The quest for a unified view of bacterial land colonization

12 Pages·2014·1.97 MB·English

by  Hao Wu

TheISMEJournal(2014)8,1358–1369 OPEN
&2014InternationalSocietyforMicrobialEcology Allrightsreserved 1751-7362/14
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ORIGINAL ARTICLE
The quest for a unified view of bacterial land
colonization
Hao Wu1, Yongjun Fang2, Jun Yu1 and Zhang Zhang1
1CASKeyLaboratoryofGenomeSciences andInformation,BeijingInstituteofGenomics,ChineseAcademy
of Sciences, Beijing, China and 2Key Lab of Rubber Biology, Ministry of Agriculture and Rubber Research
Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, China
Exploring molecular mechanismsunderlying bacterial water-to-land transition representsa critical
start toward a better understanding of the functioning and stability of the terrestrial ecosystems.
Here, we perform comprehensive analyses based on a large variety of bacteria by integrating
taxonomic, phylogenetic and metagenomic data, in the quest for a unified view that elucidates
genomic,evolutionaryandecologicaldynamicsofthemarineprogenitorsinadaptingtononaquatic
environments.Wehypothesizethatbacteriallandcolonizationisdominatedbyasingle-genesweep,
that is, the emergence of dnaE2 derived from an early duplication event of the primordial dnaE,
followedbyaseriesofniche-specificgenomicadaptations,includingGCcontentincrease,intensive
horizontalgenetransferandconstantgenomeexpansion.Inaddition,earlybacterialradiationmay
be stimulated by an explosion of land-borne hosts (for example, plants and animals) after initial
land colonization events.
The ISME Journal(2014)8, 1358–1369;doi:10.1038/ismej.2013.247; publishedonline23 January 2014
Subject Category: Microbial population and community ecology
Keywords: adaptivemutagenesis;bacteriallandcolonization; GCcontent; genomeexpansion;HGT;
metagenomics
Introduction cope with this new environment. For instance, soil
microbes are intensively reported to possess high
Terrestrial ecosystems must have pressured the
metabolic versatility, such as metal-reducing ability
primordial microbial life with diverse and less
(Venkateswaran et al., 1998), abundance of nitrous
movable microhabitats, scarce resources and other
oxidereductase(Sanfordetal.,2012)andantibiotic-
environmental hazards (for example, turbulence
resistant genes (Riesenfeld et al., 2004). In addition,
of pH and temperature, ultraviolet radiation and
soil-borne bacteria have been found to have the
desiccation). Soil bacteria represent the majority of
highest number of associations with diverse
biodiversity in terrestrial ecosystems and are essen-
hostsascomparedwithotherenvironment-dwelling
tiallyinvolvedintheestablishmentandevolutionof
bacteria (Hooper et al., 2009) and even the most
primary elements in these ecosystems, such as
co-occurring partnerswithin soil environmentitself
carbon sequestration, nitrogen fixation and element
(Freilich et al., 2010), together making terrestrial
cycling (Vogel et al., 2009; Madsen, 2011; He et al.,
environment the most important yet complicated
2012; Sanford et al., 2012; Yergeau et al., 2012).
system.
Therefore, bacterial land colonization is arguably
The next-generation sequencing technology has
notonlyoneofthemostchallenging,butalsooneof
made the bulk generation of pangenomic and
themostfundamentalandseminalecologicaltransi-
metagenomic data sets possible for studying phylo-
tions in bacterial evolution. The conquest of the
genetic and taxonomic biogeography of soil micro-
Earth’s solid surface must have entailed significant
bial communities (Fierer and Jackson, 2006; Fierer
genomic changes and genetic innovations in the
et al., 2012a, b). Currently, major efforts have been
overall architecture, flexible configuration and
focusedoninvestigatingenvironment-specificgenes
necessary elements of bacterial chromosomes to
involved in various metabolic pathways (Sanford
etal.,2012;Barretetal.,2013)thatarehypothesized
toberesponsibleforbacterialniche-specificadapta-
Correspondence:JYuorZZhang,CASKeyLaboratoryofGenome
SciencesandInformation,BeijingInstituteofGenomics,Chinese tions (Hacker and Carniel, 2001; Konstantinidis
Academy of Sciences, No. 1 Beichen West Road, Chaoyang and Tiedje, 2005; Shapiro et al., 2009; Coleman
District,Beijing100101,China. and Chisholm, 2010). However, such investigations
E-mail:[email protected]@big.ac.cn
mayonlygiveusaglimpseoftheemergenceofsome
Received24June 2013; revised 15November2013; accepted12
December2013;publishedonline23January2014 specific metabolic features under limited nutrients
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or resources instead of providing global pictures of Detection of DnaE2 in metagenomic data sets
bacterial evolution. Taking bacterial land coloniza- The metagenomic data for the Sargasso Sea are
tion as an example, there has been limited progress collected from a previous publication (Venter et al.,
in revealing how bacterial communities have 2004). The newly released metagenomic data from
conquered the land, survived in the new and harsh Peru Margin sediment are also collected (Orsi etal.,
environments and formed unique patterns of taxo- 2013). All other metagenomic data are from
nomic diversity distinct from other communities, JGI metagenomics program (http://genome.jgi.
