Diversity and distribution of Actinobacteria associated with reef coral Porites lutea

13 Pages·2015·1.6 MB·English

by  Lijuan Long

ORIGINALRESEARCH
published:21October2015
doi:10.3389/fmicb.2015.01094
Diversity and distribution of
Actinobacteria associated with reef
coral Porites lutea
WeiqiKuang1,2†,JieLi1†,SiZhang1andLijuanLong1*
1CASKeyLaboratoryofTropicalMarineBio-ResourcesandEcology,RNAMCenterforMarineMicrobiology,SouthChina
SeaInstituteofOceanology,ChineseAcademyofSciences,Guangzhou,China,2CollegeofEarthScience,Universityof
ChineseAcademyofSciences,Beijing,China
Actinobacteria is a ubiquitous major group in coral holobiont. The diversity and spatial
and temporal distribution of actinobacteria have been rarely documented. In this
Editedby:
ShengQin, study, diversity of actinobacteria associated with mucus, tissue and skeleton of Porites
JiangsuNormalUniversity,China
lutea and in the surrounding seawater were examined every 3 months for 1 year on
Reviewedby:
Luhuitoufringingreef.ThepopulationstructuresoftheP.lutea-associatedactinobacteria
SyedGulamDastager,
NationalCollectionofIndustrial were analyzed using phylogenetic analysis of 16S rRNA gene clone libraries, which
MicroorganismsResourceCenter, demonstrated highly diverse actinobacteria profiles in P. lutea. A total of 25 described
India
families and 10 unnamed families were determined in the populations, and 12 genera
WeiSun,
ShanghaiJiaoTongUniversity,China were firstly detected in corals. The Actinobacteria diversity was significantly different
P.Nithyanand,
between the P. lutea and the surrounding seawater. Only 10 OTUs were shared by
SASTRAUniversity,India
the seawater and coral samples. Redundancy and hierarchical cluster analyses were
*Correspondence:
LijuanLong, performedtoanalyzethecorrelationbetweenthevariationsofactinobacteriapopulation
CASKeyLaboratoryofTropical within the divergent compartments of P. lutea, seasonal changes, and environmental
MarineBio-ResourcesandEcology,
factors. The actinobacteria communities in the same coral compartment tended to
RNAMCenterforMarine
Microbiology,SouthChinaSea clustertogether.Evenso,anextremelysmallfractionofOTUswascommoninallthree
InstituteofOceanology,Chinese
P.luteacompartments.Analysisoftherelationshipbetweenactinobacteriaassemblages
AcademyofSciences,Xingangxi
Road164,Guangzhou510301,China and the environmental parameters showed that several genera were closely related to
[email protected] specificenvironmentalfactors.Thisstudyhighlightsthatcoral-associatedactinobacteria
†Theseauthorshavecontributed populationsarehighlydiverse,andspatiallystructuredwithinP.lutea,andtheyaredistinct
equallytothiswork.
fromwhichintheambientseawater.
Specialtysection: Keywords:actinobacteria,Poriteslutea,diversity,temporalandspatialdistribution,16SrRNAgene
Thisarticlewassubmittedto
ExtremeMicrobiology,
asectionofthejournal Introduction
FrontiersinMicrobiology
Received:10July2015 Coralreefecosystemisoneofthemostimportanttropicalmarineecosystems,mainlydistributed
Accepted:22September2015 intheIndo-WestPacific,EasternPacific,WesternAtlantic,andtheEasternAtlantic(Mobergand
Published:21October2015
Folke,1999).Coralsprovidehabitatsfornumerousbacteriaintheirmucuslayer,tissue,andcalcium
Citation: carbonateskeleton,aswellasthedinoflagellates,fungi,archaea,andviruses(Rosenbergetal.,2007).
KuangWQ,LiJ,ZhangSandLong Coral-associatedbacterianotonlytakepartincarbon,nitrogen,andsulfurbiogeochemicalcycles
LJ(2015)Diversityanddistributionof
and provide necessary nutrient for coral, but also keep corals from being infected by pathogens
Actinobacteriaassociatedwithreef
(Rosenbergetal.,2007;Rainaetal.,2009;BourneandWebster,2013).
coralPoriteslutea.
Front.Microbiol.6:1094. Highlydiverseandheterogeneousbacterialcommunitieshavebeenrevealedindifferentcoral
doi:10.3389/fmicb.2015.01094 speciesatvariouslocations(Rohweretal.,2002;Lietal.,2013).Actinobacteriaisgenerallyaccepted
FrontiersinMicrobiology|www.frontiersin.org 1 October2015|Volume6|Article1094
Kuangetal. Poriteslutea-associatedactinobacteria
as a ubiquitous major group in corals (Bourne and Munn, the coral colonies was collected, and filtered through 0.22µm-
2005; Carlos et al., 2013; Li et al., 2013, 2014a). Yang pore-size filter membrane (Millipore). The filter membranes
et al. (2013) detected 19 Actinobacteria genera in soft coral were stored at −80◦C until DNA extraction. As the samples
Alcyonium gracllimum and stony coral Tubastraea coccinea in were collected at the same time, environmental parameters
the East China Sea through analysis of 16S rRNA gene clone including water temperature, salinity, dissolved oxygen, pH
libraries. Some actinobacterial genera were previously detected value,ultravioletradiationintensity,andrainfallwerecitedfrom
in corals by using the culture-dependent method (Lampert thepublisheddata(Lietal.,2014a).
