Download That bacteria may be able to mutate in response to

DIRECTED MUTATIONS
As
by Billy Goodman
That bacteria may be
able to mutate
in response to
environmental cues
could add a wrinkle to
evolutionary theory
but is likely to do
it no serious damage.
24 MOSAIC Volume 23 Number 1 Spring 1992
sentences in scientific papers
go, this was guaranteed to raise
eyebrows: "We describe here
.
experiments and some circumstantial evidence suggesting that
bacteria can choose which mutations
they should produce."
The sentence appeared in a 1988 Nature paper, 'The Origin of Mutants," by
John Cairns, Julie Overbaugh, and Stephan Miller of the Harvard School of
Public Health. The paper inflamed passions in the genetics and evolutionarybiology communities and reopened an
issue that biologists had considered
long settled. Must mutations arise spontaneously, independent of natural selection and without regard to their potential
usefulness? the Harvard researchers
asked. Or do mutations sometimes arise
as a specific response to the current
needs of an organism?
For nearly half a century, the unequivocal answer has been that mutations are
spontaneous and random. According to
the neo-Darwinian view of evolution,
variation created in this fashion is then
acted on by the forces of evolution,
chiefly natural selection, to produce
change. One blunt sentence and what
the authors referred to as "three or four
rather ambiguous experiments" are not
enough to overturn the mutational cornerstone of neo-Darwinism, in the minds
of most biologists. After all, the alternative view, that mutations happen in direct response to need, harks back to the
theories of Jean Baptiste de Lamarck,
the French forerunner of Charles Darwin. Lamarck's views often are caricatured simplistically as "inheritence of acquired characteristics."
No one in this debate about mutation
is abandoning natural selection as the
prime shaper of evolution. But Cairns
and some supporters suggest that evolutionary theory must incorporate a new
wrinkle. They say that some mutations
may occur more often when they are
advantageous than when they are not.
(Some call this contention neo-Lamarckian, but Cairns professes no interest in
such a label)
Cairns's report in Nature was provocative but short on experimental details
and data. Several biologists have since
obtained experimental results, mainly
with the bacterium Escherichia coli, that
they say support Cairns and what has
come to be called directed mutation.
Meanwhile, some criticize the results on
theoretical grounds. A few have produced experimental results that contradict at least some of the observations
supporting directed mutation.
The blunt language of Cairns's paper
and its challenge to long-held views
stirred controversy and debate that continue with each new paper published.
For Sahotra Sarkar, a Boston University
philosopher of biology, the uproar is
appropriate: "Science working as it
should; claims have led to skepticism,
which has sparked people to do further
experiments."
The Luria-Delbruck distribution
Demonstration of the spontaneity of
mutation was one of the triumphs that
ushered in the modern age of molecular
biology, ten years before the elucidation
of the structure of deoxyribonucleic
acid, or DNA, by James Watson and Francis Crick. The credit goes largely to Salvadore Luria at Indiana University and
physicist-by-training Max Delbriick at
Vanderbilt University, who were studying bacterial viruses called phages.
MOSAIC Volume 23 Number 1 Spring 1992 25
In the early 1940s, bacteriologists
were largely in the dark about the nature
of heredity and mutation in bacteria.
However, they easily,could select for
mutants, such as bacteria resistant to attack by phages. When abacterial culture
was spread on a plate containing an
abundance of phage particles, typically
only a rare colony, representing a single
resistant bacterium and its descendants,
would arise after incubation.
The obvious question was: Did contact with the phages cause bacteria to
become resistant, at some low frequency? Or did bacteria spontaneously
become resistant while growing in the
test tube before contact with the
phages? In that case, phages simply
would be revealing preexisting mutants
by killing sensitive cells.
The method of proof was indirect.
They relied on a statistical argument.
Luria said later that the argument occurred to him when, as he watched a
slot machine at a faculty dance,; he noticed that most coins fed into the machine yielded nothing. Occasionally,
small returns were forthcoming. Rarely,
one coin produced a jackpot.
Luria realized that the slot machine
might be a model for spontaneous mutation, in which a mutation to phage resistance could occur at any time during
the history of a growing culture. If a mutation occurred early, subsequent cell
division; would lead to a large clone of
resistant cells. When plated in the presence of phages, many resistant colonies
would appear, even though only one mutation had occurred. Such a situation
would mimic the rare jackpot from the
slot machine. Late-occurring spontaneous mutations would produce smaller
clones. A series of cultures would show
great variability in the number of resistant cells. This clonal statistical distribution is generally called the LuriaDelbriick distribution.
