UNIVERSITE PARIS.DIDEROT (Paris 7) ECOLE DOCTORALE – COMPLEXITE DU VIVANT DOCTORAT Discipline – Sciences de la vie Spécialité – Génétique Guillaume CAMBRAY – EVOLUTIVITE – LE CAS DES INTEGRONS ET UTILISATION DE SEQUENCES SYNONYMES EN EVOLUTION DIRIGEE Thèse dirigée par le Dr. Didier MAZEL Soutenue le 10 Juillet 2009 JURY Mme. la Pr. Isabelle Martin-Verstraete Président M. le Pr. Pierre Capy Rapporteur M. le Pr. Fernando De La Cruz Rapporteur M. le Dr. Antoine Danchin Examinateur M. le Dr. Ivan Matic Examinateur M. le Dr. Didier Mazel Directeur de thèse
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EVOLVABILITY – THE INTEGRON CASE AND THE USE OF SYNONYMOUS SEQUENCES FOR DIRECTED EVOLUTION
Phenotypic stability is essential to the success of organisms evolving under steady conditions. However, the environment is subjected to perpetual stochastic variations, to which living beings must constantly adapt. Evolvability characterizes the ability of a population to respond to such selective pressures through the generation of heritable phenotypic changes. Most mutations being deleterious, processes enabling the confinement of mutations to periods of stress, or to specific loci and well-defined phenotypes, have been selected over evolution. Integrons constitute a particularily sophisticated illustration of such processes. Initially identified through their involvement in multi-resistance to antibiotics, these bacterial genetic systems are specialized in the exchange and stockpiling of accessory genes and therefore con-stitute an important source of genetic diversity. This work shows that integrons are directly coupled with the SOS system, a major bacterial stress response. By allowing the generation of significant phenotypic diversity during periods of stress without impacting the rest of the ge-nome, integrons hence constitute a paradigmatic example of evolvability. Another aspect of this work demonstrates that synonymous coding sequences – although specifying identical proteins – can access different area of the phenotypic space through ponctual mutations. When properly exploited, this property can enhance the evolva-bility of any protein in the context of biotechnological applications.
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UNIVERSITE PARIS.DIDEROT (Paris 7)
ECOLE DOCTORALE – COMPLEXITE DU VIVANT
DOCTORAT
Discipline – Sciences de la vie
Spécialité – Génétique
Guillaume CAMBRAY
– EVOLUTIVITE –
LE CAS DES INTEGRONS ET UTILISATION DE SEQUENCES
SYNONYMES EN EVOLUTION DIRIGEE
Thèse dirigée par le Dr. Didier MAZEL
Soutenue le 10 Juillet 2009
JURY
Mme. la Pr. Isabelle Martin-Verstraete Président
M. le Pr. Pierre Capy Rapporteur
M. le Pr. Fernando De La Cruz Rapporteur
M. le Dr. Antoine Danchin Examinateur
M. le Dr. Ivan Matic Examinateur
M. le Dr. Didier Mazel Directeur de thèse
– EVOLVABILITY –
THE INTEGRON CASE AND THE USE OF SYNONYMOUS
SEQUENCES FOR DIRECTED EVOLUTION
“The beauty of the cosmos is given not only by unity into diversity,
but also by diversity into unity.”
– Umberto Eco, in The Name of the Rose
“La science a en fait deux aspects.
Ce qu’on pourrait appeler science de jour et science de nuit.
La science de jour met en jeu des raisonnements qui s’articulent comme des engrenages, des
résultats qui ont la force de la certitude. On en admire la majestueuse ordonnance comme
celle d’un tableau de Vinci ou d’une fugue de Bach. On s’y promène comme un jardin à la
française. Consciente de sa démarche, fière de son passé, sûre de son avenir, la science de jour
avance dans la lumière et la gloire.
La science de nuit, au contraire, erre à l’aveugle. Elle hésite, trébuche, recule, transpire, se
réveille en sursaut. Doutant de tout, elle se cherche, s’interroge, se réprend sans cesse. C’est
une sorte d’atelier du possible où s’élabore ce qui deviendra le matériau de la science…
Ce qui guide l’esprit alors, c’est l’instinct, l’intuition. C’est le besoin d’y voir clair. C’est
l’acharnement à vivre. C’est le courage…”
– François Jacob
7
RESUME
La stabilité phénotypique est essentielle au succès d’organismes évoluant sous des
conditions constantes. L’environnement est néanmoins soumis à de perpétuelles variations
stochastiques, auxquelles les êtres vivants doivent sans cesse s’adapter. L’évolutivité
caractérise la capacité d’une population à répondre à de telles pressions sélectives par la
génération de modifications phénotypiques héritables. La majorité des mutations étant
délétères, des processus permettant de limiter la production de telles variations aux seules
périodes de stress, ou de la confiner à des loci et phénotypes bien définis, ont été sélectionnés
au cours de l'évolution.
Les intégrons en constituent une illustration particulièrement sophistiquée.
Initialement identifiés comme vecteurs de résistance à de multiples antibiotiques, ces
systèmes génétiques bactériens spécialisés dans l’échange, la collecte et l’expression de gènes
accesoires constituent une importante source de diversité génétique. Ce travail montre que les
intégrons sont directement couplés à une voie majeure de réponse au stress chez les bactéries,
le système SOS. En permettant de générer de la variabilité phénotypique en période de stress
sans affecter le reste du génome, les intégrons constituent ainsi un exemple paradigmatique
d’évolutivité.
Un autre aspect de ce travail démontre que des séquences codantes synonymes – bien
que spécifiant des protéines identiques – peuvent accéder par mutations ponctuelles à des
régions différentes de l’espace phénotypique. Utilisée de manière adéquate, cette propriété
permet d’étendre l’évolutivité d’une protéine quelconque dans le cadre d’applications
TABLE OF CONTENTS ..................................................................................................................................... 13
Results and discussion.......................................................................................................................... 171
Article II ................................................................................................................................................... 172
Article III ................................................................................................................................................. 172
III. Intrinsic evolutionary potential of genes .................................................................................212
Results and discussion.......................................................................................................................... 212
Article IV ................................................................................................................................................. 213
Integrons are complex genetic structures, which are notably responsible for most of the
antibiotics multi-resistance phenotypes that threaten our control over pathogenic bacteria. The
system maintains an array of unexpressed gene cassettes that stands as a reservoir of potential
genetic variation. Silent cassettes can be randomly recombined at an expression site by a
dedicated integrase. This establishes a switching mechanism that allows instantaneous expres-
sion of potentially adaptive functions. Here, we model the evolution of integrase-mediated re-
combination rate in a stochastically fluctuating environment. The integrase gene is a modifier
locus at which alleles can change the turnover rate of the expressed cassettes. Simulations
show that the mean recombination rate of a population would tend to fit the environmental
change rate. Cassette-borne genes are under relaxed selective constraints when not expressed.
We show that deleterious mutations tend to accumulate in the unexpressed part of the cassette
array. While this process does not affect the mutation rate in large populations, it may pro-
mote the functional diversification of cassette-encoded functions. We suggest that a stress re-
sponsive control of recombination rate may be an efficient alternative to a constitutively
determined bet-hedging strategy. This work highlights the importance of integrons as a major
bacterial adaptive system through its effect on evolvability.
Results – Evolution of recombination rate in integrons
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Introduction
Bacteria are one of the major successful life form (Gould, 1996). They have been iso-
lated from a wide range of natural environments, some of which quite extremes. However, di-
rect observation of natural microbial communities is uneasy and little is known about the
actual ecology of bacterial populations. Indeed, a great majority of the bacterial species
(>99%) cannot be cultivated, rendering their detection and studies only possible through me-
tagenomic approaches (Streit and Schmitz, 2004; Rusch et al., 2007). Despite the inherent dif-
ficulties in characterizing the ecological specificities of individual bacterial species, it is safe
to generally regard those organisms as essentially sessile on a macroscopic scale. As a conse-
quence they cannot track their environment in space when it changes, and hence experience a
wide variety of environmental variations, be they physical, chemical or biotic (Andrews,
1998).
The ubiquity of bacteria underlies the remarkable diversity of metabolisms developed
over evolutionary times. The ability of bacterial populations to adapt rapidly to new and ever-
changing environments has been documented in both experimental and natural conditions.
The long-term evolution experiment initiated in 1988 by R. Lenski and colleagues monitored
the adaptation of twelve replicate populations of Escherichia coli to a regime of exponential
growth in a nutrient-limited environment (Lenski et al., 1991; Lenski and Travisano, 1994).
Since then, numerous fitness-enhancing phenotypic variations occurred more or less repeat-
edly in the different populations. These variations include morphological diversification
(Lenski and Mongold, 2000; Philippe et al., 2009), topological modification of DNA (Crozat
et al., 2005), global change in gene expression (Cooper et al., 2003; Pelosi et al., 2006; Phil-
ippe et al., 2007; Cooper et al., 2008), specialization and diversification of metabolic abilities
(Cooper and Lenski, 2000; Cooper et al., 2001; Blount et al., 2008). Adaptive changes occur-
ring in natural conditions are more difficult to identify and most examples involve medically
or economically relevant pathogenic bacteria. The most striking illustration is certainly the
ever more rapid development of resistance phenotype consecutive to the introduction of new
antibiotics (Hawkey, 2008).
According to Fisher’s fundamental theorem of natural selection (Fisher, 1930), the rate
of adaptation in a population equals the heritable variance in fitness in this population. The
speed of adaptive evolution can thus theoretically be increased by increasing the variance in
fitness in the population – its evolvability. The most straightforward way to achieve this is
through a genome-wide increase in mutation rate. Mutator strains are indeed found at non-
Results – Evolution of recombination rate in integrons
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negligible frequencies (0.1% to >60%) in pathogenic bacterial isolates (Denamur and Matic,
2006). Besides, a mutator phenotype reached fixation in 3 out of the 12 replicate lines per-
petuated in Lenski’s long-term evolution experiment (Sniegowski et al., 1997). These strains
are generally affected in their ability to perform mismatch repair, a major system of DNA re-
pair also involved in recombination. Particularly, mutations in the mutS and mutL genes re-
sults in up to 100-fold increase in mutation rates (Denamur and Matic, 2006).
Genes that can affect the mutation rate by their activity can be abstracted as modifier
loci which are subjected to indirect selection by hitchhiking with the mutations they contrib-
uted to generate (Kondrashov, 1995). Because most mutations are deleterious (Eyre-Walker
and Keightley, 2007), a general mutator is generally counter-selected. Nevertheless, the pro-
duction rate of advantageous mutations increases with the initial maladaptation of the popula-
tion (Silander et al., 2007; Martin and Lenormand, 2006). Hence, increased mutation rate are
more likely to be beneficial in fitness-compromising conditions. Indeed, mutator readily pro-
vides short-term adaptive advantage in new, changing or heterogeneous environments (Giraud
et al., 2001). The rise in frequency of a mutator genetically linked to a beneficial mutation is
only the consequence of adaptation, and do not constitute an adaptation in itself (Sniegowski
et al., 2000). Instead, the accumulation of deleterious mutations over time hampers the long-
term success of mutator populations (Funchain et al., 2000; Cooper and Lenski, 2000; Zeyl et
al., 2001). After adaptation occurred, a modifier locus experiences a strong selective pressure
toward lower mutation rate if the environment remains constant (De Visser, 2002). As a re-
sult, the spontaneous mutation rates observed in various microbes are surprisingly steady and
are though to be actively maintained at the lowest level afforded by the cost of fidelity (Drake
et al., 1998).
The evolution of increased evolvability through an increase in the genome-wide muta-
tion rate is thus heavily constrained by the prevalence of deleterious mutations. To circumvent
this limitation, some loci are subjected to frequent, stochastic and heritable modifications me-
diated by dedicated genetic or epigenetic mechanisms (van der Woude and Baumler, 2004;
and see Discussion). These specific mutations are easily reversible, which promotes the con-
stitutive wavering between well defined phenotypic states. Such localized increase in muta-
tion allows the combinatorial diversification of target functions while limiting the potential
deleterious effects of mutations at loci that do not need to evolve.
Integrons constitute a particularly sophisticated example of such systems. A typical in-
tegron consists of a stable platform associated with a variable array of dedicated gene-
Results – Evolution of recombination rate in integrons
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cassettes. The functional platform constitutes a tightly packed locus comprising an intI gene,
coding for a site-specific recombinase, a primary recombination site attI and a promoter Pc
oriented toward attI (figure 1). The gene cassettes integrated in integron arrays are generally
composed of a single and promoterless ORF flanked by two attC recombination sites (Mazel,
2006). The integrase catalyses the recombination of cassettes through a cut-and-paste mecha-
nism whereby cassettes are randomly excised from the cassette array (attC x attC recombina-
tion) to be preferentially integrated in attI downstream of the Pc promoter (Collis et al., 1993;
Collis et al., 2001). This oriented process ensures instantaneous expression of the mobilized
cassettes, while previously integrated cassette are progressively moved away from the Pc
(Collis and Hall, 1995). Overall, only the few first cassettes in the array are expressed and
hence subjected to selection, while the others constitute a silent reservoir of potential genetic
variation (figure 1). Exogenous cassettes uptaken from the environment or brought about by
mobile elements can be incorporated in the array, thereby enriching the repertoire of available
functions (Holmes et al., 2003; Biskri et al., 2005).
