The σE stress response is required for stress‐induced mutation and amplification in Escherichia coli

Article (PDF Available)inMolecular Microbiology 77(2):415-30 · May 2010with36 Reads
DOI: 10.1111/j.1365-2958.2010.07213.x · Source: PubMed
Abstract
Pathways of mutagenesis are induced in microbes under adverse conditions controlled by stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e. are stressed. Stress-induced mutagenesis in the Escherichia coli Lac assay occurs either by 'point' mutation or gene amplification. Point mutagenesis is associated with DNA double-strand-break (DSB) repair and requires DinB error-prone DNA polymerase and the SOS DNA-damage- and RpoS general-stress responses. We report that the RpoE envelope-protein-stress response is also required. In a screen for mutagenesis-defective mutants, we isolated a transposon insertion in the rpoE P2 promoter. The insertion prevents rpoE induction during stress, but leaves constitutive expression intact, and allows cell viability. rpoE insertion and suppressed null mutants display reduced point mutagenesis and maintenance of amplified DNA. Furthermore, sigma(E) acts independently of stress responses previously implicated: SOS/DinB and RpoS, and of sigma(32), which was postulated to affect mutagenesis. I-SceI-induced DSBs alleviated much of the rpoE phenotype, implying that sigma(E) promoted DSB formation. Thus, a third stress response and stress input regulate DSB-repair-associated stress-induced mutagenesis. This provides the first report of mutagenesis promoted by sigma(E), and implies that extracytoplasmic stressors may affect genome integrity and, potentially, the ability to evolve.
The s
E
stress response is required for stress-induced
mutation and amplification in
Escherichia coli
mmi_7213 415..430
Janet L. Gibson,
1
Mary-Jane Lombardo,
1†
Philip C. Thornton,
1
Kenneth H. Hu,
1‡
Rodrigo S. Galhardo,
Bernadette Beadle,
2
Anand Habib,
Daniel B. Magner,
1
** Laura S. Frost,
2
Christophe Herman,
1,3
P. J. Hastings
1
and
Susan M. Rosenberg
1,3,4,5
*
1
Department of Molecular and Human Genetics,
3
Department of Molecular Virology and Microbiology,
4
Department of Biochemistry and Molecular Biology,
and
5
The Dan L Duncan Cancer Center, Baylor College
of Medicine, Houston, TX 77030-3411, USA.
2
Department of Biological Sciences, University of
Alberta, Edmonton, Alberta T6G 2E9, Canada.
Summary
Pathways of mutagenesis are induced in microbes
under adverse conditions controlled by stress
responses. Control of mutagenesis by stress
responses may accelerate evolution specifically
when cells are maladapted to their environments, i.e.
are stressed. Stress-induced mutagenesis in the
Escherichia coli Lac assay occurs either by ‘point’
mutation or gene amplification. Point mutagenesis is
associated with DNA double-strand-break (DSB)
repair and requires DinB error-prone DNA polymerase
and the SOS DNA-damage- and RpoS general-stress
responses. We report that the RpoE envelope-protein-
stress response is also required. In a screen for
mutagenesis-defective mutants, we isolated a trans-
poson insertion in the rpoE P2 promoter. The inser-
tion prevents rpoE induction during stress, but leaves
constitutive expression intact, and allows cell
viability. rpoE insertion and suppressed null mutants
display reduced point mutagenesis and maintenance
of amplified DNA. Furthermore, s
E
acts independently
of stress responses previously implicated: SOS/DinB
and RpoS, and of s
32
, which was postulated to affect
mutagenesis. I-SceI-induced DSBs alleviated much of
the rpoE phenotype, implying that s
E
promoted DSB
formation. Thus, a third stress response and stress
input regulate DSB-repair-associated stress-induced
mutagenesis. This provides the first report of
mutagenesis promoted by s
E
, and implies that extra-
cytoplasmic stressors may affect genome integrity
and, potentially, the ability to evolve.
Introduction
Stress-induced mutagenesis is a collection of mecha-
nisms observed in bacterial, yeast and human cells in
which mutagenesis pathways are activated in response to
adverse conditions, such as starvation or antibiotic
stresses (reviewed, Galhardo et al., 2007). These mecha-
nisms enhance mutagenesis specifically during times of
stress, and thus have the potential to increase genetic
diversity upon which natural selection acts, potentially
accelerating evolution, specifically when cells are mal-
adapted to their environments, i.e. are stressed. These
mechanisms are potentially important models for
mutagenesis that drives antibiotic resistance (Cirz and
Romesberg, 2007; Galhardo et al., 2007; Lopez and
Blazquez, 2009; Petrosino et al., 2009; Kohanski et al.,
2010) and pathogen–host evolutionary arms races (e.g.
Prieto et al., 2006; Boles and Singh, 2008).
Although there appear to be multiple molecular mecha-
nisms of stress-induced mutagenesis observed in different
strains, organisms and environmental conditions, a strong
common theme among them is the requirement for one or
more cellular stress response(s). For example, induction of
the SOS DNA-damage response is required for SOS
‘untargeted’ mutation of undamaged DNA (Witkin and Wer-
mundsen, 1979), stress-induced reversion of a mutant lac
gene in Escherichia coli cells starving on lactose medium
(McKenzie et al., 2000), ciprofloxacin-induced resistance
mutagenesis in E. coli (Cirz et al., 2005), bile-induced
resistance mutagenesis in Salmonella enterica (Prieto
et al., 2004; 2006), and mutagenesis in aging E. coli colo-
nies (Taddei et al., 1995). Similarly, the RpoS (s
S
) general-
or starvation-stress response is required for most of the
pathways listed above, except ciprofloxacin-induced
Accepted 9 May, 2010. *For correspondence. E-mail smr@bcm.edu;
Tel. (+1) 713 798 6924; Fax (+1) 713 798 8967. Present addresses:
The J. Craig Venter Institute, San Diego, CA 92121, USA;
Massa-
chusetts Institute of Technology, Cambridge, MA, USA;
§
Department
of Microbiology, University of Sao Paulo, Sao Paulo, Brazil;
Stanford
University, Palo Alto, CA, USA; **Max-Plank-Institute for Biology of
Ageing, Gleueler Straße 50a, D-50931 Cologne, Germany.
Re-use of this article is permitted in accordance with the Terms
and Conditions set out at http://www3.interscience.wiley.com/
authorresources/onlineopen.html
Molecular Microbiology (2010) 77(2), 415–430 doi:10.1111/j.1365-2958.2010.07213.x
First published online 7 June 2010
© 2010 Blackwell Publishing Ltd
mutagenesis (Bjedov et al., 2003; Layton and Foster,
2003; Lombardo et al., 2004; J. Casadesus, pers. comm.)
and additionally for stress-induced gene amplifications
during starvation in E. coli (Lombardo et al., 2004), stress-
induced excisions of coliphage Mu (Gomez-Gomez et al.,
1997; Lamrani et al., 1999), and stress-induced point
mutagenesis (Saumaa et al., 2002) and transposon move-
ment (Ilves et al., 2001) in Pseudomonas putida. The
stringent response to amino acid starvation in E. coli
(Wright et al., 1999) and Bacillus subtilis (Rudner et al.,
1999b), and the ComK competence response to starvation
in B. subtilis (Sung and Yasbin, 2002), are required for
mutation pathways induced by starvation stresses. In
human cells, two different stress responses to hypoxia
provoke two separate mechanisms of genome instability
(Bindra et al., 2007; Huang et al., 2007). Coupling of muta-
tion pathways to cellular stress responses appears to be
how mutagenesis is targeted specifically to times of stress.
The stress responses are thus the cornerstone of the
regulation of these mutation pathways, and their identities
provide windows into the biological/environmental stres-
sors that elicit the mutagenesis responses.