et al., 2006; Nithyanand and Pandian, 2009; Nithyanand
et al., 2011b; Zhang et al., 2013; Li et al., 2014b). Among DNAExtractionandPCRAmplification
these culturable actinobacteria, Streptomyces, Verrucosispora, The coral tissue and skeleton samples were homogenized
Rhodococcus,Micromonospora,Nocardia,Jiangella,Nocardiopsis, thoroughly in liquid nitrogen with sterile mortar and pestle
Pseudonocardia, and Salinispora showed antibacterial activities, beforeaddedtothePowerBeadTubes.Thefiltermembraneswith
which were considered to contribute to coral health (Ritchie, adsorbed microbial cells were cut into pieces, and then added
2006;Nithyanandetal.,2011a;Kredietetal.,2013;Zhangetal., to the PowerBead Tubes. Total DNA was extracted using the
2013;Lietal.,2014b). PowerSoilDNAIsolationKit(MoBio,SolanaBeach,CA,USA)
Environmental conditions, coral species, colony physiology, accordingtothemanufacturer’sinstruction.
and seasonal variation are considerable influencing factors on 16S rRNA genes were nest PCR amplified, the first
the coral-associated bacterial community (Hong et al., 2009). PCR reactions using the combination of universal bacterial
Moreover, due to various microhabitats provided by corals’ primers27F(5′-AGAGTTTGATCMTGGCTCAG-3′)and1492R
biological structures, the spatial heterogeneity has been proved (5′-TACGGYTACCTTGTTACGACTT-3′). PCR amplifications
in bacterial communities associated with a single coral colony were performed in a Mastercycler pro (Eppendorf, Hamburg,
(Rohweretal.,2002;Sweetetal.,2011;Lietal.,2014a).Asamajor Germany)inafinalvolumeof50µL,containing2µL(10µM)
coral-associated bacterial group, how actinobacteria is spatially each primer, 1µL (10–20ng) template DNA and 25µL premix
andtemporallyorganizedincorals,andwhatistheconnection Ex Taq mixture (Takara, Dalian). The PCR conditions were
betweentheactinobacteriacommunitiesincoralsandinseawater as follows: 94◦C for 5min; 30 cycles of 94◦C for 30s, 54◦C
remainspoorlyunderstood.Comprehensiveinvestigationofthe for 30s, 72◦C for 90s; followed by 72◦C for 10min. In the
distribution of this ubiquitous group at spatial and temporal secondPCRreactions,theactinobacteria-specificprimerpairs,S-
scales will help understanding the variation of coral associated C-Ac-0325-a-S-20(5′-CGCGCCTATCAGCTTGTTG-3′)andS-
bacteria and the potential function of actinobacteria, and will C-Act-0878-a-A-19 (5′-CCGTATCCCCAGGCGGGG-3′), were
contribute a lot to bioprospect the actinobacteria resources for used to amplify the V3-V5 regions (about 640bp) of the
utilizationasnovelsourcesforbioactivenaturalproducts. actinobacteria 16S rRNA gene (Stach et al., 2003). In the PCR
Coral reefs are widely distributed in the South China Sea reactions,5µLof1:10dilutionofthefirstroundPCRproduct
(Liu et al., 2009; Wang et al., 2014). Porites lutea is the was used as DNA template, the PCR mixture (50µL) contain
dominant, typical coral species in the Luhuitou fringing reef, 2µL (10µM) each primer, 25µL premix Ex Taq mixture, the
whichislocatedinthesouthendoftheHainanprovince(Zhao PCR conditions were as follows: 95◦C for 5min; 30 cycles of
et al., 2008). In this study, the diversity and distribution of 95◦Cfor45s,68◦Cfor45s,72◦Cfor60s;followedby72◦Cfor
actinobacteria were investigated in coral P. lutea and in the 10min. Each genomic DNA sample was amplified in triplicate
surrounding seawater every 3 months for 1 year using culture- PCR reactions. Amplicons from the same sample were pooled
independent method for the first time. We aimed to reveal the and purified using the E.Z.N.A.(cid:13)R Gel Extraction Kit (Omega
coral-associated actinobacteria community structures in three Bio-Tek,China).
divergentcoralcompartmentsindifferentmonths,comparethe
actinobacterialcommunitiesinthecoralandinthesurrounding GeneLibraryConstructionandSequencing
seawater, and research the actinobacteria community variation Sixteen clone libraries of actinobacterial 16S rRNA genes
respondstotheenvironmentalfactors. were constructed using the pMD18-T Vector Cloning Kit
and E. coli DH5α competent cells (Takara, Dalian) following
the manufacturer′s instructions. The positive clones from
Materials and Methods each library inoculated on MacConkey agar with ampicillin
(100µg/ml) were randomly picked and sequenced using M13F
SampleCollection (−47) primer on ABI 3730xl capillary sequencers (Applied
The coral and surrounding sea water samples were collected in Biosystems,USA).
