Assessment of Bacterial bph Gene in Amazonian Dark Earth and Their Adjacent Soils

12 Pages·2014·1.23 MB·English

Assessment of Bacterial bphGene in Amazonian Dark
Earth and Their Adjacent Soils
MariaJuliadeLimaBrossi1*,LucasWilliamMendes1,MarianaGomesGermano2,AmandaBarbosaLima1,
Siu Mui Tsai1
1CellularandMolecularBiologyLaboratory,CenterforNuclearEnergyinAgriculture,UniversityofSa˜oPaulo,Piracicaba,SP,Brazil,2BrazilianAgriculturalResearch
Corporation,EmbrapaSoybean,Londrina,PR,Brazil
Abstract
Amazonian Anthrosols are known to harbour distinct and highly diverse microbial communities. As most of the current
assessments of these communities are based on taxonomic profiles, the functional gene structure of these communities,
such as those responsible for key steps in the carbon cycle, mostly remain elusive. To gain insights into the diversity of
catabolic genes involved in the degradation of hydrocarbons in anthropogenic horizons, we analysed the bacterial bph
gene community structure, composition and abundance using T-RFLP, 454-pyrosequencing and quantitative PCR essays,
respectively. Soil samples were collected in two Brazilian Amazon Dark Earth (ADE) sites and at their corresponding non-
anthropogenicadjacentsoils(ADJ),undertwodifferentlandusesystems,secondaryforest(SF)andmanioccultivation(M).
Redundancy analysis ofT-RFLP data revealed differences in bphgene structure according to both soiltype and landuse.
Chemical properties of ADE soils, such as high organic carbon and organic matter, as well as effective cation exchange
capacityandpH,weresignificantlycorrelatedwiththestructureofbphcommunities.Also,thetaxonomicaffiliationofbph
gene sequences revealed the segregation of community composition according to the soil type. Sequences at ADE sites
were mostly affiliated to aromatic hydrocarbon degraders belonging to the genera Streptomyces, Sphingomonas,
Rhodococcus,Mycobacterium,ConexibacterandBurkholderia.Inbothlandusesites,shannon’sdiversityindicesbasedonthe
bphgenedatawerehigherinADEthanADJsoils.Collectively,ourfindingsprovideevidencethatspecificpropertiesinADE
soils shape the structure and composition of bph communities. These results provide a basis for further investigations
focusingon thebio-exploration ofnovel enzymeswith potentialuse inthe biotechnology/biodegradation industry.
Citation:BrossiMJdL,MendesLW,GermanoMG,LimaAB,TsaiSM(2014)AssessmentofBacterialbphGeneinAmazonianDarkEarthandTheirAdjacent
Soils.PLoSONE9(6):e99597.doi:10.1371/journal.pone.0099597
Editor:NiyazAhmed,UniversityofHyderabad,India
ReceivedJanuary22,2014;AcceptedMay16,2014;PublishedJune13,2014
Copyright: (cid:2) 2014 Brossi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:Fundingprovidedby‘ConselhoNacionaldeDesenvolvimentoCient´ıficoeTecnolo´gico’andthe‘Fundac¸a˜odeAmparoa`PesquisadoEstadodeSa˜o
Paulo’(FAPESP/Biota2011/50914-3).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction ADJsitespresentdifferencesinmicrobialcommunitycomposition,
andbacterialdiversityhasbeenreportedtobehigheratADEsites
Amazonian Dark Earth (ADE), locally termed ‘Terra Preta de
[4,5,6,7,8].Mostofthesestudiesrelyoncomparisonsbetweenthe
I´ndio’, are anthropogenic soil horizons built-up by the Pre-
taxonomic profiles of these communities (i.e., based on the
Colombian Indians between 500and 8,700 years ago. These soil
taxonomic bacterial 16S rRNA gene). In this sense, theextent to
sites were formed by the progressive deposit of materials and
whichthelocalenvironmentshapesthefunctionalprofilesofthese
organic compounds, such as charcoal, bone, and pottery sheds,
communities, and influences their performance, remains mostly
which gradually shifted the natural physical and chemical
elusive [9,10,11,12,13].
properties of thesoil. As a result, relatively infertileAmazon soils
Soil is one of the most biodiverse ecosystems on Earth, being
were progressively converted into highly fertile spots through
able to support communities from multiple trophic levels, which
processes like increasing the cation exchange capacity and the
areconstantlyperformingthemetabolismofdiverseandcomplex
nutrient content,as wellas promotingthestabilization of thesoil
substrates.Theextremespatialandtemporalheterogeneityofthe
physicalstructure[1,2].Substantialincrementsoforganicmaterial
soil matrix, paired with the myriad of internal and external
in these sites gradually increased the carbon content, yielding to
feedbacks, are known to determine the structure and function of
theformationofsoilspotswithahighproportionofincompletely
thesecommunities[14].Microbesareinvolvedinmanyecosystem
combusted biomass (biochar). These spots have been reported to
processes, including biodegradation, decomposition and mineral-
reachuptoa70-foldhigheramountofcarbonthannativesoilsat
ization,inorganicnutrientcycling,diseasecausationandsuppres-
adjacent locations (ADJ) [3].
sion,andpollutantremoval.Soildisturbancesareknowntocause
The existence of ADE sites close to their natural ADJ soil
shiftsinmicrobialactivities,shiftingtherateoftheseprocessesand
locations, which present the same geological history, provides a
triggering impacts on the ecosystem performance [15]. Several
unique opportunity to investigate the role of biotic and abiotic
environmental factors are known to affect microbial community
factors influencing the microbial community assembly and
composition in the soil, including soil temperature, moisture,
dynamics at these sites. Previous studies revealed that ADE and
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BacterialbphGeneinAmazonianDarkEarth
texture,carboncontent,nutrientavailability,pH,landusehistory, 10cm), and the overlaying litter was discarded. Each sample
seasonality, and the content of incompletely combusted biomass, containedapproximately300gofsoilandwastransportedtothe
such asbiochar [13,16,17,18,19]. laboratory at4uCtofurtherprocessing(,24h).Aportionofthe
The biochar content is the major physical distinction between samples were frozen (220uC) for total DNA extraction while the
ADE and their ADJ soils, which is also known to play an other portionwas kept at 4uCforchemical measurements.