The last bastion
By contrast, if mutations to phage resistance occur only after exposure to
phages, results of plating replicate cultures would be quite different. If each
cell has the same small chance of being
made resistant and if roughly the same
number of cells are exposed on each
plate, then each plate would contain similar numbers of mutants and no jackpots.
The mutants would represent a Poisson
distribution around a mean value. That
value is the product of the number of
bacteria exposed and their probability of
becoming resistant.
This is known as the fluctuation test,
The answer came in a series of elebecause it examines the fluctuation in
gantly simple experiments conceived by
the number of mutants per plate. The
Luria, analyzed mathematically by Delresults clearly demonstrated the exisbriick, and widely regarded as the birth
tence of jackpots, so mutations must
of bacterial genetics. Luria and Delhave been occurring spontaneously durbriick presented their conclusions in a
famous 1943 report in Genetics: "We con- ing the growth of the cultures before
they were challenged with phages. The
sider the . . . results as proof that in our
Luria-Delbriick analysis contained clear
case the resistance to virus is due to a
caveats ("in our case" and "it remains to
heritable change of the bacterial cell
be seen"). It also stimulated others to
which occurs independently of the aclook for more direct ways of distinguishtion of the virus. It remains to be seen
ing spontaneous from environmentally
whether or not this is the general rule."
induced adaptive mutations.
Goodman is a free-lance science writer
Joshua Lederberg, then at the Univerbased in Brooklyn, New York. He was also sity of Wisconsin, confirmed and exthe author of'Toward a Pump, Not a Filtended the Luria-Delbriick results.
ter," Mosaic Volume 22 Number 2.
Lederberg and his colleagues showed
26 MOSAIC Volume 23 Number 1 Spring 1992
that mutations for resistance to some antibiotics occurred spontaneously. The
researchers ingeniously revealed that
cells that never had been exposed to the
antibiotic still could be resistant.
Lederberg says that his work and that
of Luria, Delbriick, and others "brought
bacteria into the [neo-Darwinian] fold.
Bacteria had been the last bastion of
Lamarckian ideas." Since these famous
experiments of the 1940s and 1950s, almost no one seriously had believed that
mutations may occur in response to the
need of an organism.
No one, that is, until John Cairns.
Challenge...
A molecular geneticist interested in
the rate-limiting steps of mutations that
lead to cancer, Cairns recognized that
Luria, Delbriick, and the others never
had really tested whether the environment could selectively induce mutations. For example, for a bacterial cell to
become resistant to phages, the cell
must have a mutation before it comes in
contact with the virus. Phages bind to
cell-surface receptors and kill sensitive
cells immediately, before they replicate
and have a chance to mutate. When a
mutation makes a cell genotypically resistant, its surface still will bear phage
receptors that had been made previously. The cell will remain sensitive.
Only after several cell divisions, which
eliminate the receptors by dilution, will
the progeny be phenotypically resistant.
As a result of phenotypic lag, "these
classical experiments could not have detected (and certainly did not exclude)
the existence of a non-random, possibly
product-oriented form of mutation,"
Cairns and his colleagues wrote.
"If you want to know whether creatures can turn on a mutation they need,"
John Cairns says, "it's not fair to kill
them. You want to know if they can turn
on a mutation by worrying about the
environment."
To give bacteria something to worry
about, and time to respond adaptively,
Cairns and his colleagues imposed nonlethal selection. In separate experiments, they plated strains of E. coli that
shared an inability to use lactose as an
energy source (and thus are designated
Lac-) on agar plates with lactose as the
only energy source. The conditions
were sharply selective for Lac+ cells.
Under such conditions Lac- cells do
not die, however. They enter a starvation
state known as the stationary phase. It
has been known for several decades that
mutations can occur in stationary-phase
bacteria. This is paradoxical. For a mutation to become part of the genome, a
damaged DNA strand—the incipient mutation—must be replicated before it is
corrected by DNA repair systems. Yet stationary-phase bacteria no longer replicate their DNA or divide. How can mutations happen in such bacteria? Stanford
University's Philip Hanawalt says that
limited DNA repair activity is believed to
take place in stationary-phase bacteria.
