Sampling Terrestrial Environments for Bacterial Polyketides

49 Pages·2017·8.21 MB·English

molecules
Review
Sampling Terrestrial Environments for
Bacterial Polyketides
PatrickHill1,GrahamW.Heberlig2andChristopherN.Boddy1,2,*
1 DepartmentofBiology,UniversityofOttawa,Ottawa,ONK1N6N5,Canada;[email protected]
2 DepartmentofChemistryandBiomolecularSciences,UniversityofOttawa,Ottawa,ONK1N6N5,
Canada;[email protected]
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:KiraJ.Weissman
Received:28January2017;Accepted:18April2017;Published:29April2017
Abstract: Bacterialpolyketidesarehighlybiologicallyactivemoleculesthatarefrequentlyusedas
drugs,particularlyasantibioticsandanticanceragents,thusthediscoveryofnewpolyketidesisof
majorinterest. Sincethe1980sdiscoveryofpolyketideshassloweddramaticallydueinlargepartto
therepeatedrediscoveryofknowncompounds. Whilerecentscientificandtechnicaladvanceshave
improvedourabilitytodiscovernewpolyketides,onekeyareahasbeenunderaddressed,namelythe
distributionofpolyketide-producingbacteriaintheenvironment. Identifyingenvironmentswhere
producingbacteriaareabundantanddiverseshouldimproveourabilitytodiscover(bioprospect)
newpolyketides. Thisreviewsummarizesforthebioprospectorthestate-of-the-fieldinterrestrial
microbial ecology. It provides insight into the scientific and technical challenges limiting the
applicationofmicrobialecologydiscoveriesforbioprospectingandsummarizeskeydevelopments
inthefieldthatwillenablemoreeffectivebioprospecting. Themajorrecenteffortsbyresearchersto
samplenewenvironmentsforpolyketidediscoveryisalsoreviewedandkeyemergingenvironments
suchasinsectassociatedbacteria,desertsoils,diseasesuppressivesoils,andcavesarehighlighted.
Finallystrategiesfortakingandcharacterizingterrestrialsamplestohelpmaximizediscoveryefforts
areproposedandtheinclusionofnon-actinomycetalbacteriainanyterrestrialdiscoverystrategy
isrecommended.
Keywords: polyketides;bioprospecting;microbialecology
1. Introduction
Polyketide natural products are exquisite molecules, often with extraordinary and diverse
structuresrangingfrommacrolidestopolyethersandpolyphenols. Theytypicallybindwithhigh
affinity and selectivity to a biological target (usually a protein or RNA) and can cross biologically
relevantmembranes,enablingthemtomodulatethebiologyoforganisms. Thesefeatureshavemade
polyketidenaturalproductsanindispensableresourceforthediscoveryanddevelopmentofnew
pharmacologicalagentsincludingtheantibiotics, likeerythromycinAandtetracycline, antitumor
agents,likedoxorubicin;andtheanti-fungaldrugs,likeamphotericinB[1].
While structurally diverse, all polyketides are related by their highly conserved biosynthetic
origins. The polyketide backbone is assembled, analogously to fatty acid biosynthesis, through
sequential additions of two carbon building blocks (ketide units) derived from malonyl-CoA.
Thisassemblyiscatalyzedbyenzymescalledpolyketidesynthases(PKS).Inbacteriathesepolyketide
synthasestypicallyoccurwithoneoftwodifferentarchitectures,modulartypeIpolyketidesynthases
andtheiterativetypeIIpolyketidesynthases.
ThetypeIPKSpathwayscontainmultiplemodulesconsistingofketosynthase(KS)domains,
whichcatalyzetheadditionofketideunitstothegrowingpolyketidechainviadecarboxylativeClaisen
Molecules2017,22,707;doi:10.3390/molecules22050707 www.mdpi.com/journal/molecules
Molecules2017,22,707 2of49
condensationofanacylcarrierprotein(ACP)-linkedmalonyl[2,3]. InadditiontotheKSandACP
manytypeIPKSpathwaysalsopossessanacyltransferase(AT)domainembeddedintoeachmodule
whichloadstheappropriatemalonylextenderunitontotheACP.ThetransATtypeIPKSpathways
possessaseparatestandaloneATdomainresponsibleforloadingtheACPdomains[4]. Inaddition
tothesecatalyticdomainswhichextendthepolyketidechain,typeIPKSpathwayscanalsopossess
reductivedomainssuchasaketoreductase(KR),dehydratase(DH),andenoylreductase(ER)domains
thatreducethegrowingpolyketideintermediate,similartomammalianfattyacidbiosynthesisprior
tothenextroundofpolyketideelongation[2,3]. Elongationcontinuesuntileventualthecompleted
polyketidesisreleasefromtheACPbyathioesterase(TE)domain[5]. TypeIPKSareresponsiblefor
theproductionofawidevarietyofbioactivecompoundsincludingtheantibioticerythromycinand
theantifungalamphotericinB.
TypeIIpolyketidesynthasesaremostfrequentlyfoundintheorderofbacteriaactinomycetales
andfunctionsimilarlytobacterialfattyacidsynthases. LiketypeIPKS,thesesynthasesrelyonaKS
domain,whichheterodimerizeswithaproteincalledchainlengthfactor(CLF),andanACPdomain.
Amalonylacyltransferase(MAT)domainisusedtoloadmalonylontotheACPfordecarboxylative
condensation with the growing KS-bound polyketide chain. Unlike type I PKS, these pathways
typically use these four catalytic domains iteratively and possess a single reductive domain [3,6].
TypicalproductsfromtypeIIPKSpathwaysarearomaticcompoundsthatareoftenhighlytailoredto
generatecomplexmoleculessuchastheantibiotictetracyclineortheanticanceragentdoxorubicin.
OftentypeIpolyketidesynthasesformhybridpathwayswithnon-ribosomalpeptidesynthetases,
producing natural products containing both polyketide portions and peptide portions [7–9].
TheanticanceragentepothiloneBisaprimeexampleofaproductfromthistypeofpathway. Inthis
reviewarticlewebroadlydefinebacterialpolyketidesascompoundsproducedbytypeIandtypeII
polyketidesynthasebiosyntheticpathwaysaswellashybridpolyketide-non-ribosomalpeptidepathways.
Thediscoveryofnewpolyketideshasslowednoticeablysincethelate1980sasthetraditional
methodofdiscovery,isolatingandcultivatingbacteria(usuallyActinomycetes,andespeciallytheir
genusStreptomycetes[1])fromsoil,foundfewerandfewernewpolyketides. Atthesametimebacterial
resistancetoantibioticshasbeenincreasing. Torestartpolyketidediscovery,bioprospectorsareusing
methodsthatimproveorreplacebacterialcultivationandsamplingalternativeenvironmentstosoil.
Bothoftheseapproachesmeanthatitisimportanttoknowhowbacterialpolyketidesaredistributed
tosamplethemefficiently.
