REVIEWS

Evolutionary microbial genomics:

insights into bacterial host adaptation
Christina Toft and Siv G. E. Andersson
Abstract | Host-adapted bacteria include mutualists and pathogens of animals, plants and
insects. Their study is therefore important for biotechnology, biodiversity and human health.
The recent rapid expansion in bacterial genome data has provided insights into the adaptive,
diversifying and reductive evolutionary processes that occur during host adaptation. The
results have challenged many pre-existing concepts built from studies of laboratory bacterial
strains. Furthermore, recent studies have revealed genetic changes associated with transitions
from parasitism to mutualism and opened new research avenues to understand the functional
reshaping of bacteria as they adapt to growth in the cytoplasm of a eukaryotic host.

Mutualistic relationship
A symbiosis in which both
species increase their fitness.
Pathogenic relationship
A symbiosis in which one
species increases its fitness
while the fitness of the other
species is adversely affected.
Commensal relationship
A symbiosis in which
one partner increases its
fitness without affecting
the other species.
Department of Molecular
Evolution, Uppsala University,
752 36 Uppsala, Sweden.
e-mails:
christina.toft@ebc.uu.se;
siv.andersson@ebc.uu.se
doi:10.1038/nrg2798
Published online 2 June 2010
Van Leeuwenhoek opened the gates to the microbial
world. Since then, there has been a race to uncover
its secrets because of the importance of understand-
ing microbes to human health. The enormous genetic
diversity of the bacterial world compared with the
animal and plant worlds also makes bacteria excellent
subjects for studies of the principles of genetics and
evolution. Interactions between species have driven
the emergence of complex life forms and many other
major evolutionary changes. Bacteria have contributed
substantially to these events through the establishment
of mutualistic, pathogenic or commensal relationships with
eukaryotic hosts.
The population dynamics of bacteria that repli-
cate inside eukaryotic cells have yielded some of the
smallest 1,2, most stable3,4, most deteriorated5–7, most
highly repeated8 and most highly recombined bacterial
genomes9 sequenced to date. Studying these extreme
examples can provide broad lessons about the evolu-
tionary dynamics in bacteria by revealing ‘hidden’ proc-
esses, such as massive gene amplifications, deletions and
recombination events, that are slightly deleterious
and would therefore be rapidly eliminated during
selection for rapid growth in large free-living bacterial
populations. Identifying the evolutionary dynamics
of intracellular genomes is also important because it
may reveal how mutational alterations in the bacterial
genome influence host phenotypes.
The recent leap in sequencing technology has allowed
the sequencing of numerous species from natural popu-
lations, and progress has been made in deciphering the
associations between genome composition, bacterial
diversity and lifestyle. Our new-found ability to study
a much wider range of species from diverse habitats
has yielded substantial insights into fundamental ques-
tions such as how and why host adaptation alters the
bacterial genome (for a recent review of the genomics
of prokaryotic symbionts of animals see Ref. 10). This
Review focuses specifically on host adaptation in rela-
tion to bacteria that have established an intracellular
relationship. We discuss the emerging picture of phy-
logenetic distributions of host adaptation and possible
factors that influence this, the different stages of host
adaptation and the selective forces that operate at these
stages. Particular focuses are on the transitions between
selfish and cooperative behaviour in host-adaptive
processes and factors that may act to stabilize mutu-
alistic relationships and prevent them from breaking
down into parasitism or autonomy.
Ecological shifts to host-adaptive lifestyles
Bacteria show astonishing diversity in genome content
and metabolic capabilities, which explains their ubiquity
in many, if not all, ecological niches. Making the transi-
tion to an intracellular lifestyle is a dramatic ecological
change, therefore we may ask whether some bacterial
groups are more likely to make such switches and, if so,
why this might happen.
Phylogenetic distributions. Many bacterial species
have been selected for genome projects because of
their importance to agriculture and human health,
thereby providing an extensive collection of genomes
for bacteria associated with invertebrates, plants
and animals. Overall, approximately one-third of all
genomes sequenced to date are from bacteria that live