In the E. coli Lac assay for starvation-stress-induced
mutagenesis (Cairns and Foster, 1991), either reversion of
a frameshift mutation by compensatory frameshift muta-
tion (Foster and Trimarchi, 1994; Rosenberg et al., 1994)
in an F-borne lac gene, or amplification of the leaky lac
allele to multiple copies (Hastings et al., 2000; Powell and
Wartell, 2001), allows growth of cells starving on lactose
medium. The frameshift (‘point’ mutation) pathway requires
double-strand-break (DSB)-repair proteins (Harris et al.,
1994; 1996; Foster et al., 1996), TraI, an F-encoded single-
strand endonuclease or an I-SceI-generated DSB (Ponder
et al., 2005), the DinB error-prone DNA polymerase
(Foster, 2000; McKenzie et al., 2001), and two stress
responses: the SOS DNA-damage response (McKenzie
et al., 2000) and the general-stress response regulated by
RpoS (Layton and Foster, 2003; Lombardo et al., 2004),
both of which upregulate dinB transcription. Our lab has
provided evidence that supports a mechanism in which the
point mutations are formed in acts of error-prone DSB
repair. The normally high-fidelity DSB-repair mechanism
switches to an error-prone mutagenic pathway that uses
DinB during the SOS and RpoS responses (Ponder et al.,
2005). [Alternative models discussed by Roth et al. (2006)
and Galhardo et al. (2007) and below]. The GroEL chap-
erone also modulates DinB activity (Layton and Foster,
2005). GroEL is an essential protein complex that is both
constitutively expressed and upregulated during the RpoH/
s
32
-controlled cytoplasmic unfolded-protein-stress or heat-
shock response, and also directly regulates the activity of
s
32
(Guisbert et al., 2004).
The rpoE gene encodes the s
E
transcription factor, which
positively regulates the envelope-stress response to extra-
cytoplasmic unfolded proteins. Originally identified as a
heat-shock factor, s
E
is essential for growth in E. coli at all
temperatures although the exact nature of the s
E
require-
ment for growth is not known (Hiratsu et al., 1995; Rou-
viere et al., 1995; De Las Penas et al., 1997b). In E. coli, s
E
expression during either exponential or stationary phase
up- or downregulates as many as 200 genes involved in all
areas of metabolism (Kabir et al., 2004; Rhodius et al.,
2006). Although many s
E
-regulated genes encode proteins
that promote either maintenance or synthesis of the cell
envelope, a significant number encode cytoplasmic pro-
teins that function in transcription, translation, and DNA
synthesis and repair (Rhodius et al., 2006).
The regulation of s
E
, and thus the stress response that
it controls, is complex and entails at least two independent
pathways (Ades, 2004). First, RseA, the s
E
anti-sigma
factor, is an integral transmembrane protein that binds s
E
,
sequestering it at the cytoplasmic face of the inner mem-
brane (Missiakas et al., 1997; De Las Penas et al., 1997a;
Campbell et al., 2003). An inner membrane protease
cascade (Ades, 2008), part of a regulatory pathway
termed RIP (regulatory intramembrane proteolysis) con-
served from bacteria to mammals (Rudner et al., 1999a;
Brown et al., 2000), is responsible for degradation of
RseA. Upon stress, unfolded outer membrane proteins
trigger RseA proteolysis thereby releasing s
E
into the
cytoplasm where it can associate with RNA polymerase
and activate transcription of the s
E
-regulon genes (Kane-
hara et al., 2003; Walsh et al., 2003). Turnover of RseA is
continuous, but upon stress, degradation accelerates until
the stress is removed, allowing increased s
E
levels in the
cytoplasm and induction of transcription of the s
E
regulon
(Ades et al., 1999; Alba et al., 2001; 2002; Kanehara
et al., 2003; Grigorova et al., 2004).
In addition to activation by unfolded membrane pro-
teins, s
E
is also regulated growth-phase-dependently by
the stringent response, via a less well-defined pathway.
This results in stationary-phase induction of s
E
in
response to the alarmone, ppGpp (Costanzo and Ades,
2006; Costanzo et al., 2008).
In this study, we show that activation of the s
E
stress
response is required for stress-induced mutagenesis in
the E. coli Lac system. We describe a transposon inser-
tion that disrupts s
E
expression during stress. We show
that stress-inducible rpoE expression (the s
E
stress
response) is not required for viability but the constitutive
rpoE expression is, and we correlate the stress response,
rather than the constitutive essential function of s
E
, with
formation of stress-induced point mutants and with main-
tenance (and thus accumulation of) DNA amplification.
We show that s
E
plays at least two roles in the point-
mutation pathway, one of them involving formation of
DSBs. This adds a third stress response to those control-
ling stress-induced point mutagenesis, a second to that
416
J. L. Gibson
et al
.
© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 415–430
controlling amplification, and additionally adds genetic
and genomic instability to the repertoire of consequences
of the s
E
extracytoplasmic protein stress response in
E. coli.
Results
rpoE2072::Tn10dCam is a non-null
stress–response-defective mutation that allows cell
viability without extragenic suppressor mutations
We developed a genetic screen, used it with transposon
mutagenesis to identify mutants defective in stress-
induced mutagenesis in the Lac assay (Experimental pro-
cedures in Supporting information), and identified a new
mutation that affects the rpoE gene, designated
rpoE2072::Tn10dCam. rpoE2072::Tn10dCam was moved
by P1 transduction into a ‘clean’ (non-mutagenized) assay
strain and characterized.
We find that the rpoE2072::Tn10dCam insertion lies
between the -10 and -35 regions of s
E
-dependent (P2)
gene-proximal promoter (Fig. 1A). Such an insertion might
be expected to block transcription entirely and, given that
rpoE is an essential gene (De Las Penas et al., 1997b;
Alba and Gross, 2004), allow growth only of isolates with
additional suppressor mutations that restore viability. rpoE
null mutants acquire unlinked suppressor mutations (De
Las Penas et al., 1997b). Using co-transduction experi-
ments, we show that rpoE2072::Tn10dCam insertion
mutant does not contain or require unlinked suppressor
mutations for its viability (Fig. S1). We used a kanamycin-
resistance marker in yfhH, a non-essential gene partially
linked with rpoE2072::Tn10dCam, to transduce cells car-
rying neither the rpoE nor yfhH::Kan mutation with phage
grown on the yfhH::Kan rpoE2072::Tn10dCam donor
(Fig. S1). Co-transductant frequencies of 51 4% Cam
R
/
Kan
R
transductants, and 67 2% Kan
R
/Cam
R
transduc-
tants (Fig. S1) show that although there is a slight
bias against acquisition of the rpoE2072 allele, the
rpoE2072::Tn10dCam insertion does not induce lethality
when transduced into a clean (suppressor-mutation-free)
strain background (Fig. S1). Thus unlinked suppressor
mutations are not required for the mutant’s growth.
Although rpoE2072::Tn10dCam is not a null mutation
(Fig. S1) this allele causes a slight temperature-sensitive
phenotype with similar numbers of cfu at 30°C and 37°C,
but about twofold fewer at 42°C relative to 30°C (not
shown).
Two likely explanations for the viability of rpoE2072
cells are either that the 3 end of the transposon supplies
a poorly conserved -35 sequence (Fig. 1B) or that tran-
scription is being initiated from an outward-reading pro-
moter within the transposon allowing some s
E
expression.
Data below indicate that the insertion affects the expres-
sion from the s
E
-dependent P2 promoter.
We find that rpoE2072::Tn10dCam cells do not induce
the s
E
response to a stress peptide signal. We tested
whether the transposon insertion in P2 affected stress
induction of rpoE specifically by using an rpoHP3::lacZ
fusion that is dependent on s
E
for activity (Mecsas et al.,
1993). b-Galactosidase activity was assayed over time
following initiation of the s
E
stress response by induction of
plasmid-borne YYF, a peptide homologous to the unfolded
outer membrane protein-stress signal that activates DegS
protease to degrade RseA (anti-s
E
), liberating s
E
to induce
transcription of the stress response genes (Walsh et al.,
2003). Our data show that stress induction of s
E
-regulon
genes is impaired in the rpoE2072::Tn10dCam mutant as
follows. Following IPTG induction of the YYF stress-signal
peptide from plasmid pBA166, b-galactosidase activity
expressed from the rpoHP3–lacZ fusion increased
approximately fivefold in rpoE
+
cultures coincident with an
increase in growth (Fig. 2A and B), whereas in the
rpoE2072::Tn10dCam strain, b-galactosidase activity
increased only slightly and the cultures stopped growing
(Fig. 2A and B). The data show an inability of
rpoE2072::Tn10dCam cells to induce the s
E
stress
response in response to inducer peptide.
