Vol 462 | 24/31 December 2009 | doi:10.

1038/nature08656

LETTERS
A phylogeny-driven genomic encyclopaedia of
Bacteria and Archaea
Dongying Wu1,2, Philip Hugenholtz1, Konstantinos Mavromatis1, Rüdiger Pukall3, Eileen Dalin1, Natalia N. Ivanova1,
Victor Kunin1, Lynne Goodwin4, Martin Wu5, Brian J. Tindall3, Sean D. Hooper1, Amrita Pati1, Athanasios Lykidis1,
Stefan Spring3, Iain J. Anderson1, Patrik D’haeseleer1,6, Adam Zemla6, Mitchell Singer2, Alla Lapidus1, Matt Nolan1,
Alex Copeland1, Cliff Han4, Feng Chen1, Jan-Fang Cheng1, Susan Lucas1, Cheryl Kerfeld1, Elke Lang3,
Sabine Gronow3, Patrick Chain1,4, David Bruce4, Edward M. Rubin1, Nikos C. Kyrpides1, Hans-Peter Klenk3
& Jonathan A. Eisen1,2

Sequencing of bacterial and archaeal genomes has revolutionized our
understanding of the many roles played by microorganisms1. There
are now nearly 1,000 completed bacterial and archaeal genomes
available2, most of which were chosen for sequencing on the basis
of their physiology. As a result, the perspective provided by
the currently available genomes is limited by a highly biased phylo-
genetic distribution3–5. To explore the value added by choosing
microbial genomes for sequencing on the basis of their evolutionary
relationships, we have sequenced and analysed the genomes of 56
culturable species of Bacteria and Archaea selected to maximize
phylogenetic coverage. Analysis of these genomes demonstrated
pronounced benefits (compared to an equivalent set of genomes
randomly selected from the existing database) in diverse areas includ-
ing the reconstruction of phylogenetic history, the discovery of
new protein families and biological properties, and the prediction
of functions for known genes from other organisms. Our results
strongly support the need for systematic ‘phylogenomic’ efforts to
compile a phylogeny-driven ‘Genomic Encyclopedia of Bacteria
and Archaea’ in order to derive maximum knowledge from exist-
ing microbial genome data as well as from genome sequences to
come.
Since the publication of the first complete bacterial genome,
sequencing of the microbial world has accelerated beyond expecta-
tions. The inventory of bacterial and archaeal isolates with complete or
draft sequences is approaching the two thousand mark2. Most of these
genome sequences are the product of studies in which one or a few
isolates were targeted because of an interest in a specific characteristic
of the organism. Although large-scale multi-isolate genome sequen-
cing studies have been performed, they have tended to be focused on
particular habitats or on the relatives of specific organisms. This over-
all lack of broad phylogenetic considerations in the selection of micro-
bial genomes for sequencing, combined with a cultivation bottleneck6,
has led to a strongly biased representation of recognized microbial
phylogenetic diversity3–5. Although some projects have attempted to
correct this (for example, see ref. 5), they have all been small in scope.
To evaluate the potential benefits of a more systematic effort, we
embarked on a pilot project to sequence approximately 100 genomes
selected solely for their phylogenetic novelty: the ‘Genomic
Encyclopedia of Bacteria and Archaea’ (GEBA).
Organisms were selected on the basis of their position in a phylo-
genetic tree of small subunit (SSU) ribosomal RNA, the best sampled
1
DOE Joint Genome Institute, Walnut Creek, California 94598, USA. 2University of California, Davis, Davis, California 95616, USA. 3DSMZ, German Collection of Microorganisms and
Cell Cultures, 38124 Braunschweig, Germany. 4DOE Joint Genome Institute-Los Alamos National Laboratory, Los Alamos, California 87545, USA. 5University of Virginia,
Charlottesville, Virginia 22904, USA. 6Lawrence Livermore National Laboratory, Livermore, California 94550, USA.
gene from across the tree of life7. Working from the root to the tips of
the tree, we identified the most divergent lineages that lacked repre-
sentatives with sequenced genomes (completed or in progress)8 and
for which a species has been formally described9 and a type strain
designated and deposited in a publicly accessible culture collection10.
From hundreds of candidates, 200 type strains were selected both to
obtain broad coverage across Bacteria and Archaea and to perform
in-depth sampling of a single phylum. The Gram-positive bacterial
phylum Actinobacteria was chosen for the latter purpose because of
the availability of many phylogenetically and phenotypically diverse
cultured strains, and because it had the lowest percentage of sequenced
isolates of any phylum (1% versus an average of 2.3%)11. Of the 200
targeted isolates, 159 were designated as ‘high’ priority primarily on the
basis of phylum-level novelty and the ability to obtain microgram quan-
tities of high quality DNA. The genomes of these 159 are being
sequenced, assembled, annotated (including recommended metadata12)
and finished, and relevant data are being released through a dedicated
Integrated Microbial Genomes database portal13 and deposited into
GenBank. Currently, data from 106 genomes (62 of which are finished)
are available.
To assess the ramifications of this tree-based selection of organisms,
we focused our analyses on the first 56 genomes for which the shotgun
phase of sequencing was completed. The 53 bacteria and 3 archaea
(Supplementary Table 1) represent both a broad sampling of bacterial
diversity and a deeper sampling of the phylum Actinobacteria (26
GEBA genomes). An initial question we addressed was whether selec-
tion on the basis of phylogenetic novelty of SSU rRNA genes reliably
identifies genomes that are phylogenetically novel on the basis of other
criteria. This question arises because it is known that single genes, even
SSU rRNA genes, do not perfectly predict genome-wide phylogenetic
patterns14,15. To investigate this, we created a ‘genome tree’ (ref. 16) of
completed bacterial genomes (Fig. 1) and then measured the relative
contribution of the GEBA project using the phylogenetic diversity
metric17. We found that the 53 GEBA bacteria accounted for 2.8–4.4
times more phylogenetic diversity than randomly sampled subsets of
53 non-GEBA bacterial genomes. A similar degree of improvement in
phylogenetic diversity was seen for the more intensively sampled acti-
nobacteria (Table 1). These analyses indicate that although SSU rRNA
genes are not a perfect indicator of organismal evolution, their phylo-
genetic relationships are a sound predictor of phylogenetic novelty
within the universal gene core present in bacterial genomes.

