Proc. Natl. Acad. Sci. USA Vol. 96, pp. 5089–5094, April 1999 Evolution

Hypermutation in derepressed of Escherichia coli K12 (stringent response͞transcription͞mutations)

BARBARA E. WRIGHT*, ANGELIKA LONGACRE, AND JACQUELINE M. REIMERS

Division of Biological Sciences, University of Montana, Missoula, MT 59812

Communicated by E. R. Stadtman, National Heart, Lung and Blood Institute, Bethesda, MD, February 16, 1999 (received for review December 15, 1998)

ABSTRACT This article presents evidence that starva- the concentration of single-stranded DNA (ssDNA), which is tion for leucine in an Escherichia coli auxotroph triggers more vulnerable to mutations than double-stranded DNA. metabolic activities that specifically target the leu for Although the mutations per se are random, as described above derepression, increased rates of , and mutation. for background mutations, the mechanisms that target oper- Derepression of the leu operon was a prerequisite for its ons for increased rates of transcription are highly specific. This activation by the signal nucleotide, guanosine tetraphosphate, specificity is not compatible with current neo-Darwinian which accumulates in response to nutritional stress (the dogma. And yet, evidence in the literature supports the two stringent response). A quantitative correlation was estab- major assumptions on which our hypothesis is based: (i) -lished between leuB mRNA abundance and leuB؊ reversion ssDNA is more vulnerable to mutagenesis than double rates. To further demonstrate that derepression increased stranded DNA; increased rates of transcription will, therefore, mutation rates, the chromosomal leu operon was placed under increase rates of mutation; and (ii) derepression and activation the control of the inducible tac . When the leu operon of an amino acid biosynthetic operon occur specifically in was induced by isopropyl-D-thiogalactoside, both leuB mRNA response to starvation for that amino acid. abundance and leuB؊ reversion rates increased. These inves- Evolution implies stress; a perfectly adapted organism in a tigations suggest that guanosine tetraphosphate may contrib- stable environment would not need to evolve. In fact, evidence ute as much as attenuation in regulating leu operon expression from marine fossil communities indicates that environmental and that higher rates of mutation are specifically associated stress accelerates the rate of evolution (7). In microorganisms, with the derepressed leu operon. prolonged nutritional stress results in genome-wide hypermu- tation, i.e., high mutation rates in various genes located in The mechanisms of evolution have been the subject of many chromosomes, episomes, transposons, or insertion elements controversies and speculations for some 200 years (1, 2). (8–14). Such effects likely contribute to the evolutionary Clearly, selection of the fittest occurs, but does the environ- process by increasing the size of the mutant population. ment also play a role in generating the fittest? Do all of the Indeed, hypermutable genes apparently have been selected variants selected result from mutations that are completely during evolution as uniquely advantageous to the survival of ‘‘random’’? Background mutations are loosely referred to as the organism (15). The question then arises: By what mecha- random even though they do not occur with equal probability, nism(s) related to stress could specific genes become hyper- but at different and characteristic rates because of DNA mutable? Transcription-enhanced hypermutation is a plausi- context and variables such as the intrinsic instability of cyto- ble mechanism by which a direct causal relationship can be sine, giving rise to the (most frequent) C-to-T transition established between higher mutation rates in specific genes mutations (3, 4) or the presence of tandem repeats, resulting and the selective conditions of stress that evoke them. There in frameshift mutations (5). Moreover, environmental condi- is an accepted biochemical basis for the stimulation of tran- tions such as thymidine starvation can selectively increase the scription by starvation (derepression); for example, the lack of an amino acid results in the derepression of the operon rate of particular kinds of mutation (6). However, in an controlling its synthesis. Because increased rates of transcrip- evolutionary context, ‘‘random’’ has a very specific meaning: tion can cause higher mutation rates in specific operons (see Neo-Darwinism holds that the spectrum of background mu- Discussion), a direct causal relationship appears to exist be- tations and the frequency with which they occur are random tween starvation for a particular amino acid and higher (undirected) with respect to selective conditions of the envi- mutation rates in genes of the operon encoding the enzymes ronment. Another ambiguous word, ‘‘mechanism,’’ can mean that catalyze its synthesis. one thing when applied to evolution and another when applied The metabolism of starvation is called the stringent response to mutations. There are mechanisms by which particular kinds (17). After deprivation of any essential nutrient, the cell of mutations occur (e.g., base substitution, deletion, frame- restructures its metabolism from one geared to growth and cell shift), and there are mechanisms by which the rates of many division under favorable nutritional conditions to one allowing kinds of background mutations are stimulated (e.g., replica- survival under conditions of starvation. This abrupt shift in tion, UV irradiation, defective repair, transcription). It is the metabolism results in the rapid accumulation (16, 17) of latter sort of mechanism that applies to evolution because guanosine tetraphosphate (ppGpp), a signal nucleotide that stimulating mutation rates increases the availability of variants appears to be the most dominant primary regulator of nutri- on which evolution depends. Our data indicate that transcrip- tional distress (17, 18). It is synthesized by ppGpp synthase I, tion (starvation-induced derepression) is unique in augment- the relA gene product, and is degraded by the spoT gene ing variant availability in a specific manner, i.e., by stimulating product. Recent investigations have demonstrated that this rates of transcription (and associated phenomena such as RNA signal nucleotide binds to the ␤-subunit of RNA polymerase polymerase pausing) in targeted operons, thereby increasing (19) and affects both transcription initiation (20) and poly- merase pausing (21). By recognizing particular promoter The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: IPTG, isopropyl-D-thiogalactoside; ssDNA, single- accordance with 18 U.S.C. §1734 solely to indicate this fact. stranded DNA; ppGpp, guanosine tetraphosphate. PNAS is available online at www.pnas.org. *To whom reprint requests should be addressed.

