Polyketide synthase pathways identified from a metagenomic library are derived from soil ...

12 Pages·2011·0.54 MB·English

RESEARCH ARTICLE
Polyketidesynthasepathwaysidenti¢edfromametagenomic
libraryarederivedfromsoilAcidobacteria
LarissaC.Parsley1,JonathanLinneman2,AnnM.Goode1,KristenBecklund3,IsabelleGeorge4,
RobertM.Goodman5,NicoleB.Lopanik2&MarkR.Liles1
1DepartmentofBiologicalSciences,AuburnUniversity,Auburn,AL,USA;2DepartmentofBiology,GeorgiaStateUniversity,Atlanta,GA,USA;
3DepartmentofPlantPathology,UniversityofWisconsin,Madison,WI,USA;4Unite´deGe´nieBiologique,Universite´CatholiquedeLouvain,Louvain-la-
Neuve,Belgium;and5SchoolofEnvironmentalandBiologicalSciences,Rutgers,TheStateUniversityofNewJersey,NewBrunswick,NJ,USA
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Correspondence:MarkR.Liles,Department Abstract fro
ofBiologicalSciences,AuburnUniversity,101 m
LifeSciencesBuilding,120W.SamfordAve., Panotliybkieottiidceosraarentsitcraunccteurraalcltyivditiyv.erTsyepeseIcomnoddaurylarmpeotalybkoelittiedse,smynatnhyasoef(wPKhiSc)hghenavees http
Auburn,AL36849,USA.Tel.:[phone] s
1656;fax:[phone]; are typically large and encode repeating enzymatic domains that elongate and ://a
c
e-mail:[email protected] modify thenascentpolyketidechain.Afosmidmetagenomic libraryconstructed a
d
e
from an agricultural soil was arrayed and the macroarray was screened for the m
Received10September2010;revised22April presence of conserved ketosynthase [b-ketoacyl synthase (KS)] domains, enzy- ic.o
2011;accepted26April2011. maticdomains present inPKSs. Thirty-fourclones containing KS domains were up
Finalversionpublishedonline23May2011. .c
identified bySouthern hybridization. Manyof theKSdomains contained within o
m
metagenomicclonessharedsignificantsimilaritytoPKSornonribosomalpeptide /fe
DOI:10.1111/j.1574-6941.2011.01122.x m
synthesis genes from members of the Cyanobacteria or the Proteobacteria phyla. s
e
Editor:ChristophTebbe However, analysis of complete clone insert sequences indicated that the BLAST c/a
analysis for KS domains did not reflect the true phylogenetic origin of many of rtic
Y Keywords these metagenomic clones that had a %G1C content and significant sequence le-a
G pkeotlyokseyntitdheassyen;tmhaestaeg;esoniol;mmicasc;rAoacirdraoyb;acteria. sainmAilcairditoybatoctegreinaeosrfirgoimnfmoremsebveerraslocflothneesphwyalusmfurAthciedrosbuapctpeorirat.edThbiysceovindcelunscieonthoatf bstrac
O cultured soil Acidobacteria from different subdivisions have genetic loci closely t/7
8
L related to PKS domains contained within metagenomic clones, suggesting that /1/1
O Acidobacteriamaybeasourceofnovelpolyketides.Thisstudyalsodemonstrates 76
theutilityofcombiningdatafromculture-dependentand-independentinvestiga- /52
C 1
tionsinexpandingourcollectiveknowledgeofmicrobialgenomicdiversity. 1
7
E 9
b
y
Y g
u
e
G s
Introduction tionofametagenomicapproachtoisolatelarge,contiguous t o
O regionsofDNAfromunculturedmicroorganismsisaviable n 0
6
L The isolation of secondary metabolites produced by soil strategy toaccessintact(ornearly intact)geneticpathways A
p
O mideicnrtoifoyrignagninsmovselhacsomhipstoournicdasllywbitehenaanntimefifcercotibviealmaecatnivsitoyf. tshtuadtieasrehaivnevodlevmedoninstrnataetdurtahlepurtoildituycotfssycnrteheensiins.gPenrevvirioonu-s ril 20
BI Soil environments contain an impressive 1010microbial mental metagenomic libraries to identify bioactive mole- 19
O cellsg(cid:2)1, while only a relatively small fraction of the extant cules(Courtoisetal.,2003),proteolyticsystems(Bejaetal.,
microorganisms has been cultivated in the laboratory 2000), and polyketide synthases (PKS) (Moffitt & Neilan,
R
(Hugenholtzetal.,1998;Curtis&Sloan,2005).Whilethis 2003; Ginolhac et al., 2004; Schirmer et al., 2005; Wawrik
C
small percentage of cultured soil microorganisms has al- etal.,2005).
I
M readyillustratedthephylogeneticdiversityofthesecommu- Polyketides are a diverse group of bioactive secondary
nitiesandhasprovidedmanyimportantantibioticstodate, metabolites produced by bacteria, fungi, and plants and
a potentially richer resource for novel antibiotic discovery include several therapeutically important drugs such as
resides within as yet uncultured bacteria (Rondon et al., erythromycin, tetracyclines, immunosuppressants FK506
2000;Gillespieetal.,2002;Handelsman,2004).Theapplica- and rapamycin, and antitumor compounds doxorubicin
(cid:3)c2011FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol78(2011)176–187
PublishedbyBlackwellPublishingLtd.Allrightsreserved
PolyketidesynthasepathwaysfromsoilAcidobacteria 177
andmithramycin(Staunton&Weissman,2001).Polyketide this type of biosynthetic pathway, such as members of the
biosynthetic pathways are generally classified into three phylumAcidobacteria.
majorgroups:TypeI,TypeII,orTypeIII.TypeIIPKSsare
composed of individual proteins that perform one enzy- Materialsand methods
matic activity iteratively to catalyze the formation of aro-
matic polyketides such as the antibiotic actinorhodin
Soil collection andDNA isolation
(Hertweck et al., 2007). Type III PKSs participate in the
Soilsampleswerecollectedfromsoilcores(10–50cmdepth)
assembly of small aromatic compounds and are found in
from an agricultural soil in the Central Sands area of
plantsandbacteria(Moore&Hopke,2001).TypeIPKSsare
Wisconsin at the University of Wisconsin-Madison’s Han-
large,modular,multifunctionalproteinscomposedofenzy-
cockAgriculturalResearchStation(HARS)inHancock,WI.