fourdifferentmonths(February,May,August,andNovember)in
2012fromtheLuhuitoufringingreef(109◦28′E,18◦13′N).Coral LibrariesAnalysis
fragments (approximately 10×10cm) were collected from the The vector sequences were screened by the VecScreen
sideofthreehealthyP.luteacoloniesatthedepthof3–5meach tool provided in NCBI (http://www.ncbi.nlm.nih.gov/tools/
timeusingpunchandhammer.Coralmucus,tissuesandskeleton vecscreen/).Chimeraswerecheckedbyrunningchimera.uchime
were separated and stored according to the method described packagedinMothur(Schlossetal.,2009),andpotentialchimeras
previously (Li et al., 2014a). One liter of seawater adjacent to were removed. All valid sequences were deposited in GenBank
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Kuangetal. Poriteslutea-associatedactinobacteria
(accession numbers were shown in Data S1). All qualified Shannonindicesinmucuscollectedindifferentmonthsranged
sequences were identified by using the classify.seqs command from2.32to3.44,from2.45to3.55intissues,from1.82to3.35
in Mothur with Silva reference alignment database (http:// in skeleton, and from 1.53 to 2.82 in sea water (Table1), and
www.mothur.org/wiki/Silva_reference_files, Release 119) at a the diversity in the actinobacterial community associated with
confidencelevelof80%.Thesequences,whichdonotbelongto P. lutea was higher than which in the surrounding sea water
Actinobacteria, were removed from further analysis. Sequences (P=0.045).
were clustered into operational taxonomic units (OTUs) with
a 97% threshold using the cluster command in Mothur. The Coral-associatedActinobacterialCommunity
relationships among actinobacterial communities associated Composition
withdifferentcoralcompartmentsandintheambientseawater
At a confidence threshold of 80%, 2403 qualified reads were
in different months were analyzed by hierarchical cluster
assigned to four classes, i.e., Acidimicrobiia, Actinobacteria,
analysis. Based on Bray-Curtis similarity estimated from the
Thermoleophilia,andKIST-JJY010.Amongthem,Acidimicrobiia
OTU matrix, clustering was generated by using the complete
and Actinobacteria were ubiquitous and dominant in P. lutea
linkagemethodwiththePRIMER5software(Clarke,1993).The
andintheseawatersamples.Thermoleophiliawasnotdetectedin
shared OTUs were determined by using the online tool venny
coralscollectedinFebruary,inthemucusandseawaterinMay,
(Oliveros, 2007–2015, http://bioinfogp.cnb.csic.es/tools/venny/
and in the mucus in August, while accounted for 0.5–48.8% in
index.html).
allothersamples.KIST-JJY010wasdetectedonlyinthemucusin
ThecorrelationsbetweenActinobacteriaassemblagesofcoral
November(0.6%),andintheskeletoninAugust(2.6%).
samples and the environmental factors were analyzed by using
Twenty-five described families and 10 unnamed families
the software package CANOCO 4.5.1 (ter Braak and Šmilauer,
were detected in the 16 libraries (Figure2). OM1_clade
2002).Redundancyanalysis(RDA)wascarriedouttodetermine
and Propionibacteriaceae (genera Friedmanniella and
the relationship between the actinobacteria community and
Propionibacterium) were ubiquitous, major groups in P. lutea.
the environmental factors including temperature, salinity,
Meanwhile, OM1_clade was not detected in the seawater in
dissolved oxygen, pH value, rainfall, and UV radiation and in
combination with two nominal variables including the coral
divergentcompartmentsandthedifferentsamplingmonths.The
significanceoftherelationbetweentheexplanatoryvariablesand
the actinobacterial community compositions was tested using
MonteCarlopermutationtests(9999unrestrictedpermutations,
P<0.05).
Results
Coral-associatedActinobacteriaDiversity
Atotalof2403sequenceswereobtainedfromsixteen16SrRNA
geneclonelibraries,resultingin395OTUs(stringencyat97%).
Therarefactionanalysisresultshowedthatmostofthecurvesdid
notflattentoasymptote,butclimbedlesssteeply(Figure1).The
coveragesrangedfrom0.69to0.97in16libraries,andtheaverage
coverage was 0.83 (Table1). The highest number of OTUs was
FIGURE1|RarefactioncurvesofActinobacteria16SrRNAgene
found in the tissue collected in May, while the lowest OTUs
sequences.
wasfoundintheskeletoncollectedinNovember(Table1).The
TABLE1|NumberofsequencesandOTUs(97%)anddiversityestimatesoftheActinobacterialibrariesinP.luteaandintheambientseawater.
Index A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 C3 C4 D1 D2 D3 D4
No.ofSeq. 153 133 150 185 105 151 134 181 132 146 153 179 149 109 172 171
OTUs 56 42 41 25 46 66 31 29 37 41 54 44 40 33 17 43
Chao 343.00 147.60 69.88 34.43 108.14 201.13 44.00 55.25 63.86 64.75 124.13 106.14 55.83 48.17 19.50 66.00
ACE 600.00 756.54 131.72 56.36 194.33 388.96 61.80 63.38 535.51 93.22 182.95 108.04 68.08 46.83 21.10 114.38
Shannon 3.33 2.45 3.08 1.53 3.44 3.55 2.70 1.89 2.32 3.07 3.35 2.68 2.89 3.11 1.82 2.84
Coverage 0.73 0.75 0.85 0.94 0.71 0.69 0.90 0.92 0.79 0.86 0.78 0.83 0.87 0.87 0.97 0.86
A1,mucusinFebruary;A2,tissueinFebruary;A3,skeletoninFebruary;A4,seawaterinFebruary;B1,mucusinMay;B2,tissueinMay;B3,skeletoninMay;B4,seawaterinMay;C1,
mucusinAugust;C2,tissueinAugust;C3,skeletoninAugust;C4,seawaterinAugust;D1,mucusinNovember;D2,tissueinNovember;D3,skeletoninNovember;D4,seawaterin
November.