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sequences (17, 22), ppGpp serves three essential roles for the RNA Probe Preparation. The E. coli leuB, pyrD, and glpK starving cells. The first two occur regardless of the starvation genes were PCR amplified and cloned into a vector containing regimen and consist of the inhibition of DNA, rRNA, nucle- opposable T3 and T7 promoter sites (pBluescript II SK(ϩ), otide, and phospholipid synthesis, thereby arresting cell divi- Statagene). For each gene, plasmid DNA was isolated (Quan- sion and the activation of metabolic pathways that protect the tumPrep mini-prep kit, Bio-Rad) and cut with a restriction vulnerable cells from environmental extremes such as heat, enzyme to produce a 100- to 600-bp section of the gene with desiccation, and oxidative damage (17, 18). The third essential either the T3 or T7 promoter at one end. Antisense RNA role is starvation regimen-dependent in that ppGpp activates probes were produced by in vitro transcription (Ampliscribe T3 only those genes that are specifically derepressed by the type and T7 in vitro transcription kit, Epicentre Technologies, of starvation imposed. For example, the expression of amino Madison, WI). The probes were labeled with biotin (BrightStar acid biosynthetic operons requires both derepression (removal Psoralen-Biotin kit, Ambion) and were purified by using of end product inhibition by attenuation or repression) and denaturing polyacrylamide gel electrophoresis. The probes Ј activation by ppGpp. Starvation for tryptophan as well as other contained some vector sequence at the 5 end that was amino acids elicits the stringent response, but only tryptophan nonhomologous to the target mRNA, producing protected starvation increases trp expression (23); the absence of thre- hybrid fragments that were shorter than the full-length probe onine, but not arginine or histidine, leads to derepression of and separable by gel electrophoresis. This difference in pro- the thr operon (24). However, regulation in some operons tected vs. full length probe is evidence of a working assay. As apparently may be triggered as a general consequence of the a standard, positive-sense mRNA was generated for each gene stringent response (17). from the same plasmid but using the other promoter. Nuclease Protection Assay and Densitometry. The biotin- ylated antisense RNA probe of defined length was hybridized MATERIALS AND METHODS overnight to the target mRNA in solution at 50°C. After Bacterial Strains and Mutation Rate Experiments. The digestion of single strand nucleic acids by T2 ribonuclease, the bacteria used in this study are two multiple auxotrophs of hybrids were precipitated, were separated by denaturing poly- Escherichia coli K12, CP78 and CP79, isogenic except for relA, acrylamide gel electrophoresis, and were transferred to posi- and a construct of CP78, called 78AL (see below). The tively charged nylon membrane by semidry electroblotting. The biotinylated probe was conjugated to streptavidin-alkaline conditions for growing CP78 and CP79 and for determining phosphatase and CDP-Star by using Ambion’s Bright-Star mutation rates in these strains has been described (25, 26). To BioDetect kit. Chemiluminescent probes were detected with determine mutation rates in 78AL, the minimal medium used x-ray film. Standards were run with each sample, and relative was limiting for arginine or histidine, rather than leucine, to ϩ intensities of different mRNA species within a single RNA avoid selection of Leu revertants. When the cultures reached extract were compared by densitometry using an Astra (Soder- stationary phase (Ϸ24 h), they were washed twice with leucine- talje, Sweden) 1200S document scanner with transparency free minimal medium to remove all residual leucine and adapter and Scanalytics (Bellerica, MA) ONE-DSCAN software. isopropyl-D-thiogalactoside (IPTG) and were plated (at a Ϸ ϫ 8 The probe molecules were present in 4- to 10-fold molar excess concentration of 1 10 ) on the same medium. Thereafter, of the target mRNA to demonstrate linearity between the total cell numbers (determined at 24, 48, and 72 h) were amount of RNA present and the amount of binding to the constant and comparable, regardless of prior exposure to probe. IPTG. Mutation rates were determined by the ‘‘zero’’ method Replacing the leu with the tac Promoter (Strain 78AL). A of Luria and Delbruck (27). The values given in Tables 1 and suicide vector, pBanz2, containing the tac promoter (tacP), a 2 are averages of 5–8 experiments. kanamycin resistance cassette, and ends that are homologous RNA Isolation. Cells were collected and lysed according to to the E. coli chromosome flanking the leu promoter was Emory and Belasco (28). The lysate was extracted twice with generated (see Fig. 1) and then was used to transform E. coli ͞ ͞ Ϫ phenol chloroform isoamyl alcohol (50:49:1) and once with CP79 (relA2, leuB ). Double crossover homologous recombi- chloroform. The RNA was precipitated with 0.5 M ammonium nation between the chromosome and the plasmid replaced the Ϫ acetate and 2.5 volumes ethanol overnight at 20°C, was chromosomal leu promoter with the tac promoter and kana- pelleted and washed with 70% ethanol, and was resuspended mycin resistance cassette (Fig. 1). The construct was generated in diethyl pyrocarbonate water. It was reprecipitated with 2.5 as follows: (i) leu upstream and leuA regions were PCR M LiCl at Ϫ20°C for 1 h and was pelleted. After this, the pellet amplified, with specific restriction sites in the primers from the was washed with ethanol and was resuspended in diethyl CP78 chromosome, and were cloned into T-Vectors (29); (ii) pyrocarbonate water, and the RNA was treated with 10 units the leu upstream and the leuA fragments were recovered from of RNase-free DNase (Promega). Phenol͞chloroform͞ the T-Vectors by restriction digestion; (iii) the kanamycin isoamyl alcohol extraction was used to remove Dnase, and the cassette was acquired by restriction digest of plasmid pBSL15 final RNA was precipitated with ammonium acetate and (29); (iv) the fragments were sequentially cloned into plasmid ethanol as above. RNA concentration was determined by pKK223–3 (tacp, ampr; Amersham Pharmacia); and (v) the reading the OD at 260 nm. 3.6-kilobase construct was recovered by PCR amplification by