matic domains that typically perform a single reaction in
D
polyketide chain assembly (Fischbach &Walsh, 2006). The The soil was a Plainfield loamy sand taken from the plow ow
zone at HARS. Bacterial cells were collected after soil n
biosynthesisofTypeIpolyketidesproceedsinanassembly- lo
line fashion: the starter unit is loaded onto the loading homogenization by differential centrifugation and then ad
e
embeddedandlysedwithinanagaroseplug.High-molecu- d
moduleand transferred to thefirst extendermodule. After lar-weightmetagenomicDNAwasrecoveredandpurifiedas fro
extensionandmodificationbythefirstmodule,thenascent m
polyketideispassedontothenextextendermoduleuntilit describedpreviously(Lilesetal.,2008,2009). http
reeitahcehresathcyectlhizieodesoterraaselindeoamrafionr,mw.hIinchmreolsetasceasseths,emchoadinuliens Fosmid libraryconstruction s://ac
a
havethreeenzymaticdomainsthatarenecessarytocatalyze ThemetagenomiclibrarywasconstructedinthepCC1FOS dem
one cycle of chain elongation: b-ketoacyl synthase (KS), vector with the CopyControlTM fosmid library system ic
acyltransferase, and acyl carrier protein (ACP). The action according to the manufacturer’s instructions (Epicentre, .oup
ofoptionalb-ketoreductivedomains(ketoreductase,dehy- Madison, WI). Briefly, purified metagenomic DNA was .co
dratase,andenoylreductase)contributestothediversityof m
polyketide natural products. Tailoring enzymes such as raagnardoosmeglyelsbheefaorreedligaantidonsiiznetostehleecpteCdC1frFoOmSvaecptourls.eLdig-fiateeldd /fem
s
glycosyl transferases, methyltransferases, and monooxy- e
DNA was then packaged (MaxPlax Lambda Packaging c
greelneaasseesdcfraonmfuthrtehPeKrSm(oRdixifyettahl.e,2m00et2a)b.oTlhiteesmaofdteurlatrhiteyyaanrde E(Exptriacecnt)traen)datnitderepdlaotnedto othnetoEPLIu10ri0aT–MB-eTrt1anciloncionngtasitnrianing /article
colinearity of enzyme and product of Type I PKSs make 12.5mgmL(cid:2)1chloramphenicolfortheselectionofrecombi- -ab
s
them attractive for genetic engineering and combinatorial nantclones.Toinducetheproductionofhigherfosmidcopy tra
biosynthesis to produce novel metabolites (Kittendorf & numbers, arabinose (0.01% v/v) was added to the media ct/7
Sherman,2006). with the targeted clones. The recombinant Escherichia coli 8/1
Whilethesynthesisofpolyketidesisoftenassociatedwith /1
cloneswereroboticallypickedinduplicateontonylonmem- 7
6
bacteria within the phylum Actinobacteria (Staunton & branesattheClemsonUniversityGenomicsInstitute,result- /5
2
Weissman, 2001; Stinear et al., 2004), PKS genes and 1
ing in duplicate clone macroarrays. Macroarrays were 1
metabolites have also been identified in a diverse group of generated with and without arabinose induction of fosmid 79 b
bacteria, including speciesofPseudomonas (El-Sayed etal., y
copy number and contained 9216 fosmid clones with an g
2003), Stigmatella (Beyer et al., 1999), Sorangium (Schupp u
averageinsertsizeof42kb(c.100E.coligenomeequivalents). e
s
etal.,1995;Beyeretal.,1999;Julienetal.,2000),Myxococcus t o
(Simunovic et al., 2006), Bacillus (Chen et al., 2006), and Screening offosmid macroarrays n 06
Burkholderia (Partida-Martinez & Hertweck, 2005). The A
p
wbeicdaeuspsereaadddidtiiostnraiblurteisoenrvooifrsPoKfSpoplaytkhewtidayess iins soofilimntiecrreos-t PgeonoeleradtefoasmPiKdSDpNroAbesefrovreddoawsnastrteeammpllaitberairny ascrPeCenRintgo. ril 20
1
organisms likely exist, but have remained uncharacterized The b-ketosynthase (KS) domain was targeted with the 9
dueto cultivationbiases.Inthisstudy,weuseddegenerate degenerateprimerset5LLand4UU(Table1)(D.Sherman,
primersandprobestoidentifypotentiallynovelPKSpath- UniversityofMichigan,pers.commun.).Theamplification
ways from a soil metagenomic library via macroarray was conducted with a touchdown PCR, with a 2-min
hybridization.TheisolationandmanipulationofPKSpath- denaturationat951C,followedby14cyclesofatouchdown
waysusingametagenomicapproachprovidesaccesstothe PCR (951C for 30s, annealing beginning at 631C and
uncharacterized PKS pathways of as yet uncultured soil ramping down 11C per cycle to 501C for 30s, and 721C
microorganisms (Schirmer et al., 2005; Brady et al., 2009) for1min)andthen30cyclesofamplification(951Cfor30s,
andallowsforthetargeteddetectionofsuchpathwaysfrom 501Cfor30s,and721Cfor1min),andthen5minat721C.
