Download Patchy distribution of flexible genetic elements in bacterial

MINIREVIEW
Patchy distribution of £exible genetic elements in bacterial
populations mediates robustness to environmental uncertainty
Holger Heuer1, Zaid Abdo2 & Kornelia Smalla1
1
Federal Research Centre for Cultivated Plants, Julius Kühn-Institute (JKI), Braunschweig, Germany; and 2Departments of Mathematics and Statistics
and Initiative of Bioinformatics and Evolutionary Studies, University of Idaho, Moscow, ID, USA
Correspondence: Holger Heuer, JKI, Institute
for Epidemiology and Pathogen Diagnostics,
Messeweg 11-12, D-38104 Braunschweig,
Germany. Tel.: 149 531 299 3831; fax: 149
531 299 3006; e-mail:
[email protected]
Received 13 December 2007; revised 23 May
2008; accepted 23 May 2008.
First published online 8 July 2008.
DOI:10.1111/j.1574-6941.2008.00539.x
Editor: Jim Prosser
Keywords
flexible genome; bacterial population
heterogeneity; broad host-range plasmids;
evolvability; robustness; mobile genetic
elements.
Abstract
The generation and maintenance of genetic variation seems to be a general
ecological strategy of bacterial populations. Thereby they gain robustness to
irregular environmental change, which is primarily the result of the dynamic
evolution of biotic interactions. A benefit of maintaining population heterogeneity
is that only a fraction of the population has to bear the cost of not (yet) beneficial
deviation. On evolutionary time frames, an added value of the underlying
mechanisms is evolvability, i.e. the heritable ability of an evolutionary lineage to
generate and maintain genetic variants that are potentially adaptive in the course
of evolution. Horizontal gene transfer is an important mechanism that can lead to
differences between individuals within bacterial populations. Broad host-range
plasmids foster this heterogeneity because they are typically present in only a
fraction of the population and provide individual cells with genetic modules newly
acquired from other populations or species. We postulate that the benefit of
robustness on population level could balance the cost of transfer and replication
functions that plasmids impose on their hosts. Consequently, mechanisms that
make a subpopulation conducive to specific conjugative plasmids may have
evolved, which could explain the persistence of even cryptic plasmids that do not
encode any traits.
Introduction
One of the key findings of comparative genome analyses was
the large difference between closely related strains. With
each sequenced genome of eight strains of Streptococcus
agalactiae, or seven strains of Escherichia coli, a significant
number of new genes were found (Abby & Daubin, 2007).
These results suggest that the pan genome, comprised of the
different genes of all strains of a species, is typically several
times larger than the core genome, which is shared by the
majority of conspecific strains. One part of the core genome
evolves by optimization of the intracellular networks for
maintenance and replication (Fig. 1). The other part of the
core genome, which forms the basis of the ecotype, is shaped
by long-term correlations of environmental conditions in
the primary habitats of the species (Fig. 1). The counterpart
of the core genome is called the flexible genome (Hacker &
Carniel, 2001). It is determined by the high plasticity and
modularity of its genetic elements and a high rate of gene
FEMS Microbiol Ecol 65 (2008) 361–371
acquisition and loss (Fig. 1). Comparative genomics of freeliving bacteria indicated that horizontal gene transfer is the
primary source of intraspecies genomic diversity (Lerat
et al., 2005). It seems that by transformation, transduction
or conjugation most bacteria constantly acquire genes from
a large available gene pool. However, the majority of the
acquired genes do not persist within lineages over evolutionary times. Free-living bacteria in particular have a high
incidence of horizontal gene transfer but relatively few
mobile genetic elements in their genomes because, due to
their large effective population sizes, selection efficiently
removes acquired genes (Ochman & Davalos, 2006). Over
evolutionary times, foreign DNA is more likely to be
deleterious than to increase the fitness of the recipient and
consequently become fixed in the lineage (Kurland, 2005;
Hao & Golding, 2006). Over ecologically relevant time
periods, the contribution of evolutionarily successful gene
acquisition by the core genome to bacterial population
fitness seems to be small. For E. coli K-12, it was estimated
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362
Fig. 1. Components of the population metagenome that differ in
flexibility as a result of variation in continuity of the shaping selective
forces.
