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\DOI{10.5802/crbiol.201}
\datereceived{2026-03-04}
\daterevised{2026-06-11}
\dateaccepted{2026-06-12}
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\COI{The authors do not work for, advise, own shares in, or receive
funds from any organization that could benefit from this article, and
have declared no affiliations other than their research organization.}

\dateposted{2026-08-06}
\begin{document}

%\dateposted{2026-02-16}

\begin{noXML}

\CDRsetmeta{articletype}{research-article} 

\editornote{Article submitted by invitation. A Chinese translation of this article is available on the open archive HAL at \url{https://hal.science/view/index/docid/5696532}}
\alteditornote{Article soumis sur invitation. Une traduction en chinois de cet article est disponible sur l'archive ouverte HAL \`a l'adresse \url{https://hal.science/view/index/docid/5696532}}

\title{The origin of the logic of adaptive mutations: standing by to
grow again}

\alttitle{\`{A} l'origine de la logique des mutations adaptatives :
veiller pour rena\^{i}tre}

\author{\firstname{Antoine} \lastname{Danchin}\CDRorcid{0000-0002-6350-5001}\IsCorresp}
\address{School of Biomedical Sciences, Li KaShing Faculty of Medicine,
Hong Kong University, Pokfulam, SAR Hong Kong, China}
\email[A. Danchin]{antoine.danchin@academie-sciences.fr}

\author{\firstname{Agnieskza} \lastname{Sekowska}}
\addressSameAs{1}{School of Biomedical Sciences, Li KaShing Faculty of
Medicine, Hong Kong University, Pokfulam, SAR Hong Kong, China}
\email[A. Sekowska]{a.sekowska77@gmail.com}

\keywords{\kwd{Evolution theory}
\kwd{Protein-L-isoaspartate(D-aspartate)-O-methyltransferase}
\kwd{CRP}
\kwd{Cytidine triphosphate}
\kwd{Methylglyoxal}}

\altkeywords{\kwd{Th\'{e}orie de l'\'{e}volution}
\kwd{Prot\'{e}ine-L-isoaspartate(D-aspartate)-O-m\'{e}thyltransf\'{e}rase}
\kwd{CRP}
\kwd{Cytidine triphosphate}
\kwd{M\'{e}thylglyoxal}}

\begin{abstract} 
For decades, discussions about the evolution of species overlooked
microorganisms. Over a century ago, Neisser and Massini isolated a
coliform bacterium that appeared to acquire mutations adapting it to
its environment, naming it \textit{Bacterium coli mutabile} to reflect
this feature. With the advent of molecular biology, these widely
debated experiments were subsequently forgotten. Here, we present the
history of an experiment that reproduces their observations in a modern
context where it has become possible to identify the nature of these
mutations down to the nucleotide level. Its findings demonstrate that
the transcription of gene families that ensure the long-term
maintenance of the metabolism of ageing cells is a direct source of
adaptive mutations: this process enables bacteria to identify
previously unexploited environmental factors that can now support
growth. We propose that the driving force behind this adaptation is the
spontaneous dehydration/deamidation of polypeptide chains, which
dictates an intrinsic lifespan for every protein. This universal
mechanism of inevitable protein ageing necessitates their re-synthesis
to maintain their function; however the transcription process, which
involves opening the DNA double helix, is locally mutagenic. Thus, as
bacteria age, the continuous re-synthesis of some of the proteins that
perform the functions enabling survival triggers a local mutagenic
process. This yields genetic variants, some of which may be beneficial
and are therefore retained.
\end{abstract}

\begin{altabstract}
Longtemps la r\'{e}flexion sur l'\'{e}volution des esp\`{e}ces a
ignor\'{e} les microorganismes. Il y a plus de cent ans, Neisser et
Massini isolaient un colibacille qui semblait acqu\'{e}rir des
mutations l'adaptant \`{a} son environnement et le nommaient pour cette
raison \textit{Bacterium coli
mutabile}. Ces exp\'{e}riences tr\`{e}s
discut\'{e}es ont \'{e}t\'{e} oubli\'{e}es avec l'av\`{e}nement de la
biologie mol\'{e}culaire. Nous rapportons ici l'histoire d'une
exp\'{e}rience qui reproduit leurs observations dans un contexte
moderne o\`{u} il est devenu possible d'identifier au nucl\'{e}otide
pr\`{e}s la nature de ces mutations. Ses r\'{e}sultats font
appara\^{i}tre que la transcription de familles de g\`{e}nes assurant
le maintien \`{a} long terme du m\'{e}tabolisme de la cellule
vieillissante est source de mutations qui lui permettent de s'adapter
en d\'{e}couvrant dans l'environnement ce qui lui permet de cro\^{i}tre
\`{a} nouveau. Le ressort de cette adaptation est la
d\'{e}shydratation/d\'{e}samidation spontan\'{e}e des cha\^{i}nes
polypeptidiques qui conf\`{e}re une dur\'{e}e de vie intrins\`{e}que
\`{a} chaque prot\'{e}ine. C'est que ce m\'{e}canisme universel du
vieillissement in\'{e}vitable des prot\'{e}ines n\'{e}cessite leur
resynth\`{e}se pour assurer leur fonction, alors que le processus de
transcription, ouvrant la double h\'{e}lice de l'ADN est localement
mutag\`{e}ne. Ainsi, en vieillissant, les prot\'{e}ines qui assurent
les fonctions permettant \`{a} la bact\'{e}rie de survivre
d\'{e}clenchent un processus de mutagen\`{e}se qui en produit des
variants dont certains peuvent \^{e}tre b\'{e}n\'{e}fiques et sont
alors retenus.
\end{altabstract}

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\defcitealias{Massini1907}{ibid.}
\defcitealias{Frendorf2019}{ibid.}

\section{Introduction}\label{sec1}

Putting man in its proper place as just another animal, ideas about the
evolution of species have long reflected an anthropocentric view. Based
on the history of multicellular organisms, any species of interest kept
a scale matching that of man. Yet the cell, almost always visible only
through a microscope, is the atom of life. If evolution by natural
selection was universal, it was necessary to consider whether the
processes proposed to explain the fate of macroscopic organisms
remained valid for microbes, which are normally unicellular and very
small. This was all the more important because, even at a time when the
existence and nature of genes were not yet understood, the accepted
rules for explaining evolution soon added a component other than size:
the sexual distinction between individuals. However, it was not obvious
that this distinction, which was quite clear in microbes such as yeast,
was also valid for bacteria. Very early on, one of the implicit
\mbox{questions} postulating the existence of a sexual cycle in bacteria had
triggered the study of this important {process} \citep{Sherman1937}, but
it was only in parallel with the discovery of the role of DNA as a
vector of heredity that the mechanisms governing it were understood
\citep{Tatum1947}. Furthermore, singularised by their pleomorphism,
microbes have phenotypes that vary considerably depending on how they
are cultured. Until the details of heredity were understood, this gave
rise to many unfounded speculations. Early microbiologists thus
believed that the heredity of microbes obeyed different laws than those
of plants and animals \citep{Rodet1894,Enderlein1925,Hadley1927}. To go
further, it was first necessary to choose model organisms that would
allow for the construction of conclusive experiments and explore the
evolution of their descendants.

\section{Early observations of adaptive mutations in bacteria}

As early as 1859, to explain why broths and other infusions were
quickly invaded by microbes, Louis Pasteur demonstrated that this
process required the prior presence of living organisms. Life does not
arise spontaneously. His famous opponent F\'{e}lix Pouchet, a proponent
of the traditional animistic view, vigorously challenged him, claiming
to have found evidence contrary to Pasteur's. A fierce battle ensued.
What was Pouchet's ``evidence''? Pasteur used heated yeast extract as
sterilised broth, while Pouchet showed that he recovered microorganisms
after boiling containers containing water in which hay had been infused
\citep{RollHansen1979}. We now know that herbaceous plants are the
natural environment in which the ``hay bacteria'' characterised in 1872
by Ferdinand Cohn and now known as \textit{Bacillus subtilis} multiply.
These bacteria form spores that are highly resistant to heat
\citep{Nicholson2002}, which explains the observations made by Pouchet,
who had poorly sterilised his culture medium. One of the models chosen
by Jacques Monod to study bacterial physiology, this organism is
nowadays once again being used to analyse the mechanisms of their
evolution \citep{Zeigler2017}. However, it is another bacterium, also
used by Monod in his thesis, the coliform bacterium originally named
\textit{Bacterium coli commune}, which has since become the
best-understood living organism \citep{Meric2016}.
\mbox{Discovered} in 1885 in the colons of infants and renamed
\textit{Escherichia coli} in honour of its discoverer, German
paediatrician Theodor Escherich, it has served as a model for studying
the mechanisms of bacterial evolution since its earliest day.
Unfortunately, this work has been largely forgotten. Indeed---and this
amnesia is exaggerated because what is known to the general public must
be easily accessible on the web and is therefore often very recent---,
many crucial experiments that were discussed even before the discovery
of molecular mechanisms of heredity are overlooked. Rediscovering them,
illuminated by our current knowledge, brings to light old controversies
that reveal unexpected evolutionary behaviours, the explanation of
which opens up new perspectives on the mechanisms of species evolution.

