Genome-Based Comparative Analyses of Antarctic and Temperate Species of Paenibacillus

12 Pages·2014·0.91 MB·English

Genome-Based Comparative Analyses of Antarctic and
Temperate Species of Paenibacillus
Melissa Dsouza1*, Michael W. Taylor1, Susan J. Turner1,2, Jackie Aislabie3
1CentreforMicrobialInnovation,SchoolofBiologicalSciences,UniversityofAuckland,Auckland,NewZealand,2BioDiscoveryNewZealandLimited,Parnell,Auckland,
NewZealand,3LandcareResearch,Hamilton,NewZealand
Abstract
Antarctic soils represent a unique environment characterised by extremes of temperature, salinity, elevated UV radiation,
lownutrientandlowwatercontent.Despitetheharshnessofthisenvironment,membersof15bacterialphylahavebeen
identifiedinsoilsoftheRossSeaRegion(RSR).However,thesurvivalmechanismsandecologicalrolesofthesephylaare
largelyunknown.TheaimofthisstudywastoinvestigatewhetherstrainsofPaenibacillusdarwinianusowetheirresilience
to substantial genomic changes. For this, genome-based comparative analyses were performed on three P. darwinianus
strains, isolated from gamma-irradiated RSR soils, together with nine temperate, soil-dwelling Paenibacillus spp. The
genome of each strain was sequenced to over 1,000-fold coverage, then assembled into contigs totalling approximately
3Mbp per genome. Based on the occurrence of essential, single-copy genes, genome completeness was estimated at
approximately88%.Genomeanalysisrevealedbetween3,043–3,091protein-codingsequences(CDSs),primarilyassociated
with two-component systems, sigma factors, transporters, sporulation and genes induced by cold-shock, oxidative and
osmotic stresses. These comparative analyses provide an insight into the metabolic potential of P. darwinianus, revealing
potentialadaptivemechanismsforsurvivalinAntarcticsoils.However,alargeproportionofthesemechanismswerealso
identified in temperate Paenibacillus spp., suggesting that these mechanisms are beneficial for growth and survival in a
rangeofsoilenvironments.TheseanalyseshavealsorevealedthattheP.darwinianusgenomescontainsignificantlyfewer
CDSsandhavealowerparalogouscontent.Notwithstandingtheincompletenessoftheassemblies,thelargedifferencesin
genomesizes,determinedbythenumberofgenesinparalogousclustersandtheCDScontent,areindicativeofgenome
content scaling. Finally, these sequences are a resource for further investigations into the expression of physiological
attributesthatenablesurvivalunderextremeconditionsandselectionprocessesthataffectprokaryoticgenomeevolution.
Citation:DsouzaM,TaylorMW,TurnerSJ,AislabieJ(2014)Genome-BasedComparativeAnalysesofAntarcticandTemperateSpeciesofPaenibacillus.PLoS
ONE9(10):e108009.doi:10.1371/journal.pone.0108009
Editor:PatrickC.Y.Woo,TheUniversityofHongKong,HongKong
ReceivedApril1,2014;AcceptedAugust21,2014;PublishedOctober6,2014
Copyright: (cid:2)2014 Dsouza et al.Thisisan open-access article distributed under theterms oftheCreative Commons AttributionLicense, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
DataAvailability:Theauthorsconfirmthatalldataunderlyingthefindingsarefullyavailablewithoutrestriction.Thiswholegenomeshotgunprojecthasbeen
depositedatDDBJ/EMBL/GenBankunderthefollowingaccessionnumbers:JFHT00000000forstrainBr,JFHU00000000forstrainCE1,andJFHV00000000forMB1.
Funding:ThisresearchwassupportedbyCorefundingforCrownResearchInstitutesfromtheMinistryofBusiness,InnovationandEmployment’sScienceand
InnovationGroup,NewZealand(www.mbie.govt.nz).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationof
themanuscript.
CompetingInterests:Ihavereadthejournal’spolicyandtheauthorsofthismanuscripthavethefollowingcompetinginterests:oneormoreoftheauthorsare
employedbyacommercialcompany-BioDiscoveryNewZealandLimited.ThisdoesnotalterouradherencetoPLOSONEpoliciesonsharingdataandmaterials.
*Email:[email protected]
Introduction species [16]. Members of this genus have largely been isolated
fromterrestrialenvironmentsincludingcoldsoilsoftheAntarctic
The phylum Firmicutes represents one of the most abundant Peninsula [17], the Transantarctic Mountains, the Kafni glacier,
and ubiquitous bacterial groups in the environment. Members of Himalayas [18], Alaska [19] and from temperate soil environ-
thisphylumhavebeenidentifiedinawidevarietyofhabitatsthat ments,particularlythoserichinhumusandplantmaterial[20,21].
vary in physical and biochemical characteristics, including the Their ability to successfully colonise these environments can be
vertebrate gut [1], activated sludge [2], soil [3,4], sediment [5], attributed to common physiological traits including formation of
ocean waters [6], and lakewater [7]. Within soil, Firmicutes stress-resistant endospores,secretionofextracellular enzymesand
typically form a minor, yet consistent, component of microbial
anti-microbial compounds (that suppress growth of competing
communities [8–11]. In addition, members of Firmicutes can be microorganisms), and the ability to hydrolyse a variety of
locallyabundant,asobservedingrasslandsoilsofTheNetherlands
carbohydrates including cellulose, starch, and xylan. They are
[12], and significantly more abundant in compacted forest [13]
also noted for their ability to form complex colonial patterns
and arid soils [14] as compared to unimpacted, control soils.
illustrating complex multicellular-like behaviours and for the
Within Firmicutes, members of the genera Bacillus, Clostridium, production of phytohormones, antifungal compounds and nutri-
and Paenibacillus are commonly identified in soil microbial ents including nitrate that promote plant health [16]. Whole
communities [15].