that only one transfer event in 400 000 years gets fixed in the
lineage (Lawrence & Ochman, 1998). Nevertheless, mobile
genetic elements, the agents of horizontal gene transfer, are
abundant in bacterial populations of many habitats, which
points to a major ecological function. Horizontally transferred genes can persist for a long time in a fraction of a
bacterial population even if they are slightly deleterious
(Novozhilov et al., 2005). Thereby, mobile genetic elements
create standing genetic variation, by which adaptation to a
rapid environmental change or habitat expansion can more
likely be achieved rather than by new mutations (Jain et al.,
2003; Hermisson & Pennings, 2005). In this minireview, we
discuss whether the primary benefit of bacterial populations
to maintain mobile genetic elements is to gain robustness to
uncertain environmental change, and whether a secondary
benefit is evolvability, i.e. the heritable ability of an evolutionary lineage to generate and maintain genetic variants
that are potentially adaptive in the course of evolution. We
summarize evidence for the generation of population heterogeneity by mobile genetic elements as a basis for robustness in fluctuating environments. We place special emphasis
on conjugative broad host-range plasmids, as they are major
vehicles of frequent interspecific gene transfer, which may be
the basis for their evolutionary success.
Broad host-range conjugative plasmids
Plasmids are typically composed of (1) conserved backbone
modules coding for replication, maintenance and transfer
functions, and (2) variable accessory modules. By definition,
they do not carry genes that are essential for growth of their
host, except in special environmental situations. Most
evidence points to plasmids being unable to survive as
purely parasitic elements or by only temporarily providing
beneficial traits to the cell (Bergstrom et al., 2000). Even if a
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H. Heuer et al.
plasmid carries beneficial accessory modules, these could
easily be integrated into the chromosome to minimize the
cost associated with plasmid backbone modules (Thomas,
2004). In the absence of sexual reproduction, a genetic
element that lowers the fitness of its host and initially resides
in only a fraction of the population will inevitably be lost by
natural selection (Johnson & Brookfield, 2002). Plasmidbearing cells will be outcompeted by plasmid-free relatives if
the cost of carrying the plasmid backbone is not balanced by
a benefit for the cell. This problem is especially evident for
broad host-range plasmids, because their genetic load may
not be adaptive in the ecological niche of a new host after
transfer, even if it was at some point beneficial for the
previous one. Some of their accessory genes may be temporarily beneficial for many hosts in special environments.
However, evidence from plasmid genome comparisons
found that broad host-range IncP-1 plasmids even without
any accessory genes existed in microbial communities over
evolutionary times and occasionally acquired accessory
elements at different insertion sites (Heuer et al., 2004).
The recently sequenced IncP-1 plasmids pA1 and pBP136
are thought to resemble such a cryptic plasmid without
accessory genes (Harada et al., 2006; Kamachi et al., 2006).
However, each carries two ORFs of unknown function in
addition to the backbone. The effects of these accessory
genes and of the plasmid backbone on fitness of the host
have not yet been investigated. The long-term existence of
cryptic plasmids could be explained if bacterial populations
actively maintain a type of plasmid backbone, regardless of
accessory elements, thus taking advantage of their capacity
to capture genetic modules, which could increase phenotypic diversity, and thereby the chance to respond to uncertain
environmental change or to grasp an opportunity for
transient niche expansion.
Heterogeneous distribution of plasmids
in bacterial populations
The incidence of plasmids in bacterial isolates from diverse
habitats, or in collections of strains, range from nearly 100%
to o 10% (Sobecky, 1999; Cook et al., 2001; Malik et al.,
2002; Thomas, 2004). In a survey of 521 diazotrophic
isolates from four salt marsh grass rhizoplanes, the proportion of plasmid-containing strains was between 12% and
30% (Beeson et al., 2002). The analysed strains belonged to
more than six genera, and 71 different plasmids, which
ranged in size from 2 to 4 100 kbp, were detected in 134 of
the strains. These data suggest that plasmids are abundant in
many populations, but cannot determine estimates on the
plasmid distribution within populations. An examination of
74 strains of the fish pathogen Photobacterium damselae ssp.
piscicida isolated from amberjacks (Seriola spp.) in Japan
found that all strains had several plasmids (Kijima-Tanaka
FEMS Microbiol Ecol 65 (2008) 361–371
363
Population-level robustness through genome flexibility
et al., 2007). Even though this locally emerging population
showed only subtle genomic differences among strains, the
distribution of plasmids was heterogeneous. There was no
analysis of plasmid types and no discussion of the potential
involvement of these plasmids in pathogenicity that could
explain the high incidence in this population. Data on the
frequency of a particular plasmid type, as defined by its
backbone, and to what extent it confers phenotypic diversity
by varying accessory elements in an environmental population of bacteria are scarce. A glimpse into the natural
heterogeneity of soil populations due to conjugative plasmids was given by a recent study by Kuhn et al. (2008). The
incidence and diversity of pSmeSM11a-like plasmids was
analysed in isolates of Sinorhizobium meliloti from two field
sites, representing two populations. Five of 21 strains
isolated from one field, and four of 16 strains from the other
field contained plasmids with pSmeSM11a-like backbone
genes, while the majority of isolates from both populations
did not carry this type of plasmid. The pSmeSM11a-like
plasmids differed in size and accessory gene content. Thus,
the isolates represent two natural field populations that are
considerably heterogeneous with respect to their plasmids.