important role in global carbon biogeochemistry [20]. In Chemical analyses were performed at Amazon Embrapa
anthrosols, such as ADE, it is predicted that distinct microbial (Manaus, Brazil), according to instructions provided by the
communitiescanperformuniqueprocesses,suchastheretention Embrapa protocol [30]. Briefly, soil samples were analysed in
of high-labile carbon [5]. Despite the unique and specialized triplicate for pH (H O, 1:2.5); H+Al (calcium extractor 0.5 mol
2
capabilitiesofthesesoils,functionalassessmentsoftheirmicrobial L21, pH 7.0); sum of bases (SB); soil organic matter (SOM); soil
community, particularly those of genes encoding important steps organiccarbon(SOC;Walkely-Blackmethod);extractablefraction
in the carbon cycle, are still scarce. Biodegradation through of Al, Ca, and Mg (1 M KCl); extractable fraction of P and K
bacterialactivityisoneofthemostimportantprocessesoccurring (double acid solution of 0.025M sulphuric acid and 0.05M
in soils regarding organic matter recycling. This process involves hydrochloric acid Mehlich 1); and effective cation exchange
genes actingon key stepsin thecarboncycle, fortheturnover of capacity (eCEC).
more recalcitrant organic carbon, as well as for pollutant
degradation in the ecosystem [21]. This process, along with Total soil DNA extraction and PCR amplifications for
biosynthesis,largelygovernsthecarboncycleintheenvironment, T-RFLP
which is dependent on microbial enzymatic activities that most
Total DNA was extracted using 0.25g of soil as an initial
often use organic compounds asa primary energysource[22].
material. Extractions were carried out in triplicate for each site,
Theprimarystepinvolvedintheaerobicmicrobialdegradation using the PowerSoil DNA isolation kit (MoBio Laboratories,
of aromatic hydrocarbons is an oxidative attack [23,24], where Carlsbad, CA, USA), according to the manufacturer’s protocol.
enzymes named oxygenases are responsible for the insertion of DNAqualityandquantityweremeasuredspectrophotometrically
molecular oxygen into aromatic benzene rings [25]. Genes using NanoDrop1000(Thermo Scientific, Waltham, EUA).
encoding these enzymes have been characterized in Rhodococcus,
For T-RFLP analyses the bacterial 16S rRNA gene was
Acinetobacter, Pseudomonas, Mycobacterium, Burkhoderia, inter alia amplified with the primer set 27F - FAM labelled (59 AGA
[25,26]. The a-subunit of oxigenases is known as the catalytic GTT TGA TCC TGG CTC AG 39) and 1492r (59 ACC TTG
domaininvolvedinthetransferofelectronstooxygenmolecules. TTA CGA CTT 39) [31]. The bph gene was amplified with the
Due its DNA sequence conservation, this subunit has been primer set BPHD F1– FAM labelled (59 TAY ATG GGB GAR
currentlyusedasatargetgeneforthedetectionofsuchenzymesin GAYCCIGT39)andBPHDR0(59ACCCAGTTYTCICCR
complex communities[21,27,28,29]. TCG TC 39) [21]. For 16S rRNA gene amplification, PCR
In this study, we evaluated the structure, composition and reactionswerecarriedoutinavolumeof25 mLcontaining2.5mL
abundance of the bacterial catabolic gene Biphenyl Dioxygenase reaction buffer 106 (Invitrogen, Carslbad, CA, USA), 1.5mL
(bph)involvedinaromatichydrocarbondegradationinAmazonian MgCl (50 mM),1 mLofeachprimer(5pmolmL21),0.2mL(5 U)
2
Dark Earth and their adjacent soil locations. We aimed to of Platinum Taq DNA polymerase (Invitrogen), 0.5 mL of
determinetheroleofanthropogenicactioninthediversity ofthe deoxyribonucleotide triphosphate mixture (2.5 mM), 0.25mL of
bphgeneinsoilbacterialcommunities.Understandingthediversity bovine serum albumin (1 ng mL21), 1 mL of DNA template (ca.
ofspecificbacterialgenesinADEshouldledtofuturestudiesthat 10ng) and 18.05mL of sterilized ultrapure water. Amplifications
investigate the microbial ecology of anthropogenic altered soils, were performed in the GeneAmp PCR System 9700 thermal
especially in regards to their potential source of novel enzymes. cycler (Applied Biosystems, Foster City, CA, USA). Reaction
Collectively,thisstudycharacterizedthiscatabolicgeneoccurring conditionswere94uCfor3 min,followedby35cyclesof94uCfor
in Amazonian Dark Earth, and compared the profiles with those 30s,59uCfor45s,and72uCfor1 minwithafinalextensionstep
obtained from adjacent sites, as well as under distinct land use at72uCfor15min.Forthebphgeneamplification,PCRreactions
systems. werecarriedoutinavolumeof25 mLcontaining2.5 mLreaction
buffer 106 (Invitrogen, Carslbad, CA, USA), 1.5mL MgCl
2
Materials and Methods (50 mM),1.25mLofeachprimer(5pmolmL21),0.5mL(5U)of
Platinum Taq DNA polymerase (Invitrogen), 0.3mL of deoxyri-
Ethic statement
bonucleotide triphosphate mixture (2.5 mM), 1 mL of DNA
Nospecificpermitswererequiredforthedescribedfieldstudies. template (ca. 10ng) and 16.7mL of sterilized ultrapure water.