genome sequencing can provide an insight into the molecular
The genus Paenibacillus contains a monophyletic lineage of mechanismsofthesephysiologicalattributesand,moregenerally,
endospore-forming bacteria represented by over 100 described elucidate the metabolic potential and ecological role of these
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ComparativeAnalysesofAntarcticandTemperatePaenibacillusspp.
species.Todate,67Paenibacillusgenomeshavebeensequenced, of Paenibacillus, yet significantly different to that of the nine
of which two, those of P. larvae [22,23] and P. vortex [24], are temperatespecies(P,0.01).Thegenomescontained3,101–3,123
accompanied by publications [25]. P. larvae was investigated for open reading frames with an average coding density of 87.4%.
its ability to cause American foulbrood, a disease of honey bees, Thesegenomescomprisedapprox.3,000proteincodingsequences
and P. vortex for its social organisation and complex pattern- (CDSs). For each genome, approx. 77% of the total CDSs were
forming behaviours. assigned to clusters of orthologous groups (COG) categories.
Here, we report genome analyses of three P. darwinianus Notably, the highest proportion of genes was assigned to COG
strains, isolated from gamma-irradiated soils of the Ross Sea categories including amino acid transport and metabolism [E],
Region (RSR), Antarctica [26]. Soils of the RSR represent a carbohydrate transport and metabolism [G], transcription [K],
unique environment characterised by physical extremes of low andtranslation,ribosomal structure andbiogenesis[J](Figure 1).
temperature, elevated ultraviolet radiation and geochemical Transmembrane proteins constituted approx. 25% of the total
extremes of high salinity, low water and low nutrient availability CDSsidentified.Putativehorizontallytransferredgenesconstitut-
[27]. Despite the harshness of this environment, many bacterial ed 1.87–2.29% of the total genes observed. Significantly fewer
species prevail, with 16S rRNA gene pyrosequencing- and clone copies of the 16S rRNA gene were identified in P. darwinianus
library-based studies identifying members of 15 bacterial phyla, genomes as compared to the temperate Paenibacillus spp. (P,
namely Acidobacteria, Actinobacteria, Armatimonadetes, Bacteroi- 0.01). Temperature range data indicated that P. darwinianus
detes, Chloroflexi, Cyanobacteria, Deinococcus-Thermus, Firmi- strains are psychrotolerant with optimal, minimal and maximal
cutes, Gemmatimonadetes, Nitrospira, Planctomycetes, Proteobac- growth occurring at 18, 10 and 37uC, respectively [26]. The
teria, Spirochaetes, Verrucomicrobia and Candidate ‘TM7’ [28– temperatePaenibacillusspp.exhibitgrowthoverawiderangeof
35]. Of these, Firmicutes were identified in most soil microbial temperatures (10–45uC) with optimal growth occurring between
communities,buttheirrelativeabundancewasvariable,andthey 25and30uC [16,20,41,42].
typicallyformedaminorcomponentofthecommunity.However,
they can be abundant, as observed in ornithogenic soils of Cape General genome comparisons
Hallett [36] and in mineral soils of the Wright Valley [31]. In
General comparisons between genomes of the three P.
addition, a pyrosequencing-based diversity study of airborne
darwinianus strains and nine temperate Paenibacillus spp. were
bacteria over Miers Valley identified members of the family
carried out using CMG-Biotools [43]. Firstly, the amino acid
Paenibacillaceae (of phylum Firmicutes) amongst the most
composition of protein sequences from the 12 Paenibacillus spp.
abundant OTUs (operational taxonomic units)[37].
was determined. This analysis revealed broad similarities in
Members of the genus Paenibacillus are commonly cultured
genome-wide amino acid usage profiles for all 12 Paenibacillus
and identified in polar and permafrost soil communities. Despite
spp.withAla,Leu,Gly,andValthemostfrequentlyusedamino
this,onlysixnovelPaenibacillusspp.,namelyP.antarcticus[38],
acids across all genomes (Figure 2). Predicted proteome compar-
P. cineris, P. cookii [39], P. darwinianus [26], P. macquariensis
isons and a pan- and core-genome plot analysis were also
[40], and P. wynnii [17] have been isolated from the Antarctic
performed on all 12 Paenibacillus genomes using CMG-Biotools
environment,andlittleisknownaboutthepotentialfunctions,or
[43]. Proteomes were predicted using Prodigal [44] and then
the metabolic contributions, of these bacteria in the Antarctic
BLAST algorithm (Basic Local Alignment Search Tool)-based
environment.AkeyquestioniswhetherstrainsofP.darwinianus
proteome comparisons were performed to identify whether
owe their resilience to substantial genomic changes compared to
proteinsaresharedbetweenpredictedproteomes[45].Themain
Paenibacillus spp. isolated from temperate soil environments.
part of the matrix (shaded green) (Figure 3) consists of pairwise
Therefore,theobjectiveofthisstudywastocomparegenomesof
proteomecomparisonsandthebottomrow(shadedred)represents
threestrainsofP.darwinianuswithninetemperate,soil-dwelling
a self-comparison where a hit within the proteome to a protein
Paenibacillus spp., focusing on traits that may contribute to
other than the query is identified as an internal homolog or a
survival and growth in soil, including signal transduction
paralog. The BLAST matrix illustrates that the conservation
pathways, sigma factors, sporulation, motility and their ability to
betweengenomesishigherwithinspeciesthanwithinagenus.P.
cope with oxidative and osmotic stress. This study has demon-
darwinianusstrainsexhibitedahighlevelofconservationofgene
strated that, while the genomic content investigated is largely
families(89.2–90.3%).Thisobservationwasalsosupportedbythe
similaracrossall12Paenibacillusisolates,thereissomeevidence
pan- and core-genome analysis, as the P. darwinianus core- and
for genomecontentscaling.
