Download Increased transcription rates correlate with increased reversion rates

Microbiology (2004), 150, 1457–1466
DOI 10.1099/mic.0.26954-0
Increased transcription rates correlate with
increased reversion rates in leuB and argH
Escherichia coli auxotrophs
Jacqueline M. Reimers, Karen H. Schmidt, Angelika Longacre,3
Dennis K. Reschke and Barbara E. Wright
Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA
Correspondence
Barbara E. Wright
[email protected]
Received 1 December 2003
Revised 15 January 2004
Accepted 20 January 2004
Escherichia coli auxotrophs of leuB and argH were examined to determine if higher rates of
transcription in derepressed genes were correlated with increased reversion rates. Rates of
leuB and argH mRNA synthesis were determined using half-lives and concentrations, during
exponential growth and at several time points during 30 min of amino acid starvation. Changes
in mRNA concentration were primarily due to increased mRNA synthesis and not to increased
stability. Four strains of E. coli amino acid auxotrophs, isogenic except for relA and argR, were
examined. In both the leuB and argH genes, rates of transcription and mutation were compared.
In general, strains able to activate transcription with guanosine tetraphosphate (ppGpp) had
higher rates of mRNA synthesis and mutation than those lacking ppGpp (relA2 mutants). argR
knockout strains were constructed in relA+ and relA mutant strains, and rates of both argH
reversion and mRNA synthesis were significantly higher in the argR knockouts than in the
regulated strains. A statistically significant linear correlation between increased rates of
transcription and mutation was found for data from both genes. In general, changes in mRNA
half-lives were less than threefold, whereas changes in rates of mRNA synthesis were often
two orders of magnitude. The results suggest that specific starvation conditions target the
biosynthetic genes for derepression and increased rates of transcription and mutation.
INTRODUCTION
Nutritional stress results in the derepression, activation
and transcription of specific genes, and therefore may be
a major cause of mutations. Transcription can lead to
mutation by driving localized supercoiling and the accumulation of DNA secondary structures containing unpaired
and/or mispaired bases known to be vulnerable to mutations (reviewed by Wright, 2000). Using a new computer
algorithm, it has been possible to predict mutation frequencies in derepressed genes of Escherichia coli auxotrophs
and in the human p53 cancer gene (Wright et al., 2002,
2003).
In E. coli, amino acid biosynthetic operons are repressed
in the presence of their end-product amino acid. However,
when the cells are starved for a required amino acid, the
biosynthetic operon (or regulon) is deattenuated (leucine
operon) or derepressed (arginine regulon) and activated.
During leucine starvation, for example, leucine-charged
tRNAs become limiting, causing ribosomes to stall at
one of the leucine control codons. This deattenuates the
leu operon, allowing transcription to continue into the
3Present address: Department of Medicine/Section of Nephrology,
University of Chicago, Chicago, IL 60637, USA.
0002-6954 G 2004 SGM
Printed in Great Britain
structural genes of the operon. The stalled ribosomes and
accumulated uncharged tRNALeu also simultaneously trigger
the stringent response and the synthesis of the alarmone
guanosine tetraphosphate (ppGpp). In turn, ppGpp activates specific derepressed genes encoding enzymes required
to overcome the starvation conditions. The stringent response also redirects the cell’s resources toward survival by
decreasing the synthesis of stable RNA, nucleotides and
other metabolites required for cell replication. During the
stringent response, ppGpp synthesis is catalysed by the
relA gene product (ppGpp synthase I) and ppGpp begins
accumulating immediately following starvation for any
amino acid (Cashel et al., 1996). The amount of ppGpp
produced depends upon the identity of the absent amino
acid (Donini et al., 1978). Activation by ppGpp is essential
to enhanced expression of the deattenuated leu operon
(Wright et al., 1999), as well as a number of other amino
acid operons (Morse & Morse, 1976; Perel’man & Shakulov,
1981; Smolin & Umbarger, 1975; Stephens et al., 1975;
Zidwick et al., 1984).
Amino acid biosynthetic genes regulated by repression
are also activated by ppGpp in relA+ cells (Zidwick et al.,
1984). The arginine regulon consists of 11 genes, all of
which are repressed at the operator site by arginine when
1457
J. M. Reimers and others
combined with the protein encoded by argR. Positive
regulation by ppGpp occurs at both the transcription and
the translation level (Williams & Rogers, 1987; Zidwick
et al., 1984).
Starving an auxotroph for its required amino acid will
derepress/deattenuate the operon (including the defective
gene) and reversion rates to wild-type should increase if
transcription enhances mutations. Cells with the ability to
activate transcription in the presence of ppGpp (relA+)
would be expected to have higher rates of transcription
and mutation than cells that can not activate derepressed
genes (relA mutants). In addition, inactivation of a repressor would also be expected to increase transcription and
thereby affect mutation rates. Unlike the situation in
regulated strains, mutations could occur during growth in
repressor knockouts because transcription is not repressed
by the presence of the required amino acid.