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REVIEWS
Endosymbiont
An intracellular organism that
contributes to the survival of
the host cell and depends
on the host for its own
persistence. The relationship
can be either mutualistic or
commensalistic.
Protozoan
A single-celled (usually
microscopic) eukaryotic
organism. The name originates
from the Greek words ‘proton’
and ‘zoa’, meaning first and
animals, respectively.
Horizontal gene transfer
The transfer of genetic
material between the
genomes of two organisms
not through the normal
parent–progeny transmission
during cell division.
466 | juLy 2010 | VOLuMe 11
in association with eukaryotes. By mapping the host-
associated bacteria onto a phylogeny that represents the
variability spectrum of sequenced genomes it becomes
noticeable that these bacteria have a broad phylogenetic
distribution (fIG. 1). Such diversity is bidirectional, as
hosts for these bacteria belong to orders that span the
tree of life from protists to plants and animals10,11.
However, the success and abundance of host-adapted
bacteria differs among phylogenetic groups. For exam-
ple, only a few phylogenetic clades — predominantly the
α-, β-, and γ-proteobacteria, with the notable exceptions
of Chlamydiae — contain species that have evolved to
the intracellular stage. Bacterial species that have obligate
mutualistic relationships with their hosts are even more
biased in their phylogenetic distribution pattern and are
almost exclusively found among α- and γ-proteobacteria
(fIG. 1). This overrepresentation might be an effect of
sampling bias owing to the greater incentive to sequence
endosymbionts that are of immediate concern to
human society than those with other host interactions.
It is possible, however, that certain bacterial groups
are predisposed to evolve intimate interactions with
eukaryotic host cells. Many species in the α- and
γ-proteobacteria classes are abundant in soils, lakes and
oceans, and they have large and dynamic genomes with
access to a big pool of mobile genes present in other
bacteria in these environments12,13. In some clades of
the α-proteobacteria, genes for symbiotic and environ-
mental interactions are located on plasmids that can be
several megabases in size14, which can facilitate rapid
adaptive responses and the transfer of host-association
genes in single events. Therefore, both ecological factors,
such as abundance and physical contacts with eukaryo-
tic hosts, and genomic factors, such as ease of adap-
tation, might have contributed to the many successful
interactions between Proteobacteria and eukaryotes.
Influence of the host. It is also important to consider
the influence of differences among hosts on bacterial
adaptation. For example, host dietary restriction is an
important selective force that drives the evolution of
mutualism. Indeed, mutualistic relationships in which
the bacteria supply compounds that the host cannot
get from its food source have mostly been identified in
invertebrates15. In vertebrates, the extracellular commu-
nal gut flora is also known to have a dietary role16. an
extreme case of this is the giant panda, the bamboo diet
of which seems to be directed more by its gut bacteria
than by its genetic composition17. Thus, the rarity or lack
of obligate mutualistic bacteria associated with verte-
brates seems not to be because vertebrates have no need
for symbiotic relationships, but because members of the
gut flora do not develop an obligate mutualistic lifestyle.
Likewise, soil bacteria provide beneficial functions such
as nitrogen fixation to plants, but to our knowledge there
are no obligate intracellular mutualists of plants. This
suggests that there might be other barriers to obligate
mutualism that are difficult to overcome in plants and
animals, such as the cell wall of plants, adaptive immu-
nity in animals and difficulties in infecting the germ line
in these organisms.
© 2010 Macmillan Publishers Limited. All rights reserved
Protozoa contribute to bacterial adaptation. Many
intracellular pathogens can survive in amoeba18 and it
has been suggested that protozoa act as a ‘nursery’ from
which pathogens of higher eukaryotes emerge19,20. For
example, it has been suggested that Rickettsia spp. and
Chlamydia spp. used protozoa as a first stage in their
adaptation to the intracellular lifecycle21. Strategies to
avoid predation by free-living protists in the environ-
ment may result in the evolution of mechanisms to avoid
resistance to macrophage destruction and the ability to
replicate intracellularly, as suggested for mycobacteria22.
Furthermore, horizontal gene transfer (HGT) of genes from
co-infecting bacteria can enhance the infection potential
of the emerging pathogen21,23. Obligate intracellular rela-
tionships with free-living amoeba have been observed in
α- and β-proteobacteria24–27 as well as among species in
Bacteroidetes28 and Chlamydiae29,30. The described cases
are probably just the tip of the iceberg of the true diversity
of such relationships in nature. an interesting topic for
future studies will be to search for intracellular parasit-
ism and mutualism of microbial eukaryotes in a system-
atic manner. It also remains to be seen whether a similar
diversity of relationships is found in other phyla.
Evolutionary stages in host-adaptation processes
By comparing the genomes of phylogenetically distinct
intracellular bacteria (fIG. 1), we can learn how the chal-
lenges presented by the environment of a hostile eukary-
ote have been solved. Terms such as ‘host adaptation’ and
‘intracellularity’ are in essence rather broad because they
encompass bacteria that have the potential for intracel-
lular survival as well as bacteria that have multiplied in
the host cell environment for hundreds of millions of
years18. fIGURe 2 shows a schematic overview of the dif-
ferent stages of the integration process and the genomic
changes that occur at each stage, and below we discuss
the selective forces that drive these processes.
Figure 1 | Global phylogeny of host-associated ▶
bacteria. A modified version of the maximum likelihood
phylogenetic tree that was obtained in a previous study
using 350 bacterial species, the genomes of which have
been sequenced128. In that study, the authors selected 350
species to show maximum phylogenetic coverage and
used a concatenated data set derived from 31 highly
conserved genes for their phylogenetic inference128.
Coloured branches label the different phyla and taxa
are coloured according to the nature of their association
with the host (see Supplementary information S1 (figure)
for taxa names). These classifications should only be seen
as a guide and not as the only association possible for the
bacterium. The stage of intracellular association (described
further in fIG. 2) is also colour coded and representative
examples are shown in the outermost layer of the
phylogeny (see Supplementary information S2 (table) for
further details). Rare cases of obligate mutualistic
associations in the α-proteobacterial genera, which
otherwise shows commensal or pathogenic relationships
with hosts, are indicated (*). Species names that are under
the category Candidatus are indicated (**). Image is
modified, with permission, from Nature Ref. 128 © (2009)
Macmillan Publishers Ltd. All rights reserved.
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 REVIEWS

Wigglesworthia glossinidia
Baumannia cicadellinicola

s asymbiotica
Intracellularity

fensa**
Facultative

Blochmannia spp.**

ius

ii **
ila
**
Obligate

Sodalis glossinid

Fran lla spp eumoph

lla ruddii

ica kutan
Hamitonella de
Buchnera spp.

us
Obligate mutualistic

Photohabdu

ari
Coxie nella pn i
r

o
Legio lla flexne

alle ecess
ma socius
Rut icomy spp.
Carsone

n
g n if
Ves isella

eri cter

i
o
Host interactions

Shige

am
a
c
Pathogenic

rkh cleob
h ia
Mutualistic

old
ynu
Commensal m
eu

Pol

la
in

ico
Bu

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ia spp
c
zu

ad
kin la
m
riu

dg nel
γ-proteobacteria te

Ho rto
c
ba

Ba
β-proteobacteria ylo
en
α-proteobacteria Ph
δ-proteobacteria p.
sp
s ma pp. p.*
Acidobacteria la s .
ap ia a sp spp i
ε-proteobacteria An rlich achi ttsia .* mush
Eh olb icke ssp uga
r
W eo ttsi sut a s
Aquificae ris
N icke tia t llula
R rien trace
Bacteroidetes O ia i
n
son
Chlorobi Law
Chlamydiae and Verrucomicrobia γ
Planctomycetes
β
Spirochaetes
Actinobacteria
Cyanobacteria α
Chloroflexi
Firmicutes
Tenericutes spp.
Blattabacterium
Fusobacteria Sulcia muelleri**
Synergistetes
Thermotogae Amoebophilus asiaticus**
Deinococcus and Thermus
Chlamydia spp.
Chlamyd
Protoch ophila spp.
lamydia
amoebo
phila**