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Kuangetal. Poriteslutea-associatedactinobacteria
FIGURE2|Actinobacteriacompositionprofiles.Taxonomicclassificationofactinobacteriasequencesintofamilyidentifiedbyusingtheclassify.seqscommand
inMothurusingSilvareferencealignmentdatabase(http://www.mothur.org/wiki/Silva_reference_files,Release119)withaconfidencelevelof80%wereappliedfor
classification.A1,mucusinFebruary;A2,tissueinFebruary;A3,skeletoninFebruary;A4,seawaterinFebruary;B1,mucusinMay;B2,tissueinMay;B3,skeletonin
May;B4,seawaterinMay;C1,mucusinAugust;C2,tissueinAugust;C3,skeletoninAugust;C4,seawaterinAugust;D1,mucusinNovember;D2,tissuein
November;D3,skeletoninNovember;D4,seawaterinNovember.
FebruaryandMay,andrareintheothertwoseawaterlibraries, the surrounding seawater. In reverse, Microbacteriaceae and
andPropionibacteriaceaewasabsentinalltheseawaterlibraries. Ilumatobacter were major groups in sea water, while they were
Micromonosporaceaewasthemostabundantgroupinthetissue lessabundantinP.lutea.
in February (47.4%) and in the mucus in August (46.2%), in
which most of the reads were affiliated with an unclassified SpatialandTemporalDistributionof
group. Nonetheless, Micromonosporaceae was absent in all P.lutea-associatedActinobacteria
other coral and seawater samples. Sva0996_marine_group was Results of hierarchical cluster analysis showed that the
detected in all coral samples (5.2–50%) except in the skeleton actinobacteriacommunitiesweresignificantlydifferentbetween
collected in November, and which also was abundant in the inthecoralandinthesurroundingseawatersamples(p = 0.01,
ambient sea water (21.9–80%). Micrococcaceae was absent in R=0.993).Theactinobacterialcommunitiesassociatedwiththe
the coral skeleton collected in August and in November, and samecoralcompartmentstendedtoclustertogether(Figure3).
in the sea water samples. Group 480-2 was abundant in the Theseasonfactordidnotsignificantlyinfluencethevariationin
coraltissueinAugust(24.7%),aswellasintheskeletoninMay theactinobacteriacommunities.TheRDAresultsindicatedthat
(26.9%) and in November (48.8%), but it was nearly absent in 38.9%ofthetotalvarianceinthecoral-associatedactinobacterial
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Kuangetal. Poriteslutea-associatedactinobacteria
FIGURE3|HierarchicalclusteranalysisofactinobacteriacommunitiesassociatedwithP.lutea.ClusteringwasbasedonBray-Curtissimilarityestimated
fromtheOTUsmatrixbyusingthecompletelinkagemethod.
composition was explained by the environmental, spatial libraries, 7.6%). The secondary abundance OTU0004 affiliated
and temporal factors (Figure4). The first and second axes withFriedmanniellawaspresentinalllibrariesexceptinskeleton
differentiatedtheactinobacteriaassemblagesinthedistinctcoral collectedinNovember.
compartments (Figure4, Table S1). This result was consistent
with the hierarchical cluster analysis. None of the environment Discussion
parameters analyzed in this study was determined as the
significant influencing factor in the variation of the P. lutea HighlyDiverseActinobacteriaAssociatedwith
associatedactinobacteriacommunities.Atriplotmapillustrated P.lutea
the relationship between major actinobacterial groups, with In comparison with previously reported results (Lampert
abundance more than 1%, and the environmental parameters et al., 2006, 2008; Bruck et al., 2007; Kageyama et al., 2007;
(Figure4). Friedmanniella and Micrococcus were positively Santiago-Vázquezetal.,2007;Ben-Dovetal.,2009;Nithyanand
related with the salinity. Microbacterium, Propionibacterium, and Pandian, 2009; Seemann et al., 2009; Shnit-Orland and
and group 480-2 were positively correlated with seawater Kushmaro, 2009; de Castro et al., 2010; Thomas et al., 2010;
temperature,butnegativelycorrelatedwithdissolvedoxygen. Nithyanand et al., 2011a,b; Cardenas et al., 2012; Chiu et al.,
ToinvestigatethedistributionofOTUsinthethreedivergent 2012; Sun et al., 2012, 2014; Zhang et al., 2012, 2013; Yang
coral compartments (mucus, tissue, and skeleton) and in et al., 2013; Chen et al., 2014; Li et al., 2014a,b; EIAhwany
the surrounding seawater, a venn diagram was constructed. et al., 2015; Sarmiento-Vizcaíno et al., 2015), 12 genera
The results showed that only 5 OTUs were present in all of including Actinopolyspora, Blastococcus, Candidatus_Aquiluna,
P. lutea mucus, tissue and skeleton, and in sea water, which Demetria, Fodinicola, Friedmanniella, Geodermatophilus,
were identified as Sva0996_marine_group, Ilumatobacter, Iamia, Modestobacter, Ornithinimicrobium, Tersicoccus, and
Corynebacterium, OM1_clade and Microbacterium (Table2, Yonghaparkia were firstly detected in corals in this study
Figure S1A). Another 17 OTUs, which were identified as (Table4).Furthermore,manyunclassifiedgroupsweredetected
Candidatus_Microthrix, Corynebacteriales, Friedmanniella, in P. lutea, including even the group at the class taxon level.