Previous investigations with multiple auxotrophs of E. coli
(CP78 and CP79, which are isogenic except for relA) have
demonstrated a positive correlation between reversion rates
in the leuB gene, ppGpp levels, and the concentration of
leuB mRNA after 15 min of leucine starvation (Wright
et al., 1999; Wright & Minnick, 1997). Increases in leuB
and argH mRNA levels are known to be specific to the
starvation conditions, i.e. leuB mRNA accumulates during
leucine, but not arginine or threonine starvation, and argH
mRNA accumulates during arginine, but not histidine
starvation (Wright et al., 1999). These observed changes in
mRNA levels in response to nutritional stress could result
from a change in the rate of mRNA synthesis, degradation
(stability), or both.
It is commonly assumed that mRNA concentration reflects
the rate of transcription. This relationship has been documented in a number of studies in which transcription
increased in response to environmental conditions (Meyer
& Schottel, 1991; Pease & Wolf, 1994). However, very few
mRNA stability studies have been published. In fact,
Bernstein et al. (2002) found published reports of RNA
half-life for less than 0?5 % of the 4288 predicted ORFs in
the E. coli genome. This small number of investigations
could reflect the costly and labour-intensive nature of such
work. Several of these mRNA stability studies do reveal
that high levels of gene expression are due either solely or
partially to increased stability of the mRNA (Bricker &
Belasco, 1999; Georgellis et al., 1993; Zgurskaya et al., 1997).
Currently, DNA microarray studies are widely used to
indicate changes in gene expression when cells are exposed
to a new condition or event. While microarrays measure
mRNA abundance, they should not be used as an indication of gene ‘expression’ unless combined with a study to
determine if the changes in quantity are due to increased
stability of the message or increased rates of mRNA
synthesis.
In this study, mRNA concentrations and half-lives were
analysed and transcription rates were calculated at several
1458
points during growth and starvation to determine whether
increased stability, increased synthesis or both accounted
for the increase in concentration of mRNA. These transcription rates were then compared with mutation rates to
determine if increased synthesis of mRNA is correlated with
increased mutation rates.
METHODS
Bacterial strains and growth conditions. The bacterial strains,
CP78 and CP79, used in this study are multiple auxotrophs of E. coli
K-12, requiring leucine, arginine, threonine and histidine (Wright &
Minnick, 1997). They are isogenic, except that CP78 is relA+ and
CP79 is relA2. The argR mutants (DargR : : cat), DR78 and DR79
(derived from CP78 and CP79 respectively) were obtained by homologous recombination using the pWM91 suicide vector (Metcalf
et al., 1996) containing the entire argR gene disrupted with a cat
cassette. Successful recombination resulted in chloramphenicolresistant cells. PCR analysis verified the presence of the cat cassette
within the argR gene. Derepression of the argCBH genes was confirmed by comparing the activity of the argB enzyme in our argR
knockout strain to that of a known argR mutant (MA1030: CGSC
no. 1184).
Cells were grown as previously described at 37 uC (Wright & Minnick,
1997). Growth was monitored by following the OD550 of the cultures
(Perkin Elmer model 35 spectrophotometer). Total RNA was isolated
during growth by removing aliquots from exponentially growing
cultures. For isolation of RNA during starvation, cells were washed out
of minimal medium by centrifuging for 1 min at 10 000 g and
resuspended in pre-warmed 37 uC minimal medium lacking leucine
or arginine. Starved cultures were incubated as before and aliquots
were taken at specified time points.
Mutation rate determinations. Conditions for growth and deter-
mination of mutation rates have been previously described (Wright,
1996; Wright & Minnick, 1997; Wright et al., 1999). A large culture
was inoculated with cells from a 7 h old nutrient agar plate and
1?5 ml aliquots were distributed into 40 2 cm diameter test tubes,
which were shaken at a 45u angle at 37 uC until the supply of the
limiting amino acid was exhausted and growth ceased. Each entire
culture was plated onto selective medium and incubated at 37 uC.
Several identical cultures were diluted and plated onto nutrient agar
plates to determine viable cell numbers. The number of selective
plates without revertants was counted after 48, 65 and 72 h. Mutation
rates were estimated by the ‘zero method’ of Luria & Delbrück
(1943) according to the expression MR = (2ln2) (ln P0/N), where
P0 is the proportion of cultures with no revertants, and N is the
total number of cells per culture. The incubation period chosen for
each strain on selective media depended upon a number of variables.
