Sequence-Based Analysis of Secondary-Metabolite Biosynthesis in Marine Actinobacteria

13 Pages·2010·1.7 MB·English

APPLIEDANDENVIRONMENTALMICROBIOLOGY,Apr.2010,p.2487–2499 Vol.76,No.8
0099-2240/10/$12.00 doi:10.1128/AEM.02852-09
Copyright©2010,AmericanSocietyforMicrobiology.AllRightsReserved.
Sequence-Based Analysis of Secondary-Metabolite Biosynthesis
(cid:1)
in Marine Actinobacteria ‡
Erin A. Gontang,† Susana P. Gaudˆencio, William Fenical, and Paul R. Jensen*
CenterforMarineBiotechnologyandBiomedicine,ScrippsInstitutionofOceanography,
UniversityofCalifornia,SanDiego,LaJolla,California92093-0204
Received25November2009/Accepted3February2010
A diverse collection of 60 marine-sediment-derived Actinobacteria representing 52 operational taxonomic
units was screened by PCR for genes associated with secondary-metabolite biosynthesis. Three primer sets
D
were employed to specifically target adenylation domains associated with nonribosomal peptide synthetases
o
(NRPSs) and ketosynthase (KS) domains associated with type I modular, iterative, hybrid, and enediyne w
polyketidesynthases(PKSs).Intotal,two-thirdsofthestrainsyieldedasequence-verifiedPCRproductforat n
least one of these biosynthetic types. Genes associated with enediyne biosynthesis were detected in only two lo
a
genera,while88%oftheketosynthasesequencessharedgreatesthomologywithmodularPKSs.Positivestrains d
includedrepresentativesoffamiliesnottraditionallyassociatedwithsecondary-metaboliteproduction,includ- e
d
ing the Corynebacteriaceae, Gordoniaceae, Intrasporangiaceae, and Micrococcaceae. In four of five cases where
f
phylogenetic analyses of KS sequences revealed close evolutionary relationships to genes associated with ro
experimentally characterized biosynthetic pathways, secondary-metabolite production was accurately pre- m
dicted.SequenceclusteringpatternswereusedtoprovideanestimateofPKSpathwaydiversityandtoassess h
thebiosyntheticrichnessofindividualstrains.ThedetectionofhighlysimilarKSsequencesindistantlyrelated tt
p
strains provided evidence of horizontal gene transfer, while control experiments designed to amplify KS :
/
sequences from Salinispora arenicola strain CNS-205, for which a genome sequence is available, led to the /a
detectionof70%ofthetargetedPKSpathways.Theresultsprovideabioinformaticassessmentofsecondary- e
m
metabolite biosynthetic potential that can be applied in the absence of fully assembled pathways or genome
.
sequences. The rapid identification of strains that possess the greatest potential to produce new secondary a
s
metabolitesalongwiththosethatproduceknowncompoundscanbeusedtoimprovetheprocessofnatural- m
productdiscoverybyprovidingamethodtoprioritizestrainsforfermentationstudiesandchemicalanalysis. .
o
r
g
/
Microbial natural products represent the primary resource tionsthereof(17).Althoughactinomyceteshavebeenstudied o
n
fromwhichnewmedicinesarederived,accountingforapprox- extensively in the past, it is estimated that only 3% of the

J
imatelyhalfoftheantibioticsdiscoveredasof2002(6).Over natural-productpotentialofeventhewell-studiedgenusStrep- a
thepastseveraldecades,however,drugdiscoveryeffortshave tomyces has been realized (46), thus leaving considerable op- n
u
moved away from microbial products (29), in part due to a portunity for new discovery. In addition, strains derived from a
r
reductionintheratioofnewchemicalentitiesdiscoveredrel- poorlystudiedenvironments,includingmarinesamples(8,15, y

ative to the isolation of known metabolites (3) and the chal- 33),haveproventobeaproductivesourceofnewcompounds. 8
,

lenges associated with developing effective “dereplication” This potential has helped drive calls for the development of 2
0
methods to improve the efficiency of the discovery process. newapproachestonatural-productdiscoverythatincludemin- 1
More recently, the rise in drug-resistant pathogens has left imizing the isolation of previously described compounds (4). 9