Micrococcus, Mycobacterium, OM1_clade, Propionibacterium, These results suggested that highly diverse and abundant
Sva0996_marine_group,Yonghaparkiaand480-2werecommon known actinobacteria were associated with P. lutea as well as
in mucus, tissue, and skeleton (Table2, Figure S1A). Twelve unknown groups. It was also noticed that many actinobacterial
OTUs distributed in Propionibacterium, Friedmanniella, groups were only detected by the culture-independent method
OM1_clade, Sva0996_marine_group, Kocuria, Mycobacterium, (Table4), and some of them were ubiquitous and abundant,
Corynebacteriales, Brevibacterium, and Brachybacterium were such as Friedmanniella, Ilumatobacter, and OM1_clade. Their
present in coral libraries in all four different months (Table3, physiologicalpropertiesandecologicalsignificanceareworthyof
FigureS1B).ThemostabundantOTU0003,whichwasclassified deepresearch.Forthispurpose,thedevelopmentandinnovation
as Propionibacterium, was present in all coral samples with oftheisolationandcultivationmethodsinordertoobtainpure
a high abundance (128 out of total 1687 reads in the coral culturesfromthecoralholobiontisparticularlyimportant.
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Kuangetal. Poriteslutea-associatedactinobacteria
FIGURE4|RDAordinationtriplotshowingtherelationshipamongtheenvironmentalvariables,coralsamples,andactinobacterialcomponents.
CorrelationsbetweenenvironmentalvariablesandthefirsttwoRDAaxesarerepresentedbythelengthsandanglesofthearrows(environmental-factorvectors).Only
abundantactinobacterialgroups(>1%)wereshowedinthetriplot.UV,ultravioletradiationintensity;Temp,seawatertemperature;DO,dissolvedoxygen.
According to our summary (Table4), genera Agrococcus, consideredasthebioactivecompoundsproducers(Fiedleretal.,
Amycolatopsis, Arthrobacter, Brachybacterium, Brevibacterium, 2005;Tabaresetal.,2011;Margasseryetal.,2012;Vicenteetal.,
Candidatus_Microthrix, Corynebacterium, Cellulosimicrobium, 2013;Manivasaganetal.,2014;Valliappanetal.,2014;EIAhwany
Cellulomonas, Dermatophilus, Dietzia, Gordonia, Janibacter, etal.,2015),andprobablytakepartinnitrogen(Suetal.,2013)
Jiangella,Kocuria,Kytococcus,Microbacterium,Micromonospora, and phosphorus (Sabarathnam et al., 2010) biogeochemical
Micrococcus, Mycobacterium, Nocardioides, Nocardiopsis, cycles.Whethertheyplaythesefunctionalrolesincoralsinsitu
Propionibacterium, Pseudonocardia, Rhodococcus, Rothia, and needtobefurtherinvestigated.
Streptomyces were detected in diverse coral species including
scleractiniancorals,suchasAcroporadigitifera(Nithyanandand ComparisonofActinobacterialCommunitiesin
Pandian,2009;Nithyanandetal.,2011b),P.lutea(Lietal.,2014b; theCoralsandintheAmbientSeawater
Sun et al., 2014) and Galaxea fascicularis (Li et al., 2014b), and ComparingtheactinobacteriacommunitiesbetweeninP.lutea
gorgoniancorals,Siderastreasidereal(Cardenasetal.,2012)and andinthesurroundingseawaterwillhelpustounderstandthe
Platygyracarnosus(Chiuetal.,2012).Mostofthemwerepresent source of coral associated actinobacteria, and the interaction
also in other marine organisms, such as sponges (Kim and between thebacteria in sea water and in corals. Consisted with
Fuerst,2006;Zhangetal.,2006;Selvinetal.,2009;Abdelmohsen previous study on bacteria communities (Li et al., 2014a), the
et al., 2010, 2014; Schneemann et al., 2010; Sun et al., 2010; P.luteaassociatedactinobacteriacommunitiesweresignificantly
Webster and Taylor, 2012; Vicente et al., 2013), mollusks different from which in the ambient seawater (Figure3).
(Romanenkoetal.,2008;Peraudetal.,2009),fishes(Sheejaetal., Groups such as Propionibacteriaceae, Micromonosporaceae, and
2011),seaweeds(Lee,2008;Singh andReddy,2013),seagrasses Micrococcaceae,werespecificallyassociatedwiththecoralsrather
(Ravikumar et al., 2012), and sea cucumber (Kurahashi et al., than in the ambient seawater, where they originated from
2009). Moreover, some of these widely distributed groups were should be in doubt. Whether the wide distributed groups such
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Kuangetal. Poriteslutea-associatedactinobacteria
TABLE2|OTUspresentedinallofthecoralandseawaterlibraries,or TABLE3|OTUspresentedinP.luteacollectedinfourdifferentmonths.
presentedinallthreedivergentcompartmentsofP.lutea.