We can understand the cessation of growth in the
rpoE2072 mutant upon induction of YYF (Fig. 2B) as
follows. YYF is expected to activate all s
E
-regulated pro-
moters, including the promoter immediately upstream of
the rseA anti-sigma factor gene, but not the transposon-
disrupted rpoEP2. This would be expected to result in
induction of rseA but not rpoE, shifting the s
E
:RseA ratio to
favour RseA and decreasing available s
E
. We suggest
Fig. 1. Location and consequences of a Tn10dCam insertion in
the s
E
-dependent rpoE P2 promoter.
A. The Tn10dCam insertion site determined by sequence analysis,
the transcription start site (*), and the –10 and –35 sequences
(underlined) are indicated (Rouviere et al., 1995). P
1
and P
2
denote
the two previously mapped s
E
promoters (Rouviere et al., 1995).
Also shown is the location of the promoter between rpoE and rseA
(Rhodius et al., 2006). (+) indicates positive transcriptional
regulation by s
E
.
B. A putative –35 sequence (underlined) is supplied by the
transposon. Location of the Tn10dCam insertion site is indicated by
symbol (). Sequence to left of symbol (Italics) is Tn10 sequence
and to the right (large type) is 5 sequence of rpoE.
s
E
stress response in mutagenesis
417
© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 415–430
that an inability to elevate transcription of the
s
E
-dependent P2 promoter of rpoE in response to stress
may result in an imbalance between sigma factor and
anti-sigma factor, leading to depletion of active s
E
and
thus the observed cessation of growth, because s
E
func-
tion is required for growth (Hiratsu et al., 1995; Rouviere
et al., 1995; De Las Penas et al., 1997b).
Stress-induced mutagenesis defect of
rpoE2072::Tn10dCam and null strains
First, quantitative stress-induced mutagenesis assays
confirmed that rpoE2072::Tn10dCam decreases stress-
induced Lac
+
colony formation dramatically at 30°C
(Fig. 3A) and 37°C (Fig. 3C). This decrease cannot be
explained by net death of the population in that the
overall survival of the population is decreased little
during the stress-induced mutagenesis experiments
(results range from no decrease, Fig. 3B, to at most a
fourfold decrease at later time points in some experi-
ments on amplification below). The Lac
+
reversion rate
from multiple experiments was approximately 11-fold
lower in the rpoE2072::Tn10dCam than wild-type cells
(Fig. 3C).
Second, an rpoE null mutation (in cells with extragenic
suppressor mutations) causes a similar severe stress-
induced mutagenesis defect (Fig. S2). Stress-induced
mutagenesis assays were performed at 30°C, because
even with its suppressor mutation, the rpoE null mutant is
temperature-sensitive and cannot grow at 37°C (De Las
Penas et al., 1997b; Alba and Gross, 2004). Nearly iden-
tically to the rpoE2072::Tn10dCam mutant, accumulation
of stress-induced Lac
+
mutant colonies was greatly
reduced in the rpoE null mutant (Fig. S2). Similar results
were obtained in another strain set in which the rpoE
+
, the
rpoE null and the rpoE2072::Tn10dCam strains all carried
DydcQ, a known rpoE suppressor mutation (Button et al.,
2007), to ensure their isogenicity (Fig. S2). DydcQ
increased the rate of accumulation of Lac
+
mutant colo-
nies in all strains but did not affect the relative reduction in
the null mutant compared with the rpoE
+
(Fig. S2). These
results generalize the conclusion that functional s
E
is
required for appearance of stress-induced mutants in the
Lac assay.
Third, the apparent decrease in Lac
+
colonies in the
stress-induced mutagenesis assay could result either
from a failure to form Lac
+
mutations or from an inability of
the Lac
+
mutant colonies carrying an rpoE mutation to
form colonies under the assay conditions. We performed
reconstruction experiments the results of which show that
cells carrying rpoE2072::Tn10dCam and a Lac
+
mutation
form colonies nearly normally under selective conditions
(Table S1). Therefore, we conclude that formation of the
mutants, not their subsequent growth on the selective
medium, is impaired by the rpoE2072::Tn10dCam allele.
The results shown in Tables S1 and S2 also address
a specific hypothesis for why s
E
might be required
for stress-induced mutagenesis. Lac
+
stress-induced
mutagenesis is associated with a cell subpopulation with
transiently increased mutation rates (a hypermutable cell
subpopulation) as seen by data showing that Lac
+
mutants possess higher levels of other mutations through-
out their genomes than stressed cells that did not become
Lac
+
(Torkelson et al., 1997; Rosche and Foster, 1999;
Godoy and Fox, 2000), and that the mutagenesis process
is not easily uncoupled from generation of these unse-
lected ‘secondary’ mutations (Gonzalez et al., 2008). The
s
E
stress response might have been required to allow
normal growth rates of cells carrying additional unse-
lected chromosomal mutations, perhaps by upregulating
chaperones and proteases in response to resulting mis-
Fig. 2. Stress induction of a s
E
-regulated promoter is inhibited in
rpoE2072::Tn10dCam cells. b-Galactosidase activity expressed
from the rpoH P3 promoter was measured following induction of
the OMP C-terminal peptide, YYF, from pBA166, or with the control
vector plasmid pTrc99. The experiment was repeated twice with
similar results.
A. b-Galactosidase activity plotted over time following IPTG
addition to LBH cultures. Strains are: SMR8843, rpoE::Tn[pYYF]
(
); SMR8844 rpoE::Tn[pTrc] (); SMR8846, rpoE
+
[pYYF] ();
SMR8845, rpoE
+
[pTrc] ().
B. Growth curve following IPTG addition. Symbols as in (A).
418
J. L. Gibson
et al
.
© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 415–430
folded proteins. However, the data in Table S2 show that
placing the rpoE2072::Tn10dCam allele into five different
Lac
+
revertants with accumulated unselected secondary
mutations did not inhibit their colony-forming ability. The
slightly longer times to form colonies overall reflect the
lower growth temperature of 32°C used in these experi-
ments (Table S2), whereas the slightly increased time for
colony formation under lactose-selective conditions at
37°C (Table S1) appears to be the result of the rpoE
mutation alone. Therefore, failure to ameliorate mutant
proteins appears not to underlie the strong mutagenesis
defect of the rpoE2072::Tn10dCam mutant (Fig. 3);
rather, mutagenesis itself is implicated.
Maintenance of gene amplification requires
the s
E
response
Stress-induced Lac
+
colonies arise via either ‘point muta-
tion’ (compensatory lac frameshift mutation) (Foster and
Trimarchi, 1994; Rosenberg et al., 1994) or via amplifica-
tion of the leaky lac allele, with lac amplification represent-
ing a minority of the Lac
+
colonies until after day 8 of
starvation on lactose plates (Hastings et al., 2000; Powell
and Wartell, 2001). We found that accumulation of lac-
amplified colonies was decreased 320-fold in the
rpoE2072::Tn10dCam mutant relative to the rpoE
+
strain
(Fig. 4A and C). In these experiments, in which lac-
amplified cfu were separated from the point mutants,
the point mutation rate was decreased ~15-fold by
rpoE2072::Tn10dCam relative to rpoE
+
(Fig. 4D).
Although there is some loss of cell viability in the rpoE
strain on later days in some experiments (e.g. Fig. 4B),
the at most 75% ( 4-fold) loss of viability does not
explain the ~300-fold deficiency in accumulation of lac-
amplified colonies.
Reconstruction of rpoE2072::Tn10dCam lac-amplified
strains showed that the decrease in viable cfu (Fig. 4B)
was not caused by slow growth, but rather an inability of
rpoE2072::Tn10dCam strains to maintain amplification:
first, on average, the transconjugants of lac-amplified
DNA into rpoE2072::Tn10dCam recipients were 32-fold
less able to form colonies on lactose medium than the
rpoE
+
transconjugants (Table 1, columns 2–4). Second,
this lower recovery was not due to slower growth
(5.1 0.2 days for rpoE
+
versus 4.9 0.3 days for
rpoE2072::Tn10dCam cells to form colonies, Table 1), or
to lower efficiency of conjugation (Table 1, last two
columns). Third, when F factors carrying a Lac
+
point
Fig. 3. The rpoE2072::Tn10dCam mutation decreases stress-induced Lac
+
reversion. Strains are rpoE
+
, SMR4562 ( );
rpoE2072::Tn10dCam, SMR5236 ().
A. Representative experiment performed at 30°C. Values are means one standard error of the mean (SEM) for eight independent cultures
of each strain. Where not visible, error bars are smaller than the symbol. A second experiment at 30°C gave similar results.