manycurrentantibioticsobsolete,whilethelimitedsuccessof Advances in our understanding of the molecular genetics of by
alternativediscoverystrategies,suchascombinatorialchemis- natural-productbiosynthesiscoupledwithincreasingaccessto g
try,havecreatedavoidinthepipelineofnewdrugleads(39). DNAsequencingnowcreateunparalleledopportunitiestoin- ue
Theresponsetothisneedincludesrenewedinterestinagroup corporatesequence-basedapproachesintotheprocessofnat- s
t
of actinobacteria commonly called actinomycetes (defined ural-productdiscovery.
hereasbacteriawithintheorderActinomycetales),whichhave Polyketides, nonribosomal peptides, and polyketide/nonri-
long been recognized as a prolific source of natural products, bosomal peptide hybrids are synthesized by the coordinated
including polyketides, nonribosomal peptides, and combina- actions of enzymatic assembly lines, which conduct the itera-
tivechemicalcondensationofmonomericunits,includingcar-
boxylic acid and/or amino acid monomers (10, 17, 45). The
*Corresponding author. Mailing address: Scripps Institution of orderandidentityofeachdomainwithinagivenassemblyline
Oceanography, University of California, San Diego, 9500 Gilman
specifythesequenceofmonomeractivationandincorporation,
Drive, La Jolla, CA 92093-0204. Phone: [phone]. Fax: (858)
534-1318.E-mail:[email protected]. thechemicalreactionsthatoccurateachstepintheassembly
†Presentaddress:DepartmentofMicrobiologyandMolecularGe- process, and the length and functionality of the product re-
netics, Harvard Medical School, 200 Longwood Ave., Boston, MA leased(17).InthecaseoftypeIpolyketidesynthases(PKSs),
02115.
the condensation of a carboxylic acid monomer to a growing
‡Supplementalmaterialforthisarticlemaybefoundathttp://aem
.asm.org/. acyl chain is accomplished via ketosynthase (KS), acyltrans-
(cid:1)Publishedaheadofprinton12February2010. ferase, and acyl carrier protein domains that act either itera-
2487
2488 GONTANG ET AL. APPL.ENVIRON.MICROBIOL.
tively, for the biosynthesis of aromatic polyketides, or, more evaluated. Phylogenetic analyses were used to assess the sim-
commonly,inanonredundant,assembly-linefashion.Follow- ilarityofKSsequencestothoseassociatedwithexperimentally
ingeachcondensationreaction,thepresenceofketoreductase, characterizedpathwaysand,infivecaseswhereclosematches
dehydratase,andenoylreductasedomainsdeterminestheoxi- were detected, to predict the types of products that would be
dation state of the beta-carbonyl (25). The incorporation of produced. The results reveal that pathways associated with
aminoacidmonomersintopeptidesvianonribosomalpeptide secondary-metabolite biosynthesis are widely distributed
synthetases (NRPSs) is accomplished via the condensation, among the Actinomycetales and that bioinformatic analyses
adenylation (A), and peptidyl carrier protein domains associ- provideapowerfulmethodofdereplicationthathasconsider-
ated with the enzyme (17). When present, epimerization, able potential to improve the efficiency with which secondary
methyltransferase, and oxidase domains tailor the amino acid metabolitesarediscovered.
being added. The immense structural complexity and func-
tional diversity of polyketides, nonribosomal peptides, and
MATERIALSANDMETHODS
compoundsofhybridbiosyntheticoriginaregeneratedbothby
Nucleicacidextraction,PCRamplification,cloning,andsequencing.Atotal
the intricately coordinated organization of these enzymatic of60marine-sediment-derivedactinomycetestrains,representing52OTUscul- D
assembly lines and by the specific tailoring enzymes responsi- turedduringaresearchexpeditiontotheRepublicofPalau(20),wereexamined. o
w
bleforpostassemblymodifications(10,17). GenomicDNAwasextractedfromeachisolateaccordingtotheDNeasypro-
n
In an effort to identify new sources of bioactive secondary tocol(QiagenInc.,Valencia,CA),withseveralmodifications.AfterRNaseA(2 lo
metabolites, degenerate PCR primers have been used to mg/ml)wasaddedtotheenzymaticlysisbuffer,theresuspendedbacterialpellet a
wasincubatedfor2hat37°C.FollowingtheadditionofproteinaseKandlysis d
screen for the presence of genes associated with PKS and bufferAL,thesamplewasheldfor1hat70°C.GenomicDNAwaselutedfrom e
NRPSpathwaysinDNAderivedfromsoil(18,47,48),sponge thespincolumnwith100(cid:1)lofelutionbufferAEforimmediateuseorstorage d
tissues(16,41),andavarietyofculturedorganisms,including at(cid:2)20°C. fro
cyanobacteria(9,14,37),dinoflagellates(37),andGram-pos- Ketosynthase(KS)domainsoftypeIpolyketidesynthase(PKS)geneswere m
itivebacteria(1,2,21,23,28).Whencoupledwithhomology- PCCGRCSaTmSpYliTfieSdCTfrSoGmAg-e3n(cid:3))omanicdDKNS-ARu(s5i(cid:3)n-gGtThSeCpCriSmGeTrsSCKCS-GFT(G5(cid:3)S-CGCYSSCTACGSASA-3G(cid:3)) h
t
basedsearchesandphylogeneticanalyses(18),sequence-based (18).ThesedegenerateprimersweredesignedtoamplifyKSdomainsassociated tp
approaches offer an opportunity to predict which isolates or withmodular,iterative,andNRPShybridtypeIPKSgenes.The50-(cid:1)lPCR :/
environments harbor the greatest potential to produce inter- mixturecontained20to50ngofDNA,800pmolofeachprimer,10(cid:4)PCRbuffer /a
II(AppliedBiosciences,FosterCity,CA),2.5mMMgCl (AppliedBiosciences, e
esting new secondary metabolites. Phylogenetic analyses are 2 m
Foster City, CA), 1.5 U of AmpliTaq Gold DNA polymerase (Applied Bio-
particularly useful in that clustering patterns can be used to sciences,FosterCity,CA),400(cid:1)Mdeoxynucleosidetriphosphatemixture,and .a
predict the potential number of biosynthetic pathways in a 7%dimethylsulfoxide(DMSO).ThePCRprotocolconsistedofa15-minde- s
m
strainandifthebiosyntheticlogicofthePKSgeneismodular, naturationat95°C;1cycleof1minat95°C,1minat65°C,anda1-minextension .
at72°C;35cyclesof1minat95°C,1minat62°C,anda1-minextensionat72°C; o
iterative,ortypicalofaPKS-NRPShybrid(19,25).Inthecase r
followedby10minat72°C. g
ofmodularorganization,whichisfrequentlytheresultofdu- Duetoahighlevelofsequencedivergence,adistinctsetoftypeIPKSprimers /
plicationevents(22,24),KSlocifromthesametypeIPKScan wereusedtoscreenforsequencesassociatedwiththebiosynthesisofthecore on
often be recognized as a cluster of closely related sequences. (“warhead”) portion of enediyne secondary metabolites. The primers EdyA J