such as the aquatic and the host associated doe.gov/programs/metagenomes/index.jsf). We collect
(Madigan et al., 2011). Therefore, identification 7334298 protein sequences from aquatic environ-
of adaptive and diversifying evolutionary processes ment (including marine and fresh water) and
inassociation with thewater-to-land transition is of 14303396 protein sequences from soil environment
critical importance in systematically understanding for the detection of DnaE polymerases. The detailed
bacterial radiation, host–pathogen coevolution and identification procedures are described in
ecosystem stability. (Supplementary Figure S1). We first extract all
One of our recent studies has clarified the peptide sequences annotated as ‘DNA polymerase
relationship between error-prone DNA synthesis III alpha subunit’ (DnaE/PolC polymerases) with
and GC content variations, showing that a paralog length of 4100 amino acids and then recruit these
of replicative DnaE polymerase—DnaE2—is respon- candidate DnaE/PolC sequences for further HMM
sible for bacterial GC increase, whereas other (Hidden Markov Model) profiling using HMMER
mutator genes only play fine-tuning roles in this (version 3.0) (Finn et al., 2011). Three different
process, and that DnaE2-containing bacteria are DnaE/PolC HMM profiling matrices (from DnaE1þ
found to be specifically enriched in soil environ- DnaE3, DnaE2 and PolC, respectively) are built
ments (Wu et al., 2012). We conceive that DnaE2 (hmmbuild) based on our previous curation (Wu
may play a major role in the bacterial water-to-land et al., 2012). These three HMM matrices are then
transition,asitismoreprevalentinbacterialspecies used to search each candidate DnaE polymerase
found in terrestrial than aquatic environments. extracted from the metagenomic data sets
To test this hypothesis, here we perform (hmmsearch) ending with three different HMM
comprehensive comparative analyses based on a profiling scores and relative E-values. The scores
large quantity of bacteria by combining evidence reflecting the identity of the candidate sequence
from taxonomic, metagenomic and phylogenetic with each of the three HMM profiling matrices are
analyses. We also provide a unified view on then compared for further classification. The candi-
bacterial land colonization by invoking GC content date sequence is identified as DnaE2 only when its
variation and genome size expansion. identity with DnaE2 HMM matrix is the largest and
meantime 1.5 times larger than that with the
DnaE1þDnaE3 HMM matrix. The DnaE1þDnaE3
Materials and methods polymerase is also identified in the same way (as
long as the score with DnaE1þDnaE3 is the largest
Taxonomic structure analysis and also 1.5 times larger than the score with the
The taxonomic structure of all Proteobacteria is DnaE2 HMM matrix), whereas a polymerase is
estimated from the National Center for Biotechno- identifiedasPolCaslongasithasthebestsimilarity
logy Information (NCBI) taxonomy website score with PolC HMM matrix, as PolC is very
(http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/ distinct from other polymerases. All other poly-
wwwtax.cgi?id=1224). Only bacteria with available merases are grouped as unclassified for better data
full genomic sequences are included in this quality and excluded from further analysis. Three
analysis, that is, 1620 in total (on 19 March 2013) mutator genes (mutT, mutYand mutM) that partici-
after the exclusion of Epsilon-proteobacteria. pate in DNA repair and contribute to GC content
AnalternativesubsetofProteobacteriawithdetailed variation(lossofmutTincreasesGC,whereaslossof
annotation of bacterial habitats and DnaE2 poly- mutY/MincreasesAT)(Garcia-Gonzalezetal.,2012;
merases is collected from our previous study (195 Wu et al., 2012) are also collected in order to
genomes in total and 84 are DnaE2-containing examine their relative abundances in terrestrial
bacteria) (Wu et al., 2012) and also recruited for versus aquatic environment. As these repair genes
taxonomicanalysis.Thedatasetusedforestimating tend to have shorter lengths (encoding B200 amino
taxonomicstructureofsoilbacteriaisobtainedfrom acids), only sequences with HMM scores X50 and
a previous collection (Madigan et al., 2011). The E-value X1e(cid:2)10 are extracted for further analysis.
presence/absence ofDnaE2 in98 randomly selected Besides, DnaE sequences in 12 freshwater samples
terrestrial and aquatic bacteria, which are grouped and20FACE(Free-AirCarbonDioxideEnrichment)
based on both taxonomic positions and lifestyles, is soil samples are used for sequencing saturation
further reannotated (Supplementary Table S1). The analysis and the relative abundance of DnaE2 in six
detailed metadata are retrieved from Genomes samples of different depths (5, 30, 50, 70, 91, and
Online Database (http://www.genomesonline.org/; 159m) from Peru Margin marine sediment are also
Pagani et al., 2012). examined. The proportion of DnaE2 (DnaE2%) is
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calculated as follows (based on the fact that Table1 NumberofDnaE2-containingbacteriaineachphylum
DnaE1þDnaE3 sequences are generally single cop-
ied and thus their numbers can roughly reflect the Group Bacteria Numberof
dnaE2bacteria
total number of bacteria):
No: of DnaE2 Terrabacteria Actinobacteria 202
DnaE2 % ¼ (cid:3)100 % Chlorofexi 4
No: of DnaE1þDnaE3 Cyanobacteria 0
Deinococcus 3
Firmicutes 5
Hydrobacteria Acidobacteria 6
Phylogenetic tree construction Aquificae 0
Bacteroidetes 9
We build all phylogenetic trees using MEGA 5.05
Chlamydiae 0
(Tamura et al., 2011) under JTTþ!4 model. The Chlorobi 0
phylogeny of 137 DnaE2 sequences is built using Fusobacteria 0
neighbor-joining method with 100 bootstraps. Gemmatimonadetes 1
A total of 10 random selected DnaE1 sequences Planctomycetes 6
Proteobacteria 369
from Actinobacteria is used as the outgroup. The
Spirochaetes 0
main topology of DnaE2 tree is also validated by a Synergistetes 0
more comprehensive data set of outgroup including Tenericutes 0
25 DnaE1 sequences from five different taxonomic Thermotogae 0
Verrucomicrobia 5
groups using both neighbor joining (Supplementary
Figure S2A) and more robust maximum likelihood
BacteriainboldaretheDnaE2-containingbacteria.