For the argH reversions a compromise (i.e. 48 h) was necessary to
read the plates when the majority of true revertants were expressed
while the minimum numbers of suppressors were observed. In the
case of leuB reversions, suppressors were negligible and at 65 h of
incubation, 90 % of the revertants expressed were true. To correct
for the presence of intergenic suppressors of the argH mutation, at
least 20 revertants from each strain were isolated at various times
after plating. These were sequenced to find the ratio of true revertants to suppressors. The percentage of true revertants was used to
correct the P0 component of each mutation rate calculation.
Sequencing showed that the leuB mutation was a C-to-T transition
changing TCG (serine) to TTG (leucine), and revertants were either
TCG (serine) or GTG (valine) (Wright & Minnick, 1997). The argH
mutation was a G-to-A transition changing TGG (tryptophan) to TGA
(stop). The revertants appearing most often were TGG (tryptophan),
Microbiology 150
Transcription rates and mutation in E. coli
TTA (leucine), TGC (cysteine), AGA (arginine) and CGA (arginine).
TGT (cysteine), TCA (serine) and GGA (glycine) were observed less
frequently.
RNA isolation. Total cellular RNA was isolated according to the
hot phenol method of Tsui et al. (1994). Cells were added directly
to lysis buffer at 100 uC, extracted twice with phenol at 65 uC, once
with phenol/chloroform/isoamyl alcohol (25 : 24 : 1, by vol.) at room
temperature, and once with chloroform/isoamyl alcohol (24 : 1, v/v).
After two diethyl ether extractions, RNA was precipitated with
2-propanol at 220 uC overnight. This pellet was washed with 70 %
ethanol, resuspended in water and precipitated with 0?5 vol. 7?5 M
LiCl at 220 uC. After a 70 % ethanol wash, the RNA was treated
with 5 U RQ1-RNase-free DNase (Promega) for 15 min at 37 uC.
DNase was removed by phenol/chloroform/isoamyl alcohol extraction followed by chloroform extraction. The RNA was precipitated
with 0?1 vol. 5 M ammonium acetate and 2?5 vols 100 % ethanol
overnight at 220 uC. After centrifugation at 16 000 g for 20 min at
4 uC, the final RNA pellet was resuspended in 200 ml water. Total
RNA concentration was determined by reading the A260 (Beckman
DU 650 spectrophotometer).
S1-nuclease protection assays. A 4- to 10-fold molar excess of
biotinylated antisense RNA probe (Wright et al., 1999), 0?5–100 mg
total cellular RNA and 10 mg yeast tRNA were precipitated with 0?1
vol. 5 M ammonium acetate and 3 vols 100 % ethanol at 220 uC for
30 min. After centrifugation at 16 000 g for 15 min at 4 uC, the
pellets were air-dried for 30 min and resuspended in 10 ml RPAII
hybridization buffer (Ambion). The samples were hybridized overnight at 50 uC for leuB or 52 uC for argH. Unhybridized RNA and
probe were digested with 300 U S1-nuclease (Promega) at 42 uC for
45 min. The digest was stopped by adding 0?2 vol. stop buffer [4 M
ammonium acetate, 30 mM EDTA, 170 mg tRNA ml21 and 0?7 mg
GlycoBlue ml21 (Ambion)] and the protected hybrids were precipitated with 2?5 vols 100 % ethanol. After precipitation at 220 uC for
30 min and centrifugation at 16 000 g for 15 min at 4 uC, the pellets
were air-dried for 30 min and suspended in 11 ml Gel Loading
Buffer II (Ambion). The samples were boiled for 4 min, loaded onto
pre-run 5 % (w/v) polyacrylamide gel (19 : 1, w/w, acrylamide/bisacrylamide) containing 8 M urea and 16 TBE and electrophoresed
at 40 V cm21 until the bromphenol blue band ran off the gel. The
gels were electroblotted onto nylon membranes; the hybrids were
cross-linked to the membrane with UV light, washed and bound
with streptavidin–alkaline phosphatase. Chemiluminescent reagent,
CDP-Star (Ambion), was added and hybrids were detected with
X-ray film as described previously (Wright et al., 1999). Scanning
densitometry and comparison to known amounts of standards were
used to determine specific mRNA concentrations.
Half-life determinations. Decay rates of specific mRNAs, at speci-
fic times of starvation, were measured by inhibiting transcription
initiation with 300 mg rifampicin ml21. Aliquots of cells were
removed immediately before and at several time points after rifampicin addition. Total cellular RNA was extracted, hybridized to
gene-specific probes and the amounts of specific mRNAs were
determined by S1-nuclease protection assays. The half-life was
calculated from the slope of a least-squares regression line of a semilogarithmic plot of percentage mRNA remaining as a function of
time. The half-life was calculated using time points determined
when the decay rate was exponential.