pan-genome comprised 2,735 and 3,194 gene families, respec-
tively. A large proportion of genes in the P. darwinianus core-
Results and Discussion
genomewereassignedtoCOGcategoriesincludingcarbohydrate
Genome overview transport and metabolism [G], amino acid transport and
The three P. darwinianus genomes are composed of the metabolism[E],andtranscription[K].Incontrast,thetemperate
chromosome(approx.3 Mbp),eachcontainingavaryingnumber Paenibacilluscore-andpan-genomecomprised1,139and25,493
of DNA scaffolds ranging from 107 to 111. The genome gene families, respectively. Genes in the temperate Paenibacillus
completeness was approx. 88%, assessed by the occurrence of core-genome were assigned to COG categories including transla-
essential, single-copy genes. Due to this incompleteness, we have tion,ribosomalstructureandbiogenesis[J],aminoacidtransport
regarded the apparent absence or low copy number of a given andmetabolism[E],andtranscription[K].Overall,thetemperate
genewithcaution.GeneralgenomefeaturesoftheP.darwinianus and Antarctic core- and pan-genome comprised 998 and 26,612
strainscomparedwithninetemperatePaenibacillusspp.arelisted genefamilies,respectively.Finally,upto6%oftheCDSswerein
in Table 1. Dot plot diagrams comparing levels of homology paralogous clusters for temperate Paenibacillus spp., whereas
betweenthethreeP.darwinianusgenomesweregeneratedonthe approx. 2.4% of the CDSs were in paralogous clusters for all P.
IMGwebsite.Theseplotsrevealedhighlevelsofsyntenybetween darwinianus strains(Figure 3).
the three strains (Figure S1). The genome G+C content for the
threestrainswasapprox.56%,inaccordancewithothermembers
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ComparativeAnalysesofAntarcticandTemperatePaenibacillusspp.
Figure1.ComparisonofgenecontentintemperatePaenibacillusspp.andP.darwinianusstrainsbyCOGcategories.
doi:10.1371/journal.pone.0108009.g001
Genomic features linked to survival in stressful hydrolases(enzymesthathavetheabilitytodegradebacterialcell
conditions wall), was not identified in P. darwinianus genomes [53]. In the
Two-componentsignaltransductionsystems. Two-com- psychrophilic bacterium, Flavobacterium psychrophilum, expres-
ponent signal transduction systems (TCS) represent a primary sion of the sensor kinase LytS was cold-induced, with expression
meansbywhichabacterialcellsensesandrespondstoavarietyof significantly upregulated at 8uC compared to its expression at
stresses and to the changing environment. P. darwinianus 20uC [54]. In the Antarctic soil environment, regulated autolysis
genomes contained 78–82 genes associated with TCS including mayallowfortherecyclingofcellularcomponents,thusproviding
approx. 40 genes for membrane-bound histidine kinases and 41 bacterial communities withnutrients.
for response regulators (Table S1). A large number of genes for Withtheexceptionofornithogenicsoilsformedunderpenguin
TCSwerealsoidentifiedinthepsychrophilicarchaeonMethano- rookeries, RSR soils typically contain low concentrations of
coccoides burtonii [46] and bacterium Desulfotalea psychrophila organic carbon, ranging from 0.01 to 0.96 mgC g21 soil [55].
[47], further illustrating the importance of signal transduction ThereforetheabilitytodetectlabileCsourcessuchasdicarboxylic
systems for growth and survival in cold environments. In P. acidscanbecrucialforsurvivalintheAntarcticsoilenvironment.
darwinianus genomes, approx. 2.6% of the total CDSs can be Organic compounds including C4-dicarboxylates, oxalate and
attributed to TCS. A similar proportion of genes (approx. 3% of succinatehavebeenidentifiedinaerosolparticlesovercoastalEast
the total CDSs) were attributed to TCS in genomes of the Antarctica [56], Showa Station [57] and Finnish Station Aboa
temperate Paenibacillus species. Genes for TCS, DesK-DesR for [58] in Queen Maud Land. Genes for TCS, DctS-DctR (for the
low temperature [48], CheA-CheY for chemotaxis [49], PhoR- detection of aerobic C4-dicarboxylates, namely succinate, fuma-
PhoP for phosphate regulation [50], ResE-ResD for oxygen rate, malate, and oxaloacetate) were identified in the P.
limitation [51], DegS-DegU for exoprotease production, compe- darwinianus genomes and in the genomes of just two temperate
tence development, biofilm, flagellum and complex colony Paenibacillus spp., P. mucilaginosus and P. terrae. Genes
formation [52], were identified in all 12 Paenibacillus genomes encoding additional proteins required for the function of sensor
(TableS2).Notably,genesforTCS,LytS-LytR(fortheregulation kinase,DctSincludingDctAandDctB,werealsoidentifiedinall
ofautolysis),wereidentifiedinallP.darwinianusgenomesandin P.darwinianus genomes [59].
the genome of just one temperate Paenibacillus species, P. Sigma factors. Sigma factors are dissociable units of RNA
mucilaginosus.However,thelrgABoperon,inducedbythelytSR polymerasethatactivatetheconditionalexpressionofaspecificset
operon and responsible for blocking the activity of murein of genes in response to a particular stress or stimulus, thus
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ComparativeAnalysesofAntarcticandTemperatePaenibacillusspp.
e
12 6,760,575 5,851,762(86.56) 48.53 45 105 64 8 26 6,169 6,604(98.30) 4,754(77.06) 1,310(21.24) 4,443(72.02) 283(4.59) - Daejeon,SouthKorea Rhizospheresoil %15–37 45;10,P.terra
3
1
11 5,247,653 4,508,951(85.92) 44.79 20 100 68 8 16 4,779 4,679(97.91) 3,692(77.52) 987(20.65) 3,461(72.42) 101(2.11) - Wu-Feng,Taiwan Farmlandsoil 10–45 acisoliDSM2
n
3) 8) 4) 7) pa
10 6,083,395 5,167,317(84.94) 46.77 1 117 89 9 - 5,642 5,525(97.9 3,768(66.7 1,757(31.1 4,117(72.9 - - GaraMountains, Soil 10–40* 21567;9,P.