Isolated from a case of enteritis by Max Neisser in 1906, a strain of
\textit{B.~coli commune} thus became the subject of heated
debate on evolution. Neisser had selected it after observing a curious
phenomenon: unlike the reference strain that ferments lactose, this
strain did not. However, when the bacteria were left at 37~\textdegree
C for 72 to 96~h on Petri dishes containing lactose and a
colourless indicator that turns red in the acidic environment created
by fermentation, red daughter colonies appeared on the surface of
``white'' parent colonies that did not ferment it. These microbes had
therefore acquired this ability after being exposed to the sugar, and
did so in a stable and apparently irreversible manner. This led Neisser
to name it \textit{B.~coli mutabile} to emphasise its instability. In
the original article describing the strain, Neisser suggests, without
discussing the cause, that the variation observed in \textit{B.~coli
mutabile} results from a mutation in the sense understood by De~Vries
in plants in the early 1900s \citep{Neisser1906}. Unifying what was
understood about heredity, this seemed to reveal a case of heredity of
an acquired trait, an observation that was anathema to proponents of a
narrow interpretation of Darwin and Wallace's theories. This may
explain the long silence that obscured these observations.\looseness=-1

Neisser had suggested to his student Rudolf Massini that he made this
the subject of his thesis. Massini showed in great detail, starting
with individual bacteria that he allowed to multiply on plates, how,
when cultured on a solid medium containing a specific dye, they first
form white colonies. After several days, ``papillae'' appear on these
colonies and turn red. Subcultures made from these papillae on media
containing lactose never produce white offspring on the same sugar,
even after prolonged subculturing on lactose-free media
\citep{Massini1907}. We reproduce here an excerpt of the original text
of Massini's observations because they are identical to our later
description using a bacterial model with known genetic and molecular
characteristics, which could not have been the case at the time 
(Box~\Custref{1}{box1}).\looseness=1

{

\pagebreak

\onecolumngrid

\bigskip\bigskip
\unskip\noindent\begin{frtextbox}{\textbf{Box 1. Papillae fermenting lactose maintain this ability over
generations.}}
\enlabel{box1}
\mbox{}

{\begin{quote}

\vspace*{-10pt}

{Die zweite Eigenart unseres Bakteriums
besteht darin dafs, nicht alle Keime zu gleichm\"{a}fsigen Kolonien
auswachsen sondern, dafs unter bestimmten Bedingungen einzelne Keime
variieren und ihre neu erhaltene Eigent\"{u}mlichkeit sofort konstant
weiter vererben. Wird das Koli auf Endoplatten weiter gez\"{u}chtet, so
dafs der neue Satz immer von einer isolierten Kolonie des
vorhergehenden Satzes ausgeht, so sieht man nach einigen
Umz\"{u}chtungen neben den weifsen Kolonien rote auftreten) Diese
letzteren sind nach 16 Stunden nicht von den fr\"{u}her beschriebenen
farblosen Kolonien zu unterscheiden nach 18--20 Stunden aber erhalten
sie einen roten Nabel und nach 36--48 Stunden sind sie ganz rot und
zeigen den f\"{u}r das Bacterium coli commune typischen metallischen
gr\"{u}nen Fuchsinglanz. Am 2 und 3 Tage sind sie durchschnittlich
gr\"{o}fser als die weifsen Kolonien, bleiben aber in den
n\"{a}chstfolgenden Tagen an Gr\"{o}fse hinter den letzteren bedeutend
zur\"{u}ck. Die roten Kolonien erhalten nie Kn\"{o}tchen wie ich sie im
ersten Teil der Arbeit als, f\"{u}r die weifsen Kolonien
charakteristisch beschrieben habe Wird nun von einer roten Kolonie
wieder abgeimpft und ein neuer Endosatz ausgestrichen, so entstehen nur
rote Kolonien keine einzige weifse und auch bei weiterer Verimpfung
bleiben alle folgenden Generationen rein rot es gelingt nie mehr auf
Endo eine weifse Kolonie zu erhalten.} \citepalias{Massini1907}
\end{quote}}

The second peculiarity of our bacterium is that not all germs grow into
uniform colonies, but that under certain conditions individual germs
vary and immediately pass on their newly acquired peculiarity
constantly. If the \textit{coli} is further cultivated on Endo plates,
the new set always originates from an isolated colony from the previous
set, after a few re-cultivations, red colonies appear alongside the
white ones. After 16~h, the latter are indistinguishable from the
colourless colonies described earlier, but after 18--20~h, they develop
a red navel, and after 36--48~h they are completely red and show
the metallic green fuchsine luster typical of the \textit{Bacterium
coli commune}. On days~2 and 3, they are on average larger than the
white colonies, but in the following days they remain significantly
smaller than the latter. The red colonies never develop nodules as I
described in the first part of this paper as characteristic of the
white colonies. If a red colony is now inoculated again and a new Endo
plate is spread, only red colonies develop, not a single white one, and
even with further inoculation, all subsequent generations remain purely
red; it is never possible to obtain a white colony on Endo.
\end{frtextbox}

\vspace*{14pt}

\twocolumngrid

}

How should we interpret these observations---along with many others,
such as the heritable adaptive emergence of a ``capsule'' in pathogenic
bacteria \citep{Stewart1926}---, at a time when the physical nature of
genes remained a mystery? Few questions have sparked as much
debate---often heated---as that of the origins of these mutations (see,
for example, the discussions sparked by the generation of adaptive
mutations in \textit{E.~coli} in 1988 \citep{Cairns1988}). Similarly,
few questions have brought to light as many prejudices rooted in
ignorance of the fundamental biological mechanisms underlying the
\mbox{variability} of bacterial phenotypes, whatever its origins and extent.
Are they adaptive mutations or temporary adaptation to the environment?

This question, which Monod addressed in his study of bacterial
adaptation to growth on various carbon sources \citep{Monod1949},
enabled him to understand enzymatic adaptation through gene expression.
In his study, it takes only a few hours for lactose-fermenting
\textit{E.~coli} to appear, and this adaptation is reversible. This
implies a mechanism that does not alter the nature of the genes in the
cells involved. In contrast, in the experiments by Neisser and Massini,
it takes several days, and this adaptation is irreversible. The
extensive literature on this subject consists almost entirely of
observations lacking a common guiding principle, other than revisiting
the question of the inheritance of acquired characteristics. The puzzle
still presented itself in this way in 1938: 

\begin{quote}
{In the case of the
citrate ``mutant'', a smear on citrated agar usually shows no sign of a
colony for at least three days and frequently this time interval may be
four, five, or six days. Why the delay? Secondary colonies of}
\textit{Bacterium coli-mutabile} {appear on lactose indicator agar only
after a lapse of days. Typical cultures of this organism sown in
lactose broth may show no visible acidity for more than a week.}
\textbf{We felt that we shall never have the exact answers to
what actually does go on in strains like these until methods better
than those now in use have been developed} [emphasis added]
\citep{Parr1938}. 
\end{quote}

These observations have long been cited and discussed, particularly in
relation to the possible existence of sexual reproduction and the
general mechanisms of heredity in bacteria \citep{Luria1946}, but after
the discovery of DNA's role in heredity, they were quickly forgotten.
Every genetic mutation must have a physical basis, observable in DNA,
but it was only very recently that it became possible to identify them.
To go further, it was indeed necessary not only to start with a very
well-known genetic model---which became easy once genomic sequencing
experiments became commonplace---but also to consider what types of
experiments to pursue, considering the life cycle of organisms. We
explore first this point, rarely emphasized.

\section{The ages of life: which phase of the life cycle should be
chosen to study evolution?}

When considering evolution, we often overlook the fact that the course
of life is not uniform. The existence of individuals or species, from
birth to death, unfolds in several phases (Figure~\ref{fig1}). We tend to focus
on the periods when the organism is young, since evolution involves the
generation of progeny. Yet cells---whether isolated or forming a
multicellular organism---go through a progression in which this phase
represents only a part, often a brief one, of their entire lifespan.
However, the nature and fate of the ages of life certainly play a key
role in how they will affect the evolution of the entity of \mbox{interest}.
This is because the selective processes involved in this evolution
cannot be the same if the entity is at the beginning of its life cycle,
if it is growing, in a stationary state, ageing, or near death. Only
the final phase is unlikely to have a significant influence on the
production of its progeny, and even then, it may affect the survival of
those already born \citep{Travers2021}.

\begin{figure}
\includegraphics{fig01}
\caption{\label{fig1}{The ages of life}. For convenience, most
experiments on evolution in the laboratory tend to avoid what happens
in the long run. In chemostats, cells grow for a long time, but in a
stable environment. Here we show that during the stationary
phase---when the maturation of certain compounds transitions into
ageing---coordinated mutations begin to appear. At the end of this
period, populations age and cells die one after another, initially
following Gompertz's law \citep{Kirkwood2015}, and then with a
probability that tends to be constant \citep{Steiner2021}. In the case
of single-celled organisms that divide by binary fission, death only
occurs after a number of generations---usually unknown---which means
that, in their case, the universality of this concept remains, for the
time being, a conjecture. In the stationary phase, when there is no
growth, death may only occur after a very long time
\citep{Pechter2017}. The term ``senescence'' is used here in Steiner's
sense. The blue arrow represents the conjecture presented in this
historical review, which explores the idea of an adaptive reset
triggered by the renewal of proteins that have become altered over
time.}
\end{figure}