FIG. 1. Construct map of 78AL and 79AL. Recombination with the suicide vector pBanz2, which contained the above construct, resulted in the arrangement of genes into the genomes of CP78 and CP79 as shown. The PCR primers described are depicted in their respective approximate positions and in their direction of polymerization. The recombination event introduced a kanamycin resistance gene cassette and the tac promoter, replacing the endogenous leu promoter. Downloaded by guest on October 3, 2021 Evolution: Wright et al. Proc. Natl. Acad. Sci. USA 96 (1999) 5091

using the Expand High Fidelity PCR System of Boehringer- Mannheim with primers LeuA4 and LeuA2 (Fig. 1; see Primers) and was cloned into suicide vector pWM91 [oriR6K, ampr, sucroses due to sacB (30)] to generate plasmid pBanz2, which was introduced into CP79 by electroporation. Plasmid pBanz2 is propagated only in E. coli strains that produce the ␭ Pir protein and will not replicate in CP78 or CP79, therefore rendering pBanz2 a suicide vector for these strains. When introduced into CP79 by electroporation, single- crossover homologous recombination resulted in integration of the suicide vector into the leu operon. An integrant (CP79- integrant) was selected by resistance to kanamycin and ampi- cillin. Double-crossover homologous recombinants that main- tained the 3.6-kilobase fragment (kanr) but that lost the remainder of the suicide vector containing the toxic sacB gene were selected by sucrose and kanamycin resistance and were screened for ampicillin sensitivity. P1vir transduction was used wt Ϫ to transfer the recombinant promoter to CP78 (relA , leuB ), ϩ ϩ FIG.2. leuB mRNA abundance in E. coli CP78 (relA , ppGpp ) resulting in the recombinant 78AL. A PCR fragment, using a and CP79 (relAϪ, ppGppϪ). Cells were grown to log phase (log) in primer set that flanked the construct sequences in the E. coli minimal medium or were grown to log phase and were washed and chromosome (see Primers), was amplified from 78AL and was transferred to minimal medium without arginine (Ϫarg), threonine sequenced to confirm replacement of the leuP with the tacP. (Ϫthr), or leucine (Ϫleu) for 15 min. Total RNA was recovered, and Primers. The 572-bp leu upstream region was amplified with the level of leuB transcript was determined by nuclease protection forward primer LeuA4 (5ЈAAGGCGCATGCTCAGTA- assay and densitometry (see Materials and Methods). Ј ACGGGC-3 ), including a SphI site (underlined) and reverse ϩ Ϫ primer LeuA5 (5Ј-CAACGGCTAGCACGCATGGTCGA- leucine-starved relA compared with the relA strain may be Ј attributed to derepression (36) plus ppGpp activation in the 3 ) with a NheI site (underlined). The 1,589-bp leuA gene was ϩ Ј case of the relA strain and to continued repression in the amplified with forward primer LeuA3 (5 -GCCGGAAT- Ϫ TCATGAGCCAGC-3Ј, including the leuA start codon (un- relA strain. Although starvation for either arginine or thre- derlined and bold) and an EcoRI site (underlined), and reverse onine evokes the stringent response and high ppGpp levels primer LeuA2 (5Ј-ATCCTGCAGCACACGGTTTC-3Ј) with (16), it does not activate the leu operon because of the presence of leucine. The data suggest that, in the absence of a PstI site (underlined). Ϫ Primers used to amplify the regions surrounding the inte- ppGpp (i.e., the relA mutant), removal of inhibition by gration site (see Fig. 1) were as follows: The forward primer, attenuation alone (i.e., leucine starvation) does not allow leu from upstream of the leu upstream region, was SeqA (5Ј- operon expression at 15 min starvation because transcript CACGCTAAGTACGCTCATC-3Ј), and the reverse primer, levels stay the same. originating within the leuB gene downstream of the leuA gene, Mutation Rates Are Correlated with leuB mRNA Abun- Ј Ј dance. As shown in