Genome Features of the Endophytic Actinobacterium Micromonospora lupini Strain Lupac 08

19 Pages·2014·7.48 MB·English

Genome Features of the Endophytic Actinobacterium
MicromonosporalupiniStrain Lupac 08: On the Process
of Adaptation to an Endophytic Life Style?
MarthaE.Trujillo1*,RodrigoBacigalupe1,PetarPujic2,YasuhiroIgarashi3,PatriciaBenito1,Rau´lRiesco1,
Claudine Me´digue4, Philippe Normand2
1DepartamentodeMicrobiolog´ıayGene´tica,EdificioDepartamental,CampusMigueldeUnamuno,UniversidaddeSalamanca,Salamanca,Spain,2Universite´ Lyon1,
Universite´ de Lyon, CNRS-UMR5557 Ecologie Microbienne, Villeurbanne, France, 3Biotechnology Research Center, Toyama Prefectural University, Kurokawa, Imizu,
Toyama,Japan,4Genoscope,CNRS-UMR8030,AtelierdeGe´nomiqueComparative,Evry,France
Abstract
Endophyticmicroorganismsliveinsideplantsforatleastpartoftheirlifecycle.Accordingtotheirlifestrategies,bacterial
endophytes can be classified as ‘‘obligate’’ or ‘‘facultative’’. Reports that members of the genus Micromonospora, Gram-
positive Actinobacteria, are normal occupants of nitrogen-fixing nodules has opened up a question as to what is the
ecologicalroleofthesebacteriaininteractionswithnitrogen-fixingplantsandwhetheritisinaprocessofadaptationfrom
aterrestrialtoafacultativeendophyticlife.TheaimofthisworkwastoanalysethegenomesequenceofMicromonospora
lupiniLupac08isolatedfromanitrogenfixingnoduleofthelegumeLupinusangustifoliusandtoidentifygenomictraitsthat
provideinformationonthisnewplant-microbeinteraction.ThegenomeofM.lupinicontainsadiversearrayofgenesthat
mayhelpitssurvivalinsoilorinplanttissues,whilethehighnumberofputativeplantdegradingenzymegenesidentifiedis
quite surprising since this bacterium is not considered a plant-pathogen. Functionality of several of these genes was
demonstrated invitro, showing that Lupac 08 degraded carboxymethylcellulose, starch and xylan. In addition, the
production of chitinases detected invitro, indicates that strain Lupac 08 may also confer protection to the plant.
Micromonosporaspeciesappearsasnewcandidatesinplant-microbeinteractionswithanimportantpotentialinagriculture
and biotechnology. Thecurrent data strongly suggeststhata beneficial effect isproduced onthe host-plant.
Citation:TrujilloME,BacigalupeR,PujicP,IgarashiY,BenitoP,etal.(2014)GenomeFeaturesoftheEndophyticActinobacteriumMicromonosporalupiniStrain
Lupac08:OntheProcessofAdaptationtoanEndophyticLifeStyle?PLoSONE9(9):e108522.doi:10.1371/journal.pone.0108522
Editor:HolgerBru¨ggemann,AarhusUniversity,Denmark
ReceivedJune27,2014;AcceptedAugust22,2014;PublishedSeptember30,2014
Copyright: (cid:2)2014 Trujillo etal. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
DataAvailability:Theauthorsconfirmthatalldataunderlyingthefindingsarefullyavailablewithoutrestriction.Allrelevantdataarewithinthepaperandits
supporting information files. This Whole Genome Shotgun project has been deposited at European Nucleotide Archive under accession number
NZ_CAIE00000000.01.
Funding:METreceivedfinancialsupportfromtheSpanishMinisteriodeEconom´ıayCompetitividadunderprojectCGL2009-07287.PNacknowledgesfinancial
supportfromtheANR(Sesam).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisio´ntopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*Email:[email protected]
Background co-workers [6] recently showed how a pathogen can evolve in a
few generations to become a symbiotic endophyte by losing
For a long time, it was considered that a healthy plant was a specific transporters andregulators linkedtopathogenesis.
plant without microbes within its tissues. However, this view has
Micromonospora is a genus of Gram-positive Actinobacteria
startedtochangewithnewapproachestoallowstrainstogrowfor
thatwasfirstisolatedfromsoil[7].Thisbacteriumhasreceiveda
alongertimeuponisolationaswellastheuseofNGS,whichhas
lotofattentionduringnaturalproductscreeningprograms,given
permittedtheidentificationofseveralstrainspresentinthetissues
its ability to produce a very rich array of secondary metabolites
of healthyplants, inparticular several actinobacteria [1,2].
[8,9,10].ThedistributionofmembersofMicromonosporaiswide-
Endophyticmicroorganismsliveinsideplantsforatleastpartof
ranging since these bacteria have been isolated from different
their life cycle. According to their life strategies, bacterial
geographical zones. In addition, its habitats are also diverse and
endophytes can be classified as ‘‘obligate’’ or ‘‘facultative’’.
include: soil, freshwater and marine sediments, mangrove soils,
Obligate endophytes are strictly dependent on the host plant for
rocks, and nitrogen fixing nodules of both leguminous and
theirgrowthandsurvivalwhilefacultativeendophyteshaveastage
actinorhizal plants [11,12,13]. The recent report [13] that
in their life cycle during which they exist outside host plants [3].