Diversification of an introduced population by acquisition
of naturally occurring conjugative plasmids was shown in a
field experiment, using a genetically marked Pseudomonad
as a recipient that colonized sugar beet plants (Lilley &
Bailey, 1997b). At least four different large mercury-resistance plasmids were captured at frequencies between 10 6
and 1 for a few samples. One of the captured plasmids, the
330-kbp mercury-resistance plasmid pQBR103, was further
studied in a greenhouse experiment. It conferred a transient
fitness advantage to the plasmid-bearing cells compared
with the plasmid-free subpopulation of Pseudomonas fluorescens SBW25 in the phytosphere of mature sugar beet plants
(Lilley & Bailey, 1997a). The detoxification of mercury
encoded by this plasmid could result in the coexistence of
plasmid-free and plasmid-bearing cells at a certain range of
mercury concentrations (Ellis et al., 2007). The higher the
frequency of plasmid-bearing cells, the more the neighbouring
plasmid-free cells benefit from detoxification without paying
the cost of plasmid carriage. However, this kind of stabilizing
frequency-dependent selection (Levin, 1988) cannot be a
general explanation for the observed plasmid incidences in
bacterial populations, but only in those special cases where a
plasmid-encoded gene product alters the external environment in a way that plasmid-free cells benefit. Such gene
products are occasionally encoded by accessory elements of
plasmids, but occur chromosomally encoded as well.
In conjugation experiments with close contact of plasmid-containing donor cells and plasmid-free recipient cells,
only a fraction of cells receive a plasmid even under
optimized conditions, i.e. in filter matings on nutrient
media. In the natural environment, much lower transfer
FEMS Microbiol Ecol 65 (2008) 361–371
rates are expected. In the rhizosphere of pea, which was
shown to be a hot spot of plasmid transfer, the IncP-1
plasmid pKJK5 could transfer only to a tiny fraction of an
introduced recipient strain or indigenous bacteria even if the
potential recipients were heavily surrounded by plasmidcontaining cells (Mølbak et al., 2003, 2007). In contrast to
this and many other studies, in a recent mating experiment
the plasmid pKJK5 could completely invade plasmid-free
populations of an E. coli and a Kluyvera sp. strain in bacterial
mats without providing any obvious selective advantage
(Bahl et al., 2007). This exceptional result could be due to
the special conditions of the bacterial mat and also to the
recipient strains applied. Often environmental strains are
surprisingly reluctant to receive an IncP-1 plasmid in
biparental mating (Heuer et al., 2007a), while conjugative
transfer from a donor to cells of a bacterial community, e.g.
from the rhizosphere or activated sludge, resulted in diverse
transconjugants (Pukall et al., 1996; DeGelder et al., 2005).
Selected recipient strains like Pseudomonas putida KT2442
consistently show high frequencies of plasmid capture.
Pseudomonas putida KT2442 was originally isolated as the
plasmid-bearing strain mt-2, which was then cured of the
TOL plasmid but may have retained or even increased its
conduciveness to plasmids through a long history of cultivation and mutation (Regenhardt et al., 2002). Recent evidence suggests the conduciveness for a plasmid is strain
specific rather than species specific in P. putida (Heuer et al.,
2007a), which was subsequently confirmed also for other
species (DeGelder et al., 2007). In P. putida strain H2, IncP-1
plasmids were very unstable and conferred a high fitness
cost. However, analysis of plasmid stability in eight lineages
derived from single cells revealed high instability of the
plasmid in only six of the lineages in batch culture (Heuer
et al., 2007a). This population heterogeneity was still significant after 1000 generations of adaptive evolution, while
plasmid stability and cost ameliorated. Large differences in
the expression profiles of the plasmid between a conducive
E. coli host and the unfavourable P. putida host H2 suggested
considerable host–plasmid interactions, but could not give a
mechanistic explanation for the differences in cost and
stability of the plasmid. A specific mechanism of interaction
between plasmid and chromosome was found in strain
P. putida KT2440, which is known to be conducive for
plasmids. Transcriptome analysis revealed that the chromosomal gene parI is positively regulated by the plasmid-encoded
ParA and by ParI itself (Miyakoshi et al., 2007). This might
support plasmid partitioning and maintenance in this strain.