B. Relative viability of the Lac
-
population monitored per Harris et al. (1996) beginning on the day after plating (day 1) for the experiment
presented in (A). Values are means SEM for data from six selection plates. Because Lac
+
mutant cells form colonies that are visible 2 days
later (McKenzie et al., 1998), the day 3 Lac
+
colony counts pertain to the day 1 viable cell measurements, and day 5 Lac
+
colonies to the day
3 viable cells, etc. To make this comparison easier, we have shifted the viability data (B) 2 days rightward (the day 1 viability data are
presented on day 3, etc.) for easier comparison with (A).
C. Lac
+
colony formation rates at 37° from multiple experiments. Lac
+
colonies per day were calculated from colonies appearing from days 3–5
for seven independent stress-induced mutation assays and fold-difference between rates for SMR4562, rpoE
+
and SMR5236,
rpoE2072::Tn10dCam presented. Viability of all cultures was monitored per Harris et al. (1996). Mean SEM for the seven experiments is
shown in last row of table. As observed previously, overall mutation rates are higher at 30°C than 37°C, although mutations that decrease
mutagenesis do so similarly at both temperatures [Ponder et al. (2005) and A versus C].
s
E
stress response in mutagenesis
419
© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 415–430
mutation were conjugated into rpoE
+
and
rpoE2072::Tn10dCam F
-
cells (Table 1), there was no
difference in conjugation efficiency between rpoE
+
and
rpoE2072::Tn10dCam strains, showing that the poor
plating efficiency of rpoE cells plated on lactose is unique
to amplification (Table 1). Thus, we conclude that this
decrease represents a failure to maintain the amplified
arrays. In addition to the ~30-fold defect in maintenance
of amplified arrays (Table 1) there is a variable 75%
( 4-fold) decrease in viable cell counts of the
rpoE2072::Tn10dCam in late days in some experiments
(Fig. 4B). These two factors can account for
30 ¥ 4 = about 120-fold of the decrease observed in accu-
mulation of lac-amplified cfu during stress-induced
mutagenesis experiments. Because the decrease in
accumulation of stress-induced lac-amplified colonies is
~300-fold (Fig. 4C), it appears likely that, in addition to
inability to maintain gene amplifications once formed,
there was also a decrease in amplification formation
caused by the rpoE2072::Tn10dCam mutation.
s
E
induction is not generally mutagenic in growing cells
Fluctuation tests were performed to determine whether
induction of the s
E
response provokes mutagenesis
generally in growing cells, as, for example, induction of
the RpoS regulon does (Yang et al., 2004). We measured
mutation to rifampicin resistance, which occurs by any of
a few specific base-substitution mutations in the rpoB
gene (Jin and Gross, 1988), and used the plasmid-borne
gene for the YYF peptide to induce expression of
s
E
-regulated promoters in rpoE
+
cells. Expression of YYF
had no effect on mutation rate per generation indicating
that the s
E
response does not provoke mutagenesis in
general but its requirement is specific for stress-induced
mutagenesis (Table S3).
Recombination proficiency in rpoE2072::Tn10dCam
cells
s
E
upregulates transcription of homologous-recombination
genes recB, recD, recJ and recR (Rezuchova et al., 2003;
Kabir et al., 2005; Rhodius et al., 2006). Because recB
mutants (Harris et al., 1994) and recD recJ double mutants
(Foster and Rosche, 1999) are defective in stress-induced
mutagenesis, possible loss of homologous-recombination
proficiency in rpoE2072::Tn10dCam cells might explain
their reduced stress-induced mutagenesis. However, first,
whereas recB single mutants and recD recJ double
mutants are hypersensitive to ultraviolet (UV) irradiation
Fig. 4. RpoE is required for stress-induced lac amplification.
A. lac-amplified colonies, identified by sectored-colony morphology on LBH medium with X-gal, during a stress-induced mutagenesis
experiment. Strains, rpoE
+
SMR4562; rpoE::Tn SMR5236. Means for four cultures 1 SEM. Similar results were obtained from two additional
experiments.
B. Viable cell measurements were performed as described in Fig. 3. Means for four cultures 1 SEM.
C. lac amplification rates from days 2–7 in three experiments.
D. Lac
+
point mutation rates from days 2–7 in three experiments. Point mutants were distinguished from lac-amplified clones by their pure
blue colony morphology on LBH X-gal.
420
J. L. Gibson
et al
.
© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 415–430
(Lovett et al., 1988), rpoE2072::Tn10dCam cells are not
(Fig. S3), implying that levels of RecB, RecD and RecJ are
not, in general, drastically reduced in this strain. These
experiments cannot rule out insufficiency of RecB, RecD or
RecJ under a particular stress condition that elicits a s
E
response. Second, quantitative Hfr-mediated conjugation
experiments (Table S4) and quantitative P1 transduction
experiments (Fig. S4) showed the rpoE2072::Tn10dCam
mutant to be as recombination-proficient as its isogenic
rpoE
+
parent. These results cannot exclude a possible
recombination defect specific to recombination during cel-
lular stress such as may occur during stress-induced
mutagenesis. Third, if stress-induced mutagenesis
required wild-type stress-inducible s
E
solely to produce
sufficient levels of RecB, then we would expect stress-
induced mutagenesis to be s
E
-independent if a different
homologous-recombination (HR) pathway was used to
substitute for RecBC. Poteete et al. showed that the phage
l Red HR system can substitute for recBC in stress-
induced mutagenesis in the Lac assay (Poteete et al.,
2002). As shown previously (Poteete et al., 2002), muta-
tion rate was stimulated more than eightfold in DrecBC-
D::red
+
relative to isogenic recBC
+
cells (Fig. S5A).
However, functional RpoE was still strongly required for
stress-induced mutagenesis in DrecBCD::red
+
cells
(Fig. S5A). Therefore, the primary cause of the mutagen-
esis defect of rpoE cells is not deficient expression of recB
or recD. Fourth, E. coli recJ mutant cells are not impaired in
stress-induced mutagenesis (Harris et al., 1994; Foster
and Rosche, 1999), and we find that stress-induced
mutagenesis was unchanged in the recJ or recR single
mutants as well as in the recJ recR double mutant relative
to the isogenic rec
+
strain (Fig. S5B). Therefore, dimin-
ished RecJ and/or RecR production is not the cause of the
stress-induced mutagenesis defect of rpoE cells.
The role of the s
E
response in mutagenesis is
independent of SOS and DinB upregulation, cytoplasmic
heat-shock- and RpoS-stress responses
We wished to distinguish whether the s
E
response is an
independent stress input into mutation, or feeds into the
SOS, RpoS or cytoplasmic heat-shock stress responses.
We first examined its effect on error-prone DNA poly-
merase, Pol IV or DinB, which is required for DSB-
associated stress-induced point mutagenesis (Foster,
2000; McKenzie et al., 2001) and is upregulated transcrip-
tionally by both the SOS (Kim et al., 1997; Courcelle et al.,
2001) and RpoS (Layton and Foster, 2003) responses.
dinB upregulation is the only role of the SOS response in
stress-induced mutation (Galhardo et al., 2009). Although
dinB upregulation by s
E
has not been reported (Rhodius
et al., 2006), a modest increase might have been
overlooked.
Table 1. Decreased ability to maintain lac amplification in rpoE2072::Tn10dCam cells.
Expt. No.