Table 1, a quantitative correlation exists was SeqZ (5 -GGCGATACGTTCGATCTCA-3 ). The size of Ϫ the amplified fragment from this set of primers was designed between leuB reversion rates and transcript abundance [the ϭ to allow parents and recombinants to be distinguished: The Pearson correlation 0.967, significant at the 0.01 level parental product was 3,683 bp, and the recombinant product (2-tailed)]. This correlation is particularly meaningful in that was 4,254 bp (see Fig. 1). the mutation rates were determined in the past (25, 26, 32) and, in effect, predicted the mRNA levels that have now been found. Preliminary data suggest that the leuB mRNA accu- RESULTS mulation pattern follows that of ppGpp; that is, the peak is very The present study concerns two isogenic E. coli multiple sharp and occurs within the first 10 min (16). To obtain the auxotrophs (leuBϪ, argHϪ, thrϪ, hisϪ) differing only in relA data for Table 1, cells were starved for 15 min before mea- (and therefore ppGpp levels). Investigations have concen- suring leuB mRNA levels, which were too variable at earlier Ϫ time points to be useful for comparative purposes. trated on the leuB point mutation, which is a C-to-T transition Ϫ resulting in a serine-to-leucine substitution at amino acid In fluctuation tests, to determine leuB reversion rates in residue 286 of the LeuB protein (26). In a typical mutation rate cells grown with limiting levels of leucine (25, 32), cells first experience starvation in the liquid minicultures; therefore, leu experiment, 80% of the colonies counted proved to be true Ϫ revertants (26). Most mutations occurred immediately when operon derepression and leuB reversions presumably occur starvation was imposed (25), coinciding with the burst in before plating (it is not possible to distinguish between mu- ppGpp levels (16); a positive correlation was established tations that occur at the end of growth and mutations that between leuBϪ reversion rates and the concentration of ppGpp occur within a few hours after plating). If cells are grown with (26). Reversion rates (25, 26) of the leuBϪ, argHϪ, and thrϪ limiting levels of threonine or arginine, the stringent response mutant alleles in response to amino acid starvation were higher also occurs in the liquid culture at the end of growth, but in the relAϩ strain (CP78) than in the relAϪ strain (CP79), in derepression of the leu operon does not; leu operon expression Ϫ contrast to mutations in genes regulating maltose catabolism and leuB reversions must then occur after plating to the or pyrimidine biosynthesis (32). Stringent response mutations, selective medium lacking leucine. It is not possible to measure like transcription-induced lacϪ reversions (33), are recA- reversion rates without derepressing the operon (16, 25, 32). independent (data not shown), distinguishing them from pro- Derepression of the leu Operon Is Specific Within the longed stress-induced ‘‘adaptive’’ mutations (8–14) and from Genome. Three biotin-labeled probes were prepared to quan- DNA damage-induced “SOS” mutagenesis, both of which tify mRNA from genes involved in different metabolic path- require recA (34, 35). ways: leuB (␤-isopropylmalate dehydrogenase, EC 1.1.1.85), Derepression of the leu Operon Is Specific to Leucine pyrD (dihydroorotate oxidase, EC 1.3.3.1), and glpK (glycerol Starvation. Fig. 2 illustrates the specificity of leuB gene kinase, EC 2.7.1.30). Fig. 3 illustrates the results of hybridizing expression resulting from starvation for leucine, arginine, or these probes to RNA isolated from log and from 60 min threonine. The difference between leuB mRNA levels in the leucine-starved relAϩ and relAϪ cells. As anticipated, based on Downloaded by guest on October 3, 2021 5092 Evolution: Wright et al. Proc. Natl. Acad. Sci. USA 96 (1999)