methods (Supplementary Figure S2B). Four DnaE2
sequences (one from Nitrospirae, one from Verruco-
microbia and two from Delta Proteobacteria)
identified previously by HMM profiling method
are excluded for further analysis as they are
phylogenetically classified as DnaE1 (Supple-
mentary Figure S2). The phylogenies of three case
studies referring to Chlamydiae-Verrucomicrobia,
Azospirillum (Alpha Proteobacteria) and Beta
Proteobacteria are built by DnaE1 sequences using
neighbor-joining method with 500 bootstraps and
visualized with the help of the online iTOL tool
(Letunic and Bork, 2011).
Results
Evidence from the taxonomic structure
Diverse soil bacterial communities are often
characterized by the dominance of Proteobacteria
(mainly within Gamma, Beta and Alpha class),
Actinobacteria, Acidobacteria, Planctomycetes and
Verrucomicrobia (Dedysh et al., 2006; Zhou et al.,
2009; Bergmann et al., 2011; Montana et al., 2012).
Community-based analyses in paleosols also con-
Figure 1 Taxonomic structure of DnaE2-containing and soil-
firm this distinct taxonomic distribution (Chandler
dwelling bacteria. ‘P_sub’ stands for a subset of Proteobacteria
et al., 1998; Hart et al., 2011). Coincidently, we collectedfromourpreviousstudy(Wuetal.,2012)thatingeneral
notice that these dominant bacterial phyla in soil reflectsthetaxonomystructureofallsequencedProteobacteriain
are, intriguingly, also DnaE2 bearing, with very few theNCBIdatabase(P_NCBI).Thedatasetforthe‘Soil’bacteriais
fromMadiganetal.(2011).‘DnaE2’includesallDnaE2-contain-
exceptions (Table 1). For further validation, we
ingbacteriain‘P_sub’.
compare thetaxonomicstructure ofDnaE2-containg
Proteobacteria withthat ofterrestrial Proteobacteria
(giventhatProteobacteriacontainthemostavailable variable as compared with the entire Proteobacteria
genome sequences representing an unbiased population (Figure 1). Interestingly, Epsilon-proteo-
sampling). Our result demonstrates that these bacteria are undetected or nearly absent in either
two groups of bacteria have very similar taxo- datasets. The similarcommunitystructure between
nomic structure—both underrepresentation DnaE2-bearing and soil-dwelling bacteria strongly
of Gamma-proteobacteria but overrepresentation indicates that the appearance of dnaE2 plays an
of Beta-proteobacteria, whereas the proportions of important role in shaping the biogeographic pattern
Alpha- and Delta-proteobacteria are not much of terrestrial bacteria. Moreover, the presence of
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DnaE2 in most bacteria is associated with terrestrial DnaE2 (on average, only 42.4% are DnaE2-contain-
environment, whereas the absence of DnaE2 is ing bacteria), as compared with the deep marine
found to be common in aquatic bacteria even when sediment below 50m (at depths of 50, 70, 91 and
comparingwithinthesamephylum(Supplementary 159m) where higher GC contents (B53.5%) are
Table S1), suggesting that it is DnaE2 not taxonomy found to be correlated with higher proportion of
that is linked to environmental adaptations. DnaE2-containing bacteria (84.5%) (Supplementary
Table S2), confirming the major role of DnaE2 to
bacterialGCincrease.Wecan onlyroughly estimate
Evidence from metagenomic study the relative abundance of three mutator genes
We further explore the abundance of DnaE2 using because of lack of benchmarking gene of similar
the abundant metagenomic data, as metagenomes length (Supplementary Table S3). The results indi-
enable systematic understanding of gene content, cate that in all three samples of aquatic
functional relevance and genomic plasticity
in natural microbial communities. We argue that
DnaE2 is more prevalent among terrestrial than
aquatic bacteria and the presence of DnaE2
contributes considerably to the success of bacterial
land colonization. To test this proposition, we
collect six different metagenomic samples: three
data sets generated from aquatic environment DNA
and three from terrestrial DNA. Consistent with our
expectations, a clear enrichment of DnaE2 was
identified in terrestrial (B55–68% DnaE2-contain-
ingbacteria)thaninaquatic(onlyB11–21%DnaE2-
containingbacteria)environments(Table2).Wealso
detect the proportion of DnaE2-containing bacteria
in each of the 12 freshwater and 20 FACE soil
samples. Intriguingly, we find that the sequencing
depth (indicated by the abundance of DnaE poly-
merases) correlates linearly with the proportion of
DnaE2 (Figure 2). Specifically, soil samples present
astrongupwardtrend,implyingthattheproportion
of DnaE2-containing bacteria may constitute 470%
of total bacterial species at least in the FACE soil
samples (R¼0.81, Po0.0001), whereas freshwater
samples exhibit a strong descending trend,
indicating that DnaE2-containing bacteria in this
environmentareclearlyunderrepresented(aslowas
B10%; R¼ (cid:2)0.58, Po0.05). Taken together, these
results clearly suggest that dnaE2 is one key Figure2 CorrelationbetweenDnaE2(%)andsequencingdepth.