Transfer of lac-amplified
DNA (Lac
+
transconjugants/total
transconjugants, mean SEM)
a
Transfer of Lac
+
point mutations
(Lac
+
transconjugants/total
transconjugants, mean SEM)
b
Average days to Lac
+
colony
formation of lac amplification
carriers (mean SEM)
c
Efficiency of conjugation
(Pro
+
Tet
R
transconjugants/
recipient cell)
d
rpoE
+
rpoE::Tn rpoE
+
/rpoE::Tn rpoE
+
rpoE::Tn rpoE
+
/ rpoE::Tn rpoE
+
rpoE::Tn rpoE
+
rpoE::Tn
1 0.20 0.02 (3.2 0.8) ¥ 10
-3
63 1.05 0.65 1.62 4.6 0.5 5.0 0.3 1.9 ¥ 10
-3
2.9 ¥ 10
-3
2 0.67 0.05 (8.8 1.8) ¥ 10
-2
7.7 0.99 0.90 1.10 5.3 0.2 4.9 0.2 1.8 ¥ 10
-3
1.4 ¥ 10
-3
3 0.53 0.07 (2.1 0.7) ¥ 10
-2
25 0.93 0.83 1.12 5.3 0.2 4.9 0.1 1.4 ¥ 10
-3
0.96 ¥ 10
-3
Mean SEM 0.47 0.14 3.7 ¥ 10
-2
2.6 ¥ 10
-2
32 16 0.99 0.04 0.79 0.07 1.3 0.17 5.1 0.2 4.9 0.3 1.7 ¥ 10
-3
0.2 ¥ 10
-3
1.8 ¥ 10
-3
0.6 ¥ 10
-3
a. F plasmids carrying lac-amplified DNA and a pro
+
marker were conjugated from donor strains PJH18, PJH51, PJH64, PJH69 and PJH74 into D(lac-pro)Tet
R
recipients carrying either rpoE
+
(PJH479) or
rpoE2072::Tn10dCam (PJH480) and the ability of the transconjugants to form colonies was determined on minimal lactose Tet medium (selecting the multiple lac copies, Pro
+
, and the recipient Tet
R
marker)
and on minimal Tet medium (selecting Pro
+
Tet
R
transconjugants whether or not they maintain the lac-amplified DNA). The ratios of amplification-bearing (Lac
+
Pro
+
Tet
R
) to total (Pro
+
Tet
R
) are shown. Transfer
of plasmids carrying lac amplification into rpoE2072::Tn10dCam cells is 32-fold lower than into rpoE
+
cells.
b. Control experiments show no such bias against conjugation of Lac
+
point mutations.
c. Control experiments show no meaningful difference in the number of days required to form colonies of lac amplification bearers between rpoE
+
and rpoE2072::Tn10dCam cells in reconstruction experiments
discussed in the text.
d. Control experiments show similar efficiencies of conjugation for these strains when amplification is not selected.
s
E
stress response in mutagenesis
421
© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 415–430
First, we found that s
E
does not generally upregulate
dinB transcription (Fig. 5A). We measured b-galactosidase
activity in cells carrying a plasmid-borne dinB promoter
fused to lacZ in rpoE
+
and rpoE2072::Tn10dCam cells. No
difference in activity was observed in either SOS-proficient
lexA
+
or lexA(Def) strains in which the SOS response is
constitutively derepressed, suggesting that dinB transcrip-
tion is not controlled by s
E
(Fig. 5A). Second, Western
immunoblot analyses showed that DinB protein levels
were unchanged in the rpoE2072::Tn10dCam mutant rela-
tive to rpoE
+
cell extracts (Fig. 5B). Third, we asked
whether the s
E
stress response might function by simply
activating the SOS response, which is required for stress-
induced mutagenesis (McKenzie et al., 2000), as follows.
lexA(Def) (null) mutants are constitutively derepressed for
the SOS/LexA regulon genes resulting in higher levels of
SOS-controlled proteins including DinB (Friedberg et al.,
2005). The factor(s) regulated by s
E
appear not to be DinB
or any other protein induced by the SOS response because
the lexA(Def) allele did not restore the ability to mutate to
rpoE2072::Tn10dCam cells (Fig. 5C). Fourth, we used two
operator-constitutive mutations of dinB that produce SOS-
induced levels of DinB constitutively (Galhardo et al.,
2009), and found that these do not restore normal levels of
mutagenesis to rpoE2072::Tn10dCam cells (Fig. 5D),
although they do to SOS-induction-defective cells (Gal-
hardo et al., 2009). These results show that SOS induction
and dinB expression are not the limiting factor for stress-
induced mutagenesis in the rpoE2072::Tn10dCam cells
defective for inducing the s
E
response.
The s
E
stress response upregulates s
32
, the transcrip-
tional activator of the cytoplasmic unfolded-protein or
heat-shock response, which in turn increases transcrip-
tion of the genes encoding the GroELS chaperone (Lund,
2001), which is required for efficient stress-induced Lac
reversion (Layton and Foster, 2005). We tested whether
the requirement for the s
E
response induction in mutagen-
esis was in fact a requirement for inducing the s
32
regulon
by examining dnaK756 mutant cells, in which the s
32
regulon is expressed at induced levels constitutively
Fig. 5. The role of the s
E
response in stress-induced mutagenesis is independent of SOS induction and dinB upregulation.
A. Expression from the dinB promoter is not reduced in rpoE mutant cells. b-Galactosidase activity assayed in rpoE
+
and
rpoE2072::Tn10dCam cells carrying a plasmid-encoded P
dinB
::lacZ fusion. Strains are: lexA(Def)rpoE::Tn, SMR10475; lexA(Def), SMR10474;
rpoE
+
, SMR10472; rpoE::Tn, SMR10479. b-Galactosidase activity is expressed as Miller units per 0.5 ml culture and is the average of two
experiments range.
B. DinB protein levels are not reduced in rpoE2072::Tn10dCam cells. Western immunoblot of rpoE2072::Tn10dCam (rpoE::Tn, SMR5236) and
rpoE
+
(SMR4562) cells using antibodies against DinB. Proteins separated by SDS-PAGE were blotted to PVDF membranes and reacted with
antibodies as described in Experimental procedures. A separate experiment gave similar results.
C. Constitutive expression of SOS/LexA regulon genes does not alleviate the requirement for an inducible s
E
response in stress-induced
mutagenesis. Rates of stress-induced Lac
+
colony formation at 37°C calculated from colonies arising from days 3–5 from three separate
experiments. Mean SEM. Strains are: lexA(Def), SMR10369; lexA(Def)rpoE::Tn, SMR10370; rpoE
+
, SMR4562; rpoE::Tn, SMR5236.
lexA(Def) strains also carry mutations in sulA (required for cell viability) and psiB (inactivating an SOS-upregulated inhibitor of mutation) per
McKenzie et al. (2000).
D. SOS-induced levels of DinB do not substitute for a stress-inducible s
E
in stress-induced mutagenesis. Experimental details as in (C).
Strains are: rpoE
+
, SMR4562; rpoE::Tn, SMR5236; dinBO
c1
, SMR10464; dinBO
c2
, SMR10465; rpoE::Tn dinBO
c1
, SMR10466; rpoE::Tn
dinBO
c2
, SMR10467.
422
J. L. Gibson
et al
.
© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 415–430
(Straus et al., 1990). The data show that the dnaK muta-
tion does not substitute for a functional s
E
response as
follows (Fig. 6). First, the dnaK mutation decreases
stress-induced Lac reversion (Fig. 6A). However, a func-
tional RpoE response is still required for the mutagenesis
that remains, as seen by the severe decrease in mutagen-
esis in the dnaK rpoE2072::Tn10dCam double mutant
relative to the dnaK mutant cells (Fig. 6A). These data
indicate that the s
E
stress response provides function(s)
for mutagenesis other than or in addition to those upre-
gulated by the s
32
-activated cytoplasmic heat-shock
response.
The general- or stationary-phase-stress response
controlled by s
S
(RpoS) is required for both stress-
induced point mutagenesis (Layton and Foster, 2003;
Lombardo et al., 2004) and amplification (Lombardo
et al., 2004); however, two lines of evidence imply
that a defect in inducing the RpoS stress response is not
the primary cause of diminished mutagenesis in rpoE
mutant cells. First, because katE is induced RpoS-
dependently in stationary phase (Mulvey et al., 1990),
we assayed b-galactosidase activity in rpoE
+
and
rpoE2072::Tn10dCam saturated cultures that contained
the katE::lacZ fusion. We find that rpoE2072::Tn10dCam
cells induce the fusion gene similarly to isogenic rpoE
+
cells and more than the rpoS deletion mutant in which
b-galactosidase activity is severely reduced (Fig. 6C).
Second, the requirement for rpoS in stress-induced
mutagenesis is not mitigated by provision of DNA DSBs
using a restriction enzyme in vivo, indicating that RpoS
acts downstream of the production of DSBs in the
mutagenic mechanism (Ponder et al., 2005). We show in
the following section that much of the requirement for s
E
is
alleviated by introduction of enzyme-generated DSBs.
