusterasfortheapncluster ofB.subtilisCU1065. r
g
suggestedthatPaeIwasresponsibleforpaenibacillinimmunity. ImmunityprovidedbyApnIisspecifictowardsubtilomycin. /

Furthermore,weobtainedmoreuncharacterizedputativelantibi- Sincethesoleputativetransmembraneprotein,ApnI,conferred o
n
otic gene clusters from the Bacillus cereus group (including 5 B. significantresistancetothesensitivehostCU1065,wenextevalu-
A
cereusstrainsand5Bacillusthuringiensisstrains)basedonrunning ated whether ApnI could confer cross-resistance against other p
r
protein BLAST using the sequences of ApnA and ApnI on the bacteriocins.Towardthisend,weselectedthefollowingpeptide il
NCBI website. Comparison of these gene clusters with the apn antibiotics based on their relationship with subtilomycin, listed 4
,

cluster showed that they shared the same gene arrangement, from more distantly to closely related: vancomycin, nisin, and 2
0
lanAPBCfollowedbylanIandlanT.TheLanIproteinsfromtheB. paenibacillin.Wefirstcarriedoutasensitivitytestwithvancomy- 1
cereusgroupshowedlowsimilarity(27to29%)toApnI.TheLanIs cinandnisin.AsshowninFig.7A,CU1065withandwithoutthe 9

from these gene clusters were predicted to have six transmem- integratedgeneapnI(withthepro -apnIfragment)showedan b
apnA y
branedomains,whichhadsimilartopologywithApnI.Accord- inhibition zone of the same size with various vancomycin and g
ingly, we named these LanI proteins as ApnI-like proteins and nisin concentrations, suggesting that ApnI could not recognize u
e
predictedthattheywereresponsiblefortheimmunityofthecor- vancomycinandnisin. s
t
respondinglantibiotics. Since subtilomycin and paenibacillin showed high similarity
WepredictedthatthepaenibacillinstructuralgenepaeAwould (54%)andhavethesamefive-thioetherringbackbone(Fig.2B),
also exist in the genome sequence of Paenibacillus strain ATCC we next evaluated whether ApnI and PaeI could confer cross-
842, which is the strain most closely related genetically to the resistance against paenibacillin and subtilomycin, respectively.
paenibacillin producer strain P. polymyxa OSY-DF (28), and Thesensitivitytestsshowedthatdespitethefactthattheseantibi-
paenibacillinproductionwasdetectedfromthecrudeextractsof oticsarestructurallysimilar,nocross-resistancewasobservedbe-
the24-h-culturedsupernatantofstrainATCC842,accordingtoa tweenthepro -apnIandthepro -paeIrecombinants(Fig.
apnA apnA
previouslyreportedmethod(seeFig.S3inthesupplementalma- 7B).Together,theseresultsimplythatApnIprovidesimmunity
terial)(14).Weestimatedthatthepurityofpaenibacillininthe thatmayinvolveaspecificsequencerecognitionmechanism.
crudeextractswasabout8%basedontherelativeabundanceof TherecombinantB.subtilisexpressingApnIcouldbindsub-
thepaenibacillinpeakunderthedetectionofUV254nmbyLC- tilomycin.WecharacterizedthefunctionofApnIbyconductinga
MS.TovalidatethefunctionofpaeI,whichencodesanApnI-like quantitativepeptide-bindingassayusing15(cid:5)g/mland25(cid:5)g/ml
protein, we cloned the fragment containing the paeI and the ofsubtilomycin(Fig.8).AsadditionofNaCltotheassaysolution
downstream promoter regions of apnA (pro ) and paeA coulddecreasetheamountofcell-bindinglantibioticsbyreducing
apnA
(pro ) into the integrated vector pDG1730, respectively, and thelantibioticaffinitytothecytoplasmicmembrane(21),toin-
paeA
6310 aem.asm.org AppliedandEnvironmentalMicrobiology
SubtilomycinGeneandLantibioticImmunity
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
a
e
m
.
a
s
m
.
o
r
g
/