Two distinct forms of integrons are generally distinguished in the literature. Mobile in-
tegrons (MI) were the first to be identified through their involvement in antibiotic multi-
resistance phenotype (Martinez and de la Cruz, 1988; Stokes and Hall, 1989). They are lo-
cated on mobile genetic elements such as ICEs, plasmids and transposons, which permit their
dissemination and potentially make them efficient shuttles for the transfer of cassette between
genomes (Biskri et al., 2005). They comprise only few cassettes (up to 8 (Naas et al., 2001b)),
which typically encode antibiotic resistance proteins (Fluit and Schmitz, 2004). Chromosomal
integrons (CI), in contrast, are essentially sedentary. They have been identified in around 10%
of bacterial genomes sequenced to date (Boucher et al., 2007). A subset of these CIs, often re-
ferred to as superintegrons, comprise large array that can span hundreds of cassettes and are
hypothesized to play a major role in the generation of cassettes (Mazel, 2006), a process
which otherwise remains unraveled. Most CI’s cassettes harbor genes of unknown functions.
Nevertheless, the functions that can be predicted are very diverse and a substantial part of it is
involved in substrate modification (acethyltransferases) or interactions with biotic factors
(virulence factors and DNA modification) (Boucher et al., 2007). Besides, 10 to 30% of the
genes potentially encode protein carrying a signal peptide region for either membrane associa-
tion or export from the cell (Koenig et al., 2008). Altogether, these data suggest that cassette-
encoded genes can mediate adaptation to wide range of environmental conditions. Both the
functional platform and the cassettes of MIs are though to derive from CIs (Mazel, 2006;
Results – Evolution of recombination rate in integrons
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158
Labbate et al., 2009). In this light, the impressive ability of bacteria to rapidly overcome such
drastic environmental changes as those imposed by the human use of antibiotics heavily relies
on the recruitment of pre-existing integrons. This illustrates the capacity of the system to cope
with ever changing environments.
Obviously, the shuffling of gene-cassette introduces variability in the integron regard-
ing which traits are expressed or not, and this process is directly dependent on the system’s
recombination rate. The functionality of integron integrases has been demonstrated experi-
mentally over a wide range of substrates and model systems. However, the integrase was al-
ways artificially overexpressed in these studies and spontaneous recombination events have
never been observed in controlled conditions. Thorough epidemiological studies designed to
monitor the spread of multi-resistant MIs evidenced a continuum of cassette arrangements,
evidencing their effective diversification in naturae (Moura et al., 2009). Considerable vari-
ability has been observed in CIs, even between closely related bacterial species and strains
(Boucher et al., 2006). The integron locus is actually one of the most variable genomic loci, a
feature that has been used to finely resolve phylogenetic relationships between otherwise
identical isolates (Labbate et al., 2007). One of the most precise examples to date identified
numerous rearrangements between three pandemic V. cholerae strains over a one century pe-
riod. More accurate estimations of recombination dynamics would rely on the comparison of
very closely related arrays, which is difficult to achieve in practice through the sequencing of
random natural isolates. Hence, although the evolvability bestowed by integron relies on cas-
sette rearrangement, the recombination rate in these systems remains enigmatic.
To shed light on this question, we model the evolution of site-specific recombination
in an integron subjected to a fluctuating environment, which entails the need to constantly
evolve. By essence, environmental changes in nature are stochastic. Their effects on natural
selection are difficult to capture in a purely analytical model without tremendous assumptions.
To avoid oversimplification and to accurately describe the integron system, we develop a
Monte-Carlo simulations scheme incorporating a quantitative-genetic-based modeling of fit-
ness. We show that the selected recombination rate tend to fit the rate of environmental shifts.
Moreover, we highlight that mutations tend to accumulate in non-expressed cassette, particu-
larly under slowly fluctuating conditions.
Results – Evolution of recombination rate in integrons
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Methods
We used Monte-Carlo simulations to model the evolution of the site-specific recombi-
nation rate in an integron, in a randomly fluctuating environment. An integron with C cas-
settes was modeled such that, for each cassette ci, its genetic value zi was drawn in a uniform
distribution of variancec². The fitness of each cassette relative to the best possible genotype
was then assigned by applying a stabilizing fitness function, such that for a genotype of value
z, its relative fitness is: 2
2
( ( ))
0 0( ) (1 )z t
W z W W e
.
The Gaussian term in this function (2
2
( ( ))z t
e
) refers to selection for an optimum genotype
whose value (t) can change in time. The term W0 (0<W0<1) is a constant representing the
basal fitness of the organism irrespective of which cassette is currently expressed. This latter
term can be viewed as the dispensability of the integron: when W0 is equal to 1, the fitness of
the individual is maximal whatever the expressed cassette in the integron, such that the inte-
gron does not improve the fitness of the individuals; in contrast when it equals 0, the fitness of
individuals varies much according to which cassette is express by the integron. For simplicity,
a single cassette in the array – the one integrated at the expression site attI – is considered to
be expressed. The remaining C-1 cassettes are silent and constitute the reservoir of genetic
variation. Besides the expressed and unexpressed cassettes, integrons bear an integrase locus,
the product of which is responsible for cassette excision and subsequent integration. This lo-
cus determine the site-specific recombination rate, and hence the turnover pace of expressed
cassettes. Mutations at this locus can potentially impact the mean cassette turnover rate, mak-
ing it a modifier of the system, just as in models of modifiers of homologous recombination,
segregation or mutation for instance (Kondrashov, 1995). Polymorphism on the recombina-
tion rate trait was allowed at this locus, such that there could be up to I different alleles, the
recombination rate of each allele inti being ri. At the beginning of each run of the simulation,
we set r1=0 (no recombination), while the recombination rate ri of each of the other alleles
was drawn randomly. Specifically, we used ri=10i where i was drawn uniformly between 1
and 4.5, so that a wide range of recombination values were explored. During the course of the
simulation, mutations were then allowed to occur at the integrase locus at equal rate µ among
all pairs of alleles. To model the effect of the integrase locus on the recombination rate, a pro-
portion ri of the expressed cassettes of individuals carrying allele inti at the integrase locus, were
replaced by other cassettes in the unexpressed pool of the same individuals at each generation.
Results – Evolution of recombination rate in integrons
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Fluctuations in the environment were modeled as changes in the optimal genetic value
(t). These changes happened randomly in time at rate e. A new genetic value was drawn in a
uniform distribution of variance e²=c², such that the potential values of the optimum and
those of the actual genetic values of cassettes fully overlapped; we assumed no autocorrela-
tion, meaning that (t) was independent of (t-1). To improve computer-time efficiency in
cases where e<1/20, environment shifts were modeled as a Poisson process, such that the time
in generations between two changes was drawn in an exponential distribution of parameter e.
When mentioned, mutation was also allowed inside the cassettes at a rate . We assumed
those mutations had deleterious effects, as a consequence of either pleiotropic effects on traits
not considered in the model, or of a general decrease in the efficiency of the protein encoded
by the affected cassette. The Gaussian term in the fitness of a cassette affected by m mutations
was then multiplied by an amount (1- s)m if m < mmax, and 0 if m ≥ mmax. Practically, mmax
was set to 0 or 1 in this work to model the absence of mutations and the occurrence of drasti-
cally deleterious mutations.
To model genetic drift, we used a genotype-based framework with multinomial sam-
pling adapted from that of Tenaillon et al. (Tenaillon et al., 1999). This framework has the
caveat that it imposes prior knowledge of all possible genotypes, but it is effective in term of
computer time and efficient to model very large populations for many generations.
We aim at understanding how the genetic properties of the integron, such as the turn-
over rate of expressed cassettes, change as an adaptation to the environmental fluctuations. At
each run of simulation, a burn-in period of 10 environmental changes was let to elapse in or-
der to allow the system to reach its dynamical equilibrium, and the population was then left to
evolve for another 100 environmental changes, during which the mean recombination rate r
and the mean fitness of the population were recorded at each generation, and averaged over
the 100 environmental changes.
Results
We developed a simulation framework to study the evolution of recombination rate in
a stochastically fluctuating environment. To provide an overview of the model behavior, we
draw the evolution of the frequency of integrase alleles in a population of integrons over a pe-
riod of time spanning 75 environmental changes in one run of simulation (figure 2A). The
population consisted in N=108 integrons and comprised I=6 different integrase alleles associ-
ated with an array of C=5 different gene cassettes, of which only one is expressed in each in-
Results – Evolution of recombination rate in integrons
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161
dividual. All 30 possible genotypes are initially introduced in equal frequency in the popula-
tion. Environmental shifts occur stochastically according to a predefined rate. To highlight the
dynamic of integrase allele in diverse contexts, the rate of environmental change was initially
set to 10-2.5 and was decreased by a factor of 10-0.5 every 25 shifts, resulting in 3 successive
regimes of selection.
Before any environmental shift occurs, the genotype expressing the fittest cassette as-
sociated to the non-functional integrase (no recombination) is transiently favored. This allele
is strongly counter-selected by the first shift, because it does not allow the generation of di-
versity necessary to adapt to new conditions. The two alleles with highest recombination rates
were also rapidly counter-selected for the exact opposite rationale: their associated array is not
stable enough to sustain selection in steady environments. In contrast the three integrase al-
leles characterized by intermediate recombination rate rose in frequency. A burst of succes-
sive environmental variations quickly led to the drop of the allele with the lowest rate. Then,
one allele predominated in this regime, with few occasional take over by the allele with im-
mediate lower rate correlating with period of relative environmental stasis. After a lag, the
passage to the next regime of environmental change indirectly drove the fixation of this latter
allele. Similarly, the slowest fluctuating regime led to the rise of the allele with the lowest re-
combination rate, which was previously counter-selected. We also calculated the mean re-
combination rate in the population at each generation. As illustrated in figure 2B, the mean
recombination rate selected indirectly via the fitness effect of gene cassettes tends to stabilize
in each regime to fit the imposed fluctuation rate. Overall, each regime promotes either the
fixation of one specific allele, or the maintenance of a polymorphism at the integrase locus,
resulting in a relatively stable recombination rate over the long term; this steady-state recom-
bination rate changes with the speed of environmental fluctuations.
Prompted by this observation, we undertook a more systematic approach to monitor
the evolution of the mean recombination rate r over a range of different environmental change
rates e, for different values of specificity and dispensability. Overall, the results confirmed the
existence of a linear relationship between these two variables (figure 3). Each point represents
the average of 100 independent runs. In each run, different cassette values and recombination
alleles were randomly sampled and the mean recombination rate over 100 stochastic envi-
ronmental shifts was calculated. Despite the high level of noise imposed by this method, the
mean recombination rate remarkably conform to the fluctuation rate corrected by the expecta-
tion of the number of switch necessary to recombine the best cassette (figure 3). The dispen-
Results – Evolution of recombination rate in integrons
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sability of the integrons, i.e. its contribution to the global fitness, does not seem to influence
this result on its own. In contrast, low cassette specificity – which is modeled by a wider
Gaussian fitness curve – results in lower recombination rates, especially at high rates of envi-
ronmental change. This can be understood as a consequence of clonal interference (Gerrish
and Lenski, 1998; De Visser et al., 1999). The simultaneous occurrence of cassette with simi-
lar effect on fitness decrease their relative selection coefficient and results in slower evolu-
tionary dynamics. Under rapid environmental change, the frequency of cassettes does not
change fast enough to allow adaptation, even when the best cassette has been reached by re-
combination. In this context, there is no advantage in increasing the mutation rate. Moreover,
it has been suggested that under very rapid environmental change, it may be advantageous to
decrease evolvability, since a genetic response in one generation often decreases adaptation in
the next generation (Kawecki, 2000). The combination of these two factors may explain why
recombination rate decreases at high rates of environmental change and weak selection, a re-
sult that has not been described in pervious models of mutators. Note also that the dispensa-
bility of cassettes does reduce the mean recombination rate under high specificity of cassettes.
In a given integron only the cassettes proximal to the Pc promoter are expressed and
thus subjected to directional selection. The remaining cassettes experience relaxed selective
pressure and may accumulate deleterious mutations, because those cannot be efficiently
purged by natural selection. This should produce a decrease in mean fitness – a genetic load –
that is different from the one directly caused by mutation itself, and more similar to the drift
load (Hartl and Taubes, 1998; Poon and Otto, 2000). We term it the silencing load. To address
the importance of this silencing load, we incorporated a rate of deleterious mutations in the
previous framework. We considered a drastic mutational model wherein a single mutation
leads to gene inactivation. We carried the same simulation as described previously, and moni-
tored the mean frequency of inactivated genes in the cassette reservoir L.