soil-specific gene. In addition, we notice that The total number of DnaE sequences (DnaE1þDnaE2þDnaE3)
the lower GC contents (B48%) of bacteria found in and the proportion of DnaE2-containing bacteria (DnaE2%) are
indicatedinthexandyaxes,respectively,andtheformercanbe
theupperlevelofthemarinesediment(atdepthsof
roughlyusedasameasureofsequencingdepths.Thereare20soil
5 and 30m) are associated with lower abundance of (a)and12freshwater(b)samplesrecruitedforthisanalysis.
Table2 SummaryofDnaEandPolCpolymerases(X100aminoacids)insixmetagenomicsamples
Samples DnaE1þ DnaE2 PolC unclassified Total DnaE2%
DnaE3
NorthPacificOcean 1414 307 209 543 2473 21.71
Sargassosea 1353 283 798 110 2544 20.92
Freshwatera 872 96 85 388 1441 11.01
PeruMarginsedimentb 92 52 29 19 192 56.52
MinnesotaFarm 66 45 5 40 156 68.18
FACEc 4406 2450 393 2034 9283 55.61
Thenumberofpolymerasesislistedineachgroupforeachsample.
aTwelvesamplesfromfreshwaterareallrecruitedforfurtherdatasaturationanalysisinFigure2.
bSixsamplesofmarinesedimentofdifferentdepthsarefurthercomparedinordertoclarifytheassociationbetweenDnaE2%andGCcontent
variationsinSupplementaryTableS2.
cTwentysamplesfromFACE(Free-AirCarbonDioxideEnrichment)sitesarealsorecruitedforfurtherdatasaturationanalysisinFigure2.
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environment, mutT is indeed more abundant than horizontal gene transfer, for example, dnaE2 of
mutY/M. But in terrestrial environment, only bacteria in Planctomycetes and Bacteroidetes. How-
sample from FACE site has a slightly higher ever, the most striking finding is that DnaE2 in
enrichment of mutY/M than mutT, whereas the terrabacteria (that is, Actinobacteria, Firmicutes,
other two samples unexpectedly have more mutT Chloroflexi and Deinococcus-Thermus) (Battistuzzi
genes. Currently, we are not sure whether this et al., 2004; Battistuzzi and Hedges, 2009) are more
unexpected pattern in two soil samples is a result closely related to DnaE1, implying that dnaE2 might
of lower level of sequencing depths or alternatively first appear in terrabacteria, which further confirms
itstandsforacommonobservationforgenesthatare our idea about the unrecognized outstanding contri-
playing subsidiary roles in altering genomic GC bution of DnaE2 to bacterial water-to-land transition.
contents.
Case studies
Evidence from phylogenetic analysis Chlamydiae and Verrucomicrobia are known to be
We also construct the phylogenetic tree of DnaE2 closely related (Wagner and Horn, 2006; Griffiths
polymerase (Figure 3) using 10 DnaE1 sequences as and Gupta, 2007), but it remains unclear why they
anoutgroup.WefindthatdnaE2isofteninvolvedin havedistinctecologicalnichesandgenomicfeatures
Figure 3 Phylogenetic tree of DnaE2. The tree is constructed by using MEGA 5.0 under JTTþ!4 model (with 100 bootstraps). Ten
randomly selected DnaE1 sequences from Actinobacteria are used as outgroup (in gray color). Terrabacteria are colored in blue.
HorizontallytransferredDnaE2sequencesarecoloredinred.ThemaintopologyofDnaE2treeisvalidatedbyamorecomprehensivedata
setofoutgroupusingbothneighbor-joining(NJ)andmaximumlikelihood(ML)methods(SupplementaryFigureS2).
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Figure 4 The gain and loss of dnaE2 and its correlation with bacterial land colonization. Bacteria in Chlamydiae-Verrucomicrobia
(a,withbranchescoloredinmagentaandlightblue)andRhodospirillaceae(b)areusedforthiscasestudy.Thephylogenetictreesare
constructed by using MEGA 5 under JTTþ!4 model (with 500 bootstraps). Red circles mapped on the trees are proportional to the
genomesizeofeachbacterium.BacterianameslabeledinbrowncolorstandforDnaE2-containingbacteriaandthegreenbarsintheright
panel are proportional to the GC content of each bacterium. Although the non-Azospirillum bacteria are correlated with aquatic
environment, most of them actually dwell in the boundaries or mixtures of soil and water, such as water sediment (for example,
M.magnetotacticum,M.gryphiswaldenseandT.profundimaris)andditchmud(forexample,R.rubrumandP.molischianum).