Transcription rates. Rates of mRNA synthesis (KS) were calculated
from the measured values of mRNA concentration ([mRNA]) and halflife (t1/2) according to the following equation: KS=(ln2 [mRNA])/t1/2
(Zgurskaya et al., 1997).
Determination of ppGpp. Cells were grown in minimal medium
as previously described (Wright, 1996) with 0?05 M MES (pH 6?5)
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and 0?05 M KH2PO4 substituted for 0?04 M sodium phosphate
buffer (pH 6?5). When cells were to be starved for an amino acid, it
was present at 0?2 mM in the growth medium. When cultures
reached an OD550 of 0?1, they were labelled with 100 mCi (3?7 MBq)
H332PO4 ml21 (ICN; 3?76106 Bq ml21) and then grown at 37 uC to
an OD550 of 0?3. Growth was monitored in an unlabelled companion culture. The cells were then starved for either arginine or
leucine by centrifuging at 16 000 g for 1 min, and resuspending the
cell pellet in radioactive minimal medium lacking arginine or
leucine. Immediately before and at several time points during starvation, 50 ml aliquots of cells were removed and added to 17 ml
23?6 M formic acid on ice, quickly mixed and frozen in a dry ice/
ethanol bath. When samples were collected at all time points, the
samples were freeze–thawed twice and centrifuged for 1 min.
Aliquots (2 and 5 ml) were spotted onto washed PEI cellulose plates.
Forty nanomoles of GTP and ppGpp (Trilink Biotechnologies)
treated in the same manner were spotted as standards. Ascending
chromatography in 1?5 M KH2PO4 (pH 3?4) was run at 5 uC until
the solvent front was 16 cm from the origin. The radioactive spots
were located by autoradiography. The radioactive areas corresponding to ppGpp were excised from the plate, wetted with 0?25 ml
water and their radioactivity measured in 5 ml Ecolite (ICN). The
concentration of ppGpp [nmol (OD550 unit)21] was based on the
specific radioactivity of the phosphate in the medium (Wright &
Minnick, 1997).
RESULTS AND DISCUSSION
In the leu operon and arg regulon, reversions of mutants
would not be expected to occur during growth in the
presence of their end product, when transcription rates are
low. During starvation, when transcription is activated
by deattenuation or derepression, reversion rates should
increase. In contrast, in the arginine repressor knockout
strains (unregulated), reversions could occur during growth
in the presence of arginine, because transcription rates
are high due to the absence of the repressor protein. The
uniform size of revertant colonies formed on selective
plates indicated that mutations occurred more or less
synchronously. Although this suggests that the reversions
occurred over a brief period of time, the exact time is
unknown. Therefore, transcription rates were calculated
for periods encompassing the most likely times reversions
could occur, namely: (1) growth, (2) peak mRNA synthesis
and (3) starvation steady state. In regulated strains, transcription during growth would not be expected to contribute
significantly to mRNA synthesis. However, in unregulated
strains, transcription during growth would be expected to
make a significant contribution to the overall transcription
rate. In order to compare transcription rates, the same
time periods of growth and starvation were analysed for all
strains.
During experiments to determine mRNA concentrations
and half-lives, cells grew exponentially for approximately
4 h before enough cells were present to harvest or to
initiate starvation experiments. However, when calculating
transcription rates, a more conservative estimate of 2 h
for the duration of exponential growth was chosen. The
effects of starvation were very dynamic in the first few
minutes, but after 30 min mRNA levels appeared to approximate a steady state. Therefore, during starvation, mRNA
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J. M. Reimers and others
The concentration of leuB mRNA in CP78 increased 60fold above the level during growth by 10 min of starvation,
then declined to a level 24-fold of that during growth and
remained at that level for at least 20 min. Preliminary
experiments (not shown) indicated that the level of
mRNA reached by 30 min of starvation was maintained
for approximately 8 h. In CP79 the concentration of leuB
mRNA was increased 45-fold by 4 min of starvation, after
which there was a rapid decline, and very little mRNA
remained after 15 min.
Fig. 1. Concentration of leuB mRNA (a) and ppGpp (b) in
CP78 ($) and CP79 (#) strains during growth and starvation
for leucine. Leucine was removed from cell cultures at 0 min
(see Methods). The mRNA concentration data are the means of
two to four independent experiments and the error bars indicate
SD. The ppGpp concentration data represent the means of
duplicate samples which differed by no more than 20 %.
concentrations and half-lives were measured at peak mRNA
levels and at the 30 min starvation steady state. Assuming
that reversions occurred within this 2?5 h period of growth
plus starvation, transcription rates and amounts of mRNA
produced during each phase were determined and a mean
rate of transcription for each gene was calculated.
Levels of leuB mRNA and ppGpp in CP78 and
CP79
Concentrations of leuB mRNA were measured every few
minutes during growth and starvation for leucine (Fig. 1a).