M
60) DS
[100]. 9 6,326,414 5,389,440(85.19) 48.22 43 127 87 7 26 5,718 5,591(97.78) 4,389(76.76) 1,202(21.02) 4,151(72. 99(1.73) - PocheonProvince,SouthKorea Soil 15–45* P.terrigena
p. 8,
p 1;
acilluss 8 6,361,561 5,583,508(87.77) 46.08 40 118 71 10 27 5,932 5,814(98.01) 4,777(80.53) 1,037(17.48) 4,468(75.32) 240(4.05) - Chiba,Japan Soil #4–32 DSM1549
enib ensis
wellingPa 7 7,464,058 6,619,323(88.68) 53.22 36 103 61 7 29 6,575 6,472(98.43) 5,199(79.07) 1,273(19.36) 4,887(74.33) 302(4.59) - Daejeon,SouthKorea Soil 30** 7,P.daejeon
d 2;
perate,soil- 6 6,241,931 5,308,414(85.04) 44.62 2 196 14 14 - 6,228 6,032(96.85) 4,463(71.66) 1,569(25.19) 3,906(62.72) 257(4.13) - China Rhizospheresoil 30** P.polymyxaSC
m 6,
s.ninete 5 8,770,140 7,182,175(81.89) 58.24 1 224 189 13 - 7,476 7,252(97) 4,389(58.71) 2,863(38.30) 5,362(71.72) - - - Soil 10–45* aginousK02;
y)v mucil
(thisstud 4 5,452,778 4,835,968(88.69) 51.94 40 133 101 10 - 4,957 4,824(97.32) 3,488(70.37) 1,336(26.95) 3,390(68.39) 398(8.03) - Kobecity,Japan Soil 30** usYK9;5,P.
P.darwinianusgenomes 23 3,056,1613,051,566 2,672,1232,663,886(87.43)(87.30) 56.1456.08 108107 5372 2134 45 2426 3,1233,163 3,0703,091(98.30)(97.72) 2,5402,535(81.33)(80.15) 530556(16.97)(17.58) 2,4312,426(77.84)(76.70) 5959(1.87)(1.89) 8888 CapeEvans,MinnaBluff,RossSeaRossSeaRegionRegion Gamma-Gamma-irradiatedirradiatedsoilsoil 10–3710–37 winianusMB1;4,P.curdlanolytichumiDSM23905.
of darpini
GeneralgenomefeaturesTable1. 1 Genomedata Genomesize(bp)3,021,550 DNAcodingregion(%)2,645,264(87.55) GCcontent(%)56.23+ Scaffoldcount111 TotalRNAgenes58 tRNAgenes23 16SrRNAgenes4 OtherRNAgenes29 Totalnumberofgenes3,101 TotalproteinCDSs(%)3,043(98.13) Withfunctionprediction2,487(%)(80.20) Withoutfunctionprediction556(%)(17.93) WithCOGs(%)2,389(77.04) Codingfortransmembrane71(2.29)proteins(%) GenomeCompleteness(%)87 Metadata IsolationsourceBritannia,DarwinMountains HabitatGamma-irradiatedsoil Temperaturerange(C)10–37u 1,P.darwinianusBr;2,P.darwinianusCE1;3,P.HPL-003;11,P.taiwanensisDSM18679;12,P.*,Datafrom[16];**,Optimaltemperaturereported[16];#,Datafrom[41];,Datafrom[42];%,Datafrom[20].doi:10.1371/journal.pone.0108009.t001
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ComparativeAnalysesofAntarcticandTemperatePaenibacillusspp.
Figure2.AminoacidusageheatmapoftemperatePaenibacillusspp.andP.darwinianusstrains.
doi:10.1371/journal.pone.0108009.g002
Figure3.ABLASTmatrixofanallagainstallproteincomparisonof12Paenibacillusgenomes.
doi:10.1371/journal.pone.0108009.g003
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ComparativeAnalysesofAntarcticandTemperatePaenibacillusspp.
implementing compensatory physiological changes. P. darwinia- this transporter were also identified in genomes of all temperate
nusgenomescontainedgenesassociatedwithadiversesetofsigma Paenibacillus spp. (TableS4).
factors similar to the multiple copies observed in psychrophilic Transporter genes. Antarctic Paenibacillus genomes con-
bacteria Planococcus halocryophilus [60] and Psychromonas tained 411–422 membrane transport protein-related genes that
ingrahamii [61] (Table S3). These included the primary house- constituteapprox.14%ofthetotalCDSs(TableS4).Genomesof
keepingfactor,sAandalternativesigmafactorsincludinggeneral thetemperatePaenibacillusspp.containedasimilarproportionof
stress response factor, sB, chemotaxis and flagellar motility membranetransport-relatedgenes.GenesencodingATP-binding
associated factor,sD,cellenvelopestressassociated factor,sECF, cassette (ABC)-type transporters associated with amino acid,
coldshockresponsefactor,sL,andsporulation-specificfactors,sE antimicrobial peptide, sugar, nitrate and proline/glycine betaine
sF, sG, and sH [62,63]. Among these factors, sB is crucial as it transport, comprised approx. 60% of the membrane transporter
controls the expression of an estimated 150 or more genes in genes(TableS5).Itisnotablethatsignificantlyfewercopiesofthe
response toa wide range of stress and starvation conditions [64]. ABC-type multidrug transport system were identified in P.