Regulation of DSB formation by s
E
A fundamental requirement for stress-induced mutagen-
esis is a DSB, which, in the F, is thought to originate most
frequently from a single-strand nick made at oriT by the
F-encoded TraI endonuclease (Foster and Trimarchi,
1995; Rosenberg et al., 1995). This single-strand break is
thought to become a DSB during replication (Kuzminov,
1995; Rosenberg et al., 1995; Rodriguez et al., 2002).
TraI is required for stress-induced mutation of lac in the F
but this requirement can be bypassed entirely, and
mutagenesis stimulated an additional ~70-fold more, by
DSBs made near lac by the I-SceI double-strand endonu-
Fig. 6. rpoE2072::Tn10dCam inhibits stress-induced mutagenesis independently of effects on the s
32
cytoplasmic heat-shock- or RpoS-stress
responses.
A. Constitutive activation of the s
32
response genes in dnaK mutant cells does not substitute for a functional s
E
response in stress-induced
mutagenesis. Strains are: SMR4562 rpoE
+
( ); SMR8862 dnaK (); SMR5236 rpoE::Tn (); SMR8863 rpoE::Tn dnaK (). Assay was
performed at 37°C as described in Experimental procedures. Values are means one SEM for six independent cultures of each strain in one
experiment. Three experiments gave similar results.
B. Viability of all cultures was monitored per Harris et al. (1996). Strains and symbols are as in (A) but with open symbols.
C. Activity of the RpoS-dependent katE promoter is not diminished by rpoE2072::Tn10dCam. b-Galactosidase activity from a katE::lacZ fusion
was measured in saturated LBH cultures in strains SL590, rpoE
+
; SMR8919, rpoE2072::Tn; and CH1761, rpoS::FRTKan. The means range
of two experiments are shown. Error bars are too small to see for the rpoS strain.
s
E
stress response in mutagenesis
423
© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 415–430
clease in vivo (Ponder et al., 2005). I-SceI-induced DSBs
made near lac increased mutation rate ~6000-fold in the
absence of traI and ~70-fold in its presence (Ponder et al.,
2005). Unlike TraI, the requirements for DSB-repair pro-
teins, an inducible SOS response, RpoS and DinB could
not be substituted for by I-SceI cuts. The substitution of
I-SceI cuts specifically for TraI supports the model that the
role of TraI in mutagenesis is the ultimate generation of a
DSB (Ponder et al., 2005).
We found that I-SceI-induced DSBs near lac could
relieve much of the mutagenesis defect caused by
rpoE2072::Tn10dCam (Fig. 7A). First, in the left panel in
Fig. 7A, we see that rpoE2072::Tn10dCam depresses
the mutation rate 15-fold in cells that do not make
I-SceI-induced DSBs because they have either only
the chromosomal inducible I-SceI gene but no cutsite
(ISceI and rpoE::Tn ISceI), or have neither enzyme
nor cutsite. Second, when DSBs were induced in the
rpoE2072::Tn10dCam mutant (Fig. 7A, right panel), the
stress-induced Lac
+
reversion rate increased nearly 500-
fold relative to the control strain that expressed I-SceI in
the absence of the I-SceI cutsite (Fig. 7A, left panel). In
the DSB-inducing strain (Fig. 7A, right panel), the
rpoE2072::Tn10dCam mutation caused only a threefold
depression of mutation rate, compared with its 15-fold
defect in the absence of I-SceI-made DSBs (Fig. 7A, left
panel). This constitutes an ~80% alleviation of the
rpoE2072::Tn10dCam mutagenesis defect (3/15 = 20%
of the mutagenesis defect remaining). Thus much of the
requirement for the s
E
response in stress-induced
mutagenesis is a requirement for creation of the DSBs
that provoke mutagenesis, implying that the s
E
response
promotes DSB formation in F. This numerical compari-
son assumes that the mutation mechanism is the
same with I-SceI cuts as with TraI. In all ways testable,
this was shown to be so previously: lac mutation
sequence spectrum, requirements for RecA, RecB, Ruv,
SOS, RpoS and DinB, and fraction amplified and point
mutant (Ponder et al., 2005). Thus, the comparison
appears justified. Western analyses show that TraI
protein levels are reduced only about 30 0.6% in the
rpoE2072::Tn10dCam mutant relative to isogenic rpoE
+
cells (Fig. 7B). Thus, although DSB formation appears to
be limiting in the rpoE2072::Tn10dCam background, it is
probably not due to lowered expression of the F plasmid
traI. We cannot rule out an effect on TraI activity;
however, unaltered conjugation frequency in the
rpoE2072::Tn10dCam mutant (data not shown) argues
against this possibility. Other possibilities are considered
below.
Discussion
Separation of the essential and stress–response
functions of s
E
One interesting aspect of the data presented is the dis-
covery that rpoE2072::Tn10dCam, a mutation that allows
constitutive expression but not induction of the s
E
stress
response by the YYF inducing peptide (Fig. 2), retains
viability in the absence of unlinked suppressor mutations
(Fig. S1). This implies that the essential function of s
E
relates to one or more of the functions it controls consti-
tutively, not to transient expression of the stress response.
Fig. 7. I-SceI-generated DSBs relieve much of the rpoE2072::Tn10dCam defect in stress-induced mutagenesis.
A. Stress-induced mutation rates in the presence of I-SceI generated DSBs. Expression of I-SceI enzyme in cells with no I-SceI cutsite (left
panel) does not affect the requirement for functional rpoE in stress-induced mutagenesis. However when I-SceI is expressed in cells with a
cutsite near lac (DSBs, right panel), the rpoE2072::Tn10dCam phenotype is greatly reduced. Rates are Lac
+
cfu per 10
8
cells per day between
days 3 and 5 and represent the averages of four experiments SEM. Experiments were performed at 37°C as described in Experimental
procedures, and viability monitored per Harris et al. (1996). Strains are: rpoE
+
, SMR4562; rpoE::Tn, SMR5236; rpoE
+
I-SceI (enzyme, no
cutsite), SMR6276; rpoE::Tn I-SceI (enzyme, no cutsite), SMR9191; rpoE
+
DSB (enzyme + cutsite), SMR6280; and rpoE::Tn DSB (enzyme +
cutsite), SMR10168.
B. Western immunoblot using antibodies against TraI. Proteins separated by SDS-PAGE as described in Experimental procedures were
blotted to nitrocellulose membranes and probed with anti-TraI. MC4100 [F
+
]; rpoE
+
[F], SMR4562; rpoE::Tn[F], SMR5236. Two separate
experiments gave similar results.
424
J. L. Gibson
et al
.
© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 415–430
The rpoE2072::Tn10dCam allele is likely to provide a
useful reagent for future genetic studies of the effects of
loss of the stress response, which can now be achieved
both specifically, without simultaneous loss of the essen-
tial function, and cleanly, in cells that do not also carry
suppressor mutations.
Control of genomic instability by the s
E
stress response
and s
E
-activating stressors
The data presented show that the stress–response func-
tion of s
E
is required for stress-induced point mutagenesis
(Figs 3 and 4D, Tables S1 and S2) and gene amplification
(Fig. 4, Table 1). Moreover s
E
constitutes an independent
stress and stress–response input to mutation, indepen-
dent of known SOS and s
S
and postulated s
32
heat-shock
response involvement (Figs 5–7). Therefore, first, stress-
induced point mutagenesis and amplification are con-
trolled by multiple independent stress responses: the
SOS DNA-damage (McKenzie et al., 2000), RpoS
general-stress (Layton and Foster, 2003; Lombardo et al.,
2004) and s
E
responses for point mutation, and RpoS
(Lombardo et al., 2004) and s
E
for amplification. Input of
multiple stress responses is important in regulating the
circumstances under which cells increase genetic diver-
sity and their potential to evolve. Second, the results
indicate that the s
E
response influences genome (in)sta-
bility generally.
Although both s
E
and s
S
responses are necessary for
the genomic instability studied here, many stressors are
expected to activate both, including starvation (via
ppGpp) (Costanzo and Ades, 2006; Costanzo et al.,
2008), but also many antibiotics, which may trigger mem-
brane stress (such as b-lactams) and all of which appear
to induce reactive oxygen species (reviewed, Kohanski
et al., 2010) and so oxidative stress, an inducer of s
S
. The
coupling of mutagenesis to antibiotic stressors is a poten-
tially serious problem with resistance mechanisms insti-
gated by the antibiotics (Cirz and Romesberg, 2007;
Galhardo et al., 2007; Lopez and Blazquez, 2009; Cohen
and Walker, 2010; Kohanski et al., 2010).