Thelocationsarenotprotected.Thefieldstudiesdidnotinvolve Forbphgene,similarPCRcyclingconditionswereused,exceptthe
endangered or protected species. annealing temperature that was set at 60uC. Negative PCR
controls (without DNA template) and positive controls (using the
Study sites, sample collection and soil chemical analyses EscherichiacoliATCC25922DNAforthe16SrRNAgeneandthe
StudiedsitesarelocatedattheCaldeira˜oExperimentalStation DSM6899PseudomonasputidaDNAforbph)wereruninparallelfor
of Amazon Brazilian Agricultural Research Corporation (Em- both amplifications. After the amplifications, 5 mL of obtained
brapa) in Iranduba County in the Brazilian Central Amazon products (ca. 60ng) was digested with the endonuclease HhaI
(03u26900" S, 60u23900" W). A detailed description of the soil (Invitrogen) in 15mL reaction for 3 h at 37uC. Obtained
samplinglocationsisgivenbyTaketanietal[8].Briefly,thefour fragments were further purified using sodium acetate/EDTA
sitessampledarecomposedoftwoAmazonianDarkEarth(ADE) precipitation and then mixed with 0.25mL of the Genescan 500
and their two correspondent adjacent soils (Haplic Acrisol, ADJ). ROXsizestandard(AppliedBiosystems)and9.75 mLofdeionized
These sites are under a ,35 year-old secondary forest (SF) or formamide.Priortofragmentanalysis,samplesweredenaturedat
under manioc (Manihot esculenta) cultivation (M). Hereafter these 95uCfor5 minandchilledonice.Analysisofterminalrestriction
sites are termed as ADE-SF, ADJ-SF, ADE-M and ADJ-M. Soil fragment (T-RF) sizes and quantities was performed on an ABI
samples were taken in triplicate from the topsoil layer (ca. top PRISM3100genetic analyzer(Applied Biosystems).
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BacterialbphGeneinAmazonianDarkEarth
T-RFLP profiles were analysed using PeakScanner v1.0 at position 351of thereference sequence of Pseudomonasputida F1
software (Applied Biosystems, Foster City, CA, USA). Terminal (YP_001268196). The total valid sequences were rarefied to the
restriction fragments (T-RFs) of less than 25bp were excluded smallest number of sequences per sample in order to minimize
priortotheanalysis.ThetotalvaluesofT-RFsforeachsoilsample effects of samplingeffortupon analysis.
were pulled together to construct a Venn’s diagram showing Distance matrices were constructed using the MOTHUR
sharedT-RFsamongsamples.Therelativeabundanceofasingle software [34]. The resulting matrices were used to estimate the
T-RF was calculated as percent fluorescence intensity relative to number of operational protein families (OPF) (i.e. group of
totalfluorescenceintensityofthepeaks[32].Datafromindividual proteinsthatshareacommonevolutionaryorigin)andtoestimate
sampleswerecombinedtosoilchemicalparametersandsubjected richness(i.e.Chao1,Jackknife,andACEindices)anddiversity(i.e.
to multivariate analysis using Canoco 4.5 (Biometris, Wagenin- Shannon and Simpson indices). Rarefaction curves were con-
gen,The Netherlands) and Primer6 (PrimerE, Ivybridge, United structed at a cutoff level of 94% of amino acid identity.
Kingdom). All matrices were initially analysed using de-trended MOTHUR was also used to perform #-Libshuff comparisons
correspondence analysis (DCA) to evaluate the length of the betweenthefourstudiedsites.TheGood’scoverageestimatorwas
gradientofthespeciesdistribution;thisanalysisindicatedlinearly usedtocalculatethesamplecoverageusingtheformulaC= 1-(n/
i
distributeddata(lengthofgradient,3),revealingthatthebest-fit N),whereNisthetotalnumberofsequencesanalysedandn isthe
i
mathematical model for the data was the redundancy analysis numberofreadsthatoccursonlyonceamongthetotalnumberof
(RDA).Forwardselection(FS)andtheMonteCarlopermutation readsanalysedatacutoffvalueof94%ofaminoacididentity[35].
test were applied with 1,000 random permutations to verify the UnweightedUniFracdistancesamongcommunitieswereestimat-
significance of soil chemical properties upon the microbial ed using a tree constructed de novo using FastTree. One
community. In addition to P values for the significance of each representative sequence per OPF was selected and subjected to
soil chemical property, RDA and Monte Carlo permutation test taxonomic affiliation by the comparison tool of NCBI Tblastx
supplied informationabout themarginal effects of environmental (GenBank) using Blast2Go [36].
variables, quantifying the amount of variance explained by each Sequence data generated by 454-pyrosequencing are available
factor.WeusedANOSIMbasedonrelativeabundanceofT-RFs attheMG-RASTserver(http://metagenomics.anl.gov)underthe
totest forstatistical differencesbetween samples. project ‘Diversity of bhp gene in Amazon soils’ (ID 8489) and
accession numbers 4557319.3 (ADE_SF), 4557318.3 (ADE_M),
454-Pyrosequencing analyses of the bacterial bph gene 4557321.3(ADJ_SF) and4557320.3(ADJ_M).
A partial region of the bph gene was amplified for the 454-
pyrosequencing using the primer set BPHD F3 (59 ACT GGA Quantitative PCR (qPCR) of the bacterial 16S rRNA and
ARTTYGCIGCVGA39)andBPHDR0(59ACCCAGTTY bph genes
TCI CCR TCG TC 39) [20] containing specific Roche 454- Thebacterial16SrRNAgenewasamplifiedwiththeprimerset
pyrosequencing adaptors and barcodes of 8 bp. The expected U968F (5’ AAC GCG AAG AAC CTT AC 3 ’) and R1387 (5’
fragment size was ca. 520bp. Three independent amplifications CGG TGT GTA CAA GGC CCG GGA ACG 3 ’) [37], which
were performed for each sample. The 20 mL PCR mixture amplify a fragment of approximately 400bp. The 520bp
contained 16 FastStart High Fidelity Reaction Buffer (Roche fragmentofthebphgenewasamplifiedwiththeprimersetBPHD
Diagnostics,Basel,Switzerland),1.25mMofeachprimer,150ng F3(59ACTGGAARTTYGCIGCVGA39)andBPHDR0(59
mL21ofbovineserumalbumin(NewEnglandBioLabs,Ipswich, ACC CAG TTY TCI CCR TCG TC 39) [21]. qPCR reactions
MA,USA),0.2mMofdNTPs,0.5mL(2.5U)ofFastStartHigh were performed in 10mL containing 5 mL of SYBR green PCR
FidelityPCRSystemEnzymeBlend(RocheDiagnostics)and4 ng master mix (Fermentas, Brazil), 1mL of each primer (5 pmol
oftemplateDNA.ThePCRconditionswereoptimizedusingthe mL21), 1mL of DNA template (ca. 10ng) and 2 mL of sterilized
genomicDNAofBurkholderiaxenovoransLB400[33],whichcarries ultrapure water. Thermocycling conditions for the 16S rRNA
one of the target dioxygenase genes. Amplifications were gene were set as follows: 94uC for 10min; 40 cycles of 94uC for
performed as follows: 95uC for 3min, 30 cycles of 95uC for 30s, 56uC for 30s and 72uC for 40s. Amplification specificity
45s,60uCfor45sand72uCfor40s,withthefinalextensionof was checked by a melting curve and the data collection was
72uCfor4 min.TriplicatePCRproductscontainingtheexpected performed at every 0.7uC. qPCR reactions for the bph gene were
fragmentsizewerepurifiedusingtheQIAquickGelExtractionKit performed at similar conditions, except for the annealing
(Qiagen, Hilden, Germany) and QIAquick PCR Purification Kit temperature set at 60uC. Reactions were performed in a
(Qiagen). DNA concentrations were determined using the StepOnePlus system (Applied Biosytems). The Cts values (cycle
NanoDrop ND-1000 spectrophotometer (NanoDrop Technolo- threshold) were used as standers for determining the amount of
gies,Wilmington,DE,USA).PurifiedPCRproductswerepooled DNAtemplateineachsample.Standardcurveswereproducedfor
andsubjectedtopyrosequencingusingtheFLXsequencingsystem the 16S rRNA and bph genes using specific cloned fragments.