Microbial models of evolution have primarily focused on growth in
liquid culture, which is easy to set up. This is also when the number
of descendants is highest, making it suitable for robust statistical
analyses \citep{Dworkin2022}. In this context, long-term evolutionary
experiments use either growth in a chemostat \citep{Marliere2011}, or
the daily dilution of cells into fresh medium, \mbox{allowing} them to grow
again \citep{Papadopoulos1999}. These approaches are typically
anthropocentric, as they are designed to facilitate work in the
laboratory. While they do not allow for a realistic exploration of the
phenomenon of bacterial evolution, they have nevertheless brought to
light the important role of an often-neglected parameter: ageing. It
should be noted here that, just as with the term ``senescence'', this
is a ``prospective'' concept, in John Myhill's sense, inevitably
sparking much debate \citep{Myhill1952}. We therefore use these terms
while retaining the imprecision of their commonsense meanings. In
liquid culture, the bacterial population creates a competitive
situation among individuals that allows for the rapid emergence of
mutants capable of gaining a growth advantage over other members of the
population when it enters the stationary phase, the predominant phase
of its life cycle. This phenomenon of ``growth advantage in the
stationary phase'' (GASP) has, for example, highlighted the key role of
an RNA polymerase subunit, RpoS, whose involvement we discuss later
\citep{Zinser1999,Abram2021}.

Other studies, designed to explain the observations made by Cairns and
his colleagues using Petri dishes, have sought to understand the
mutations that arise during the stationary phase on solid media. These
experiments are based on the premise that ageing, rather than being
recognised as a necessary component of life with its own distinct
characteristics, is merely one of the many ``stresses'' that the cell
must contend with, thereby acting as a stimulator of the background
level of mutagenesis \citep{Lansch2024}. As was very common at the
time, the observed mutations were therefore interpreted as the result
of a partial loss of activity in the DNA mismatch repair system and/or
the DNA recombination process \citep{Taddei1997,Bjedov2003}.

\section{An ``intelligent'' \textit{E.~coli} to explore the origin of
adaptive mutations}

During the stationary phase, the organism ages while restoring, to the
best of its ability, what is no longer functional in order to survive.
The consequences of the mutations that occur at that time become
apparent during evolution as soon as the organism finds a way to
generate progeny. This is not merely a theoretical view: preserving
dormant bacteria on solid media or in ``deep agar'' often reveals, when
used to restart a new culture, unexpected variants carrying mutations
\citep{Faure2004,Nahku2011}. This fact is, unfortunately, little known
despite the many difficulties that arise when comparing the work of
laboratories that claim to use the same strain \citep{Soupene2003}.
Highlighting the importance of understanding what happens during
survival, this is another significant---yet neglected---reason that
casts doubt on the reproducibility of many biological experiments
\citep{Cobey2024}.

In the mid-1980s, our laboratory launched an investigation designed to
test these observations experimentally. To this aim, we used a strain
of \textit{E.~coli} with impaired catabolic repression, a research
topic familiar to Jacques Monod's laboratory and to Agn\`{e}s Ullmann,
with whom we were collaborating. These bacteria carried a mutation that
eliminated the \textit{cya} gene encoding adenylate cyclase, rendering
them incapable of utilising a wide range of carbon sources. We then let
a few hundred individual bacteria grow on Petri dishes. These plates
contained a rich medium that allowed them to grow as small colonies
until they had used up all the carbon sources they could find. When the
medium was supplemented with maltose, could these bacteria generate
progeny? The prevailing wisdom holds that only pre-existing mutations
would allow new functions to emerge. This should, therefore, not be
possible, but the experiment was worth trying. The medium, designed to
be selective for enterobacteria, also contained a dye meant to indicate
acid production (MacConkey medium \citep{MacConkey1905}) and was
supplemented with maltose. These plates, incubated at 37~\textdegree C
for 24~h, were then left for several days at laboratory temperature
(18--22~\textdegree C). As expected, small white colonies
were initially observed, which eventually stopped\break growing.

We could have considered the matter closed and discarded the plates.
However, waiting a little while was not difficult, and our patience was
rewarded: a few days later, red papillae appeared on their surface, the
exact counterpart to what Neisser and Massini had described (Figure~\ref{fig2}).
Since this experiment was highly reproducible and could be completed in
less than a month, it served as the basis for one of the General
Microbiology teaching courses organised by Agn\`{e}s Ullmann at the
Pasteur Institute \citep{Ullmann1986}. Only one genetic conclusion
could be drawn from these early experiments: contrary to the widespread
hypothesis that recombination was \mbox{involved} in explaining the phenomenon
of adaptive mutations, we showed that inactivation of the \textit{recA}
gene merely slowed growth, without preventing the appearance of the
papillae \citep{Danchin2007}. However, since, at the time, we were
unable to determine whether the papillae carried meaningful mutations,
this work was not further developed.

\begin{figure}
\includegraphics{fig02}
\caption{\label{fig2}Growing papillae on MacConkey agar plates in the
presence of maltose. The small colonies of the strain unable to
produce cAMP are occasionally invaded by adaptive mutants that can
utilise this sugar.}
\end{figure}

More than twenty-five years after these initial experiments, we
developped a model based on the archetypal \textit{E.~coli} K12 strain (strain
MG1655), whose genome was first sequenced, in order to identify the
mechanisms that shape evolution when progeny generation occurs during
the survival phase \citep{Blattner1997}. To serve as a reference, the
genome sequence of the \textit{cya} mutant, AMB1655, was determined. We also
established that this strain does not produce more rifampicin-resistant
mutants (an antibiotic that inhibits transcription) than its parent
strain MG1655 \citep{Sekowska2016}. Its distinctive feature is that it
cannot grow on many sugars because the transcription of the genes
involved in their catabolism requires the presence of cyclic AMP
(cAMP), a mediator of gene expression for several hundred genes in
\textit{E.~coli} \citep{Kolb1993}, whereas the \textit{cya} gene
encoding adenylate cyclase had been deleted in the strain. This allowed
us to explore what happens when cells are left to survive in a rich
medium, where they initially find enough to produce small colonies but
stop growing once they have used up the carbon sources available to
them. We then fed them with one or another of these sources known to be
unusable in the absence\break of cAMP.

Allowing these bacteria to grow and enter the stationary phase, we
aimed at sequencing the genomes of the expected mutants. The colonies
formed after 24~h remained unchanged, but after three to five days, red
papillae began to appear on the surface of some of them---at a rate of
about one papilla per hundred white colonies. These papillae kept
growing until they covered the entire plate. In an atmosphere where
humidity and temperature are kept constant, this pattern repeats
itself, and new papillae appear for at least two months
\citep{Sekowska2016}. Purified by successive passage and isolation of
red colonies on plates containing the same medium, these papillae
exhibit the characteristic properties of the mutations observed in the
Neisser and Massini experiment. Physiological analysis of mutants
derived from independent colonies that emerged throughout the
experiment allowed for the description of the kinetics of their
emergence, their behaviour in response to different carbon sources, and
their phenotype on various indicator media. When spread on a medium
with a different colour from that of the MacConkey medium (typically
EMB rich medium, containing eosin and methylene blue
\citep{Leininger2001}) or on media containing carbon sources other than
maltose, it was observed that their phenotypes were very diverse,
suggesting that their mutations were far from all identical.

\section{The contribution of genomics: adaptive mutations are not
randomly distributed across the chromosome}

The genomic revolution has transformed our understanding by enabling us
to compare individuals within a population through the base-by-base
identification of their genome sequences. In collaboration with Morten
N\o{}rholm's laboratory in Copenhagen, we extracted and sequenced DNA
from the genomes of 96 papillae retained for their distinct phenotypes.
The differences between their genome sequences and that of the parental
strain were characterised \citep{Sekowska2016}. Almost all mutants
carry one or more mutations in the \textit{crp} gene, which encodes the
cyclic AMP receptor, CRP, and these mutations tend to accumulate over
time, revealing an adaptive process discussed further below
\citep{Frendorf2019,Lauritsen2021}. To broaden the evolutionary
landscape of this protein, we also sequenced the \textit{crp} gene
separately from 594 independent papillae \citep{Frendorf2019}. When the
mutants emerge at the beginning of the experiment, there is generally
only a single mutation in the gene. Over time, other mutations also
arise in the same gene. However, since we lacked the resources to
analyse in detail the specific effects of each of these mutations, we
can assume that a number of them are neutral \citepalias{Frendorf2019}.