Soil cultivation methods can be scaled up to screen millions of isolates a year to find rare
polyketides[10].Sequencingofthegenomesofisolatedbacteriashowsthatmostpolyketidegenesare
“cryptic”,i.e.,notexpressedundernormalcultivationconditions.Insomecasesthesecrypticpathways
havebeencoaxedtoexpressbychangingthecultivationconditions[11],addingantibioticsthatstimulate
polyketideproduction[12],overexpressingregulatorygenes[13],orblockingcompetingpathways[14].
Alternativelythesecrypticpathwayscanbeexpressedinheterologoushosts,enablingnewcompound
isolation. Whilethisapproachhasanumberoftechnicalhurdles,includingensuringtranscription,
codonusage,proteinfolding,ensuringthepresenceofprecursors,andconcernsthatthepolyketide
productitselfmaykillthehost[15],therehavebeensomemajordiscoveriesinthisarea[16].
Whilemostbacteriainsoilcannotbereadilygrown,thecultivatablefractionofthesoilbacterial
communitycanbeincreasedbykeepingbacteriaincontactwiththesoilinburied“chips”forweeks[17].
Tocircumventcultivation,bacterialDNAcanbeextractedfromsoilandheterologouslyexpressedin
bacteriathatcangrowinthelaboratory[18,19].
Thesenewtechniquesarechangingthewaypolyketidesarediscovered(forreviewssee[20–25]),
buttheyrequireasmuchormoretimeandeffortforeachsampleasthelowertechisolationmethods
thattheyreplace,sosamplingefficientlyisimportant. Theproblemofhowtosampleisnolonger
limitedtosoil. Bioprospectorscanandshouldconsiderthemanyalternativestosoilsuchascaves,
insectsanddesertsoils. Bioprospectorsneedtocomparethemicrobialpolyketideproducerswithin
andbetweenenvironmentstofindthemostpolyketidediversitywiththefewestsamples.
Molecules2017,22,707 3of49
Until recently it was impossible to compare the bacterial community structure between
Molecules 2017, 22, 707 3 of 48
environments. Ithadlongbeenknownthatonlyoneofeveryhundredtothousandbacteriafromthe
environmentthatcouldbeseenthroughamicroscopecouldbegrowninthelaboratory[26]. In1990
Until recently it was impossible to compare the bacterial community structure between
DNenAviwroansmeexnttrsa. cItt ehdadfr loomngs boeiel,nd kennoawtunr tehda,t aonndlyi otsnere oafs esvoecriya thiounndtrimede tom tehaosuusraendd, baallcotewriian fgrormes ethaer chersto
esteinmvairtoentmhaetntt hthearte cwouelrde bthe eseeeqnu tihvraoluegnht ao fm4i0cr0o0scEo.pceo lciogueldn obme gersoiwnne ianc hthge rlaabmoroaftotrhye [s2o6]i.l I[n2 71]9.9T0 hiswas
onDeNoAf t wheasfi erxsttraucsteeds forfomcu slotiivl,a dteionnat-uinredde, paenndd itesn rte,anssuocclieaitcioanc tiidm-eb amseeadsumreedt,h aolldoswtiongs truesdeyarschoeilrsb acteria.
Motod eesrtnimnautec ltehiact athceidre bwaesreed thme eeqthuoivdasleunst uoaf l4l0y00u sEe. ctohlie gpenoolymmese rina seeacchh garinamr eoafc tthioe nso(iPl [C27R]). Ttohims easure
was one of the first uses of cultivation-independent, nucleic acid-based methods to study soil bacteria.
whichbacteriaareinthesoilandestimatehowdiversethebacterialcommunityis(Figure1).
Modern nucleic acid based methods usually use the polymerase chain reaction (PCR) to measure
PCRproductsamplifiedfromsoilDNAtemplatestypicallycontainthousandsormoredifferent
which bacteria are in the soil and estimate how diverse the bacterial community is (Figure 1).
sequences. Cloning and sequencing with Sanger sequencing (e.g., [28]) was used until the early
PCR products amplified from soil DNA templates typically contain thousands or more different
2000stosequencetheseamplicons.DuetotheintrinsicallylowthroughputofSangersequencingand
sequences. Cloning and sequencing with Sanger sequencing (e.g., [28]) was used until the early 2000s
thousandstohundredsofthousandsofbacterialspeciesinsoil, itwasdifficulttogetafullpicture
to sequence these amplicons. Due to the intrinsically low throughput of Sanger sequencing and
oftthhoeusbaancdtes rtioa lhuconmdrmedus noift tyhooufsaansdins golfe bsaactmerpialle s,pleectiaeslo inn esotiol, cito wmaps adrieffiscaumlt ptol egse.t Aa nfulal lpteicrtnuartei vofe wasto
septhaer baatectethrieal PcoCmRmaumniptyli ocfo an ssinbgyleg seaml eplleec, tlerot aplhonoer etos icsomtopgareet saam“cpolems. mAnu anlitteyrnfaitnivgee wrparsi ntot ”seapnardatceo mpare
fintgheer PpCriRn tampaptltiecornnss btoy ggeetl ealercoturogphhiodreesaiso tfoh goewt ab “accotmermiaulnciotym fminguenriptriienst”d iafnfedr ecodm. Gpaerlef fiinnggeerrpprriinntt ingcan
seppaarttaetrenas mtop gliecto an srosuegvhe riadlewa oafy sh,oiwnc bluacdtienrigalg croamdimenutnsitoiefst edmiffpeererdat. uGreel ofrinugreerap,riwnthinicgh csaenp saerpaatreaatem plified
amplicons several ways, including gradients of temperature or urea, which separate amplified DNA by
DNAbyguanine/cytosine(GC)content(e.g.,DenaturingGradientGelElectrophoresis,DGGE[29]),
guanine/cytosine (GC) content (e.g., Denaturing Gradient Gel Electrophoresis, DGGE [29]), or cutting
orcuttingampliconsintosmallerfragmentsbyrestrictionendonucleasesandseparatingbysize(e.g.,
amplicons into smaller fragments by restriction endonucleases and separating by size (e.g., Terminal
TerminalRestrictionFragmentLengthPolymorphism,T-RFLP[30]). Communityfingerprintinggives
Restriction Fragment Length Polymorphism, T-RFLP [30]). Community fingerprinting gives less
lessinformationthanSangersequencing,butcanquicklyandcheaplycomparemanysamples(Figure1).
information than Sanger sequencing, but can quickly and cheaply compare many samples (Figure 1).

FigFiugruere1 .1.M Miiccrroobbiiaall eeccoollooggyy aapppproraocahcehse ussueds etod etxoaemxianme binoethb coutlhtivcautlatbivlea atanbdl eunacnudltiuvnatcaublltei vfraatcatbiolen fraction
ofotfh tehem micircorobbiaiall ccoommmmuunniittyy sstrtuructcutruer. eT.hTish iinscilnucdleusd mesolmecoullearc umlaetrhmodest hliokde sshlioktgeusnh omtegtuagnemnoemtaeg enome
sesqeuqeunencicningg,, ccoommmmuunnitiyt yfinfignegrperripnrtiinngt ionf gpoolfympoelryasme cehraaisne rcehacatiinonr (ePaCcRti)o pnro(dPuCcRts), sperqoudenuccitnsg, osef PqCuRen cingof
PCpRropduroctdsu, acntsd, pahnodspphhoolisppidh ofalitptyi dacfiadt taynaalcyisdis.a nalysis.