It remains to be investigated at which scale a population is
heterogeneous with respect to conduciveness for plasmids.
Seemingly, strains of a global population that define a
particular species vary in that respect. But is this also true
for strains of a population that occupy a single and
contiguous spatial area, thereby providing a mechanism for
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364
sympatric diversification? Answering such a question requires a clear definition of a bacterial population, a task
complicated by the ecological and genetic interactions
between different bacterial strains. Population definitions
differ, depending on the scale at which a study of interest is
conducted. On the ecological scale, a population is defined
to be a group of individuals of the same species within the
same habitat at the same time that interact with one another
(Lowe et al., 2004; Waples & Gaggiotti, 2006). On the
evolutionary scale, populations are generally defined to be
all individuals connected by gene flow (Hedrick, 1999; Lowe
et al., 2004; Waples & Gaggiotti, 2006). These two definitions are coined, for the most part, for the study of sexually
reproductive populations where a clear separation can be
identified between ecological and evolutionary time scales.
Bacterial populations do not adhere to such time-scale
separation. Moreover, the asexual nature of bacteria further
complicates the task of identifying an appropriate definition
due to the level and mechanisms of gene flow that need to be
considered before identifying an appropriate population
concept. When plasmids are considered as an independent
entity acting as a parasite, or mutualist, in association with a
bacterial strain or species, they can be thought to form
populations separate from their bacterial host populations
(Bergstrom et al., 2000). In this case, the host bacterial
population can be defined based on common ancestry,
where strains close enough in their phylogenetic history are
considered to be part of the same population. A challenging
task is defining the meaning of ‘close enough’, which is
usually based on an arbitrary cut-off imposed on the genetic
distance between taxonomic units (Meyer & Paulay, 2005;
Abdo & Golding, 2007). We favour a definition where a
population includes strains that have similar genomic
structure of the core genome as defined in Fig. 1, and that
considers plasmids as a mechanism of gene flow between
these strains. This definition restricts the boundaries of the
population to include only those that share an evolutionary
history yet allows for gene flow through plasmid conjugation and other mechanisms. Plasmids can be thought of as
part of the overall gene content that contributes to the
diversity of a population. Such structure facilitates the
application of phylogenetic and population genetics modelling concepts on a higher evolutionary scale, i.e. the scale of
genes rather than nucleotides (Hao & Golding, 2006), and
facilitates the study and comparison of the diversity of
microbes with respect to their plasmid content and other
flexible genome differences. Population genetics modelling
concepts allow for studying the effects of migration, selection, and spatial and temporal structure as part of the
genome evolutionary process. To eliminate the use of
arbitrary cut-off limits in identifying strain memberships to
populations, we suggest the use of probabilistic, modelbased classification methods that provide a measure of
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H. Heuer et al.
confidence in the population membership of a strain (Abdo
& Golding, 2007). Such methods quantify the uncertainty in
grouping different strains to form a population based on
their evolutionary history and provide objective decision
rules for identifying population memberships of the different strains. Environmental data on incidence and diversity
of plasmids within a population could be integrated into
population genetics models to improve our understanding
of the contribution of plasmids to standing genetic variation
in stable or frequently perturbed environments. Coupling
the use of these models with probability theory allows for
evaluating the statistical significance of the effect of population heterogeneity, due to broad host-range plasmids, and
facilitates testing the hypothesis that such heterogeneity
contributes to the robustness of bacterial populations under
environmental uncertainty. Probability theory forms the
base for statistical inference by providing the link between
observed environmental data and population genetics models that can take into account population heterogeneity,
population and plasmid evolution, and plasmid proliferation under relevant selection pressures. This can overcome
limitations of existing studies on the incidence of plasmids,
which are difficult to interpret because the host population
was not well defined with respect to the phylogenetic
relationship of the strains, the spatial area occupied, the
likelihood of ecological and genetic interaction between
strains, and migration between populations.
Mechanisms potentially leading to
heterogeneity in bacterial populations
with respect to conduciveness to
horizontal gene transfer
The generation and maintenance of genetic variation seems to
be a general ecological strategy of bacterial populations.
Various mechanisms have been described to foster intrapopulation heterogeneity. Mutation rates can be genetically altered
in a subpopulation of mutator cells. This can be regulated in
response to environmental conditions, or focused on specific
loci in the genome to minimize deleterious effects on fitness
(Moxon & Thaler, 1997; Hallet, 2001). Furthermore, isogenic
cells exposed to the same environmental conditions can show
significant variation in molecular content and phenotypic
characteristics. Cell cycle, cell ageing and epigenetic regulation
are among the drivers of this heterogeneity (Avery, 2006). The
phenomenon of persisters is well studied, i.e. antibiotictolerant subpopulations of otherwise susceptible bacteria that
ensure survival of the population in case of antibiotic pressure
(Dhar & McKinney, 2007).