In addition to these mutations, we identified 145 mutations and 15
deletions or insertions of mobile sequences in regions known to be
unstable (\textit{ychE}, \textit{icd}, \textit{intR} in the Rac
prophage region \citep{Foster2015,Ramisetty2019,Wons2025}). These
mutations form a series of hot spots, while the bulk of the genome
remains unaffected. Isolated from independent papillae, they often
affect the same gene at different positions, highlighting the
functional link between the gene and the mutant's ability to grow.
Moreover, these mutations are almost all located within the coding
sequence of the genes, much more frequently than one might have
expected given the significant proportion of non-coding regions in the
genome. All of this demonstrates that the observed phenomenon is
specific to certain genes and does not increase overall mutagenicity.
\looseness=1

\section{Mutations in the stationary phase form coherent metabolic classes}

Analysis of these mutations shows that the affected functional classes
allow the mutants to be pre-adapted to resume growth in the environment
they face, revealing concerted metabolic activities (Table~\ref{tab1}). 

\begin{table*}
\caption{\label{tab1}Mutated genes with known functions\vspace*{4pt}}
\begin{tabular}{ccc}
\thead
\multicolumn{1}{c}{Process} & \multicolumn{1}{c}{Role} &
\multicolumn{1}{c}{Genes} \\
\endthead
Carbon metabolism & Maltose regulon & \textit{malT malP malG} \\
 & Arabinose regulon & \textit{araC setD(ydeA)} \\
 & Lactose operon & \textit{lacI lacZ lacP} \\
 & Catabolite repression & \textit{mlc(dgsA)} \\
Growth homeostasis & CTP synthesis & \textit{pyrG cmk udk}  \\
 & Degradosome & \textit{pnpA rhlE} \\
Methylglyoxal homeostasis & Methylglyoxal synthesis & \textit{mgsA} \\
 & Potassium homeostasis & \textit{khtL(ybaL) kdpAB kefA(mscK) proP} \\
Translation & Modulation of translation & \textit{rsmG proQ} \\
Transcription & Start and elongation & \textit{rpoC rpoD rpoS} \\
 & Regulation of transcription start & \textit{crl rseB} \\
Replication & DNA repair & \textit{xseA}
\botline
\end{tabular}
\tabnote{The genes
identified in the experiment are grouped into well-defined processes.
Their functions show that the mutations are systematically linked to a
role in the cell that enables it to adapt upon exiting the stationary
phase, particularly through the ability these mutations confer to grow
on carbon sources that are unusable by the parental strain. Details of
the processes of interest is presented in Figures~\ref{fig3} and~\ref{fig4}.}
\vspace*{4pt}
\end{table*}

\begin{figure*}
\vspace*{-2pt}
\includegraphics{fig03}
\vspace*{-2pt}
\caption{\label{fig3}\textit{De novo} synthesis of CTP does not involve
CDP, even though CDP is necessary for DNA synthesis.}
\vspace*{-2pt}
\end{figure*}

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Glycolysis bypass during the stationary phase.
The glycolysis by-pass involving MGO is highlighted by a green band. We
observe the two key steps of this pathway among our mutations (light
orange ovals), starting with methylglyoxal synthase and ending with the
D-lactate precursor, which stimulates cytoplasmic acidification by
modulating potassium transport. The step immediately downstream of MGO
is likely spontaneous and does not require a specific enzyme (thus
ruling out the emergence of mutations).}
\end{figure*}

Identifying the cause of this coordination is essential for
understanding the origin of these mutations and why they possess their
adaptive character. To establish the extent of this coherence, we first
analysed their functions.

\subsection{Cyclic AMP-sensitive catabolism of carbon sources}

The dominant function of the mutants is a direct consequence of the
genetic make-up of the AMB1655 strain, which involves catabolic
repression. This can be explained as follows: in a strain deficient in
\mbox{adenylate} cyclase, some changes in the cyclic AMP receptor can restore
growth. This is because it behaves as an allosteric regulator,
switching between an inactive form and an active form stabilised by
cAMP \citep{Harman2001}. Insensitive to cAMP, certain mutants, known as
CRP*, lock it into a permanent activator of the expression of catabolic
operons \citep{Garges1985}. These mutated receptors are generally able
to activate simultaneously several operons \citep{Youn2023}. What about
other mutations in the catabolism of sugars that cannot be utilised by
the parental strain? Several sugars are included in our list, in
particular lactose (mutations in the repressor and promoter of the
lactose operon, or in beta-galactosidase) and arabinose (regulation of
transcription or transport of this sugar). These mutations are
unexpected in that these sugars were not used in the experimental setup
in which the papillae appeared. Perhaps more easily accounted for
(since this sugar is present in the \mbox{selective} medium), a greater number
of mutations involving maltose are observed. Some are found in the MalT
regulator, which activates the transcription of several operons in the
maltose regulon. Here again, a mutation appears in a promoter
region---controlled by MalT---that of the \textit{malPQ} operon, which
enables the breakdown of maltodextrins. Finally, a mutation in a gene
encoding a subunit of a maltose transporter allows this sugar to enter
the cell. Linking these mutations to the \textit{crp*} mutations, the
location of the CRP activator binding site in the promoter region of
these genes is important for the recognition of transcription
initiation factors (sigma factors), whose role we discuss below.
Finally, a mutation is observed in the repressor of the MalT activator
DgsA, which also controls the expression and activity of glucose
transport---whose metabolism is insensitive to CRP---and its amino acid
derivatives (and consequently interferes with the glucose-associated
effects of catabolic repression \citep{Lengeler2015}). In \mbox{conjunction}
with the functional class described in the following paragraph, it
should also be noted that this regulator is involved in growth
control---hence its alternate name, Mlc, for ``Make Large
Cells''---through a trade-off between glucose transport and
cAMP-modulated catabolic repression \citep{Plumbridge1998}.\looseness=-1

\subsection{Synthesis of CTP, a metabolic integrator of cell growth}

During the stationary phase, a portion of the population dies through a
process resembling apoptosis \citep{Jung2015}. Allowing
bacteria to have progeny requires that this process be regulated in a
way that promotes the resumption of growth. A subunit of
exonuclease~VII, XseA, which is important for the correction of DNA
mismatches, controls cell death. Its expression
is repressed by the CRP-cAMP complex, which simultaneously activates
the \textit{guaBA} operon transcribed in the opposite direction and
responsible for the synthesis of guanine monophosphate, an essential
precursor for \textit{de novo} nucleic acid synthesis
\citep{Hutchings2000}. In a mutant expressing CRP*, it is therefore
conceivable that the role of XseA must be regulated to facilitate a
return to growth. This could explain the presence of an \textit{xseA}
gene mutant in our collection.

The coordination of metabolism that enables cells to grow is
illustrated by the discovery of a metabolic control mechanism recently
proposed to explain the non-homothetic nature of the growth of cellular
compartments \citep{Danchin2021}. Discovered in the course of the
analysis of the evolution of the SARS-CoV-2 virus, this novel control
mechanism identified the nucleotide cytidine triphosphate (CTP) as the
pivotal metabolite required to coordinate the synthesis of critical
metabolites in the cytoplasm (a three-dimensional space), in membranes
(two-dimensional), and in genomic DNA (linear). This role results from
the fact that the precursors for DNA synthesis are not nucleoside
\textit{tri}phosphates, but \textit{di}phosphates. This places cytosine
metabolites at a decisive point in metabolism because the \textit{de
novo} synthesis of CTP by the PyrG synthetase does not use CDP---it
uses UTP and glutamine---precluding straightforward synthesis of the
deoxyribonucleotide dCDP. This would necessitate the concerted action
of catabolic genes to produce the required CDP \citep{Ou2020}. Here,
the mandatory use of this \mbox{diphosphate} is strikingly reflected by the
presence of mutations affecting not only the anabolic pathway (the
\textit{pyrG} gene), but all pathways for the salvage of cytosine
derivatives leading to the bottleneck represented by CDP generation.
Highlighting the importance of this pathway, we isolated several
mutants of the \textit{cmk} gene, which encodes cytidylate kinase CMK
(Figure~\ref{fig3}).


\textit{De novo} synthesis of nucleoside triphosphates requires that
diphosphate precursors are phosphorylated. Ribonucleoside diphosphates
are necessary intermediates for the formation of deoxyribonucleosides
for DNA synthesis. However, \textit{de novo} synthesis of CTP does not
produce CDP, the corresponding ribonucleoside diphosphate. Since
nucleoside triphosphates are in large excess relative to their
diphosphate counterparts, the metabolic origin of CDP requires the
recycling of cytosine derivatives, particularly {via} RNA
degradation, either by phosphorolysis (\textit{pnpA}) or by hydrolysis
(ribonucleases). Interpreting these mutations (in a yellow oval) as
concerted is reflected by the grouping of the relevant enzymatic
reactions, even though their genes are not clustered on the chromosome.

\subsection{A paradoxical glycolysis bypass}

When they are not multiplying, bacteria repair their aged essential
compounds or break them down and then re-synthesise them. To produce
energy and ensure general maintenance in the presence of molecular
oxygen, they respire instead of building biomass. Glycolysis then
adapts to enable ATP synthesis through respiration by maintaining a
sufficient proton gradient across the cell membrane, while preventing
the accumulation of toxic phosphorylated derivatives that result from
its slowing down. This is made possible by the glycolysis bypass that
produces methylglyoxal (MGO) from dihydroxyacetone phosphate (DHAP)
\citep{Dickmanns2018}.
In the stationary phase, inorganic phosphate becomes a limiting factor.
When its concentration is low, glyceraldehyde-3-phosphate dehydrogenase
slows down, causing an accumulation of DHAP, which interrupts
glycolysis and thus the production of pyruvate, essential for energy
generation through \mbox{respiration.} DHAP and phosphorylated MGO hydrolyse
spontaneously, but too slowly to prevent the negative effects of DHAP
accumulation. In many organisms (such as \textit{E.~coli}, 
\textit{B.~subtilis}, or man), this has led to the emergence and stabilisation of
the synthesis of a specific enzyme, methylglyoxal synthase (MgsA),
which produces MGO and phosphate. The activity of this enzyme is
allosterically regulated by phosphate, maintaining its intracellular
concentration at an optimal level \citep{Saadat1999}.