Micromonospora inhabits nitrogen-fixing nodules in a systematic
These endophytes originate from soil, initially infecting the host
way, has opened up a question as to what is the potential
plant by colonizing, for instance, the cracks formed at points of
ecological role of this bacterium in the plant and whether this
emergence of lateral roots from where they quickly spread tothe
bacterium is in a process of adaptation from a terrestrial to a
intercellularspacesintheroot[4].Thus,aseriesofenvironmental
facultative endophyticlife style.
andgeneticfactorsispresumedtohavearoleinenablingaspecific
bacterium to become endophytic [5]. Conversely, Marchetti and
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Figure1.Neighbour-joiningtreebasedon16SrRNAgenesequencesshowingtherelationshipofMicromonosporaspeciesandother
membersofthefamilyMicromonosporaceae.Strainsisolatedfromplantrelatedsourcesareindicatedbyagreenarrow.
doi:10.1371/journal.pone.0108522.g001
Taxonomically, Micromonospora belongs to the family Micro- saelicesensis, Micromonospora zamorensis and Micromonospora
monosporaceae which currently contains 27 genera and includes chokoriensis. These strains were isolated from a nitrogen fixing
aerobic, non-acid fast and mesophilic microorganisms. Many nodule,therhizosphereofaPisumsativumplantandasandysoil,
strains produce mycelial carotenoid pigments giving the colonies respectively. Nevertheless, a clear picture based on the habitat
an orange to red appearance, but blue-green, brown or purple cannot emerge fromthisanalysis.
pigmented strains have also been isolated. The family Micro- M.lupiniLupac08wasshowntohaveacircularchromosome
monosporaceaealsoharborsthegenusSalinispora,whichiswidely of7,327,024 bpwithaGCcontentof71.96%andnoplasmid.A
distributed in tropical and sub-tropical marine sediments. This total of 7158 genomic objects were identified: 7,054 protein-
taxon was described as the first marine actinomycete given its coding, 10 rRNAs, 77 tRNAs, and 12 miscRNAs genes. The
inability to grow in low salinity medium. Indeed, genomic average gene length was 964bp with an average intergenic
information obtained from the genomes of Salinispora tropica distance of 126bp. After manual validation of the automatic
and Salinispora arenicola provide evidence of marine adaptation annotation, 61.5% (4338 CDSs) of the genes were assigned a
of Salinispora species [14]. Thus, it appears that Salinispora biological function while 38.5% were registered as open reading
evolvedfromaterrestrialenvironmenttoamarinehabitat.Inthe frames(ORFs)withanunknownfunction.BasedontheG+Cskew
caseofsomeMicromonosporalineages,thequestioniswhetherthis analysis and position of dnaA, the probable origin of replication
bacteriumhasfollowedacomparableevolutionprocess,changing (oriC), was mapped close to the ribosomal protein rpmH. A
froma terrestrial toanendophytic lifestyle. circular representation of the M. lupini chromosome is provided
Furtherexamplesofcloselyrelatedactinobacteriawithdifferent inFigure 2 indicating someof thefeatures describedabove.
lifestyles reflected in their genomes include, among others, the The genomic characteristics of strain Lupac 08 and three
genera Frankia, Mycobacterium and Streptomyces. In the case of additional Micromonospora genomes deposited in the public
Frankia, comparative genomic analysis of three representative databases including Micromonospora sp. strain L5 isolated from
strains,differingbylessthan2%intheir16SrRNAgenesrevealed root nodules of Casuarina equisetifolia [19]; M. aurantiaca
significantdifferencesintheirgenomesizes(5.4–9.0Mb)suggest- ATCC 27029T and Micromonospora sp. ATCC 39149 isolated
ing that these differences (e.g. gene deletion, acquisition and from soil (Table 1) were compared. An important difference
duplication, etc.) reflect their rapid adaptation to contrasted host betweenthefourstrainswasthenumberoftRNAsidentified.M.
plants and to their environments [15]. Similarly, several myco- lupini 08 contained by far the highest number with 77 tRNAs
bacterial genomes were analyzed both at the nucleotide and while the other strains had between 51 and 53. At present, M.
protein levels. One of the most striking features was lipid lupini Lupac 08 contains one of the largest numbers of tRNAs
metabolism genes with marked expansions of the number of reported among the actinobacteria sequenced. The number of
genesrelatedtosaturatedfattyacidmetabolisminthepathogenic rRNAandtRNAgenesinagenomecanbeseenasanindication
mycobacteria comparedtothesoil-dwelling strains[16]. of positive selection. A high number of rRNA genes increases
In an effort to identify the genomic traits which make possible ribosome synthesis, which in turn increases the protein synthesis
adaptation from a soil dwelling way of life to an endophytic rate [20]and growthrate [21].