CommonregulatorsofsB,includingRsbWandRsbV,werealso darwinianus strains as compared to the temperate species (P,
identified inallP. darwinianusstrains. 0.01).WhiletemperatePaenibacillusspp.containedseveralcopies
Oxidativeandosmoticstressresistance. Reactiveoxygen of genes encoding ABC-type oligopeptide, ABC-type dipeptide/
species (ROS) including superoxides (O 2), hydrogen peroxide oligopeptide/nickel, and ABC-type polysaccharide/polyol phos-
2
(H O ),hydroxylradicalmolecules(?OH)andlipidperoxides,are phate transport systems, no copies of genes associated with these
2 2
bothproducedandaccumulatedasaresultofaerobicmetabolism transportsystemswereidentifiedintheP.darwinianusgenomes.
[65]. Additionally, Antarctic soil bacteria are exposed to low However, a similar proportion of CDSs were associated with
temperatures that increase ROS stability and improve oxygen carbohydratemetabolismacrossallPaenibacillusgenomes(Table
solubility [66]. Consequently, combating free radical damage is S6). Therefore, the limited catabolic activity demonstrated by P.
essential for survival in the Antarctic soil environment. Genomes darwinianus strain BrT in Biolog’s phenotype microarray-based
of all three P. darwinianus strains contained two copies of the assay[26],maybeduetoitsinabilitytotransportpolysaccharides
catalasegene,twoofthesuperoxidedismutasegenesodAandone and peptides, suggestive of an adaptation to the nutrient-limited
of the DNA oxidative damage protectant gene dps. Additionally, Antarctic soilenvironment.
up to six copies of the peroxiredoxin genes, ahpCF, bcp and tpx Coldshockresponse. Duringsummer,diurnaltemperature
and nine copies of the thioredoxin genes, trxA and trxB were fluctuationsarecommoninAntarcticsoils.Duringthisperiod,air
identified in all P. darwinianus strains (Table2, Table S10). temperatures are often below 0uC, however surface soils can be
OtherbacteriathatalsocontainmultiplecopiesofgenesforROS heatedupto10–15uCatmiddaydependingonthepositionofthe
detoxification include Colwellia psychrerythraea [67], D. psychro- sun and cloud cover, and drop to below 0uC overnight [75,76].
phila [47], and P. halocryophilus [60]. These genes were also Low temperatures have a major impact on the structure and
identified in genomes of all temperate Paenibacillus spp. In function of cellular constituents including the membrane, the
Bacillus subtilis, the oxidative stress response is regulated by ribosome and nucleic acids by decreasing membrane fluidity,
proteins,PerRandOhrR,activatedbyperoxidesandbyproteins, reducingribosomefunction,unwindingtheDNAdoublehelixand
sB and Spx, that are in turn activated under diverse stress by stabilizing secondary structures of nucleic acids, thus reducing
conditions[68,69].GenesforregulatoryproteinsPerR,OhrRand mRNAtranscriptionandtranslation[77,78].Genomeanalysisof
sBwereidentifiedinallP.darwinianusgenomes.However,Spx P. darwinianus strains revealed the presence of 4–5 copies of
was only identified in P. darwinianus strain CE1. In genomes of genes forcold-shock proteinssimilartothemultiplecopiesfound
the temperate Paenibacillus spp., these regulatory proteins were in C. psychrerythraea [67], Psychrobacter arcticus [79] and
largely absent. Shewanella oneidensis [80]. In addition, several genes for cold-
SalinityisaprominentfeatureofAntarcticsoils,withsomesoils shock induced proteins, comparable to those identified in the
such as those in central Wright Valley containing water-soluble transcriptomeofcold-shockedB.subtilis[81]werealsoidentified
salts as high as 10g cm22 soil [70]. Accumulation of osmopro- in all P. darwinianus genomes (Table 3, Table S11). A large
tectantssuchasglycinebetaineandprolineisaneffectivestrategy proportion of these genes were also identified in temperate
tocombathyper-osmoticstress.Genesinvolvedinglycinebetaine Paenibacillus spp.
uptake(opuA)comprisingthreecomponents:OpuAA,theATPase Sporulation. Spores exhibit a high degree of resistance to
component, OpuAB, the permease component and OpuAC, the various stresses including low temperatures, frequent freeze-thaw
periplasmic component, were identified in all P. darwinianus cycles, UV and gamma radiation, extreme desiccation, and low
strains and in most temperate Paenibacillus spp. [71]. Addition- availability of nutrients that are all common features of the
ally, genes for high-affinity proline-specific uptake by the Antarctic soil environment. P. darwinianus genomes encode an
sodium:solute symporter, OpuE were identified in all P. extensivesetof63–78genes(2.3%ofthetotalCDSs)responsible
darwinianus genomes and in the genome of just one temperate for various facets of sporulation including DNA replication and
species,P.terrae.Prolinebiosynthesisenzymes,c-glutamylkinase translocation, formation of the sporulation septum, engulfment,
(proB), c-glutamyl phosphate reductase (proA) and Pyrroline-5- sporemorphogenesisandgermination(TableS7).Incomparison,
carboxylate reductase (proC) were also identified in all Paeniba- genomesoftemperatePaenibacillusspp.contained94–112genes
cillusgenomes[72](Table2,TableS10).Multiplecopiesofgenes (1.7% ofthetotal CDSs) associated with sporulation(Table S8).