Stationary-phase bacteria are hardly quiescent; many of their genes are specifically expressed.
One experiment typifies what Cairns
and his colleagues found. Some Lac+ revertants formed colonies within 48
hours of plating, providing evidence that
they probably formed spontaneously in
the growing cultures before plating.
Other revertants continued to appear on
the plates as the bacteria starved. This
is a key observation, which Cairns was
not the first to make, but which he was
the first to exploit Cairns also noted that
revertants did not accumulate when lactose was absent, a circumstance in
which the mutations would not have
conferred any advantage.!
The shape of the distribution of mu-
tants also suggested to Cairns and his
colleagues that some mutations occurred spontaneously during the growth
phase and that other mutations occurred
during the stationary phase. The shape
was similar to that predicted by models
in which mutations happen randomly
during the growth of a culture, followed
by additional mutations in the final generation, after exposure to the selective
agent has taken place.
All these facts led Cairns to conclude
that the selective agent was inducing
mutations in the stationary- phase E. coli
cells on his plates.
. . . and response
As befits a boldly written paper in a
prominent journal, Cairns's article galvanized evolutionary biologists. Critical
letters poured into Nature pointing out
that Cairns and his colleagues had failed
to rule out simpler alternatives to directed mutation. Barry Hall of the University of Rochester, who has published
a series of papers that are widely regarded as the best evidence for directed
mutation, has written that Cairns's paper
caused controversy for two reasons.
"First, it challenged the orthodox view
of the complete separation of mutation
and selection; second, it suggested the
existence of unknown mechanisms by
which the environment could somehow
instruct a cell as to which mutations
should be generated or retained," Rochester's Barry Hall wrote.
Hall was inspired by discussions with
Cairns before the publication of Cairns's
Nature article. Then at the University of
Connecticut, Hall began working with a
system that required two mutations to
occur before E. coli could use the sugar
salicin. One of the mutations, he documented in a 1988 Genetics article, is so
rare in growing cells that he failed to
detect it. But plated on salicin, Sal+ revertants appeared after a delay of about
12 days. Their frequency was orders of
magnitude greater than expected if the
two mutations were occurring independently and spontaneously.
Furthermore, the rarer spontaneous
mutation happened first This mutation
was excision of an insertion sequence.
It was followed relatively quickly by the
second mutation, which only then permitted transcription. When salicin was
absent, Sal+ revertants had no advantage, and Hall was unable to detect excision mutants.
Among Cairns's critics, Bruce Levin
of the University of Massachusetts is
typical in acknowledging that Cairns's
paper "made me aware that the alternative hypothesis to random mutation
hadn't been rejected." Levin is one of a
handful of biologists who use microbes
to probe evolution. He says that the distribution of mutants on plates can shift
from the clonal arrangement expected
of spontaneous mutations toward the
Poisson distribution expected of specifically induced mutations for a variety of
simple biological reasons. He has pursued theoretical and experimental work
on this topic with postdoctoral associate
David Gordon in his laboratory and
mathematician Frank Stewart of Brown
University in Providence, Rhode Island.
Following the lead of earlier workers
who had studied the Luria-Delbriick distribution, the trio presented what Levin
calls "a general method for incorporating virtually any form of biological reality into the distribution of mutants." The
key conclusion, he says, is that almost
any biological complication, such as mutants growing faster or slower than the
parental strain, shifts the distribution t o
ward the Poisson. "The implication of
this is that one can't, or shouldn't, use
MOSAIC Volume 23 Number 1 Spring 1992 27
deviations from Luria-Delbruck distributions as arguments for directed mutation, as Cairns and colleagues did."
Nonetheless
However, Levin's arguments about
statistics do not address Cairns's observation of accumulation of mutants on
plates. Levin suspects that this accumulation was not a result of mutations in
specific response to the selection pressure. Instead, most of the mutants on
the plates were replication-dependent,
spontaneous mutants. For a variety of
physiological and ecological reasons,
some occurred in the growing cultures
but were delayed in their appearance on
the plates. Others occurred on the plates
themselves as a result of growth by
cross feeding. For example, a Lac- cell
can divide on a plate containing lactose
as a carbon source, by using metabolites
excreted by Lac+ colonies that are already on the plate.