OTUs Coralsamples Abundance Phylogeneticaffiliation
OTUs Observedinsamples Abundance Phylogeneticaffiliation
OTU0003a Feb,May,Aug,Nov 128 Propionibacterium
OTU0001 Mucus,Tissue, 303 Sva0996_marine_group OTU0004 Feb,May,Aug,Nov 122 Friedmanniella
Skeleton,Seawater
OTU0013 Feb,May,Aug,Nov 40 OM1_clade
OTU0007 Mucus,Tissue, 63 Ilumatobacter
OTU0014 Feb,May,Aug,Nov 40 Sva0996_marine_group
Skeleton,Seawater
OTU0015 Feb,May,Aug,Nov 39 Kocuria
OTU0011 Mucus,Tissue, 46 Corynebacterium
Skeleton,Seawater OTU0017 Feb,May,Aug,Nov 33 OM1_clade
OTU0017 Mucus,Tissue, 33 OM1_clade OTU0022 Feb,May,Aug,Nov 21 Sva0996_marine_group
Skeleton,Seawater OTU0025 Feb,May,Aug,Nov 18 OM1_clade
OTU0020 Mucus,Tissue, 24 Microbacterium OTU0027 Feb,May,Aug,Nov 18 Mycobacterium
Skeleton,Seawater OTU0028 Feb,May,Aug,Nov 17 Corynebacteriales
OTU0002 Mucus,Tissue,Skeleton 186 480-2
OTU0033 Feb,May,Aug,Nov 13 Brevibacterium
OTU0003 Mucus,Tissue,Skeleton 128 Propionibacterium
OTU0059 Feb,May,Aug,Nov 5 Brachybacterium
OTU0004 Mucus,Tissue,Skeleton 122 Friedmanniella
OTU0009 Mucus,Tissue,Skeleton 52 Candidatus_Microthrix aOTU0003waspresentinall12libraries.TheotherOTUslistedinthistablewerepresent
ineitherofthecompartmentmucus,tissueandskeletonofcoralscollectedinfourdifferent
OTU0012 Mucus,Tissue,Skeleton 43 OM1_clade
months.
OTU0013 Mucus,Tissue,Skeleton 40 OM1_clade
OTU0014 Mucus,Tissue,Skeleton 40 Sva0996_marine_group
theseassociatesandcoralsshouldbeaddressedfromafunctional
OTU0023 Mucus,Tissue,Skeleton 21 Micrococcus
perspective.
OTU0025 Mucus,Tissue,Skeleton 18 OM1_clade
OTU0027 Mucus,Tissue,Skeleton 18 Mycobacterium Relationshipofenvironmentalfactorsandthe
OTU0028 Mucus,Tissue,Skeleton 17 Corynebacteriales
P.lutea-associatedActinobacteria
OTU0030 Mucus,Tissue,Skeleton 15 Propionibacterium
It is different from previous conclusion of the distribution of
OTU0032 Mucus,Tissue,Skeleton 13 Mycobacterium
coral-associated bacteria (Chen et al., 2011; Li et al., 2014a),
OTU0034 Mucus,Tissue,Skeleton 12 Sva0996_marine_group
actinobacteriaassociatedwithP.luteadidnotshowtheapparent
OTU0035 Mucus,Tissue,Skeleton 12 Sva0996_marine_group
seasonaldynamicvariations.Wesuggestthattheactinobacteria
OTU0042 Mucus,Tissue,Skeleton 8 Sva0996_marine_group
compositions are relatively stable in distinct compartments in
OTU0056 Mucus,Tissue,Skeleton 5 Yonghaparkia
P.lutea.Inaddition,noneoftheenvironmentalfactorsanalyzed
in this study was determined as the most significant influence
on the actinobacteria communities. Even so, some genera were
as Sva0996_marine_group, OM1_clade, Microbacteriaceae and foundcloselycorrelatedwithspecificenvironmentalfactors.For
Ilumatobactertravelbetweentheambientseawaterandthecorals instance, Propionibacterium showed negatively correlation with
needtobeinvestigated. dissolved oxygen, probably due to its capability of living in the
When researchers make a general observation of the anaerobic conditions (Patrick and McDowell, 2012). Moreover,
wholebacterialcommunities,whichwereobservedsignificantly the OTUs0003 and 0004 affiliated with Propionibacteriaceae
different in coral mucus, tissue, and skeleton (Rohwer et al., was present in almost all 12 clone libraries with a very high
2002; Bourne and Munn, 2005; Sweet et al., 2011; Lee abundance.Whethertheyaretruesymbionts,andwhatfunctions
et al., 2012). However, it is unclear whether actinobacteria theyplayareworthfurtherresearch.
has a similar distribution pattern. In this study, both the
hierarchical cluster analysis (Figure3) and the RDA analysis Conclusion
(Figure4) showed that the actinobacteria communities from
the same compartment tended to cluster together. The distinct Thediversityanddistributionofcoral-associatedactinobacteria
physiochemicalmicroenvironmentsprovidedbycoralsprobably were first comprehensively investigated in this study. Highly
is one of the causes (Le Tissier, 1990; Brown and Bythell, diverseactinobacteriawasrevealedinthe16SrRNAgeneclone
2005; Sweet et al., 2011; Tremblay et al., 2011). Only a libraries of scleractinian coral P. lutea in the South China Sea.
small fraction of OTUs (22 out of 299 OTUs in the coral Twelve Actinobacteria genera were detected in corals for the
libraries) was common in the coral mucus, tissue, and skeleton first time as well as a large number of unclassified groups.
librariesinthisstudy(Table2).Thisresultsuggestedthatthese TheactinobacterialcommunitycompositionsweredistinctinP.
members might have capabilities to adapt to different micro- luteaandinthesurroundingseawater.Furthermore,thehigher
environments in divergent compartments of P. lutea. A large similarity of actinobacteria composition was observed in the
amount of the OTUs was specifically associated with a certain same compartment (i.e., mucus, tissue, or skeleton) of P. lutea.
coralcompartment.Whetherandhowthepropertiesofdistinct This study will help attracting the attentions on the ecological
actinobacteria assemblages in different coral compartments role of actinobacteria in corals besides the natural products
actuallycontributetothecloserelationshipconstructedbetween bioprospecting.