Although KS domains within the same biosynthetic pathway (5(cid:3)-CCCCGCVCACATCACSGSCCTCGCSGTGAACATGCT-3(cid:3)) and EdyE a
(5(cid:3)-GCAGGCKCCGTCSACSGTGTABCCGCCGCC-3(cid:3))(31)targetedtheN- n
arenotalwaysinthesamecluster,thosefromunrelatedstrains terminalregionoftheenediynePKSgene,inclusiveoftheKSdomain.The50-(cid:1)l ua
thatproducethesameproductgenerallyhavesimilarcluster- PCRmixturecontained20to50ngofDNA,500pmolofeachprimer,10(cid:4)PCR r
y
ing patterns. By following this logic, a KS sequence from a bufferII(AppliedBiosciences,FosterCity,CA),1.5mMMgCl2(AppliedBio- 8
chemicallyunknownstrainthatshowsahighlevelofsequence sciences,FosterCity,CA),1UofAmpliTaqGoldDNApolymerase(Applied ,
Biosciences,FosterCity,CA),200(cid:1)Mdeoxynucleosidetriphosphatemixture, 2
identity to an experimentally characterized pathway can be and5%DMSO.PCRamplificationwasperformedaccordingtothefollowing 0
usedtopredictthattheunknownstrainhasthegeneticpoten- 1
protocol:a5-mindenaturationat94°C,30cyclesof45sat94°C,90sat62°C,and 9
tial to produce secondary metabolites related to those previ- a2-minextensionat72°C,followedby7minat72°C. b
ouslyreportedfromthatpathway(see,e.g.,reference28).This AdenylationdomainsofNRPSpathwayswerePCRamplifiedusingtheprim- y
predictive capability provides a rapid method to avoid the ersNRPS-A3(5(cid:3)-GCSTACSYSATSTACACSTCSGG-3(cid:3))andNRPS-A7R(5(cid:3)- g
SASGTCVCCSGTSCGGTAS-3(cid:3))(2).The50-(cid:1)lPCRmixturecontained20to u
isolation of known compounds or to identify strains that pro- 50 ng of DNA, 400 pmol of each primer, 10(cid:4) PCR buffer II (Applied Bio- es
ducecompoundswithinadesiredstructuralclass.Likewise,KS sciences,FosterCity,CA),2.5mMMgCl (AppliedBiosciences,FosterCity, t
2
sequences that do not cluster with characterized biosynthetic CA),1.5UofAmpliTaqGoldDNApolymerase(AppliedBiosciences,Foster
pathways will have a greater probability of yielding new sec- City, CA), 400 (cid:1)M deoxynucleoside triphosphate mixture, and 10% DMSO.
PCRamplificationofNRPSadenylationdomainswasperformedaccordingto
ondarymetabolites,whilethenumberofdistinctKSsequence
thefollowingPCRprotocol:a5-mindenaturationat95°C,35cyclesof30sat
clustersprovidesanestimateofthemaximumnumberofPKS
95°C,90sat59°C,anda1-minextensionat72°C,followedby10minat72°C.All
pathwaysthatanindividualstrainmaypossess. PCRmixtureswereexaminedbygelelectrophoresis,andbandscorrespondingto
ByaPCR-basedapproach,culturedstrainsfrom52Actino- KS domains (expected size, (cid:5)670 bp), N-terminal regions of enediyne PKS
mycetalesoperationaltaxonomicunits(OTUs)recoveredfrom pathways (expected size, (cid:5)1.4 kb), and adenylation domains (expected size,
(cid:5)700 bp) were excised using a sterile scalpel and purified using Qiagen’s
marine sediments collected in the Republic of Palau were
QIAquickgelextractionkitaccordingtothemanufacturer’ssuggestedprotocol
screened for the presence of A domain sequences associated (QiagenInc.,Valencia,CA).
with NRPS pathways and KS loci associated with modular, AllcorrectlysizedPCRproductswereimmediatelyclonedusingtheTOPO
iterative, hybrid (PKS-NRPS), and enediyne PKSs (type I). TAcloningkit(InvitrogenCo.,Carlsbad,CA)accordingtothemanufacturer’s
suggested protocol with the following modifications. The 6-(cid:1)l TOPO cloning
Strainsfromfamiliesthatarewellknowntoproducesecondary
reactionmixtureconsistedof1(cid:1)lfreshPCRproduct,1(cid:1)lsaltsolution,3(cid:1)l
metaboliteswerescreened,andinaddition,taxanotgenerally sterile water, and 1 (cid:1)l TOPO vector. The reactions were mixed gently and
associatedwiththebiosynthesisofthesecompoundswerealso allowedtoincubateatroomtemperaturefor15min.Oncetheligationreaction
VOL.76,2010 SECONDARY-METABOLITE BIOSYNTHESIS 2489
TABLE 1. PCRscreeningofmarineactinobacteria
PKS
Family Genus OTU Strain(NCBIaccessionno.) NRPS
TypeIa Enediyne
Brevibacteriaceae Brevibacterium 1 CNJ737(DQ448693) (cid:2) (cid:2) (cid:2)
Corynebacteriaceae Corynebacterium 2 CNJ954(DQ448694) (cid:2) (cid:2) (cid:6)
Dermacoccaceae Kytococcus 3 CNJ855(DQ448695) (cid:2) (cid:2) (cid:2)
Dietziaceae Dietzia 4 CNJ898(DQ448696) (cid:2) (cid:2) (cid:2)
Geodermatophilaceae Blastococcus 5 CNJ868(DQ448697) (cid:2) (cid:2) (cid:2)
Modestobacter 6 CNJ793(DQ448698) (cid:2) (cid:2) (cid:2)
Modestobacter 6 CNJ794(DQ448774) (cid:2) (cid:2) (cid:2)
Gordoniaceae Gordonia 7 CNJ756(DQ448699) (cid:2) (cid:2) (cid:6)
Gordonia 8 CNJ863(DQ448700) (cid:6) (cid:2) (cid:6)
Gordonia 9 CNJ754(DQ448701) (cid:2) (cid:2) (cid:6) D
Gordonia 10 CNJ752(DQ448702) (cid:2) (cid:2) (cid:6) ow
Intrasporangiaceae Ornithinimicrobium 11 CNJ824(DQ448703) (cid:2) (cid:2) (cid:2) n
Serinicoccus 12 CNJ927(DQ448704) (cid:6) (cid:2) (cid:2) lo
Microbacteriaceae Agromyces 13 CNJ745(DQ448705) (cid:2) (cid:2) (cid:2) a
Microbacterium 14 CNJ930(DQ448706) (cid:2) (cid:2) (cid:2) de
Microbacterium 15 CNJ743(DQ448707) (cid:2) (cid:2) (cid:2) d
Microbacterium 16 CNJ797(DQ448708) (cid:2) (cid:2) (cid:2)
f
r
Micrococcaceae Kocuria 17 CNJ723(DQ448709) (cid:2) (cid:2) (cid:2) om
Kocuria 17 CNJ928(DQ448783) (cid:2) (cid:2) (cid:2)
Kocuria 18 CNJ900(DQ448710) (cid:2) (cid:2) (cid:2) h
Kocuria 19 CNJ770(DQ448711) (cid:2) (cid:2) (cid:6) tt
Kocuria 19 CNJ787(DQ448773) (cid:2) (cid:2) (cid:6) p
Micrococcus 20 CNJ719(DQ448712) (cid:2) (cid:2) (cid:2) ://
a
Micromonosporaceae Micromonospora 21 CNS326(DQ448713) (cid:2) (cid:2) (cid:6) e
Micromonospora 22 CNJ878(DQ448714) (cid:6) (cid:6) (cid:6) m
Salinispora 23 CNS051(DQ448715) (cid:6) (cid:6) (cid:6) .a
Salinispora 23 CNS205(CP000850) (cid:6) (cid:6) (cid:6) s
Salinispora 24 CNS055(DQ224159) (cid:6) (cid:2) (cid:6) m
Salinispora 24 CNS143(DQ092624) (cid:6) (cid:6) (cid:6) .
Salinispora 24 CNS237(DQ318246) (cid:6) (cid:6) (cid:6) or
g
Mycobacteriaceae Mycobacterium 25 CNJ859(DQ448716) (cid:6) (cid:2) (cid:6) /
Mycobacterium 26 CNJ823(DQ448717) (cid:6) (cid:2) (cid:6) o
n
Nocardiaceae Nocardia 27 CNS044(DQ448718) (cid:6) (cid:2) (cid:6) J
a
Nocardioidaceae Aeromicrobium 28 CNJ889(DQ448719) (cid:2) (cid:2) (cid:2) n
Marmoricola 29 CNJ780(DQ448720) (cid:2) (cid:2) (cid:2) u
a
Marmoricola 30 CNJ872(DQ448721) (cid:2) (cid:2) (cid:6) r
Nocardioides 31 CNJ892(DQ448722) (cid:2) (cid:2) (cid:2) y