In the 2000s next generation sequencing replaced Sanger sequencing and fingerprinting of PCR
Inthe2000snextgenerationsequencingreplacedSangersequencingandfingerprintingofPCR
products. With next generation sequencing thousands to hundreds of thousands of different amplicons
procoduulcdt sb.eW seiqthuennecxetdg fernomer aat isoinngslee qsuamenpclien agntdh omuasnayn sdasmtoplhesu ncodurledd bseo mftuhlotiupsleaxnedds toogfedtihfefer rteon gtiavme plicons
couenldormbeouses qduatean sceetsd [3fr1o].m Thais siinncgrelaessedam thpe lveoalunmdem ofa sneyquseanmcipngle rsescuolutsl danbde thme unlutmipbleerx oefd setoqgueenthceedr togive
engoernmoomuess.d Raetasesaertcsh[e3r1s ]c.oTuhldis eivnecnr esaesqeudenthcee uvnoalummpleifoiefds eDqNuAen fcrionmg trhees uslotisl amnedtatgheennomume (bFeirguorfes 1e)q uenced
geannodm eessti.mRaetes ewahrcichhe prshycloougledneetvice gnroseuqpus eitn bceelounngaemd tpol [i3fi2e]d. DNAfromthesoilmetagenome(Figure1)
andestNimucalteeicw achiidcsh apreh ynolot gtheen eotniclyg briooumposleictubleeslo tnhgate cdanto b[e3 2u]s.ed to measure microbial community
structure. Analysis of phospholipid fatty acids (PLFA) from the cell membranes of soil microbial
Nucleicacidsarenottheonlybiomoleculesthatcanbeusedtomeasuremicrobialcommunity
communities began in the 1980s (Figure 1) [33]. Like nucleic acid methods, it provides information
structure. Analysis of phospholipid fatty acids (PLFA) from the cell membranes of soil microbial
on the uncultivated majority of bacteria. While PLFA has largely been replaced by next generation
communitiesbeganinthe1980s(Figure1)[33]. Likenucleicacidmethods,itprovidesinformation
sequencing, there is a lot of PLFA data in the literature. Unlike nucleic acid sequencing, PLFA cannot
ontheuncultivatedmajorityofbacteria. WhilePLFAhaslargelybeenreplacedbynextgeneration
identify bacteria at the genus or species level and is often specific to groups that are even broader
seqthuaenn pcihnygla, t(he.egr.e, Gisraamlo-ptoosfitPivLeF bAacdteartiaa)i.n Int hsepiltiet eorfa tthuisr et.hUerne lairkee anduvcalnetiacgaecs itdo sPeLqFuAe.n Ict iins gc,hPeaLpFeAr cannot
ide ntifybacteriaatthegenusorspecieslevelandisoftenspecifictogroupsthatareevenbroaderthan
Molecules2017,22,707 4of49
phyla(e.g.,Gram-positivebacteria). InspiteofthisthereareadvantagestoPLFA.Itischeaperand
fasterthannucleicacidmethodsandgivesabetterpictureoffungaltobacterialratios. Resultsfrom
PLFAcommunityanalysisshowedthatsoilpHcontrolledbacterialcommunitystructurewellbefore
thiswasfoundbynucleicacid-basedmethods[34]. Ithasalsobeensuggestedthatphospholipidfatty
acidsarequicklydegradedinsoil,unlikeDNA,soPLFAmaygiveabetterpictureofthelivebacterial
communitythanDNAmethodsthatalsocapturerelicDNA[35].
The most important bacterial group for polyketide production is the Gram-positive phylum
Actinobacteria. Withinthisphylum,theActinomycetalesorder,haveproducedmorepharmaceutically
useful polyketides than all the rest of the bacteria [36]. This review will use both the terms
Actinobacteria and Actinomycete. This is as molecular microbial ecology studies usually use the
termActinobacteriawhilecultivationstudiestypicallyusuallyusethetermActinomycetales.
The reader might think that deciding where to sample for discovery of new polyketides is
straightforward. ReviewthemicrobialecologyliteraturetofindenvironmentswhereActinomyces
arediverseandalargefractionofthebacterialcommunity. Whilethereareamanystudiesthatuse
molecularbiologymethodstocharacterizethemicrobialcommunities,usingthisliteraturetodecide
wheretosampletofindnewpolyketidesisdifficult. Thereareseveralreasonsforthis.
MostmodernmicrobialecologystudiesusePCRtoamplifyfromnucleicacidsextractedfromthe
environment. TheresultsfromPCR-basedmethodsdependsignificantlyonthechoiceofextraction
methods,PCRprimers,andreactionconditions. Manystudiesuseauniqueversionofeachofthese,
soresultsareoftennotcomparablebetweenstudies. Recentlyseverallargescaleprojects,suchasThe
EarthMicrobiomeproject[37],havebeenapplyingstandardizedmethodsonsamplesfromacross
theplanet.
Bacterialhabitatsdonotfollowthesamepatternasthehabitatsofeukaryoticlifethatwesee
around us. Environments that to the human eye seem very different, can have similar bacterial
communities [38]. In addition the actinobacterial community may not be controlled by the same
factorsasthewholebacterialcommunity. FurthermoretheActinobacteria,whichhavesofarsupplied
mostofourmedicinallyusefulpolyketides,maynotproducemostofthetypeIpolyketidesinthe
environment[39]. Understandingwhatcontrolsbacterialcommunitystructureandhowthisaffects
thedistributionofpolyketideproducingbacteriaisstillunresolved.
Finally the major ecological question of whether bacterial distribution is controlled by
biogeography has still not been settled. Biogeography, the pattern of species distribution across
geographical area, affects where multicellular eukaryotes such as plants and animals are found,
however it is not clear if this is true for bacteria as a whole or for polyketide producing bacteria
specifically. Questionssuchas“Arethesamebacterialcommunitiesfoundwherevertherearethe
sameconditions?”decidewhetherbioprospectorsshouldsampleparticularenvironmentsinmany
placesovertheearthormanyenvironmentsthatmaybeinthesamearea.
Wereviewwheretosampleforbacterialpolyketidesinterrestrialenvironments. Thefirstsection
ofthisreviewdiscussesproblemsinusingthecurrentscientificliteratureforbioprospectingasbriefly
outlinedabove.Becauseterrestrialbioprospectingfornewpolyketidesisalreadyunderway,thesecond
partofthisreviewdiscussesseveraloftheterrestrialenvironmentswherenewpolyketideshavebeen
foundorwhichhavebeensuggestedforbioprospecting.
Thefinalsectionsuggestshowtosampletofindnewpolyketidesinterrestrialenvironments.
Werecommendsamplingdifferentenvironmentsandmeasuringsamplespropertiesinseveralstages.