FrontiersinMicrobiology|www.frontiersin.org 7 October2015|Volume6|Article1094
Kuangetal. Poriteslutea-associatedactinobacteria
TABLE4|SummaryoftheActinobacteriaassociatedwithcorals.
Family Genus Sourcecoral Isolate/clone References
Acidimicrobiaceae Ilumatobacter Poriteslutea Clone Chenetal.,2014
Poriteslutea Clone Thisstudy
Iamiaceae Iamia Poriteslutea Clone Thisstudy
Actinopolysporaceae Actinopolyspora Poriteslutea Clone Thisstudy
Actinospicaceae Actinospica ZoanthidPalythoaaustraliae Clone Sunetal.,2014
Brevibacteriaceae Brevibacterium Acroporadigitifera Isolate NithyanandandPandian,
2009
Tubastraeacoccinea Clone Yangetal.,2013
Acroporamillepora Isolate Lietal.,2014b
Galaxeafascicularis Isolate Lietal.,2014b
Poriteslutea Isolate Lietal.,2014b
Poriteslutea Clone Thisstudy
Dermacoccaceae Demetria Poriteslutea Clone Thisstudy
Dermacoccus Tubastraeacoccinea Clone Yangetal.,2013
Kytococcus Fungiascutaria Isolate Lampertetal.,2006
Poriteslutea Clone Thisstudy
Dietziaceae Dietzia Leptogorgiaminimata Isolate Brucketal.,2007
Scleronephthyasp. Isolate Sunetal.,2012
Alcyoniumgracllimum Clone Yangetal.,2013
Tubastraeacoccinea Clone Yangetal.,2013
ZoanthidPalythoaaustraliae Clone Sunetal.,2014
Poriteslutea Clone Thisstudy
Geodermatophilaceae Blastococcus Poriteslutea Clone Thisstudy
Geodermatophilus Poriteslutea Clone Thisstudy
Modestobacter Poriteslutea Clone Thisstudy
Intrasporangiaceae Janibacter Acroporagemmifera Isolate Kageyamaetal.,2007
Alcyoniugracllimum Clone Yangetal.,2013
Acroporagemmifera Isolate Valliappanetal.,2014
Poriteslutea Clone Thisstudy
Ornithinimicrobium Poriteslutea Clone Thisstudy
Serinicoccus Tubastraeacoccinea Clone Yangetal.,2013
Mycobacteriaceae Mycobacterium Sinulariasp. Isolate Thomasetal.,2010
Scleronephthyasp. Isolate Sunetal.,2012
Alcyoniugracllimum Clone Yangetal.,2013
Tubastraeacoccinea Clone Yangetal.,2013
Poriteslutea Isolate Lietal.,2014b
Poriteslutea Clone Thisstudy
Nocardiaceae Rhodococcus Iciligorgiaschrammi Isolate Brucketal.,2007
Scleronephthyasp. Isolate Sunetal.,2012
Tubastraeacoccinea Clone Yangetal.,2013
Nocardioidaceae Nocardioides Palythoacaribaeorum Isolate Seemannetal.,2009
Scleronephthyasp. Isolate Sunetal.,2012
Tubastraeacoccinea Clone Yangetal.,2013
Poriteslutea Clone Thisstudy
Nocardiopsaceae Nocardiopsis Platygyralamellina Clone Lampertetal.,2008
Acroporamillepora Isolate Lietal.,2014b
Galaxeafascicularis Isolate Lietal.,2014b
Poriteslutea Isolate Lietal.,2014b
Poriteslutea Clone Thisstudy
(Continued)
FrontiersinMicrobiology|www.frontiersin.org 8 October2015|Volume6|Article1094
Kuangetal. Poriteslutea-associatedactinobacteria
TABLE4|Continued
Family Genus Sourcecoral Isolate/clone References
Propionibacteriaceae Friedmanniella Poriteslutea Clone Thisstudy
Propionibacterium Cirrhipiatheslutkeni Isolate Santiago-Vázquezetal.,2007
Mussimiliahispida Isolate deCastroetal.,2010
Acroporadigitifera Isolate Nithyanandetal.,2011b
ZoanthidPalythoaaustraliae Clone Sunetal.,2014
Poriteslutea Clone Thisstudy
Tessaracoccus Poriteslutea Clone Chenetal.,2014
Pseudonocardiaceae Pseudonocardia Acroporamillepora Isolate Lietal.,2014b
Galaxeafascicularis Isolate Lietal.,2014b
ZoanthidPalythoaaustraliae Clone Sunetal.,2014
Poriteslutea Clone Thisstudy
Amycolatopsis Galaxeafascicularis Isolate Lietal.,2014b
ZoanthidPalythoaaustraliae Clone Sunetal.,2014
Prauserella Galaxeafascicularis Isolate Lietal.,2014b
Saccharomonospora Antipathesdichotoma Isolate Seemannetal.,2009
Streptomycetaceae Streptomyces Iciligorgiaschrammi Isolate Brucketal.,2007
Acroporadigitifera Isolate Nithyanandetal.,2011b
Antipathesdichotoma Isolate Zhangetal.,2012
Scleronephthyasp. Isolate Sunetal.,2012
Alcyoniumgracllimum Clone Yangetal.,2013
Tubastraeacoccinea Clone Yangetal.,2013