Myc
oba
cter
ium
lepra
e

Ren
iba
cte
riu
m
sal
mo
nin
Tr aru
op m
he
ry
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aw
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i
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eria
.
sma spp
s
a penetran

mo
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Phytopla

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0.2
gen
Mycoplasm

es

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REVIEWS

1 Free-living and 2 Facultative intracellular 3 Obligate intracellular 4 Obligate intracellular 5 Organelle

extracellular (early stage) (advanced stage) mutualist
(extreme stage)

Plasmid Genomic island Recombination Functional divergence Gene loss

Genome
Phage Rearrangement Duplication Non-functionalization Transfer to host genome

Figure 2 | stages of host adaptation. The genome dynamics for different host-adaptation stages: free-living (1),
facultative intracellular (2), obligate intracellular (3), obligate intracellular mutualist (4) and organelle (5).
Arrows that point directly to the genomes indicate the acquisition of genes by horizontal gene Nature Reviews
transfer | Genetics
(through
plasmids, genomic islands and/or bacteriophages). Arrows that loop back to the genome indicate changes within
the genome (rearrangements, gene duplication, recombination, functional divergence (shifts) and non-functionalization).
Arrows that point away from the genome indicate gene loss or gene transfer to the host genome. The relative
influence of each of these types of events at the different intracellular stages is shown by the weight of the arrow.
See text for more details.

Pseudogenization
The process whereby changes
in the coding region of a gene
that disrupt the function of the
gene lead to its inactivation.
Endosymbiotic bacterium
A non-pathogenic bacterium
that lives inside host cells.
Free-living to intracellular lifestyle. The early stage
of intracellularity (fIG. 2; step 2) is exemplified by fac-
ultative intracellular bacteria such as Legionella spp.,
Francisella spp. and Bartonella spp., which grow in the
cytoplasm of their natural hosts but have retained the
ability to grow in a free-living mode31–33. This stage
is characterized by dramatic changes in genes cod-
ing for outer surface structures, including horizontal
acquisition of new genes and rapid modification of
these by duplication and recombination events. at the
advanced stage of host interaction (fIG. 2; step 3) ,
the ability to grow on artificial media is lost, as exem-
plified by the obligate intracellular pathogens Rickettsia
spp.34, Chlamydia spp.35,36, and Coxiella spp.37. This stage
often involves specialization to a restricted set of host
species and is characterized by extensive pseudogenization
and gene loss.
Intracellular lifestyle to mutualistic endosymbiosis.
Once a strong interaction and an intracellular lifestyle
with a particular host are secured, the loss or modifi-
cation of bacterial genes and metabolic reactions can
enable the transition to an obligatory mutualistic life-
style (fIG. 2; step 4). Such relationships impose dramatic
changes in both the host and the bacterium10. For
example, the ontogeny of an invertebrate host might be
reprogrammed to produce cells to house endosymbiotic
bacteria . at this extreme stage of host adaptation,
the endosymbiont genome starts co-evolving with the
host nuclear genome under selection to benefit
the host. The best-studied example is the large group
of obligate mutualists in the γ-proteobacteria that
includes Buchnera spp., Wigglesworthia glossinidia and
Blochmannia spp. (fIG. 1), which are estimated to have
diverged from their free-living relatives several hundred
million years ago38–40. Bacteria that have reached this
level of integration with their hosts are characterized
by extreme reductive genome evolution to minimize
genetic (see Supplementary information S2 (table)) and
hence metabolic redundancy. In the final stages, exem-
plified by organelles, endosymbiont genes are either
transferred to the host nuclear genome or replaced by
functions encoded by host nuclear genes (fIG. 2; step 5).
Selective forces in host-adapted bacteria
until recently, concepts in bacterial genetics were mostly
based on studies of free-living species that are easy to
culture, maintain and manipulate in the laboratory, such
as Escherichia coli and Bacillus subtilis, which have been
selected for maximum growth rates and maximum effi-
ciency of protein synthesis41. The main focus in experi-
mental evolutionary studies in the laboratory has been
on the mechanisms and selective forces driving the
fixation of adaptive mutations and the purification of
deleterious changes under these growth regimes42–46.
In nature, however, translational power is highly
variable among bacteria and depends on the availability
of nutrients and the ecological strategy 47. In particular,
the intracellular milieu is very different from the growth
regime in the laboratory. Studies in natural populations
are now challenging the concepts based on laboratory
strains. Below, we discuss selective forces that drive
adaptive processes and how these have shaped the
bacterial genome.