Differences within a population in conduciveness to
horizontal gene transfer can be the result of genetic or
epigenetic variation in defence against foreign DNA or in
DNA uptake. Mechanisms that confine DNA uptake to a
FEMS Microbiol Ecol 65 (2008) 361–371
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Population-level robustness through genome flexibility
subpopulation could minimize the cost for integration of
potentially not (yet) beneficial foreign DNA. Evidence for
variation in conduciveness to horizontal gene transfer was
recently found within a global collection of E. coli isolates.
Lineages were identified by multilocus sequence typing,
which bore specific pathogen types arising several times
independently through horizontal gene transfer, whereas in
other lineages few pathogenic strains have arisen (Wirth et al.,
2006). It is plausible that the pathogen-bearing lineages
evolved a higher or more variable conduciveness to mobile
genetic elements, which resulted in episodic selection of
strains that transiently expanded their ecological niche by
escaping the host immune response. Pathogenicity-related
genes were often found on genomic islands, i.e. large chromosomal regions, in a subset of strains of the same species,
that contain genes for chromosomal integration and excision
and other mobility-related genes (Dobrindt et al., 2004). They
were shown to contribute to strain diversity in populations of
Mezorhizobium and Pseudomonas aeruginosa (Klockgether
et al., 2007; Nandasena et al., 2007). Site-specific excision
can compromise the maintenance of genomic islands in
individual cells. As shown for uropathogenic E. coli, excision
is stimulated by low temperature and high cell density
(Middendorf et al., 2004), which could induce patches of
cells without genomic islands in environmental biofilms.
Also, epigenetic mechanisms can be the basis for heterogeneity in conduciveness to horizontal gene transfer. Noise
in gene expression, when subject to positive feedback
regulation, can result in bistability, i.e. the expression of
two different genetic programmes within a clonal population
of bacteria under identical external conditions (Dubnau &
Losick, 2006). Bistability was shown, for different species, to
direct uptake of foreign DNA to a fraction of the population.
Steinmoen et al. (2003) showed that the partitioning of
Streptococcus pneumoniae populations into lysing ‘donor’
and DNA-uptaking ‘recipient’ subpopulations is achieved
epigenetically by the ComCDE signal transduction pathway.
Kaern et al. (2005) found that in populations of Bacillus
subtilis, a minority of cells became competent in DNA
uptake due to intrinsically random fluctuations in comK
mRNA production and degradation. ComK is the master
regulator for competence and positively autoregulates comK
expression. It was suggested that the cellular processes,
which modulate the level of noise in gene expression and
thereby the proportion of competent cells in a population,
evolved under selection to maintain a well-tolerated rate of
diversification (Maamar et al., 2007).
Various barriers limit the transfer, uptake and stabilization
of foreign DNA in bacteria (Thomas & Nielsen, 2005). These
limitations could be relaxed in a fraction of a population,
leading to a subpopulation with an increased likelihood of
extant DNA acquisition. As an example, bacterial strains with
increased potential for genomic integration of foreign DNA
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by recombination have been found in natural populations at
proportions of 1–20%, which was in particular caused by
altered methyl-directed DNA repair (references in Thomas &
Nielsen, 2005). Frequencies of DNA uptake by natural
transformation can be highly variable within populations of
Pseudomonas stutzeri, B. subtilis or Haemophilus influenzae
(Lorenz & Sikorski, 2000, and references therein). Only a
fraction of the strains analysed could be transformed, and
frequencies varied over more than three orders of magnitude.
In addition, natural competence for transformation is under
physiological and environmental modulation, so that individual cells of a population in a spatially heterogeneous
environment will differ in transformation potential.
Some of the mechanisms that limit the transfer, uptake and
stabilization of foreign DNA may also be the basis for
differential conduciveness to plasmids within natural populations of bacteria. Restriction–modification (R–M) systems
play an important role in protection against foreign DNA by
significantly decreasing the efficiencies of conjugation, transformation and phage infection (Thomas & Nielsen, 2005;
Hoskisson & Smith, 2007). R–M systems are ubiquitous in
bacteria and archaea, but they can vary among strains of the
same species, and are often part of the flexible genome
(Naderer et al., 2002; Bishop et al., 2005). Even more, R–M
systems were found to be phase variable in a variety of species,
including Helicobacter pylori, Mycoplasma pulmonis, Pasteurella hemolytica, Neisseria meningitidis, Streptomyces coelicolor
and H. influenzae (Hoskisson & Smith, 2007), i.e. R–M
systems can be switched on and off in individual cells of a
population and thereby affect plasmid uptake. In populations
of H. influenzae, the high-frequency phase variation of the
R–M system HindI was shown to be mediated by tetra- and
pentanucleotide repeats (Zaleski et al., 2005).