Physical/chemicalpropertiessuchaspH,organiccarbonortexturecanbemeasuredforabroadrange
of samples. The microbial community of a subset of these samples can be determined with rapid,
lowcostcommunityfingerprinting,beforenextgenerationsequencingofsubsetswithcontrasting
fingerprints. An advantage of using community fingerprinting is that they give results quickly.
Ifcertainsamplesgiveunusualorpromisingfingerprints,theenvironmentorareawheretheyare
fromcanberesampled. Bioprospectorsshouldalsotrytofindnon-actinomycetalbacterialpolyketides
astheyaremorelikelytobenovel.
Molecules2017,22,707 5of49
2. UnresolvedIssuesinMicrobialEcologythatLimitBioprospectingforNewPolyketides
Molecularbiologicalmethodshavechangedourviewofsoilbacterialcommunities. SomePhyla,
suchastheAcidobacteria,areamuchlargerfractionofsoilbacteriathanwaspreviouslyknown[40].
SoilpropeMrtoileecusless u20c17h, 2a2,s 70t7e xtureandpHareoftenmoreimportantindeterminingcommu5 nof i4t8y structure
thanvegetationorclimate[41]. Whileweknowmuchmoreaboutbacterialcommunitiesthanwedid
2. Unresolved Issues in Microbial Ecology that Limit Bioprospecting for New Polyketides
20yearsago,therearestillgapsinourknowledgethatmakeitdifficulttoknowwheretobioprospect
Molecular biological methods have changed our view of soil bacterial communities. Some Phyla,
fornewbacterialpolyketides.
such as the Acidobacteria, are a much larger fraction of soil bacteria than was previously known [40].
Soil properties such as texture and pH are often more important in determining community structure
2.1. DataComparability
than vegetation or climate [41]. While we know much more about bacterial communities than we did
20 years ago, there are still gaps in our knowledge that make it difficult to know where to bioprospect
Many microbial ecology studies produced data that cannot be compared with other studies.
for new bacterial polyketides.
Studiesoftenlookatasingleenvironmentandlookatchangesinthemicrobialcommunitycaused
bytreatme2.n1.t sD,astau Ccohmapasrathbielitye f fectofdifferenttillageonsoilbacteria[42],thechangesinthemicrobial
communityduMrainngy cmoicmropboiaslt eincoglo[g4y3 ]s,tuodritehs eprboadcutceerdia dfaotau nthdati ncadnnifofte rbeen ctokmipnadresdo wfoitfhfi octehedru sstut.dTiehs.e sestudies
extractDNStAudfireos mofttehn eloeonk vaitr ao nsimngelen etn,vaimropnmlifeynti atnwd iltohokp arti mchearnsgeasn idn tahne amlyiczroebtihale cofimnmgeurnpitryi ncatsusoerd sequences
by treatments, such as the effect of different tillage on soil bacteria [42], the changes in the microbial
of the amplicons. They often use a unique combination of extraction method, primers, and
community during composting [43], or the bacteria found in different kinds of office dust. These
amplification conditions. Changing any one of these steps can significantly change the results.
studies extract DNA from the environment, amplify it with primers and analyze the fingerprints or
Forexampseleq,uDeneclems oof nthtee atmapl.li[c4on4s]. cTohmeyp oaftreend usae raa unngiqeuoe fcoDmNbiAnateioxntr oafc etxiotrnactmioent mhoetdhosdo, nprtimweorsg, arnadss landsoils
andfoundamthpaltifitchaetiyong caovnedivtieornys. dCihfafenrgeinngt apniyc tounree sofo tfhethsee smtepics rcoabn isailgncoifimcamntluyn cihtayn.gHe othneg reestualtls.. [F4o5r ]usedtwo
example, Delmont et al. [44] compared a range of DNA extraction methods on two grassland soils
extractionmethodsandtwoprimersetstomeasurethebacterialcommunityofabeachsandsample,
and found that they gave very different pictures of the microbial community. Hong et al. [45] used
only11of1098operationaltaxonomicunits(OTUs,definedas16SribosomalRNAgenesthathaveless
two extraction methods and two primer sets to measure the bacterial community of a beach sand
than99%ssaimmpillea,r oitnyly) s1e1 qouf e10n9c8e ospwereartieonfaolu tanxdonionmailcl utnhirtse e(OdTaUtsa, sdeetfisne(Fd iagsu 1r6eS 2r)ib.oIsnomoualr RoNwAn gwenoesr ktwosets
of“actinobthaact thearviea llessps tehcainfi 9c9”%a snimdiolanrietys) esetqoufenecuebs awcetreer fioauln1d6 iSn palrl itmhreeer sdaatma spetlsi fi(Feigduvree 2r)y. Idn iofufer roewnnt groupsof
work two sets of “actinobacterial specific” and one set of eubacterial 16S primers amplified very
theactinobacteria[46]. Evenwhenthesamemethodsarecarriedoutindifferentlaboratoriestherecan
different groups of the actinobacteria [46]. Even when the same methods are carried out in different
besignificantdifferencesinresults[47].
laboratories there can be significant differences in results [47].

Figure 2. The overlap between Sanger sequencing results of three 16S clone libraries at 99% identity
Figure2.TheoverlapbetweenSangersequencingresultsofthree16Sclonelibrariesat99%identity
from a beach sand using two DNA extraction methods and two primer pairs. Clone library 1
fromabeachsandusingtwoDNAextractionmethodsandtwoprimerpairs.Clonelibrary1Extraction
Extraction method 1 PCR primer pair 1, Clone library II Extraction method 2 PCR primer pair 1, Clone
method1lPibCraRryp IrIiIm IIe Erxptraaicrtio1n, Cmleotnhoedl i1b rPaCrRy IpIriEmxetrr apcatirio 2n. Rmeeptrhinotedd 2byP CpeRrmpirsismione rfrpoami rM1a,cCmliollnane libraryIII
IIExtractiPounblmisheetrhs oLdtd:1 ISPMCER J. p[4r5i]m, ceorpypraigihrt 2(2.0R09e)p. rintedbypermissionfromMacmillanPublishersLtd:
ISMEJ.[45],copyright(2009).
Bioprospectors need to know where to sample. The most useful results will come from studies
that use the same method over a broad range of samples from different environments. As methods
Bioproof sapnaelcytsoisr asren ecoendtintouakllny oimwprwovhinegr,e thtoe ssaammplpesle (.alTonhge wmitoh sintfuorsmefautilonr easbuoultts wwheilrle caondm wehferno mstudies
they were collected and what they are like) should be stored for reanalysis as new methods come
thatusethesamemethodoverabroadrangeofsamplesfromdifferentenvironments. Asmethodsof
online [48]. Several large collaborative projects have been set up to do exactly this [37,49–51], and this
analysisarecontinuallyimproving,thesamples(alongwithinformationaboutwhereandwhenthey
is an area of research that is expanding [52].
werecollectedandwhattheyarelike)shouldbestoredforreanalysisasnewmethodscomeonline[48].