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a γ-proteobacteria b All groups
CV = 0.46
Spearman correlation: Spearman correlation:
6 ρ = –0.764; P < 1.4 × 10–7 6 ρ = –0.760; P < 2.2 × 10–16
CV = 0.39
5 5
4 4
Genome size (Mb)
Genome size (Mb)
3 3 CV = 0.33
CV = 0.25
2 2
CV = 0.15
1 1
0 0
Early Advanced Extreme Early Advanced
Intracellular stage Intracellular stage
Figure 3 | Genome size in bacteria correlates with the intracellular stage.
Nature Reviews | Genetics
Genome size variations for different host-adaptation stages: facultative intracellular
(early), obligate intracellular (advanced) and obligate intracellular mutualist (extreme).
a | Non-parametric correlation analyses indicate that genome size is significantly
anti-correlated to the intracellular stage in γ-proteobacteria. b | The same trend of
correlation between genome size and intracellular stage is seen if all bacterial groups
of the phylogeny are included in the analysis. The coefficient of variation (CV = sd/mean)
shows a clear declining trend as the relationship becomes more intimate, indicating a
hypothetical minimum genome size for these bacteria. However, caution should be
exercised when estimating the CV in γ-proteobacteria because six out of ten of the
sequenced genomes are of the same species (Buchnera aphidicola). Further sequencing
efforts may confirm the observation made here. The raw data for this small analysis can
be found in Supplementary information S2 (table).
Population dynamics in the intracellular environment.
Intracellular bacteria evolve under selection for host-
interaction processes and they show very different pop-
ulation dynamics compared to free-living bacteria48,49.
Importantly, the effective population size is dramati-
cally reduced because it is constrained by the number of
hosts, the number of infected cells and the cellular space
available for growth. This makes selection less efficient
in countering the accumulation of slightly deleterious
mutations. Furthermore, the transmission dynam-
ics among hosts may introduce strong bottlenecks on
the effective population size and therefore reduce the
genetic variability in the next generation compared
Bacteriocyte with the original (ancestral) bacterial population. This
A specialised host cell
that houses obligate
can contribute to the loss of favoured alleles and lead
mutualistic bacteria. to increased divergence among populations50. There
is also less opportunity for innovation as HGT occurs
Conjugation less frequently than in free-living bacteria51. Finally, the
The physical joining of two
metabolite-rich intracellular environment relaxes
bacterial cells to transfer
genetic material from the the selective constraints on metabolic genes, which
donor cell to the recipient leads to the accumulation of mutations, gradual loss of
cell through a pilus. function in these genes and gene loss4.
For all of these reasons, a decline of genome size is
Coefficient of variation
A normalized measure
expected. Indeed, the intracellular environment is the
of dispersion of a main factor that correlates with genome size in bacteria.
probability distribution. In γ-proteobacteria, there is strong correlation between
naTuRe ReVIeWS | Genetics
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genome size and the different stages of host adaptation
(fIG. 3a) and this is also seen when intracellular bacte-
ria from different clades are analysed together (fIG. 3b).
although it has been known for some time that the
stronger the level of host dependency the smaller
the genome, our comparison shows that the genome
size variability also decreases. a high fixation rate for
nucleotide substitutions is another characteristic feature
of intracellular bacterial populations, as underscored by
exceptionally long branches leading to these lineages
(fIG. 1). Functional divergence of the molecular chap-
eronin GroeL (also known as Cpn60) is seen for sev-
eral obligate intracellular lineages, such as Chlamydiae,
anaplasmataceae and Mycobacteria, and this may
CV = 0.27 reflect an adaptation to the higher mutational load of the
intracellular lifestyle52.
Selection for microdiversity in surface structures. For
Extreme the intracellular lifestyle, bacteria must overcome prob-
lems such as attaching to host cells, entering the cyto-
plasm, multiplying, exiting and being transmitted to new
host individuals without being recognized by the host
immune system. acquiring these ‘skills’ may involve the
acquisition of novel genes for host-interaction processes
by HGT (reviewed in Ref. 43) or changes in the func-
tion of existing genes53. extreme sequence divergence
among closely related species is often seen in proteins
that directly mediate the interactions with the host envi-
ronment, such as secretion systems, lipopolysaccharides
and other outer surface structures (for a summary of
different selection models, see BOX 1).
Horizontal gene transfer and functional divergence.
Symbiotic and pathogenic bacteria express and secrete
a wide range of proteins to enable them to interact with
their hosts. The best-characterized secretion systems,
both functionally and structurally, are the type III and
type IV secretion systems (T3SS and T4SS, respectively),
which secrete effector molecules into the host cell (for
recent reviews see Refs 54,55) (BOX 2). Despite their
ubiquity and importance as potential drug targets, we
still know little about the molecular evolutionary forces
and mechanisms that shape these systems in response
to the interplay with the host. Phylogenetic studies of
the T3SSs and T4SSs have revealed frequent exchanges
by HGT56,57, often across rather distantly related phyla.
Functional divergence is also widespread and is linked
to lifestyle shifts58. For example, the T3SS in endosym-
biotic bacteria of insects has been remodelled by gene
loss59 and functional divergence60, possibly to mediate
the invasion of host bacteriocytes, ovaries and embryos,
and thereby to ensure transmission to host offspring 10.
Similarly, HGT of genes for the T4SSs into intracellular
species have been associated with functional shifts from
bacterial conjugation to host-cell binding 57.
The horizontally acquired genes are incorporated into
existing gene networks in intriguing ways. For example,
in Bartonella henselae a vertically inherited regulatory
system that senses changes in pH in free-living bacteria
is used to control the expression of horizontally acquired
target genes such as a T4SS61. This results in upregulation
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Box 1 | Measuring and modelling selection in host-adaptive bacteria