8
Nocardiopsaceae Nocardiopsis 32 CNR923(DQ448723) (cid:6) (cid:2) (cid:6) ,
2
Promicromonosporaceae Promicromonospora 33 CNJ734(DQ448724) (cid:2) (cid:2) (cid:2) 0
1
Pseudonocardiaceae Pseudonocardia 34 CNJ888(DQ448725) (cid:2) (cid:2) (cid:6) 9
Pseudonocardia 35 CNS139(DQ448726) (cid:2) (cid:2) (cid:6) b
Pseudonocardia 36 CNS004(DQ448727) (cid:2) (cid:2) (cid:6) y

g
Streptomycetaceae Streptomyces 37 CNJ962(DQ448737) (cid:6) (cid:2) (cid:6) u
Streptomyces 38 CNR872(DQ448734) (cid:6) (cid:2) (cid:6) e
Streptomyces 39 CNR877(DQ448740) (cid:2) (cid:2) (cid:6) st
Streptomyces 40 CNR880(DQ448735) (cid:2) (cid:2) (cid:2)
Streptomyces 41 CNR881(DQ448730) (cid:6) (cid:2) (cid:6)
Streptomyces 42 CNR884(DQ448728) (cid:2) (cid:2) (cid:6)
Streptomyces 43 CNR879(DQ448785) (cid:6) (cid:2) (cid:6)
Streptomyces 43 CNR885(DQ448739) (cid:6) (cid:2) (cid:6)
Streptomyces 44 CNR887(DQ448738) (cid:6) (cid:2) (cid:6)
Streptomyces 45 CNR918(DQ448731) (cid:6) (cid:2) (cid:6)
Streptomyces 46 CNR924(DQ448732) (cid:6) (cid:2) (cid:6)
Streptomyces 47 CNR925(DQ448742) (cid:6) (cid:2) (cid:6)
Streptomyces 48 CNR926(DQ448729) (cid:2) (cid:2) (cid:6)
Streptomyces 49 CNR927(DQ448786) (cid:6) (cid:2) (cid:6)
Streptomyces 49 CNR940(DQ448741) (cid:6) (cid:2) (cid:6)
Streptomyces 50 CNS177(DQ448736) (cid:6) (cid:2) (cid:6)
Streptomyces 51 CNR876(DQ448784) (cid:6) (cid:2) (cid:6)
Thermomonosporaceae Actinomadura 52 CNU125(DQ448743) (cid:6) (cid:2) (cid:2)
Total 26 5 38
aPrimerstargetedmodular,iterative,andhybridbutnotenediynePKSs.
2490 GONTANG ET AL. APPL.ENVIRON.MICROBIOL.
b.
of Hy 010000000000000000000000000000010000000000000100
ofclonescKStype: Iter. 100000000011000011100000000000000000000000010001
o.P d.
N o 001111111100111100011111111111101111111111101010
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by
%ntit 584560634975716651525361878986687555607577727172737272747068505057716971716869687573505356545452
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BLASTmatchpathwayproduct ChlorothricinMicrocystinMyxothiazolNystatinJamaicamideChlorothricinAmphotericinCandicidinJamaicamideMicrocystinChlorothricinChlorothricinRifamycinRifamycinRifamycinMeridamycinChlorothricinChlorothricinChlorothricinVicenistatinVicenistatinVicenistatinVicenistatinVicenistatinVicenistatinVicenistatinVicenistatinVicenistatinVicenistatinMeridamycinMeridamycinCuracinCuracinECO-02301ECO-02301ECO-02301ECO-02301ECO-02301ECO-02301ECO-02301SpinosadSpinosadMicrocystinChlorothricinJamaicamideJamaicamideJamaicamideChlorothricin Downloa
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aBLE2.KSaminoacidsequences TopBLASTmatch(sourceorganism),acloneidentifier chlB1Streptomycesantibioticus()mcyGMicrocystisaeruginosaKS1(),1mtaBStigmatellaaurantiaca()nysIStreptomycesnoursei()jamLLyngbyamajuscula()chlA2S.antibioticus()amphCStreptomycesnodosus()fcsCStreptomyces(sp.strainFR-008)jamJLyngbyamajuscula()mcyDMicrocystisaeruginosa()chlB1S.antibioticusKS1(),1chlB1S.antibioticus()rifBAmycolatopsismediterraneiKS2(),1247rifBKS3Amycolatopsismediterranei(),1247rifEAmycolatopsismediterraneiKS1(),1250merCStreptomycesviolaceusnigerKS1(),315chlB1S.antibioticusKS1(),2029chlB1S.antibioticusKS1(),2407chlB1S.antibioticusKS1(),4951vicAStreptomyceshalstedii()vicAS.halstedii()vicBS.halstedii()vicCS.halstedii()vicDS.halstedii()vicAS.halstedii()vicAS.halstedii()vicBS.halstedii()vicDS.halstedii()vicDS.halstedii()merCS.violaceusniger()mcyEMicrocystisaeruginosa()curGLyngbyamajuscula()curILyngbyamajuscula()ecoCStreptomycesaizunensis()ecoCS.aizunensis()ecoCS.aizunensis()ecoCS.aizunensis()ecoCS.aizunensis()ecoCS.aizunensis()ecoGS.aizunensis()spnDSaccharopolysporaspinosa()spnDSaccharopolysporaspinosa()mcyEMicrocystisaeruginosa()chlB1S.antibioticusKS1(),1jamKLyngbyamajuscula()jamPLyngbyamajuscula()jamKLyngbyamajuscula()chlB1S.antibioticus() on Janhttp://aem.asm.org/m
A u
T NCBIccessionno. FJ844611FJ844612FJ844613FJ844614FJ844615FJ844616FJ844617FJ844618FJ844619FJ844620FJ844621FJ844622FJ844623FJ844624FJ844625FJ844626FJ844627FJ844628FJ844629FJ844630FJ844631FJ844632FJ844633FJ844634FJ844635FJ844636FJ844637FJ844638FJ844639FJ844640FJ844641FJ844642FJ844643FJ844644FJ844645FJ844646FJ844647FJ844648FJ844649FJ844650FJ844651FJ844652FJ844653FJ844654FJ844655FJ844656FJ844657FJ844658 ary 8,
a 2
0
No.ofuniqueclones 112 114 117 5 8 11 1112 19 b
y
No.ofclonesequenced 2262 1213 3298 45 40 40 1123 gues
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a
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Nearesttypestrain ActinomaduracremeaGordonianitidaMicromonosporaendolithi MycobacteriumbrisbanensMycobacteriumporiferaeNocardiaarthritidis NocardiopsislucentensisSalinisporaarenicolaSalinisporaarenicola Salinisporapacifica SalinisporapacificaA SalinisporapacificaB SerinicoccusmarinusStreptomycesaureofaciensStreptomycesbikiniensisStreptomycescaviscabies
Strain NU-125NJ-863NJ-878 NJ-859NJ-823NS-044 NR-923NS-051NS-205 NS-143 NS-055 NS-237 NJ-927NR-876NR-881NR-918
CCC CCC CCC C C C CCCC
VOL.76,2010 SECONDARY-METABOLITE BIOSYNTHESIS 2491
000000000000000000000000000000000000000000 3
000000000000000000000000000000000000000000 8
111111111111111111111111111111111111111111 9
7
121121112122222233445561223441122234111223
589423107914540164623526277782356316548439
765777777888888977779979999995577867777675
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d
n