In CP78, the increase in leuB mRNA levels was slightly
preceded by and correlated with increased ppGpp levels,
as previously reported, while CP79 (relA2) produced
minimal amounts of ppGpp (Fig. 1b; Donini et al., 1978).
Deattenuation was probably responsible for operon expression during the first few minutes of leucine starvation,
since leuB mRNA accumulated immediately in both CP78
(+ppGpp) and CP79 (2ppGpp). However, as starvation
continued, only CP78 synthesized leuB mRNA, and by
20 min of starvation, 96 % of leuB mRNA synthesis was
ppGpp-dependent. CP78, with its higher levels of ppGpp,
was able to maintain higher levels of mRNA for a much
longer time than CP79 (Fig. 1a, Table 1).
Half-lives and synthesis rates of leuB mRNA in
CP78 and CP79
S1-nuclease protection assays were used to determine
mRNA concentrations and decay rates in the presence of
rifampicin (1) during exponential growth, (2) at peak
mRNA concentration during leucine starvation, and (3)
after 30 min of leucine starvation, during the new starvation steady-state. Transcripts of leuB became two to three
times more stable during starvation (Fig. 2a, b, Table 1)
and CP79 leuB mRNA showed an increase in stability over
CP78. Transcription rates were calculated (see Methods)
using the concentration and half-life data obtained at each
of the above time points. A low level of mRNA synthesis
was maintained during growth in both CP78 and CP79
(Table 1). Within 10 min of leucine starvation, there was a
60-fold increase in synthesis rate in CP78. After this peak
level was reached, the synthesis rate decreased, but CP78
Table 1. Concentrations, half-lives and synthesis rates of leuB mRNA during growth and during leucine starvation
The values given are the means of three to six independent determinations±SD; see Methods for details.
Strain
Relevant
genotype
CP78
leuB6 relA+
CP79
1460
leuB6 relA2
Time of
determination
Growth
Starved for
Starved for
Growth
Starved for
Starved for
10 min
30 min
4 min
30 min
leuB mRNA concn
[pg leuB mRNA
(mg total RNA)”1]
Half-life
(min)
Synthesis rate
[pg leuB mRNA
(mg total RNA)”1 min”1]
8±2
485±29
193±20
7±3
319±70
10±4
1?1±0?4
1?1±0?3
1?8±0?4
1?0±0?2
1?9±0?2
2?8±0?5
5
306
74
5
116
2
Microbiology 150
Transcription rates and mutation in E. coli
Fig. 2. Decay of mRNA during exponential growth (m), at peak mRNA concentration ($) and at 30 min of starvation (&), for
leuB mRNA in CP78 (a) and CP79 (b), and for argH mRNA in CP78 (c), CP79 (d), DR78 (e) and DR79 (f). The data
represent the means of two or three samples which differed by no more than 20 %. The half-life values determined from these
data are shown in Tables 1 and 3.
continued to transcribe at a rate 15-fold higher than that
during growth. In CP79, the rate of synthesis reached a
maximum at 4 min of starvation, but rapidly declined to a
level equivalent to that during growth. Even at its maximal
rate, CP79 synthesized mRNA at one-third the rate of CP78
(Table 1).
Correlation of leuB transcription with reversion
rates
The mean leuB mRNA transcription rate (Table 2) during
the period that the reversions were expected to occur was
calculated by determining the amount of mRNA produced
during growth (2 h) and starvation (30 min) and dividing
by the total time (150 min). The amount of mRNA produced during each time period was calculated by determining the time each rate was in effect from the profiles of
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the graphs in Fig. 1(a) and multiplying by the transcription rate during each period. The mean rate of leuB
mRNA synthesis was fourfold higher in CP78 than in CP79
(Table 2).
Reversion rates for amino acid auxotrophs were determined previously (Wright, 1996) by growing the cells to
stationary phase in a limiting amount of the required
amino acid and then plating to minimal medium lacking
that amino acid. Only those cells that revert to prototrophy
survive. When cells were starved for leucine, the reversion
rate of leuB6 in CP78 was sevenfold higher than that in
CP79 (Table 2; Wright, 1996). By comparing strains isogenic except for relA, we have shown that the strain able to
activate leuB transcription with ppGpp had a higher mutation rate. Table 2 also shows the correlation between leuB
reversion rates and transcription rates during the 2?5 h
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J. M. Reimers and others
Table 2. leuB mRNA synthesis rates and mutation rates in CP78 and CP79
Synthesis rates from Table 1, duration of rate from Fig. 1(a) and mutation rates from Wright (1996).