MGO is a toxic metabolite: it is a highly reactive dicarbonyl that
damages proteins, nucleic acids, and numerous metabolites. The cell
must therefore find a way to counteract its toxicity. This is all the
more necessary in the case of our experiments, since \textit{crp*}
mutations have long been known to overproduce this metabolite
\citep{Botsford1981,Melton1981}. As shown in Figure~\ref{fig4}, several
processes address this issue: (1) the use of oxidoreductases that
convert MGO into (\textit{S})-lactaldehyde; (2) degradation; and finally,
(3)~a change in the reactivity of this electrophile, which loses its
reactivity in an acidic environment. Pathway~1 exists but merely shifts
the problem due to the intrinsic reactivity of aldehydes, which is not
very different from that of MGO. The pathway must therefore be
supplemented by an aldehyde dehydrogenase. Pathway~2 is widespread in
aerobic organisms. It exploits MGO's affinity for nucleophiles---in
this case, sulfhydryl groups---to make it react with glutathione (or
its analogs, such as bacillithiol in Bacillaceae or mycothiol in
Mycobacteria). The thiol group of these metabolites protects the cell
against many toxic compounds, particularly reactive oxygen species.
With MGO, glutathione forms a hemithioacetal converted by a glyoxalase
into (\textit{S})-lactoylglutathione, which is then hydrolysed into
D-lactate \citep{Iacometti2022}. Finally, pathway~3 mobilises proton
transport into the cell. Many transport systems import protons, but a
preferred pathway for MGO detoxification uses the inverse transport of
potassium and protons as a means. This is illustrated, for example, in
\textit{Yersinia enterocolitica}, where the relatively acidic
intracellular environment mitigates the toxicity of MGO
\citep{Bengoechea2000}.


In our experiments, the latter pathways manifest as a set of mutations
forming a coherent functional unit, starting with the \textit{mgsA}
gene mutation. Cytoplasmic acidification involves the potassium ion
(K$^{+}$), which must be imported. To this end, KdpFABC, a control valve
composed of four subunits, uses ATP in a gating mechanism to maintain a
high intracellular potassium concentration, whereas it is generally low
outside the cell. By associating, KdpA and KdpB form a tunnel through
which several water molecules and a K$^{+}$ ion move together into the
cell when its intracellular concentration falls below a threshold,
triggering ATP hydrolysis \citep{Greie2011,Boel2019,Valia2026}.
Substantiating this function, we observed mutations in the
\textit{kdpA} and \textit{kdpB} genes. The coupling between potassium
export and proton import, meanwhile, is implicated through a mutation
in the \textit{khtL} (\textit{ybaL}) gene, which encodes a cation/proton
antiporter. This ion exchanger links pathways~2 and 3, as its homolog
in \textit{B.~subtilis}, \textit{khtU}, encodes a transporter that
exports K$^{+}$ in response to activation by
($S$)-D-lactoyl-bacillithiol, leading to cytoplasmic
acidification that is both necessary and sufficient for maximum
protection against MGO \citep{Chandrangsu2014}. Finally, a gene
identified in our study, \textit{kefA} (\textit{mcsK}), directs the
synthesis of a diaphragm that opens when osmotic pressure increases,
but only in the presence of potassium in the cell's periplasm
\citep{Li2002}. Its expression is normally repressed during the
stationary phase. Furthermore, the KefA protein associates with the
degradosome, which organises RNA degradation through both hydrolysis
and phosphorolysis \citep{Regonesi2006}. This function is necessary for
the CTP metabolism described above, which substantiates our
interpretations.

The logic behind this set of functions is further established when we
consider the \textit{proP} mutation in the promoter of the ProP
transporter gene, which maintains intracellular osmolarity through the
simultaneous import of protons along with proline or glycine betaine
\citep{Ozturk2023}. This coherence is supported by another mutation
affecting the ProQ molecular chaperone, which stabilises the ProP
messenger RNA, as well as the messenger RNA for the periplasmic protein
MalM of the maltose regulon, whose function remains unknown to date
\citep{Stein2020}. It is merely known that the efficiency of
\textit{malM} gene translation is highly sensitive to the presence of
protons \citep{Schumacher2023}. This thus links the maltose regulon to
the role of protons in MGO resistance. Finally, it is worth noting that
the transcription of the previously mentioned \textit{khtL}
(\textit{ybaL}) gene depends on DNA supercoiling, which is highly
sensitive to the osmotic stress response. It is further controlled by
sigma factor~38 (RpoS), linking this entire complex to the
transcription function we must now consider, since here too, numerous
mutations in the corresponding machinery have been isolated in our
experiment.

\subsection{Transcription machinery}

Mutating the transcription mechanism is the most direct way for a cell
to optimise its metabolism in response to environmental changes. A
general involvement of this process is obvious, considering the
mutations in RNA polymerase and factors that control the initiation of
its activity \citep{Cohen2022}. \textit{Escherichia coli} RNA
polymerase consists of several subunits: two ${\upalpha}$ subunits
(RpoA), one ${\upbeta}$ subunit (RpoB), and one ${\upbeta}'$ subunit
(RpoC). Mutants of these subunits were present in our experiments. To
enable it to recognise a promoter and initiate transcription, RNA
polymerase uses an additional subunit, a sigma factor of which there
are several types. The most important of these, sigma~70 (RpoD), is the
factor that directs the enzyme towards the transcription of
housekeeping genes. Mutants of this proteins are also found among our
mutants. However, the RpoS factor (sigma~38), which is specific to the
stationary phase \citep{Schwartz2025}, best characterises our mutations
associated with the transcription process.

RpoS regulates the transcription of most genes involved in the cell's
management of physiological or metabolic transitions, as well as its
entry into the stationary phase \citep{Hengge2011}. The mutations
observed in our study often lead to its inactivation, but not always.
This may be related to the fact that its expression is repressed by CRP
in the presence of cAMP during growth phase transitions
\citep{Guo2015}. Furthermore, the corresponding gene, \textit{rpoS}, is
known for its instability, as it is inactive or has been lost in many
laboratory strains \citep{Spira2011}. Finally, the inactivation of RpoS
in ageing colonies confers a selective advantage for survival by
allowing the accumulation of acetate during the stationary phase
\citep{Bergman2014}. These features are reflected here by the increase
over time in the number of mutations within the \textit{rpoS} gene,
appearing more frequently in late papillae \citep{Sekowska2016}.
Responding to many types of transitions, RpoS is a \mbox{fragile} protein that
is easily degraded by proteolysis. An adaptor, RssB, transports it to
the discriminating protease ClpXP. In stressful conditions, the
\mbox{degradation} of RpoS is suspended due to the sequestration of RssB by
anti-adaptors, each of which is induced in response to a specific
stress \citep{Brugger2023}. Apart from RpoS itself, we have not
identified any mutants of these factors, but it goes without saying
that, despite the scope of the experiment, we have explored only a
fraction of the evolutionary landscape. As a case in point, we
identified a mutant of the Crl factor, which specifically binds to the
complex between RNA polymerase and RpoS and stabilises it, thereby
playing an essential role in restoring the RpoS basal level
\citep{Bougdour2004}.

Sigma E (RpoE), a factor specific to the expression of genes involved
in cell envelope metabolism, undergoes a fate similar to that of RpoS.
In this case, the anti-sigma factor RseA is sequentially cleaved by the
proteases DegS, RseP, and cytoplasmic proteases to release sigma E in
response to a dysfunction in outer membrane biogenesis
\citep{Konovalova2018}. This proteolysis is regulated because the
direct interaction between RseA and another factor, RseB, inhibits the
cleavage of RseA by DegS \citep{Chaba2011}. The proteolytic activation
of DegS and the disruption of the binding of RseB---a factor for which
we have isolated a mutation---are therefore both necessary to trigger
the extracellular stress response via sigma E. At this current
qualitative and descriptive stage, we can observe that it is genes
preferentially expressed in the stationary phase that are the target of
specific mutations.