We found that L is inversely proportional to e (figure 4). Under rapidly cycling envi-
ronments (e=10-4), the non-expressed compartment had the time to accumulate up to 8% of
inactivated cassettes, irrespective of the set of parameter used. As the frequencies of environ-
mental shifts decreases, simulations ran with a low cassette specificity (σ=0.8) progressively
cumulate more deleterious mutations than their counterparts. Similarly, simulations ran with
higher dispensability (ω0=0.5) display an increased silencing load. Because both of these pa-
rameters decrease the intensity of selection, these data strongly suggest that natural selection
is involved in purging the silencing load. In rapidly fluctuating environments, cassettes ex-
Results – Evolution of recombination rate in integrons
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perience environments in which they prove adaptive at a higher rate and thus tend to spend
less time in the non-expressed compartment. The silencing load clearly reflects the frequency
at which deleterious mutations are purged by selection when cassettes are put under expres-
sion in a favorable environment. Overall, the silencing load does not affect the population fit-
ness. Although heavy loads are essentially cumulated in slowly fluctuating environments,
these conditions also provide the sustained periods of stasis required for efficient purifying se-
lection. In these conditions, the dynamic of natural selection is fast enough to mediate effi-
cient adaptation. In rapidly changing environments, the efficiency of selection is reduced, but
favorable environment occur fast enough to limit the impact of deleterious mutations on fit-
ness. The introduction of deleterious mutation had no impact on the selected recombination
rate (figure 3).
Discussion
To be written.
Essential points that will be raised include:
Comparison of the integron system with other loci subjected to diversity-generating
mechanisms (e.g. SSRs, gene conversion, epigenetic switches and other systems relying on
site-specific recombination). Highlight the general scope of integron with respect to these sys-
tems.
Similar relationships between the optimal rate of phenotypic switches and the rate of
environmental variations have been reported in theoretical (Kimura, 1967; Lachmann and
Jablonka, 1996; Kussell et al., 2005) and experimental (Acar et al., 2008) studies. However,
these models only consider two phenotypic states in binary environments that change with a
constant period. Discuss the advantage of this stochastic model to address the complex case of
integron without such simplification.
Discuss the control of phenotypic plasticity by recombination-mediated expression.
Contrast it with classical physiological regulation.
Discuss the bet-hedging strategy.
Discuss the benefit of stress-responsive regulation of integrase expression with respect
to constitutive regulation.
Highlight the impact of the system (silencing load) on cassette diversification.
Results – Evolution of recombination rate in integrons
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Figures
Figure 1 - Schematic organization of the integron locus
Integrons forms integrated genetic systems. The intI gene encodes a site-specific tyrosine re-
combinase capable of mobilizing dedicated gene cassettes. Most gene cassettes in the array
are unexpressed. Excision of non-replicative cassette intermediate occurs through random
attC x attC recombination mediated by IntI. Such intermediates are preferentially recombined
in attI through IntI-mediated attC x attI recombination. Newly integrated cassettes are thus
directly put under expression by the Pc promoter. These specific properties enable a versatile
switching mechanism whereby recombination affects the expression of potentially adaptive
traits.
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Fig
ure
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Results – Evolution of recombination rate in integrons
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.
Figure 3 - Impact of the environmental change rate on the selection of recombination rate
Each data point corresponds to the recombination rate averaged over 100 simulation runs. In
each run, the mean mutation rate selected over 100 environmental changes is selected. Envi-
ronmental changes occur stochastically according to a predefined rate. Filled triangles and
filled squares indicate whether deleterious mutations were allowed or not, respectively. Col-
ors distinguish different combination of dispensability ω0 and specificity σ as follow: dark
blue, W0=0 and σ=0.2; light blue, W0=0 and σ=0.8; violet, W0=0.5 and σ=0.2; and red,
W0=0.5 and σ=0.8. The black line correspond to y=(C-1).x, where C-1 is the number of unex-
pressed cassette in the array.
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Figure 4 - Accumulation of deleterious mutations in unexpressed gene cassettes
(silencing load)
The silencing load is defined as the frequency of unexpressed cassette inactivated by deleteri-
ous mutations. Each data point corresponds to the silencing load averaged over 100 simula-
tions. In each run, the mean mutation rate selected over 100 environmental changes is
selected. Environmental changes occur stochastically according to a predefined rate. Deleteri-
ous mutations occurred at a rate of 10-6 per generation. Colors distinguish different combina-
tion of dispensability ω0 and specificity σ as follow: dark blue, W0=0 and σ=0.2; light blue,
W0=0 and σ=0.8; violet, W0=0.5 and σ=0.2; and red, W0=0.5 and σ=0.8.
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II. RECOMBINATION IN INTEGRONS IS
CONTROLED BY THE SOS RESPONSE TO
STRESS
BACKGROUND
Theoretical considerations strongly suggest that the optimal recombination rate in in-
tegron must match the average rate of environmental changes (see Article 1, p153). Two dis-
tinct strategies can be encompassed to implement such a relationship: i) the recombination
rate could be constitutively coded in the integrase, in which it can be slowly fine-tuned
through mutations affecting the protein activity and/or its expression level; and ii) the expres-
sion of the integrase could be responsively regulated by environmental changes. So far, the
expression pattern of integrases resisted experimental analysis (see p146), which is consistent
with the second hypothesis. We thus undertook to identify stress-responses capable of modu-
lating the expression of the integrases.
METHODS
The promoter region of all intI genes deposited in GenBank were recovered and ana-
lyzed for the presence of specific sequence motifs using custom scripts, leading to the identi-
fication of LexA binding sites. To investigate the involvement of the SOS response, electro-
mobility shift assays (EMSA) were performed with material from V. cholerae N16961. The
expression of the integrase in different genetic background and stressing conditions was
monitored using LacZ reporters in V. cholerae and a class 1 integron in E. coli. We devised a
positively selectable reporter of recombination in order to further examine the link between
integrase induction and recombination rate.
RESULTS AND DISCUSSION
We identified a LexA binding motif in the promoter region of most integron inte-
grases. The site is effectively bound by LexA in V. cholerae. The expression of the integrase
is induced by classical trigger of the SOS response, including widely used antibiotics, in both
V. cholerae and E. coli. In contrast, no induction was measured when the SOS response is im-
Results – Recombination in integrons is controled by the SOS response to stress
172
paired. The induction of the integrase has a neat functional impact and strongly increases the
recombination rate. By ensuring diversification in response to a wide range of environmental
challenges, the regulation of recombination rate promotes the evolvability of the organism.
This complex adaptive phenotype arises from the coupling of two simpler genetic modules
and only involves few mutations. Mapping of the LexA binding sites identified in silico to the
IntI phylogenetic tree revealed that the SOS control of recombination pervades marine spe-
cies. In contrast, this trait appears very sporadically in soil and freshwater species, suggesting
different selective pressure in these niches. Strikingly, all clinically relevant multi-resistance
integrons are subjected to SOS control, irrespective of their phylogenetic relationships. This
observation is particularly meaningful in the light of antibiotic-mediated induction of the inte-
grase.
ARTICLE II
This article has been published as a brevia in Science and concisely reports the SOS
control of recombination rate (pp 154-186)
ARTICLE III
This manuscript is in preparation to be submitted to Nucleic Acids Research. It further
discusses the implication of the coupling between the SOS and integron system and its phy-
logenetic distribution (pp 186-211).
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A manuscript to NAR
SOS control of recombination in integron is a primeval feature
Guillaume Cambray1*, Neus Sanchez-Alberola2*, Ivan Erill3*, Susana Campoy3,
Émilie Guerin4, Sandra Da Re4, Bruno Gonzales-Zorn5, Marie-Cécile Ploy4, Jordi Barbé3,
Didier Mazel1
1 Institut Pasteur, Unité de Plasticité du Génome Bactérien, CNRS URA 2171, 75015 Paris,
France 2 Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Barcelona,
Spain 3 Department of Biological Sciences, University of Maryland Baltimore County, Baltimore
21228, USA.
4 Université de Limoges, Faculté de Médecine, EA3175, INSERM, Equipe Avenir, Limoges
87000, France 5 Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de
Madrid, 28040 Madrid, Spain.
*: equal contribution
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SUMMARY
Integrons are found in the genome of hundreds of environmental bacterial species, but
are mainly known as the genetic agents responsible for the capture and spread of antibiotic re-
sistance determinants among Gram-negative pathogens. The SOS response is a regulatory
network under control of the repressor protein LexA and is targeted at repairing and bypass-
ing DNA damages, thus promoting genetic variation in time of stress. We recently reported a
direct link between the SOS response and the expression of integron integrases in Vibrio
cholerae and a plasmid-borne class 1 mobile integron. . Here we conduct a systematic study
of all integron integrase promoter regions available in genomic databases and we show that
LexA controls the expression of most integron integrases. We also provide experimental vali-
dation of integrase LexA control for another Vibrio chromosomal integron and a multi-
resistance plasmid harbouring two integrons. By mapping the distribution of predicted LexA-
binding sites onto an IntI phylogeny, we propose that SOS control arose early and was proba-
bly the ancestral state in integron evolution. Importantly, these data indicates that SOS regula-
tion has been positively selected for in mobile integrons. The coupling of both genetic
systems enhances the potential for cassette swapping and capture in cells undergoing stress
and changing conditions, while freezing the cassette arrangement in steady environments. In
agreement with this, we find a strong correlation between the lack of LexA control and inte-
grase inactivation by mutation, which suggests that unregulated integrase activity may be
deleterious. This discovery highlights the role of integrons and the SOS response as integrated
adaptive systems and will likely have important implications for antibiotic treatment policies.
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Integrons are bacterial genetic elements capable of incorporating exogenous and pro-
moter-less open reading frames (ORF), referred to as gene cassettes, by site-specific recombi-
nation (Figure 1). First described in the late 1980’s in connection to the emergence of
antibiotic resistance (Stokes and Hall, 1989), integrons always contain three functional com-
ponents: an integrase gene (intI), which mediates recombination, a primary recombination site
(attI) and an outward-orientated promoter (Pc) (Mazel, 2006a). Cassette integrations mainly
occur at the attI site (Collis et al., 2002), ensuring the correct expression of mobilized cas-
settes’ genes by placing them under the control of Pc (Levesque et al., 1994). To date, two
main subsets of integrons have been described. On the one hand, mobile integrons, also re-
ferred to as resistance integrons, contain relatively few (2-8) cassettes and encode resistance
to a broad spectrum of antibiotics (Rowe-Magnus and Mazel, 2002; Fluit and Schmitz, 2004;
Partridge et al., 2009). They have been conventionally divided into five different classes ac-
cording to their intI gene sequence (Mazel, 2006a). These are typically associated with mobile
elements, such as transposons and conjugative plasmids, ensuring their dissemination across
bacterial species. They are present mostly in the Proteobacteria, but have also been reported in
other bacterial phyla, such as Gram-positive bacteria (Mazel, 2006a). On the other hand,
chromosomal integrons have been identified in the genomes of many bacterial species
(Boucher et al., 2007). Although many chromosomal integrons comprise a limited number of
cassettes (ref ACID), a subset of them – termed superintegrons (SI) – exhibits large arrays
spanning hundreds of cassettes (Mazel, 2006a). SIs have been specifically identified in the
Vibrionaceae and, to some extent, in the Xanthomonadaceae and Pseudomonadaceae (Mazel
et al., 1998; Rowe-Magnus et al., 1999; Rowe-Magnus et al., 2001; Vaisvila et al., 2001;
Rowe-Magnus et al., 2003; Gillings et al., 2005) and seem to be ancient residents of the host
genome (Rowe-Magnus et al., 2001). Contrasting with mobile integrons, most cassette in a
given SIs display recombination site (attC) that are typical of the species, suggesting that SI
Results – Recombination in integrons is controled by the SOS response to stress
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harbouring bacteria are implicated in cassette genesis (Rowe-Magnus et al., 2003). Most cas-
sette-borne genes in chromosomal integrons are of unknown function (Boucher et al., 2007),
though some of them are related to existing resistance cassettes (Rowe-Magnus et al., 2002;
Melano et al., 2002; Petroni et al., 2004). While stable under laboratory conditions, superinte-
grons have been reported to be the most variable loci among V. cholerae natural isolates
(Rowe-Magnus et al., 1999; Labbate et al., 2007).
Despite the importance of integrons in the acquisition and spread of antibiotic resis-
tance determinants and – from a broader perspective – in bacterial adaptation, little was
known on the dynamics of cassette recombination. Integron integrases mediate recombination
by interacting with single-stranded (ss) attC sites present in all reported cassettes, employing
a unique site-specific recombination process (MacDonald et al., 2006; Bouvier et al.,
2005)xxx(Bouvier, submited). However, the level and control of integrase expression, which
are central to this process, remained enigmatic until recently, when we reported that expres-
sion of the integrases of the V. cholerae superintegron and of a class 1 mobile integron were
controlled by the SOS response (Guerin et al., 2009).