habitat,theaimofthisworkwastopresentthegenomesequence
analysis of a representative strain, Micromonospora lupini Lupac Comparative genome analysis
08,isolated froma nitrogenfixingnoduleofthelegumeLupinus COG distribution. Nearly 70% of the CDS were classified
angustifolius.Thisstrainispartofacollectionofmorethan2000 intoclustersoforthologousgroups(COGs,TableS1).Thus,4873
strainsisolatedfromnitrogenfixingrootnodulesofdiverselegume out of 7054 CDS were assigned to 24 different categories,
[17,18]andactinorhizalspecies[13].StrainLupac08wasselected including those for amino acid transport and metabolism (E,
asitshowedgoodplantgrowthpromotion,wasusedpreviouslyfor 12.7%), transcription (K, 10.8%), carbohydrate transport and
insitulocalizationstudiesinplanta[11]andproducedseveralnew metabolism(G,9.7%),inorganiciontransportandmetabolism(P,
secondarymetabolites[9,10].Theresultspresentedhereshowthat 8.7%), energy production and conversion (C, 5.5%), and signal
the genome of M. lupini Lupac 08 contains a diverse array of transduction mechanisms (5.5%).
genesthatmayhelpitssurvivalinsoilsorinplanttissues,whilethe TheCOGdistributionofM.lupiniwassimilartothatobserved
highnumberofputativeplantdegradingenzymegenesidentified in other bacteria in the family Micromonosporaceae, however
in its genome is quite surprising since this bacterium is not variousdifferencesweredetectedsuchastheabundanceofgenes
considered a plant-pathogen and may instead reflect their ability related to carbohydrate transport and metabolism. Among the
tobind toplant structural compounds. Micromonosporagenomescurrentlyavailable,M.lupiniLupac08
contained the highest percentage of genes (9.7%, 685) related to
Results thiscategory,followedbyMicromonosporasp.L5(8.9%,598)and
M. aurantiaca ATCC 27029 (8.5%, 576). The gene contents (in
Phylogenetic position of M. lupini Lupac 08 and general
the same COG category) of other bacterial genomes classified in
genome features the family Micromonosporaceae were lower as in the case of S.
The phylogenetic position based on 16S rRNA gene sequence tropica CNS-205 (7.4%, 391) and S. arenicola CNH-643 (6.4%,
analysis of strain Lupac 08 with respect to currently described 374) two obligate marine actinomycetes. On the other hand, the
Micromonospora species and other members of the family overall COG profiles of Verrucosispora maris AB-18-032T
Micromonosporaceae is presented in Figure 1. Those strains (genome size 6.7Mb) and M. lupini Lupac 08 were very similar
associated with plant/rhizosphere sources are highlighted. Strain and no clear differences were found. Although V. maris was
Lupac08wasclearlypositionedwithinthegenusMicromonospora isolated from a sea sediment, it does not require sea salts for
and forms a subgroup together with the species Micromonospora growthanditisnotconsideredanobligatemarinemicroorganism
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Figure2.Circularrepresentation ofMicromonosporalupiniLupac08.Circlesdisplayedfrom theoutsidein: 1. Cellulose-bindinggenesin
black, chitin-binding genes in red, lectin genes in lavender blue; 2. Genomecoordinates; 3. MW; 4. GC% (linearrange between 65 and 80%); 5.
RegionsofgenomeplasticityaccordingtotheRGP_Findermethod(Mageplatform)basedonsyntenybreaksbetweenthequerygenome(Lupac08)
andclosegenomes(MicromonosporaaurantiacaATCC27029T,Micromonosporasp.L5andVerrucosisporamarisAB-18-032T)correlatedwithHGT
features(tRNAhotspot,DNArepeats,mobilitygenes),andcompositionalbiasandGCdeviationcomputation.C1toC15indicatethepositionofthe
15clustersofgenescodingforsecondarymetabolitesofTable4.
doi:10.1371/journal.pone.0108522.g002
unlikeS.tropicaandS.arenicola.Thus,itsmetabolismsuggestsa carbonsource[24,25].Thereforetheabundanceofthesegenesin
terrestrial life style. Micromonosporae are well known for their the genome of strain Lupac 08, at first glance may not seem
abilitytodegradecomplexpolysaccharidessuchascellulose,chitin surprising, however, the value of 9.7% is comparable to that of
andlignin[22,23].Inparticular,celluloseisfrequentlyutilizedasa highly active cellulolytic microorganisms such as Cellulomonas
Table1. Comparative genomiccharacteristics ofM.lupini Lupac08 andthree Micromonospora genomes publiclyavailable.
Feature M.lupiniLupac08 M.aurantiacaATCC27029T Micromonosporasp.L5 Micromonosporasp.ATCC39149
Size(Mb) 7.3 7.0 6.9 6.8
GC% 72 73 73 72
rRNAOperon 10 9 9 6
tRNA 77 52 53 51
CDSnumber 7054 6676 6617 5633
Averagegenesize(kb) 946 964 969 975
Protein-codingdensity(%) 90.1 90.4 90.4 89.9
GenesinCOGs(%) 70.2% 68.3% 69% nd
nd,notdetermined.
doi:10.1371/journal.pone.0108522.t001
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flavigena134T(9.5%)andThermobifidafuscaXY(7.9%),which plasmidscanberelatedtotheplasticityofagenome.StrainLupac
are abundant in cellulose enriched environments such as soil, or 08 contained 49 CDSs (0.7%, of total CDSs) related to gene
plant tissues. exchange including eight integrases and eleven recombinases.