There are two general models to explain the co-evolutionary processes taking place between bacteria and their hosts.
The trench warfare model96, also called the balanced polymorphism model, predicts constant diversity in the population
owing to selective pressures to maintain polymorphisms, for example to evade the host immune system. The
co-evolutionary arms race model97, also called the red queen model98, suggests that there is back-and-forth evolution
between the bacterial protein and the targeted eukaryotic molecules. Positive selection on favourable alleles yields
genetic variation over time, but reduces polymorphisms in the population. For example, the effector proteins associated
with the type III secretion system (T3SS) in Pseudomonas spp. have been suggested to evolve according to this model.
Mechanistically, chimeric genes for these effector proteins are created through random genetic fusions of gene
segments in a process called terminal reassortment99. Under both models, polymorphisms should be considered adaptive.
Identification of adaptive processes in bacteria and their hosts may provide information that is important to
understanding their interaction dynamics. One way to identify these adaptive processes at the molecular level is through
the estimation of the ratio between the numbers of amino acid-replacing codon changes (non-synonymous changes; N)
and synonymous changes (S). The ratio between these rates (ω = dN/dS) is the most common way to measure selection in
protein-coding genes. Values of ω = 1, ω <1 and ω > 1 indicate neutral evolution, purifying and positive selection,
respectively100,101. However, several exceptions undermine ω as a valid measure of selection. For instance, synonymous
sites evolve under purifying selection in free-living bacterial populations to match the use of tRNA isoacceptors or under
positive selection in RNA viruses, where secondary structure is essential for viral stability. The ω values can also be
misleading in bacteria with substantial recombination rates, such as Neisseria spp. or Wolbachia spp., where these values
do not necessarily reflect actual nucleotide substitution. Furthermore, obligate intracellular bacteria tend to have strong
nucleotide composition biases (generally AT rich) and small effective population sizes. This leads to relaxed constraints
on protein sequences, yielding increased fixation rates of slightly deleterious mutations and therefore inflated ω values.
These values can be mistakenly interpreted as evidence of positive selection because patterns of ω values produced by
genetic drift are, under some circumstances, indistinguishable from those yielded by adaptive processes.
Functional divergence in some lineages in a particular protein region is another way to identify adaptive processes.
Many methods have been developed to either identify functional divergence of a single protein102–104 or to explore
complete genomes for such evidence52,58. Identification of genes evolving under positive selection during host
adaptation would therefore require exhaustive testing using various methods. Knowledge of the biochemical and
metabolic interactions between the bacterium and the host, and the population parameters underlying these
interactions, will be essential to understand the adaptive processes involved.

Genomic island
A region of the genome that
has been acquired through
a horizontal event.
Bacteriophage
A virus that infects bacteria
and can serve as a vector
of novel genetic material.
Prophage
The genome of a
bacteriophage when
it is integrated into
the chromosome of the
host bacterium.
Secondary symbiont
A facultative bacterial
symbiont that is not
essential for host survival
and reproduction.
tRNA isoacceptor
One of a group of tRNA
species that can bind to
different codons for the
same amino acid residue.
of the T4SS and the associated secreted proteins owing
to altered pH of the cytoplasm following infection of the
mammalian host cell.
Duplication and diversification. Surface proteins that
are involved in host–pathogen interactions often show
greater diversity in both sequence and patterns of pres-
ence or absence among closely related strains than
sequences that evolve in a neutral manner. These proc-
esses may be speeded up by recombination between
duplicated genes in and among genomes. a particu-
larly illustrative example is provided by a tandemly
duplicated array of three genes in a gene cluster for a
conjugative T4SS, which mediates binding to eryth-
rocytes in Bartonella spp.62. Two of the genes encode
proteins located on the cytoplasmic side of the mem-
brane and these evolve by purifying selection. By con-
trast, one of the genes in the duplicated array encodes
a surface-exposed protein and this gene was found to
be highly variable across strains63. It was postulated
that selection drives sequence divergence to continu-
ally generate new variants of the pilus proteins and
thereby match a divergent set of target molecules on the
erythrocyte surfaces.
The role of mobile elements. Genes important for host
adaptation, such as those encoding secretion systems, tend
to be located in genomic islands or near to mobile elements
such as plasmids and bacteriophages64. Consistent with a
high degree of mobility and variability during the emer-
gence of host-adaptive strategies, facultative intracellular
bacteria contain four or five times more mobile Dna
than obligate intracellular bacteria51. Bacteriophages are
one of the most effective ways to transmit foreign Dna
between species and have great potential for genome
diversification and the creation of new host interaction
systems. For instance, genes located in some prophages of
secondary symbionts may be involved in controlling host
reproduction and defending the host against eukaryotic
parasites (reviewed in Ref. 65).
In Bartonella spp. there is a region several hundred
kilobases in length that contains duplicated gene clus-
ters for the T3SS, T4SS and T5SS, which is amplified
and packaged into bacteriophage particles66. The ampli-
fication process generates single-stranded Dna ends,
thereby facilitating recombination between the ampli-
fied sequences and the duplicated genes in the genome.
The genes for a gene transfer agent that packages the
Dna and the genomic sequence spanning the putative
phage replication initiation site from which the ampli-
fication process is started are highly conserved among
all Bartonella genomes sequenced to date66. This sug-
gests that the amplification mechanism is maintained by
selection that favours the generation of variability, irre-
spective of the specific interactions provided by each of
the encoded secretion systems.
The maternally inherited Wolbachia species that
use insects as hosts (discussed further below) have the
genomic feature of 30–50 genes that encode eukaryotic-
like ankyrin repeat proteins, some of which are presum-
ably secreted into the host cell9,67,68. Several of these genes
are located in the immediate vicinity to prophages. a