The SOS response is a global regulatory network governed by a repressor protein
(LexA) and principally targeted at addressing DNA damage (Walker, 1984; Erill et al., 2007).
LexA represses SOS genes by binding to highly specific binding sites present in their pro-
moter regions. In E. coli and most β- and γ-Proteobacteria these sites consist of a 16 bp long
palindromic motif (5’-CTGTatatatatACAG-3’), commonly known as LexA box (Walker,
1984). The SOS response is typically induced by the presence of single stranded DNA frag-
ments (ssDNA), which can arise from a number of environmental stresses (Aertsen and Mi-
chiels, 2006), but is normally linked to replication-fork stall due to DNA lesions. These
ssDNA fragments bind non-specifically to the universal RecA protein (Sassanfar and Roberts,
1990), enabling it to promote LexA inactivation by autocatalytic cleavage (Little, 1991) and
Results – Recombination in integrons is controled by the SOS response to stress
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thus inducing the SOS response. Up to 40 genes have been shown to be directly regulated by
LexA in E. coli (Fernandez De Henestrosa et al., 2000; Courcelle et al., 2001), encoding pro-
teins to stabilize the replication fork, repair DNA, promote translesion synthesis and arrest
cell division. Following its initial description in E. coli (Walker, 1984), the SOS response has
been characterized in many other bacterial classes and phyla and LexA has been shown to
bind very different motifs in different phyla (Erill et al., 2007).
In recent years, the SOS response has been linked to clinically relevant phenotypes,
such as the activation and dissemination of virulence factors carried in bacteriophages (Kim-
mitt et al., 1999; Waldor and Friedman, 2005), transposons, pathogenicity islands and inte-
grating conjugative elements encoding antibiotic resistance genes (Erill et al., 2007; Kelley,
2006). Moreover, it has recently become established that some widely used antibiotics, such
as fluoroquinolones, trimethoprim and β-lactams are able to trigger SOS induction and are
thus able to promote the dissemination of antibiotic resistance genes (Erill et al., 2007; Kel-
ley, 2006) or the generation of resistant alleles (Cirz et al., 2005). This puts forward a positive
feedback loop that has been postulated to have important consequences for the emergence and
dissemination of antibiotic resistance (Avison, 2005). Our recent work demonstrating a direct
link between the SOS response and integrase-mediated recombination further reinforces this
line of reasoning, as it provides bacteria with an antibiotic-induced mechanism for gene ac-
quisition, functional expression and dispersal (Guerin et al., 2009). Here we expand on this
recent connection by means of a systematic study of integron integrase promoter regions. Pu-
tative LexA-binding sites are found in the majority of integron integrase promoters, suggest-
ing that the SOS response control recombination in most integrons. We provide further
experimental validation of this control in the Vibrio parahaemolyticus chromosomal integron
and in the integrons of E. coli multi-resistance plasmid pMUR (Gonzalez-Zorn et al., 2005).
The phylogenetic distribution of the LexA controlled integrases suggests that SOS control
Results – Recombination in integrons is controled by the SOS response to stress
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evolved in the ancestor of chromosomal integrons, and that it has been positively maintained
in mobile integrons. We also find a correlation between the loss of LexA control and integrase
inactivation by mutation, indicating that unregulated recombination may be deleterious in
these genetic elements. The only exceptions to this rule appear to be multi-resistance mobile
integrons, in which SOS deregulation leads to the creation of a secondary cassette promoter.
We discuss these findings for the adaptive dynamics of integrons and their implications on the
antibiotic resistance genes acquisition and dissemination.
Results
Identification of LexA-binding sites in intI promoters
We recently identified Escherichia coli-like LexA binding sites overlapping the pro-
moter of the integrase genes intI from all clinically relevant mobile integrons and intIA from
the V. cholerae SI (Figure 2A). We have shown that intI expression was indeed controlled by
the SOS response, eventually resulting in heightened rates of integrase-mediated recombina-
tion upon SOS induction (Guerin et al., 2009).
To gain insight into the general relevance of this observation, we undertook an exhaus-
tive in silico study. Using BLASTP, we identified 296 homologues of intIA in the GenBank
database and systematically searched the nucleotide sequences corresponding to their coding
region plus 501 bp upstream. We conducted independent searches for each of the described
LexA-binding motifs (Erill et al., 2007). Putative LexA-binding sites were detected in 66%
(196) of the 296 sequences (Table S1) All the identified LexA-binding sites corresponded to
the motif found in E. coli and most β/γ-Proteobacteria,. This suggests that the putative LexA
regulation of intI genes probably originated after the split of the α- and β/γ -Proteobacteria
subclasses, since the LexA-binding motif of α-Proteobacteria is markedly divergent from the
E. coli one (Tapias and Barbe, 1998). When we examined the core 16 bp of the identified E.
Results – Recombination in integrons is controled by the SOS response to stress
the LexA-binding sites exhibit a high level of conservation, as reflected in their joint informa-
tion content logo (Figure 2C), which contrast with the immediately surrounding sequences.
This strongly support the functionality of these motifs. Importantly, E. coli-like LexA sites
were detected in all but one of the mobile integron classes and in almost all Vibrionaceae su-
per-integrons (Figure 2B), evidencing that putative LexA regulation of intI genes is a wide-
spread phenomenon pervading all integron divisions.
Predicted LexA-binding sites correspond to functional transcriptional control elements.
We have previously shown that LexA regulates the expression of intI in V. cholerae,
and our in-silico search identified LexA-binding sites in the promoter region of intI for all se-
quenced Vibrio species but V. fischeri (Table S1). To further assess the overall functionality
of the in silico predicted LexA-binding sites, we evaluated integrase LexA regulation in V.
parahaemolyticus ATCC 17802, which harbours a LexA-binding site upstream of its intIA
gene in a genetic context that is substantially different from the one of V. cholerae (Figure
2A). Using RT-PCR, we determined the intIA expression level in both the wild-type strain
and its lexA(Def) derivative. We found an expression ratio of 6.18, revealing a strong LexA
regulation of the intIA gene expression (Figure 3A).
In several class 1 integrons, heightened expression of the cassette genes has been
shown to rely on a secondary cassette promoter called P2, located just upstream of the intI1
gene (Figure S1). P2 is enabled by a CCC insertion that increases the distance between a -35
box sequence and a sequence resembling the -10 box consensus from 13 to 16 bp, thereby
generating a functional 70 promoter (Kim et al., 2007; Collis and Hall, 1995). In all its re-
ported instances, this CCC insertion takes place in what appears to be a disrupted LexA-
binding site. Therefore, the CCC insertion that enables P2 should simultaneously abolish inte-
grase regulation by LexA. Here we tested this hypothesis using the E. coli multi-resistant
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plasmid pMUR050 (Gonzalez-Zorn et al., 2005). This plasmid provides an ideal material to
address this issue because it harbours two integrons with inactivated copies of the intI1 gene
(Figure S1). However, only one of these intI genes contains a functional LexA-binding site in
its promoter, while the other presents a CCC insertion disrupting the LexA-binding site (Fig-
ure S1). Using EMSA, we found that the CCC insertion effectively prevents LexA-binding
(Figure 3B). Furthermore, RT-PCR in WT and lexA(Def) backgrounds confirmed that LexA
regulation was only observed in the integron carrying an intact LexA-binding site, with a
strong deregulation (6.55 ratio) in the lexA(Def) background (Figure 3A). Thus, the CCC in-
sert does not only enable the secondary cassette promoter P2, but concomitantly disrupts the
LexA-binding site of the integrase promoter. Evidence of increased cassette expression due to
the CCC insert was obtained by comparing RT-PCR expression profiles for the first cassette
gene of both pMUR integrons, and this increase was found to be independent of the lexA(Def)
background (data not shown). In-silico searches for disrupted LexA-binding sites revealed 31
such instances in integrons from a wide variety of species (Table 1). Furthermore, all the
identified CCC insertions corresponded to multi-resistance mobile integrons. Together, these
results suggest that LexA regulation may be eventually lost under heavy selection to promote
higher basal levels of the antibiotic resistance transcript.
Analysis of LexA-binding sites distribution
The presence of confirmed LexA regulation in V. cholerae and V. parahaemolyticus
SIs suggested that SOS induction of intI genes probably originated very early in the evolu-
tionary history of integrons. To further explore this hypothesis, we mapped the in silico iden-
tified LexA-binding sites onto a phylogenetic tree of IntI protein sequences. The tree shown
in Figure 4 is in overall agreement with previously published IntI phylogenies (Diaz-Mejia et
al., 2008; Mazel, 2006a; Nemergut et al., 2008; Boucher et al., 2007). It distinguishes two
major ecological groups. Integrons borne by marine species form a monophyletic clade, with
Results – Recombination in integrons is controled by the SOS response to stress
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the Vibrionaceae super-integrons sitting at the root of the tree. Integrons from soil and fresh-
water bacteria, on the other hand, seem to constitute a more recent branch. As has been noted
previously, the tree also put forward that multiresistance mobile integrons probably arose sev-
eral times in both ecological groups (classes 2, 4 and 5 [green panel] and classes 1 and 3 [or-
ange panel] in Figure 4).
The distribution of identified LexA boxes in Figure 4 shows that LexA regulation of
intI genes is prevalent among marine chromosomal integrons and their cognate mobile rela-
tives. Conversely, no functional LexA-binding sites can be identified in the chromosomal in-
tegrons from soil and freshwater species. Nonetheless, the mobile integrons branching off
from soil and freshwater bacteria do contain functional LexA-binding sites, hinting that LexA
regulation could have been lost in most non-marine chromosomal integrons but has been pre-
served in their related mobile counterparts.
DISCUSSION
Coupling of integrons with the SOS response
We have recently demonstrated that the SOS response regulates the expression of two
integron integrase genes, leading to heightened recombination rates upon SOS induction, both
in a class 1 mobile integron and in the V. cholerae SI (Guerin et al., 2009). The extensive in
silico search reported here shows that about two thirds of the available integron integrase se-
quences are putatively regulated by LexA, and this regulation has been confirmed here for ad-
ditional integrase genes. In hindsight, the coupling of genetic elements capable of cassette
integration with a global response to stress comes out as an elegant and powerful pairing. As
illustrated in Figure 1, integrons can be seen as stockpiling agents of genetic diversity, which
in addition, can tap into a huge and variable pool of cassettes through horizontal gene transfer
from the surrounding bacterial communities (Boucher et al., 2007; Michael et al., 2004;
Koenig et al., 2008). Nonetheless, the efficient expression of these acquired traits is highly
Results – Recombination in integrons is controled by the SOS response to stress
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dependent on integrase-mediated recombination. Newly integrated cassettes sitting in the
proximal region of the integron are highly expressed by the Pc promoter, but they can be
moved to distal parts of the integron and thus progressively put away from expression by con-
secutive recombination events (Figure 1), which may also reinstate formerly acquired cas-
settes under full expression.
The SOS response comes thus as an obvious choice for regulation of integrase activ-
ity, as it is already a key component of adaptive mutagenesis in bacteria, triggering both tran-
slesion synthesis and activation of transposable elements (Bjedov et al., 2003; Ubeda et al.,
2007). Furthermore, SOS induction is carefully timed to those periods of stress in which adap-
tive mutagenesis can be particularly advantageous. In the early chromosomal integrons, where
SOS regulation apparently arose, LexA repression of the intI gene may have contributed to
integron stability by minimizing the basal expression levels of intI and thus decreasing the
rates of integrase-mediated recombination. Then again, SOS regulation would have ensured
that both the occasional cassette reordering and the acquisition of exogenous cassettes took
place at a time of need for innovation, such as in reaction to antibiotic exposure. Therefore,
regulation of integrase activity by the SOS response comes as a natural way to optimize inte-
gron-mediated adaptation without incurring in excessive integron destabilization or in the
possible toxic effects of sustained integrase expression.
Loss and persistence of integrase LexA-regulation
The phylogenetic distribution of LexA-binding sites reveals an apparent loss of LexA
regulation in several instances. LexA regulation of intI genes is clearly prevalent among ma-
rine species. Loss of LexA regulation is only observed in the SXT integrating-conjugative
element, for which SOS-dependent transfer has been reported (Kelley, 2006) and in Vibrio
fischeri. Curiously enough, in V. fischeri LexA shares its binding motif with the LuxR quo-
rum sensing regulator (Shadel et al., 1990), suggesting that LexA regulation of intI may have
Results – Recombination in integrons is controled by the SOS response to stress
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been lost in this species to prevent interference with the lux regulon.