1056
©2009 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 462 | 24/31 December 2009 LETTERS

365
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Acidobacteria Spirochaetes Fusobacteria

Figure 1 | Maximum-likelihood phylogenetic tree of the bacterial domain based on a concatenated alignment of 31 broadly conserved protein-coding
genes16. Phyla are distinguished by colour of the branch and GEBA genomes are indicated in red in the outer circle of species names.

The discovery and characterization of new gene families and their as ecological niche; nevertheless, higher rates of novel protein family
associated novel functions provide one incentive for sequencing discovery were found in the more phylogenetically diverse taxa
additional genomes, analysis of which has helped to redefine the (Fig. 2). In addition, of the 16,797 families identified in the 56
protein family universe18. We explored the quantitative effect of GEBA genomes, 1,768 showed no significant sequence similarity to
tree-based genome selection on the pace of discovery of novel proteins any proteins, indicating the presence of novel functional diversity.
and functions. Specifically, we compared the rate of discovery of These results highlight the utility of tree-based genome selection as
novel protein families when progressively adding more closely related a means to maximize the identification of novel protein families and
genomes versus when adding more distantly related ones (Fig. 2). argues against lateral gene transfer significantly redistributing genetic
Granted, many factors contribute to protein family diversity, such novelty between distantly related lineages.
1057
©2009 Macmillan Publishers Limited. All rights reserved
LETTERS NATURE | Vol 462 | 24/31 December 2009