Patricia Foster, a bacterial geneticist
at the Boston University School of Medicine who has done some further experiments with John Cairns, says that
28 MOSAIC Volume 23 Number 1 Spring 1992
growth on plates does not account for
the accumulation of all revertants. In her
work, the bacterial population on the
plate never grows enough to account for
the number of revertants seen.
Another possible explanation for the
observations is that the stress of starvation could cause a general increase in
mutation rates, rather than a specific directed response. That is what was suspected by Richard Lenski, an evolutionary biologist at the University of
California at Irvine, and his graduate student, John Mittler. "We're not convinced
by the control experiments published so
far or by the bookkeeping that certain
mutations are occurring more often in
the presence of favored selection than
not," Lenski says. Shortly after Cairns's
paper appeared, Lenski and Mittler took
aim at one of the key examples.
That example was based largely on a
1984 paper by University of Chicago microbiologist James Shapiro. Shapiro, in
fact, is the unsung protagonist of this
entire field. His paper foreshadowed
Cairns's 1988 work, referring explicitly
to the problem that phenotypic lag pre-
sented for the Luria-Delbrtick paradigm.
Using the techniques of Chicago colleague Malcolm Casadaban, Shapiro
worked with an E. coli strain which was
engineered to study hybrid proteins.
Shapiro's construct had an arabinose
regulatory gene followed by an arabinose structural gene into which was inserted a Mu prophage. Further downstream was the lacZgem that codes for
beta-galactosidase, the enzyme that degrades lactose. This arrangement, in
which the strain could grow on neither
arabinose nor lactose, is designated
Lac (Am)-. However, appropriate excision of the Mu element fused the initial
region of the ara gene to the beginning
of the lacZgene, allowing the bacterium
to grow as long as lactose and arabinose
are present.
Shapiro demonstrated that ara~lac fusions occurred only after plating on a
selective medium, with both arabinose
and lactose, and only after a long delay.
Cairns confirmed these results. The interpretation of Cairns's work on this system is still debated by his supporters
and critics. As Boston University's Foster sees it, "Cairns showed that saturated cultures that had exhausted their
carbon source did not produce fusions
unless and until lactose (and arabinose)
was added." To Cairns and his coauthors, the events seem to be "another
example of the production of appropriate mutations in response to selection."
On the other hand, John Mittler says,
he and Richard Lenski were concerned
that the experiments of Shapiro and
Cairns did not adequately distinguish
between the effects on the rate of Mu
excision caused by cell starvation and
those caused by the presence of lactose
and arabinose.
Mittler and Lenski therefore designed
an experiment to focus on the effect of
starvation on the rate of Mu excisions.
They plated Lac (Ara) - cells on plates
with minimal nutrients and no added
sugars. They sprayed some plates immediately with lactose and arabinose.
Any Lac (Ara) + colonies that appeared
within two days of spraying were considered to have been preexisting and to
have formed spontaneously. When the
plates were sprayed at the time of plating, no Lac(Ara)+ colonies appeared
within two days. However, the longer
the bacteria were allowed to starve on
the plates, the more Lac (Ara) + colonies
were revealed within two days. After
controlling for cell death and growth,
Mittler and Lenski concluded that the
increased rate of excision of Mu elements could be attributed entirely to the
stress of starvation. Among starving
cells excision mutations occurred at
comparable rates in the presence or absence of lactose and arabinose; there
was no need to invoke directed mutation
to explain the excisions.
Another route
After his salicin paper appeared, Hall
chose to work on E. coli with base-substitution mutations that rendered the
bacteria unable to synthesize the amino
acid tryptophan. Bacteria that are unable
to synthesize an amino acid are called
auxotrophs because they must be supplied with a nutrient to grow, tryptophan
in this case. In growing culture with tryptophan supplied, the bacteria that Hall
used reverted to prototrophy at the rate
of about one every ten billion cell divisions. When Trp- cells were incubated
on plates without tryptophan, some revertants appeared within four days.
These were judged to be preexisting. Revertants continued to appear after the
fourth day, for as long as ten days (since
extended to 28 days).
In an important experiment, Hall used
an E. coli strain that was auxotrophic for
the amino acids tryptophan and cysteine
to test the specificity of the mutagenic
response to starvation. Colonies that
were Trp-Cys- were starved for either
tryptophan or cysteine. Those starved
for tryptophan reverted to Trp+, but did
not revert to Cys+. Those starved for
cysteine reverted to Cys+ but did not revert to Trp+. These results demonstrated that the delayed mutations were
specific to the selection that was imposed upon them.