Conversely, loss of LexA regulation seems to be the norm among soil and freshwater
species harbouring chromosomal integrons. In some cases, this loss of regulation has an obvi-
ous explanation. Some families, like the Nitrosomonadaceae and the Chromatiaceae, simply
do not possess any LexA homologues, thus explaining the absence of this motif upstream of
their intI genes (Erill et al., 2007). A similar, yet less powerful argument can be made for the
Xanthomonadaceae, in which neither of the two identified LexA proteins recognizes the β/γ-
Proteobacteria LexA-binding site (Yang et al., 2002),. However, the main mechanism associ-
ated with the loss of LexA-regulation appears to be the inactivation of the integrase gene. The
majority of Xanthomonadaceae chromosomal integrases, for instance, are inactivated by di-
verse types of mutations and deletions (Gillings et al., 2005). There is also evidence that
frame-shift mutations may have inactivated most of the remaining intI genes lacking apparent
LexA regulation (Nemergut et al., 2008). Thus, it seems that many species may have opted
for inactivating their intI gene upon loss of LexA regulation, or that accidental inactivation of
intI has made LexA regulation superfluous. Both lines of reasoning strongly suggest that un-
regulated intI expression must be somehow detrimental to the cell, thereby introducing an ad-
ditional selective pressure towards the initial emergence of LexA regulation of integron
integrase genes. Further strengthening this conclusion, it is a well known informal observation
for worker in the integron field that experimental overexpression of the integrase is deleteri-
ous to the cell.
In contrast to their soil and freshwater chromosomal relatives, most class 1 and class 3
integrases are both LexA regulated and functional. This indicates that the capability of cas-
sette uptake and shuffling is a useful trait in mobile integrons, since it would allow their hosts
to express novel phenotypes in selective environments. This parallels the persistent regulation
by LexA of functional integrase genes in marine species, in which reorganization of the su-
Results – Recombination in integrons is controled by the SOS response to stress
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perintegrons has been evidenced by comparative genomics (Labbate et al., 2007)(+ref feng
2008). In any case, the preservation of LexA regulation in integrons harbouring functional in-
tegrases suggests again that, if not mandatory, LexA regulation of intI genes must be overtly
beneficial for integron hosts when the product of the intI gene is a functional protein. The
only exception to this general rule appears to be deregulation mediated by a CCC insertion
that disrupts the LexA-binding site. This same CCC insert, however, enables a secondary cas-
sette promoter (P2) that enhances cassette expression, and in silico search results evidence
that this insert is only found in multi-resistance plasmids. This suggests that the detrimental
effects of unregulated intI expression trade-off with the selective pressure towards increased
expression of multi-resistance phenotypes. Nonetheless, as the pMUR case illustrates, en-
hanced cassette promoter may be maintained with a subsequent inactivation of the integrase,
in this case by a IS26 insertion.
Clinical implications of SOS-induced integrase activity
Both integrons and the SOS response have been previously singled out as elements of
clinical importance and have therefore been the focus of abundant research in the fight against
antibiotic resistance and antibacterial drug development (Weldhagen, 2004; Nijssen et al.,
2005; Cirz et al., 2005). Beyond its fundamental relevance to bacterial adaptation, serious
clinical implications emerge from the discovery of a direct link between SOS induction and
integrase activity. Since most multi-resistant Gram-negative bacteria carry mobile integrons,
this establishes a generic system for genetic interchange under control of a general stress re-
sponse shared by a large group of human and animal pathogens. In this setting, it is important
to note that integron cassettes encoding resistance to several antibiotics known to induce the
SOS response, such as trimethoprim, quinolones and β-lactams, are common today (Rowe-
Magnus and Mazel, 2002; Fonseca et al., 2008). This suggests that the indirect triggering ef-
fect of these antibiotics on the capture of resistance cassettes has been very efficient.
Results – Recombination in integrons is controled by the SOS response to stress
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A less obvious consequence of integrase SOS regulation is its repercussion on antibi-
otic resistance policies. Current policies in the fight against antibiotic resistance rely largely
on the detrimental effects most resistance mechanisms inflict on bacteria, which eventually
lead to loss of resistance genes in the absence of antibiotic exposure (Andersson, 2006). Since
most cassettes are promoter-less, the most ancient cassettes (located at the distal part of the
integron) are subject to severe polar effects, leading to rare or non-existent protein products
(see Figure 1) (Collis and Hall, 1995). In this context, the incorporation of SOS regulation in
integrons puts forward a mechanism by which antibiotic-resistance genes and other useful ad-
aptations can be silently set aside, while current adaptive traits are steadily kept under expres-
sion. In time of stress, such as exposure to antibiotics, the relevant resistance cassette can be
called upon by integrase-mediated translocation, and thus selected for only when its expres-
sion is required. Furthermore, cassette’s genes temporarily relegated to distal positions in in-
tegrons may also sustain increased evolution rates, generating a substantial pool of variability
from which to draw on when the appropriate selective pressures resurface. Therefore, SOS
mediated regulation of integron integrases should be taken into account regarding the time
spans currently being considered for spontaneous loss of antibiotic resistance in restrictive use
policies and, ultimately, concerning the future development and assessment of antibiotic
guidelines.
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MATERIALS AND METHODS
In silico searches and phylogenetic analyses were made on sequences deposited in
GenBank as described previously (Abella et al., 2007; Mazel, 2006b). The electro-mobility
shift assays were performed as described before (Abella et al., 2007). The different lacZ re-
porter constructions were made by fusion at the initiation codon of the tested genes and -
galactosidase activities were measured in Miller units. The full list of strains and plasmids is
available as Table S7, and oligonucleotide primers are listed in Table S8. Full methods and
associated references are described in the supplementary text.
ACKNOWLEDGEMENTS
We thank Mike C. O’Neill for his careful reading and comments on the different ver-
sions of this manuscript. This work was supported by grants from the Ministère de la Recher-
che et de l’Enseignement supérieur, the Conseil Régional du Limousin, the Fondation pour la
Recherche médicale (FRM) and from the Institut National de la Santé et de la Recherche
Médicale (Inserm) for the Ploy lab; by the Institut Pasteur, the Centre National de la Recher-
che Scientifique (CNRS-URA 2171), the FRM and the EU (STREP CRAB, LSHM-CT-2005-
019023, and NoE EuroPathoGenomics, LSHB-CT-2005-512061), for the Mazel lab; and by
grants BFU2008-01078/BMC from the Ministerio de Ciencia e Innovación de España and
2005SGR-533 from the Generalitat de Catalunya, for the Jordi lab.
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Figures
Figure 1 – Schematic organization of integrons
The functional platform of integrons is constituted by an intI gene encoding an integrase, a
cassette promoter Pc and a primary recombination site attI. The system maintains an array
that can consist in more than 200 cassettes in chromosomal superintegrons. Only the few first
cassettes are expressed by the Pc, a feature represented by the fading filling color. The rest of
the array can be seen as a reservoir of standing genetic variation. A cassette is generally con-
stituted of a promoterless ORF flanked by two recombination sites termed attCs. Cassettes
can be excised from any position in the array through attC x attC recombination mediated by
the integrase. The resulting circular intermediate can then be integrated by the integrase, pref-
erentially at attI bringing the cassette under control of Pc. Note that exogenous circular inter-
mediate can be integrated owing to the low specificity of the integrase activity, rendering the
system prone to horizontal transfer. In the present study, the integrase promoter Pint is shown
to be under the control of the SOS system, thus conditioning recombination to periods of SOS
inducing stress.
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Figure 2 – In silico analysis of integrases promoter
(A) Alignment of representative promoter regions of Vibrionaceae intIA homologues. Puta-
tive LexA-binding sequences are boxed, while putative σ70 promoter elements (-35 and -10)
are underlined and the translation start site of intIA is boxed and highlighted in bold type. (B)
Representative examples of LexA-binding sites identified upstream of different integrase
genes. MI stands for mobile integron, while SI stands for superintegron and the subsequent
number (1-5) denotes integrase class. The provided accessors correspond to IntI proteins
Results – Intrinsic evolutionary potential of genes
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224
Figure S2: Alignment of aacWT and aacELP sequences
While encoding identical proteins, aacWT and aacELP only share 61% identity. In this figure,
different bases are highlighted in red. Overall, 119 codons out of 184 are different between
the two sequences.
Figure S3: Number of mutations and protein space exploration
Min. number of mutations 1 2 3
From single codon 30 51 19 Average %
of aa acces-
sible From all synonymous codons 40 53 7
No amino acid shows more than four codons with different REP. Thus, at any position, a set
of four ELP-designed sequences accesses the same evolutionary landscape as do all the syn-
onymous codons corresponding to the position considered. We compared the minimum num-
ber of mutations necessary to reach the other 19 aa from either a single codon or all
synonymous codons. This figure summarizes the percentage of amino acid accessible in 1, 2
or 3 mutations averaged over the 61 single codon or the 20 sets of synonymous codons. The
use of four ELP-designed sequences, achieves a shift toward a lower number of mutations. It
drastically decreases the number of substitutions requiring three mutations by codon.
A T G A C C A A C A G C A A C G A T T C C G T C A C A C T G C G C C T C A T G A C T G A G C A T G A C C T T G C G
A T G A C G A A C T C G A A T G A C A G C G T G A C C C T C A G A T T G A T G A C G G A A C A C G A T T T G G C C
A T G C T C T A T G A G T G G C T A A A T C G A T C T C A T A T C G T C G A G T G G T G G G G C G G A G A A G A AA T G T T G T A C G A A T G G T T G A A C A G A A G T C A C A T T G T G G A A T G G T G G G G G G G T G A G G A G
G C A C G C C C G A C A C T T G C T G A C G T A C A G G A A C A G T A C T T G C C A A G C G T T T T A G C G C A AG C T A G A C C C A C T T T G G C A G A T G T C C A A G A G C A A T A T C T T C C C T C G G T G C T G G C C C A G
G A G T C C G T C A C T C C A T A C A T T G C A A T G C T G A A T G G A G A G C C G A T T G G G T A T G C C C A GG A A A G T G T G A C G C C C T A T A T C G C T A T G C T T A A C G G T G A A C C C A T C G G T T A C G C A C A A
T C G T A C G T T G C T C T T G G A A G C G G G G A C G G A T G G T G G G A A G A A G A A A C C G A T C C A G G AA G T T A T G T G G C A T T G G G T T C G G G T G A T G G T T G G T G G G A G G A G G A G A C G G A C C C C G G T
G T A C G C G G A A T A G A C C A G T T A C T G G C G A A T G C A T C A C A A C T G G G C A A A G G C T T G G G AG T C A G A G G T A T T G A T C A A C T G C T T G C C A A C G C T A G T C A G C T T G G G A A G G G G C T T G G T
A C C A A G C T G G T T C G A G C T C T G G T T G A G T T G C T G T T C A A T G A T C C C G A G G T C A C C A A GA C G A A A T T A G T G A G A G C A T T A G T G G A A C T T C T T T T T A A C G A C C C A G A A G T G A C G A A A
A T C C A A A C G G A C C C G T C G C C G A G C A A C T T G C G A G C G A T C C G A T G C T A C G A G A A A G C GA T T C A G A C T G A T C C C A G T C C C T C G A A T C T T A G A G C C A T T A G A T G T T A T G A A A A G G C C
G G G T T T G A G A G G C A A G G T A C C G T A A C C A C C C C A G A T G G T C C A G C C G T G T A C A T G G T TG G T T T C G A A C G T C A G G G G A C G G T C A C G A C G C C C G A C G G G C C C G C A G T T T A T A T G G T G
C A A A C A C G C C A G G C A T T C G A G C G A A C A C G C A G T G A T G C C T A A aac_wt C A G A C T A G A C A A G C T T T T G A A A G A A C T A G A T C G G A C G C A T G A aac_syn
aac WT aac ELP
Results – Intrinsic evolutionary potential of genes
1 AACelps were designed to construct the synthetic gene aacELP 2 AACmuts were used to amplify cloned genes 3 The letter p indicates phosphorylation
Results – Intrinsic evolutionary potential of genes
Article IV
226
able S2: Properties of the mutant libraries
1 Libraries were generated by error-prone PCR from each version of the gene aac(6’)-Ib. 2 Each pool contained approximately the same number of clones, as estimated on plates be-
fore selection. 3 The mean mutation rate is 2.5 mut./kb for aacWT and 2 mut./kb for aacELP
# Size2 Mut. Rate
1 >106 0.52 >106 1.33 >106 3.1 aa
c WT
1
4 >106 5.21 >106 1.32 >106 0.93 >106 2.5aa
c EL
P1
4 >106 3.2
Results – Intrinsic evolutionary potential of genes
Article IV
227
Text S1: Modeling of the relationship between protein space exploration and library size
Let us assume that the sequence is L nucleotides long and that any modification in a fraction f
of its positions is not lethal (i.e. leads to properly folded proteins [1]).
The probability that a sequence codes for a properly folded proteins after m independent mu-
tations is:
!
)!(.
)!(
)!()( .
L
mL
mLf
Lf
C
CmP
mL
mfL
f
(1)
The denominator is the total number of mutants bearing m mutations, while the numerator is
the number of combinations in which these mutations does not adversely affect protein func-
tion. Assuming the sequence length L is much larger than the number of introduced mutations
m (L >> m), this equation simplifies into:
mf fmP )( (2)
which is consistent with several studies [1, 2].