o
n

A
p
r
il
4
,

2
0
1
9

b
FIG4FunctionalanalysisofsubtilomycinimmunitybygeneexpressioninB.subtilisCU1065andgeneinterruptioninB.subtilisBSn5.(A)Mapdisplaying y

essentialDNAfragmentsforsubtilomycinimmunity.EachconstructionwasclonedintothevectorpDG1730andintegratedintotheamyElocusofthe g
u
chromosomeofthesubtilomycin-sensitivestrainB.subtilisCU1065.ThepredictedfragmentscontainingtheapnAandapnTpromoterregionsareindicatedby e
grayarrows.(B)SubtilomycinsensitivitiesofB.subtilisCU1065strainsharboringdifferentfragmentsofthesubtilomycingeneclusterwereinvestigatedinagar s
diffusiontests.B.subtilisCU1065anditsrecombinantsweretransformedwiththevectorcontainingpro -apnI-T,pro -apnI,pro -apnT,orthewhole t
apnA apnA apnA
subcluster.Startingfromthearrowandmovingclockwise,thesubtilomycinconcentrationsappliedwereasfollows:0.125(cid:5)M,0.25(cid:5)M,0.5(cid:5)M,1(cid:5)M,2(cid:5)M,
and8(cid:5)M.Theasteriskindicatesablurryinhibitionzone.(C)LineardependenciesbetweenthesquareoftheradiusofthehalosshowninpanelBandthenatural
logarithmoftheappliedsubtilomycinconcentrationwith60(cid:5)lofsubtilomycinanddiffusionforatleast4h.ResultsforB.subtilisCU1065transformedwith
vectorscontainingapnI,apnT,apnI-apnT,andsubgeneclustersareshown.(D)SubtilomycinimmunityofstrainBSn5(plate1)withinterruptionofapnI(plate
2)andapnT(plate3).Startingfromthearrowandmovingclockwise,theappliedconcentrationsofsubtilomycinwere3.125(cid:5)M,12.5(cid:5)M,25(cid:5)M,50(cid:5)M,100
(cid:5)M,and200(cid:5)M.Thewellinthemiddleoftheplatewasnotcounted.(E)SubtilomycinimmunityofBSn5withinterruptionofapnTandapnI.Theimmunity
responsewascalculatedcorrespondingtothesquareoftheradiusofthehalosshowninpanelD.
vestigatethetargetspecificityofApnIagainstsubtilomycin,the geststhattheapnI-expressingstrainconfersresistancebyseques-
assaywasperformedinthepresenceof1MNaCl.ForB.subtilis teringsubtilomycin.
CU1065expressingapnI,(cid:9)7(cid:5)gofsubtilomycinwasfoundtobe
attachedtothecellsindependentlyofthequantityofappliedsub- DISCUSSION
tilomycin(15or25(cid:5)g/ml)(Fig.8).Correspondingly,theamount Although a link between subtilomycin and its gene cluster was
ofsubtilomycinremaininginthesupernatantoftheapnI-express- proposedbasedonamatchbetweentheN-terminalregionanda
ingrecombinantdecreasedsignificantly(Fig.8).Thisresultsug- functionalgeneatthe5=end,itsbiosyntheticgeneclusterhasnot
October2014 Volume80 Number20 aem.asm.org 6311
Dengetal.
FIG5ThepotentialApnI-likeimmunityprotein-encodinggenefromtheuncharacterizedlantibioticgeneclusters.Genesarecolor-codedbythepredicted
functionoftheirproducts:genesforprecursorpeptides(black),genesforproteases(yellow),genesformodifyingenzymes(green),genesfortransporters
(purple),andgenesforimmunity(red).Thebarrepresents1kbp.Thenumberofindependentisolatesthathavethepotentialclusterisshown.Detailsforthese
sequencesareshowninTableS2inthesupplementalmaterial.
D
o
w
yetbeenfunctionallyvalidated.Inthisstudy,weexperimentally validated homolog could be found. We thus first determined n
identifiedtheapnclusterresponsibleforsubtilomycinbiosynthe- themechanismofself-resistanceofalantibioticproducerto- lo
a
sisandprovidedevidencethatapnIdoesindeedconfersubtilomy- wardPep5,whichishomologouswithsubtilomycin.Theself- d
cin immunity on its host. When we BLAST searched the NCBI protectionagainstPep5issolelydependentonfunctionalex- e
d
database using the ApnI amino acid sequence, no functionally pressionofthe69-amino-acidPepI,whichwasdemonstrated
f
r
throughaseriesofcloningexperiments(29,30).Furtherfunc- o
m
tionalanalysisindicatedthatPepIisalipoproteinwithanapo-