(for example, GC contents, genome sizes). We thus We also examine bacteria of the genus Azospir-
use them for a further dissection of the enigmatic illum that are reported to have transitioned from
relationship among GC content variation, genome marine to terrestrial environments (Wisniewski-Dye´
size expansion and ecological shifts. Our compara- et al., 2011). Our results demonstrate that all the
tive analysis reveals that most of the Verrucomicro- foursoil-dwellingbacteriaofthisgenus,havinghigh
bia bacteria have gained dnaE2 gene, presumably GC contents and large genome sizes, are indeed
provoking higher GC content and better fitness in DnaE2-containing bacteria (Figure 4b). However,
soil environment. In contrast, Chlamydiae, lacking we find it is very unconvincing that bacterial land
dnaE2, are evolving toward a very different destiny, colonization may begin from this genus
namely, they are involved in a series of ecological (Wisniewski-Dye´ et al., 2011), given the fact that
shifts (for example, from environment to animals or Alpha-proteobacteria are abundant in soil. As in
from animals to humans) and host–pathogen coevo- this case, we notice that most bacteria of the non-
lution events (Horn et al., 2004; Roulis et al., 2012), Azospirillum within the Rhodospirillaceae family
followed by dramatic genome reduction and GC have comparable high GC contents with that of the
decrease (Figure 4a). Notably, there are only three four bacteria in Azospirillum (except Candidatus
bacteria (Methylacidiphilum infernorum V4, Akker- Endolissoclinum patella L2 that has experienced
mansia muciniphila and Opitutaceae bacterium striking genome reduction because of its ancient
TAV2)inVerrucomicrobiafoundtohavelostdnaE2. symbiotic relationship with marine tunicate; Kwan
TheabsenceofdnaE2inthefirsttwobacteriacanbe et al., 2012). Therefore, we infer that dnaE2 should
better indicated by their dramatic genome reduc- notonlyappearinAzospirillumbutalsobecommon
tions, and the loss of dnaE2 in Opitutaceae among non-Azospirillum bacteria. Our genome
bacterium TAV2 (or Diplosphaera colitermitum screening indeed show that at least five of these
TAV2) still needs further examination owing to its non-Azospirillum bacteria also possess dnaE2, and
incomplete genome sequence. thus we further wonder why these water-associated
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non-Azospirillum bacteria (Wisniewski-Dye´ et al., thermophilus in Chloroflexi is also found to be very
2011) are DnaE2-containing bacteria if DnaE2 is the differentfromitscloserelativesbyhavinghigherGC
decisive element of bacterial land colonization. content and living in terrestrial environment
Our following ecological survey in the DSMZ (originally isolated from sewage sludge; Pati et al.,
(Deutsche Sammlung von Mikroorganismen 2010). Our result from genome analysis indicates
und Zellkulturen GmbH) microbial collections that this bacterium also possess dnaE2. The only
(http://www.dsmz.de/catalogues/catalogue-micro exception is Cyanobacteria that belong to terrabac-
organisms.html) indicates that although most of the teria yet with no evidence of possessing dnaE2.
non-Azospirillum bacteriaare associatedwith aqua- However, we believe one of the earliest lineage of
tic environments, they actually dwell in the bound- Cyanobacteria may once have had dnaE2 and
ary zones between water and soil environments, conquered the land environment as evidenced by
such as water sediment (for example, Magnetospir- current phylogenetic analyses that tend to root
illum magnetotacticum, M. gryphiswaldense Cyanobacteria at Gloeobacter violaceus (Nakamura
and Thalassospira profundimaris) and ditch et al., 2003), a high GC bacterium that prefers
mud (for example, Rhodospirillum rubrum terrestrial environment (SA´Nchez-Baracaldo et al.,
and Phaeospirillum molischianum). In addition, 2005). That is to say, the current marine habitant
Tistrella bauzanensis that forms a coherent cluster of Cyanobacteria may actually be a back-to-the-
with DnaE2-bearing T. mobilis is reported to be sea event, which is in good agreement with
soil dwelling (Zhang et al., 2011). Therefore, previous studies (SA´Nchez-Baracaldo et al., 2005;
we argue that not only the Azospirillum genus, but Wisniewski-Dye´ et al., 2011).
also bacteria of the entire Rhodospirillaceae family
should have already begun their journey toward
land colonization. GC increase vs genome expansion in relation to
environmental adaptations
We have provided evidence in our previous
(Wu et al., 2012) and present studies to support
Discussion
theproposition that DnaE2 plays themajorrole ina
The gain and loss of dnaE2 and its association with ‘dice-casting’ of bacterial evolution, although deba-
bacterial land colonization table in detailed molecular mechanisms of bacterial
We have reported previously that B68% of the GCincrease.WefurtherarguethatGCincreaseisthe
terrestrial bacteria are DnaE2-containing bacteria prerequisite of genome expansion based on the
(Wu et al., 2012). Here we combine evidence following grounds. First, it has long been known
from taxonomic, metagenomic and phylogenetic that GC content increase is linearly correlated
analyses, revealing that the emergence of dnaE2 is with genome size expansion (Musto et al., 2006).