Hall believes that he eliminated the
possibility of growth on plates by using
ampicillin to kill growing cells. He measured the death rate of cells on selective
plates in the presence and absence of
ampicillin. With ampicillin, cells could
die, but new cells could not arise by cell
division. The observed death rate equals
the actual death rate minus the growth
rate. If growth were occurring, the observed death rate would be greater in
the presence of ampicillin than in its
absence. However, the observed death
rates were identical.
Despite strong confirmation of preferential production of advantageous mutations, Hall was not ready to accept a truly
directed mechanism. In the 1988 Nature
paper, Cairns and his colleagues had
sketched three possible mechanisms for
their observations. One required some
form of information flow from protein
back to the DNA, counter to the central
dogma of molecular biology.
Cairns speculated that a bacterium
under stress might make many mistakes
in transcribing its DNA. For any gene, a
variable set of messenger RNAS would
exist Using a still-unidentified organelle, the cell then would sense which
mRNA made the best protein and would
reverse- transcribe the messenger.
Aside from the fact that no direct evidence has been found to support such
a model, there are "about 47 problems"
with it, quips Hall.
"I accepted [John Cairns's] argument
about the limits of the Luria-Delbriick
work, but I was firmly rooted in conventional dogma," Hall says. John raised the
question of a specific response to the
environment. I kept asking, 'Could an
underlying random process account for
what I'm seeing?'" In fact, Cairns and
his coauthors had suggested random
processes for their second and third
mechanisms, in a single sentence that
attracted much less attention than the
more speculative first mechanism.
Error-prone stages
Franklin Stahl, of the Institute of Molecular Biology at the University of Oregon, gave a more detailed version of
one of Cairns's random mechanisms in
a "News and Views" report that accompanied Cairns's paper in Nature. Stahl's
model is based on the knowledge that
the first steps of DNA synthesis are error
prone. Those steps create thousands of
mispairings and other incipient mutations. Mismatch-repair systems largely
correct those errors before replication
can proceed to stabilize them as mutations. In the absence of mismatch repair,
DNA synthesis has an error rate of about
one per million bases, which shrinks to
about one per billion bases after mismatch repair.
Stationary cells do not replicate their
DNA, but there is thought to be limited
DNA turnover or repair synthesis, as polymerases remove stretches of DNA and
put in patches. Mistakes occur during
MOSAIC Volume 23 Number 1 Spring 1992 29
soned that transcription Itself might be
mutagenic, because the double helix of
DNA unzips partly during transcription in
order to permit one strand to form a template In making mRNA. "Single-stranded
DNA is much more vulnerable to mutation," Davis says.
"During transcription there is always
a region of about 12 single-stranded
bases behind the RNA polymerase," he
says. Normally, transcription proceeds
at a pace of about 45 bases a second, but
Davis speculates that It may be much
slower as a cell starves for nutrients,
leaving the window of single-stranded
bases open longer.
What would account for mutations being directed at just the gene or operon
that a cell needs fixed? Take the lactose
operon as an example. Although a mutation In the lacZ gene may render the
enzyme inoperative, its operator should
still function to turn the operon on. According to the Davis model, if bacteria
are plated on lactose, their lac- operons
should hold temporary regions of singlestranded DNA.
A trigger
this process. In what Stahl terms the one
speculation of his model, he suggests
that mismatch repair in starving cells Is
sluggish and depends on a slow trickle
of energy, perhaps from cannibalizing
the cell's own building blocks.
If by chance a useful error occurs, the
cell Is able to grow In Its current environment. Then the cell will replicate Its
DNA and divide. If replication occurs
faster than mismatch repair can correct
the error, the mutation becomes a permanent part of the cell's genome. The
30 MOSAIC Volume 23 Number 1 Spring 1992
sluggishness, of the repair system has
no consequence for incipient mutations
that do not permit the cell to grow, Stahl
says. As long as the cell remains In stationary phase, such mutations eventually will be repaired.
Another model, again relying on wellknown molecular biology, was proposed
In 1989 In the Proceedings of the National
Academy of Sciences by bacterial physiologist Bernard Davis, retired from the
Harvard Medical School. Davis calls his
mechanism transcriptional bias. He rea-
In a 1990 Genetics paper, Hall proposed his own heuristic model along the
lines of the Stahl and Davis models. Essentially, according to Hall's model,
when life is tough, cells mutate like
crazy, in hopes that something good will
happen. Hall wrote that the problem "is
to explain how selective conditions
could Increase the frequency of useful
mutations without increasing the frequency of mutations at other loci."