Table 1 | Effect of SSU rRNA tree-based selection of organisms on compar-
ative genomic metrics
Comparative genomic metric
Genome tree phylogenetic diversity17
Bacteria (domain)
Actinobacteria (phylum)
New protein family links 46 3 6 4 (5) 6.6 to .15.3
Genes in new chromosomal cassettes 71,579 16,579 6 5,523 (20) 3.2–6.5
New gene fusions 433 65 6 31 (20) 4.5–12.7
GEBA genomes were compared to equivalently sized random sets of reference genomes to
quantify the effect of phylogenetic selection.
Novel proteins also can serve to link distantly related homologues
whose relatedness would otherwise go undetected. Forty-six such
links were identified in the 56 GEBA genomes compared to an average
of only three new links in equivalent sets of randomly sampled non-
GEBA genomes (Table 1). A useful complement to homology-based
predictions of gene function are ‘non-homology methods’ (ref. 19)
such as gene context-based inference that relies on the conserved
clustering of functionally related genes across multiple genomes, often
in operons or as gene fusions20. We identified over 70,000 genes in new
chromosomal cassettes of two or more genes in the GEBA genomes.
families, respectively. Halorhabdus utahensis, a halophilic archaeon
known to have b-xylanase and b-xylosidase activities21, has a chromo-
GEBA set Random sets Fold somal cluster including two GH10 family b-xylanases and six novel
(number of resamplings) improvement GH5 family proteins of unknown specificity.
The enrichment of genetic diversity is also seen within families of
non-coding RNAs, transposable elements, and other cellular compo-
11.0 3.2 6 0.7 (100) 2.8–4.4
4.3 1.4 6 0.3 (100) 2.5–3.9 nents. For example, the genome of the marine myxobacterium
Haliangium ochraceum contains 807 CRISPR (clustered regularly
interspaced short palindromic repeats) units including the largest
single CRISPR array known, comprising 382 spacer/repeat units.
CRISPR is a newly recognized, but ancient and widespread, system
in bacteria and archaea that confers resistance to viruses and other
invading foreign DNAs22.
Results from the GEBA pilot project challenge our current under-
standing for the taxonomic distribution of known gene families. The
most striking example of which is the discovery of an actin homo-
logue in H. ochraceum. Actin and its close relatives are structural
components of the eukaryotic cytoskeleton that are found in every
eukaryote and only in eukaryotes. Bacteria and archaea encode
instead the shape-determining protein MreB. Although MreBs have
some functional and structural similarities to eukaryotic actins, they
are regarded, at best, distantly related homologues23 and possibly not

RNase treated
This represents a three- to sixfold increase over equivalent sets of non- a b
GEBA genomes (Table 1). Similarly, the number of new gene fusions

Markers
Peptidase M4
identified in the GEBA genomes is 4 to ,13 times greater than in thermolysin

DNA

RNA
randomly selected genome sets (Table 1). Because the GEBA data
set produced a several-fold improvement over random sets for all
metrics examined (Table 1), we predict that other aspects of Hypothetical protein
sequence-based biological discovery will similarly benefit from tree-
based genome sequencing. Hypothetical protein 1.6 kb
The GEBA genomes also show significant phylogenetic expansions 1.0 kb
within known protein families. For example, although only two of the Hypothetical protein
0.5 kb
56 GEBA organisms are known cellulose degraders, we identified in the
set of genomes a variety of glycoside hydrolase (GH) genes that may BARP
participate in the breakdown of cellulose and hemicelluloses. Among
Phosphatase c
these are 28 and 7 phylogenetically divergent members of the endo- 1 kb
glucanase- and processive exoglucanase-containing GH6 and GH48 Oxidoreductase

70,000 Conserved hypothetical protein

Hypothetical protein
Methyltransferase
(including families with single members)

60,000 Bacteria from GEBA project

(1,060.6 new families/genome) d
1C0F_A D G E D V Q A L V I D N G S G M C K A G F A GD D A P R A V F P S I VG R P R H T G K DS Y V . . .
50,000 BARP S S . . . . P I I I H P G S D T L Q A G L A D E E H P G S I F P N I VG R H K L A G L M E W V D Q R
Protein family number