A widely distributed and highly flexible defence system
against foreign DNA might be associated with clustered
regularly interspaced short palindromic repeats (CRISPRs),
which were identified in the genomes of a wide range of
bacterial and archaeal genera (Godde & Bickerton, 2006).
CRISPR loci consist of direct repeats separated by stretches
of variable sequences called spacers. Bacteria can become
immune against specific foreign genetic elements when
they acquire fragments of the foreign DNA into the spacers.
This is mediated by the function of CRISPR-associated cas
genes (Barrangou et al., 2007), probably via a mechanism
based on RNA interference (Makarova et al., 2006). It was
shown that variation of the CRISPR region is fast enough
to promote individuality in otherwise nearly clonal populations of Leptospirillum sp. (Tyson & Banfield, 2008). This
hypervariability most likely will cause microdiversity in
DNA acquisition within natural populations of bacteria.
Among others, spacer sequences from plasmids were also
found (Makarova et al., 2006), but experimental evidence
for interference with plasmid stability is still missing.
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366
H. Heuer et al.
Table 1. Terms and definitions
Environmental uncertainty
Sympatric diversification
Bacterial population
Pan/core genome
Flexible genome
Mobile genetic elements
Flexible genetic elements
Evolvability
Frequency-dependent selection
Bistability
Phase variation
Surface exclusion
Entry exclusion
Plasmid incompatibility
Broad host-range plasmids
Irregular external change that challenge the adaptive potential of a population to withstand adverse conditions,
or to transiently expand its ecological niche
Development of genetically encoded phenotypic variation in a population that occupies a single and contiguous
spatial area
Bacterial cells of the same species that live in the same habitat patch and therefore have a chance to interact
ecologically and genetically
The sum of all genes that occur in any/each strain of a species
Counterpart of the core genome that varies between strains of a species, determined by its high rate of
acquisition, deletion and recombination of genetic elements
Plasmids, transposons, IS elements, integrative conjugative elements, gene cassettes, bacteriophages and other
genetic units that can relocate
Mobile genetic elements and other genetic structures like repetitive sequences that foster genetic variation
in a population
The heritable ability of an evolutionary lineage to generate and maintain genetic variants, which are potentially
adaptive in the course of evolution
In stabilizing (negative) frequency-dependent selection, the fitness of a phenotype decreases relative to other
phenotypes in a given population as it becomes more common
Nonunimodal phenotypic variation due to the expression of two different genetic programmes within a clonal
population of bacteria under identical external conditions
ON2OFF switching of a phenotype on the DNA level, e.g. by reversible recombinase-mediated inversion of a
DNA segment, leading to a heterogeneous bacterial population
Inhibition of mating pair formation in conjugative DNA transfer (mediated by the gene traT of IncF plasmids)
Inhibition of plasmid transfer despite mating pair formation (e.g. mediated by genes eexAB of IncP-1 plasmids)
Inability of related plasmids with interfering partitioning or replication systems to stably coexist in the same
host cell
Plasmids that transfer by conjugation between many species and that are stably inherited by hosts belonging
to distant phylogenetic branches, e.g. from different subclasses of Proteobacteria. The potential host-range of
IncP-1 plasmids comprises virtually all Gram-negative bacteria
Furthermore, variation in conduciveness to plasmids
among strains of the same species may be based on the
molecular mechanisms underlying surface exclusion, entry
exclusion or plasmid incompatibility when these traits were
acquired by the chromosome of some strains of a population (for an explanation see Table 1). As an example, the
acquisition of a 16-bp palindrome sequence, which is the
binding site of the partitioning protein ParB, was sufficient
to induce incompatibility towards a group of plasmids from
Alphaproteobacteria represented by the symbiotic plasmid
p42d of Rhizobium etli CE3 (Sóberon et al., 2004).
Population heterogeneity with respect to mobile genetic
elements could also be achieved by mechanisms leading to
variable maintenance of acquired DNA. Enterobacteriaceae
and probably also other Proteobacteria can selectively silence
the expression of foreign DNA by binding of the nucleoidassociated protein H-NS (Navarre et al., 2007). This could
compromise the replication and maintenance of plasmids,
thus increasing the rate of segregational loss. In contrast,
silencing of accessory elements of the plasmid could decrease the metabolic burden for the cell. Individual cells of a
population could have lower levels of H-NS, or activate one
of the mechanisms of countersilencing, e.g. mediated by the
alternative sigma factor RpoS under conditions of stress.