ZoanthidPalythoaaustraliae Clone Sunetal.,2014
Acroporamillepora Isolate Lietal.,2014b
Galaxeafascicularis Isolate Lietal.,2014b
Poriteslutea Isolate Lietal.,2014b
Sarcophytonglaucum Isolate EIAhwanyetal.,2015
Poriteslutea Clone Thisstudy
Cellulomonadaceae Cellulomonas Scleronephthyasp. Isolate Sunetal.,2012
Alcyomumgracllimum Clone Yangetal.,2013
ZoanthidPalythoaaustraliae Clone Sunetal.,2014
Dermatophilaceae Dermatophilus Fungiascutaria Isolate Lampertetal.,2006
Alcyoniumgracllimum Clone Yangetal.,2013
ZoanthidPalythoaaustraliae Clone Sunetal.,2014
Micromonosporaceae Micromonospora Fungiascutaria Clone Lampertetal.,2008
Platygyralamellina Clone Lampertetal.,2008
Antipathesdichotoma Isolate Zhangetal.,2012
Tubastraeacoccinea Clone Yangetal.,2013
Acroporamillepora Isolate Lietal.,2014b
Galaxeafascicularis Isolate Lietal.,2014b
Poriteslutea Isolate Lietal.,2014b
Scleronephthyasp. Isolate Sunetal.,2012
Poriteslutea Clone Thisstudy
Verrucosispora gorgoniancorals Isolate Zhangetal.,2013
Salinispora Nephtheasp. Isolate Maetal.,2013
Acidimicrobiales_Incertae_Sedis Candidatus_Microthrix Alcyoniumgracllimum Clone Yangetal.,2013
Tubastraeacoccinea Clone Yangetal.,2013
Poriteslutea Clone Thisstudy
(Continued)
FrontiersinMicrobiology|www.frontiersin.org 9 October2015|Volume6|Article1094
Kuangetal. Poriteslutea-associatedactinobacteria
TABLE4|Continued
Family Genus Sourcecoral Isolate/clone References
Corynebacteriaceae Corynebacterium Fungiagranulose Isolate Ben-Dovetal.,2009
Alcyoniumgracllimum Clone Yangetal.,2013
Tubastraeacoccinea Clone Yangetal.,2013
ZoanthidPalythoaaustraliae Clone Sunetal.,2014
Poriteslutea Clone Thisstudy
Cryptosporangiaceae Fodinicola Poriteslutea Clone Thisstudy
Dermabacteraceae Brachybacterium Acroporadigitifera Isolate NithyanandandPandian,
2009
Galaxeafascicularis Isolate Lietal.,2014b
Poriteslutea Isolate Lietal.,2014b
Poriteslutea Clone Thisstudy
Microbacteriaceae Agrococcus gorgoniancorals Isolate Zhangetal.,2013
Poriteslutea Clone Thisstudy
Candidatus_Aquiluna Poriteslutea Clone Thisstudy
Curtobacterium Acroporadigitifera Isolate Nithyanandetal.,2011b
Leucobacter Siderastreasidereal Isolate Cardenasetal.,2012
Microbacterium Siderastreasidereal Isolate Cardenasetal.,2012
Tubastraeacoccinea Clone Yangetal.,2013
Poriteslutea Isolate Chenetal.,2014
Acroporamillepora Isolate Lietal.,2014b
Galaxeafascicularis Isolate Lietal.,2014b
Poriteslutea Clone Thisstudy
Yonghaparkia Poriteslutea Clone Thisstudy
Micrococcaceae Arthrobacter Stonycoral Isolate Shnit-OrlandandKushmaro,
2009
Poriteslutea Clone Thisstudy
Kocuria Acroporadigitifera Isolate Nithyanandetal.,2011b
Poriteslutea Isolate Chenetal.,2014
ZoanthidPalythoaAustralia Clone Sunetal.,2014
Poriteslutea Clone Thisstudy
Micrococcus Acroporadigitifera Isolate Nithyanandetal.,2011b
Galaxeafascicularis Isolate Lietal.,2014b
Poriteslutea Clone Thisstudy
Rothia Platygyracarnosus Isolate Chiuetal.,2012
Poriteslutea Clone Thisstudy
Tersicoccus Poriteslutea Clone Thisstudy
Gordoniaceae Gordonia Scleronephthyasp. Isolate Sunetal.,2012
Alcyoniumgracllimum Clone Yangetal.,2013
Tubastraeacoccinea Clone Yangetal.,2013
Galaxeafascicularis Isolate Lietal.,2014b
Acroporamillepora Isolate Lietal.,2014b
Poriteslutea Isolate Lietal.,2014b
Jiangellaceae Jiangella Acroporamillepora Isolate Lietal.,2014b
Galaxeafascicularis Isolate Lietal.,2014b
Promicromonosporaceae Cellulosimicrobium Acroporamillepora Isolate Lietal.,2014b
Poriteslutea Isolate Lietal.,2014b
Myceligenerans Fam.Caryophillidae Isolate Sarmiento-Vizcaínoetal.,
2015
Tsukamurellaceae Tsukamurella Galaxeafascicularis Isolate Lietal.,2014b
Thegenerafirstlyreportedinthisstudywereshowninbold.
FrontiersinMicrobiology|www.frontiersin.org 10 October2015|Volume6|Article1094