Synteny. The genome sequence of strain Lupac 08 was ExceptforsevenCDSs,mostofthesegenesweregroupedinto20
aligned with those of Micromonospora sp. L5, M. aurantiaca clusters.Interestingly, eightof thesemobile element clusters were
ATCC 27029T and Micromonospora sp. ATCC 39149T (Fig.3). found near genes related to carbohydrate transport and metab-
Although the four genomes share a significant amount of genetic olism.
characteristics, they have undergone various inversions and Metabolic Features. A metabolic pathway reconstruction
translocations and M. lupini Lupac 08 contains the highest was performed between the genome of strain Lupac 08 and 20
number of non-conserved regions. In addition, this alignment additional strains among which plant pathogens, symbiotic and
shows a high homology between strains Micromonospora sp. L5 saprophyticbacteriawereincluded.Thedistributionandgrouping
and M. aurantiaca ATCC 27029T confirming their close of the microorganisms analyzed using 798 metabolic routes are
phylogenetic relationship as suggested by 16S rRNA gene presented in Figure5. A good correlation was obtained between
phylogeny(Fig.1);nevertheless,strainL5showsalargeinversion the microorganisms, their life style and phylogeny. Two main
event. Thus, although the four Micromonospora genomes share groups were obtained, the proteobacteria and actinobacteria.
many commonfeatures, it isalso evident that M.lupini contains Within the actinobacteria, three clusters were clearly identified:
unique genomic regions as compared to M. aurantiaca ATCC the first one contained strains that belonged to the family
27029Tor Micromonospora sp. L5. Micromonosporaceae, the second cluster corresponded to various
streptomycetes and the third cluster included the three Frankia
DiversityofMicromonosporae:corevs.flexiblegenepool genomes.Surprisingly,MicromonosporalupiniLupac08showeda
UsingtheMicromonosporagenomesofstrainsM.lupiniLupac closer metabolic relationship with the three Frankia strains
08,M.aurantiacaATCC27029TandthatofMicromonosporasp. (ACN14a, CcI3 and EAN1pec) than with the other two
L5 available in the NCBI [19], the core genome was calculated Micromonospora genomes.
usingtheSiLixsoftware[26].Thecoregenomewascomposedof
2294 CDSs, which correspond to approximately 32% of the Plant/Soil-associated life style
predicted proteome. In addition, M. lupini Lupac 08 contained Transport systems. Organisms living in endophytic associ-
thehighestnumberofstrainspecificCDSs,4702(66.6%),whichis ations need to share resources with their host. Membrane
averyhighvaluewhencomparedtoMicromonosporasp.L5and transport systems play essential roles in cellular metabolism and
M. aurantiaca ATCC 27029T (13–14%, Figure 4), which both activities.Currentdatasuggestacorrelationoftransporterprofiles
share a highgenesimilarity (85–86%). to both evolutionary history and the overall physiology and
Horizontalgenetransferisuniversallyrecognizedasanefficient lifestyles of organisms[27].
mechanism for microorganisms to acquire functions that enable A total of 631 CDSs were located in the genome of M. lupini
them to adapt to environments with different selective pressures. coding for a large diversity of transporters, representing approx-
Thereforeinsertionelements,transposases,integratedphages,and imately8.9%ofthegenome.ThemajorityofCDSswererelated
Figure3.MAUVEalignmentofthegenomesequencesofMicromonosporalupiniLupac08,Micromonosporasp.L5,Micromonospora
aurantiacaATCC27029TandMicromonosporasp.ATCC39149.Whenboxeshavethesamecolour,thisindicatessyntenicregions.Boxesbelow
thehorizontallineindicateinvertedregions.Rearrangementsareshownbycolouredlines.Scaleisinnucleotides.
doi:10.1371/journal.pone.0108522.g003
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Figure4.Venndiagramshowingthenumberofclustersoforthologousgenes,sharedandunique,betweenM.lupiniLupac08,
Micromonosporasp.L5andM.aurantiacaATCC27029T.
doi:10.1371/journal.pone.0108522.g004
Figure5.BiclusterplotofthemetabolicprofilesofMlupiniLupac08and20otherbacterialgenomes.
doi:10.1371/journal.pone.0108522.g005
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to ATP-binding (dependent) transporters of which 362 corre- kexin sedolisin (MiLup08_40382). Finally a third cluster contains
spondedtoABCtransporters;thenextmostabundant(215CDSs) the genes eccC (MiLup08_46744), eccD (MiLup08_46743) and
codedforsecondarytransporters,with105classifiedintheMajor mycP (MiLup08_46745).
Facilitator Superfamily –MFS-); 17 transporters belonged to ion Gram-negative bacteria use the type II secretion system to
channels and 20 were unclassified. The number of transporters transportalargenumberofsecretedproteinsfromtheperiplasmic
determinedinM.aurantiacaATCC27029TandMicromonospora space into the extracellular environment. Many of the secreted
sp. L5were lowerwith 575and587,respectively (Table 2). proteins are major virulence factors in plants and animals [33].
The number of transporters identified in the genome of M. Type II secretion systems have been found in all completely
lupini Lupac 08 is correlated with those in other bacteria with a sequenced plant pathogenic bacterial genomes, except in Agro-
plant/soilassociatedlifestyle,whichrequiresanefficientnutrient bacterium tumefaciens. In addition, other bacteria have been
uptake system to obtain nutrients produced by the host plant, in showntousesecretionsystemsforthedeliveryoftoxins,proteases,
addition to those found in the rhizosphere and the soil (Table 2) cellulases and lipases [34–37]. Genes coding for this system have
[27,28]. However, the number of transporters identified in strain also beenreported for thethreesymbiotic strains Frankia[38].