\section{Transcription is locally mutagenic}

The explanations proposed for the adaptive mutagenesis of
beta-galactosidase reported by Cairns and colleagues have sparked a
long-standing controversy \citep{Cairns1988,Brisson2003}. Apart from
fanciful interpretations or those invoking the mundane and purely
phenomenological role of mechanisms associated with DNA replication,
several studies have proposed that they originate from the
transcription process {via} a mechanism known as
``retromutagenesis'', the molecular explanation of which has evolved
over time \citep{Holmquist2002,Callegari2016}. Initially, the proposed
hypothesis remained dependent on the traditional direct involvement of
DNA, assuming that, during the transcription of DNA regions altered by
physico-chemical agents, the messenger could bypass the lesions while
triggering \mbox{local} \mbox{replication}, preventing repair and thereby fixing
local alterations in the genome. Since this would correspond to regions
transcribed in response to environmental changes, the result would be
adaptive \citep{Doetsch1999}. Later, at the very time we were reporting
on what we are discussing here, the authors of this hypothesis used the
directed reversion of a \textit{lacZ} gene mutation as a model and
modified the explanation by proposing a new concept in which the
molecular process of transcription played a central role. The DNA
double helix covered by messenger RNA forms a ``bubble'' where
single-stranded polynucleotides become accessible to reactive agents,
thereby significantly increasing the probability of local mutagenesis
\citep{Morreall2015}. Can these very limited studies be extended to
cover the entire genome? Our experiments demonstrate that the targeted
process of retromutagenesis can be applied to a population, where it
often results in the simultaneous presence of multiple mutations in the
genome or even within the same gene, always in the same strand.

This can be identified because knowing the gene sequence gives us a
handle to reveal not only the exact nature of the mutation, but
also---given the direction of gene transcription---which DNA strand has
been mutated. The vast majority of our mutations result from two types
of events: either transversions, which change G{\textbullet}C pairs to
T{\textbullet}A, or transitions, in which cytosine is replaced by
thymine. In the first case, they result from the oxidation of guanine
to 8-oxoguanine in the presence of reactive oxygen species. In the
second case, they result from the deamination of cytosines. The mutated
base is primarily present in the transcribed strand within the gene's
coding sequences (84\% for G transpositions and 93\% for C
deaminations), which establishes the role of transcription in the
process. The frequency is even higher in the \textit{crp} gene, since
in this gene isolated from 594 independent papillae, 99\% of G
transpositions and C deaminations occurred in the transcribed strand
\citep{Sekowska2016,Frendorf2019,Lauritsen2021}.

In our experiments, a papilla appears in a colony containing
approximately one million bacteria. This represents an enriched local
bias as compared to that observed in the parental strain AMB1655, which
exhibits a normal spontaneous mutation rate of less than two mutations
per billion bacteria \citep{Wu2017}. Given that these mutations make
hot spots and are restricted to two types, they can only be the result
of some well-defined molecular processes, such as the transcription of
a gene into messenger RNA. By opening the DNA double helix,
transcription exposes the bases protected within the helix to chemical
damage. In the presence of molecular oxygen, guanine is known to be
easily oxidised into a series of derivatives, such as 8-oxoguanine
\citep{Hori2011}. The unwinding of DNA also exposes cytosine to an
intrinsic chemical alteration: in equilibrium between several
tautomeric forms, its exposure to the reactivity of water promotes its
deamination to uracil \citep{Bhagwat2016}. It should be emphasised that
this latter cause of mutations is independent of the presence of
molecular oxygen.

Viewed in this way, transcription is inherently mutagenic, and the
simultaneous presence of mutations in multiple genes implies that they
are transcribed in concert. This phenomenon upends the evolutionary
landscape of the species. It exposes the process of gene expression
itself to an inevitable selection pressure that is quite different from
that generally proposed to explain how species evolve. Evolution is
supposed to result from pre-existing mutations involving directly one
or more of the organism's phenotypic traits. If the
transcription-related mechanism we uncovered is general, it remains to
be explained why the mutations we observe form clusters that alter
functions grouped into metabolic pathways corresponding to coherent
functional units.

\section{Concerted, adaptive mutagenesis results from the concurrent
transcription of genes involved in survival and growth}

How can we explain this functional coordination? We must first assume
that the genes expressed during this age when the cell just survives
serve to keep the cell alive. Only a small fraction of the functions
encoded by the genome are involved. Survival requires a basal
metabolism in which the cell recycles everything it can. This activity
is illustrated here by mutations in the MGO-dependent glycolysis
bypass. If the genes of this pathway appear in our study, it is because
they are transcribed. The most plausible explanation is therefore that
the proteins involved, \mbox{having} aged, aggregate or are degraded, losing
their function, which requires them to be synthesised anew. The
consistency of the whole is evident in the fact that the pathway using
MGO is strictly subordinate to the pathway that uses glutathione to
produce D-lactate, then pyruvate \citep{Iacometti2022}. These functions
are all part of the set of mutations obtained. The associated proteins
must therefore change at a comparable rate, adapting their ageing
process to their role in metabolic surveillance, which enables
survival.

A second constraint also follows: the cells must be ready to resume
growth without being subjected to the unbearable mechanical forces
resulting from the differential growth of their components. Poor tuning
would lead to their lysis. This requires the maintenance of the cell's
spatial homoeostasis to be anticipated when it begins to grow again. In
a completely different context, we have demonstrated that the cell
utilises the nucleotide CTP for this purpose, through both its
\textit{de novo} synthesis and the recycling of all compounds
containing cytosine derivatives, particularly RNAs. Once again, we must
assume that the proteins decay at a similar rate so that they can be
re-synthesised in a coordinated manner, enabling cells with the
relevant mutations to grow. Unfortunately, there are few studies that
explicitly identify the degradation of the enzymes involved in the
biosynthesis of nucleotides in this family, aside from work showing,
{in vitro}, that they are susceptible to proteolysis (e.g.\ 
\citep{Simard2003} for PyrG and \citep{Ofiteru2007} for Cmk).

Does this also apply to the metabolism of carbohydrates regulated by
cAMP? The ultimate reason for their use seems here quite
straightforward: the very fact that the cell contains a constitutively
active activator, CRP*, means that most, if not all, the operons
positively regulated by CRP are activated. We must, however, explain
why the \textit{crp} gene is transcribed during survival. This amounts
to determining whether CRP decays in a time-dependent manner or, at the
very least, is an unstable protein. Most studies of this receptor
concern not the protein's lifespan, but that of its messenger RNA. An
earlier study, however, shows that CRP is naturally degraded, likely
quite rapidly, and that this depends on its interaction with the
machinery that controls its folding, assisted by the molecular
chaperone DnaKJ \citep{Ohki1992}.\looseness=1

There are few studies exploring the lifespan of proteins in 
\textit{E.~coli}. The most detailed study analysed the lifespan of 3200
of these
proteins when the bacteria are subjected to various types of
starvation. \mbox{However}, the experiment was conducted in a chemostat. It is
therefore limited to reporting what occurs during growth in liquid
medium, far removed from the conditions of survival on solid substrates
\citep{Gupta2024}. A notable finding of this study was, however, that
none of the proteolytic mechanisms identified to date appear to be
involved in the degradation of these proteins. This shows that our
knowledge of intracellular proteolysis is lacking essential elements.
It is, of course, a very important experiment, which can be used to
understand the selective mechanisms that have linked the function of
these proteins to their more or less rapid turnover during growth. A
second observation from this study is  that, frequently, regulators of
gene expression have a short lifespan, well suited to their regulatory
function. This could also apply to the regulators identified in our own
study. This work, however, does not account for the ages of life other
than the growth phase, emphasising the lack of attention paid to the
universal yet poorly understood mechanism of protein ageing that we are
now describing.

\section{Is programmed protein ageing the source of adaptive mutations?}

The mutation outcome of our experiments naturally leads to the
hypothesis that a cell in survival mode remains on standby, ready to
resume division as soon as conditions become favourable---even though
the cells are deteriorating in a time-dependent manner. What mechanism
did evolution adopt to make the most of this standby period, and what
are its consequences? As always, the core mechanism underlying natural
selection is to make use of whatever is available and to recruit what
is already present as an agent of some stabilising function
\citep{Schmalhausen1949}, even if this necessitates refining this
initially awkward capability over generations. As time goes by, the
proteins that maintain the cell's functions inevitably deteriorate.
Their effectiveness gradually declines. Cells must, therefore, replace
those that no longer function and synthesise them anew. This requires
the expression of their genes, beginning with their transcription. If
the very process of ageing can be subject to natural selection, we will
have identified the primary cause triggering the general process that
leads to adaptive mutagenesis.

If programmed, protein ageing could serve as a \mbox{biological} clock,
linking survival-essential functions of a subset of key
proteins---which may differ in different organisms---to the exploration
of new behaviours better adapted to the environment thanks to the
mutagenesis induced by their re-synthesis. What do we know about the
fundamental mechanisms of protein ageing? Due to passing fads and the
irrepressible quest for youth, simplistic ideas with lucrative
implications dominate the scene, far removed from scientific
rationality that tells us that the human life span is considerably
constrained by our genome \citep{Shenhar2026}. As illustrated by the
plethora of advertisements for fanciful remedies, one cause of cellular
alterations results from chemical reactions due to short-lived reactive
compounds (for example, the famous ``reactive oxygen species'',
consequences of oxidative stress) or from metabolic or even physical
accidents, because matter is subjected to various types of radiation.
However, while these alterations are ubiquitous, their sites of action
and intensity vary considerably. Since they arise from accidents---by
definition random---it is challenging to incorporate them into a
universal law of evolution. For this reason, they mask an underlying
intrinsic phenomenon that, in turn, marks the passage of time, but now
in a constant and inevitable manner. Proteins are particularly
affected, but their individual lifespan remains difficult to predict,
if only because they rarely exist in isolation, as they play a major
role in multi-protein complexes, which often also include nucleic acids
\citep{McShane2016,Sagawa2024}.