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Rhizobium more extreme case is the genome of the scrub typhus
One of a group of pathogen, Orientia tsutsugamushi (a maternally inher-
nitrogen-fixing bacteria that ited bacterium of mites that infects rodents and other
have symbiotic associations mammals), which contains more than 20 repeated gene
with plants.
clusters for putative effector proteins. These include
ankyrin repeat and tetratrico peptide repeat proteins
that are located immediately downstream of genes for
the conjugative T4SS8. Thus, in both organisms there
is a linkage between mobile elements and genes for
eukaryotic-like proteins that may be secreted into the
cytoplasm to manipulate the molecular processes in
the host cell. The use of two different types of mobile
elements — plasmids and bacteriophages — for the
transfer processes suggests that these associations have
arisen independently.
The physical linkage between mobile elements and
genes for secreted proteins was also seen in a study of 21
genomes of free-living bacteria69. It was suggested that
such linkage might help resolve the problem of how to
avoid the emergence of ‘cheaters’ in the bacterial popu-
lation, which benefit from the effects of the secreted
proteins on the host cell but do not participate in their
production. The authors of this study suggest that mobile
elements are prevalent because they carry social traits and
are thereby generators of social networks among bacterial
cells. The invasion of mobile elements into cheaters that
have lost their genes for the secreted proteins enforces
cooperation through co-transfer of the cooperative
genes69. Genes for mobile elements and secretion sys-
tems are thereby inextricably linked to ecological
innovation and evolution of host-adaptive strategies in
these species.
Box 2 | Virulence and host-adaptability features
There are numerous mechanisms that pathogenic and symbiotic bacteria use to
interact with their host. The mechanisms include complexes that inject proteins
(effectors) into the host cell cytoplasm and excretion of exotoxins to the extracellular
medium. There are many differences between toxins and effectors. For example, toxins
are delivered exogenously and can act directly on the host cell, whereas effectors
require protein complexes for delivery, and toxins inhibit cellular functions, whereas
effectors can produce a coordinated modulation of host cell molecules.
Pore-forming toxins form the largest class of the bacterial toxin proteins105.
These secreted proteins are water soluble and capable of binding to the surface of the
host cell. After binding, they multimerize and insert into the amphiphatic host cell
membrane, forming a pore106. The bacterial type III and type IV secretion systems (T3SS
and T4SS, respectively) are membrane-spanning complexes through which effector
molecules are secreted107,108. These structures each contain over 20 different proteins
and consist of a basal body, which spans the membrane of the bacterial cell wall, and
a long hollow tube called the pilus (reviewed in Refs. 54 and 55). The membrane-
spanning part of the T3SSs shows an ancestral relationship with the flagellum109,
which is used for motility in bacteria. The T4SSs consist of membrane-spanning
protein complexes with diverse functions such as conjugation, effector translocation
and DNA uptake110.
Effectors can be broadly classified onto two main categories. One includes effectors
that mimic host endogenous cellular proteins111 and the other comprises effectors that
covalently modify host cell proteins112. Ankyrin B of Legionella pneumophila provides an
example of the mimicking activity of bacterial effectors: it reproduces molecular and
functional activities of eukaryotic F-box proteins, thereby exploiting the conserved
polyubiquitylation machinery for intracellular proliferation113. Conversely,
some modifying effectors, such as Vibrio outer protein S (VopS) from the Vibrio
parahaemolyticus T3SS, inhibit Rho guanosine triphosphatases (GTPases), which leads
to cell rounding and the collapse of the actin cytoskeleton114.
naTuRe ReVIeWS | Genetics
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Influence of host population dynamics. For genera with
several different hosts there seems to be a correlation
between host range and population size and the reper-
toire of bacterial outer surface proteins. For example,
human pathogen specialists in the genera Rickettsia and
Bartonella tend to have fewer and less variable surface
proteins than those that infect larger populations such
as rodents. In principle, as there is a mutational bias
for deletions in bacterial genomes70, there will be pseu-
dogenization and gene loss unless there is selection for
high copy numbers. The expansion of genes for surface
proteins in bacteria that are adapted to rodents suggests
that the amplification processes are driven by selection
in these hosts. This might explain the ease with which
human pathogens emerge from bacteria that are natu-
rally adapted to other reservoirs; once a bacterium has
acquired the necessary surface components for inva-
sion in one mammalian host, the infection may spread
to new host species simply by selection for further
sequence changes in the duplicated genes.
Transitions between parasitism and mutualism
Mutualistic bacterial symbionts are widespread in
nature and such relationships can evolve quickly
in the laboratory, as exemplified by the co-evolution of
two taxonomically and functionally unrelated organ-
isms, Desulphovibrio vulgaris and Methanococcus
maripaludis 71. However, evolutionary theory predicts
that such interactions should be unstable owing to con-
flict of interest between the interacting partners72,73.
Mutualism is thus expected in most cases to break
down into parasitic relationships or revert to auton-
omy. Phylogenetic studies have shown that shifts from
mutualism to parasitism occur only rarely, but that
many cooperative biological systems have reverted to
autonomy in the evolutionary past 74. In this section,
we discuss genomic changes associated with transitions
from parasitic to mutualistic relationships and suggest
that extreme genome reduction and increased host
dependence may prevent the breakdown and reversal of
such relationships.
Requirements for parasitic or symbiotic relationships.
The development of an intracellular mutualistic rela-
tionship is dependent both on genes required for the
establishment of the intracellular lifestyle and symbiosis
genes directly involved in the symbiotic relationship.
Symbiosis genes, in Rhizobia for example, are typically
carried on large plasmids that can spread by HGT.
However, for these transfers to be productive, the appro-
priate genetic background and ecological context have
to exist and/or evolve in parallel thereafter. This was
elegantly shown by the conversion of the root-infecting
pathogen Ralstonia solanacearum to an intracellular
symbiont of plant nodules by a two-step process75. The
inactivation of a master regulator enabled the intracel-
lular infection of nodule cells, whereas mutations in a
gene for a T3SS caused nodulation and early infection75.