specific bacterial taxa that arenot typically associated with Seven other Type I and Type II PKS-specific or NRPS-
FEMSMicrobiolEcol78(2011)176–187 (cid:3)c2011FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
178 L.C.Parsleyetal.
Table1. Oligonucleotideprimersusedinthisstudy Table2. ListofAcidobacteriastrains,theirphylogeneticclassification,
1 andthepresenceorabsenceofgeneticlociassociatedwithtypeIPKS
A3 GCSTACSYSATSTACACSTCSGG pathways
A7R SASGTCVCCSGTSCGGTAS Acidobacteria Acidobacteria KS mtaD
2 strain subdivision domain domain
K1 TSAAGTCSAACATCGGBCA
IGE012 1 1 (cid:2)
M6R CGCAGGTTSCSGTACCAGTA
IGE013 1 1 (cid:2)
3
IGE015 3 (cid:2) 1
KSLF CCSCAGSAGCGCSTSYTSCTSGA
IGE017 4 (cid:2) 1
KSLR GTSCCSGTSCCGTGSGYSTCSA
IGE001 6 (cid:2) 1
4
IGE002 6 1 1
KSDPQQF MGNGARGCNNWNSMNATGGAYCCNCARCANMG
D
KSHGTGR GGRTCNCCNARNSWNGTNCCNGTNCCRTG The Acidobacteria cultured isolates (George et al., manuscript in pre- ow
5 paration)werescreenedbyPCRusingeitherKSdomain(Table1,primer nlo
5LL GGRTCNCCIARYTGIGTICCIGTICCRTGIGC set5)ormtaD-specific(Table1,primerset9)primersets.Ineverycaseof ad
4UU MGIGARGCIYTICARATGGAYCCICARCARMG anegativePCRresult,acontrolPCRwasperformedandthegenomic ed
6 DNA template did provide a 16S rRNA gene amplicon using the fro
FR TTSGGGCASATNGCCCTGTGCGCAGYAGACBSCACTGCCT ‘puhnyilvoegrseanly’oBfatchteerAiacipdroimbaecrtser2ia7FKSanodrm1t4a9D2gRen(Leatniceloectiaarle.,p1re9s8e5n)t.edThine m http
7 540F GGITGCACSTCIGGIMTSGAC Figs2and4,respectively. s://a
c
1100R CCGATSGCICCSAGIGAGTG ad
Verificationandsequencing ofKS e
8 m
kSALPHA TTCGGCGGXTTCCAGTCXGCCATG domainsfrom positive clonesand ic
.o
ACP CCXATGCTCAGCXACCGCGACGACCT Acidobacteria-cultured isolates up
9 .c
o
mtaD-F GTGGTGCTGGAGCAGTCG To confirm that the 34 hybridization-positive clones were m
mtaD-R TCACCGCTCAGCGATGTC unique and to estimate insert size, restriction fragment /fem
length polymorphism (RFLP) profiles were generated for se
Primersets1through8wereusedinthegenerationofketosynthase- c
eachcloneusingtherestrictionenzymeBamHI.Theresulting /a
sGpiencoilfihcacpreotbaels.,(S2e0o0w4;eAtyaul.s,o1-9S9a7ci;dMoe&tsGa-eKneitlleoluade,t2a0l.0,52;00W2a;wPireikl,2et00a2l.;, digest reactions were electrophoresed on a 0.8% (w/v) 1(cid:4) rticle
2005).Primerset5,specifictoTypeIKSdomains,wastheonlyprimerset TAE pulsed-field agarose gel at 5Vcm(cid:2)1 with a 1–15-s -a
b
thatyielded aPCRproductusing apooled metagenomic library DNA switchtimefor16h. stra
DteNmAplatetemwplhaitlee.noPtrigmiveirngseatsi9miwlaarsprsopdeuccifitcwittohEthscehemritcahDiachoolmigoelnoogmouics Twenty-one of the KS-positive clones produced a KS ct/7
amplicon following PCR with the 5LL/4UU primer set to 8
sequencepresentinfosmidcloneA12andwasusedtoidentifymtaD /1
verifythepresenceofaKSdomain.Toobtainsequencedata /1
genesequencesfromculturedAcidobacteria. 7
from these clones, the KS primers were modified with T3 6/5
(5LL)andT7(4UU)primerrecognitionsequencesatthe50- 21
1
specificprimer setswerealsoevaluated forprobesynthesis endofeachrespectiveprimerandusedinasecondaryPCR 79
(Table1),butwerenotusedbecausetheyeitheryieldedPCR togenerateKSampliconsforsequencing.TheKSgeneswere by
g
productsfromcontrolreactionscontainingE.coligenomic thensequencedusingprimersT3andT7,whichresultedin u
e
DNA as a template or did not yield the expected PCR higherqualitysequencedatathanusingthedegenerate5LL st o
product based on sequences of representative amplicons. or 4UU primers. All 34 KS-positive clones were end n 0
The Type I KS domain amplicon resulting from PCR with sequencedusingthevectorprimersEpiFOSFandEpiFOSR. 6 A
tuhseingprtihmeeDrsIG5LPLCRanSdyn4tUhUesiswsaysstleambe(leRdocwhiet,hInddigiaonxaigpeonliisn, bacKteSridaogmenaionmsewseoreutalosof tPhCeRsixamApcliidfioebdacftreormia tihsorleaeteAsctidhoa-t pril 20
1
IN)andusedasaprobeinmacroarrayscreeningbycolony weretested(I.F.George,M.Hartmann,M.R.Liles&S.N. 9
blot hybridization at 421C with medium hybridization Agathos, unpublished data) (Table 2). Each Acidobacteria
washing stringency as per the manufacturer’s instructions. isolate was grown on 1/100th strength nutrient agar for 3
The hybridization was performed using the digoxigenin weeksandbacterialcellswererecoveredfromtheplateusing
detection system (Roche) with detection mediated by the asterileswab.GenomicDNAwasextractedfromAcidobac-
CSPD chemiluminescent substrate. The detection of posi- teriaculturesusingagenomicDNAisolationkit(Promega).
tivecloneswasaccomplishedaftermultipleexposuretimes The5LL/4UUprimersetwasusedinaPCRwithAcidobac-
for each blot. Clones that exhibited chemiluminescence in teria genomic DNA as a template, as described above.