(454 Life Sciences,Branford, CT,USA). Gene fragments were quantified in a spectrophotometer (190 a
Raw data was filtered for valid sequences using the FunGene 840nm - NanoDrop ND-1000) and diluted (107 to 103 genes
Pipeline Repository (http://fungene.cme.msu.edu/ mL21forthe16SrRNAgeneand106to102genesmL21forbph)to
FunGenePipeline/). Quality sequences were translated in the generateeachspecificstandardcurves.Thegenecopynumbersin
correct frame of aminoacids using the RDP FrameBot tool. The different soil samples were expressed as log copy numbers of the
RDP pipeline extracted a set of representative sequences from gene per gram of soil. Statistical comparisons were performed
known bph sequences to use as subject sequences with FrameBot. using one-way ANOVA(Tukey’s test).
TheFrameBotproducesanoptimalalignmentbetweenthequery
andthesubjectsequencesinthepresenceofframeshifts.Onlythe Results
protein pairwise alignment with the best score was reported.
ProteinsequencespassingFrameBotwerealignedwithHMMER Variation in soil chemical properties
usingamodeltrainedonthesamesetofrepresentativesequences Soil chemical properties were measured for each individual
used by FrameBot. The aligned protein sequences were chopped samplecollectedinADEandADJsites(foradetaileddescription
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BacterialbphGeneinAmazonianDarkEarth
see Table S1). Statistical differences were observed using Tukey’s mostly with pH, eCEC, SOM and SOC, while sites at ADJ-FS
test.Overall,soilchemicalpropertiesofADE-SFwerechemically presented asignificant correlation toH+Al.
similar to ADE-M. Likewise, ADJ-SF chemical properties were ANOSIM analysis indicated statistical differences between the
also very similar to ADJ-M. As expected, major differences were twosoiltypesandlandusesystems(Table1).R-valuesrevealeda
attributed mostly tosoil typerather than theland use history. clear segregation of bph gene structures (R.0.75) in ADJ soils,
Higher soil pHvalueswereobserved inADEratherthan ADJ while the 16S rRNA gene differed to a lower extent across sites
sites.WhileADJsoilswereveryacidicwithapHof3.53(ADJ-SF) (R,0.2).
and3.74(ADJ-M),ADEsiteswereonlyweaklyacidicwithapHof
5.51 (ADE-SF) and 5.41 (ADE-M). Sites at ADE showed lower Diversity of the bacterial bph gene across soil types and
total and exchangeable Al (H+Al), a phenomenon that is likely
land uses
directly connectedtoobserved variations insoil pH.
Toaccessthecompositionofbphgene,samplesweresequenced
Soil organic carbon (SOC), soil organic matter (SOM) and
using 454-pyrosequencing. A total of 7,710 reads matched the
effective cation exchange capacity (eCEC) were higher in ADE
barcodes,ofwhich6,877readspassedtheinitialfiltering(89.2%)
sites.DifferentlandusesdidnotinfluencethesepropertiesinADJ
and 5,965 (86.7%) were effectively translated into amino acid
soils; however, the same properties showed significantly higher
sequences using the FrameBot. A total of 4,690 valid amino acid
values in the site under secondary forest rather than in manioc
sequenceswerefurtherrarifiedtothedepthof750sequencesper
cultivation, in ADE soil locations. In detail, SOC, SOM and
sample(theminimuminasinglesample)forcomparativeanalysis.
eCECvalueswereapproximately30%higherintheADE-SFsite
The diversity indices for the bph gene (Table 2) revealed that
whencompared totheADE-M.
ADEsites(SFandM)(H9=4.24and4.05,L9=0.024and0.028,
respectively) were more diverse than ADJ sites (SF and M)
Assessment of community structures based on the
(H9=3.19 and 3.17, L9=0.098 and 0.107, respectively). These
bacterial 16S rRNA and bph genes
indices also showed that sites under SF were more diverse than
T-RFLP analyses for the bacterial 16S rRNA and bph genes
sites under M. Richness estimators (i.e. Chao1, ACE and
wereperformedforthefoursites.Theobtainedprofileswereused
Jackknife) also revealed ADE sites (SF and M) (Chao 1=238
todeterminetherichnessofterminalrestrictionfragments(T-RFs)
and 229, ACE =248 and 286, Jackknife =271 and 255,
andtoperformthemultivariateanalyses.Atotalof152,144,147
respectively) to present higher values than ADJ (Chao 1=151
and141T-RFswereobtainedfortheanalysisofthebacterial16S
and 127, ACE =210 and 166, Jackknife =170 and 130,
rRNA gene in ADE-SF, ADE-M, ADJ-SF and ADJ-M sites,
respectively), withSF sites being alsohigherthan sites underM.
respectively. There were 14 T-RFs detected as dominant
ADEsitespresentedatotalof159and129OPFsforSFandM
throughoutallsites,accountingfor.50%ofthetotalfluorescence
sites,respectively.Conversely,thesevaluesforADJwerelower(95
detected for the 16S rRNA gene analyses. Tukey’s test (P.0.05)
and 90, for SF and M, respectively). These sites also presented a
indicatednodifferencebetweensitesintherichnessofT-RFsfor
differentnumberofsingletons(numberofuniquereadsperOPF):
theobtained profiles ofbacterial 16S rRNAgene.