ECO-02301EpothiloneJamaicamideCandicidinMeridamycinLipomycinConcanamycinMeridamycinVicenistatinKijanimicinKijanimicinKijanimicinKijanimicinKijanimicinKijanimicinKijanimicinMeridamycinMeridamycinSpinosadSpinosadTautomycinTautomycinKijanimicinActinofuranoneActinofuranoneActinofuranoneTetronomycinTetronomycinTetronomycinCuracinCuracinECO-02301ECO-02301ECO-02301JamaicamideVicenistatinECO-02301ECO-02301ECO-02301SpinosadSpinosadEpothilone presentedinFig.1a Download
ecoAS.aizunensis()epoDPolyangiumcellulosum()jamLLyngbyamajuscula()fcsCStreptomyces(sp.FR-008)merCS.violaceusniger()lipCS.aureofaciens()conBStreptomycesneyagawaensis()merBS.violaceusniger()vicAS.halstedii()kijDActinomadurakijaniataKS2(),1kijAA.kijaniataKS4(),2kijAA.kijaniataKS4(),3kijCA.kijaniataKS1(),4kijCA.kijaniataKS1(),5kijAActinomadurakijaniataKS4(),6kijBA.kijaniataKS2(),7merAS.violaceusnigerKS3(),8merAS.violaceusnigerKS3(),9spnASaccharopolysporaspinosaKS1(),12spnASaccharopolysporaspinosaKS1(),13ttmCStreptomycesspiroverticillatusKS1(),10ttmCS.spiroverticillatusKS2(),11kijAA.kijaniataKS2(),14furAS.aculeolatuKS3(s),1furAS.aculeolatusKS2(),2furDS.aculeolatusKS1(),3tmnAIStreptomycesKS3(sp.strainNRRL11266),4tmnAIVStreptomycesKS1(sp.NRRL11266),5tmnAIVStreptomycesKS2(sp.NRRL11266),6curALyngbyamajuscula()curALyngbyamajuscula()ecoIS.aizunensis()ecoHS.aizunensis()ecoHS.aizunensis()jamLLyngbyamajuscula()vicDS.halstedii()ecoAS.aizunensis()ecoAS.aizunensis()ecoAS.aizunensis()spnESaccharopolysporaspinosa()spnESaccharopolysporaspinosa()epoDPolyangiumcellulosum() biosyntheticpathways.Cloneidentifierscorrespondtothoseareinbold. on Janhttp://aem.asm.org/ed from
edes u
FJ844659FJ844660FJ844661FJ844662FJ844663FJ844664FJ844665FJ844666FJ844667FJ844668FJ844669FJ844670FJ844671FJ844672FJ844673FJ844674FJ844675FJ844676FJ844677FJ844678FJ844679FJ844680FJ844681FJ844682FJ844683FJ844684FJ844685FJ844686FJ844687FJ844688FJ844689FJ844690FJ844691FJ844692FJ844693FJ844694FJ844695FJ844696FJ844697FJ844698FJ844699FJ844700 characterizASTmatch ary 8,
yL 2
allB 0
2 12 112 14 6 7 6 erimentthetop 19 b
3 23 112 41 14 8 35 edwithexpidentityto y gues
StreptomyceschartreusisR-924 StreptomyceshebeiensisR-872StreptomyceslydicusS-177 StreptomycessampsoniiJ-962S.sampsoniiR-887StreptomycestendaeR-879 S.tendaeR-885 StreptomycesthermocoprophilusR-925 S.thermocoprophilusR-927 S.thermocoprophilusR-940 al TopBLASTmatchesaretoKSdomainsassociat(cid:1)KSaminoacidsequenceswith85%aminoacidMod.,modular;iter.,iterative;hyb.,hybrid. t
N NN NNN N N N N ot a b c
C CC CCC C C C C T
2492 GONTANG ET AL. APPL.ENVIRON.MICROBIOL.
mixturewasaddedtothecompetentcells,thecellswereincubatedonicefor30 matography(HPLC),resultinginpurifiedactinofuranoneB,whichwasanalyzed
min,heatshockedat42°Cfor30s,andthenallowedtorecoverduring1hof byHRESI-MS.
shakingat37°Cin250(cid:1)lofSOCmedium(Invitrogen).Followingrecovery,25 Totestfortheproductionofcompoundsinthekijanimicinclass,strainCNR-
(cid:1)lofthetransformedcellswereplatedontoLBagarcontainingX-Gal(5-bromo- 885 was cultured in 20 2.8-liter Fernbach flasks, each containing 1 liter of
4-chloro-3-indolyl-(cid:7)-D-galactopyranoside) and kanamycin (50 (cid:1)g/ml). Following mediumA1BFe(cid:6)Cbyfollowingthepreviouslydescribedscale-upprocedures.
overnightincubationat37°C,vectorswereisolatedusingQiagen’sQIAprepSpin TheculturewasextractedwithAmberliteXAD-16resinonday7aspreviously
Miniprepkitaccordingtothemanufacturer’ssuggestedprotocol(QiagenInc.,Va- described,andfollowingremovaloftheacetone,theresultingaqueouslayerwas
lencia,CA).Followingisolation,thevectorinsertsweresizedusingtherestriction freeze-dried. The resulting material was fractionated using silica gel column
enzymeEcoRI(NewEnglandBiolabs,Inc.,Beverly,MA)andsequencedusingthe chromatographywithastepgradientofisooctane,ethylacetate,andmethanol.
M13FprimeronanABI3100DNAsequencerattheDNASequencingShared ThemethanolfractionwasfurtherpurifiedusingC chromatographywithastep
18
Resource,UCSDCancerCenter(fundedinpartbyNCICancerCentersupport gradientofwaterandacetonitrilefollowedbyreversed-phaseHPLCusing55%
grant2P30CA23100-18). aqueousacetonitrile.Thisresultedinapartiallypurifiedcompoundthatpos-
Sequenceanalyses.From1to98clonesweresequencedfromeachPCRthat sessedLC-MScharacteristicsindicativeofthekijanimicinclass.Thiscompound
yieldedacorrectlysizedproduct.Allnucleotidesequenceswereanalyzedand wasfurtheranalyzedbyHRESI-MSandbycarbonnuclearmagneticresonance
manually edited using the Sequencher software package (version 4.5; Gene (NMR)(CDCl3)usinga300-MHzVarianInovaNMRspectrometer.
CodesCo.,AnnArbor,MI),andsequenceswereverifiedusingtheNCBI(http: Methodvalidation.KSsequencesfromtypeIPKSpathwayswereobtained
//www.ncbi.nlm.nih.gov/)BasicLocalAlignmentSearchTool(BLAST).TypeI fromthegenomesequenceofSalinisporaarenicolastrainCNS-205(NCBIac- D
KSdomainsequencesweretranslatedusingtheSequenceManipulationSuite cessionno.NC009953)byusingKSdomainsfromtherifamycin(CAA11035) o
(http://www.bioinformatics.org/sms2/) and compared to sequences within the and calicheamicin (AAM94794) biosynthetic pathways to query the genome w
NCBIproteindatabaseusingBLASTp.ThetopBLASTpmatchesreportedare sequenceusingtheJointGenomeInstitute(JGI)BLASTsearchengine(http: n
forKSsequencesassociatedwithexperimentallycharacterizedbiosyntheticpath- //genome.ornl.gov/microbial/sare/).Theresultingsequenceswerealignedusing lo
a
ways. ClustalX version 1.83 (42) and manually edited using MacClade version 4.07 d
AreferencedatasetofKSsequenceswasassembledfromPKSgenesassoci- (32),andadistanceneighbor-joiningtreewasconstructedusingPAUP(33).To e
atedwith36experimentallycharacterizedpathways(22weredescribedinpre- testtheeffectivenessofthetypeIPKSprimers,KSdomainsfromstrainCNS-205 d