Role of s
E
response in stress-induced mutagenesis
One major role of the s
E
response in stress-induced
mutagenesis is apparent: ~80% of its function is substi-
tuted by an I-Sce-induced DSB (Fig. 7), implying that
somehow the s
E
response promotes TraI-generated
double-strand-end (DSE) formation, for which I-SceI also
substitutes (Ponder et al., 2005). Previous work indicates
that the point mutagenesis occurs in acts of error-prone
DSB repair, and that in the F, most of the DSBs originate
from the action of TraI endonuclease (Ponder et al.,
2005). The breaks are repaired non-mutagenically in
unstressed cells and mutagenically, using DinB error-
prone DNA polymerase if RpoS is induced either by stress
or artificially (Ponder et al., 2005). The s
E
response could
promote either the nicking by TraI or the replication into
that nick. Although TraI levels were affected only modestly
in the rpoE2072::Tn10dCam mutant (Fig. 7B), it is pos-
sible that replication or specifically F replication might be
affected by s
E
, and the effect of s
E
might be F-specific.
The mechanism of the remaining ~20% of the
s
E
-response effect is unknown. Given that one of the
ways that the s
E
response is induced is via starvation
using ppGpp and the stringent response (Costanzo and
Ades, 2006; Costanzo et al., 2008), it might, like RpoS,
facilitate the switch to mutagenic repair of DSBs under
stress.
Alternative amplification-mutagenesis model
Others favour a model of mutation in the Lac system that
does not involve stress or stress responses but instead
invokes growth of cells carrying an amplified lac array,
which produces more b-galactosidase activity from the
weakly functional mutant lac gene (Roth et al., 2006;
Roth, 2010). In this model, when about 10
5
cells with
many lac copies are present in a microcolony, a Lac
+
point
mutation occurs spontaneously, and the point mutant then
overgrows the colony. We do not favour this model for
several reasons. First, its prediction that point mutants
arise from lac-amplified clones was contradicted by
experiments showing that: (i) Lac
+
microcolonies, as early
as the two-cell stage, are pure point mutants, not mostly
amplified with point mutants arising later point mutants
did not arise in amplified young colonies (Hastings et al.,
2004), (ii) lac-amplified microcolonies do not produce
point mutants efficiently under selective conditions (Hast-
ings et al., 2004), and (iii) mutation of the DNA poly-
merase I gene obliterates amplification without
diminishing point mutation (Hastings et al., 2004; Slack
et al., 2006). This would be impossible if amplification
were a precursor to point mutation, as the model
specifies. As far as we are aware, alternative interpreta-
tions of these data have not been offered. Other data also
contradict this model (Stumpf et al., 2007). Second, the
fact that three stress responses are required for point
mutation [SOS (McKenzie et al., 2000), RpoS/s
S
(Layton
and Foster, 2003; Lombardo et al., 2004) and s
E
] and two
for amplification [s
S
(Lombardo et al., 2004) and s
E
]isnot
compatible with the amplification model, which does not
involve stress or stress responses. Neither are results,
reviewed above, that showed that DSB repair switches to
a mutagenic mode using DinB if RpoS is expressed, even
in the absence of any external stress or selection (Ponder
et al., 2005). We do not know of alternative interpretations
for those data, and favour their obvious interpretation: that
s
E
stress response in mutagenesis
425
© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 415–430
stress responses upregulate mutagenesis when cells are
stressed.
Mutation as a stress response and the regulation
of evolvability
The coupling of mutagenesis to stress responses means
that cells turn up mutation rate specifically when they are
maladapted to their environment, i.e. are stressed, poten-
tially accelerating evolution specifically then. This can be
a powerful device for adaptation to adversity including
antibiotics and host defences in bacteria (McKenzie and
Rosenberg, 2001; Cirz et al., 2005; Galhardo et al.,
2007), and hypoxia (Bindra et al., 2007; Huang et al.,
2007) and chemotherapeutic drugs in developing
cancers. More than 80% of E. coli natural isolates
respond to stress with mutagenesis, and modelling sup-
ports the potential benefits in enhanced evolvability of this
response (Bjedov et al., 2003).
The requirement for (at least) three stress responses for
point mutagenesis (SOS, s
S
and s
E
) and two for amplifi-
cation (s
S
and s
E
) implies that cells do not instigate poten-
tially dangerous programmes of genomic instability
until they sense multiple independent stressors. Similarly
in B. subtilis, both the stringent response (Rudner
et al., 1999b) and ComK-controlled competence stress
response (Sung and Yasbin, 2002) are required for
starvation-induced mutagenesis. In Salmonella, bile-
induced resistance mutagenesis requires both SOS and
s
S
responses (Prieto et al., 2006; J. Casadesus, pers.
comm.). Coupling genome instability to more than one
stress response, any of which might not be turned on in all
cells in a population, may further restrict or regulate
mutagenesis to a cell subpopulation (Galhardo et al.,
2007; Gonzalez et al., 2008), providing one way to
achieve a potential bet-hedging mechanism such as is
seen in bistable subpopulations critical to many bacterial
survival strategies (Veening et al., 2008). These are highly
regulated programmes, exquisitely tuned to cellular
stresses, which regulate mutagenesis, and thus the ability
to evolve, temporally. Understanding and targeting the
regulatory components is likely to provide powerful new
antibiotic strategies.
Experimental procedures
Bacterial strains and growth conditions
Strains used are listed in Table S5. Standard genetic tech-
niques were used in strain constructions (Miller, 1992). All M9
minimal media (Miller, 1992) had carbon sources added at
0.1% and thiamine (vitamin B1) at 10 mgml
-1
. LBH medium is
per, for example, Torkelson et al. (1997). Antibiotic and other
additives were used at the following final concentrations:
ampicillin, 100 mgml
-1
; chloramphenicol, 25 mgml
-1
;
kanamycin, 50 mgml
-1
; tetracycline, 10 mgml
-1
; rifampicin,
100 mgml
-1
; 5-bromo-4-chloro-3-indolyl-b-D-galactoside
(X-gal), 40 mgml
-1
; sodium citrate, 20 mM.
RpoE reconstruction experiments
Lac
+
strains carrying rpoE2072::Tn10dCam were constructed
and their growth on lactose quantified (Table S1). The rpoE
allele was moved by P1 transduction into three Lac
+
day 5
stress-induced mutants that had been characterized with
respect to colony-forming ability on lactose medium (Rosen-
berg et al., 1998). To measure the days required for colony
formation on lactose minimal medium, growth and plating of
cells was identical to that described for stress-induced
mutagenesis experiments, including the addition of lac-
deleted scavenger cells in the same numbers. Additionally,
the rpoE2072::Tn10dCam allele was transferred by P1 trans-
duction into five Lac
+
stress-induced mutants known to carry
secondary mutations (Torkelson et al., 1997) and the ability to
form colonies in exact reconstruction of experimental selec-
tion conditions measured. These experiments were carried
out at 32°C due to the temperature-sensitive phenotype of
one of the mutants.
Generation-dependent mutagenesis assays
Generation-dependent mutation rates to rifampicin resistance
were measured in MG1655 cells harbouring either pBA166 or
the vector pTrc99a. Single colonies grown on LBH-amp plates
were inoculated into tubes containing 5 ml of LBH-amp broth
with either 0.1% glucose or 0.1 mM IPTG to repress or induce
expression of YYF from pBA166 respectively. Cultures were
incubated overnight at 37°C prior to plating on LBH plates
containing rifampicin. For each determination, 19 cultures
were used and median mutant frequencies used to estimate
mutation rates using a modified method of the median (Lea
and Coulson, 1949; Von Borstel, 1978).
Mutation assays, quantitative conjugation and
P1 transduction
Stress-induced mutagenesis assays were performed as
described at 30°C or 37°C unless otherwise indicated (Harris
et al., 1996). SMR5236 cultures were concentrated 10-fold
before plating to obtain sufficient numbers of Lac
+
colonies. In
some experiments amplification of the lac region was quan-
tified as described (Hastings et al., 2004).
In reconstruction experiments for amplification, rpoE
+
and
rpoE2072::Tn10dCam F
-
cells were plated under the condi-
tions of stress-induced mutagenesis experiments after con-
jugation with the same set of five F factors carrying lac
amplification. The ratio of donor to recipient cells was 20:1.