66, 57, 43 and 36 for ADE-SF, ADE-M, ADJ-SF and ADJ-M,
For the bph gene analysis no dominant T-RFs were found,
respectively.
possiblyduetothehighheterogeneityofthisgene.Therewere90,
Statisticaldifferencesamongsitesforthecompositionofthebph
78, 73 and 69 T-RFs in ADE-SF, ADE-M, ADJ-SF and ADJ-M
gene were confirmed by #-Libshuff (P,0.001). Venn’s diagrams
sites, respectively. Samples from ADE sites showed statistically
highlight the number of shared OPFs among samples (Figure 4).
higherrichnessofT-RFs(Tukey’stest)thantheobservedat ADJ
The number of shared OPFs between ADE soils under different
sites (P.0.05).
land uses systems (SF and M) was 84 (41% of the total OPFs
The Venn’s diagram according to soil type showed that ADE
presented inADEsites).Conversely,thenumberofsharedOPFs
and ADJsoils shared more commonT-RFs forthe bacterial 16S
between ADJ soils under different land uses was 55 (42% of the
rRNAratherthanforbphgene.Also,thenumberofuniqueT-RFs
totalOPFsinADJsites).SoilsunderSFpresentedhighernumbers
was higher in ADE sites for both assessed genes (Figure 1a).
ofuniqueOPFsforbothsoils(ADEandADJ)(75and40unique
Conversely, Venn’s diagram combining the four sites showed a
OPFs for SF sites and 45 and 35 unique OPFs for ADE sites,
corecontaining122T-RFsforthebacterial16SrRNAgene,while
the distribution of T-RFs for the bph gene was more site specific, respectively).
and only6T-RFs comprised a commoncore(Figure 1b). The estimation of Good’s coverage revealed higher values for
ClusteringanalysisofT-RFLPdataforthebphgenesegregated ADJ sites (0.86 for ADJ-SF and 0.87 for ADJ-M) than for ADE
samples according to soil type and land use (Figure 2). This sites(0.79forADE-SFand0.80forADE-M),suggestingahighest
analysis revealed the formation of two main clusters: the first number of unique sequences in ADE sites. Rarefaction curves
cluster (a) included samples from ADE soils (SF and M) and the (FigureS1)indicatethat ADE(SFandMsites)presented amore
second (b) samples from ADJsoils (SF and M). Thisanalysis also diverse community than ADJ (SF and M). Sites under SF (ADE
revealedthatADEandADJsitessegregatedat12%ofsimilarity. and ADJ) were also comparatively more diverse than sites under
Concerning the ADE sites, land use systems separated different M (ADE and ADJ). For all comparative analysis the sampling
landusesat27%ofsimilarity.Conversely,forADJsites,landuse effortdidnotcoveredtherichnessofbphgene.Theexceptionwas
systems differedat 20% similarity (Figure2). observed for samples from ADJ sites, where a trend towards a
Redundancy analysis based on T-RFLP data explained 78.1% ‘‘plateau’’ was observed.
of the variation in the first two axes, thus confirming the ThePrincipalCoordinateAnalysis(PCoA)basedonUnweight-
segregation of sites primaryaccording to soil type, andfurther in ed UniFrac distances revealed distinct patterns in phylogenetic
relation to different land use types (Figure 3). Replicates within community composition (Figure 5). The first axis explained
each soil site were very consistent,evidenced by theformation of 59.59% of the data variation, and this axis separated samples
concise clusters. We also observed that different soil types also according to soil type. The second axis explained 22.63% of the
correlated differently to measured chemical parameters. More datavariation,andthisaxissegregatedsamplesaccordingtoland
precisely, the bph community structure from ADE-SF correlated use system.
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BacterialbphGeneinAmazonianDarkEarth
Figure1.Venn’sdiagramofT-RFsfor16SrRNAandbphgenesaccordingto(a)soiltypesand(b)soiltypesandlanduses.ADE =
AmazonDarkEarth;ADJ = Adjacentsoils;SF = SecondaryForest;M = Manioccultivation.
doi:10.1371/journal.pone.0099597.g001
Figure2.ClusteringanalysisofT-RFLPdatabasedonBray-Curtissimilarityforthebphgene.‘a’and‘b’indicatethesegregationpatterns
accordingtosoiltype.ADE = AmazonDarkEarth;ADJ = Adjacentsoils;SF = SecondaryForest;M= Manioccultivation.
doi:10.1371/journal.pone.0099597.g002
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BacterialbphGeneinAmazonianDarkEarth
Figure3.Redundancyanalysis(RDA)basedonT-RFLPdataobtainedforthebphgene,andsoilproperties,atthefourstudiedsites.
Arrowsindicatecorrelationbetweenthechemicalparametersandcommunitystructureofsamples.Thesignificanceofcorrelationswasevaluatedvia
MonteCarlopermutationtestanditisindicatedasfollows:*p,0.05.ADE = AmazonDarkEarth;ADJ = Adjacentsoils;SF = SecondaryForest;M=
Manioccultivation.
doi:10.1371/journal.pone.0099597.g003
Taxonomic composition of the bph gene affiliation of the reads also revealed the predominance of the
Each obtained OPF were further compared to sequences from dioxygenase sequence belonging to unculturable organisms (rdh
the GenBank database for taxonomic assignment. All analyzed cf33) in ADE sites, and the predominance of the dioxygenase
sequences matched translated proteins described as dioxygenases sequences similar to those previously described at Australian soil
or putative dioxygenases, with E-values ,1023. The most (od16) inADJsites.
abundantdifferencesindioxygenases(establishedasdioxygenases
at least ten-fold higher in one site than another) are shown in Quantification of the bacterial 16S rRNA and bph genes
Figure 6. Sequence matches were associated with aromatic Variations in bacterial 16SrRNA geneabundances were clear
hydrocarbon degradation genes belonging mostly to the genera amongtheevaluatedsites.Bacterial16SrRNAgenerangedfrom
Streptomyces,Sphingomonas,Rhodococcus,Mycobacterium,Conexibacterand 2.76107(ADJ-M)upto9.76107(ADE-SF)copiespergramofsoil.