1C0F_A . . . . G D E A Q S K R G I L T L K Y P I E HG I V T N W D D M E K IW H H T F Y N E LR V A P E E
BARP V L C V G Q E A I D Q S A T V L L R H P V W SG I V G D W E A F A A VL R H T F Y R A LW V A P E E
40,000
Actinobacteria 1C0F_A H P V L L T E A P L . . . N P K A N R E K M TQ I M F E T F N T P A MY V A I Q A V L SL Y A S G R
BARP H P I V V T E S P H V Y R S F Q L R R E Q L TR L L F E T F H A P Q VA V C S E A A M SL Y A C G L
(649.6 new families/genome)
30,000 1C0F_A T T G I V M D S G D G V S H T V P I Y E G Y AL P H A I L R L D L A GR D L T D Y M M KI L T E R G
BARP D T G L V V S L G D F V S Y V A P V H R G A IV D A G L T F L E P D GR S I T E Y L S RL L L E R G
Enterobacteriaceae 1C0F_A Y S F T T T A E R E I V R D I K E K L A Y V AL D F E A E M Q T A A SS S A L E K S Y EL P D G Q V
20,000 (307.7 new families/genome) BARP H V F T S P E A L R L V R D I K E T L C Y V AD D V A K E . . A A R NA D S V E A T Y LL P N G E T

1C0F_A I T I G N E R F R C P E A L F Q P S F L G M ES A G I H E T T Y N S IM K C D V D I R KD L Y G N V
10,000 BARP L V L G N E R F R C P E V L F H P D L L G W ES P G L T D A V C N A IM K C D P S L Q AE L F G N I
Streptococcus agalactiae 1C0F_A V L S G G T T M F P G I A D R M N K E L T A LA P S T M K I K I I A PP E R K Y S V W IG G S I L A
(121.3 new families/genome) BARP V V T G G G S L F P G L S E R L Q R E L E Q RA P A E A P V H L L T RD D R R H L P W KG A A R F A
0
0 10 20 30 40 50 60 70 80 1C0F_A S L S T F Q Q M W I S K E E Y D E S G P S I VH R K C F
BARP R D A Q F A G F A L T R Q A Y E R H G A E L IY Q M . .
Genome number