However, the effects of silencing on plasmid maintenance
have not been investigated yet.
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We listed various mechanisms that plausibly lead
to population heterogeneity through plasmids or other
horizontally transferred DNA. However, it needs to
be shown which mechanisms are relevant for natural populations of bacteria. In addition, the relative importance
of specific mechanisms that cause heterogeneity in environmental populations still needs to be investigated. Horizontal
gene transfer clearly contributes to intrapopulation
diversity, but the ecological significance is not well
documented.
Diversity within a population increases
robustness
Bacteria evolved adaptive responses to regular variations in
their habitats. However, fluctuations of the environment are
often irregular and stochastic. In these cases, adaptation
through gene regulation may not be sufficient because the
repertoire of sensing and response mechanisms is limited, or
the time needed for sensing and response may be too short
for some threats such as antibiotics. In addition, there is a
cost associated with maintaining a sensory machinery to
detect external change. In contrast, the generation of population diversity, from which variants are selected in response
to environmental change, enables adaptation to challenges
for which no sensing mechanism has evolved, which are
FEMS Microbiol Ecol 65 (2008) 361–371
367
Population-level robustness through genome flexibility
precipitated, or which occur so infrequently in the habitat of
the population that maintaining a specific sensor does not
pay. The generation of population diversity can be favoured
by challenging environmental conditions, e.g. if the underlying cellular mechanism is enhanced in response to stress
(Aertsen & Michiels, 2005).
According to the insurance hypothesis of biodiversity
(Yachi & Loreau, 1999), resistance and resilience of ecosystem functioning increase with species richness because a
greater number of species can express a greater range of
responses to environmental perturbation. Analogously, genetic diversification of a bacterial population could increase
the chance that a subpopulation might be prepared for
irregular adverse conditions (Boles et al., 2004). This ecological strategy is known as bet-hedging in population genetics. It was predicted by simulations that in fluctuating
environments heterogeneous populations can have a larger
net growth rate than homogeneous populations (Thattai &
van Oudenaarden, 2004; Gander et al., 2007). Population
heterogeneity by stochastic switching is favoured as an
adaptive strategy when environments change infrequently,
while for adaptation in regularly varying environments it is
beneficial to maintain an active sensor for detecting a
specific environmental change (Kussell & Leibler, 2005).
The longer the environment remains constant, the less it
pays to have a sensor. The advantage of stochastic switching
will be further increased when individuals remember the last
few phenotypic switches and the fluctuating environment
exhibits longer correlations (Kussell & Leibler, 2005). Such a
stochastic switching with memory can be the horizontal
acquisition of a mobile genetic element that confers an
adaptive trait under infrequently occurring conditions. As
an example of stochastic switching with memory, a bacterial
subpopulation may switch the phenotype by acquisition of
a plasmid. The plasmid has a history of selection in other
cells, for which it has occasionally been beneficial, thereby
increasing its abundance and chance of horizontal spread.
Environmental uncertainty is typically associated with
biotic interactions, because these are dynamically evolving,
in contrast to abiotic conditions (Smets & Barkay, 2005). An
obvious example of environmental uncertainty is the production of new antibiotic compounds by cohabitants or
hosts (including humans), which resulted in the enrichment
of subpopulations that acquired mobile genetic elements
with antibiotic resistance genes (Witte, 1998; McManus
et al., 2002). Environmental uncertainty is not necessarily
associated with adverse conditions. It could also be the
opportunity to access previously unavailable resources.
Examples of the contribution of mobile genetic elements to
niche expansion of subpopulations are the escape of the host
immune system through acquisition of genomic islands
associated with either pathogenic or symbiotic interactions
(Hochhut et al., 2006; Arnold et al., 2007), or the degradaFEMS Microbiol Ecol 65 (2008) 361–371
tion of recalcitrant compounds through pathway assembly
(Top & Springael, 2003).
Inheritance of, and selection for,
evolvability
Evolutionary successful bacteria must have a balance between both resisting genetic change and evolvability (Lenski
et al., 2006). Evolvability may be defined as the heritable
ability of an evolutionary lineage to generate and maintain
potentially adaptive variants, upon which selection can act.