duplicate (n=34) were considered KS positive and sub- Because the yield of genomic DNA was low for each
jectedtofurtheranalyses. Acidobacteriaisolate,acontrolPCRtargetingthe16SrRNA
(cid:3)c2011FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol78(2011)176–187
PublishedbyBlackwellPublishingLtd.Allrightsreserved
PolyketidesynthasepathwaysfromsoilAcidobacteria 179
gene was performed for each DNA sample using the structions.Onehalfofapyrosequencingplatewasdevoted
‘universal bacteria’ primers 27F (50-AGAGTTTGATC tothefosmidclones,providingatleast47-foldcoveragefor
MTGGCTCAG-30) and 1492R (50-GYTACCTTGTTAC each fosmid DNA insert. Primer walking and Sanger
GACTT-30)(Laneetal.,1985),andonlyisolatesthatyielded sequencingwasusedtoextendDNAsequencesfromfosmid
a16SrRNAgeneampliconwereincludedinthisstudy.KS clone contigs. DNA sequencing reactions were conducted
domain PCR products were cloned intothe pCR2.1vector usingafosmidDNAtemplatewithspecificoligonucleotide
(Invitrogen, Carlsbad, CA) and sequenced using vector primers at the Lucigen Corporation or the Georgia State
primers.AconsensusDNAsequencewasgeneratedforeach University DNA/Protein Core facility (Atlanta, GA). The
respective KS amplicon derived from an Acidobacteria insert sequences for clones A2, A3, A11, A12, and C5
culture. The KS domain sequences were submitted to were submitted to GenBank under the accession numbers
GenBankundertheaccessionnumbersJF342575–JF342588. ofJF342589–JF342593,respectively.
D
o
w
n
KS domain phylogenetic analysis Bioinformatic analysis offosmid-derivedORFs lo
a
d
KSgenesequencesfrom19PCR-positivefosmidclonesand Fosmid DNA sequences generated by 454 sequencing were ed
three Acidobacteria isolates were aligned with KS domain assembled into contiguous fragments (contigs) using the fro
m
sequences from other bacterial sources usingCLUSTALX soft- CLC Genomics Workbench (Cambridge, MA) assembly h
ware. A minimum evolution phylogenetic tree was gener- algorithm, and contigs were analyzed for the presence of ttps
atedandbootstrapanalysis(5000iterations)wasconducted putative PKS-related genes. ORFs were identified using a ://a
c
usingMEGA4(Tamuraetal.,2007).NotethattheKSdomains GENEMARKheuristicapproachforgenepredictioninprokar- ad
e
from two of the 21 PCR-positive fosmid clones were yotes (http://opal.biology.gatech.edu/GeneMark/). Addi- m
excludedduetopoorsequencequality. tionally, GLIMMER 3.02 and NCBI’s ORF FINDER (http://www. ic.o
u
ncbi.nlm.nih.gov/gorf/gorf.html) were utilized to corrobo- p
.c
Southern hybridization of putativeKS-positive rate the predicted ORFs obtained from GENMARK analysis. om
clones The prediction of gene function was accomplished by /fe
m
comparing each ORF sequence against the GenBank nr/nt s
e
To investigate the 13 clones that were KS-positive after database using the BLASTP and BLASTN algorithms. Any pre- c/a
colony blot hybridization, but failed to yield a KS PCR dictedORFslackinganysignificanthomologytoothergene rtic
product,aSouthernhybridizationwasperformedusingthe products in GenBank (E value 40.001) were further le-a
same digoxigenin-labeled heterogeneous KS probe as that investigated for secondary structures (profiles, patterns, bs
used in previous experiments. Fosmid DNA from each of blocks, motifs, and protein families) using a web server tra
c
the13cloneswasrestrictiondigestedwithBamHI,electro- (http://motif.genome.jp/). Additional assembly and ORF t/7
8
phoresed through a 1% (w/v) 1(cid:4) TAE agarose gel, identificationforcloneA3wereperformedusingLASERGENE /1/1
transferred to a nylon membrane using the Whatman software (DNASTAR, Madison, WI). Putative gene func- 76
TurboBlotter system (Kent, UK), and hybridized with the tions were identified using BLASTX and BLASTPalgorithms in /52
1
KSprobe.DNAfragmentshybridizedtotheKSgeneprobe the GenBank nr/nt database, with further motif analysis 1
7
werevisualizedusingtheNBT/BCIPcolorimetricdetection generated by the ExPASy Proteomics Server (http://ca. 9 b
y
method(Roche). expasy.org). g
u
e
s
Insert sequencing offosmid DNA clones Analysis of cloneA12 t o
n
0
6
Ten clones wereselected forcomplete insert sequencing by Preliminary sequence data from clone A12 indicated that A
p
4an54dpfuynroctsieoqnuaelndcionmgabinasse.dFoosnmtihdeDirNpAredwiacsteedxtprhaycltoedgefnreotmic tShoilsibcalcotnereumsiataythuasvfreosmignthifiecpahnytluhmomAocliodgoybatoctetrhiea.gAendodmitieono-f ril 20
1
clonesA2,A3,A11,A12,B1,B8,B10,B11,C1,andC5using ally, a sequence with significant similarity to an MtaD-like 9
the Qiagen Large-Construct DNA Isolation kit (Qiagen, protein involved in polyketide (myxothiazol) biosynthesis
Valencia,CA),withanadditionalplasmid-safeexonuclease was detected on this clone. Because the fosmid clone A12
digestion step for 4h at 371C to remove contaminating insertsequencewasnotcomplete,aSouthernblotusingan
genomic DNA. Purified DNA was sent to the Lucigen mtaD probe was conducted to verify its presence on the
Corporation (Middleton, WI) for bar-coded shotgun clone. Purified DNA from fosmid A12 was restriction
subclone library construction and then sequenced at the digested with the enzymes BamHI, ClaI, EcoRI, EcoRV,
EnGenCore Center at the University of South Carolina Hind III, NotI, PstI, SalI, SmaI, SphI, or XbaIand electro-
(Columbia, SC) using a Genome Sequencer FLX system phoresed through a 0.8% (w/v) 1(cid:4) TAE agarose gel. The
(Roche, Nutley, NJ) according to the manufacturer’s in- digested DNA was transferred to a nylon membrane and
FEMSMicrobiolEcol78(2011)176–187 (cid:3)c2011FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
180 L.C.Parsleyetal.
hybridized with the mtaD homolog probe as described primer set 4UU/5LL that is specific to the KS domain, the
abovefortheKSdomainprobe. mostconservedregionofTypeIPKSpathways(Bejaetal.,
2000; Hertweck et al., 2007). The KS domain amplicon
Detection ofmtaDhomologsfrom cultured resultingfromapooledmetagenomiclibraryDNAtemplate
Acidobacteria wasselectedfordigoxigeninlabelingandsubsequentmacro-
arrayhybridization.