vious studies [18, 19], and 14 are described in Table S1 in the supplemental wereamplified,cloned,andsequencedasdescribedabove.Toassesstheeffectof fr
o
material).The368KSsequencesinthisreferencedatasetwerealignedusing PCRbias,thecloningexperimentswereperformedfollowingasinglePCRand m
ClustalX version 1.83 (34) and manually edited using MacClade version 4.07 after three separate PCR mixtures were pooled. In addition, nondegenerate

(28).Toestimatethenumberofwithin-strainKSsequenceclusters,allunique primersspecificfortheKSdomainsofSare1248,Sare1249,Sare3151,andthe h
t
KSaminoacidsequencesobtainedinthisstudywerealignedwiththereference thirdmoduleofSare3156,whichwerenotdetectedinthecloningexperiments, tp
datasetandusedtocreatephylogenetictrees(datanotshown)constructedusing werealsotested(seeTableS2inthesupplementalmaterial). :
/
the neighbor-joining, unweighted-pair group method using average linkages /a
(UPGMA),andmaximum-parsimonymethodsinPAUP(33).Clusteringpat- e
m
ternswithinthesetreeswereexamined,andincaseswheretheKSdomainsfrom RESULTS
anindividualstrainwereinthesamecladeandhadtopBLASThitstothesame .a
pathway, those sequences were assigned to the same cluster. Stand-alone KS Distribution of biosynthetic genes. Sixty diverse actinomy- s
m
sequences were also assigned a distinct cluster number. KS sequences were ceteisolatesrepresenting18familylevelgroupings,25genera, .
identified as being associated with either modular, iterative, or hybrid PKSs o
basedonthecharacterizedfunctionofthetopBLASTmatchinthereference and 52 OTUs cultured as part of a prior study (20) were rg
dataset.ThehybriddesignationwasappliedonlyincaseswheretheKSdomain screened by PCR for the presence of KS domains associated /
wasdirectlydownstreamofanNRPSmoduleinthegeneclusterfromwhichthe with type I PKS (including enediyne) biosynthetic pathways o
n
referencedatasetwasderived. and A domains associated with NRPS pathways. In total, all J
Fermentationandchemicalanalysis.StrainCNR-925wasinoculatedfroma but 20 of the strains yielded sequence-verified products asso- a
frozenstockinto25mlofmediumA1(10gsolublestarch,4gyeastextract,2g n
ciated with at least one of the three targeted pathway types u
peptone,750mlnaturalseawater,and250mldeionized[DI]water).Theculture a
wasincubatedat27°Cwithshakingat230rpmfor3daysandthentransferred (Table1).Targetedlociwereobservedfromrepresentativesof r
y
toa2.8-literFernbachflaskcontaining1literofthesamemedium.Following 12 (66.7%) of the families and 13 (52.0%) of the genera
8
anotherthreedaysofincubation,25mlofculturewasaddedto1literofmedium screened.Morespecifically,typeIPKSlociweredetectedin26 ,

E1(10gglucose,10gsolublestarch,50mlcornsteepliquor,3gNaCl,1g 2
MgSO (cid:1)7HO, 5 g CaCO, 1 liter of DI water, pH 7.0), which is known to strains representing 22 of 52 OTUs, enediyne PKS loci were 0
4 2 3 detectedinfivestrainsrepresentingthreeOTUs,whileNRPS 1
supporttetronomycin(Tmn)production(27).Ondays3,5,and7,25mlwas 9
removed and extracted with 50 ml of ethyl acetate. The organic layers were loci were detected in 38 strains representing 32 OTUs.
b
separated, dried over anhydrous sodium sulfate, decanted, and concentrated Enediyne PKS loci were detected only in strains belonging to y
under vacuum. The resulting crude extracts were dissolved in methanol and thefamilyMicromonosporaceaeandrepresentedtheleastcom- g
analyzedat254and350nminthepositivemodeusinganAgilent1100liquid u
monofthethreebiosynthetictypestargetedinthisstudy. e
chromatography-massspectrometer(LC-MS)andagradientfrom10%aceto-
s
nitrileinwaterto100%acetonitrile.Fractionscontainingmassescharacteristic Sequenceanalysis.Foreachcorrectlysizedinsert,thetop t
oftetronomycinwereexaminedbyhigh-resolution(HR)electrosprayionization BLASTmatchwastoaKSorAdomain,confirmingthatthe
(ESI)-massspectrometry(MS)inpositivemodeusingaThermoScientificLTQ correctlocihadbeenamplified.Additionalsequencing(8to
OrbitrapXLmassspectrometer.Totestforactinofuranoneproduction,strain
98clones)oftheKSlibrarieswasperformedfor10ofthe26
CNR-925wasadditionallyinoculatedinto5litersofmediumA1BFe(cid:6)C[10g
starch, 4 g yeast extract, 2 g peptone, 1 g CaCO, 5 g Fe(SO)(cid:1)4HO, 5 g PCR-positive strains in an effort to assess genome-level se-
3 2 4 2
potassiumbromide(KBr),1liternaturalseawater,pH8.0]byfollowingasimilar quence diversity (biosynthetic richness). These strains were
scale-upprocedure.SterilizedAmberliteXAD-7resin(20g/liter)wasaddedon selected to include a mix of genera that are either recog-
day7andtheresultingmixtureshakenforanadditional4h.Theresinwasthen nized(e.g.,Streptomyces,Salinispora)ornotrecognized(e.g.,
collectedbyfiltrationthroughcheesecloth,washedwithDIwater,andextracted
Gordonia)asarichsourceofsecondarymetabolites.Attempts
overnightwithacetone.Theacetonewasthenseparatedfromtheresin,concen-
tratedinvacuo,andpartitionedbetweenwaterandethylacetate,andtheorganic were made to sequence these libraries until saturation, al-
fractionwasdriedinvacuo.Thecrudeextractwasthenseparatedusingasilica thoughinsomecases(e.g.,CNR-927),thismaynothavebeen
gelflashcolumn,elutingwithastepgradientofisooctanetoethylacetate.All achieved.Thedeeplysequencedlibrariesrevealedarangeof1
fractionswereanalyzedbyLC-MS,andthefractioncontainingacompoundwith
(CNJ-863)to14(CNR-885)uniqueKSalleles(Table2).Phy-
themolecularweightandUVpropertiesconsistentwithactinofuranoneBwas
logenetic analyses (data not shown) placed these sequences
identified.Thisfractionwasthensubjectedtofurtherchromatographicpurifi-
cationusingsemipreparativereversed-phase(C)high-performanceliquidchro- intoasmanyassixdistinctclusters,indicatingthatsomestrains
8
VOL.76,2010 SECONDARY-METABOLITE BIOSYNTHESIS 2493
may possess as many as six different PKS biosynthetic path- maybearichsourceofdiverse,polyketide-derivedsecondary
ways. metabolites.
The vast majority (88%) of the KS sequences are most TheremainingKSsequencesdetectedinthisstudyhadtop
closelyassociatedwithmodulartypeIPKSs,basedonBLAST BLASTmatchesthatrangedfrom45to84%aminoacididen-
and phylogenetic analyses (Table 2). Of the 90 unique KS tity to KS domains from 15 different biosynthetic pathways.
sequences that were identified, 14 shared (cid:1)85% amino acid ThemostcommontopBLASTmatchesweretoKSsequences
identity with their top BLAST matches (Table 2). These 14 associated with chlorothricin and jamaicamide biosynthesis,
sequences were derived from strains CNS-205 (3 sequences), which was observed in each of seven strains. Interestingly,
CNR-885(5sequences),andCNR-925(6sequences).Thetop strainsCNJ-927(Serinicoccusmarinus)andCNR-923(Nocar-
BLAST matches for these 14 sequences are to genes respon- diopsis lucentensis) belong to different families within the
sibleforthebiosynthesisoffivedistinctsecondarymetabolites Actinomycetales yet possess KS sequences that cluster closely
(Table2).CNS-205wasincludedlargelyformethodvalidation together(Fig.1),suggestingthattheyhaveundergonearecent
(see below), as a genome sequence is available for this strain horizontal gene transfer (HGT) event. The pathways associ-
(40)andithaspreviouslybeenshowntoproducePKS-derived atedwiththesetwosequenceslikelyproducethesamemetab- D
metabolites in the rifamycin class (26). As expected, KS se- olites, although they are not likely to be closely related to o
w
quences with a high percent identity (86 to 89%) to those chlorothricingiventhelowlevelofsequenceidentity(53%)to n
associatedwithrifamycinbiosynthesisweredetectedfromthis thispathway(Table2).Itisalsoofinterestthattherewaslittle lo
overlap in the top BLAST hits for Salinispora pacifica strains a
strain(Table2). d
CNS-143 and CNS-055 with those obtained for the “B” phy- e
The six KS sequences obtained from strain CNR-925 all
d
shared(cid:8)90%aminoacididentitywithhomologoussequences lotypeofthisspecies(CNS-237),suggestingthatdifferentphy- f
from experimentally characterized pathways. Three of these lotypes within the same species may produce distinct sets of ro
shared (cid:8)95% identity to sequences associated with the tet- polyketide-derivedsecondarymetabolites. m
InadditiontobeingdetectedamongmembersoftheStrep- h
ronomycin (Tmn) biosynthetic gene cluster (13), while the t
remaining three had an equally high level of identity to se- tomycetaceae,typeIPKSsequencesweredetectedamongac- tp
quences associated with the biosynthesis of the metabolite tinomycete taxa that are not traditionally recognized for the ://
production of secondary metabolites. These included strain a
E-837,whichwasconcurrentlyreportedfrombothStreptomy- e
CNJ-863,whichismostcloselyrelatedtotheunicellularacti- m
ces aculeolatus (5) and a marine-sample-derived Streptomyces
nomyceteGordonianitida,agenusforwhichnopriorreports .a
sp. (strain CNQ-766) under the name actinofuranone (12).
of secondary-metabolite production could be found. The KS s
Interestingly,theE-837-producingS.aculeolatusstrain(NRRL m
18422) was also reported to produce tetronomycin. A phylo- s(eshqaureinncge(cid:8)s9a9s%socaimatiendoawciitdhidthenistitsyt)r,atinhuwsienrdeiclaatrigneglythiadteinttmicaayl .or
genetic analysis revealed the close evolutionary relationships g
possess only one type I PKS pathway consisting of a single /
of the CNR-925 KS sequences with the two experimentally module. BLAST (Table 2) and phylogenetic (Fig. 1) analyses o
characterizedpathways(Fig.1)andledtothehypothesisthat n
revealedthattheseKSsweremostcloselyrelatedtothehybrid J
this strain had the genetic potential to produce secondary PKS-NRPS pathway responsible for microcystin biosynthesis a
metabolites related to both actinofuranone and tetronomycin n
(43).Thesesequences,however,sharedonly45%aminoacid u
(see “Fermentation studies,” below). For both biosynthetic a
identity,indicatingthatacloselyrelatedbiosyntheticpathway r
pathways,theKSsequencesfellintotwoseparateclusters(Fig. y
hasyettobecharacterized.Chemicalstudiesofthisstrainhave
1;Table2),providingexamplesinwhichtheKSdomainsfrom 8
yettoyieldanyhybridPKS-NRPSsecondarymetabolites. ,