Let us now consider a given target optimal genotype that is k mutations away from the
reference one. Among sequences with m mutations, the probability that the k desired muta-
tions are present is:
otherwise 0 andk m if ,)|(
kkL
km
mL
kmkL
(m-k)!L
m!
C
C
C
CmutationsmsolutionP (3)
The probability that a sequence with m mutations encodes a properly folded protein and con-
tains the k desired mutations directly stems from equation (2) and (3):
otherwise 0 andk m if ,)|( k
m
(m-k)!L
m!fmutationsmfoldedandsolutionP (4)
Results – Intrinsic evolutionary potential of genes
Article IV
228
If we assume, as usual, that a library is composed of sequences with a Poisson distributed
number of mutations with mean X, then the probability to find the target sequence coding for
a properly folded protein is:
k
m
km
mX
Lkm
mf
m
XeaverageonmutationsXfoldedandsolutionP
)!(
!
!)|(
(5)
which simplifies into:
k
fX
L
fXeaverageonmutationsXfoldedandsolutionP
)1()|( (6)
The inverse of (6) is the mean library size required to generate one target clone.
Deriving equation (6) with respect to X gives the optimal mean mutation rate respective to
targets k mutations away
f
kX opt
1
(7)
The graph below displays the inverses of equation (6) for target variants at k=1 (red), k=2
(orange) and k=3 (yellow) mutations away from the template. We assumed a standard bacte-
rial gene length (L=1000) and a conservative proportion of non-deleterious mutations at the
DNA level (f=3/4, corresponding to 1/3 of lethal aa substitutions (Dawid et al.)). Numbers on
the left side scale are obtained by calculating the inverse of equation (6) for X equal to Xopt
from equation (7).
Results – Intrinsic evolutionary potential of genes
Article IV
229
The increase in required size between a library covering a mutational distance k+i and one
targeting k mutations is:
i
fX
L
averageonmutationsXfoldedandiksolutionP
averageonmutationsXfoldedandksolutionP)
.(
)|(
)|(
(8)
The larger the mean number of mutations, the higher the chance to recover a target further
away. However, optimal mutation rate for error-prone PCR derived libraries are predicted to
be rather low, even when subtle advantages of high mutation rate are taken into account [2].
The following graph displays equation (8) for i=1 (red) and i=2 (orange). A substantial in-
crease in library size is required to fully explore possibilities, even with a somewhat high mu-
tation rate of 4 mutations on average per gene (dotted line). As the occurrence of several
mutations in the same codon is very rare using error-prone PCR, these curves can be inter-
preted as lower bounds to the increase in library size necessary to obtain a 2 or 3 mutations in
the same codon instead of 1.
Results – Intrinsic evolutionary potential of genes
Article IV
230
The overall picture could have been worse if we had assumed a cumulative effect of muta-
tions: due to negative epistasis neutral mutations may become deleterious when they accumu-
late [4].
1. Bloom JD, Silberg JJ, Wilke CO, Drummond DA, Adami C, Arnold FH (2005) Thermody-
namic prediction of protein neutrality. PNAS :606–611.
2. Drummond DA, Iverson BL, Georgiou G, Arnold FH (2005) Why High-error-rate Random
Mutagenesis Libraries are Enriched in Functional and Improved Proteins. J. Mol. Biology
350: 806-816.
3. Guo HH, Choe J, Loeb LA (2004) Protein tolerance to random amino acid change. PNAS
101: 9205-9210.
4. Bershtein S, Segal M, Bekerman R, Tokuriki N, Tawfik DS (2006) Robustness-epistasis
link shapes the fitness landscape of a randomly drifting protein. Nature 444: 929.
Results – Intrinsic evolutionary potential of genes
Article IV
231
Data S1: Alignment of the aac(6')-Ib homologs identified by BlastP
The protein sequence AAC(6')-Ib was blasted again the NCBI nr protein database as of
2007/08/26. Corresponding nucleotide sequences were fetched, sorted and aligned using a
dedicated BioPerl script.
The file can be downloaded on the PLoS Genetics server
shtein et al., 2006; Weinreich et al., 2006) and the fluorescent proteins – such as GFP
(Crameri et al., 1996; Sacchetti et al., 2000; Miyawaki et al., 2005; Shaner et al., 2007) –
would be privileged candidates in this respect.
III.2. Synthetic integrons
While the ELP principle can speed up protein evolution through point mutations, the
integrons system may prove useful to engineer whole metabolic pathways. Indeed, integrons
basically perform combinatorial rearrangement of silent gene cassettes under the control of a
single promoter, thereby providing the opportunity to select for the best arrangement of ex-
pressed cassette under appropriate conditions.
We developed a directed evolution protocol whereby a library of synthetic cassettes
harboring genes of interest is introduced in an E.coli strain containing an inducible intI1 gene
and a chromosomal attI site associated with a strong inducible promoter. Upon induction of
the integrase, cassettes from the library are randomly recombined at the attI site – so that ex-
tensive variability is generated at the population level. Expression of the array can then be
turned on to screen individual cells for desired properties. Successive rounds of recombina-
tion-selection may be chained until no further improvements could be detected. As a proof of
principle, we introduced the five genes of the E. coli tryptophan operon into separate cas-
settes. These functional cassettes – interspersed with three inappropriate ones (one containing
lacZ and two harboring a transcription terminator) – were cloned in disarray into a library
plasmid. Cells transformed by this plasmid were selected for growth in minimal medium after
thay have been subjected to IntI1-mediated shuffling. Preliminary data indicate that several
functional arrangements can readily be selected (D. Bikard, unpublished results). Although
the involvement of the few first cassettes of a natural integron in the same functional path-
ways has never been reported (but see Elsaied et al., 2007), these data suggest that integrons
may facilitate the emergence of operons.
The synthetic cassettes have been constructed according to a standardized, fast, and ef-
ficient procedure inspired by the rise of synthetic biology standards. This integron-based
combinatorial strategy would be easily applicable to the optimization of artificial biochemical
pathways, when competing candidate genetic elements are available. Alternatively, the design
of attC sites with primary sequences coding for flexible polypeptides would permit to use this
system to shuffle protein domains. Such a tool may prove particularly valuable in the genera-
Discussion – Biotechnological considerations
250
tion of the multi-modular enzymes which are responsible for the synthesis of polyketides
(Menzella and Reeves, 2007). In this perspective, the very system that initially led to the rise
of antibiotic resistances would ironically be subverted to produce brand new antiobiotics.
251
252
253
APPENDIX
Appendix – Epistemological considerations on the role of variations in biology
254
EPISTEMOLOGICAL CONSIDERATIONS ON THE
ROLE OF VARIATIONS IN BIOLOGY
Maintenance versus variability: a major evolutionary
trade-off
Cats do not make dogs and children tend to resemble their parents. These simple ob-
servations are accessible to everyone’s immediate experience. They nonetheless underlie two
related, essential and long mysterious biological processes: the maintenance of species char-
acteristics and the inheritance of individual traits over time. Another compelling observation
is that, beyond the astonishing diversity of living forms on Earth, some species exhibit patent
similarities between each others. This allowed generation of naturalists since Aristotle to un-
dertake systematic classifications of animals and vegetals. These two concepts are seemingly
difficult to reconcile: on the one hand, heredity entails the transfer of unchanged information
from parent to offspring within species, while on the other hand the study of diversity unveils
the profound link between species.
For ages, at least in occident, the most successful explanation of this paradox was one
coupling the platonic’s idea of essential types to the existence of an intelligent and omnipotent
agent responsible for their creation and embodiment. Essentialism holds that, for any specific
kind of entity, there is a set of permanent, unalterable, and eternal characteristics, all of which
any entity of that kind must possess. Real entities then stand as imperfect manifestations of
their ideal essence. This view goes along well with the concept of biological species. Indeed,
if there are some accidental differences between individuals, none had ever observed modifi-
cation of a species’ representative traits over a human lifetime. The belief that an intelligent
force, a demiurge, created the essences reaches back to Plato (ca. 428-348 BC) and may
somehow account for the observed relationship between species. Suffice it to say that, along
its initiative, the demiurge was inspired by its former creations and accordingly developed a
range of resembling forms. Some pre-Socratic philosophers, e.g. Empedocles (ca. 490–430
BC) and Democritus (ca. 460-370 BC) and their followers, such as Epicurus (ca. 341-270 BC)
and Lucretius (ca. 99-55 BC) rejected these deterministic ideas and let much space to chance
and contingency in their world views. Such metaphysical edifices are not meant to satisfy the
scientific principle of objectivity, but rather appear as a posteriori constructions justifying
Maintenance versus variability: a major evolutionary trade-off
255
ethical and political beliefs. The essentialist view established itself in the Eastern and Middle-
Eastern thoughts because it fitted well the precepts of the Abrahamic religions (essentially Ju-
daism, Christianity and Islam). These religions had, and still have, a profound impact on hu-
man societies, ethics and sciences. A famous illustration, on which we will come back later, is
the natural theologism and fixism of Carl Linnaeus. The Swedish taxonomist believed his
classification scheme to reveal the divine order of God's creation. In his own words: “There
are as many species as the number of different forms created by the Infinite Being in the be-
ginning. These forms have then according to the inherent laws of creation always produced
offspring like themselves, so that we do not now find more species than have previously ex-
isted. Thus, there are as many species as there are different forms or structures if we exclude
the non-essential deviations (varieties) that are conditioned by the habitat or by fortuities”
(As cited in (Gustafsson, 1979)). Beyond the three main monotheisms and to the best of my
knowledge, all civilizations developed cosmogonies to account for the biosphere by the ab ni-
hilo appearance of demiurge-like entities and the subsequent transformations, emanations or
creations of living forms.
Because of complex socio-cultural factors, a lot a people still believe in these types of
metaphysical explanations. However, the last 150 years have seen the birth and development
of a consistent and powerful body of knowledge, i.e. a theory, the modern evolutionary syn-
thesis, which allow biologists to account for these natural facts in an objective, scientific, and
much more satisfactory manner. We now know the nature and mechanisms of transmission of
the genetic material responsible for heredity. We also know that all diverse living forms, be-
yond their perceptible macroscopic similarities, dwell in a profound mechanistic unity and re-
late to each other by common descent.
Given the aforementioned fixist grasp of life, the explanation of relationship between
species by way of common descent was difficult to admit. Indeed, the concept of common de-
scent supposes the apparition and perpetuation of modifications into species. When consid-
ered separately, the ideas of modification and perpetuation were not so problematic. For
instance, the appearance of viable variations in cultivated plants is so common that they can-
not be unnoticed. Regarding this issue, Linnaeus wrote: “Let a garden be sown with a thou-
sand different seeds, let to these be given the incessant care of the Gardener in producing
abnormal forms, and in a few years it will contain six thousand varieties, which the common
herd of Botanists calls species. And so I distinguish the species of the almighty Creator which
are true from the abnormal varieties of the gardener: the former I reckon of the highest im-
portance because of their author, the latter I reject because of their authors. The former
Appendix – Epistemological considerations on the role of variations in biology
256
persist and have persisted from the beginning of the world, the latter, being monstrosities, can
boast of but a brief life” (as cited in (Gouyon et al., 2002)). This way, modifications were
usually seen as fortuitous anomalies that could only be perpetuated by artificial selection, but
would be quickly eliminated otherwise. The real trouble arose when naturally occurring and
heritably stable variants were discovered. Linnaeus was once confronted with a mutant of the
otherwise well described Linaria vulgaris species (see Figure 40), in which the fundamental
symmetry of the flower is changed from bilateral to radial. The specimen was in complete
contradiction with the botanist’s classifying system, which is grounded on flower morphol-
ogy. This naturally led him to name the plant Linaria peloria, i.e. monster in ancient Greek
(Gustafsson, 1979). The case troubled the taxonomist’s faith, and eventually had him embrace
the possibility that “all species be-
longing to the same genus originally
formed a single species which diversi-
fied by hybridization” (as cited in
(Gouyon et al., 2002)). In other
words, the constancy of divine crea-
tion might only hold until genera. We
now know that the flower’s altered
phenotype is the consequence of an
epimutation (Cubas et al., 1999), i.e.
due to an epigenetic phenomenon (see
Epigenetics, p89). As we will see
throughout this work, bacteria are
particularly prone to genetic modifications. It is interesting to muse that, if basic techniques of
microbiology were available before L. Pasteur (1822-1895) and colleagues set those up, a lot
of spontaneous and self-perpetuating variations could have been observed. In hindsight and
despite its falseness, the fixist paradigm initially subtended the edification of classifications
and may thus be considered as a necessary epistemological intermediate. Indeed, the devel-
opment of systematic classifications drove a fantastic accumulation of specimens, such as
peloria, which in turn reinforced the idea of evolution.