He suggested that during prolonged
starvation, some cells in a colony enter
a hypermutable state. In this condition,
both extensive random DNA damage and
error-prone repair occur. By chance, an
error introduced into the DNA during this
process might solve the problem facing
the cell at the time. The cell then would
replicate Its DNA and stabilize the advantageous mutation. The cell then would
exit the hypermutable state.
If none of the incipient mutations
proved useful, the cell would die. It
wouldn't "hang around for you to look
for other, nonselected mutations," Hall
says. Foster points out that Hall's hypermutable-state model, Stahl's slow-repair
model, and one of the random models
proposed In Cairns's 1988 paper share
the same underlying idea: "There is a
reversible process for creating variation,
that only results In permanent genetic
change if the cell is successful."
The hypermutable-state model predicts that mutations at nonselected loci
should be found In successful mutants.
Hall screened 110 trp+ revertants and
discovered two other auxotrophic mutations. This frequency of auxotrophic
mutations "Is about 600-fold higher than
expected If the two mutations (trp reversion and an auxotrophic mutation) were
independent events," Hall wrote.
Joshua Lederberg, who played a lead-
ing role In starting the field of microbial
genetics nearly a half century ago, was
one of those Inspired to investigate the
molecular biology underlying apparently directed mutation. Until a few
years ago, Lederberg was president of
Rockefeller University. Now he and a
colleague, David Thaler, are beginning
to examine the Issue from a mechanistic
perspective.
Mechanistic
Lederberg finds Davis's views "very
congenial" to his own. He suggests that
there is "a good case that differences in
secondary structure of DNA will result In
differential mutability." In other words,
the DNA double helix Is hardly homogeneous. There are all manner of landmarks along Its length that could be targets for mutagenesis. Those landmarks
are related to its supercoiling and the
proteins with which It interacts. In
Davis's model, It Is the single-stranded
region around the RNA polymerase that
is the target.
MOSAIC Volume 23 Number 1 Spring 1992 31
The little evidence that addresses the
transcriptional-activation model is not
supportive. Cairns says he excluded it
with an experiment reported in 17 words
in the 1988 paper. The experiment employed isopropyl beta-D-thiogalactoside
(IPTG) . This substance is known as a gratuitous inducer because it can induce
the lactose operon to begin transcription, but it cannot be degraded by betagalactosidase. Thus it provides no energy to the cell Cairns reported that
Lac+ mutants did not accumulate when
Lac- cells were grown in the presence
of IPTG. If transcriptional activation alone
were enough to stimulate mutation,
Lac+ revertants should have accumulated with IPTG.
Cairns returned to the laboratory with
Patricia Foster. In 1991, the pair published work on reversion to Lac+ in a
strain of E. coli that contains a frame
shift rendering it Lac- but in which the
lactose operon is permanently switched
on. Foster says their work disproved
transcriptional activation in this case.
The operon was always transcribed,
whether or not lactose was present, and
yet Lac+ revertants accumulated only
when lactose was present.
Foster and Cairns found preliminary
evidence that what they call adaptive
mutation depends on a function of the
reck gene, which is part of the recombination-repair system of DNA. In their
experiments, mutations in reck had no
effect on spontaneous mutation during
32 MOSAIC Volume 23 Number 1 Spring 1992
growth. However, the mutations in reck
greatly reduced the occurrence of mutations appearing on the plates after lactose had been added.
Transcription
Meanwhile, others remain convinced
that transcription is playing a role in the
phenomenon. Thomas Cebula, chief of
the molecular biology branch of the division of microbiology of the United
States Food and Drug Administration,
and Michael Prival of the agency's genetic toxicology branch, have found evidence for late-appearing, apparently directed mutations in the common
bacterium Salmonella typhimurium.
They studied missense mutations in
the histidine operon. Reversions to His+
could occur intragenically, by base substitutions in the codon for a crucial
amino acid. Alternatively, reversions
could occur extragenically, by a suppressor mutation that causes transfer
RNA to make a mistake in translation that
cancels out the mutation- causing histidine auxotrophy. Cebula says that both
kinds of reversions appear to have happened spontaneously in growing cultures. However, only intragenic events
were observed in colonies that had
starved for more than two days.