Lupac 08 was lower than those present in other bacteria such as FifteengenesinM.lupiniwereannotatedascomponentsofthe
Bradyrhizobium japonicum USDA6T (1138, 11.8%), Mesorhizo- Type II secretion system, grouped into clusters of three to five
bium loti MAF303099 (968, 14.2%), Sinorhizobium meliloti genes (Table 3). Nine of these genes were annotated as Type II
Sm1021(1024,16.4%)andRhizobiumleguminosariumbv.trifolii secretion system proteins including protein E and protein F; four
WSM 3125 (1087, 15.5%), which form a very close interaction were recorded as TadE family proteins and Milup08_40403 was
withlegumes.Nevertheless,theoveralldistribution(types)andthe annotated as an uncharacterized protein closest to one found in
percentagesofthesevaluesweresimilar.Anadditionaldifference theFrankiasymbiont of Datiscaglomerata.
was the absence of phosphotransferase system transporters (PTS) The secretion systems III and IV which are commonly related
in M. lupini as compared to the strains mentioned above and to plant-associated bacteria transport a wide variety of effector
othersoil/plantbacteriaincludedinTable 2.Ontheotherhand, proteins into the extracellular medium or into the cytoplasm of
theoverallprofileofM.lupiniLupac08wasverysimilartothose eukaryotic host cells thus affecting the interaction [39]. In
of Frankia sp. ACN14a and Frankia sp. CcI3 which also lack a addition, a functional type IV system has been described in the
PTS system. plantsymbiontM.lotistrainR7A[40].AgeneannotatedasvirB4
Secretion systems. Secreted proteins play a number of and related to secretion system IV was located in Lupac 08
essentialrolesinbacteria,includingthecolonizationofnichesand (MiLup08_42651), this ORF is surrounded by proteins with
host–pathogen interactions. In Gram-positive bacteria, the unknown function related to those present in the genomes of
majority of proteins are exported out of the cytosol by the Micromonospora sp.L5andM. aurantiacaATCC 27029T.
conserved Sec translocase system or, alternately, by the twin- Survival against plant defenses. Reactive oxygen species
argininetranslocationsystem.Inaddition,auniqueproteinexport (ROS) play a major role in plant defense against pathogens. In
system, the type VII or ESX secretion system also exists in some response to attempted invasion, plants mount a broad range of
Gram positivebacteria [29]. defenseresponses,includingthesynthesisofROS.M.lupinineeds
The genome of M. lupini Lupac 08 encodes for 537 (7.6%) to survive under an oxidative environment in the rhizosphere
secreted proteins including several protein secretion systems beforeitcancolonizeplantrootsanditsgenomerevealedseveral
(Table 3). All genes related to the Sec-dependent pathway were genes encoding proteins to neutralize oxidative stress. The
locatedandincludedtheSecYandSecEproteinswhichformthe followinggeneswereidentified:threesodgenes(MiLup08_45788,
membrane channel and interact with the cytoplasmic membrane MiLup08_46012 and MiLup08_46604) that code for superoxide
proteinSecG;theauxiliaryproteinsSecD,YajCandtheATPase dismutases; a catalase HPII katE (MiLup08_44247); a catalase-
SecA. In addition, the heterodimer Ffh-FtsY (MiLup08_41486 peroxidase (katG, MiLup08_44435) and a catalase hydroperox-
andMilup08_41460)wasalsopresent.AsinotherGram-positive idase (katA, MiLup08_45857); four hydroperoxide reductases
bacteria, M. lupini Lupac 08 lacks homologs of SecB, the (MiLup08_40110, MiLup08_40293, MiLup08_41393,
chaperone that targets proteins to the Sec translocon for passage MiLup08_45407); a chloroperoxidase (MiLup08_44157) and a
through thecytoplasmic membrane [30]. thiolperoxidase (MiLup08_43629).
Genes related to the Sec-independent twin-arginine transloca- Inaddition,aputativeorganichydroperoxideresistanceprotein
tionpathyway(TAT),whichexportsprefoldedproteinsacrossthe (Ohr, MiLup08_45098); a 4-hydroxyphenylpyruvate dioxygenase
cytoplasmicmembraneusingthetransmembraneprotongradient (Hpd, MiLup08_46664) and a homogentisate 1,2-dioxygenase
as the main driving force for translocation were also located in (MiLup08_46677) were identified. Other enzymes include a
strain Lupac 08 (Table3). Homologs of TatA and TatC were glutathione peroxidase (MiLup08_45173); two glutathione trans-
identified, however no homolog for TatB was found. Similar to ferases(MiLup08_46358andMiLup08_41529)andfourglutathi-
otheractinobacteria(e.g.Frankiasp.ACN14a)thetatAgenewas one-S-transferases (fdh, MiLup08_42270, MiLup08_42834,
found next to tatC. Only an ORF encoding TatC was located in MiLup08_44416 and 45648). Experimental data indicated that
thegenomesofMicromonosporasp.L5andM.aurantiacaATCC M.lupiniindeedyieldsacatalasepositivereaction[17]confirming
27029Twhile nocopies oftatA ortatB werefound. thefunctionalityofsomeofthesegenes.Therefore,tosuccessfully
AsetoffifteengenesidentifiedaspartofthetypeVIIsecretion reach the internal plant tissues, these genes may defend the
system were located in M. lupini Lupac 08 (Table3). These are bacterium againsta ROS releaseby theplant.