a key genetic innovation underlying the success of Second, GC content is well recognized as one of the
bacterial land colonization. Given the strong corre- important barriers for horizontal gene transfers in
lation between dnaE2-bearing and soil-dwelling recent studies as bacterial genic GC contents have
bacteria,weproposethatbacteriallandcolonization not much deviated from the GC content of the main
is an ongoing process occurring successfully only chromosomes (Popa et al., 2011; Nishida, 2012a, b;
when the bacterium acquires a dnaE2 gene and has Hayek,2013).Third,bacteriacanselectivelysilence
nothing to do with taxonomic affiliations. This foreign genes whose GC content is lower than the
inference explains why the water-to-land transition host genomic GC content (Navarre et al., 2006;
of the Azospirillum genus (Wisniewski-Dye´ et al., Navarre et al., 2007). Fourth, according to the
2011) has occurred much later than the suggested phylogenetic analysis of dnaE2, this gene may first
divergence of hydrobacteria and terrabacteria appear in terrabacteria that are estimated to have
(Battistuzzi et al., 2004; Battistuzzi and Hedges, begun colonizing land as early as 3.54 to 2.83Gyr
2009). The horizontal transfer of dnaE2 to Azospir- (Battistuzzi and Hedges, 2009). Therefore, there is a
illum genus should have occurred much later, possibility that the emergence of dnaE2 (and
potentially consistent with the radiation of vascular subsequent GC increase) has happened before the
plantsonlandassuggestedbyWisniewski-Dye´ etal. burst of de novo gene-family birth (B3.33–2.85Gyr)
(2011), whereas terrabacteria might be one of the oratleastattheearlystageofthis‘Archaeangenetic
first groups of bacteria that has gained dnaE2. Our expansion’ when extensive genome expansions
results also provide insights into the strikingly have not been summoned (David and Alm, 2011).
different evolutionary scenarios of the two closely Takentogether,thegainofexogenousenvironmental
related groups—Chlamydiae and Verrucomicrobia. DNA/genes of high GC content rarely happens
The ancestor of Verrucomicrobia has gained dnaE2 without an initial GC content increase of the host
followed by GC increase, genome expansion and genome.
land colonization, whereas the dnaE2-deficient Genome expansion stimulated by GC increase
earlylineagesofChlamydiaehavebuiltanancestral enablesthesuccessofbacteriallandcolonizationby
relationship with diverse hosts and experienced providing bacteria with higher rate of distant
dramatic genomic reduction. Sphaerobacter horizontal transfers from donors of similar genome
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sizes (Cordero and Hogeweg, 2009) and with more A unified view of bacterial land colonization
genesinvolvedinregulation,signalingorsecondary Our hypothesis, for a better display, is illustrated in
metabolism (Cases et al., 2003; Konstantinidis Figure 5. The marine ancestral dnaE1-containing
andTiedje,2004).Withinthiscontext,wecanbetter bacteria(step1)gainanactivecopyofdnaE2(evolved
understand why environment pressures are mis- from a dnaE1 duplicate) followed by GC increase
leadingly reported to shape bacterial GC contents (step 2), genome expansion and land colonization
based on the finding that terrestrial bacteria gen- (step3).Startingfromstep3,therearethreedifferent
erally have higher GC content than aquatic bacteria possible evolutionary scenarios. First, some bacteria
(Foerstneretal.,2005).Inaddition,wenotethatthe in this stage further experience niche-restricted
RcGTA (Rhodobacter capsulatus gene transfer adaptations because of ecological shifts (to mammals
agent) (Lang and Beatty, 2007) that has been for example), lose dnaE2 (step 4) and continue to
reported to be able to boost the efficiency of evolve generally in the form of GC decrease (step 5)
horizontal gene transfers (McDaniel et al., 2010) is and genome reduction (step 6) owing to host jump
enriched in the soil than in the marine environ- (for example, from environment to insects). Second,
ments (Supplementary Figure S3), providing addi- theremaybealsosomethathaveexperiencedstriking
tional evidence to explain bacterial genome genome reduction but still possess dnaE2 and thus
expansion in terrestrial environment. keephighGC content (step 7). Third, others possibly
Based on the immense metabolic flexibility of reinvade the marine environment (step 8 and 9) and
high-GC DnaE2-containing bacteria, we conclude further spread to marine-borne hosts (step 10). In
that there must be ample unusual metabolic features addition, pathogenic/symbiotic bacteria in various
because of their enhanced ability to gain exogenous land- and water-dwelling organisms may also evolve
genetic materials. To take Symbiobacterium thermo- from lineages derived directly from ancestral dnaE1-
philumasanexample,thisDnaE2-containingbacter- containing bacteria.
ium is known to possess a variety of respiratory In summary, the bacterial water-to-land transition
systems found only in Gram-negative bacteria (Ueda is characterized by a new pathway of adaptive
etal., 2004); DnaE2-containing Silicibacter pomeroyi mutagenesis that arms the bacteria with a genome
is also found to have some unusual metabolic of higher GC content, larger genome size and an
pathways to deal with nutrient-poor habitats open pan-genome so that they have better ability to
(Moran et al., 2004); there is even one DnaE2- deal with strange and hostile soil environments.