In fact, one cause---long recognised but largely ignored---must be the
primary factor: in water, all proteins change \textit{spontaneously},
each with its own intrinsic decay time (half-life), acting as genuine
biological clocks \citep{Robinson2004}. This constraint comes into play
as early as the translation step, even before the protein can interact
with other cellular components. This is because the polypeptide chain
is naturally unstable due to the presence of mainly two amino acids,
asparagine and aspartate (though not exclusively these). Depending on
the structural context (sequence and spatial structure), L-aspartate-
and especially L-asparagine-containing motifs form an L-succinimide
intermediate through dehydration (or deamidation in the case of
asparagine). This intermediate rehydrates and tends to isomerise into
L-isoaspartate. The chronological evolution of the polypeptide thus
modified can even subsequently lead to a change in chirality, with
L-aspartate becoming D-aspartate (Figure~\ref{fig5}). All of this
distorts the backbone of the protein's peptide bonds, altering or
destroying its function \citep{Robinson1970,Aswad2000}. This process,
depending on physico-chemical conditions (temperature and water
activity in particular), has a characteristic lifetime for each
protein, ranging from a few minutes to several decades or even
centuries \citep{McKerrow1979}. If this process is subject to natural
selection, it could then serve all sorts of functions that have so far
gone completely unnoticed.

\begin{figure*}
\vspace*{-1pt}     
\includegraphics{fig05}
\vspace*{-2pt}     
\caption{\label{fig5}Spontaneous cyclisation and fate of aspartate and asparagine residues
in polypeptides. Isoaspartate residues can be methylated, and the
methyl group is then hydrolysed, which can result in the formation of
an aspartate residue, but only at a low rate.}
\vspace*{-2pt}     
\end{figure*}

Very often, but not always, this isomerisation is followed by the
repair of L-isoaspartate residues, catalysed by a methyltransferase
that uses \mbox{S-adenosylmethionine} (AdoMet) and restores an \mbox{L-aspartate}
residue. If the starting point is an \mbox{L-aspartate} residue in the native
chain, the polypeptide returns to its initial state. However, the
repair process replaces L-asparagine residues with L-aspartate
residues, which alters many of the protein's properties, particularly
its electrical charge. Furthermore, this process is imperfect and
metabolically very costly, as it requires three ATP molecules to
produce one AdoMet, and only a fraction of the methylated
L-isoaspartate yields L-aspartate, while the rest returns to the
incorrect motif the rest of the time (\citep{Lowenson1991,Danchin2011}
and Figure~\ref{fig5}). In \textit{E.~coli}, this remediation is carried
out by the L-isoaspartate(D-aspartate)-O-methyltransferase Pcm protein,
known for its role in the survival of ageing bacteria
\citep{Visick1998}. It goes without saying, however, that most aged
proteins will not be repaired but degraded. If they are important for
survival, they will then be replaced by ``young'' proteins through the
expression of the corresponding genes, which involves a transcription
step---a source of adaptive mutations.

To take this a step further and set up an initial experiment to
determine the role of this repair mechanism, we needed to establish
whether the proteins we had identified as mutation targets were
susceptible to ageing. This was all the more important given that, in
\textit{E.~coli}, at least two proteins evolve into a sequence
containing an isoaspartate motif while \mbox{remaining} functional. These are
the ribosomal S11 protein and the MurA protein, which is key to
bacterial cell wall synthesis. The modification of the S11 protein
occurs so rapidly (in the order of a few minutes) that we must assume
it is a functional modification \citep{David1999}. This is also the
case for UDP-N-acetylglucosamine~1-carboxyvinyltransferase MurA, which
supports the first step in bacterial cell wall synthesis and for which
the presence of a functional isoaspartate residue has been established
\citep{Zhang2020}. This is therefore not a matter of ageing but of true
post-translational maturation. Furthermore, it is reasonable to
consider that some of these isomerisations lead to functional
deamidations, particularly to adapt certain proteins for survival by
modifying some of their physico-chemical properties. This could
specifically affect the resilience of the transcription machinery,
which must remain functional over the long term \citep{Danchin2011}.

Unfortunately, as we have seen, the details of spontaneous protein
ageing have been little studied. We do know, however, that the
expression of the \textit{crp} gene results in a finely tuned content
of the protein CRP in the cell. Its transcription is negatively
regulated by the CRP-cAMP complex, which binds immediately downstream
of the transcription start site, preventing the formation of the
corresponding mRNA \citep{Hanamura1991}. Furthermore, this
transcription itself is positively regulated by this complex
\citep{Hanamura1992}. This results in maintaining the receptor
concentration at an \mbox{optimal} level for the cell. Thus, if we find a way
to trigger the degradation of CRP, its transcription will be
immediately activated, carrying over all the consequences of adaptive
mutagenesis that we have identified. With some luck, if CRP ages due to
the isomerisation of certain aspartate or asparagine residues and if
this ageing is subject to repair, inactivating the \textit{pcm} gene in
our model could therefore teach us a little more about the role of
cellular ageing by accelerating the degradation and re-synthesis of
CRP. 

\begin{figure*}
\vspace*{3pt}
\includegraphics{fig06}
\vspace*{3pt}
\caption{\label{fig6}Papillae observed on colonies of strain AMB1655
carrying a mutation that inactivates the \textit{pcm}
gene. The figure displays the outcome of two independent
experiments, two days after spreading a few hundred bacteria onto
MacConkey's maltose medium. Almost all the colonies developed a red
papilla.}
\vspace*{3pt}
\end{figure*}

We therefore replaced the \textit{pcm} gene in strain AMB1655 with its
inactive counterpart from the reference collection of all inactivated
\textit{E.~coli} genes (Keio collection \citep{Baba2006}) and verified
that the resulting strain does not have a mutator phenotype. In the
set-up used to monitor the generation of adaptive mutations, the result
of this construct is spectacular, as shown in Figure~\ref{fig6}. After
48~h, almost all the white colonies had produced a red papilla, which
corresponds to a presumably local mutation rate at least two hundred
times higher than that observed with the parental strain. It also
becomes very difficult to maintain the basic construct, which must be
systematically re-isolated from a white colony. 

The demonstration of the role of spontaneous chemical evolution of
proteins in the origin of the adaptive mutations reported has remained
at a very preliminary stage so far, due to a lack of financial support.
However, as this article aims to show, this work opens up a new avenue
for understanding what occurs during survival, particularly with regard
to its contribution to species evolution. We hope this will stimulate
further research.

\section{Perspectives: how evolution anticipates future events}

The evolution of a population towards differentiation into several
species is based on the functional triad: variation, selection, and
amplification. The most widely accepted premise underpinning the theory
of evolution by natural selection is that variation, which is in
practice random, cannot favour any particular future direction for
evolution. It is assumed to affect the genomes of the individuals
making up the population at random, notwithstanding the internal
constraints linked to the biochemical nature of the DNA that
constitutes them. The population, \mbox{facing} an uncertain environment, will
retain---select---a certain number of descendants who will multiply
and, over time, produce a divergent tree of successive descendants
whose adaptation to the environment will be the result of happy
accidents. There is a discordant but ideal scenario that is rarely
considered \mbox{because} it is easily misappropriated to justify eugenic
approaches \citep{Weindling2012}. If the mechanisms of evolution
allowed individuals to anticipate certain characteristics of the
environment their progeny will face, this would go a long way towards
ensuring the long-term survival of new species. But chance is blind.
Taking this feature of evolution into account is, therefore, generally
not considered acceptable. 

We know, however, that at least one mechanism of anticipation already
exists. While ubiquitous, its role remains vastly underestimated and
obscured in most phylogenetic trees---largely due to the reservations
just mentioned, as well as the ``adamist'' bias that insists on a
single origin for any entity evolving by descent. This process is the
acquisition of genes present in the environment through horizontal gene
transfer, a major pathway of genomic innovation \citep{Soucy2015}. This
is because sampling these genes---which reflect the many adaptive
strategies employed by the communities present---and incorporating them
into its genome enables an \mbox{individual} to respond effectively to
conditions which, for its parents, were unattainable novelties. This
certainly plays a crucial role in speciation 
\citep{Medigue1991,Thomas2017,Munshi2025}. Furthermore, given that the
action of chance can differ according to the age of life, the emergence
of adaptive mutations could provide a rational basis for a new type of
anticipation.