Burkholderia, and uncultured bacterial clones. The taxonomic The results indicated that, soil type and land use influenced the
Table1. ANOSIMtest forthe bacterial 16SrRNA andbph genebasedon T-RFLPdata ofthe Amazonian Dark Earth (ADE) and
Adjacentsoil(ADJ) undersecondary forest(SF) and undermanioc cultivation(M).
16SrRNAa bpha
Rvalues pvalues Rvalues pvalues
ADE6ADJ 0.17 ,0.001 1.00 ,0.001
ADE-SF6ADE-CULT 0.11 ,0.001 1.00 ,0.001
ADJ-SF6ADJ-CULT 0.03 ,0.001 1.00 ,0.001
aSampleswerecomparedusingT-RFpeakheightasameasureofabundance.
doi:10.1371/journal.pone.0099597.t001
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BacterialbphGeneinAmazonianDarkEarth
ntity. abundanceofbacteriaintheanalyzedsamples,andhighervalues
e were foundat ADE-SFsite (Figure7a).
d
ei Theabundanceofthebphgenerangedfrom1.66106(ADJ-M)
c
en to 2.96106 (ADE SF) copies per gram of soil. Conversely to the
bC 9 0 6 7 equ dataobtainedforthetotalbacteria(i.e.16SrRNAgenedata),the
S 7 8 8 8 s
E 0, 0, 0, 0, of abundance of the bph gene was also higher at ADE-SF site
4% (Figure 7b).
9
of
ue Discussion
al
v
off The aim of this study was to assess bacterial hydrocarbon
cut degrading genes in anthropogenic sites from Brazilian Amazon
a
at comparativelytotheiradjacentlocations,undertwodifferentland
9L) ed use systems. Recent studies have described the high fertility of
Simpson( 0.024 0.028 0.098 0.107 erecalculat A(eAxDDchEJa),nsgomeilsocsawtplyhaecnibtyecc,oanumustperaieronefdt ctthooneitraednjitancacenrnedatsiensodciolsmpHipnl,ettehhleiyghcsaeormmebcuaastirtoeenda
w biomass [3,6,8,28,38]. Although it is well-known that the
e
g taxonomic composition of bacterial communities is strongly
a
ver influenced by pH [39,40], the pH variation in our data indicate
o
c thatthisisalsoastrongpredictorofthecompositionanddiversity
e
mpl of the bacterial bph gene. Variations in soil pH, together with
sa eCEC, SOC and SOM, collectively accounted for 78.1% of the
d
ate totalvariationexplainedintheRDAplotbasedonT-RFLPdata.
y 9n(H) bestim HseeiginhgerthPatvlaolwueasciinditAyDsoEilssiateres kanreowlinketloyitnocrbeeaseantheeffPecstoolufbpilHity,
Diversit Shanno 4.24 4.05 3.19 3.17 pson)and [ain2mt8re]i.nnsdiAecdlscohw,arittahhcetebrhoisinstitecosroiacfanthldisfovsreymgsteaetmtaitoi,onwnhoibfcuhArcnDoinuEgldsapitcoetsisvsiibt(cileyosn)esxtipasnlaatilnny
m
Si thehighcontentofphosphorus.Thereisalsoadirectrelationship
nd between soil P content and eCEC values in ADE sites mainly
a
n because the P adsorption decreases due to the formation of
o
nn complexcompoundsbetweenPandtheorganicmatterpresentin
ne. (Sha theupperlayerofthesoil[41].ThevaluesofeCECinoursamples
hge nife dicesation. wwearse2m-foolsdtlyhicgohrerrelianteAdDtoEtthheanorAgaDnJicsimteas.ttTerhecsoenfcienndtirnagtsioanr,ewlihkieclhy
forthebp Jackk 271 255 170 130 e),diversityinManioccultiv eadsuxnurptefhlaatrcoioenpeiatodsrgeseapbne[yi2cci0fts,ihi4ctee2ss,u.4hr3Bifg]ai.ohcIecenhraasnhradomisrnot,keutngnhaotetwicvonoeflflcoebhcriatoirrvcegehteaacidrhneeinnfmogsiuitcsnyaodliplpenrirnouuptrnetihrietteniseotessf
ors knifM= intrinsic of ADE sites may play an important role in their high
estimat ACE 248 286 210 166 andJackyforest; lceovnetlesxot,fwnuethriyepnotthaveasiizlaebtihliatytsauncdh,fuerlttiimlitaythelays,ctahuesiredfetrhtieliAtyD.IEnstohiilss
ness Chao1condar taodjhaacrebnotrsoailhs.igAhgerricmuilcturorabliaplrdaicvteicresistycaanndalfsuonaclttieornasoliitlyptrhoapnerthtieeisr,
ch CE,Se mostly by interfering on the biochemistry of the organic matter
ndri ess 1 ors(ASF= ainvatihlaeblemienastuhreemsyestnetms o[4f4S].OOCu,rSdOatMareavnedaleedCsEuCch, awnhiicnhfluwenecree
ComparisonofdiversityindicesaTable2. RichnaSiteOPFs Chao ADE-SF159238 ADE-M129229 ADJ-SF95151 ADJ-M90127 aTheoperationalproteinfamily(OPFs),richnessestimatADE=AmazonDarkEarthsoils;ADJ=Adjacentsoils;doi:10.1371/journal.pone.0099597.t002 suadubpcm[dsAc(Ria4poosninaigNffmih6mmtSffnddc=ptee]Oroeeeip.Hmmrrfog0rrriielSleocensb.I,uuntm1naIsrnseannccSMg7netfneahiiBaw)o.totttsndelyynhrfyeamiMaaiioerpnt[ssmnshenmc4ihadspicdve5ctitosaecsrgdetrt]nbcluuetuaoha,itoifrgeclomdlbeefwtdnettteirsyaiwudtviasoarhn,hlraetleuiaiiateebcntnneTenbishactnorritu-woctgainAlRbhnactoamnclaeheraeDdntfFsisretmfnnyadLaiwiPEmascednmmautP,ealsuceilsnecnsooMsceumsonriietorsonsttotes.eyegmfgalbssysuOinduTAinmatssadihlr(eunt-tftRDlehufrARedsndegucmnepJd=DrFuocrdioeHietstlmnEin0payaucrda+b.ntchurm1ne(aAereesd1eld1ndaeenstum6lwgtco,wdncSibfortioeadoiiiceyAnAttnnenrraisyhrddnRDstDleihaAslwvoNpbEtoAnJteroenDrreiycoADatusotaahtaiElcp)tblaEsfdli.ets-oneoincusetSslHrriiaoi.aafrlgbFtteistnenailyonnsiayesdtdweicssvotofigeditteietffctsAeycsiirhvuvaorpattsDeabehhiclnuntealrnnhoyyeeJssst,l
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BacterialbphGeneinAmazonianDarkEarth
Figure4.Venn’sdiagramofbphdatabelongingtooperationalproteinfamilies(OPFs)fordifferentsoiltypesunderdifferentland
uses.ADE = AmazonDarkEarth;ADJ = Adjacentsoils;SF = SecondaryForest;M= Manioccultivation.SequencesweregroupedintoOPFsbased
onsequenceidentityof94%.