Purebacterialculturesrepresentingsubdivisions1,3,4,and
6ofAcidobacteria(Table2)werescreenedformtaDhomo-
Identification ofKS domain-containing clones
logous sequences by PCR using mtaD-specific primers
from thefosmid library
developed from the fosmid A12 sequence (Table 1). The
following touchdown thermal cycling program was used: Atotalof34clonesfromthearabinose-inducedmacroarray
initialdenaturingat941Cfor2min,followedby14cyclesof exhibited chemiluminescence after hybridization with the D
o
denaturingat941Cfor30s,stepwiseannealingfrom63to digoxigenin-labeledTypeIKSprobe(induplicate),indicat- wn
501C for 30s, and extension at 721C for 2min, and an ing the presence of a KS domain and therefore a possible loa
d
additional 30 cycles of the above with a 501C annealing PKSpathway(Fig.1a).Only19KS-containingcloneswere ed
temperature.Ampliconsofthepredictedsizeof482bpwere identifiedfromthemacroarraypreparedwithoutarabinose fro
m
subclonedintothepCR2.1vector(Invitrogen)accordingto induction(datanotshown),whichwerealsodetectedinthe h
the manufacturer’s instructions and inserts were amplified arabinose-induced macroarray in addition to 15 other ttp
s
viacolonyPCRusingthevectorprimersM13FandM13R. clones. The position of the duplicate clones relative to one ://a
c
The resulting PCR amplicons were purified using the another enabled the determination of the specific fosmid a
d
PromegaWizardPCRClean-upSystem(Madison,WI)and cloneidentityandRFLPprofilesofthe34clonesconfirmed em
submitted to the Auburn University Genetic Analysis that they contained unique inserts (Fig. 1b). Of the 34 ic.o
Laboratory for sequencing. The sequences were trimmed fosmidclonesthatwereidentifiedbyhybridization,only21 up
.c
for quality and compared with their nearest significant yielded a PCR product using primers 4UU and 5LL, o
m
neighbors in GenBank by multiple alignment (CLUSTALX) suggesting that the remaining 13 clones may contain KS /fe
m
andmaximumparsimonyanalysis(MEGA4). domains that are phylogenetically divergent from known s
e
genes or may be misidentified clones or clones with mis- c
/a
Results matcheswiththe4UU/5LLprimerset. rtic
le
-a
Generation ofaKS-specific probe Phylogenetic analysis ofKS-positive clones bs
tra
MultipleprimersetsspecifictoPKSorNRPSpathwayswere DNAsequences derivedfromKSampliconsobtainedfrom ct/7
tested for their use to prepare a probe for macroarray the fosmid clones were compared with the GenBank nr/nt 8
/1
hybridization (Table 1). In each experiment with different database and were found to have significant similarity to /1
7
primersets,apooledfosmidlibraryDNAtemplatewasused known KS genes (similarities ranging from 51 to 73%).As 6/5
2
as a template. Every primer set that generated a product the highest percent similarity in amino acid sequence 1
1
fromthelibrarytemplate(butnottheE.coliDNAtemplate) homology observed was 73%, it is likely that these KS 79
was cloned and a representative number of clones (n=8) domains and linked pathways are phylogenetically distant by
g
were sequenced to ensure that the amplicon was the from the previously characterized KS domains present in u
e
intended gene target. The only primer set that produced GenBankdatabases.Minimumevolutionanalysisoflibrary- st o
the desired PCR amplicon using a metagenomic library derivedKSdomainsandknownKSdomainsindicatesthat n 0
template and not with host genomic DNA template was the KS domains identified from the soil metagenomic 6 A
p
ril 2
0
1
9
Fig.1. (a)Clonesfromasoilmetagenomic
libraryexhibitchemiluminescenceafter
hybridizationwithadigoxigenin-labeledKS
domainampliconprobepreparedfromapooled
metagenomiclibraryDNAtemplate.Clones
arespottedinduplicate,sothatreplicate
hybridizationsignalsprovideanindicationof
positiveclonehybridization.(b)BamHIrestriction
digestsofKSdomain-containingfosmidclones
wereusedtoestimatethesizeforeach
metagenomiccloneinsert.
(cid:3)c2011FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol78(2011)176–187
PublishedbyBlackwellPublishingLtd.Allrightsreserved
PolyketidesynthasepathwaysfromsoilAcidobacteria 181
library represent diverse lineages, but are still related to transporters and translocation systems and enzymes in-
known KS domains (Fig. 2). The KS domains encoded by volved in polyketide modification [fosmid clone A3 (PCR
these fosmid clones showed similarity to those found in positive) depicted in Fig. 3; fosmid clones A2 (PCR nega-
pathwaysresponsibleforavarietyofpolyketidecompounds, tive),A11(PCRnegative),andC5(PCRpositive)shownin
such as the myxobacterial compounds epothilone and Supporting Information, Fig. S1]. Clone A3 revealed the
stigmatellin(Beyeretal.,1999),thecyanobacterialjamaica- presence of predicted genes with significant similarity to
mide(Edwardsetal.,2004),andtheactinobacterialpolyke- genes involved in both PKS and NRPS synthesis that
tide antibiotics erythromycin (Donadio et al., 1991) and suggests a hybrid pathway; in addition, on one end of the
pikromycin (Xue et al., 1998). Although many of these cloneA3insertaregeneswithsignificantsimilaritytogenes
library-derived KS domains have similarity to known KS fromtheAcidobacteriaisolateS.usitatusandahigh%G1C
domains,theresultantchemicalmoietiesproducedbythese content consistent with an Acidobacteria origin (Fig. 3).