The majority of evolutionary models limit evolution to the transmission
of genetic inheritance within a population of young individuals. It is
thought to result from a ratchet mechanism affecting the genome
following chemical or physical damage to DNA (which is a fragile
string), whether in its inactive form, during replication, or as a
result of various forms of recombination. However, such damage,
occurring before and during replication, is highly varied. In fact,
research has long led to the understanding that, although random, the
basal rate of DNA mutagenesis cannot be uniform, as it depends on the
DNA sequence and its local conformation \citep{Maki2002}. It also
depends on the biochemical processes involved. For example, replication
in bacteria is semi-conservative. This creates a fundamental asymmetry
between the two strands of the double helix and subjects each to a
different mutagenesis mechanism \citep{Snedeker2017}. The mutation rate
in a cell, of course, is also the result of the numerous processes that
ensure the maintenance of the genome during replication, immediately
afterwards, and long afterwards. Moreover, DNA is not an inert memory;
it is transcribed far more often than it is replicated. Transcribing a
gene in the same direction as replication does not have the same effect
as doing so in the opposite direction. Potential conflicts between the
orientations of the two processes mean that the distribution of genes
within the chromosome depends on their orientation relative to the
direction of replication \citep{Gao2017}.
More specifically, given that the transcription of each gene has
a start and an end, it is observed that the 5$^{\prime}$ end of genes
has an abnormally low mutation rate \citep{Radrizzani2025}.

Beyond this common way of viewing transcription's contribution to
evolution, the generally ignored hypothesis of retromutagenesis
suggests that transcription is, by its very nature, mutagenic because
the double helix unwinds to serve as a template in this initial stage
of gene expression. Mutagenesis itself remains locally random (subject
to constraints imposed by the physico-chemical mechanisms involved),
but gene transcription is not. Only a portion of the genome is
transcribed, and transcription depends on the environment and phases of
growth. One might therefore expect that the most highly transcribed
genes, especially those involved in the housekeeping machinery during
growth, would be affected first, leading to a prevalence of mutations
in the regions where they are located. However, the question of whether
this transcription, which is often high, leads to an elevated mutation
rate or vice versa, remains partly open. A key reason for this
uncertainty is that selection pressure on highly expressed housekeeping
genes could have led to restricting their sensitivity to being
transcribed because of the risk of the ``error catastrophe''
mutagenesis \citep{Orgel1963,Orgel1970}, leaving only those genes that are
less sensitive to transcription-induced mutagenesis to persist. In
fact, precisely because this is an essential mechanism, we would expect
there to be mechanisms that counteract the expected mutagenesis, and
that these have been retained over time. These mechanisms could
combine, for example, greater stability of proteins once translated
(which reduces the need for transcription, since they do not have to be
re-synthesised) with optimisation of the corresponding gene sequence to
make it more resistant to the causes of mutations \citep{Oman2022}. The
debate remains open, with experiments continuing to be interpreted as
pointing in one direction or the other \citep{Sniegowski2000,Chen2013}.
This contradiction between families of selective pressures undoubtedly
explains the many questions surrounding the role of transcription in
mutagenesis. Furthermore, what has been established regarding the
existence and nature of mutations in genes encoding proteins essential
for cell division reflects a popular view in which ageing is perceived
as the result of the accumulation of errors at all levels: replication,
transcription and translation \citep{Diggs2008}, and not as an
important or even beneficial factor in evolution. We shall not go into
further detail here, though we wish to emphasise that what is new in
our line of thinking is the involvement of a universal yet unexpected
mechanism of variation: the intrinsic spontaneous chemical modification
of proteins, the effect of which, to our knowledge, has not yet been
explicitly taken into account in evolutionary models.

Rather than focusing on growing cells, we have studied what happens
during the long duration of the final age of active life, when the cell
is merely surviving. This means taking seriously the consequences of
ageing for evolution. The role of the different ages of life is by no
means insignificant, if only because metabolism and the associated
processes are not the same at the start of growth, during its
\mbox{development}, and once it has stopped. The ageing of all cellular
components is inevitable, and this has significant consequences for the
mechanisms of mutagenesis. As we mentioned at the beginning of this
article, it took some time for it to be accepted that bacteria age and
die. Whilst this was relatively easy to understand for microbes that
reproduce by budding, as the number of buds is limited
\citep{Henderson2008}, it was more difficult when cells divide
symmetrically. The idea that bacterial division distributes ageing
entities randomly was proposed to illustrate the concept of
antifragility conceived by Nassim Taleb \citep{Danchin2011} and
revisited ten years later to account for the difficulties encountered
in accepting that bacteria age and die. In this context, the role of
proteins is central, and when cells divide, it is the fact that aged
proteins concentrate at the cell poles that creates the critical
asymmetry leading to cell death \citep{Lapinska2019,Steiner2021}.
However, what is being investigated here is not ageing during growth,
but---excluding spore formation---the duration of survival without
multiplication \citep{Pechter2017}. Whilst proteins are indeed at the
heart of the debate, could the limited lifespan of some of them be the
cause of adaptive mutations? Several deeply ingrained preconceptions
reflect the conceptual difficulties encountered during the many
discussions on this subject.

As we have seen, two families of conditions that alter proteins, which
are essential agents of growth, have dominated causal explanations for
the limits on lifespan. On the one hand, gene expression, whether
through replication, transcription or translation, is prone to
systematic errors that result in altered protein forms. On the other
hand, proteins are subject to inevitable chemical accidents. However, a
specific cause linked to their polypeptide nature has been overlooked.
Indeed, the idea that an \textit{intrinsic and inevitable}
chronological ageing of proteins contributes significantly to longevity
and the generation of offspring has not been seriously considered. To
date, this contribution has been explored only marginally, apart
through the experiments reported here, which are, of course, very
preliminary. We wished to highlight that this inevitable alteration of
proteins is associated with a process rarely invoked: mutagenesis due
to the physical implementation of transcription, triggered by the need
to resynthesise the defective proteins. However, by its very nature,
this \mbox{phenomenon} has a significant anticipatory component, whose
contribution to the evolution of species is not implausible. 

Indeed, the consequences of this inevitable time-dependent change give
the transcription of the entities of interest a recursive, and
therefore innovative, character \citep{Hofstadter1979}, inevitably
linking it to their adaptation. The fact that proteins have an
intrinsic lifespan directly linked to their polypeptide sequence is a
remarkable property, since this half-life is certainly adjustable
following mutations and is therefore a likely but unexpected target of
natural selection. While this paper presents merely a conjecture based
on a set of consistent observations, it would be interesting to
construct an abstract model to test the plausibility of the adjustment,
through natural selection, of the half-life of crucial proteins to the
reproductive lifespan of a model species. For example, we should
examine whether (and how) agents that collaborate to perform the same
function tend to have similar lifespans and be re-synthesised at the
same rate, even though they were initially subjected to purely random
evolution. This could precede or occur in parallel with the emergence
of mechanisms to regulate their co-expression (this is one of the roles
of operon regulation, for example). This type of evolution is
illustrated in our study by the presence of mutants revealing the
concerted evolution of several crucial metabolic functions. And, as
proteins can evolve by adapting their lifespan to a specific function,
this could allow natural selection to gradually adjust it to suit the
different ages of life. Living a long life has selective significance
only if it goes hand in hand with the possibility that ageing
individuals are able to produce, or at least help to produce, viable
offspring, and this explains the metabolic pathways identified in our
experiment.

Here, the march towards death is not merely an inevitable fate for
individuals. In certain contexts, it is also the means by which
numerous variants are produced, limited to specific pathways that allow
them to anticipate the emergence of unpredictable environmental traits.
This underestimated, or even ignored, aspect of the temporal
information carried by proteins should be at the centre of a new
interpretation of the theory of natural selection. An important
consequence of this approach is that, although it recognises, in
principle, the positive role of ageing, this does not mean that the
proteins involved in this process must necessarily perform identical
metabolic functions in different organisms. The only thing that matters
is that a family of important proteins contributes to evolution through
their lifespan. If our conjecture is correct, this would explain why
the precise mechanisms of aging vary from one organism to another,
which makes the study of this process particularly difficult, yet
fascinating.

Finally, those exploring the applications of synthetic biology will
have noted that variations in our proposed experimental model could be
employed to reveal unknown metabolic pathways. By using synthetic
constructs, these pathways could be directed towards new functions. For
example, artificial compounds could be added to the growth medium.
Furthermore, the deliberate and selective modification of the lifespan
of a carefully chosen protein could facilitate the identification of
new families of adaptive mutations, providing insights into the
functional cycles present within cells in both natural and synthetic
organisms.

\section*{Acknowledgments}

We dedicate this work to the memory of Agn\`{e}s Ullmann, whose
scientific contributions to the early stages of molecular biology are
often underestimated, for her unwavering support of new ideas and the
associated experiments. AD designed the experimental protocol presented
here and conducted the experiment that served as the basis for the
general microbiology course at the Pasteur Institute led by Agn\`{e}s
Ullmann, as mentioned in the text. He drafted the first version of the
manuscript. AS, while serving as director of the laboratory at Stellate
Therapeutics (aka AMAbiotics SAS), now defunct, constructed strain
AMB1655 and its derivatives, and then designed the experimental
conditions necessary to carry out, over time, the experiments for the
isolation and characterisation of adaptive mutants, which were
subsequently developed in collaboration with Morten N\o{}rholm and his
colleagues in Copenhagen. Experiments demonstrating an unexpected
effect of isoaspartate residue repair on the generation of adaptive
mutations were developed from 2011 to 2018 and presented and discussed
at several conferences held at the University of Hong Kong and BGI in
Shenzhen. AD and AS approved the final version of the manuscript. No
funding was received for the production of this article.

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