for the uptake of these osmoprotectants were also identified in Motility and chemotaxis. In soil environments, active
genomes of several cold-adapted bacteria including C. psychrer- movement by bacteria towards regions that contain higher
ythraea [67], P. halocryophilus [60], Pseudoalteromonas halo- concentrations of beneficial compounds including water and
planktis [73], and P. ingrahamii [61]. Hypo-osmotic stress, a nutrientsmaybecrucialforsurvival.Over40genesarerequired
consequence of frequent freeze-thaw cycles, is also an important forflagellarassemblyandmovement,includingstructuralsubunits
feature of the Antarctic soil environment. All P. darwinianus for the synthesis of the basal body, the hook and the filament,
strainscontainedgenesformechanosensitiveionchannels(MScL), regulatory proteins sD, FlgM and CodY, motor force generators
responsible for the release of cytoplasmic solutes [74]. Genes for MotA, MotB and chemosensory proteins [82,83]. Chemosensory
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ComparativeAnalysesofAntarcticandTemperatePaenibacillusspp.
e
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omesoftemperat Pfams pfam00199,pfam06628 pfam05067 pfam00081pfam02777 pfam00210 pfam00578 pfam08534 pfam00578,pfam08534 pfam00085 pfam13738 pfam01475 pfam01047 pfam03960 pfam00005,pfam00571,pfam00528,pfam04069 COG0591- pfam39821 pfam00696,pfam01472 pfam03807,pfam14748 mucilaginousK02;6,P.p mberarelistedinTable
ingen OGs OG0753 OG3546 OG0605 OG0783 OG0450 OG2077 OG1225 OG0526,OG3118,OG0694 OG0492 OG0735 OG1846 OG1393 OG4175,OG4176,OG2113 OG4147, OG0263 OG0345 K9;5,P. copynu
eassociatedgenes EnzymesC EC:1.11.1.6C -C EC:1.15.1.1C -C -C EC:1.11.1.15C EC:1.11.1.15C -CCC EC:1.8.1.9C -C -C -C EC:3.6.3.32CCC -C EC:1.2.1.41- EC:2.7.2.11C EC:1.5.1.2C B1;4,P.curdlanolyticusY23905.me.Locustagsforeach
stressrespons GeneSymbol katE - sodA dps ahpCF tpx bcp trxA trxB perR,fur ohrR,marR spx,arsC opuA opuE proA proB proC 3,P.darwinianusM2,P.pinihumiDSMnumberspergeno
OxidativeandosmoticTable2. Productname Oxidativestressresponse Catalase Mn-containingcatalase Superoxidedismutase DNA-bindingferritin-likeprotein Peroxiredoxin Peroxiredoxin Peroxiredoxin Thioredoxin/Thioredoxindomain-containingprotein Thioredoxinreductase Fe2/Zn2uptakeregulationproteins++ OhrR-liketranscriptionalregulator TranscriptionalregulatorSpx Osmoticstressresponse ABC-typeproline/glycinebetainetransportsystem Sodium:solutesymporterfamily c-glutamylphosphatereductase c-glutamylkinase Pyrroline-5-carboxylatereductase 1,P.darwinianusBr;2,P.darwinianusCE1;HPL-003;11,P.taiwanensisDSM18679;1Numbersineachcolumnrepresentcopydoi:10.1371/journal.pone.0108009.t002
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ComparativeAnalysesofAntarcticandTemperatePaenibacillusspp.
e
a
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bacillus m02812 m02852, m02780 m00364, nousK02; elistedin
Paeni 13 87 08,pfa 70,pfa92 76 79,pfa 98,pfa17 09 16 97 05 50 12 16 97 83 69 mucilagi mberar
mperate Pfams pfam003 pfam004 pfam002 pfam000pfam079 pfam006 pfam027 pfam001pfam028 pfam007 pfam011 pfam012 pfam000 pfam009 - pfam003 pfam010 pfam011 pfam017 pfam141 YK9;5,P. hcopynu
Cold-shockinducedgenesidentifiedingenomesofteTable3. ProductnameGenesymbolCOGs Adaptationtocold-shock ColdshockproteinscspB,cspCCOG1278 Metabolismoflipids FattyaciddesaturasedesACOG3239 Metabolismofaminoacids LeucinedehydrogenasebcdCOG0334 2-oxoisovaleratedehydrogenase(E3lpdCOG1249subunit,dihydrolipoamidedehydrogenase) 2-oxoisovaleratedehydrogenasebkdAACOG1071(E1alphasubunit) 2-oxoisovaleratedehydrogenasebkdABCOG0022(E1betasubunit) 2-oxoisovaleratedehydrogenasebkdBCOG0508(E2subunit,lipoamideacyltransferase) Metabolismofnucleotides AdenylosuccinatesynthetasepurACOG0104 Metabolismofcarbohydrates Fructose-1,6-bisphosphatealdolasefbaACOG0191 ABC-typeTransporter ABC-typeMn/Zntransportsystems,mntACOG0803bindingprotein ABC-typeMn/Zntransportsystems,mntBCOG1121ATPasecomponent ABC-typeMn/Zntransportsystems,mntCCOG1108permeasecomponent RNAsynthesis-elongation DNA-directedRNApolymerase,deltarpoECOG3343subunit Proteinsynthesis(ribosomalproteins) 30SribosomalproteinS15rpsOCOG0184 50SribosomalproteinL27rpmACOG0211 50SribosomalproteinL31rpmECOG0254 50SribosomalproteinL32rpmFCOG0333 Unknownfunction Cold-inducibleproteinYdjOydjO- 1,P.darwinianusBr;2,P.darwinianusCE1;3,P.darwinianusMB1;4,P.curdlanolyticusHPL-003;11,P.taiwanensisDSM18679;12,P.pinihumiDSM23905.Numbersineachcolumnrepresentcopynumberspergenome.Locustagsforeacdoi:10.1371/journal.pone.0108009.t003
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ComparativeAnalysesofAntarcticandTemperatePaenibacillusspp.
proteins comprise four groups, firstly a signal recognition and revealedthatalargeproportionofthesefeatureswerealsopresent
transduction group containing methyl-accepting chemotaxis pro- in genomes of temperate species. This suggests that these
teins and glutamine deamidase, CheD, secondly an excitation physiological traits, while not unique to Antarctic soils, are
groupcontaininghistidinekinase,CheA,couplingprotein,CheW, beneficialforgrowthandsurvivalinarangeofsoilenvironments.