The absence of late-arising suppressors is consistent with a transcriptionalactivation hypothesis, says Cebula. In
addition, he says, the kinds of mutational
events that they uncovered are consistent with Stahl's model of mismatch repair being slow. However, they have not
measured repair rates directly.
Philip Hanawalt, an expert on DNA repair at Stanford University, is also convinced that both transcriptional activation and DNA repair are playing some
role in the phenomenon of directed mutation. Recent work in his laboratory
shows that repair may be more efficient
in active genes than in inactive ones.
Hanawalt also suspects that the mismatch repair system is not working in
starving cells.
As Hanawalt sees it, a starving cell
may use repair synthesis to put in a
in bacteria allows at least some incipient
mutations to be incorporated into the genome more frequently when they are
advantageous than when they are not.
The production of variation continues to
be regarded as a completely random
phenomenon, although many interpreted the 1988 Nature paper as an argument for nonrandom production of variation. As they try to document and probe
the incorporation of mutations from all
angles, some biologists speculate on its
importance for evolutionary biology.
A question of significance
patch of DNA, perhaps where an RNA polymerase got hung up at a kink in the DNA.
If mismatch repair is not operating, the
error rate during repair synthesis would
be about 1,000-fold higher than during
normal DNA replication. 'The mere act
of putting in a patch entails the possibility of making a mistake, because polymerases are not perfect," Hanawalt says.
A direct test
The transcriptional-activation model
and its variants continue to attract experiments, but so far the only direct test
of Hall's model has been by Hall himself,
in experiments published in Proceedings
of the National Academy of Sciences in
1991. He measured the reversion to trp+
of a strain that was doubly mutant, trpAtrpB~. He found double mutants (revertants to tryptophan prototrophy) approximately 100 million times more
frequently than expected if the mutations were independent
Hall calculated the probability of each
base substitution occurring, given that
the cell had entered a hypermutable
state. The conditional probability for
each mutation worked out to about 0.04.
That figure should apply to other bases
in the genome, according to the hypermutable-state model. But "that's mayhem," Hall says. "That won't fly. You
can't have a base-substitution rate of one
in 25 and live to tell the tale."
Hall wondered whether such a high
rate might apply only to a small region
of the genome around the trpA and trpB
loci, so he sequenced 700 base pairs
around the two loci in 11 double revertants. The hypermutable-state model
predicts that other base substitutions
should occur among those 700 base
pairs. He failed to find any, and concludes that in its simple form, the hypermutable-state model is not correct.
Barry Hall argues that the analysis also
applies to the Stahl and Davis models,
making them incorrect in their simple
versions as well.
Many biologists, though by no means
all, are convinced that some mechanism
Cairns himself, recently retired from
the Harvard School of Public Health,
says there may be no evolutionary significance. "We're looking at reversion of
defects, which is somewhat unnatural,"
Tie declares.
~ —On this point, at least, Bruce Levin
agrees. T o accept the existence of directed mutation, we have to believe that,
in addition to random mutation, organisms have mechanisms to produce specific modifications of their genomes to
repair genes that are normally functional
and very rarely mutate to nonfunctional
states," Levin says.
Could directed mechanisms work in
organisms other than bacteria? Foster
thinks that is unlikely. "Organisms with
a germ line have a tremendous barrier,"
she says. 'The whole game is to protect
the genome from the environment." (In
late 1991, there were preliminary reports, as yet unpublished, of similar phenomena in yeast, a one-celled eucaryote.)
Nevertheless, the possibility that one
or more mechanisms may increase the
rate at which beneficial mutations occur
in starving cells has drawn increased attention to the stationary phase and new
respect for E. coli and other bacteria in
their natural habitats. Starving on a laboratory plate is far more typical of the
conditions under which most bacteria
evolved and live than the optimal laboratory conditions in which many bacteria can grow exponentially: aerated
liquid cultures in 0.2 percent glucose solutions kept at 37 degrees Celsius.
"E. coli has another life," Boston's Patricia Poster says. "It's not over when
the log phase ends."
•
The National Science Foundation has
contributed to the support of the research
described in this article principally
through its Genetics program.
MOSAIC Volume 23 Number 1 Spring 1992 33