Transconjugants were selected on M9 glycerol tetracycline
medium, which selects for Tet
R
, carried in the chromosome of
the recipient cells and for Pro
+
, bestowed by the F factor, and
also on M9 lactose tetracycline medium, which selects for the
recipient chromosome and the donor F, as above, and also
requires the maintenance of amplification of the lac locus.
Thus the fraction of the colonies on glycerol medium that
426
J. L. Gibson
et al
.
© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 415–430
forms colonies on lactose gives a measure of the ability of
cells of that genotype to support amplification.
Bacterial strains carrying the inducible DSB system con-
sisting of a chromosomally encoded I-SceI enzyme and an F
cutsite were maintained on medium containing 0.1% glucose
to repress I-SceI synthesis (Ponder et al., 2005) and experi-
ments performed as described (Ponder et al., 2005).
Quantitative conjugation and P1 transduction assays were
performed as described (Lombardo et al., 2004). P1 stocks
grown on SMR6263 were used in transduction experiments.
Transductants were selected on LBH tetracycline citrate
medium.
Co-transduction experiments (Fig. S1) were conducted
with P1 grown on SMR11044 which has a kanamycin marker
(yfhH::FRTKan) linked to rpoE2072::Tn10dCam. Following
transduction, cells were plated on either LBH-kanamycin
plates or LBH-chloramphenicol plates. Resistant colonies
were then screened for the second antibiotic resistance.
Co-transductant frequency was calculated as the number of
screened antibiotic-resistant colonies per the number of
selected antibiotic-resistant colonies. If the insertion in rpoE
confers a null phenotype, the co-transductant frequency of
chloramphenicol-resistant per kanamycin-resistant colonies
would be expected to be close to zero.
UV sensitivity was determined in saturated overnight LBH
cultures. Varying dilutions were spread on LBH plates, irradi-
ated, and the plates immediately placed in the dark and
incubated overnight at 37°C.
b-Galactosidase assays and Western immunoblot
analysis
For monitoring expression of a s
E
-regulated gene, the
rpoE2072::Tn10dCam allele was moved into strain
CAG45114, containing an rpoHP3::lacZ fusion. For moni-
toring expression of an RpoS-controlled gene,
rpoE2072::Tn10dCam was moved into SL590, containing a
katE::lacZ fusion. The presence of the transposon insertion
was verified by PCR amplification using primers flanking the
insertion, RpoE-F (5-CACTGGAAGGTGGACGACG) or
RpoE-R (5-GAGAAGTTACTGGCTGGTGG), in conjunction
with an outward-reading primer (5-GGTGGTGCGT
AACGGCAAAAG) specific for Tn10dCam.
For b-galactosidase assays saturated LBH or M9-glucose
cultures were diluted back 1:100 in fresh medium. For induc-
tion of peptide synthesis from pBA166, 1.0 mM IPTG was
added to LBH cultures at OD
600
, of 0.1 and 0.5 ml aliquots
were removed at various time intervals for b-galactosidase
assays. Assays were performed as described (Miller, 1992).
b-Galactosidase activity is expressed as a function of culture
volume. For monitoring b-galactosidase activity of the katE-
::lacZ fusion throughout the growth phase, LBH cultures were
inoculated from saturated overnight cultures. OD
600
and
b-galactosidase measurements were determined for each
sample.
Western blots were performed with polyclonal antiserum
against purified DinB as described (Galhardo et al., 2009).
TraI was examined by immunoblot analysis using a 1:5 000
dilution of polyclonal antiserum against TraI as described
(Will and Frost, 2006).
Acknowledgements
We are indebted to Virgil Rhodius and Carol Gross for
sharing unpublished data, strains and plasmids, and to
Masami Yamada and Takehiko Nohmi for the gift of DinB
antiserum. We thank Anthony Poteet for strains and John
Doan for assistance in medium preparation. R.S.G. was sup-
ported in part by a postdoctoral fellowship from the Pew Latin
American Fellows Program. This work was supported by
Canadian Institutes of Health Research Grant MT11249 (L.S.
F.), Human Frontier Science Program Young Investigator
Grant HFSP2006-0060 (C.H.), and US National Institutes of
Health Grants F32-GM19909 (M.-J.L.), R01-GM64022 (P.J.
H.) and R01-GM53158 (S.M.R.).
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    • "In the last decade many other experiments and analyses have been performed on the lac mutant of E. Coli, bringing more evidence to the hypothesis of mutations as an adaptive stress response to bad, challenging environmental conditions. It seems however to be excluded a directed mutation: a transient hypermutation under stress conditions is enough for a lac population to adapt and become a lac+, whilst it is still debated how the population is able to survive a (although temporary) huge increase in mutations (Gibson et al., 2010; Fonville, 2011; Rosenberg, 2001; Galhardo et al., 2007; Gonzalez et al., 2008). Condition-dependent mutation rates have also been found in some multicellular organisms, although more controversially (Baer, 2008; Agrawal and Wang, 2008; Cotton, 2009; Sharp and Agrawal, 2012 ), and it has also been suggested that " human cancers arise in part as an evolutionarily programmed side effect of age-and damage-inducible genetic instability affecting both somatic and germ line lineages " (Zhao and Epstein, 2008): an increase in mutation rates in the human male sperm while ageing and when experiencing environmental stress seems to lead to increased species adaptation, even though at a high cost for the single individuals. "
    [Show description] [Hide description] DESCRIPTION: MSc thesis at University of Sussex. Using evolutionary robotics methods, I've explored the question of whether mutation rate adaptivity could be beneficial in a challenging environment.
    Full-text · Research · Oct 2015 · Genetics
    • "We find, as shown in Figure 1C, that RpoS level changes regulation of both repair systems simultaneously , so both together contribute to SIM, even though different networks regulate the two processes (Al Mamun et al. 2012; Gutierrez et al. 2013). For example, other influences besides RpoS (such as from RpoE;Gibson et al. 2010) may additionally affect the overall mutation rate. Nevertheless , the mutation rate threshold in Figure 1 is correlated with the regulation of mutS and dinB in the strains set. "
    [Show abstract] [Hide abstract] ABSTRACT: Evolutionary innovations are dependent on mutations. Mutation rates are increased by adverse conditions in the laboratory but there is no evidence that stressful environments that do not directly impact on DNA leave a mutational imprint on extant genomes. Mutational spectra in the laboratory are normally determined with unstressed cells but are unavailable with stressed bacteria. To by-pass problems with viability, selection effects and growth rate differences due to stressful environments, in this study we used a set of genetically engineered strains to identify the mutational spectrum associated with nutritional stress. The strain set members each had a fixed level of the master regulator protein, RpoS, which controls the general stress response of Escherichia coli. By assessing mutations in cycA gene from 485 cycloserine resistant mutants collected from as many as independent cultures with three distinct perceived stress (RpoS) levels, we were able establish a dose-dependent relationship between stress and mutational spectra. The altered mutational patterns included base pair substitutions, single base pair indels, longer indels and transpositions of different insertion sequences. The mutational spectrum of low-RpoS cells closely matches the genome-wide spectrum previously generated in laboratory environments, while the spectra of high-RpoS, high perceived stress cells more closely matches spectra found in comparisons of extant genomes. Our results offer an explanation of the uneven mutational profiles such as the transition-transversion biases observed in extant genomes and provide a framework for assessing the contribution of stress-induced mutagenesis to evolutionary transitions and the mutational emergence of antibiotic resistance and disease states.
    Full-text · Article · Nov 2014
    • "We find, as shown in Figure 1C, that RpoS level changes regulation of both repair systems simultaneously , so both together contribute to SIM, even though different networks regulate the two processes (Al Mamun et al. 2012; Gutierrez et al. 2013 ). For example, other influences besides RpoS (such as from RpoE;Gibson et al. 2010) may additionally affect the overall mutation rate. Nevertheless , the mutation rate threshold in Figure 1is correlated with the regulation of mutS and dinB in the strains set. "
    [Show abstract] [Hide abstract] ABSTRACT: Stress-induced mutagenesis was investigated in the absence of selection for growth fitness by using synthetic biology to control perceived environmental stress in Escherichia coli. We find that controlled intracellular RpoS dosage is central to a sigmoidal, saturable 3-4 fold increase in mutation rates and associated changes in DNA repair proteins.
    Full-text · Article · Sep 2014
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