arranged in three different clusters and included the essential
proteinsforsecretionEccC,EccD,EsxAandEsxB[31].Thefirst Regulation as a means of adaptation
clustercontainseightgenes:eccC,esxA,esxB,eccD,eccB,eccEand Lifestyle can be viewed as the set of biotopes an organism can
two copies of mycP, a subtilisin-like serine protease which also thrive into and the relationships that it establishes with other
appearsessentialbutthefunctionofwhichisnotyetknown[32]. species and its abiotic components. It is one of the driving forces
The second cluster includes a copy of esxA (MiLup08_40381), that contribute to the overall characteristics of bacterial genomes
esxB (MiLup08_40380) and mycP, annotated as S8 S53 subtilin [41].
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Pseudomonassyringaepvphaseolicola1448A 5.9 670 12.5 0.11 392(58.5%) 346(88.3%) 32(4.8%) 5(0.7%) 226(33.7%) 72(31.9%) 16(7.1%) 11(1.6%)
S.scabiei8722 10 775 8.2 0.08 480(61.9%) 455(94.8%) 22(2.8%) 6(0.8%) 252(32.5%) 111(44%) 18(7.1%) 14(1.8%)
or
styles. S.coelicolerA32 9.1 798 9.7 0.09 461(57.8%) 433(93.9%) 19(2.4%) 10(1.3%) 286(35.8%) 120(42%) 15(5.2%) 21(2.6%)
ciatedlife Enterobactsp.368 4.6 662 15.6 0.14 317(49.7%) 287(90.5%) 23(3.5%) 47(7.1%) 256(38.7%) 84(32.8%) 18(7%) 14(2.1%)
o m
ss u
a arM
aplant/soil R.leguminosBvtrifoliiWS3125 7.4 1087 15.5 0.15 800(73.6%) 769(96.1%) 26(2.4%) 6(0.6%) 234(21.5%) 69(29.5%) 13(5.6%) 16(1.5%)
cteriawith B.japo-nicumTUSDA6 9.6 1138 11.8 0.12 684(60.1%) 645(94.3%) 24(2.1%) 4(0.4%) 408(35.9%) 114(27.9%) 31(7.6%) 11(1%)
ndcomparisonwithotherba F.symbiontF.alniFrankiaDasticaglomerataACN14sp.CcI3 7.55.45.3 433253300 6.44.57.1 0.060.050.06 281146210(64.9%)(57.7%)(70%) 262127(87%)189(93.2%)(90%) 127(2.8%)7(2.8%)(2.3%) ––– 1308975(25%)(30%)(35.2%) 643633(44%)(49.2%)(40.4%) 9(6.9%)7(7.9%)7(9.3%) 10118(2.7%)(2.3%)(4.3%) transportersrespectively.
a y
upac08 S.tropicaCNB440 5.2 413 7.8 0.08 247(59.8%) 228(92.3%) 11(2.7%) – 145(35.1%) 74(51%) 8(5.5%) 9(2.2%) dsecondar
L n
a
ofM.lupini S.arenicolaCNS205 5.7 405 7.1 0.07 244(60.2%) 225(92.2%) 9(2.2%) 4(1%) 9(2.2%) 65(46.8%) 7(5%) 8(2%) TPdependent
e A
thegenom Micromono-sporasp.L5 6.9 587 8.7 0.08 366(62.4%) 342(93.4%) 15(2.6%) – 192(32.7%) 70(36.5%) 7(3.6%) 13(2.2%) otalnumberof
n t
identifiedi M.aurantiacaATCCT27029 7.0 575 8.5 0.08 362(63.0%) 341(94%) 14(2.4%) – 185(32.3%) 68(36.8% 6(3.2%) 13(2.3%) relationtothe8522.t002
TransportersTable2. M.lupiniLupac08 Genome7.3size(Mb) Totaltransport631proteins Tran-8.9sporters(%) No.0.08Transporters/Mbgenome ATP379dependent(60.1%)(%oftotal) ABCfamily*362(95.5%) Ionchannels17(%oftotal)(2.7%) –Phospho-transferasesystem(PTS) Secondary215transporter(34.1%) MFSfamily*105(48.8%) RNDfamily Unclassified20(3%) *Numberandpercentageindoi:10.1371/journal.pone.010
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MicromonosporalupiniLupac08Genome
Table3. Secretionsystem genespresent inthe genomeofM.lupini Lupac08.