Furthermore, phylogenetic studies of nodulating
Bradyrhizobium strains and transmissible symbiosis
islands revealed recurrent losses of nodulation ability 76,
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Virulence
The ability to cause disease by
breaking down the protective
mechanisms of the host.
Primary endosymbiont
An obligate mutualistic
endosymbiont that is
essential for host survival
and reproduction. Primary
endosymbionts are often
located in bacteriocytes and
are maternally transmitted
between host generations.
Psyllids
small sap-sucking insects, also
known as ‘jumping plant lice’.
472 | juLy 2010 | VOLuMe 11
suggesting that reversions to autonomy occur frequently
in symbiotic systems that include free-living bacteria.
Similarly, transitions between parasitic and commensal
relationships are common during the early stages of host
adaptation. Despite the many examples of such transitions,
it has proved surprisingly difficult to identify virulence
determinants simply by comparing the genomes of strains
that have different effects on their hosts. Outer surface pro-
teins and secretion systems, once referred to as ‘virulence
factors’, are also in bacteria that have a non-pathogenic
relationship with their hosts (reviewed in Ref. 64).
Other factors, such as the immune status of the host
and regulatory systems that turn on the right genes at
the right times and places, may be as important for the
outcome of the infection as the gene complement of
the infectious agent. Importantly, infections in a new
host, where such interactions have not yet been fine-
tuned by selection, may shift the balance from a chronic
infection to an acute and highly virulent infection.
Maternal transmission. The next switch — evolution
from a pathogenic or commensal relationship to obli-
gate mutualism, in which both partners become depend-
ent on each other for survival — requires a stable and
effective transmission route among host generations.
Maternal inheritance is the mechanism most frequently
used in invertebrate hosts to ensure transmission of the
endosymbiont along with the host genomes to the next
generation. Once such a transmission route is established
in a bacterial host population, selection for functions
that benefit the host could lead to obligate mutualism.
a genus that uses various different strategies to
ensure its spread by maternal transmission is Wolbachia.
These bacteria may induce reproductive disorders in the
arthropod host populations by male killing, feminization,
parthenogenesis and cytoplasmic incompatibility 77,78.
Mutualistic relationships have been described for
Wolbachia spp. and nematodes77, in which antibiotic
treatments result in fitness decline, reduced growth rates,
delayed moulting and even death of the nematode79,80.
The genome of the Wolbachia pipientis strain wBm,
which is a mutualist of nematodes, is much smaller and
contains fewer repeats than the genomes of Wolbachia
strains that cause reproductive disorders in arthropods81.
This finding is consistent with genome reduction during
long-term mutualistic associations. More recently evolved
mutualistic relationships between Wolbachia spp. and
insects have also been discovered: it was found that an
obligate bacteriocyte-associated strain of Wolbachia pro-
vides vitamin B to its host, the common bedbug Cimex
lectularius 82. This shows that mutualistic interactions
may arise in maternally inherited lineages82.
Other examples of switches between effects on
host phenotypes are provided by the genus Rickettsia,
which comprises pathogens of vertebrates83, reproduc-
tive manipulators of arthropod hosts and bacteriocyte-
associated mutualists84. Mobile elements and genes
for outer surface structures that were acquired at the
base of the Rickettsia lineage are deteriorating and this
process is particularly rapid in the pathogenic strains5.
Rickettsia and Wolbachia are sister genera and it is
© 2010 Macmillan Publishers Limited. All rights reserved
therefore tempting to speculate that maternal transmis-
sion in arthropods was the ancestral trait, and that the
pathogenic Rickettsia species have exchanged mater-
nal inheritance for mammals that serve as the new
‘vectors’ of transmission between arthropod hosts85. If
so, there might be many maternally inherited bacteria
hidden in invertebrates that could potentially become
disease agents.
The evolutionary transition between mutualists and
organelles. Contrary to the assumed instability of
mutualistic relationships, obligate primary endosymbionts
of animals provide the proof of principle that such
relationships can be stably maintained for exten-
sive time periods10. What is the key to stability of a
cooperative biological system? Genome size minimiza-
tion is an efficient way to prevent the endosymbionts
from reverting to autonomy or changing partners, sug-
gesting that the small genome sizes may not only be an
effect of the population dynamics of intracellular bacte-
ria but are perhaps also driven by selection at the host
level. Likewise, the lack of mobile elements in obligate
mutualistic bacteria adapted to a single host species
prevents the acquisition of novel genes and counters
diversification processes51,65.
The host, however, has the option of evolving new
cooperative interactions to complement the deteriorating
endosymbiont genome. The highly reduced genome of
the Buchnera aphidicola strain BCc — an endosymbiont
of the aphid Cinara cedri — is only 420 kb and so illus-
trates genome deterioration86. This endosymbiont has
only retained a few genes located on plasmids for tryp-
tophan biosynthesis (trpE and trpG) and seems to have
lost its role as a supplier of tryptophan. Interestingly, the
‘missing’ genes (trpD, trpC, trpB and trpA) were found
in the genome of a secondary symbiont, ‘Candidatus
Serratia symbiotica’ strain SCc87. although the tiny
genome of BCc seems to be undergoing degeneration
towards extinction, its support from SCc has established
a new selection constraint that will temporarily stabilize
the triad. Interestingly, it was noted that the proteome of
BCc has a greater robustness to misfolding errors com-
pared to its sister Buchnera lineages, which have slightly
larger genomes88.
an even smaller genome is that of the endosymbiont
of psyllids, ‘Candidatus Carsonella rudii’ (Ref. 2). analysis
of its 160 kb genome has sparked speculation about the
existence of other bacteria that provide some of its seem-
ingly missing functions89. The smallest bacterial genome
sequenced to date, that of ‘Candidatus Hodgkinia’, is only
140 kb and contains no more than 169 protein-coding
genes1. This genome shares several features with mito-
chondrial genomes, such as the small size and reassign-
ment of the stop codon uGa to tryptophan owing to loss
of genes for ribosome release factors.
Other possible ways to ‘rescue’ functions that are
lost from deteriorating genomes, and thereby stabilize
a mutualistic relationship, would be to transfer endo-
symbiont genes to the nuclear genome of the host or to
evolve such functions de novo in the nuclear genome.
For example, recent findings reveal HGTs from the
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 REVIEWS