doi:10.1371/journal.pone.0099597.g004
ADE-M;andR=0.03forADJ-SFversusADJ-M;Table1).Lima is formed by an aromatic polycyclic structure, could be a factor
[47], who similarly assessed the variation of ADE-SF, ADE-M, influencing the observed differences. Ta´ncsics et al [49], by
ADJ-SF and ADJ-M sites, and Cannavan [48], who investigated analyzing the structure community of catechol 2,3-dioxygenase
changes across different ADE sites, also found differences in 16S genesinaromatichydrocarboncontaminatedenvironmentsbyT-
rRNA community structure between ADE and ADJ sites. RFLP, observed that T-RFLP chromatograms obtained from
Collectively, these studies support the idea that archaeological contaminated samples had entirely different T-RFs compared to
sites with a long history of anthropogenic activity influence the thecontrol non-contaminated sample.
inhabiting microbiota oftheir respectivesoils. Redundancy analysis revealed bph gene structure to be
Analysis of the community patterns for the bacterial bph gene correlated with different soil parameters (Figure 4). Briefly,
revealedhigherrichnessofT-RFsinADEthaninADJsites.Our ADE-SF correlated with pH, eCEC, SOM and SOC. ADJ-SF
results show that bph community changes are clear among sites showed a significant correlation with H+Al. Several studies have
(Figure2),whichcanbeobservedbychangesintheabundanceof suggested that variations in soil pH and properties related to soil
specific T-RFs across sites. In the same way, ANOSIM revealed acidity(e.g.,K,Al,basesaturation) arestrongerpredictors ofthe
significant differences in bph gene community structure between richness and diversity of inhabiting microbial communities
ADJ and ADE soils types (R=1.00) and between land uses [6,18,50,51,52,53]. We extend this concept by advocating that
(R=1.00 for ADE-SF versus ADE-M; and R=1.00 for ADJ-SF other factors also might play a role in structuring the bph gene
versusADJ-M;Table1).Wesuspect,despiteallvariablespresent communities. For instance, the quantity and/or quality of soil
inoursystem,thatthehighpresenceofbiochar,whichchemically organic matter (SOM) and its fractions are likely to regulate
Figure 5. Bar charts representing the taxonomic affiliation ofbphgene sequences. Sequences were affiliated using the TBlastX tool
availableintheGenBankdatabase.ADE = AmazonDarkEarth;ADJ = Adjacentsoils;SF = SecondaryForest;M= Manioccultivation.
doi:10.1371/journal.pone.0099597.g005
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BacterialbphGeneinAmazonianDarkEarth
genesinADEsites,peakingatADE-SF.Theseresultsindicatethat
soil characteristics of ADE, as well as their land uses has an
influence on the abundance of both analyzed genes. Ding et al
[55]analyzedtheabundanceoftotalbacteriausingthe16SrRNA
gene and the abundance of polycyclic aromatic hydrocarbons
ring-hydroxylating dioxygenase (PAH-RHD ) genes by qPCR in
a
two different phenanthrene-contaminated soils (i.e. Luvisol and
Cambisol).Theirresultsrevealedasignificantlyhigher16SrRNA
gene copy number per gram of soil in both phenanthrene-
contaminated soils compared to their controls. PAH-RHD genes
were detected only in contaminated soil samples, and the values
rangedfrom2.06107(Luvisolphenanthrene-contaminatedsoil)to
1.76106 (Cambisol phenanthrene-contaminated soil) copies per
gram of soil, showing similar values to those found in our study.
Similarly,a studyaboutthediversityofnaphthalenedioxygenase
genes (nahAc) in soil environments from the Maritime Antarctic
revealedthatthequantitiesdetectedinbulkandrhizosphericsoils
from PAH-affected sites ranged from 6.46104 to 1.76106 nahAc
gene copies per gram of soil and presented significantly higher
abundancecomparedwiththecorrespondingcounterpartsofbulk
andrhizospheric soilsfromnon-polluted sites [56].
We used 454-pyrosequencing to investigate the community
compositionofthebphgene.Theuseofahigh-throughputculture-
Figure6.PrincipalCoordinateAnalysis(PCoA)performedwith independent approach enabled an overview of the taxonomic
thesequencesobtainedforthebphgene(basedonUnweight- groups occurring in the sampled sites. Taxonomic analyses of
ed Unifrac distance). ADE = Amazon Dark Earth; ADJ = Adjacent 3,000partialsequencesofthebphgenerevealedthatthisgenewas
soils;SF = SecondaryForest;M= Manioccultivation. mostrepresented bybacteria belongingtothegenera Streptomyces,
doi:10.1371/journal.pone.0099597.g006
Sphingomonas, Rhodococcus, Mycobacterium, Conexibacter and Burk-
holderia, in addition to sequences matching uncultured bacteria
microbial community composition and associate function [54].