Severallargecollaborativeprojectshavebeensetuptodoexactlythis[37,49–51],andthisisanareaof
researchthatisexpanding[52].
Molecules2017,22,707 6of49
Theseconsortiahavethechoiceofusingmethodsthatarelabourandtimeintensivebutgive
alotofinformationforafewsamplesorusingmethodsthatarefasterandcheaper,butlessdetailed
formanysamples. ThebestexampleofthetimeandlabourintensiveapproachistheTerragenome
project[49]. Thisconsortiumbeganbysequencingthemetagenomeofafewwellcharacterizedsoil
samples,startingwithsamplesfromtheoldestfieldtrialintheworldatRothamsted. Eventuallythis
workwillprovideverydetailedinformationonpolyketidecontentinafewsoils.
Shotgun metagenome sequencing of the diverse microbial communities from soil is difficult.
Anenormousvolumeofsequencedataisneededanditisdifficulttoassemblegenomesorpolyketide
biosyntheticgeneclustersfromtheselargedatasets[53]. Terragenomeinvolvesinvestingalotoftime,
workandfundsinafewsoilsbeforeitisknownhowrepresentativetheirmicrobialcommunitiesare.
Ithasbeensuggestedthatitwouldbemoreefficienttosurveymanysoilsusingsimple16Sbacterial
communityfingerprintingmethods,beforepickingsamplesfordirectmetagenomicssequencing[54].
ThisinitialstephasbeendoneinsoilmicrobialsurveysofGreatBritainandFrance,wherehundredsof
samplesfromacrosslandscapeshavebeenfingerprinted[55,56]. Thesestudiesprovidereproducible
butlowresolutioninformationonbacterialdistributionanddiversityinsoil.
Themostambitiousoftheseprojects,whichusesidenticalmolecularmethodsonmanysamples,
istheEarthMicrobiomeproject.Thisprojectsamplesbroadlyfromenvironmentsincludingsoil,marine
sediment, and the digestive system of mammals. It uses next generation sequencing to sequence
16Sbacterialand18SfungalribosomalRNAamplicons,aswellasshotgunmetagenomesequencing.
Recentlyahostofadditionalprojectslookingatthemicrobiomeofhumanshaveusednextgeneration
sequencingof16Sampliconstocharacterizethousandsofsamples[57,58]. Thesestudiesarebeginning
toprovideaclearerpictureofbacterialcommunitystructureofmanyenvironments.
The laboratory of Brady, a pioneer in polyketide bioprospecting, has developed a database,
the Environmental Surveyor of Natural Product Diversity (eSNaPD) that includes information on
thedistributionofpolyketidesynthasesintheenvironment. Sofaritislimitedtosoilsandmarine
sedimentsfromtheSouthwesternUSandNewEngland[59].
2.2. SoilBacterialHabitatsDoNotCorrespondtoEukaryoticHabitats
For over a hundred years, terrestrial ecologists have studied how eukaryotic life on Earth is
distributed. PlantandanimallifeisdistributedovertheEarthinbiomes,suchastundra,rainforest,
prairie,etc.Thesebiomesaremorediverseinthetropicsanddiversitydecreaseswithdistancefromthe
equator[60].Whenbiomessuchastropicalrainforestareconvertedtocropsorpasture[61],biological
diversitydecreases. Givenwhatweknowaboutthesebiomes,itwouldseemreasonabletobioprospect
fornewpolyketidenaturalproductsindiversetropicalbiomesundisturbedbyhumanactivity.However
whilefungaldistributionmayfollowthesebiomes[62],bacterialdistributiondoesnot.
The plants that grow on soil are probably not the best guide to the bacteria that live in them.
Soilpropertiesbetterpredictedbacterialcommunitystructurethanvegetationinseveralearlystudies
thatlookedatsoilsandlanduses[63–65]. In2006,astudybyFierercompared88soilsundernatural
vegetationfromthePeruvianAmazonandacrossthecontinentalUnitedStates[66]. SoilDNAwas
usedfor16Scommunityfingerprinting(T-RFLP).Fingerprintsfromsoilsclosetoeachotherbutof
differentpHdifferedbutresembledthosefromthousandsofmilesawaywiththesamepH.SoilpH
alsodeterminedhowdiversethefingerprintswere,withthemostdiversityatneutralpH.Thiseffectof
pHonbacteriacommunitystructurehasbeenconfirmedbystudiesthatcomparesoilsfromnarrower
geographicalregionssuchastheArctic[38]andMalaysia[41].
TheimportanceofpHtobacterialcommunitystructureleadstonon-intuitiveconclusionsfor
microbial bioprospectors. Biomes such as tundra may have similar soil bacterial communities to
biomeswithfarmoreanddiversevegetation[38]. Tropicalrainforestsoil,underneathoneofthemost
biodiverse plant and animal ecosystems on earth, has a relatively undiverse bacterial community
comparedtosoilsfromecosystemsthathaveordersofmagnitudefewerspeciesofplantsandare
Molecules2017,22,707 7of49
thousands of miles further north [66]. Converting Amazonian tropical rainforest to cropland or
plantationmayincreasesoilbacterialdiversity[67].
WhilepHmaybethemajorcontrollingfactorforsoilbacterialcommunitystructure,itisnotthe
onlyone. WhencomparingsoilsoveranarrowerrangeofpH,othersoilpropertiesareimportant.
AstudyinTheNetherlandscompares25sitesoverabroadrangeofsoilsandlanduses. Whenthree
acidpinMeolfeocurlees s2t01s7o, 2i2l,s 70w7 ereexcluded(pH3.7–4.1),soilphosphorouscontrolledbacterial7c oof m48 munity
structureintheremainingsoils(pH5.1–7.6)[68]. OnarangeofpotatosoilsinGermanyofsimilarpH
While pH may be the major controlling factor for soil bacterial community structure, it is not the
(5.2–6.2),parentmaterialcontrolledbacterialcommunitystructure[69].
only one. When comparing soils over a narrower range of pH, other soil properties are important. A
Microbialbioprospectorscannotrelyonthevegetationthattheyseearoundthemtoknowwhere
study in The Netherlands compares 25 sites over a broad range of soils and land uses. When three
tosampalceids opiiln.eT fhoreeswt islolilnse wederet oexkcnluodweda (praHn 3g.7e–o4.f1s),o siolipl prohposeprhtioersouths actonctarnollbeed rbeaacdteirliyal dcoemtemrmuniniteyd inthe
laboratostrryucoturrteh irno tuhge hresmoailinsiungr vseoyilsin (pgH. U 5.n1–fo7.r6t)u [n68a]t. eOlyn ma raannyges otuf dpoietastoo fsobilost ihn bGaecrmtearniayl oafn sdimpiloarly ketide
distribuptiHo n(5.i2n–6s.o2i)l, pdaorennot tmiantcelruiadl ceosnotriollplerdo bpaecrtteireiasl. community structure [69].