Strain
leuB mRNA synthesis rate
[pg leuB mRNA
(mg total RNA)”1 (min)”1]
Duration of
rate (min)
Amount of leuB mRNA
produced [pg leuB mRNA
(mg total RNA)”1]
CP78
5
306
74
122*
8
20
150
122*
4
24
150
610
2448
1480
4538
610
464
48
1122
Total
CP79
Total
5
116
2
Mean transcription rate
[pg leuB mRNA
(mg total RNA)”1 min”1]
1096Mutation rate
(reversions per cell
per generation)
30?3
1?5±0?5
7?5
0?22±0?08
*Including the 2 min transition time between growth and starvation.
period of analysis. There was a sevenfold increase in mutation rate in CP78 (relA+) and a fourfold increase in transcription rate when compared to CP79 (relA2).
Levels of argH mRNA and ppGpp in CP78 and
CP79
The concentration of argH mRNA in CP78 and CP79 was
analysed during growth and during 30 min of arginine
starvation (Fig. 3a). In CP78, argH mRNA concentration
increased 200-fold after 12 min of arginine starvation and
then gradually declined to a level 78-fold higher than during growth (Table 3). In CP79, argH mRNA concentration
increased 80-fold after 12 min of arginine starvation and
continued to rise, until by 30 min it was 132-fold higher
than during growth (Table 3). This result agrees with
previous investigations, in which the amount of argCBH
mRNA was higher in a relA mutant after 30 min of arginine
downshift (Williams & Rogers, 1987).
Fig. 3. (a, b) Concentration of argH mRNA (a) and ppGpp (b) in CP78 (m) and CP79 (n) during growth and starvation for
arginine. (c, d) Concentration of argH mRNA (c) and ppGpp (d) in DR78 (&) and DR79 (%) during growth and starvation for
arginine. Arginine was removed from cell cultures at 0 min (see Methods). The mRNA concentration data are the means of two
to four independent experiments and the error bars indicate SD. The ppGpp concentration data represent the means of
duplicate samples which differed by no more than 20 %.
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Microbiology 150
Transcription rates and mutation in E. coli
Table 3. Concentrations, half-lives and synthesis rates of argH mRNA during growth and during arginine starvation in argR+
and argR knockout strains
The values given are the means of three to six independent determinations±SD; see Methods for details.
Strain
Relevant
genotype
CP78
argH46 relA+
CP79
DR78
DR79
argH46 relA2
argH46 relA+
DargR : : cat
argH46 relA2
DargR : : cat
Time of
determination
Growth
Starved for
Starved for
Growth
Starved for
Starved for
Growth
Starved for
Starved for
Growth
Starved for
Starved for
argH mRNA concn
[pg argH mRNA (mg total RNA)”1]
12 min
30 min
12 min
30 min
12 min
30 min
12 min
30 min
0?4±2
81±13
31±13
0?5±1
40±14
66±13
76±13
34±8
23±7
61±12
43±10
51±12
Half-life
(min)
Synthesis rate [pg argH mRNA
(mg total RNA)”1 min”1]
ND
(1)D
56
24
(1)D
14
38
105
34
23
70
30
17
1?0±0?2
0?9±0?4
ND
2?0±0?3
1?2±0?4
0?5±0?2
0?7±0?3
0?7±0?1
0?6±0?2
1?0±0?1
2?1±0?4
ND, Not determined due to low concentration of argH mRNA during growth.
DA synthesis rate of 1 pg argH mRNA (mg total RNA)21 min21 was estimated; see text.
The increase in argH mRNA at 12 min of arginine starvation (Fig. 3a) correlated with the increase in ppGpp in the
relA+ strain at 12 min of starvation (Fig. 3b). This is consistent with the results of Donini et al. (1978) showing that
ppGpp levels increased in CP78 at about 8 min of arginine
starvation, but only minimally in CP79. Since significant
levels of ppGpp were only present in CP78, derepression
and/or a decrease in mRNA half-life was apparently responsible for the increased concentration of argH mRNA in
CP79.
Half-lives and synthesis rates of argH mRNA in
CP78 and CP79
Half-lives and concentrations of argH mRNA were measured (1) during growth, (2) at peak mRNA synthesis during arginine starvation and (3) after 30 min of arginine
starvation in CP78 (Fig. 2c) and in CP79 (Fig. 2d). The
concentration of argH mRNA during exponential growth
was too low to determine its decay rate. However, during
arginine starvation, when half-lives could be measured,
there was no change in half-life in CP78 (Fig. 2c, Table 3),
while argH mRNA became slightly less stable in CP79
(Fig. 2d, Table 3).
Half-lives and concentrations of argH mRNA determined
above were used to calculate rates of transcription during
each time period (Table 3). Very low argH mRNA concentrations did not allow for a synthesis rate to be determined during exponential growth. However, since this
concentration was extremely low, the rate of synthesis
could not be significant even if the half-life were very
short. For example, at an mRNA concentration of 0?5 pg
mRNA (mg total RNA)21, even with a very short half-life
of 0?3 min (one-third of that measured during arginine
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starvation) the synthesis rate would be only 1 pg mRNA
(mg total RNA)21 min21. This rate was chosen as the
highest possible estimate and used below to determine the
mean transcription rate. At 12 min of arginine starvation,
the synthesis rate of argH mRNA in CP78 was fourfold
higher than in CP79 (Table 3). However by 30 min of
starvation, CP79 was transcribing at a slightly higher rate
than CP78.
Correlation of argH transcription with reversion
rates in CP78 and CP79
The mean synthesis rates of argH mRNA in CP78 and
CP79 (Table 4) were calculated as previously described for
leuB mRNA. Since an actual rate during growth could
not be determined, an estimated synthesis rate of 1 pg
mRNA (mg total RNA)21 min21 was used to calculate
mean transcription rate. The mean transcription rate of
argH mRNA was 1?4-fold higher in CP78 than in CP79.
When CP78 was starved for arginine, the argH46 mutation
reverted at a rate twofold higher than in CP79 (Table 4).
Therefore, the relA+ strain, compared to the relA2 strain,
had a higher reversion rate, indicating that the transcriptional activator ppGpp had a positive influence on the
mutation of this gene. When comparing CP78 and CP79
(Table 4), there was a qualitative correlation between the
twofold increase in mutation rate and the 1?4-fold increase
in transcription rate of the argH gene in the relA+ strain.
Reversion rates for the argH gene in CP78 and CP79
(Table 4) were determined by counting negative plates
at 48 h, instead of 72 h as previously reported (Wright,
1996). Sequencing revertants that appeared early (48 h)
as well as those that appeared later (72 h) showed that the
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J. M. Reimers and others
Table 4. argH mRNA synthesis rates and mutation rates in CP78, CP79, DR78 and DR79
Synthesis rates from Table 3; duration of rate from Figs 3(a) and (c). Mutation rate values are the means of three to eight independent
determinations±SD.
Strain
argH mRNA synthesis
rate [pg argH mRNA
(mg total RNA)”1 (min)”1]
Duration of
rate (min)
Amount of argH mRNA
produced [pg argH mRNA
(mg total RNA)”1]
CP78
(1)*
56
24
124D
8
18
150
124D
16
10
150
124D
10
16
150
124D
16
10
150
124
448
432
1 004
124
224
380
728
13 020
340
368
13 728
8 680
480
170
9 330
Total
CP79
Total
DR78
Total
DR79
Total
(1)*
14
38
105
34
23
70
30
17
Mean transcription
rate [pg argH mRNA
(mg total RNA)”1 (min)”1]
1096Mutation rate
(reversions per cell
per generation)
6?7
0?26±0?2
4?9
0?12±0?08
91?5
6?9±1?5
62?2
3?2±1?2
*A synthesis rate of 1 pg argH mRNA(mg total RNA)21 min21 was estimated; see text.
DIncluding the 4 min transition time between growth and starvation.
colonies appearing after 48 h were largely suppressors. Since
the argH46 mutation creates a stop codon, it is likely that
these later colonies were intergenic suppressors. Twenty
early (48 h) revertants of each strain were sequenced and
the percentages of true revertants were determined. In
CP78, 89 % of the scored revertants were true revertants,
while only 30 % of the revertants scored in CP79 were
true. Sequencing was necessary since colony size did not
indicate whether the colony came from a true revertant or
a suppressor. These percentages were used to calculate the
reversion rates reported in Table 4.
Levels of argH mRNA and ppGpp in the argR
knockout strains DR78 and DR79
The argR gene in CP78 and CP79 was inactivated by the
insertion of the cat gene, producing the strains DR78
(relA+ argH46 DargR : : cat) and DR79 (relA2 argH46
DargR : : cat). Since the normal transcriptional control was
removed in these knockout strains, unregulated (increased)
transcription would be expected to occur during growth in
the presence of arginine. Cunin et al. (1969) reported that
in the absence of the repressor, the enzymes encoded by
the argCBH transcriptional unit increased 60-fold in activity. Unlike the regulated (repressed) strains, high argH
mRNA levels were found during exponential growth in
both DR78 and DR79. In fact, the argR knockout strains
(Fig. 3c, Table 3) had 100- to 200-fold more argH mRNA
than CP78 and CP79 (Fig. 3a, Table 3). The argH mRNA
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levels declined as arginine starvation became more severe,
and after 30 min of starvation, the level of argH mRNA was
higher in DR79 than in DR78. Interestingly, peak concentrations of mRNA during growth of the argR knockout
strains (Fig. 3c) are similar to peak values during starvation in the regulated strains (Fig. 3a). This suggests that
removing the ArgR protein fully derepressed the argH gene.
In the argR knockout strains, DR78 and DR79, ppGpp
concentrations were analysed during arginine starvation.
In DR78 (relA+), ppGpp accumulated (Fig. 3d) while
DR79 (relA2) produced minimal amounts of ppGpp when
starved for arginine. Comparing Figs 3(c) and 3(d), there
did not appear to be a correlation between ppGpp levels
and mRNA levels; but as discussed below, the large increase
in transcription due to the inactivation of the repressor
perhaps masked a relatively small activation by ppGpp.
Half-lives and synthesis rates of argH mRNA in
DR78 and DR79
In DR78, argH mRNA half-lives remained short during
arginine starvation (Fig. 2e, Table 3), but in DR79 half-lives
increased threefold (Fig. 2f, Table 3). During exponential
growth in both DR78 and DR79, both concentrations
and synthesis rates were very high (Table 3). As starvation
continued the argH mRNA concentration and its synthesis
rate decreased. At 30 min of starvation, even though the
mRNA concentration was twofold higher in DR79, mRNA
Microbiology 150
Transcription rates and mutation in E. coli
synthesis was lower than that in DR78 because the mRNA
half-life in DR79 had increased threefold. Thus, in the case
of DR78 and DR79, increased stability did contribute to
the observed increase in concentration and therefore,
mRNA concentration was a poor indicator of the rate of
transcription.
Correlation of argH transcription with reversion
rates in DR78 and DR79
The mean synthesis rate of argH mRNA in DR78 and DR79
was calculated as previously described and is shown in
Table 4. The mean transcription rate in DR78 was 1?5-fold
higher than in DR79 (Table 4); therefore ppGpp in DR78
had a similar positive effect on transcription as in CP78.
During growth, the ArgR protein, in combination with
arginine, represses transcription of argH. If increased
transcription increases mutation rates, then the reversion
rate of argH should be higher in the (unregulated) argR
mutants than in the argR+ strains. The argR knockout
strains (DR78 and DR79) had the highest observed mutation rates, 6?961029 reversions per cell per generation in
DR78 and 3?261029 in DR79 (Table 4). In DR78, 100 %
of the revertants were true and in DR79, 72 % of the
revertants were true. Perhaps the higher percentage of
true revertants in the relA+ strains, as well as in the argR
mutants, reflected a higher reversion rate due to the higher
rate of transcription.
Table 4 shows the correlation between rates of argH
transcription and reversion rates in the argR mutants. The
transcription rate of the relA+ strain increased 1?5-fold over
that of the relA2 strain, while the mutation rate increased
approximately twofold.
Another set of correlations can be observed when comparing the regulated (CP78, CP79) and unregulated (DR78,
DR79) strains. Comparing the argR+ and argR knockout
strains, there was a 26-fold increase in mutation rates in
the absence of the repressor for both CP78 versus DR78
and CP79 versus DR79. There was also a 13-fold increase
in mean transcription rates when comparing CP78 with
DR78 and CP79 with DR79. Therefore, both transcription
and mutation rates increased by an order of magnitude
when the regulated and unregulated strains were compared. These knockout strains, lacking the repressor protein, showed the greatest effect on argH mRNA synthesis
and the highest increase in mutation rate yet observed. They
illustrate most clearly that increasing transcription increases
mutation rates.
It was surprising that in the two genes and four strains
used here, there is a single linear relationship between
increased transcription and reversion rates (Fig. 4). While
it is to be expected that such a correlation may exist for a
single gene when its level of transcription is varied, it was
not expected when comparing different genes due to the
many other variables impacting on both transcription and
mutation rates. For example, a gene’s transcription rate is
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Fig. 4. Correlation of reversion rates (reversions per cell per
generation) with transcription rates [pg mRNA (mg total
mRNA)”1 (min)”1] for the leuB (n) and argH ($) mutants;
R=0?98, P=0?02. Reversion rate data are the mean of three
to eight independent experiments. Transcription rate data are
from Tables 2 and 4.
affected by DNA-binding proteins and the level of supercoiling in its immediate environment. However, the
correlation seen in Fig. 4 suggests that variables such as
these may be similar for the leuB and argH genes.
This investigation, using mRNA half-life and concentration
data to calculate rates of transcription, established that
increased transcription rather than increased mRNA stability was primarily responsible for the increased mRNA
concentrations observed. The removal of the ArgR repressor
served to increase both transcription and mutation rates
by more than 10-fold while ppGpp had a smaller, but
consistent effect. The results of these analyses support the
hypothesis that increased mutation rates in the leuB and
argH genes are primarily due to increased rates of mRNA
synthesis.
ACKNOWLEDGEMENTS
We thank Greg St George for helpful comments on the manuscript.
This work was supported by the Eppley Foundation, the NSF EPSCoR
program, the Stella Duncan Research Institute and NIH grants
R15CA88893 and R55CA99242.
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