onepathwaydonotallhavesimilarevolutionaryhistories.For 2
Fermentationstudies.Fermentationstudieswereperformed 0
thisreason,KSclusteringpatternsprovideanestimateofthe to test the hypothesis that the production of specific secondary 1
maximum as opposed to the absolute number of distinct bio- 9
metabolites could be predicted in cases where KS sequences
b
syntheticpathwaysthatmaybepresentinastrain. shared(cid:1)85%aminoacididentitywithexperimentallycharacter- y
StrainCNR-885yieldedthelargestnumberofKSsequence izedpathways.InthecaseofCNR-925,forwhichtheproduction g
u
clusters(six)(Fig.1)anddistinctloci(14),ofwhichfiveshared of tetronomycin and actinofuranone were predicted, the strain e
(cid:1)85% amino acid identity with top BLAST hits from experi- was first cultured in the medium from which tetronomycin was st
mentally characterized biosynthetic pathways (Table 2). Of originallyisolated(27).Onday5,amolecularionthatmatched
these five sequences, three had top BLAST matches to genes themassoftetronomycinwasdetectedintheethylacetateextract
associatedwiththebiosynthesisofthepolyketide-derivedmac- of the culture. The UV absorbance maxima associated with
rolidekijanimicin(34).TwoadditionalKSsequencesformeda this compound also matched those of tetronomycin (wave-
separateclusterandhadhighlevelsofsequenceidentitywith lengths of maximum absorption [(cid:9) ], 252 and 301) (27)
max
KSs associated with the biosynthesis of the polyketide tauto- and subsequent high-resolution mass spectral data con-
mycin (30). These close affiliations led to the hypothesis that firmed the presence of the tetronomycin sodium salt in the
CNR-885hasthegeneticpotentialtoproducesecondaryme- extract(Fig.2).Totestforactinofuranoneproduction(12),
tabolites in both the kijanimicin and tautomycin classes. De- LC-MSanalysesofCNR-925extractsrevealedacompound
spitesequencingof41clonesfromthisstrain,12ofthealleles having a molecular ion and UV profile diagnostic for ac-
weredetectedonlyonceortwice,whiletwoweredetected12 tinofuranone ((cid:9) , 241 and 282) (12). Subsequent high-
max
timeseach(Fig.1),indicatingclearPCRbias.Thedetectionof resolution mass spectral analysis of the purified compound
six KS lineages in CNR-885, with closest BLAST matches to confirmed the presence of actinofuranone B in the extract
fourdifferentPKSpathways,providesevidencethatthisstrain (Fig. 2).
D
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lo
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e
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2494
VOL.76,2010 SECONDARY-METABOLITE BIOSYNTHESIS 2495
D
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lo
a
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e
d

f
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m

h
t
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FIG. 2. Tetronomycin and actinofuranone structure confirmation. (A and B) High-resolution mass (A) and structure (B) of tetronomycin a
(sodiumsalt);(CandD)high-resolutionmass(C)andstructure(D)ofactinofuranoneB.MW,molecularweight;MF,molecularformula;mmu, ry
milli-massunits. 8
,