SecretionSystem Gene(Milup08_X) Product
Sec-dependent secY(prlA)(46297) Preproteintranslocase,membranecomponent
secE(46336) PreproteintranslocasesubunitsecE
secG(44961) PreproteintranslocaseSecGsubunit
secD(42464) Protein-exportmembraneproteinsecD
secF(42465) Protein-exportmembraneproteinsecF
yajC(42463) Preproteintranslocase,YajCsubunit
secA(41087) ProteintranslocasesubunitsecA
ffh(41468) Signalrecognitionparticleprotein
scRNA(misc_RNA-12) SRP,Ribosome-nascentchaincomplex(RNC)
yidC(30220) Cytoplasmicinsertaseintomembraneprotein
yidC-like(43138) Membraneproteininsertase,YidC/Oxa1family
yidC(45964) InnermembraneproteintranslocasecomponentYidC
Milup_08_41485 SignalpeptidaseI
Milup_08_41486 SignalpeptidaseI
Milup_08_42560 Conservedproteinofunknownfuction(probablesignalpeptidaseI)
lspA(45113) Lipoproteinsignalpeptidase
lgt(45071) Prolipoproteindiacylglyceryltransferase
TAT- tatA(43424) Sec-independentproteintranslocaseproteintatA/Ehomolog
tatC(43425) Sec-independentproteintranslocaseproteintatChomolog
TypeII-(T2SS) Milup_08_40403 SimilartouncharacterizedproteinfromFrankiasymbiontofDiasticaglomerata
Milup_08_40405 Putativehelicase/secretionneighbourhoodTadE-likeprotein
tadE(40223) TadEFamilyprotein
tadE(40224) TadEFamilyprotein
tadE(42690) SimilartoTadEfamilyprotein
tadE(42691) SimilartoTadEfamilyprotein
Milup_08_40226 TypeIIsecretionsystemprotein
Milup_08_40227 TypeIIsecretionsystemprotein
Milup_08_40228 TypeIIsecretionsystemproteinE
Milup_08_40398 TypeIIsecretionsystemproteinE
Milup_08_40399 SimilartoTypeIIsecretionsystemproteinE
Milup_08_40401 SimilartoTypeIIsecretionsystemprotein
Milup_08_42693 TypeIIsecretionsystemproteinF
Milup_08_42694 TypeIIsecretionsystemproteinF
Milup_08_42695 TypeIIsecretionsystemprotein
TypeIV-(T4SS) Milup_08_42651 VirB4protein-likeprotein
TypeVII/WXG100- eccB(40554) ESX-4secretionsystemproteineccB4
eccC(40438) FtsK/SpoIIIEfamilyprotein
eccC(40557) ESX-4secretionsystemprotein/celldivisionproteinftsK/spoIIIE
eccC(46744) FtsK/SpoIIIE-liketransmembraneprotein
eccD(40556) ESX-4secretionsystemproteineccD4/Putativesecretionproteinsnm4
eccD(46743) FtsK/SpoIIIEfamilyprotein
eccE(40555) Putativeuncharacterizedprotein
esxA(40381) Putativeuncharacterizedprotein
esxA(40559) Putativeuncharacterizedprotein
esxB(40380) Putativeuncharacterizedprotein
esxB(40558) Putativeuncharacterizedprotein
mycP(40382) PeptidaseS8andS53subtilisinkexinsedolisin
mycP(40560) PeptidaseS8andS53subtilisinkexinsedolisin
mycP(40564) PeptidaseS8andS53subtilisinkexinsedolisin
mycP(46745) PeptidaseS8andS53subtilisinkexinsedolisin
TAT,twin-argininetranslocation;X,correspondstotheannotationgenenumbersgiveninparenthesis.
doi:10.1371/journal.pone.0108522.t003
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MicromonosporalupiniLupac08Genome
TheM.lupinigenomeshowsastrongemphasisonregulation, important as they serve as a means of attachment between a
with643proteins(,10%)predictedtohavearegulatoryfunction. bacterium and its host (animal or plant) and are produced by
This value is lower than that reported for the saprophytic strain either ofthetwointeracting organisms[48].
StreptomycescoelicolorA3(2)withanexclusivelyterrestriallifestyle Comparedtothe45enzymespredictedtoactonoligo-and/or
(965 proteins; 12.3%) [42], but higher than the endosymbiotic polysaccharides reported for T. fusca XY [49], the number of
strains M. loti MAFF303099 (542 proteins, 7.7%) [43]; Frankia these enzymes present in thegenomeof M. lupini issignificantly
alni ACN14a (515 proteins, 7.6%); Frankia sp. EAN1pec (555, higher.
6.1%) andFrankiasp. CcI3(244 proteins,4.3%). Cellulose metabolism. Aerobic cellulolytic actinobacteria
ThegenomecodesforvariousregulatorfamiliessuchasTetR, have been shown to use a system for cellulose degradation
AraC,LacI,ArsR,MerR,AsnC,MarR,DeoR,GntRandCrp.In consisting of sets of soluble cellulases and hemicellulases. Most of
addition, thirty-three ECF (extra-cytoplasmic function) sigma these independent cellulolytic enzymes contain one or more
factors were located. Furthermore, 147 genes were related to carbohydrate binding domains[50].
two-component regulatory systems of which 34 were LuxR Atotalof46geneswerefoundtopresentahydrolyticorbinding
proteins. These two-component systems appear to play a crucial fuctiontowardscellulose(TableS1).Severalendoglucanaseswere
role in quorum sensing of Gram-positive bacteria and a positive detected in strain Lupac 08 (e.g. C1, C2 C10 and C14), these
correlationbetweenplant-microbeinteractionsandthenumberof enzymes hydrolyze internal bonds at random positions of
LuxR proteinshas beensuggested [44,45]. amorphous regions of cellulose and generate chain ends for the
Many regulatory genes (,18%) were located near polysaccha- processive action ofcellobiohydrolases (exoglucanases). A copyof
ride related loci including those involved in plant cell wall theexoglucanasegenecbhA(C16)wasalsolocatedinthegenome.