Figure 2 | Rate of discovery of protein families as a function of
phylogenetic breadth of genomes. For each of four groupings (species,
different strains of Streptococcus agalactiae; family, Enterobacteriaceae;
phylum, Actinobacteria; domain, GEBA bacteria), all proteins from that
group were compared to each other to identify protein families. Then the
total number of protein families was calculated as genomes were
progressively sampled from the group (starting with one genome until all
were sampled). This was done multiple times for each of the four groups
using random starting seeds; the average and standard deviation were then
plotted.
1058
©2009 Macmillan Publishers Limited. All rights reserved
Figure 3 | A bacterial homologue of actin. a, Genomic context of the bacterial
actin-related protein (BARP) gene within the genome of the marine
Deltaproteobacterium H. ochraceum. Red, gene encoding BARP; white, genes
encoding hypothetical proteins; black, genes with functional annotations.
b, RT–PCR demonstration of expression of the gene encoding BARP in
H. ochraceum. c, Ribbon plot of the putative structure of BARP. d, Alignment
of BARP with actin from Dictyostelium discoideum29 with similarities in black
shaded text. Secondary structure elements (arrows, beta-strands; bars, alpha-
helices) are colour-coded as in c. A phylogenetic tree including this protein is in
Supplementary Figure 1.
NATURE | Vol 462 | 24/31 December 2009
even homologous. Like other bacteria, H. ochraceum encodes a bona
fide MreB protein, but in addition, it encodes a protein that is clearly
a member of the actin family, which we have named BARP (bacterial
actin-related protein; Fig. 3). Although we do not yet have evidence
for its precise function, BARP is expressed in H. ochraceum (Fig. 3b).
Assuming that the H. ochraceum mreB orthologue performs the same
function as in other bacteria, and given that the myxobacteria, to
which this species belongs, are known to synthesize actin-targeting
toxins24, we propose that this BARP may be a dominant-negative
inhibitor of eukaryotic actin polymerization. Regardless of its precise
function, this first—and so far only—discovery of an expressed
homologue of eukaryotic actin in a member of the Bacteria highlights
the potential for novel and surprising biological discoveries given a
wider genomic sampling of the tree of life.
We conclude that targeting microorganisms for genome sequencing
solely on the basis of phylogenetic considerations offers significant far-
reaching benefits in diverse areas. Furthermore, the benefits of phylo-
genetically driven genome sequencing show no sign of saturating with
these first 56 genomes. A key question then lies in determining how
much bacterial and archaeal diversity remains to be sampled. Using
SSU rRNA gene sequences as a proxy for organismal diversity (Fig. 4),
we estimate that sequencing the genomes of only 1,520 phylogenetically
selected isolates could encompass half of the phylogenetic diversity
represented by known cultured bacteria and archaea. Given the continu-
ing reductions in both the cost and difficulty in sequencing genomes25,
this is certainly a tractable target in the next few years.
However, the great majority of recognized bacterial and archaeal
diversity is not represented by pure cultures and an additional 9,218
genome sequences from currently uncultured species would be
required to capture 50% of this recognized diversity (Fig. 4). Such
an undertaking will require new approaches to culturing or proces-
sing of multi-species samples using methods such as metagenomics26
or physical isolation of cells from mixed populations followed by
whole genome amplification methods27. Obtaining reference gen-
omes for the uncultured microbial majority will be a natural exten-
sion of the GEBA project, the ultimate goal of which is to provide a
phylogenetically balanced genomic representation of the microbial
1,200
GEBA genomes
Pre-GEBA genomes
Organisms from the greengenes database
1,000 Organisms from the greengenes database
(excluding environmental samples)
Phylogenetic diversity
800
120
100
600
80
60
400
40
20
200
0 16.
0 400
0
0 5,000 10,000 15,000 20,000 25,000 30,000 35,000 40,000
Number of organisms
Figure 4 | Phylogenetic diversity of bacteria and archaea on the basis
of SSU rRNA genes. Using a phylogenetic tree of unique SSU rRNA gene
sequences7, phylogenetic diversity was measured for four subsets of this tree:
organisms with sequenced genomes pre-GEBA (blue), the GEBA organisms
(red), all cultured organisms (dark grey), and all available SSU rRNA genes
(light grey). For each subtree, taxa were sorted by their contribution to the
subtree phylogenetic diversity30 and the cumulative phylogenetic diversity
was plotted from maximal (left) to the least (right). The inset magnifies the
first 1,500 organisms. Comparison of the plots shows the phylogenetic ‘dark
matter’ left to be sampled.
©2009 Macmillan Publishers Limited. All rights reserved
LETTERS
tree of life. The pilot study presented here is a dedicated first step in
this direction.
METHODS SUMMARY
Starting with a phylogenetic tree of SSU rRNA genes7, we identified major
branches that had no available genome sequences but for which cultured isolates
were available in the DSMZ or ATCC culture collections. Selected isolates
(Supplementary Table 1a, b) from these branches were grown and DNA isolated