Box 3 | Applications of new technologies to studies of host-associated bacteria

Recent technological developments in genomics and synthetic biology will be paramount to further our understanding
of host-adapted bacteria.
next-generation sequencing
The whole-genome DNA amplification method has been an important development for studies of host-associated
bacteria as it allows sequencing from a small number of cells. A drawback is that this method only recovers 70– 95% of a
bacterial genome115,116 and often produces chimaeras117. However, the thousands of intracellular bacterial cells in, for
example, the bacteriocytes of insects, provide substantial amounts of bacterial DNA from a single bacterial species in
comparison to conventional single cell projects. Combined with longer read sequences to help close repeats, an
imminent future prospect is the sequencing of endosymbiont genomes from individual insect hosts.
Metagenomics
Metagenomics118,119 enables the study of bacterial communities and holds great promise for host-adapted bacteria.
The invertebrate or vertebrate host represents a semi-complex growth vessel for bacteria that is far from being as
taxonomically diverse as the open ocean or the soil. Metagenomics allows the complete bacterial gene content to be
characterized in different parts of the host body and, in the case of vector-borne bacteria, in both vectors and hosts.
Investigating the distribution of genes in the bacterial communities of different tissues can yield information on the flux of
genes across bacterial species and the extent to which cells and tissues in the host body present barriers to such a flux.
synthetic biology
Understanding the metabolic fluctuation dynamics of bacteria in their interaction with the host is essential for predicting
host-adaptive processes. Recent analyses of the transcriptome, proteome and metabolic networks of the cell-surface
parasite Mycoplasma pneumoniae have shown how complex cellular machineries can be constructed from small gene
sets through protein multi-functionality120–122. However, many obligate intracellular bacteria cannot be cultivated in the
laboratory, but synthetic biology123–125 may be able to revolutionize our understanding of naturally evolved minimal
genomes126. Designing synthetic genomes is a combinatorial problem because the order of genes being added is dependent
on the context created by the pre-existing genes. Different combinations of genes may lead to different outcomes and
this is coupled with the difficulty of predicting interactions between the bacterial cell and its host. There is not a single
solution to this problem; rather, it is equivalent to being in a fitness landscape where the initial location of the genome in
the overall evolutionary space determines its final possible composition. Here, there is much to learn from computer
simulations127 and the many independent downsizings of obligate intracellular bacterial genomes in nature. Linking
synthetic biology with studies of obligate intracellular bacteria is therefore of mutual interest to both research fields.

Metagenomics
The functional and
sequence-based analysis
of the collective microbial
genomes contained in an
environmental sample.
bacterium Wolbachia pipientis to its host Drosophila
ananassae90. It is thought that this HGT includes most of
the 1 Mb Wolbachia pipentis genome90. Similarly, trans-
fers of complete mitochondrial genomes to the nuclear
genome occur relatively frequently 91. Interestingly,
Wolbachia-like genes have recently been identified in the
nuclear genomes of aphids harbouring Buchnera endo-
symbionts92, suggesting that functional complementa-
tion may be achieved not only by direct HGTs from the
endosymbiont to the nuclear genome of its host but also
through HGTs from other infecting bacteria93.
Given that the sizes of the smallest endosymbionts
are within the range of organelles and that transfers to
the nuclear genome have been shown, could these bac-
teria eventually evolve into organelles? In order for this
to happen, the functions provided to the host must be
essential to all or most cells in the body.
Conclusions and future perspectives
The recent studies of host-adapted bacteria have given
a more nuanced view of bacterial evolution than that
obtained previously from studies of free-living bacterial
model organisms. although genome reduction is an
overriding theme, different selective forces are operat-
ing on the bacterial genome during different stages of
host–bacterium interactions. These forces can lead to
a dramatic expansion in genes for outer surface struc-
tures in the early stages, and to an extreme reduction in
genome size and an almost complete integration with the
host in the later stages.
Systematic sequencing efforts should now be initiated
to get a better resolution of phylogenetic relationships,
with a specific focus on the identification of ‘missing
links’ that may help to show previously unknown natu-
ral reservoirs for disease agents. It is equally important to
compile a catalogue of genes for outer surface structures,
the evolution and inheritance pattern of which are atypical
of the genome as a whole and therefore are associated with
a particular phenotype or putatively affected by selection.
Such knowledge is crucial to our understanding of host
switches and the difference between virulent and non-
virulent strains. It has become increasingly clear that the
acquisition of special virulence or symbioses genes is often
not enough to confer a virulence or symbiotic phenotype
to a recipient bacterium. Rather, the mutualistic or patho-
genic phenotype is dependent on genes required for pre-
adaptation to the intracellular milieu. Modifications of the
bacterial repertoire of host-adaptability genes can then
shift the balance towards mutualism or pathogenicity.
The new technologies developed in single cell genom-
ics, metagenomics and synthetic biology hold great hope for
studies of obligate intracellular bacteria (BOX 3). For exam-
ple, whole genome amplification methods will facilitate
genomic studies of host-associated bacteria for which only
limited amounts of Dna are available94. Metagenomics
will reveal how genes interact in bacterial consortia that
inhabit the same hosts and tissues and how these interac-
tions affect the outcome of the infection. exciting recent
work in synthetic biology holds promise in providing tools
to study the function of genes under non-conventional

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REVIEWS
or controlled experimental conditions. Progress in single
cell transcriptomic techniques95 will enable more detailed
analyses of host Rna expression patterns in different cells
and tissues in response to the infection. Finally, the ‘down-
loading’ of bacterial genes to the nuclear genome of the
host represents a mechanism whereby the host–symbiont
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Acknowledgements
This work was supported by grants to S.G.E.A. from the
Swedish Research Council, the Göran Gustafsson Foundation,
the Swedish Foundation for Strategic Research and the Knut
and Alice Wallenberg Foundation.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
UniProtKB: http://www.uniprot.org
GroEL | VopS
FURTHER INFORMATION
Siv G. E. Andersson’s homepage:
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SUPPLEMENTARY INFORMATION
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