Aside from these paradigmatic and religious considerations, the question of genetic
modification remains intricate and is somehow at odds with the concept of heredity. If the ex-
istence of variations is necessary for evolution to occur, their introduction in the hereditary
equation raise tricky questions concerning the control of their generation. Maintenance of ge-
The purpose of evolution
257
netic integrity is indeed essential for the continuation of important traits over time. Because
most alterations are deleterious, too much variation is likely to hinder the stability of the or-
ganism. However, too few variations might not allow sufficient evolution to changing living
conditions. In this respect, successful adaptation obviously requires an exquisite balance be-
tween stability and variability. How such a balance can be established is the central theme of
this work. Before addressing this issue, it is worth wondering why evolution is necessary,
what is exactly meant by adaptation and what constitute its fundamental mechanisms.
The purpose of evolution
The title of this section is deliberately provocative. Evolution is often presented as
blind and contingent process, and I will certainly not argue against that. Nevertheless, this de-
scription alone might be misleading and I would like to highlight the reasons for that. At the
same time, this will permit to bring out the necessity for evolutionary processes and thus the
necessity of variations.
Form, function and the watchmaker
When I was an undergraduate student, I was taught not to say that eyes are made to
see. In the same light, I was also said that an animal has sight because it has eyes, while for-
bidden to think that it has eyes because he has the need of sight. Without further explanations
(and there were not) these assertions are absolute nonsense in the light of modern biology. In
his essay Chance and Necessity, J. Monod (1910-1976) highlights “how much arbitrary and
pointless it would be to deny that the natural organ, the eye, represents the materialization of
a ‘project’ (the one of capturing image)”, and that “one of the fundamental properties com-
mon to all living beings without exception [is] that of being objects endowed with a purpose,
which at the same time they exhibit in their structure and carry out in their performance”
(Monod, 1970). The seemingly perfect adequacy between forms and functions pervade all
levels of biological organization, from whole organs to nanoscopic molecular machines,
Whether the function followed the form or the contrary (i.e. do we see because we
have eyes or have we eyes in order to see?) is an age old question again tracing back to Greek
philosophers such as Plato (ca. 428-348 BC), Democritus (ca. 460-370 BC) and Aristotle (ca.
384-322 BC). The implications of this debate extend beyond natural sciences and reach met-
physical concepts. The true question behind the alternative is to determine whether the exis-
Appendix – Epistemological considerations on the role of variations in biology
258
tence of purposeful biological structures is merely accidental or whether a force drives the de-
velopment of their intrinsic projects. That the former point must be false stands as an obvious
fact today. The functions, i.e. the adaptations of structures toward defined ends that are appar-
ent in biological entities put them at odd with other physical manifestations. It is is a statisti-
cal impossibility that structures as complex and refined as an eye, a bacteria and even a
functional enzyme can suddenly emerge with fully functional features (Salisbury, 1969;
Dawkins, 1986). Besides, the existence of spontaneous generation has been definitively ruled
out since L. Pasteur (1822-1895) (Pasteur, 1861). A rational mind feel compelled to admit the
existence of a creative force to account for the projects expressed in biological functions.
What is however the nature of this creative force? An immediate explanation would be
a theological one: the purpose apparent in living intities is the reflection of the will of the
creator. This issue is best illustrated by the so-called watchmaker analogy. This argument was
famously put forward by W. Paley (1743-1805) (Paley, 1809), but similar ideas were formerly
evoked by numerous thinkers. Let us imagine one happens to find a watch in the middle of a
virgin natural landscape. In contrast to simpler natural objects, such as stones, the obvious
complexity of the artifact, the fine and purposeful arrangement of perfectly suited mechanics
irremediably argue for the existence of a intelligent watchmaker, who designed and craft it.
Similarly, Paley argue, the complexity of living forms, their exquisite adaptations to specific
functions definitely prove the existence of an intelligent designer. There is, however, no logi-
cal demonstration in this reasoning: the long watch preamble is not a sound premise to an ar-
gument, but merely serves to establish the plausibility of the general premise one can tell,
simply by looking at something, whether or not it was the product of intelligent design, which
eventually remains unproven. This rhetorical slippage is known as the design inference and is
frequently used as an argument to the existence of God.
For a long time there was no satisfying alternative to those kinds of argument. Never-
theless, the methods of natural sciences are grounded on objectivity not projectivity and hence
do not leave room for supernatural explanations. A heuristic scientific principle, known as
Ockham razor after the logician and Franciscan friar William of Ockham (ca. 1288-1348)
holds that the explanation of any phenomenon should make as few assumptions as possible.
In this respect, the assumption of an omnipotent and omnipresent creature transcending the
law of the universe is particularily not parsimonic explanation. Before Darwin (1809-1882),
people that did not admit the divine intervention as an explanation of life were somehow
compelled to admit its spontaneous apparition.
The purpose of evolution
259
Adaptation, teleonomy and blindness
Living organisms are able to reproduce themselves in an almost identical manner, at
the exception of few variations. Because the resource of a given environment are limited, a
population of organism cannot grow indefinitely but soon become restricted. This creates ex-
trinsic selective conditions: any genetic variant that has higher chances to reproduce is stabi-
lized and increase in frequency in the population. In this light, the propagation of self appears
as the ultimate end of a living organism. It relies on proper capacity to survive, exploit the en-
vironment and reproduce. These performances are carried out by specialized devices that an
organism produce as part of is own self, which together constitute its phenotype. The pheno-
type is defined as the expression of an organism’s genetic information in a given environ-
ment. Any random phenotypic variation allowing a particular function to be performed more
efficiently is selected provided it finally results in higher prolificity. If the variation is the re-
sult of a heritable genetic mutation, the sustained selection on the phenotype leads to a rise in
frequency of the mutation in the population. When repeated iteratively, the short-sighted se-
lection of small effect mutations can progressively lead to the appearance of sophisticated
structure. This cumulative selection is a creative process that is not driven by final ends but by
the instantaneous action of the environment on the available variability. The trade-off between
productions of phenotypic variations and genetic stability mentioned earlier is essential in this
process. Sustained selection of a genetically encoded trait relies on the relative invariance of
the global phenotype. In contrast, the existence of variations is mandatory for adaptation.
The seemingly intrinsic projects that single out the phenotypes of organisms reflect
their adaptations to the environment. The adaptation is not only the static state that we can ob-
serve, and which unavoidably appears as purposeful design. Above all, it is a continuous and
dynamic process driven by the environment and resulting from the cumulative selection of
genetic determinant through their impact on the phenotype.
Unambiguously, eyes are made to see, wings to fly and at another scale, DNA poly-
merases to replicate DNA. The ambiguity does not lie in the fact of adaptation but rather in
the process of adaptation. The verb to make inevitably alludes to the existence of an almighty
watchmaker responsible for crafting the universe and its inhabitants. The modern synthesis of
evolution provides a robust and scientific framework to explain the apparent finality of bio-
logical entities. The process of cumulative selection, initially described by C. Darwin (1809-
1882) and A. R. Wallace (1823-1913) (Darwin and Wallace, 1858), fulfills the role of the
creative force that account for the finality of biological artifacts. As eloquently written by R.
Appendix – Epistemological considerations on the role of variations in biology
260
Dawkins (1941): “All appearances to the contrary, the only watchmaker in nature is the blind
forces of physics, albeit deployed in a very special way. A true watchmaker has foresight: he
designs his cogs and springs, and plans their interconnections, with a future purpose in his
mind's eye. Natural selection, the blind, unconscious, automatic process which Darwin dis-
covered, and which we now know is the explanation for the existence and apparently purpose-
ful form of all life, has no purpose in mind. It has no mind and no mind's eye. It does not plan
for the future. It has no vision, no foresight, no sight at all. If it can be said to play the role of
watchmaker in nature, it is the blind watchmaker” (Dawkins, 1986).
The decisive subtlety differentiating the artifacts produced by an intelligent watch-
maker from those generated by Dawkins’ blind watchmaker is caught between the words
teleology and teleonomy, which both refers to the issue of finality. In the one hand, teleology
refers to purposeful systems that are able to elaborate their own ends. Such systems are char-
acterized by intentionality and foresight. As described above, the concept of teleology is
closely linked to the one of theology in the context of biological systems. On the other hand,
teleonomy is a property of goal seeking systems. Such systems are not internally driven to-
ward a defined end, but result from an exploratory process composed of several round of
variation and subsequent stabilization supervised by extrinsic conditions. Biological evolution
is a perfect illustration of a goal seeking process. In this case, the goal that is sought is to
maximize the reproduction of an organism. The adaptations of this organism are both the re-
sults and the consequences of this process. In this light, the debate concerning the respective
primacy of forms over functions is pointless. Forms and functions interact in a dialectic man-
ner to results in adaptation through time. None has the primacy over the other: a given func-
tion is the consequence of the form, but the form has been selected through the function it
confers. Besides, the simplest cell is a complex network of interdependent processes that
evolved on top of each other. As a result, the expression of a given form and function relies
on preexistence of other forms and associated functions.
Impact of the environment
What is the environment?
From the standpoint of an organism, the environment represents all what is outside of
the self and comprises abiotic and biotic components. The abiotic factors correspond to the
Impact of the environment
261
physicochemical conditions experienced by the organism. They are subjected to both random
variations and regular fluctuations over a wide range of timescales. Random fluctuations typi-
cally results from meteorological phenomena: variation in temperature, drought, rain and sub-
sequent afflux of chemical… Examples of regular fluctuation include the day-night and
seasonal cycles. The biotic factors comprise all other living forms, from the same or different
species. The essential difference between the biotic and abiotic factors lies in the ability of the
formers to evolve. The co-evolution of different species results in the establishment of diverse
and interdependent relationships in the ecosystem, such as competition, symbiose or com-
mensality. The antagonistic interactions resulting from competition, predation and parasitism
are particularly interesting. They determine situations in which the survival of one species is
threatened by the existence of another species, while the survival of the latter is stricly de-
pendent on these harassments. In such contexts, every innovation developed by one camp
must be counteracted in the other resulting in an explosive mechanism of evolution, which is
often referred to as an arm race (Dawkins, 1986). The broad ecological significance of such
interactions is captured in the Red Queen analogy (Van Valen, 1973), which refers to a chap-
ter in L. Carroll's novel Through the Looking-Glass in which Alice and the Red Queen are
running in one side, while the entire world is moving in the opposite direction. As the net
movement of the protagonists is null, the Red Queen explains: “It takes all the running you
can do, to keep in the same place”. This highlights the fact that continuous adaptation is man-
datory to simply persist in an evolving biotic world.
Exposure to environmental variations depends on the actual biology of organisms.
Mobile organisms can actively forage for foods and, to some extent, can escape or avoid chal-
lenging environments, including biotic and abiotic factors. The ability to sample a larger set
of conditions renders them less dependent on local variations. In contrast, sessile organisms
are constrained to one location and condemned to undergo the vicissitude of the weather, the
food availability and cannot escape from other organisms. Although they are generally able to
move actively on a microscopic scale, microorganism can be considered as mostly sessile or-
ganisms on a macroscopic scale (Andrews, 1998).
An organism delimits a physical separation between its self and the surrounding envi-
ronment, thereby establishing an internal compartment. Apart from the particular case of
niche construction (see below), the external environment is uncontrolled by the organism. In
contrast, the internal environment is part of the individual phenotype and is subjected to so-
phisticated mechanisms to adjust its composition. This maintains a certain physicochemical
homeostasis which fundamental to the occurrence of metabolic reactions. The first replicating
Appendix – Epistemological considerations on the role of variations in biology
262
molecules to undergo evolution had to cope with direct exposure to the environment. The
evolution of the cells allowed emancipation from the hazards of the external environment.
However, this process required the coordinated association of different replicators and their
subsequent functional specialization driven by increased collective survival over evolutionary
time (Szathmary and Maynard Smith, 1997). The individuality was eventually shifted from
single independent replicators to a consortium of interdependent molecules replicating in a
coordinated fashion. In the same fashion, individuality was shifted from single cells to several
related cells during the evolution of pluricellular organisms. The progressive liberation from
the external conditions relies on the construction of a phenotype resulting from the coordi-
nated action of several entities and is accompanied by modification of the selection units. In
this view, an organism constitutes a microenvironment constructed by an assembly of genes
to cooperatively increase their survival. The coordinated action of the genes is the results of
ongoing evolution. Any genetic variation in one of these genes can be selected if it increases
the survival of the cell. However, a net survival increase may hide possibly inadequate inter-
actions between some components of the cellular environment. Hence, the phenotypic varia-
tion of one gene product may constitute a change in the internal environment for another
gene, resulting in complex epistatic relationship. The emancipation from the environment re-
lieved some constraints, while creating others.
Beyond the edification of an internal environment, an organism’s phenotype can also
significantly impact its external environment. Modified environments can affect the progeny
of the organism, resulting in ecological inheritance and influencing biological evolution. This
overlooked biotic factor is often referred to as niche construction (Laland et al., 2000).