D
pathways in their native host may be structurally distinct Preliminaryannotationoftheremainingsixcloneshasalso ow
n
fromknownpolyketidesbecausetheothermodulespresent revealed the presence of similar ORFs as well as many lo
a
inthesePKSpathwaysmaynotbesimilarinorganizationto sequenceswithnosignificanthomologsinGenBankorthat d
e
d
knownpathways. havecurrentlyunassignedfunctions(datanotshown). fro
m
h
Southern hybridization ofPCR-negativefosmid Bioinformatic analysis of cloneA12 ttp
clones Sequence data from clone A12 generated two points of s://a
c
Of the 34 fosmid clones identified by the original library interest. First, this clone appears to contain a gene with a
d
e
macroarray hybridization, 13 clones did not yield a KS 52% identity and 70% similarity to a gene that is homo- m
domain PCR product despite multiple attempts at PCR logous to mtaD, known to encode an enzyme from the ic.o
u
amplification using a variety of reaction conditions. To myxobacterium Stigmatella aurantiaca that is involved in p
.c
determinewhethereachofthesefosmidcloneDNAswould chain extension during the synthesis of the polyketide o
m
hybridize with KS domains, a Southern hybridization was myxothiazol (Silakowski etal., 1999).Second,muchofthe /fe
m
performed using a digoxigenin-labeled KS domain probe DNAcarriedoncloneA12sharessignificantsimilaritytothe s
e
generated by PCRamplification oftheKS domains from a genome of S. usitatus, a representative of the phylum c/a
poolofthe21KSdomainPCR-positivefosmidDNAs.Eight Acidobacteria(seeFig.S2). rtic
ofthe13PCR-negativeclonesyieldedKS-positiveSouthern However, the MtaD-like domain did not assemble with le-a
blot results, thus suggesting the presence of a putative KS othercontigsfromA12basedonavailablefosmidsequences; bs
domain on these clones that had a mismatch(es) with the thus, Southern hybridization was performed to verify its tra
c
4UU/5LL primer set. The five fosmid clones that were presence on this clone. Multiple bands were observed for t/7
8
originallyconsideredKS-positive by macroarrayhybridiza- cloneA12usingrestrictionenzymesthatwerenotpredicted /1
/1
tion, but have not yielded a KS PCR product or shown a tocutwithintheprobesequence,indicatingthatthismaybe 76
positive result in subsequent hybridizations may represent arepeatingdomainwithintheclone(datanotshown),and /52
1
false-positive macroarray hybridizations, misidentification explains its problematic assembly with other clone insert 1
7
9
ofthefosmidclonefromthemetagenomiclibrarystoredat sequences. The mtaD homolog gene sequence was used to b
y
(cid:2)801C, or may require lower stringency hybridization generate primer sets specific to this domain to query g
u
conditionsforthedetectionofdivergentKSdomains. culturedAcidobacteriagenomes. es
t o
n
454 sequencing offosmid clones Identification ofmtaDhomologoussequences 06
A
andKS domainsfrom culturedAcidobacteria p
Tweenrefossemleicdtecdlofnoersw(nho=le7-iPnCseRrtposesqituiveen;cnin=g3bPyC4R54nepgyartoivsee)- Using primer set #9 targeting the mtaD homolog from ril 20
1
quencingbasedontheirpredictedphylogenyandlikelihood metagenomic clone A12 or primer set #5 targeting KS 9
ofcontainingPKSpathways.Preliminaryphylogeneticana- domains(Table1),theculturedAcidobacteriawerescreened
lysis of these clones indicated that the predicted bacterial for the presence of mtaD or KS domains. Four mtaD
lineages of the clone insert sequences include members of homologs were identified from Acidobacteria strains
the phyla Acidobacteria, Cyanobacteria, and multiple sub- IGE015 (subdivision 3), IGE017 (subdivision 4), IGE002
divisionsoftheProteobacteria.Todate,fourofthe10clone (subdivision 6), and IGE001 (subdivision 6), showing at
inserts have been completely assembled and contain many least 99% identity between the mtaD homolog sequence
ORFswithsimilaritytogeneproductsfromvariousbacter- frommetagenomiccloneA12andeachoftheAcidobacteria
iallineagesassociatedwithpolyketidesynthesis,suchasacyl mtaD amplicon sequences. Minimum evolution trees of
carrier proteins and b-ketoacyl synthases, as well as cell MtaD homologous sequences and their nearest neighbors
FEMSMicrobiolEcol78(2011)176–187 (cid:3)c2011FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
182 L.C.Parsleyetal.
D
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ttp
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://a
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ic
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/fe
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1
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0
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9
Fig.2. MinimumevolutiontreebasedontheaminoacidsequencesofKSgenesequencesderivedfrommetagenomicclones,Acidobacteriacultures(in
bold),andtheGenBanknr/ntdatabase.KSdomainsequencesrecoveredfromthesoilmetagenomiclibraryareunderlinedandinbold.KSaminoacid
sequencesfromfosmidsC2andC3were98.6%identical,B5andB9were99%identical,C6andC7were100%identical,andB11andB12were
100%identical.KSsequencesfromfosmidsC4,C5,andA9groupwithacladeofKSsthatfollowNRPSenzymaticdomainsforhybridPKS/NRPS
biosysnthesisinCyanobacteriaandDeltaproteobacteria.Bootstrapanalysis(performed5000times)withpercentages 450%areindicatedatthe
nodes.Scalebarrepresents0.1aminoacidsubstitutionspersite.