Microbial bioprospectors cannot rely on the vegetation that they see around them to know where
Eukaryoticandbacterialcommunitiesdonotjustdifferinhowtheyaredistributed,bacterial
to sample soil. The will need to know a range of soil properties that can be readily determined in the
communitiesarefarmorediverse. FiererandLennondiscusswhythisis,twoofthemostimportant
laboratory or through soil surveying. Unfortunately many studies of both bacterial and polyketide
reasonsbeingphylogeneticbreadthandgeographicalscale[70]. Bacteriaareoneofthethreedomains
distribution in soil do not include soil properties.
of life so aEfauikrarcyoomticp aanrdis boanctoerfiatlh ceombamcutneritiiaesl ddoiv neorts jiutyst odfiffseor iiln whoowu ltdhebye arwe idtihstrtihbeuteddi,v bearcstietryiaol f all of
the eukcaormymotuensitliievs ianrge fainr mthoree sdoiviler(sie..e F.,ieprelra nantsd, Laennnimona dlsis,cfuussn wgih,yp throist oisz, otwaoe otfc t.h).e mMoostr iemipmorptaonrtt ant for
bioprosrpeeacsotonsr sbeiisngth pehyqlougeesnteiotinc borefasdctha laen.d Ggeroagsrsaepshiacrael sscaalme [p70le].d Baicnte1riam ares qounea oref thpel othtsreew dhoimchaincsa n have
of life so a fair comparison of the bacterial diversity of soil would be with the diversity of all of the
10,000individualgrasses,tosampleonthisscaleforbacteriamicrobiologistswouldhavetosample
eukaryotes living in the soil (i.e., plants, animals, fungi, protozoa etc.). More important for bioprospectors
asinglefinesandgrain. Tosamplegrassesonthescalethatwesamplebacteria(tenstohundreds
is the question of scale. Grasses are sampled in 1 m square plots which can have 10,000 individual
ofbilliognrsa)sspeslo, ttos swamopulled onh athvies stcoalbe efo1r 0bakcmter2i.a Gmricarsosbiionloagi1st0s wkmou2lda hreaavew too sualmdphlea av seindgilfef efirneen scaensd intheir
environgmraeinn.t ,Ttoh seamsapmlee gwraossuelsd onb etheev secnalter utheart owfeb saacmteprleia bianctesroiail ,(taencso mto phluenxderendvsi roof nbmillieonnts,) wplhotesr ethere
arechanwgoeusldin hcaoven dtoi tbieo n10s kamtt2h. Gerµamss isnc aa l1e0. km2 area would have differences in their environment, the
same would be even truer of bacteria in soil, a complex environment, where there are changes in
2.3. Acticnoonbdaitcitoenrsia alt Sthoeil µCmo mscmalue.n ityStructureMayNotFollowBacterialSoilCommunityStructure
Ac2ti.3n.o Abcaticntoebraicaterciuall Stuoirl eCdomfrmoumnitsyo Sitlrsucatrueret Mheayt Nraodt iFtoilolonwa Blascoteuriracl eSooilf Cnoemwmupnoitlyy Skteruticdtuerse . Afollowup
totheFiererstudy,whichfoundthatthesoilbacterialcommunitystructurewascontrolledbysoil
Actinobacteria cultured from soils are the traditional source of new polyketides. A follow up to
pH[26],thues Feidernere sxttugdye,n werhaicthio fnousnedq utheant cthineg sooilf b1a6cStearimal pcolimcomnusnoitbyt satirnuectdurfer owmas tchoenitrroollreidg ibnya slosial mpHp lesand
foundth[2a6t],A ucsteidn onbeaxtc tgeerniaerwatieorne smeqousetnccoinmg mofo 1n6aS tahmigphliceornps Hob[t7a1in]e(dF ifgroumre th3e).irT ohreigaincatli nsoambapcletes raianldf raction
ofthebfaocutnedri athiant cArecatisneodbafcrtoermia 5w.3er5e% maotstp cHom<m4onto a2t 4h.3ig%hear tppHH [7>1]8 (Fbiugutraec t3i)n. oTbhea catcetriniaolbdacitvereirasl itywas
fraction of the bacteria increased from 5.35% at pH < 4 to 24.3% at pH > 8 but actinobacterial diversity
greatestatanintermediatepHbetween6and7. Thesamerelationshipbetweentherelativeabundance
was greatest at an intermediate pH between 6 and 7. The same relationship between the relative
ofActinobacteriaandpHwasfoundforthebacterialcommunitiesofpolarsoils[38]. Thissuggests
abundance of Actinobacteria and pH was found for the bacterial communities of polar soils [38]. This
thatneusturgagletsotsa tlhkaat lnineuetrsaol itlos amlkaaylinbee sothilse mbeayst btea trhgee btefsot rtaarcgteitn foobr aaccttienroibaalcbteiroipalr boisopperocstpinegct.ing.

Figure 3. Bacterial 16S 454 pyrosequencing results from 88 non-agricultural soils from across North and
Figure3.Bacterial16S454pyrosequencingresultsfrom88non-agriculturalsoilsfromacrossNorthand
South America. Reproduced with permission from Lauber et al., Appl. Environ. Microbiol., published by
SouthAmerica.ReproducedwithpermissionfromLauberetal.,Appl.Environ.Microbiol.,publishedby
the American Society for Microbiology, 2009 [71].
theAmericanSocietyforMicrobiology,2009[71].
Molecules2017,22,707 8of49
HoweveractinobacterialabundanceanddistributionmaynotbecontrolledbysoilpHinthe
samewaythatthewholebacterialcommunityis. Atthecontinentalscalesoilsindryclimatesare
neutraltoalkalineandsoilsinwetclimatesareacidic. Atthelandscapelevelsoilparentmaterial
can determine soil pH. Desert soils are enriched in Actinobacteria compared to soils from wetter
climates[72]. FieldplotstudieswherethepHofaplotofsoilisadjustedoveragradientcanseparate
the effect of pH from climate. Four field plot studies have been carried out on cropped [49,73]
and pasture soils [74,75] in the United Kingdom. While they have found that the actinobacterial
community structure changes with pH [74–76], they have not found that the relative abundance
oftheactinobacterialfractiondependsonpHthroughsequencingof16Samplicons[46,47,49]and
phospholipidfattyacidabundance[48]. Intwoofthesecasestherelativeabundanceofothermajor
bacterialphylogeneticgroupschanged[46,47],inanothertherewasnochangeforanyofthebacterial
phyla, although there were changes at lower phylogenetic levels [49]. These results suggest that
actinobacterialrelativeabundanceisnotcontrolledbypHbutdonotsaywhatdoescontrolit.
Alloftheabovestudieshaveeithercomparedsoilsundernaturalvegetationorlookedatasingle
agricultural land use (cropping/pasture). Converting native vegetation to agricultural use often
increasestheactinobacterialfractionofthesoilbacterialcommunity[65,77],inparticularwhenhumid
tropicalforestisconvertedtocrops[67,78]althoughthiseffectcanbebelowstatisticalsignificance[79].