(Supplementary Table 1c) and quality checked. DNA was then used for shotgun
genome sequencing by Sanger/ABI, Roche/454 and/or Illumina/Solexa technolo-
gies (Supplementary Table 2). Sequence reads were assembled separately with
different assembly methods and the best draft assembly was used for annotation
and as a starting point for genome completion (current genome status is in
Supplementary Table 2). Annotation (gene identification, functional prediction,
etc.) was performed using the IMG system (http://img.jgi.doe.gov/geba); this was
done both after shotgun sequencing and again after genome completion. For ‘whole
genome tree’ analysis, a PHYML maximum likelihood phylogenetic tree of a con-
catenated alignment of 31 marker genes was built using AMPHORA16. Phylogenetic
diversity was calculated as the sum of branch lengths in this and other trees. Protein
families were built for various genome sets by using the Markov clustering algo-
rithm (MCL)28 to group proteins on the basis of ‘all versus all’ blastp searches. For
analysis of phylogenetic diversity of organisms, a phylogenetic tree was built for a
combined alignment of SSU rRNA sequences from published genomes and a non-
redundant subset of greengenes SSU rRNA7. Further analysis of the genomes was
done using IMG database queries and new computational analyses as described in
the main text, legends and Supplementary Methods.
Received 3 June; accepted 30 October 2009.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements . We thank the following people for assistance in aspects of the
project including planning and discussions (R. Stevens, G. Olsen, R. Edwards,
J. Bristow, N. Ward, S. Baker, T. Lowe, J. Tiedje, G. Garrity, A. Darling, S. Giovannoni),
analysis of genomes whose work could not be included in this report (B. Henrissat,
G. Xie, J. Kinney, I. Paulsen, N. Rawlings, M. Huntemann), project management
(M. Miller, M. Fenner, M. McGowen, A. Greiner), sequencing and finishing (K. Ikeda,
M. Chovatia, P. Richardson, T. Glavinadelrio, C. Detter), culture growth, DNA
extraction, and metadata (D. Gleim, E. Brambilla, S. Schneider, M. Schröder,
M. Jando, G. Gehrich-Schröter, C. Wahrenburg, K. Steenblock, S. Welnitz, M. Kopitz,
R. Fähnrich, H. Pomrenke, A. Schütze, M. Rohde, M. Göker), and manuscript editing
(M. Youle). This work was performed under the auspices of the US Department of
Energy’s Office of Science, Biological and Environmental Research Program, and by
the University of California, Lawrence Berkeley National Laboratory under contract
no. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under contract
no. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract no.
DE-AC02-06NA25396. Support for J.A.E., D.W. and M.W. was provided by the
Gordon and Betty Moore Foundation Grant no. 1660 to J.A.E. Support for work at
DSMZ was provided under DFG INST 599/1-1.
1060
©2009 Macmillan Publishers Limited. All rights reserved
NATURE | Vol 462 | 24/31 December 2009
Author Contributions D.W. (rRNA analysis, gene families, actin tree, manuscript
preparation), P.H. (selection of strains, analysis, manuscript preparation, project
coordination), L.G. and D.B. (project management), R.P., B.J.T., E.L., S.G., S.S. (strain
curation and growth), K.M., N.N.I., I.J.A., S.D.H., A.P., A.Ly. (annotation, genome
analysis), V.K. (CRISPRs, actin), M.W. (whole genome tree), P.D., C.K., A.Z. and
M.S. (actin studies), M.N., S.L., J.-F.C., F.C. and E.D. (sequencing), C.H., A.La., M.N.
and A.C. (finishing), P.C. (analysis), E.M.R. (manuscript preparation), N.C.K.
(selection of strains, annotation, analysis), H.-P.K. (strain selection and growth,
DNA preparation, manuscript preparation), J.A.E. (project lead and coordination,
analysis, manuscript preparation).
Author Information Genome sequence and annotation data is available at the JGI
IMG-GEBA page http://img.jgi.doe.gov/geba and has been submitted to GenBank
with accessions ABSZ00000000, ABTA00000000, ABTB00000000,
ABTC00000000, ABTD00000000, CP001618, ABTF00000000,
ABTG00000000, ABTH00000000, ABTI00000000, ABTJ00000000,
ABTK00000000, ABTM00000000, NZ_ABTN00000000,
NZ_ABTO00000000, ABTP00000000, ABTQ00000000,
NZ_ABTR00000000, NZ_ABTS00000000, NZ_ABTT00000000,
NZ_ABTU00000000, NZ_ABTV00000000, NZ_ABTW00000000,
NZ_ABTX00000000, NZ_ABTY00000000, NZ_ABTZ00000000,
NZ_ABUA00000000, NZ_ABUB00000000, NZ_ABUC00000000,
NZ_ABUD00000000., NZ_ABUE00000000, NZ_ABUF00000000,
NZ_ABUG00000000, NZ_ABUH00000000, NZ_ABUI00000000,
NZ_ABUJ00000000, NZ_ABUK00000000, ABUL00000000,
NZ_ABUM00000000, NZ_ABUO00000000, NZ_ABUP00000000,
ABUQ00000000, NZ_ABUR00000000, ABUS00000000,
NZ_ABUT00000000, NZ_ABUU00000000, NZ_ABUV00000000,
NZ_ABUW00000000, NZ_ABUX00000000, NZ_ABUZ00000000,
NZ_ABVA00000000, NZ_ABVB00000000 and NZ_ABVC00000000. All
strains that have been sequenced are available from the DSMZ culture collection
and culture accessions are available in Supplementary Information. This paper is
distributed under the terms of the Creative Commons
Attribution-Non-Commercial-Share Alike licence, and is freely available to all
readers at www.nature.com/nature. Reprints and permissions information is
available at www.nature.com/reprints. Further details on sequencing and genome
properties of each organism are being published in the journal Standards in
Genomic Sciences (SIGS) (http://standardsingenomics.org/). Correspondence
and requests for materials should be addressed to J.A.E. (jaeisen@ucdavis.edu).