containing bacterium reported to have some eukar- This new pathway of adaptive mutagenesis or
yotic features, for example, genes involved in sterol genetic innovation recruits the coding product of
synthesis (Pearson et al., 2003). In addition, most dnaE2 that may come from dnaE1 gene duplication
bacteria armed with compound-degrading ability are for error-prone DNA synthesis (observed as GC
also revealed to be DnaE2-containing bacteria (Phale increase). The genomic GC increase shaped by
et al., 2007; Wu et al., 2012), uncovering their great DnaE2-invloved error-prone DNA repair is then
roles in bioremediation. Furthermore, not only inferred to be the prime for subsequent bacterial
unusual but also novel metabolic pathways tend to genome expansion that in return confers bacteria
be found in DnaE2-containing bacteria (Table 3). with a fitness advantage in the soil-based environ-
Thus,itisnotsurprisingtodetectanewpathwayfor ment. Driven by positive selection, dnaE2 passes
calcification in Cyanobacteria (Couradeau et al., through one bacterium to another (by horizontal
2012) as postulated by our inference that Cyanobac- gene transfer or recombination), sweeping through
teria may once have possessed dnaE2 and dwelt different bacterial phyla and triggering new ecolo-
in terrestrial environment. Even genes involved in gical differentiations. This view is consistent with
oxygenic photosynthesis that are often involved in the model of ‘ecotype-formation mutations’ (Cohan
horizontal gene transfer (Shi and Falkowski, 2008) andPerry,2007).However,significantworkremains
may also originate during terrestrial adaptation to be done in order to reveal the detailed genetic
(Battistuzzi et al., 2004). basis of these adaptive evolutionary changes.
Table3 ExamplesofnewmetabolicpathwaysidentifiedintheDnaE2-containingbacteria
DnaE2bacteria Phylum Newpathways Reference
Burkholderiacenocepacia Actinobacteria Anoxicpersistence Sassetal.(2013)
Methylococcuscapsulatus a-Proteobacteria Gluconeogenesis;theabilitytousecopperin Wardetal.(2004)
regulation ofmethanotrophy;sterolandhopanoid
biosynthesis
Ruegeriapomeroyi a-proteobacteria Assimilationofdimethylsulfoniopropionate Reischetal.(2011)
Thermomicrobiumroseum Chloroflexi OxidizationofCOaerobically Wuetal.(2009)
CandidatusMethylomirabilisoxyfera DivisionNC10 Methaneoxidationunderanoxicconditions Ettwigetal.(2010)
Pseudomonassp.strainMT1 g-Proteobacteria 4-and5-Chlorosalicylatedegradation Nikodemetal.(2003)
Gemmatimonasaurantiaca Gemmatimonadetes Synthesisofthecarotenoids Takaichietal.(2010)
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Figure5 A unifiedmodelof bacteriallandcolonization.We propose a conceptual framework here to help clarifythe emergence of
dnaE2anditscontributiontobacteriallandcolonizationthroughaseriesofgenomicchangesincludingGCcontentincreaseandgenome
expansion. Marine-borne (for example, seaweed and fish) and land-borne organisms (for example, trees for plants, beetle for insects
and cattle for mammals) are also illustrated here for exemplification of bacterial radiation because of host diversification. The
dnaE-containing rectangles stand for different stages of bacterial evolution with the rectangle sizes proportional to bacterial genome
sizes.Therearemainlythreeroutesforbacterialevolution.Thefirstrouteisbacterialshiftsfromoceantolandowingtoemergenceof
dnaE2,GCincreaseandgenomeexpansion(blackarrows)andfurtherradiationsandadaptationsbecauseoftheexplosionofdiverseland
hosts.Thesecondrouteisthereinvasionofthemarinebacteriaafterlandcolonization(blackdashedarrows).Thelastrouteforbacterial
hostjumpsisdirectlyfromoceanictoland-borneorganisms(grayarrows).Keygenomicinnovationsandbacterialexamplesarelabeled
foreachstage(upto10).
Further radiation of the soil microbial community Cazemier et al., 2003; Ventura et al., 2007;
Based on this conceptual framework, we also find Kaltenpoth, 2009; Salem et al., 2012; Sudakaran
clues on the radiation of soil-dwelling microbial et al., 2012) have repeatedly revealed that some
communities to other land hosts. For instance, the Actinobacteria play essential roles in insect gut by
smallest Beta-proteobacterial bacterium Candidatus helping their insect hosts to use diverse plant-fiber-
Glomeribacter gigasporarum (B1815 genes), which derivedpolysaccharides.AsmostActinobacteriaare
has a surprisingly high GC content (54.8%) DnaE2-containing and particularly widespread in
(Ghignone et al., 2012), is found to maintain an the terrestrial environment, they are supposed to
ancient relationship with arbuscular mycorrhizal transit from soil to plant host and therefore are
fungi (Jargeat et al., 2004). We infer that this regularly encountered by soil-dwellinginsectsfeed-
bacterium may once have possessed dnaE2 and ing on plants (Kaltenpoth, 2009). There may also be
experienced ecological shift from soil to fungi and some Actinobacteria that have experienced soil-to-
dramatic genome reduction because most of its human host jump, for example, Turicella otitidis, a
relatives are dnaE2-containing and soil-dwelling DnaE2-containing human pathogen in skin and ear
bacteria (Supplementary Figure S4). This argument that hasasmallgenome (B1800 genes)but highGC
is also supported by another closely related fungal content(B71%)(Brinkrolfetal.,2012).Inaddition,
endosymbiont,thatis,BurkholderiarhizoxinicaHKI the shared antibiotic resistome of soil bacteria and
454, that is still holding dnaE2 and thus higher GC human pathogens (Forsberg et al., 2012) can also
content (60.7%) because of a more modest genome support the pivotal roles of soil bacterial commu-
reduction (B3936 genes) compared with Candida- nities to the bacterial radiation. Furthermore,
tus Glomeribacter gigasporarum. In addition, recent weinferthatitisaneasiershiftforbacteriatomove
metagenomic studies (Cazemier et al., 1999; fromsoiltoinlandfreshwaterthanfromsoilbackto
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marine as evidenced by limited overlaps between Battistuzzi FU, Feijao A, Hedges SB. (2004). A genomic
freshwater and marine microbial communities (for timescale of prokaryote evolution: insights into
example, soil abundant Beta-proteobacteria are also the origin of methanogenesis, phototrophy, and the
typical freshwater goers yet nearly completely colonization of land.BMCEvol Biol 4: 44.