and response regulator, CheY, an adaptation group containing Transcriptomic- and proteomic-based studies comparing the
methyltransferase,CheRandmethylesterase,CheBandfinallya expression profiles of P. darwinianus strains and temperate
signal removal group containing phosphatase, CheC [84]. While Paenibacillus spp., at relatively low versus high temperatures
approx. 50genes (1.6% of the total CDSs) encoding components may elucidate the exact mechanisms for cold-adaptation in these
oftheflagellumandchemosensorypathwayswereidentifiedinP. strains.
darwinianus genomes, temperate Paenibacillus genomes con- In conclusion, fewer CDSs, lower paralogous content and the
tained 63–98 genes (1.3% of the total CDSs) associated with limitedcatabolicactivitysuggesttheoccurrenceofgenomecontent
flagella biosynthesis and chemosensory pathways (Table S9). In scaling, offering P. darwinianus a growth advantage in the
addition, cells of P. darwinianus Br are known to possess a Antarctic soil environment. Although this study has provided an
monotrichous flagellum. However, no motility was observed in insightintothemetabolicpotentialofthisspeciesinRSRsoils,it
cells ofstrainBr,indicatingthat furtherinvestigation isnecessary drawsattentiontoourlimitedknowledge abouttheexpressionof
to identify the stimulus needed for movement [26]. A similar physiological traits that enable survival under extreme conditions
observation was made for cells of P. ingrahamii that showed no and, more importantly, to the lack of complete prokaryotic
motility despite the presence of a large cluster of flagellar genes genomes from Antarctic soil environments. Complete genomes
[61]. It ishypothesized that the lack of observed motilitymay be will not only reveal genes essential for survival in the harsh
due to a lack of appropriate stimulus or a defect in one of the Antarcticsoilenvironmentbutalsoassistwiththecharacterisation
essential flagellar proteins or in the expression or assembly of selection processes that affect prokaryotic genomes in this
processes. environment.
Conclusions Materials and Methods
Previous studies have shown that, in bacteria, an increase in Habitat and isolation strategy
genome size is often linked with an increase in metabolic Soil samples were collected from three sites: Cape Evans
complexity, allowing bacteria to produce new enzymes that (77u389S,166u249E)onRossIsland,MinnaBluff(78u309S,169uE)
exploit environmental conditions [85]. However, an increase in on the mainland coast and the Britannia Drift, Lake Wellman
complexity is linked with a quadratic increase in regulatory Region (79u55916.20S, 156u55930.70E) in south-eastern Darwin
proteins associated with transcription and two-component signal Mountains[90,91].Fieldpermitsforsamplecollectionwereissued
transduction systems [86,87]. In environments such as soil, by New Zealand Ministry of Foreign Affairs and Trade. These
efficient regulation of enzyme expression, enabling exploitation samples were irradiated with 6uCo c-rays for 80h at a dose of
of scarce yet diverse, complex nutrients can offer a selective 288Gy/handplatedontoPYGV(PeptoneYeastextractGlucose
advantage, thus lowering the penalty of slow growth, common Vitamin) gellan gum-based solid medium at 15uC for up to two
amongstdominantbacteriainsoilenvironments[88].Conversely, months[92].Followingincubationandpurification,threeisolates
in the Antarctic soil environment, organic residues are scarce yet BrT,CE1andMB1wereidentifiedasstrainsofP.darwinianusby
labile, withCandNbeingmineralisable withina relativelyshort 16SrRNAgenesequencing.P.darwinianusBrTwasdepositedat
period oftime(90 d) underoptimalconditions[89].Intheharsh the International Collection of Microorganisms from Plants
Antarctic soil environment, maintenance of metabolic versatility (ICMP), Landcare Research, New Zealand (ICMP no.19912)
comes at a higher cost and, more importantly, reproductive and at DSMZ, Germany (DSM 27245). P. darwinianus strains
efficiency (promoted by smaller genomes) is crucial for survival CE1 (ICMP no.20538) and MB1 (ICMP no.20539) were also
and growth. Comparative genomic analyses with nine soil- deposited at ICMP.
dwelling, temperate Paenibacillus spp. have revealed that P. Temperature range data were obtained for strains BrT, CE1
darwinianus genomes contained significantly fewer CDSs as andMB1 by growth onPYGV (Peptone Yeast GlucoseVitamin)
compared to the temperate species. A significantly smaller medium solidified by gellan gum [92]. All PYGV plates were
proportion of genes was identified in paralogous clusters in the incubatedfor56daysat0,5,10,15,18,20,25,37,40and45uC.
P. darwinianus genome as compared to the temperate Paeniba-
cillusgenomes(P,0.01).Inaddition,ofthetotalCDSsidentified DNA extraction and sequencing
in the P. darwinianus genomes, significantly fewer CDSs were The three Antarctic Paenibacillus strains were routinely
assignedtoCOGcategory,transcription[K](P,0.01).Finally,P. culturedonPYGVgellangumplatesat15–18uC.Highmolecular
darwinianus strain BrT showed limited catabolic activity [26], weight DNA was extracted by a modified CTAB (hexadecyltri-
indicativeofloweredmetaboliccomplexity.Itshouldbenotedthat methylammonium) and protein lysis method [93]. Briefly, cells
although smaller genomes offer a metabolic advantage during were scraped off PYGV gellan gum plates and re-suspended in
reproduction, striking a balance between minimum cellular- 740mlTEbuffer,20mllysozyme(100mg/ml)andincubatedfor
doubling time and the ability to respond to, or exploit, changing 10minatroomtemperature.Then,40mlof10%SDSand8 mlof
environmental conditions isalsocrucial. Proteinase K(10 mg/ml) wereadded andthereaction incubated
The P. darwinianus genomes contained several features that overnightat37uC.Followingincubation,100mlof5MNaCland
were also identified in genomes of cold-adapted bacteria and CTAB/NaClsolutionswereaddedtoeachreactionandincubated
archaea. These included genes for signal transduction pathways, at 65uC for 10min. Subsequently, 0.5 ml chloroform:isoamyl
sigma factors, membrane transporters, motility and sporulation alcohol (24:1) was added, and the reaction was centrifuged at
associated genes and mechanisms to deal with cold shock, 16,000 g for 15min. The aqueous phase was transferred to a
oxidative and osmotic stresses, thus suggesting their importance clean eppendorf tube containing phenol:chloroform:isoamyl alco-
in cold adaptation and survival. However, comparative analyses hol (25:24:1, pH 8) and centrifuged at 16,000g for 15min. The
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ComparativeAnalysesofAntarcticandTemperatePaenibacillusspp.