ThismaybebecauseundernativeforestthesesoilsareacidicanddeforestationraisesthesoilpH
slightly. Thisistheexplanationgivenfortheeffectoflanduse(cultivation,pasture,pineforest,and
deciduous forest) on soil bacterial community structure in a well cited paper by the Fierer group.
TheauthorsexaminedtwelvesitesinSouthCarolinaanditwasshownthatlandusechangedthe
bacterialcommunitybychangingpHratherthananyotherinherenteffectofthelanduse[77].However
therelativeabundanceofActinobacteriacanincreaseaspHdecreasesduringconversionofforested
landtocropping[80]. InourownstudyofcontrastingsoilsfromColombia,CanadaandEuropeusing
communityfingerprintingwithtwosetsofactinobacterial16Sprimers,wefoundthatfingerprints
clusteredbylanduse(cultivatedversusuncultivated),withnoeffectofpH[46](Figure4).
Thereareseveralwaysthatlandusecouldaffecttherelativeabundanceandcommunitystructure
ofsoilActinobacteria. ThehigherlevelofActinobacteriaincultivatedandpasturelandsversusforest
inAlabama[81]orpalmoilplantationversusforestinBorneo,Malaysia[82],ledresearcherstosuggest
that Actinobacteria are selected by human disturbance. A study in the savanna region of Ghana
found that soils with no or little vegetation between corn cropping seasons had a higher relative
actinobacterialfraction[83]thansoilswithmorevegetation.
Land use can also affect the bacterial community structure by adding or removing substrate
for bacterial growth. Bacteria can be classified as oligotrophs or copiotrophs, similar to K- and
r-selectedplantsandanimals. Oligotrophshaveanadvantagewhenfoodislimitedandgrowslowly.
Copiotrophsaremorecompetitivewhenfoodisplentiful. Fiereretal. measuredcarbonandnitrogen
mineralizationin71soilsandcomparedthiswiththerelativepercentageoftheActinobacteriaand
five other bacterial groups, α-Proteobacteria, β-Proteobacteria, Acidobacteria, Bacteroidetes and
Firmicutes,asmeasuredbyquantitativereal-timePCR.TheyconcludedthatwhiletheAcidobacteria
wereoligotrophsandtheBacteroidetesandβ-Proteobacteriawerecopiotrophs,theα-Proteobacteria,
FirmicutesandActinobacteriawereneither[84].
Whether Actinobacteria are oligotrophic or copiotrophic may depend on the nature of the
substrate. Additionsofcarbonrichstraworreducedtillagethatleavesstrawonthesurfaceofthesoil
reducestherelativeamountofActinobacteriainthesoilbacterialcommunity[42]. Addingnitrogen
fertilizerincreasestheactinobacterialfractionofthesoilbacteria. AnincreaseintheActinobacteria
withnitrogenwasfoundfor28soilsfromnaturalvegetationacrossthecontinentalUnitedStatesby
nextgenerationsequencingof16Samplicons[85],aswellasatagrasslandandcultivatedcornsite
throughnextgenerationsequencingof16Samplicons[86,87]andshotgunmetagenomesequencing[60].
BothnitrogenandpotassiumadditionincreasedtheactinobacterialfractionofaDutchgrasslandsoil
asmeasuredbyshotgunmetagenomesequencing[88].
Molecules2017,22,707 9of49
A100yearoldfieldtrialinAlabamacomparedtheeffectofaddinglimetoraisepHofanacid
soilversusaddinglimeandfertilizer. ThebacterialcommunitystructurewasmeasuredusingPLFA
analysis. WhilepHhadmoreofaneffectontheoverallbacterialcommunitystructurethannitrogen
fertilizer,nitrogenfertilizerhadamajoreffectontheactinobacterialandfungallevels[89]. Anincrease
Molecules 2017, 22, 707 9 of 48
intheactinobacterialfractionofthebacterialpopulationhasbeenascribedtotheflushofnutrients
producedabnyalybsuisr. nWinhigle apfHte hradd emfoorree osft aant ieoffnecitn onth thee Aovmeraalzl obnact[e5r1ia]l. community structure than nitrogen
fertilizer, nitrogen fertilizer had a major effect on the actinobacterial and fungal levels [89]. An
MoreworkisneededcomparingtherelativeimportanceofpH,moisture,landuseandnitrogen
increase in the actinobacterial fraction of the bacterial population has been ascribed to the flush of
onactinobnautcrtieenrtisa plrcoodmucemd ubyn bituyrnsintrgu acftteur rdeefaornedstartieolna tiniv theea Abmuanzdona [n5c1]e. insoil. Theremaynotbeasimple
relationbetweMeonret whoerske isf anecetdoerds .coJmusptarainsgt thhee raeclattiinveo ibmapcotretarniacel ocfo pmHm, muonistiutyre,s ltarnudc utsuer aendd noietrsogneont followthat
on actinobacterial community structure and relative abundance in soil. There may not be a simple
ofthewholebacterialcommunity,differentsubgroupsoftheActinobacteriamaybeselectedforby
relation between these factors. Just as the actinobacterial community structure does not follow that
differentfactors. Forexample,Actinobacteriacanbethelargestfractionofthebacterialcommunities
of the whole bacterial community, different sub groups of the Actinobacteria may be selected for by
ofdesertsd[i7ff2er]eantn fdactloarks.e Fowr eaxtaemrspl[e9, 0A,c9ti1n]o;bpacrtoerbiaa cbalny bde ithffee lraergnetstm fraeccthioann oifs tmhe sbaocftesriealle ccotmiomnunaitrieeso ccurringin
thesecaseos.f dTehseisrtsu [n7c2]e arntadi nlatkye mwaetaerns s[9b0i,9o1p];r porsopbeabcltyo drsiffnereeendt mtoecchoanmispmasr oef aseblercotiaodn arraen ogcceuorrfinsgo iinls ofdiffering
these cases. This uncertainty means bioprospectors need to compare a broad range of soils of differing
properties,landuses,andnutrientinputs.
properties, land uses, and nutrient inputs.

Figure 4. Forward Terminal Restriction Fragment Length Polymorphism (T-RFLP) community
Figure 4. Forward Terminal Restriction Fragment Length Polymorphism (T-RFLP) community
fingerprints of the Actinobacteria from Uncultivated soil (blue) Cultivated soils (green), animal associated
fingerprints of the Actinobacteria from Uncultivated soil (blue) Cultivated soils (green), animal
sediments (orange) and street dust (red). Reproduced from Hill et al., Microb. Ecol., published by
associatedSsperidngimer eInnttesrn(aotrioannagl eP)ubalnisdhisntgr AeeGt.,d 2u01s1t [(4r6e]d ).ReproducedfromHilletal.,Microb.Ecol.,published
bySpringerInternationalPublishingAG.,2011[46].
2.4. Non-Actinobacterial Bacterial Polyketide Producers
While most polyketides discovered by cultivation of bacteria in the laboratory so far have come
2.4. Non-ActinobacterialBacterialPolyketideProducers
from Actinobacteria, many bacterial polyketide producers in the environment are not Actinobacteria.