Battistuzzi FU, Hedges SB. (2009). A major clade of
absent in the oceans) (Philippot et al., 2010).
prokaryotes with ancient adaptations to life on land.
MolBiol Evol 26:335–343.
Bergmann GT, Bates ST, Eilers KG, Lauber CL, Caporaso
Conclusion
JG, Walters WA et al. (2011). The under-recognized
dominance of Verrucomicrobia in soil bacterial
Hereweperformcomprehensiveanalysesandtryto
communities. SoilBiol Biochem 43: 1450–1455.
put together a unified view on bacterial land Brinkrolf K, Schneider J, Knecht M, Ruckert C, Tauch A.
colonizationthathasbeenproposedtoinvolvegene (2012). Draft genome sequence of Turicella
duplication, function diversification and a series of otitidis ATCC 51513, isolated from middle ear
genomic alternations that include GC content fluid from a child with otitis media. J Bacteriol 194:
increase and genome expansion. We report that 5968–5969.
the emergence and the sweep of dnaE2 are the key CasesI,deLorenzoV,OuzounisCA.(2003).Transcription
regulation and environmental adaptation in bacteria.
genomic innovations underlying bacterial adapta-
Trends Microbiol 11:248–253.
tion to terrestrial environment based on three lines
CazemierAE,VerdoesJC,vanOoyenAJ,OpdenCampHJ.
of evidence. First, the similar taxonomic structure
(1999).Molecularandbiochemicalcharacterizationof
between dnaE2-containing and soil-dwelling bac-
two xylanase-encoding genes from Cellulomonas
teria implies that dnaE2 plays a decisive role in pachnodae.Appl EnvironMicrobiol 65:4099–4107.
shaping the unique biogeographic pattern of soil Cazemier AE, Verdoes JC, Reubsaet FA, Hackstein JH,
microbial community. Second, metagenomic data van der Drift C, Op den Camp HJ. (2003). Promicro-
screening reveal that dnaE2 is indeed soil specific. monospora pachnodae sp. nov., a member of the
Third, phylogenetic analyses indicate that dnaE2 (hemi)cellulolytichindgutfloraoflarvaeofthescarab
may first appear in terrabacteria. Taken together, beetlePachnodamarginata.AntonieVanLeeuwenhoek
83:135–148.
these results consistently and clearly show that
Chandler DP, Brockman FJ, Bailey TJ, Fredrickson JK.
dnaE2 is of great relevance to the success of
(1998). Phylogenetic diversity of archaea andbacteria
terrabacterial land colonization, providing a new
inadeepsubsurfacepaleosol.MicrobEcol36:37–50.
perspective for the study of bacterial radiation after
CohanFM,PerryEB.(2007).Asystematicsfordiscovering
landcolonization.Futurestudieswillbefocusedon thefundamentalunitsofbacterialdiversity.CurrBiol
experimental validation of this genome-based 17:R373–R386.
hypothesis. Coleman ML, Chisholm SW. (2010). Ecosystem-specific
selection pressures revealed through comparative
population genomics. Proc Natl Acad Sci USA 107:
Conflict of Interest 18634–18639.
Cordero OX, Hogeweg P. (2009). The impact of
The authors declare no conflict of interest. long-distance horizontal gene transfer on prokaryotic
genome size. Proc Natl Acad Sci USA 106:
21748–21753.
Acknowledgements CouradeauE,BenzeraraK,GerardE,MoreiraD,BernardS,
Brown GE Jr. et al. (2012). An early-branching
We thank Dr Nicola´s Pinel for providing the data set for microbialite cyanobacterium forms intracellular
analyzing the taxonomic structure of the terrestrial carbonates. Science336:459–462.
bacteria and Dr Nicolas Galtier and Dr Konstantinos T DavidLA, AlmEJ.(2011). Rapidevolutionary innovation
Konstantinidis for helpful and constructive discussions during an Archaean genetic expansion. Nature 469:
on this work. We also thank three anonymous reviewers 93–96.
for providing helpful comments on our manuscript. Dedysh SN, Pankratov TA, Belova SE, Kulichevskaya IS,
This work was supported by grants from the ‘100-Talent Liesack W. (2006). Phylogenetic analysis and in situ
Program’ of Chinese Academy of Sciences (Y1SLXb1365 identification of bacteria community composition in
to ZZ), National Programs for High Technology Research anacidicSphagnumpeatbog.ApplEnvironMicrobiol
and Development (863 Program; 2012AA020409 to ZZ) 72:2110–2117.
and the Special Foundation Work Program EttwigKF,ButlerMK,LePaslierD,PelletierE,Mangenot
(2009FY120100 to JY), the Ministry of Science and S, Kuypers MM et al. (2010). Nitrite-driven anaerobic
Technology of thePeople’s Republicof China. methane oxidation by oxygenic bacteria. Nature 464:
543–548.
Fierer N, Jackson RB. (2006). The diversity and
biogeographyofsoilbacterialcommunities.ProcNatl
AcadSci USA103: 626–631.
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