(cid:3)c2011FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol78(2011)176–187
PublishedbyBlackwellPublishingLtd.Allrightsreserved
PolyketidesynthasepathwaysfromsoilAcidobacteria 183
D
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Fig.3. PredictedORFsfrommetagenomiccloneA3aftercompleteinsertsequencing.ORFswerecategorizedbasedontheirpredictedfunctionsafter
comparisonwiththeGenBanknr/ntdatabase.(ThethirdBLASTPhitisshownforORF4inordertocorrelatethepredictedfunctionwithmotifanalysis.)
in the GenBank nr/nt database show significant bootstrap support for the monophyly of all Acidobacteria MtaD
support for the affiliation of the fosmid A12 MtaD with sequences (Fig. 4). Likewise, Acidobacteria strains IGE002
homologs in Acidobacteria subdivisions 4 and 6, as well as (subdivision 6), IGE012 (subdivision 1), and IGE013
FEMSMicrobiolEcol78(2011)176–187 (cid:3)c2011FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
184 L.C.Parsleyetal.
(subdivision 1) had KS domains with significant similarity metagenomic libraries have been constructed in bacterial
toKSdomainsequencesderivedfrommetagenomicclones, artificialchromosomevectorsthatexceedtheaverageinsert
and a minimum evolution tree reveals that several of the size of this fosmid library, but this study does report the
AcidobacteriaandmetagenomicKSdomainstogetherform detection of diverse KS-positive clones within a macroar-
coherentclades(Fig.2). rayed metagenomic library and allow an assessment of the
phylogenetic origin and PKS pathway architecture among
theseclones.
Discussion
Uponfirst comparing theKSdomainspresentonmeta-
We constructed and screened a metagenomic library from genomiccloneswiththeGenBankdatabase,itwasapparent
soil to detect the presence of Type I PKS pathways within thatthemajorityoftheKSdomain-positiveclonesencoded
large-insert clones. Probing of the fosmid library, which is KS domains mostsimilar to those fromCyanobacteria and
D
estimatedtocontainover400Mbpofclonedmetagenomic Myxobacteriataxa(amongotherphyla)andwerephylogen- ow
n
DNA, revealedthepresenceof 34 clones that were positive etically and (presumably) functionally diverse, including lo
a
foraKSdomain.Withthemetagenomiclibraryrepresent- some expected to be involved in lipopolysaccharide and d
e
d
ing approximately 100 E. coli genome equivalents and polyketide synthesis, as well as hybrid polyketide/nonribo- fro
assuming an equal relative abundance of genomes within somal peptide chain elongation. Additional bioinformatic m
thelibrary,thenatthishitfrequency,approximately40%of analysis of complete insert sequences from the KS-positive http
tdhoemgaeinno.mCoesmrpeaprreedsewntiethdpwrietvhiionutsheestliimbraatreyscoofnKtaSindeodmaaiKnS- chloomneosloignyditcoatseedqumenacneys epnrceoddicintegdKOSRdFosmwaiinths, AsiCgnPisfi,caanndt s://ac
a
positive culturedbacteria of 15–25% in different soil types enzymes involved in the postsynthesis modification of de
m
(Khatunetal.,2002),thisisahigherfrequency,suggestingat variouspolyketides(Fig.3andFigsS1andS2). ic
the very least that a culture-independent survey of PKS However, the presumed phylogenetic affiliation of KS .ou
p
pathwaysiscapableofidentifyingasignificantyieldofPKS- domains identified from some metagenomic clones with .c
o
containing clones at a frequency within the range of KSdomainsfrommembersofthephylaCyanobacteriaand m
expectation. The observed PKS-containing clone hit fre- Proteobacteria was poorly supported by weak sequence /fem
s
quency of 0.4% for this library is similar to the 0.5–0.7% similarity (maximum 73% similarity) and by weak boot- e
c
frequency observed for marine sponge metagenomic li- strap support from the phylogenetic analysis. These data /a
braries(Schirmeretal.,2005)andthe0.2%frequencyfrom calledintoquestiontheoriginofthesemetagenomicclones rticle
asoilmetagenomiclibrary(Ginolhacetal.,2004).Theother andhighlightedthegapsinexistingsequencedatabases.The -a
b
s
acknowledgedconstraintinscreeningafosmidlibraryisthe analysis of complete insert sequences (i.e. from clones A3 tra
limited insert size possible for any given clone. Other soil and A12) indicated that some of the fosmid clones may ct/7
8
/1
/1
7
Acidobacteria IGE017 6/5
FosA12 2
Acidobacteria Acidobacteria IGE002 11
7
clade Acidobacteria IGE015 9 b
Acidobacteria IGE001 y g
Microcystis aeruginosa McyG [AAX73195.1] ue
s
Pseudomonas fluorescens Type I PKS [YP_259895.1] t o
Frankia sp. beta-KS [YP_001510033.1] n
Fig.4. Minimumevolutiontreebasedonamino 0
Streptosporangium roseum Type I PKS [YP_003338144] 6
acidsequencesofMtaDhomologousgene A
Trichodesmium erythraeum beta-KS [YP_723339.1] p
Crocosphaera watsonii beta-KS [ZP_00514740] sequencesderivedfrommetagenomiccloneA12 ril 2
Stigmatella aurantiaca MtaD [AAF19812.1] (boldandunderlined),Acidobacteriacultures 01
(bold),andtheGenBanknr/ntdatabase. 9
Melittangium lichenicola MelD [CAD89775.1]
Microcoleus chthonoplastes beta-KS [ZP_05029386 ] Numbersattreenodesrepresentbootstrap
Azospirillum sp. PKS [YP_003452767] values(n=1000repetitions;onlybootstrap
Myxococcus xanthus Type I PKS [YP_632473.1] valuesover50areshown),andthetreewas
Haliangium ochraceum Type I PKS [YP_003265638] rootedwithCurHfromLyngbyamajuscula.