The inheritance of acquired characteristics
The first formal theory of evolution was proposed by Jean-Baptiste de Lamarck
(Gould, 2002). Lamarck recognized transformation of species by way of progressive modifi-
cations and highlighted the theoretical necessity for evolution (Lamarck, 1809). Following
naturalists since Aristotle, he favored the ordering of living form along a complexity scale. He
proposed that an inner complexifying force is driving evolution from the simplest living enti-
ties to the more complex. In his time, the absence of spontaneous generation was admitted for
higher animals, but the issue was not yet settled concerning microorganisms. Lamarck pro-
posed that complex organisms arose by progressive transformation of simpler one, all the way
down to microorganisms, that were conceived as simple enough to appear spontaneously (see
Impact of the environment
263
Figure 41). In this view, evolution is necessary to explain the existence of complex creatures
that cannot appear by chance alone. In Lamarck’s thought, the evolution of complexity under-
lie an idea of progress that is an inherent feature of life. This process is driven by an elusive
force referred to as “Le pouvoir de la vie”. The creative component of Lamarckian evolution
is thus teleological. Nevertheless, Lamarck clearly outlined the importance of the environ-
ment in evolution. By determining the use and disuse of phenotypic characteristics, the envi-
ronment drives the modification necessary to evolutionary change. The frequent and
continuous use of a function is expected to subtend the development of the structure carrying
this function. In contrast, a characteristic that is not used in an environment will progressively
shrink, until eventual loss. These mechanisms are grounded on the observation of diverse
phenotypic plasticity, for instance the development of the musculature upon physical exer-
cises or the deformation of certain organs subject to constant physical constraints.
Following the predominant idea of his time, Lamarck assumed that the changes affect-
ing the phenotype of the parental organisms are transmitted to their offspring. In Lamarck
own words: “All the acquisitions or losses wrought by nature on individuals, through the in-
fluence of the environment in which their race has long been placed, and hence through the
influence of the predominant use or permanent disuse of any organ; all these are preserved
by reproduction to the new individuals which arise, provided that the acquired modifications
Appendix – Epistemological considerations on the role of variations in biology
264
are common to both sexes, or at least to the individuals which produce the young” (Lamarck,
1809). At the time, the mechanisms underlying heredity were completely unknown and the
inheritance of acquired characters was a common belief. This idea was notably refuted by A.
Weismann (1834-1914), who established the distinction between germen and soma in meta-
zoan. Only a subset of cells is transmitted to the next generation, while the vast majority of
cells participates to the elaboration of the phenotype and only serve the individual. In this
context, the transmission of acquired character requires that genetic information supposedly
received by the soma be communicated to the germen. The establishment of the central
dogma of molecular biology which can be summarized as follows ADN ↔ARN→Protein –
was the ultimate proof that no information modifying the phenotype (proteins) can trace back
to the genetic information (DNA) (Monod, 1970). The reciprocal ADN ↔ARN relationship
reflects the existence of reverse transcriptase coded in retrolements.
Lamarck had a remarkable intuition concerning the role of the environment in direct-
ing adaptation of individual organisms. Nevertheless he failed to identify the actual mecha-
nisms driving this evolution. Half a century later, Darwin proposed that evolution is driven by
natural selection. In his time, the laws of heredity and the nature of the genetic information
were still unknown and his idea about the generations of variability where extremely fuzzy. In
the origins of species, he wrote: “I have hitherto sometimes spoken as if the variations… were
due to chance. This, of course, is a wholly incorrect expression, but it serves to acknowledge
plainly our ignorance of the cause of each particular variation. [The facts] lead to the con-
clusion that variability is generally related to the conditions of life to which each species has
been exposed during several successive generations” (Darwin, 1859). Ignorant of the source
of mutations, Darwin did not reject the idea that organisms may respond to environmental
conditions and furnish the gametes with information enhancing the next generation’s re-
sponse. He even suggested that stress might generate the variability upon which natural selec-
tion operates.
The idea that the environment can directly influence heritable variation is appealing
because it straightforwardly couple the rate of evolution to its immediate necessity. The whole
concept was however firmly rejected by the neo-Darwinian synthesis, which established the
unilateral primacy of selection. Any mechanisms that somehow suggest a coupling between
environment and mutation were discredited and dubbed Lamarckian.
Impact of the environment
265
The Neo-Darwinian focus on selection
A fundamental tenet of the synthetic theory of evolution is that mutations occur ran-
domly in time and genomic space. Mutations are conceived as accidental error altering the in-
tegrity of the genetic information transmitted from one generation to the other. Apart from
exposure to mutagenic conditions, the environment is considered to play no role in this proc-
ess. In contrast, selection by the environment is conceived as the sole driving force in evolu-
tion, an ordering process that sorts the preexisting random variations generated
spontaneously. No more teleological forces are required to account for the orientation of evo-
lution; the process is blindly directed by the selective action of the natural environment. Evo-
lution is essentially a random process. The shifting balance theory developed by S. Wright
contributed to show that theoretically, evolution is a short-sighted and favor immediate adap-
tation irrespective of its long term consequences (Johnson, 2008). S. Luria and M. Delbrück
provided the first experimental demonstration of the precedence of mutations over selection.
They exposed bacterial population to phage infections and carefully analyzed the distribution
of resistant variants selected in independent experiments. They showed that this distribution
was in agreement with the random accumulation of resistance mutations prior to exposure to
the phage (Luria and Delbrück, 1943). E. and J. Lederberg reached similar conclusions by
monitoring the apparition of penicillin-resistant clones (Lederberg and Lederberg, 1952).
Anticipating and responding environmental changes
The teleological idea that a mysterious force directs evolution is known as orthogene-
sis. This concept has ideological implication and as been use to legitimate the incorporation of
evolution to various doctrine. Under Stalin in URSS, Lysenko emphasizes the capacity of the
environment to direct heritable variation. The geneticist that did not agree to that position
were harassed, incarcerated or expulsed. The rising synthetic theory of evolution was seen as
a bourgeois science that denied the aspiration of the regime (Fisher, 1948). Religions rather
tend to hold that the mysterious force is the hand of God. The Jesuit T. de Chardin famously
tried to reconcile the Christian faith with the idea of evolution (Teilhard de Chardin, 1955).
Presently, an institution such as the Catholic Church officially accepts the existence of theo-
ries of evolution, but practically favors an interpretation whereby important variations are dic-
tated by God rather by contingent mutations. Other congregations are less subtle and the
belief in creationism is widespread in some countries (Miller et al., 2006; Berkman et al.,
Appendix – Epistemological considerations on the role of variations in biology
266
2008). The most moderate proponents of the Intelligent Design movement still argue that mo-
lecular machines are too complex to have evolved by cumulative selection. Re-actualizing
Paley’s design inference, they present this argument as a proof of the existence of a superior
intelligence (Behe, 1996).
Continuous pseudoscientific interpretations of evolution somehow prompted the pro-
ponents of the mainstream synthetic theory to strengthen their position concerning the pri-
macy of selection. The idea that the environment may influence another step than selection in
the evolutionary algorithm was out of the paradigm and difficult to defend in the scientific
community. A classic illustration concerns the observation of stress-induced chromosomal re-
arrangement in maize by B. McClintock (McClintock, 1950), which showed that high order
genetic changes can be elicited by the environment. It received little credit until the loci im-
plicated mere identified as transposon providing a mechanistic basis for the phenomenon
(McClintock, 1984).
However, the idea that mutations can be induced by the environment does not contra-
dict the existing evolutionary theory, but rather appear as a sound consequence of it. A huge
controversy that fuelled extensive researches in this field was initiated by the publication of a
paper by John Cairns and colleagues in 1988 (Cairns et al., 1988). In this paper and several
subsequent works, the authors established a genetic system to follow the apparition of muta-
tion in E. coli. In this setting, bacteria carrying a lacZ gene inactivated by a reversible
frameshift are selected on lactose agar plates, so that only revertants can grow. The plates
were incubated during six days. The fluctuating apparition of spontaneous revertants was ob-
served as in the Luria-Delbrück experiments during the two first days of incubation. But the
apparition of revertants continued during the following days. The late mutants were not slow
growers and their number exceeded expectations under the Luria-Delbrück model. Instead,
their distribution was consistent with apparition under selection. Overall, the observed rever-
sion rate in the selective environment exceed by 100-fold the rate measured in a non-selective
one. It was initially reported that the increased mutation rate was specifically directed to lacZ.
This process, referred to as directed mutation, supposes that a kind of molecular cognitive
system is able to predict the consequences of mutations, so that only adaptive loci are tar-
geted… Or it can easily be interpreted as evidence of orthogenesis. Subsequent studies
showed that the increased mutation rate under selection was not restricted to the lacZ gene,
but distributed over the whole genome, though the region surrounding lacZ was more vari-
able. Furthermore, the mutational signature observed under selection was found to be differ-
ent than the one observed in the absence of selection. It thus appeared that a distinct
Impact of the environment
267
mechanism is responsible for increased mutagenesis. These results fostered a comprehensive
research effort and the experimental system was fully dissected (Roth et al., 2006; Galhardo
et al., 2007).
These studies and others highlighted that the role of the environment is not restricted
to selection alone. Clearly, the results of the Luria-Delbrück (Luria and Delbruck, 1943) and
Lederberg-Lederberg (Lederberg and Lederberg, 1952) experiments rapidly gained general
acceptance because they fitted paradigmatic expectations, in spite of their restricted biological
significance (harsh selective pressure, specific type of mutations). As will be illustrated be-
low, cells evolved mechanisms to sense stressful conditions that happen to responsively con-
vert the collected information into mutations (see Stress-induced mutagenesis, pp 47-60).
Thus, the generation of variability is not necessarily constant in time but can be informed by
the environment. Moreover, mutation rates are not only variable through time but are also
variable in genomic space. Indeed, descriptions of genetic mechanisms dedicated to or favor-
ing localized and oriented mutations accumulated over the last decades. The detailed presenta-
tion of these mechanisms is covered in the introduction of this thesis (see Programed
generation of genetic variations, pp 60-99).
Collectively, these processes participate in a kind of molecular intelligence that allow
cells to anticipate changes or directs genetic changes according to the environment and are of-
ten perceived as Lamarckian. However, these discoveries do not contradict but extend the
classical synthetic theory of evolution (Thaler, 1994). In final analysis, mutations always ap-
pear in a random fashion. Genomes simply evolved the capacity to control this randomness,
so that the production of variation can be tuned to the demands of the environment. To take
up a weel put sentence, this reflects the fact that chance favors the prepared genome
(Caporale, 1999).
268
References
269
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Evolutivité – Le cas des integrons & utilisation de sequences synonymoes en évolution dirigée
La stabilité phénotypique est essentielle au succès d’organismes évoluant sous des
conditions constantes. L’environnement est néanmoins soumis à de perpétuelles variations stochastiques, auxquelles les êtres vivants doivent sans cesse s’adapter. L’évolutivité caractérise la capacité d’une population à répondre à de telles pressions sélectives par la génération de modifications phénotypiques héritables. La majorité des mutations étant délétères, des processus permettant de limiter la production de telles variations aux seules périodes de stress, ou de la confiner à des loci et phénotypes bien définis, ont été sélectionnés au cours de l'évolution.
Les intégrons en constituent une illustration particulièrement sophistiquée. Initialement identifiés comme vecteurs de résistance à de multiples antibiotiques, ces systèmes génétiques bactériens spécialisés dans l’échange, la collecte et l’expression de gènes accesoires constituent une importante source de diversité génétique. Ce travail montre que les intégrons sont directement couplés à une voie majeure de réponse au stress chez les bactéries, le système SOS. En permettant de générer de la variabilité phénotypique en période de stress sans affecter le reste du génome, les intégrons constituent ainsi un exemple paradigmatique d’évolutivité.
Un autre aspect de ce travail démontre que des séquences codantes synonymes – bien que spécifiant des protéines identiques – peuvent accéder par mutations ponctuelles à des régions différentes de l’espace phénotypique. Utilisée de manière adéquate, cette propriété permet d’étendre l’évolutivité d’une protéine quelconque dans le cadre d’applications biotechnologiques.
Evolvability – The integron case & the use of synonymous sequences for directed evolution
Phenotypic stability is essential to the success of organisms evolving under steady
conditions. However, the environment is subjected to perpetual stochastic variations, to which living beings must constantly adapt. Evolvability characterizes the ability of a population to respond to such selective pressures through the generation of heritable phenotypic changes. Most mutations being deleterious, processes enabling the confinement of mutations to periods of stress, or to specific loci and well-defined phenotypes, have been selected over evolution.
Integrons constitute a particularily sophisticated illustration of such processes. Initially identified through their involvement in multi-resistance to antibiotics, these bacterial genetic systems are specialized in the exchange and stockpiling of accessory genes and therefore con-stitute an important source of genetic diversity. This work shows that integrons are directly coupled with the SOS system, a major bacterial stress response. By allowing the generation of significant phenotypic diversity during periods of stress without impacting the rest of the ge-nome, integrons hence constitute a paradigmatic example of evolvability.
Another aspect of this work demonstrates that synonymous coding sequences – al-though specifying identical proteins – can access different area of the phenotypic space through ponctual mutations. When properly exploited, this property can enhance the evolva-bility of any protein in the context of biotechnological applications.