WhileIn mthoe scatspe oofl yarkoemtiadtice stydpei sIcI opovleyrkeetdidbesy mcoustl tpirvoadtuicoenrs oafreb liakcetlye rAiactiinnobtahcetelraiab. Goreanetos renycosdoinfag rhavecome
type II polyketide synthases (PKSII) amplified from the soils DNA either directly [92–95] or from
fromActinobacteria,manybacterialpolyketideproducersintheenvironmentarenotActinobacteria.
metagenomic libraries [96] appear to be entirely actinomycetal. The only example where PKSII
Inthecaseamopflaicroonms aaptpiecart ytop bee InIopt-oacltyinkoemtiydceetsalm is oa sstetp orfo fidveu ocuetr osf afirfetyl-sikeveelny sAeqcuteinncoesb aamctpelirfiiead. fGroemn esencoding
type II poEluyrkoepteiadne sosilys.n Itnh tahisse csas(eP thKeS PIKI)SIaI mampplliicfioends afprpoemar ttoh bee fsroomils prDotNeoAbaceteirtiha eorr fdirimreiccuttleys [[9962]. –95] or from
In contrast many studies have found environmental amplicons and clones with type I polyketide
metagenomic libraries [96] appear to be entirely actinomycetal. The only example where PKSII
synthase (PKSI) sequences that cluster with non-actinobacterial sequences. A broad range of studies
ampliconsappeartobenot-actinomycetalisasetoffiveoutoffifty-sevensequencesamplifiedfrom
have used PKSI specific primers to amplify soil DNA followed by cloning and Sanger sequencing.
EuropeanFsoor ielxsa.mInpleth, misoscta PsKeStIh seeqPueKnSceIsI farommp sloiiclso fnrosma Mppaleaaysriatno rbaienfforroemst [9p4r],o atne oAbntaacrctetirc iIaslaonrdfi [9r7m] icutes[96].
IncontrastmanystudieshavefoundenvironmentalampliconsandcloneswithtypeIpolyketide
synthase(PKSI)sequencesthatclusterwithnon-actinobacterialsequences. Abroadrangeofstudies

have used PKSI specific primers to amplify soil DNA followed by cloning and Sanger sequencing.
Forexample,mostPKSIsequencesfromsoilsfromMalaysianrainforest[94],anAntarcticIsland[97,98],
closetocucumberroots[99],andtheTibetanplateau[100]clusteredwithnon-actinobacterialsequences
whentreed.
Molecules2017,22,707 10of49
Three studies have sequenced metagenomics PKSI clones from soil that do not appear to be
actinomycetal. AspartoftheEuropeanUnionfundedMetacontrolproject,ametagenomiclibrary
wasmadeofenvironmentalDNAfromaFrenchcultivatedsoil[101]. The60,000clonesinthelibrary
werescreenedwithPKSIspecificprimersand139positiveclonesfound. Threeofthesecloneswere
fully sequenced. Two PKS domains from these clones, the ketosynthase (KS) and acyltransferase
(AT)domainsclusteredwithmyxobacterialKSandATaminoacidsequences. Afosmidlibraryfrom
acultivatedsoilintheMetagenomicprojectatthe[39]UniversityofWisconsin,wasscreenedand
29cloneswereidentifiedascontainingKSdomainsbyhybridization. Theinsertsoffiveoftheseclones
weresequenced. TheirKSdomainhomologysuggestedthattheywerefromtheProteobacteriaor
Cyanobacteria. Howeveranalysisofthefullinsert,itsGCcontent,andcomparisonwithKSdomain
sequencesfromthreeacidobacterialisolatessuggestedthattheywerefromtheAcidobacteria.
Asafinalexample,DNAwasextractedfromaBrazilianeucalyptusplantationsoil,andmade
into a fosmid clone library. A clone with a PKSI hit was sequenced [102]. The start of the clone
containedasinglePKSImodulewith20otheropenreadingframes(ORFs). MatchestotheORFwere
allnon-actinomycetal,whiletheKSandATdomainsofthePKSImoduleclusteredawayfromtheir
actinobacterial counterparts when treed with the SEARCHPKS database [103]. This database was
disproportionatelyactinomycetalpossiblybiasingtheanalysis. Howeverthisresultwasconfirmed
whenthesequenceswerereanalyzedwithabroaderrangeofnon-actinomycetalPKSdomains[104].
These environmental PKSI sequences were probably non-actinomycetal, but as only part of
theirgenomewassequencedwecannotbecertainwhichbacterialgrouptheywerefrom. Insome
environmentsmostPKSIproducersareactinomycetal.
InourworkwecomparedPKSIampliconsofKSandATfromstreetsediments[104],whichare
enriched with Actinomycetes, and soils [46]. Many soil amplicons clustered with myxobacterial
sequences. However several clades clustered with actinomycetal sequences and were specific
to samples from the streets of Ottawa (Canada), Faisalabad (Pakistan), and four European cities.
TheBradygroupcomparedPKSIKSsequencesinsoilsfromNewEnglandanddesertsoilsofthe
SouthwesternUnitedStates[105]throughpyrosequencingamplicons.KSdomaindiversitywasgreater
intheactinobacterialrichdesertsoils.
As further evidence that non-actinomycetal source may prove important in bioprospecting,
anewclassofantibiotics(albeitanon-ribosomalpeptidenaturalproduct)wasrecentlydiscovered
throughtheuseofaninsitucultivationtechniquethatgrewanovelβ-proteobacteriafromsoil[17].
Non-actinomycetalproducingorganismsmaybeimportantforfuturepolyketidediscovery.
2.5. TheQuestionofBiogeography
Thetermbiogeographyisusedintwowaysinmicrobialecology. Oftenitsimplymeanschanges
in the community over geographical distance. An example of this is the study that found that
the actinobacterial and PKSII communities of soils in Uzbekistan and New Jersey differed [93].
BiogeographyinthissensewasdiscussedintheearliersectionontheimportanceofpHandother
soil properties in the distribution of bacteria and actinobacteria (see Sections 2.2 and 2.3). In the
narrowersense,usedinthisreview,biogeographymeansthatcommunitiesinsimilarenvironments
thatareseparatedbydistanceorabarriersuchasanoceanwillhavedifferentmicrobialcommunities.
ThisdifferenceisattributedtothecommunitiesevolvingseparatelyasWallaceproposedinthe1876
GeographicDistributionofAnimals[106]. Biogeographycanhavealargeeffectonthedistributionof
largemulticellularorganisms. Forexample,marsupialsarefoundinAustraliabutnotinAfrica,even
thoughtherearesimilarenvironmentsinbothplaces.
For polyketide discovery, the more polyketide synthase gene distribution in terrestrial
environmentsiscontrolledbybiogeography,themoreimportantitistosamplesimilarhabitatsthat
areseparatedfromeachotherbydistanceorgeographicalbarriers. Ifbiogeographyisnotimportant,
itismoreimportanttosampledifferenthabitats,eveniftheyareclosetoeachother. Addressingthis
questioniscomplicatedbythedifficultyofdefiningbacterial,actinobacterialandpolyketidehabitats.