degradation.Specifically,63%ofcellulosedegradationorcellulose Exoglucanases acton theends ofcellulosepolysaccharide chains,
bindinggeneshadanearbyregulator(proximityrangedfrom2–4 liberating cellobiose as the major product. b-D-glucosidases such
genes up or downstream). In the case of xylan metabolism, as M108 and M109 which would further hydrolyze cellobiose
regulators were identified for 50% of the genes, while 43% of were also identified. In addition, several extracellular cellulase
pectinmetabolismgenesalsohadaregulatornearby.Anextended codinggeneswereidentifiedincludingcelA(C3andC6),celB(C5)
overviewoftheregulatorsandtheirassociatedcarbohydrategenes andcelD(C13).TheseresultsstronglysuggestthatstrainLupac08
ispresented inTable S2. ispotentially capable ofcompletely degrading cellulose.
StrainLupac08wastestedforin vitroproductionofcellulases.
An endophytic bacterium highly equipped with an array Very high cellulase activity was detected in minimal agar
of plant cell wall degrading enzymes supplemented with carboxymethylcellulose (CMC, 0.5%)
TheabilityofM.lupinilupac08toassimilateawiderangeof (Fig.6A). When the culture medium was supplemented with
sugarswaspreviouslyreported[19]andthisisclearlyreflectedin glucose (1%) similar results were obtained indicating that this
its genome. The range of simple and complex saccharides sugar did not repress nor derepress the expression of the genes
assimilated by this strain include cellobiose, cellulose, glucose, responsible for theproduction ofcellulases.
mannitol, starch, sucrose, trehalose, xylan and xylose among Hemicellulosic substrates. Genome analysis also revealed
others. Genomic analyses confirmed the presence of a large theabilityofM.lupinitoconvertvarioushemicellulosicsubstrates
number of genes devoted to the metabolism of carbohydrates, tosugars.Twelveputativegenesrelatedtothemetabolismofxylan
including many compounds of plant origin. Plant-polymer includedseveralcopiesofextracellularxylanases(X1,X3,X4,X5,
degrading enzymes such as cellulases, xylanases and pectinases X6, X7, X9, X10 and X12; see Table S1); an extracellular
have been suspected to play a role in internal plant colonization bifunctional xylanase/deacetylase (X8); and an arabinofuranosi-
[46].Inthecaseofplantpathogenicbacteriaandfungi,thesegain dase (X2) which work synergistically with xylanases to degrade
access by actively degrading plant cell wall compounds using xylan to its component sugars. Genes for several a-arabinofur-
glycoside hydrolases including cellulases and endoglucanases. anosidases were also identified (C17, M33, and M39); these are
However,genomicanalysesshowthatnon-pathogenicendophytic exo-acting enzymes which hydrolyze nonreducing arabinofura-
microorganismssuchasEnterobactersp.638[47],AzoarcusBH72 nose residues from arabinoxylan, pectins, and shorter oligosac-
[39]orthesymbioticactinobacteriumFrankiasp.[48]haveonlya charides.
reduced setof cell-wall degrading enzymes. InvitroxylanaseactivitywasdetectedinstrainLupac08when
The genome of M. lupini Lupac 08 revealed a significant tested in a minimal medium supplemented with xylan (1%).
number ofgenes encodingenzymespotentially involvedinplant- Production of xylanases was detected after incubation for 4 days
polymer degradation but also an important number of cellulose- increasing significantly after14days (Fig.6D).The substrate was
binding related genes. Overall, about 10% of the genome coded assayed withandwithout glucose withsimilar results.
forgenesrelatedtocarbohydratemetabolismofwhich192hada Starch degradation. Starch is a ubiquitous and easily
hydrolytic function. At least 79 genes putatively involved in accessible source of energy. In plant cells it is usually deposited
interactions with plants andwith thepotential tohydrolyzeplant aslargegranulesinthecytoplasm.Severalgenescodingforamylo-
polymerswereidentified(TableS1).Thesegeneswereplacedinto a-1,6-glucosidases(e.g.M26,M32,M44,M63,M111andM121;
the glycosyl hydrolase families GH5, GH6, GH9, GH10, GH11, Table S1) were located in addition to two amyE homologs that
GH18,GH20,GH43,GH44andGH62,orintothecarbohydrate codeforanextracellulara-amylase.Furthermore,strainLupac08
binding modules CBM2, CBM13, CBM33, CBM3, CBM46, was able to degrade this polymer under laboratory conditions
CBM42,CBM5,CBM4,CBM6andCBM32.TheCBM2family (Fig.6C) and it was previously shown that Lupac 08 can utilize
wasthemostabundantappearingin46ofthe79genesidentified. thissubstrate as a carbonsource[17].
Fourteengeneswerefurtheridentifiedaslectinsorproteinswith Pectin degradation. Pectinolytic enzymes can degrade
lectin binding domains, which presumably bind to and interact pectic substances either through hydrolysis (hydrolases) or trans-
with carbohydrates. Some of these loci (e.g. Milup_42969, elimination(lyases)[51]andareimportantvirulencemechanisms
Milup_42975, Milup_44484, and Milup_44962) appear to be in many soft-rotting and macerating pathogens [52]. Six pectate
relatedtocellulasesandxylanases,respectively.Theseproteinsare lyases (P1, P3, P4, P5 and P6; Table S1) were located in the
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