The same mechanisms underlying evolvability will generate
population heterogeneity. But only a subset of the variants
potentially enhancing robustness of a population will also be
potentially adaptive for the evolutionary lineage, because
some environmental changes may be nonrecurring or too
infrequent to shape the evolution of the lineage. It is
controversial as to whether evolvability can be favoured by
natural selection, because any cost associated with evolvability takes effect immediately while the benefit of adaptation is assumed to lie in the future (Sniegowski & Murphy,
2006). With global mutators in mind, which affect the whole
genome of a cell (like mutS defective strains), it is hard to
believe that the benefit will ever balance the cost, even
though hypermutable cells can reach high frequencies when
they are facing novel challenges from the host immune
system or chemotherapy (LeClerc et al., 1996). However,
the cost can be minimized on cell level by directing genomic
change locally (e.g. contingency loci or plasmids) and on
population level by directing genomic change to a subpopulation (heterogeneity with respect to rates of mutation or
horizontal gene transfer). Plasmids may occasionally act like
global mutators when transposable elements of the plasmid
integrate into unspecific chromosomal sites, but they will
mostly confer genetic change without major cost to their
hosts. Selectively neutral or even slightly deleterious variants
can persist for a long time in a fraction of a bacterial
population (Novozhilov et al., 2005), most likely long
enough to balance the cost with the benefit of adaptability
in an uncertain environment. Indeed, experimental data and
computer simulations suggest that evolvability is a heritable
and selectable trait, and the selective pressure for evolvability
becomes increasingly strong as the environmental conditions become more uncertain (Earl & Deem, 2004; Jones
et al., 2007). As a consequence, selective pressure of therapeutics may enhance the evolvability of pathogens. Various
mechanisms that support evolvability by affecting the probability, type and location of genetic change have been
described (Caporale, 2003). In response to environmental
uncertainty, elements of the flexible genome seem to have
evolved genetic structures that increase their rate of diversification. The effect of simple sequence repeats that can
modulate the local mutation rate by several orders of
2008 Federation of European Microbiological Societies
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c
368
magnitude has been well studied, generating phenotypic
diversity in traits that directly interact with host structures
(Moxon et al., 2006). The virulence-associated avrBs3-like
genes of plant-pathogenic Xanthomonas and Ralstonia are an
example of horizontally transferred genes with a high rate of
intra- and intergenic recombination due to a domain of direct
repeats, which was recently shown to affect population
diversity and host preference of Ralstonia solanacearum
(Heuer et al., 2007b). Also transposable elements, which are
often located on transferable plasmids, increase genetic variation and may be amenable to indirect selection for variability
(Capy et al., 2000; Poussier et al., 2003). Selection for
evolvability may partially explain the genome flexibility and
the prevalence of mobile genetic elements among bacteria.
Concluding remarks
A considerable part of the population metagenome is
attributable to the flexible genome, which is characterized
by its high plasticity, modularity and rate of horizontal
transfer (Fig. 1). The major ecological advantage of maintaining genome flexibility through horizontal gene transfer
might be that the resulting within-population variation can
offer immediate benefit in realistic situations of uncertain
environmental change by providing robustness on population level. If environmental changes stabilize to become
longer-term correlations of the environment and shape the
specific ecological niche of the ecotype, then genome flexibility can enhance evolvability. This eventually results in
new ecotype functions. In consequence, parts of the flexible
genome become fixed in the core genome (Fig. 1). Thus,
evolvability can be seen as an added value of the mechanisms
that generate variation. The flexible genome is shaped by
irregular or novel conditions, which are primarily the result
of the dynamic evolution of biotic interactions (Fig. 1).
These interactions include those between microbes and us,
having implications for human health, agriculture or recalcitrance of man-made compounds. A principal ecological
role of conjugative plasmids is probably their contribution
to make bacterial populations robust to such environmental
uncertainty. Ecosystem stability is often thought to depend
primarily on species richness, because functionally redundant species may differ in susceptibility to a particular
perturbation. However, experimental evidence in microbial
ecology is surprisingly scarce, and bacterial community
fingerprints reflecting ribotype diversity are often hardly
affected by environmental changes, e.g. effects of plants on
bacterial ribotype composition in soil (Felske & Akkermans,
1998; Heuer et al., 2002; Kielak et al., 2008). Research on
within-population diversity in the environment, rather than
ribotype diversity, is needed to clarify how the robustness of
bacterial populations contributes to ecosystem stability.
Understanding the dynamics of flexible genomes and the
2008 Federation of European Microbiological Societies
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c
H. Heuer et al.
underlying mechanisms is essential to estimate the stability
and adaptive potential of bacterial populations as a basis of
strategies to control bacterial pathogens or to manage the
sustainability of ecosystems.
Acknowledgements
H.H. was supported by the DFG, in the frame of the project
FOR566 ‘Veterinary Medicines in Soils’. We would like to
thank Jane E. Stewart for her constructive comments and for
proofreading this manuscript.
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