lar N-terminal segment for its localization at the membrane- h
t
t
cellwallinterfaceandahydrophilicCterminus,whichconfers p
:
immunity(11).However,thepredictedapnIgeneproductisa //
a
membraneproteinwithfivetransmembranedomains,whichis e
m
vastlydifferentfromPepI.
.
Otherknownmodelsforlantibioticimmunityusuallyinvolve a
s
thelanFE(G)genes,encodinganABCtransportercomplex(Fig. m
9).LanFE(G)canindependentlyprovidelantibioticimmunity, .o
suchasLctFEGforlacticin481immunity(31),orworksyner- rg
/
gistically with a membrane-associated protein, such as NisI
o
(32), or a transmembrane protein, such as NukH (9, 10) and n

LtnI (33). Lantibiotic immunity that is solely provided by A
p
transmembraneproteinsisveryrare.Wefoundonlyonereport r
of such a protein, CylI, which is different from other trans- il 4
membraneimmunityproteins,asitcontainsaconservedzinc ,
2
metalloprotease(MEROPSfamilyM50A)domain,whichsug- 0
1
geststhatitplaysaroleinimmunitybycleavingoneorbothof 9
thecytolysinsubunits(34,35). b
y
Throughbioinformaticsanalysis,manyApnI-likeproteinsin
g
theuncharacterizedlantibioticgeneclustersthatarerelatedtothe u
e
apnclusterwerefoundintheNCBIdatabase(seeTableS2inthe
s
supplemental material). Although low identity (27 to 29%) is t
presentbetweenthesequencesofApnIandtheLanIproteins,they
allarepredictedtohaveatopologicalstructuresimilartothatof
ApnI,withfiveorsixtransmembranedomains.TheLanAstruc-
turalpeptidegenesinthesegeneclusterswerealsoshowntobe
closelyrelated,withasequenceidentityof47%to58%toApnA
(Fig.5;seealsoTableS2).Intotal,21strainsofB.subtilis,Paeni-
FIG6FunctionalanalysisofthepaeIgeneinB.subtilisCU1065.(A)Paeni- bacillus,B.cereus,andB.thuringiensishavetheputativelantibiotic
bacillin sensitivity test results of B. subtilis CU1065 and the recombinants
biosyntheticgeneclusterwithApnI-likeproteins.Wesuccessfully
harboringfragmentspro -paeIandpro -paeI.Theconcentrationsof
apnA paeA introducedonesuchApnI-likeprotein,PaeI,inB.subtilisCU1065
crudepaenibacillinwereappliedwithaseriesof2-folddilutionsandwerenot
quantified.Theconcentrationsofpaenibacillinincreasedstartingfromthe fromPaenibacillusstrainATCC842andfoundthatPaeIindeed
arrowandmovingclockwise.(B)Theresponseasthelengthoftheradiiofthe confersresistancetopaenibacillin.Theseresultsindicatedthatthe
inhibitionhalosversusthecorrespondingrelativeamountofpaenibacillinas
novel model represented by ApnI for lantibiotic immunity is
showninpanelA.Twohighconcentrationsofpaenibacillin(the5thand6th
widelypresentamongtheBacillusandPaenibacillustaxa.Inaddi-
dilutions)wereremoved.Thisexperimentwasindependentlyperformedat
least3times,andtheresultswereconsistent. tion,intheB.subtilisbackground,paeIcouldbeexpressedunder
6312 aem.asm.org AppliedandEnvironmentalMicrobiology