Sorangium cellulosum TgaA [ADH04639.1] Accessionnumbersforeachsequencearein
Xanthobacter autotrophicus beta-KS [YP_001415916.1] brackets.Scalebarrepresents0.1aminoacid
Beggiatoasp. beta-KS [ZP_01997657.1] substitutionspersite.[Correctionadded1June
Lyngbya majuscula curH [AAT70103.1] 2011afteronlinepublication:Figure4replaced
withnewversiontoshowcurrentdesignationof
strains]
(cid:3)c2011FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol78(2011)176–187
PublishedbyBlackwellPublishingLtd.Allrightsreserved
PolyketidesynthasepathwaysfromsoilAcidobacteria 185
derive from soil Acidobacteria. Fortunately, the availability tion revealed that clone A12 contained several ORFs with
of cultured Acidobacteria isolates fromseveral subdivisions homology to the genome of S. usitatus fromthe Acidobac-
ofthisphylumprovidedtheabilitytoquerytheirgenomes teria phylum. Using the available Acidobacteria culture
for PKS domains. The inclusion of PKS domain sequences collection,weexploredthepresenceofMtaD-likedomains
from these recently cultured Acidobacteria indicates that fromrecentlyculturedAcidobacteria,includingtwoisolates
many of these metagenomic clones at first identified as from the previously uncultivated subdivision 6. MtaD-like
originating from Cyanobacteria or Myxobacteria taxa are sequences were identified from four Acidobacteria isolates.
rather derived from this novel phylum that is ubiquitous Therewassignificantaffiliationobservedbetweenmetagen-
and abundant in soils. These data demonstrate that the ome- and Acidobacteria-derived sequences for both MtaD
current sequence databases do not adequately reflect the and KS domains. Because these cultured isolates represent
extantdiversityofsoilbacterialPKSpathways.Someofthe multiple Acidobacteria subdivisions and were found
D
observed disparity between the predicted phylogenetic af- within soils collected in Belgium, these data indicate a ow
n
filiations of the KS domains compared with their flanking potentially widespread distribution and diversity of PKS lo
a
sequences (i.e. clone A3 in Fig. 3) may result from the pathways among Acidobacteria taxa. Further characteriza- d
e
d
paucity of Acidobacteria PKS pathways and genomes in tion of these Acidobacteria strains and their polyketide fro
GenBank. products will contribute to our understanding of natural m
A significant percentage (nearly 40%) of the 34 fosmid product discovery from previously uncultured bacterial http
cylioelndeesdidaeKntSifideodmbayinli-bsrpaercyifimcaPcCroRarpraryodhuycbtr,idleiazdatiinogntnoetvheer pidheynlati.fiTeodforuormkannowAlceiddgoeb,acntoeripaoslypk.,eatildtheopurgohdtuhcetrheassulbtseeonf s://ac
a
initialconclusionthatthesemaybefalse-positiveidentifica- onegenomesequencingstudyindicatethatmembersofthis de
m
tions or that the incorrectfosmid clones hadbeen selected phylummayharborPKS-relatedgenes(Wardetal.,2009). ic
from the 384-well formatted library. Subsequent Southern These findings reflect the scarcity of known Acidobacteria .ou
p
hybridizationanalysisindicatedthateightofthese13clones sequencesandindicatethatthesebacteriamayrepresentan .c
o
containedaKShybridizingdomain(s).Itislikelythatbase unexploredreservoirofnovelPKSpathwaysandpolyketide m
pairmismatchesbetweentheKSdomainPCRprimersand products. /fem
s
fosmidcloneDNApreventedthesuccessfulPCRamplifica- e
c
tionofKSgenes,andyettheysharedsufficientsimilarityfor /a
successful hybridization. This is strong support for the Acknowledgements rticle
utility of metagenomic macroarray hybridization for the -a
b
inclusionofmetagenomicclonesrepresentingdiversephyla WethankthemembersoftheLileslaboratoryfortheirhelp stra
such as the Acidobacteria, especially when the available in the execution of these experiments and their critical ct/7
‘universal’ primer sets designed on the basis of gene analysis of this publication. Funding for this work was 8/1
sequences derived fromculturedtaxa may not be inclusive provided by the National Institutes of Health (grant # /1
7
ofmetagenomicdiversity. 1R21AI083852-01). 6/5
2
SequenceanalysisofthePCR-negativeclonesrevealedthe 1
1
presenceofPKS-relatedORFsthatarepredictedtooriginate 79
b
from diverse bacterial lineages, such as the Acidobacteria, References y g
Cyanobacteria, Planctomycetes, and Proteobacteria phyla. u
e
Manyofthepredictedgeneproductsappeartobeinvolved Ayuso-SacidoA&GenilloudO(2005)NewPCRprimersforthe st o
in polyketide biosynthesis, postsynthesis modification, or screeningofNRPSandPKS-Isystemsinactinomycetes: n 0
detectionanddistributionofthesebiosyntheticgene 6
thetransportofsuchcompoundsoutsideofthecell.Because A
sequencesinmajortaxonomicgroups.MicrobEcol49:10–24. p
oufntlhikeeplyhtyhloagteanlletoifcathlleydpiavtehrwseaynsatwuoreulodfhthaevseebseeqenuefnucnecst,ioitni-s BejaO,SuzukiMT,KooninEVetal.(2000)Constructionand ril 20
analysisofbacterialartificialchromosomelibrariesfroma 1
ally active in E. coli, thus underscoring the advantage of a 9
marinemicrobialassemblage.EnvironMicrobiol2:516–529.
sequence-basedscreeningapproachfortheinitialidentifica-
BeyerS,KunzeB,SilakowskiB&MuellerR(1999)Metabolic
tion,tobefollowedbyresearchtoexpressPKSdomainsor
diversityinmyxobacteria:identificationofthemyxalamidand
pathwayswithinotherheterologoushost(s)predictedtobe
thestigmatellinbiosyntheticgeneclusterofStigmatella
closelyrelatedtothemicroorganismfromwhichthemeta-
aurantiacaSga15andacombinedpolyketide–(poly)peptide
genomicclonewasderived.
geneclusterfromtheepothiloneproducingstrainSorangium
Analysis of clone A12 revealed the presence of a gene cellulosumSoce90.BiochimBiophysActa1445:185–195.
whosetopBLASThitMtaDoriginatesfromthemyxobacter- BradySF,SimmonsL,KimJH&SchmidtEW(2009)
iumS.aurantiacaandisinvolvedinthehybridPKS/NRPS Metagenomicapproachestonaturalproductsfromfree-living
biosyntheticpathwayofmyxothiazol.Subsequentinvestiga- andsymbioticorganisms.NatProdRep26:1488–1503.
FEMSMicrobiolEcol78(2011)176–187 (cid:3)c2011FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved