Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA
Correspondence
Barbara E. Wright
barbara.wright{at}mso.umt.edu
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ABSTRACT |
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INTRODUCTION |
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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 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 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 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.
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METHODS |
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Cells were grown as previously described at 37 °C (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 °C 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 determination 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 45° angle at 37 °C 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 °C. 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 = (ln2) (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), 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 °C, extracted twice with phenol at 65 °C, 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 20 °C overnight. This pellet was washed with 70 % ethanol, resuspended in water and precipitated with 0·5 vol. 7·5 M LiCl at 20 °C. After a 70 % ethanol wash, the RNA was treated with 5 U RQ1-RNase-free DNase (Promega) for 15 min at 37 °C. 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 20 °C. After centrifugation at 16 000 g for 20 min at 4 °C, the final RNA pellet was resuspended in 200 µl 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), 0·5100 µg total cellular RNA and 10 µg yeast tRNA were precipitated with 0·1 vol. 5 M ammonium acetate and 3 vols 100 % ethanol at 20 °C for 30 min. After centrifugation at 16 000 g for 15 min at 4 °C, the pellets were air-dried for 30 min and resuspended in 10 µl RPAII hybridization buffer (Ambion). The samples were hybridized overnight at 50 °C for leuB or 52 °C for argH. Unhybridized RNA and probe were digested with 300 U S1-nuclease (Promega) at 42 °C for 45 min. The digest was stopped by adding 0·2 vol. stop buffer [4 M ammonium acetate, 30 mM EDTA, 170 µg tRNA ml1 and 0·7 mg GlycoBlue ml1 (Ambion)] and the protected hybrids were precipitated with 2·5 vols 100 % ethanol. After precipitation at 20 °C for 30 min and centrifugation at 16 000 g for 15 min at 4 °C, the pellets were air-dried for 30 min and suspended in 11 µl 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 1x TBE and electrophoresed at 40 V cm1 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 streptavidinalkaline 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 specific times of starvation, were measured by inhibiting transcription initiation with 300 µg rifampicin ml1. 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 semi-logarithmic 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 half-life (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) 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 µCi (3·7 MBq) H332PO4 ml1 (ICN; 3·7x106 Bq ml1) and then grown at 37 °C 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 µl aliquots of cells were removed and added to 17 µl 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 freezethawed twice and centrifuged for 1 min. Aliquots (2 and 5 µl) 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 °C 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)1] was based on the specific radioactivity of the phosphate in the medium (Wright & Minnick, 1997
).
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RESULTS AND DISCUSSION |
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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 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). The concentration of leuB mRNA in CP78 increased 60-fold 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.
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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
).
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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 (µg total RNA)1, even with a very short half-life of 0·3 min (one-third of that measured during arginine starvation) the synthesis rate would be only 1 pg mRNA (µg total RNA)1 min1. 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 (µg total RNA)1 min1 was used to calculate mean transcription rate. The mean transcription rate of argH mRNA was 1·4-fold higher in CP78 than in CP79.
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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 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 argR : : cat) and DR79 (relA2 argH46
argR : : 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 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 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, 6·9x109 reversions per cell per generation in DR78 and 3·2x109 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 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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 1 December 2003;
revised 15 January 2004;
accepted 20 January 2004.
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