2
0
InthecaseofCNR-885,theproductionofcompoundsinthe HPLC,yieldingapurecompound.Thehigh-resolutionmassof 1
9
kijanimicinandtautomycinclasseswaspredictedbasedonthe thiscompoundwas1,209.5958(m/z[M(cid:6)Na](cid:6)),anditsmolec- b
KSanalyses.FollowingcultivationinmediumA1BFe(cid:6)C,the ular formula was C H N O (Fig. 3), which corresponds y
61 90 2 21
g
acetone extract of organic materials sequestered by XAD-16 precisely to that reported for kijanimicin 53 (34). A further
u
resin was subjected to column chromatography and then analysis of the carbon NMR spectrum for this compound in e
s
t
FIG. 1. Actinobacterial KS phylogeny. Neighbor-joining distance tree constructed using aligned KS domain sequences (223 amino acid
positions)fromtypeIPKSpathwaysclonedfromstrainsCNJ-863,CNJ-927,CNR-885,CNR-923,andCNR-925(inbold).Strainnumbersare
followedbytheclone’sidentificationnumber(Table2),thenumberofidenticalclonessequenced(inbrackets),andtheaccessionnumber.Inthe
caseofCNR-885,KSclustersaredesignatedwitha“C”number,thepredictedproductofthepathway,andthepercentageofaminoacididentity
toKSsequencesfromthatpathway.ThefoursequencescomprisingclustersC3andC4wereseparatedintotwoclustersbecausetheyhavetop
BLASTmatchestodifferentbiosyntheticpathways(Table2).Forreference,theKSdomainsequencecorrespondingtothetopBLASTmatchfor
eachcloneisincludedinthetree,alongwithanyadditionalKSsequencesinthatgene.AlsoincludedforreferencearealloftheKSsequences
fromtheactinofuranone(Fur),kijanimicin(Kij),meridamycin(Mer),microcystin(Mcy),nostopeptolide(Nos),rapamycin(Rap),spinosad(Spn),
tautomycin(Ttm),andtetronomycin(Tmn)biosyntheticpathways.Referencesequencesareidentifiedbythenameoftheassociatedproduct(in
boldifthisproductispredictedbasedonahighlevelofsequenceidentity),proteinname,KSdomainnumberinthatgene,andNCBIaccession
number(inparentheses).AKSdomainfromthetypeIfattyacidsynthaseinvolvedinmycocerosicacidbiosynthesiswasusedtopositiontheroot.
Bootstrapvalues(inpercentages)calculatedfrom1,000resamplingsusingtheparsimonyfullyheuristicsearchareshownattheirrespectivenodes
for values of (cid:1)60%. Although some within-pathway branching patterns changed when neighbor-joining, UPGMA, and maximum-parsimony
treeingmethodswereapplied,theoveralltreetopologywasmaintained.
2496 GONTANG ET AL. APPL.ENVIRON.MICROBIOL.
D
o
w
n
lo
a
FIG. 3. Kijanimicin53structureconfirmation.High-resolutionmass(A)andstructure(B)ofkijanimicin53. d
e
d

f
r
o
comparison to literature values (34) confirms the identity of mentarity. For example, KS1 of Sare4951 was amplified de- m
this compound (see Table S3 in the supplemental material). spiteatotaloffiveprimer/templatemismatches,whileKS1of h
t
StrainCNR-885wasadditionallyculturedinthemediumfrom Sare3154 was not amplified even though there were only two tp
whichtautomycinwasoriginallyreported(11);however,there mismatches(seeTableS2inthesupplementalmaterial).How- :/
/
was no evidence for the production of this or related com- ever,specificprimersdesignedfortheKSdomainspresentin a
e
pounds over the course of the fermentation. In the case of Sare1248, Sare1249, Sare3151, and KS3 of Sare3156 (Table m
strain CNS-205, where KS sequences sharing (cid:1)85% amino S2), which were not observed among the original 47 clones .a
acid identity with those associated with the biosynthesis of sequenced, readily amplified these sequences, indicating that s
m
rifamycinwereobserved,theproductionofthiscompoundhas PCR bias was not due to template access. Cloning the com- .
o
already been reported (26), so no further studies were per- bined products of three individual PCRs did not increase the r
g
formed.Thus,infouroffivecaseswiththreedifferentstrains, numberofKSdomainsdetected. /

when KS sequences shared (cid:1)85% amino acid identity with o
n
homologouslocifromexperimentallycharacterizedpathways, DISCUSSION J
compounds identical or closely related to those produced by a
n
the characterized pathways were detected. No efforts were Microbialnaturalproductsrepresentaremarkablesourceof u
madetotestfortheproductsofPKSgenesthatshared(cid:10)85% small-moleculediversityfromwhichnewdrugsandotheruse- ar
y
sequenceidentitytothereferencesetofexperimentallychar- ful products have been developed. At present, contemporary
8
acterizedpathways. approachestonatural-productdiscoveryincludemethodssuch ,

2
Method validation. A total of 27 type I KS domains were asgenomescanning(49,50)andphylogeneticprediction(28), 0
obtainedfollowingabioinformaticanalysisofthegenomese- which incorporate DNA sequencing and bioinformatic analy- 1
9
quenceofSalinisporaarenicolastrainCNS-205.Asubsequent ses into the discovery process. The research described here
b
analysisplacedtheseKSsequencesintoninebiosyntheticpath- extendstheseconceptsbyprovidinganassessmentofthebio- y

ways, of which two are modular, three are iterative, two are synthetic capabilities of a taxonomically diverse collection of g
u
hybrid (PKS-NRPS), and two are associated with enediyne marine-sediment-derived actinomycetes (20). The goals were e
s
biosynthesis(40).ThetypeIKSprimersemployedformethod toscreenthiscollectionforgenesassociatedwiththeproduc- t
validation were designed to detect all but those sequences tionofspecifictypesofsecondarymetabolitesandtotestthe
associated with enediyne biosynthesis (25 in total). To test hypothesis that a relatively small amount of sequence data
their effectiveness, a single PCR and cloning experiment was from the appropriate genetic loci can be used to predict sec-
performedusinggenomicDNAfromstrainCNS-205.Despite ondary-metabolite production in cases where the sequences
sequencing47clones,onlysevenoftheexpectedKSdomains have a high level of identity to experimentally characterized
wereamplified(seeFig.S1andTableS2inthesupplemental biosyntheticpathways.
material). However, these sequences represented five of the PCR screening of 60 marine-sediment-derived actinomy-
sevenbiosyntheticpathwaystargetedbytheprimers,including cetes revealed that genes associated with secondary-metab-
both modular and all three iterative pathway types. With the olite biosynthesis were common yet unevenly distributed
exceptionsofSare3282andSare3156(KS1),thephylogenetic among different taxa. The detection of PKS and NRPS
analysis reveals a clear association of the KS sequences with genesamongstrainsrelatedtogenerasuchasGordoniaand
referencesequencesofthepredictedPKStype(seeFig.S1in Kocuriasuggeststhatthesepoorlystudiedgenerarepresent
thesupplementalmaterial).Therewasaclearbiasforcertain an underexplored resource for natural-product discovery.
KS domains that was not easily explained by primer comple- Ourinitialchemicalanalysesofthesestrains,however,have