aqueous phase was transferred to a clean eppendorf tube Table S1 List of Pfam domains associated with Two-Compo-
containing 0.6 vol isopropanol. For DNA precipitation, reactions nent Systems in genomes of temperate Paenibacillus spp. and P.
wereincubatedatroomtemperaturefor60min,thencentrifuged darwinianus strains.
at 16,000 g for 30min. The DNA pellet was washed with 70% (XLSX)
ethanol and re-suspended in TE buffer containing RNAse (99 ml
TableS2 ListofgenesassociatedwithTwo-ComponentSystems
TE buffer +1 ml RNAse (10 mg/ml)) and incubated at 37uC for in genomes of temperate Paenibacillus spp. and P. darwinianus
20min. DNA extracts were quantified by Quant-iT PicoGreen
strains.
dsDNA assay kit (Life Technologies) and their purity (A /A )
260 280 (XLSX)
was assessed on a NanoDrop ND-1000 Spectrophotometer
(Biolab). The quality of each DNA extract was tested by Table S3 List of sigma factors identified in genomes of the P.
darwinianus strains.
electrophoresis on a 1% agarose gel. Following extraction, high
(XLSX)
molecular weight DNA was sent to Macrogen (Seoul, South
Korea)forsequencingontheIlluminaHiSeq2000platformusing Table S4 List of genes associated with transporter proteins in
100bp paired end libraries. With a sequencing output of 35Gb, genomes of temperate Paenibacillus spp. and P. darwinianus
estimated coverage was over1,0006per genome. strains.
(XLSX)
De novo assembly, annotation and comparative analyses
Table S5 List of genes associated with ABC-type transport
FASTQ files obtained for each genome were trimmed by the systems in genomes of temperate Paenibacillus spp. and P.
FASTQ Trimmer tool of the FASTX-toolkit v0.0.13 [94] and darwinianus strains.
quality filtered by Sickle (https://github.com/ucdavis- (XLSX)
bioinformatics/sickle).High-qualityreads(Q.30)wereassembled
Table S6 List of COGs associated with carbohydrate metabo-
into contigs by Velvet v1.2.10 [95]. All assemblies were further
lism in genomes of temperate Paenibacillus spp. and P.
improved by tools of the PAGIT (post assembly genome
darwinianus strains.
improvement toolkit) pipeline. These included IMAGE (iterative
(XLSX)
mapping and assembly for gap elimination) for gap elimination
and iCORN (iterative correction of reference nucleotides) for TableS7 Sporulationassociated genes identified ingenomes of
sequencing error correction [96]. Improved contigs were assem- theP.darwinianus strains.
bled into scaffolds by SSPACE basic version 1.0 (stand-alone (XLSX)
scaffolder of pre-assembled contigs using paired-read data) [97].
TableS8 Sporulationassociated genes identified ingenomes of
Genepredictionandannotationwasperformedbytheautomated
temperate Paenibacillus spp.
JGI (Joint Genome Institute) Pipeline [25] and the NCBI
(XLSX)
Prokaryotic Genome Annotation Pipeline (PGAP) [98]. Genome
completeness wasassessed aspreviously described[99]. Table S9 List of flagellar assembly and chemotaxis associated
For comparative analyses, ninetemperate, soil-dwelling Paeni- genes in genomes of temperate Paenibacillus spp. and P.
bacillus species, namely P. curdlanolyticus YK9, P. daejeonensis darwinianus strains.
(XLSX)
DSM15491,P.mucilaginosusK02,P.panacisoliDSM21345,P.
pinihumi DSM 23905, P. polymyxa SC2, P. taiwanensis DSM Table S10 List of genes associated with oxidative and osmotic
18679, P. terrae HPL-003, and P. terrigena DSM 21567 were stressresponseingenomesoftemperatePaenibacillusspp.andP.
selected [100]. All analyses were performed using JGI-IMG/ER darwinianus strains.
(Integrated Microbial Genomes/Expert Review) [25] and CMG (XLSX)
biotools [43].
Table S11 List of cold-shock induced genes identified in
genomes of temperate Paenibacillus spp. and P. darwinianus
Genome Submission
strains.
This whole genome shotgun project has been deposited at (XLSX)
DDBJ/EMBL/GenBank under the following accession numbers:
JFHT00000000forstrainBr,JFHU00000000forstrainCE1,and Acknowledgments
JFHV00000000 forMB1.
We thank Antarctica New Zealand for providing logistic support during
samplecollection.
Supporting Information
Figure S1 Dot plot comparisons of the three P. darwinianus Author Contributions
strains.
Conceivedanddesignedtheexperiments:MDMWTSJTJA.Performed
(PPTX)
the experiments: MD. Analyzed the data: MD. Contributed reagents/
materials/analysistools:MDMWTSJTJA.Wrotethepaper:MD.
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