(Figure 5). Overall, these groups encompass mixed sequences of
Sincehydrocarbondegradation isonestepintothecarboncycle,
toluene/biphenyl dioxygenases, such as bphB, bphC8, bphA1,
bphdegradersmighthaveanadvantageinADEsitesbyharnessing
bphA2,bphA3,bphA4andbphD,and enzymesencoding thealpha
energy fromelevated levels of SOCandSOM.
subunitofdioxygenasesforthedegradationofPolycyclicAromatic
Quantification of total bacterial (16S rRNA) and bph gene
Hydrocarbons (PAHs), which are described as environmental
abundances (Figure 7) revealed higher copy numbers of these
widely distributed dioxygenases. Other studies targeting dioxy-
AA
aa
bb
BB
cc
dd
CC
DD
Figure7.Loggenecopynumber(yaxis)of(a)16SrRNAand(b)bphgenesacrossthestudiedsites.Sitesareindicatedinthexaxis.Error
barsrepresentthestandarddeviationofthreeindependentreplicates.ADE = AmazonDarkEarth;ADJ = Adjacentsoils;SF = SecondaryForest;M=
Manioccultivation.Differentuppercaselettersrefertodifferencesforthe16SrRNAacrosssites;whiledifferentlowercaselettersrefertodifferences
forthebphgene(P,0.01,Tukeytest).
doi:10.1371/journal.pone.0099597.g007
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BacterialbphGeneinAmazonianDarkEarth
genases have also reported sequences belonging to Sphingomonas, The taxonomic analyses of sequences also revealed that ADE
Rhodococcus, Mycobacterium, Conexibacter and Burkholderia as major sitesharboreddistinctbphphylogeneticstructurefromADJ.These
taxonomic groups [21,28,57]. Zhou et al [58] were also able to resultssuggestthattheheterogeneityofbacterialbphcommunities
isolate aromatic hydrocarbon degrading bacteria from mangrove could be related to their ability to respond to differences of land
sediments, which were classified as the genera Mycobacterium and use type and soil chemical properties. In a previous study
Sphingomonas. Members of these genera have commonly been Germano et al. [28] compared the phylogenetic structure of bph
isolatedfromdiversesedimentsandsoils[59,60],andtheyplayan sequences from ADE sites and revealed that most of the protein
important role in hydrocarbon biodegradation [61,62,63]. For clusters from these sites group apart from the main well-known
instance,Dingetal[55]whostudiedthediversityofdioxygenases dioxygenase groups previously proposedby Kweonetal [27].
usingclonelibrariesforaromaticringhydroxylatingdioxygenases Inconclusion,wehavetakenadistinctandhighlydiversesoilto
(ARHD) genes identified gene sequences corresponding to the elucidate the ecological properties and taxonomic affiliation of
phnAcgenebelongingtotheBurkholderiasp.strainEh1-1andPAH- bacterial communities characterized by the presence of the bph
RHDgenes of the Mycobacterium sp.strain JLS. gene, which is crucial to the biodegradation of aromatic
Confirming the results of T-RFLP analysis, differences among compounds. These results enable us to understand differences in
bph communities for ADEand ADJ sites and between SF and M the structure, abundance and composition of the main active
organisms in ADE soils when compared to their adjacent
landuseswerealsoobservedbydifferencesintheirShannonand
locations. Further studies focusing on the catabolic activities of
Simpsondiversityindices(Table2).Similarly,Germanoetal[28]
these communities are needed to enable a collective view of the
reported a higher diversity of ARHD genes in ADE sites under
formation, dynamics andmaintenance of functional properties in
secondary forest rather than under agricultural cultivation,
ADEsoils.
suggesting that deforestation in these sites has an influence on
thediversityofthesecatabolicgenes.Weobservedthatinbothsoil
Supporting Information
types,thenumberofOPF(Table2)washigherinSFsamples,also
indicating an influence of the land use system on the richness of Figure S1 Rarefaction curves of bacterial bph gene sequences
this gene. According to Jesus et al [51], the land conversion of weregroupedintoOPFbasedondistancesequenceof0.06.ADE
tropical forest to agricultural use modifies the size, activity and = Amazonian Dark Earth soils; ADJ = Adjacent soils; SF =
compositionofsoilmicrobialcommunities.Thisdeeplyinfluences Secondary forest; M= Manioc Cultivation.
specific bacterial functions, including those acting on organic (EPS)
matter decomposition andnutrient cycling insoils.
Table S1 Soil chemical properties of the studied sites. Amazo-
The intensive land use by agricultural practices and the
nianDarkEarth(ADE)andAdjacentsoil(ADJ).Siteswerelocated
conversion of Amazon soil into agricultural areas has been
under two different land uses: Secondary forest (SF) and under
reportedtocausesignificantshiftsinthechemicalpropertiesofthe
Manioc (Manihot esculenta) cultivation (M). Significant differences
soil,suchasvariationsinSOC,SOMandeCEC,leadingtowards
betweensitesarefollowedbydifferentletters(P,0.05,Tukeytest).
a homogenization of the inhabiting bacterial community [64].
(DOCX)
However, we hypothesized that, despite an effect of land use,
greaterdifferencesincommunitycompositionwouldbeobserved
Acknowledgments
according to soil type. We also expected communities in these
different soils to respond differently to agricultural practices. In We thank Wenceslau GeraldesTeixeira and Western AmazonEmbrapa
thiscontext,theliteraturedescribestheresiliencephenomenonas for technical support. We are grateful to ACG Souza, AK Silveira, RS
the ability of the soil to cope with external disturbances and to Macedo and TT Souza for helping with sample collection. We thank J
Quensen and the Michigan State University Genomics Technology
retain its functional capacity upon the imposition of a stress
SupportFacilityteamforthe454-pyrosequencingsupport;andJose´Elias
[65,66,67].Weobservedthathigherdifferencesinthediversityof
GomesandFa´bioDuartefortheirassistanceinmolecularanalysis.Wealso
bph occurred between the different land uses in ADJ sites rather thankCyrusA.MallonforEnglishrevisionandhelpfulcommentsonthe
thaninADEsites.Thus,landusetypeappearedtohaveastronger manuscript.
effectonthebphcommunityinADJsites,maybeduetothehigher
resilience of the ADE soil against agricultural practices. Principal Author Contributions
Coordinate Analysis (PCoA) performed for the bph gene also
Conceived and designed the experiments: MJLB MGG ABL SMT.
supportstheseresults(Figure6),revealingthatADEsites(SFand
Performedtheexperiments:MJLBMGGABL.Analyzedthedata:MJLB
M)clusteredclosertoeachotherthanobservedforADJsites(SF LWM. Contributed reagents/materials/analysis tools: SMT. Wrote the
and M). paper:MJLBSMT.
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