Table 1. Correlation between leuB mRNA levels determined as described for Fig. 2 and leuBϪ reversion rates pg leuB mRNA͞ leuBϪ reversion rate Cells relA genotype Condition :␮g of total RNA ϫ 10Ϫ9 CP79 relA unstarved 29 Ϯ 5* CP78 relAϩ unstarved 30 Ϯ 5* CP79 relA Ϫarg then Ϫ leu 17 Ϯ 6 0.15 Ϯ 0.02 CP79 relA Ϫleu 17 Ϯ 5 0.22 Ϯ 0.08† CP79pyrD relA Ϫarg then Ϫ leu 28 Ϯ 5 0.25 Ϯ 0.06‡ CP79pyrD relA Ϫleu 35 Ϯ 12 0.44 Ϯ 0.12‡ CP79⌬spoT207 relA Ϫleu 126 Ϯ 11 0.60 Ϯ 0.11§ CP78pyrD relAϩ Ϫleu 285 Ϯ 103 1.26 Ϯ 0.26‡ CP78 relAϩ Ϫleu 269 Ϯ 60 1.47 Ϯ 0.48† CP78 relAϩ Ϫarg then Ϫ leu 335 Ϯ 53 1.80 Ϯ 0.30 CP78pyrD relAϩ Ϫarg then Ϫ leu 347 Ϯ 136 2.30 Ϯ 0.70‡ CP79spoT203 relA Ϫleu 378 Ϯ 149 2.50 Ϯ 0.62§ *Not measurable. †Ref. 24. ‡Ref. 31. §Ref. 25.

previous mutation rate studies (32), leucine-starved relAϩ enhanced maximally by IPTG during growth (Table 2) and Ϫ cells had higher leuB mRNA levels than starved relA cells were recA-independent. These results are consistent with those whereas the opposite was true for pyrD mRNA levels. Thus, of Herman and Dworkin (33), which showed that recA- Ϫ both pyrD mRNA levels and reversion rates of pyrD (an independent reversions of lacϪ were enhanced Ϸ2-fold by Ϫ ϩ A-to-G transition) were higher in the relA than in the relA IPTG induction of the lac operon. strain (37). The pyrD promoter has a GC-rich discriminator sequence (38), as does pyrB1, which is inhibited by ppGpp (39, 40). As an internal control, the gene encoding glycerol kinase DISCUSSION (glpK) was examined and found to be down-regulated in the Transcription Is Known to Enhance Mutation Rates. Var- absence of growth to the same extent in the two strains when ious kinds of evidence document an effect of transcription on amino acid starvation was imposed (37). (In the experiment mutation rates; the nontranscribed ssDNA strand is more summarized in Fig. 3, glpK mRNA levels were saturating.) ϩ vulnerable to mutations than double-stranded DNA. Fix and Thus, relA strains starved for leucine (but not arginine or Glickman (41) observed that 77% of C to T transitions threonine; Fig. 2) derepressed the leuB but not the pyrD or glpK originated in the nontranscribed strand; this strand bias has genes. been confirmed in other systems (42, 43). In fact, it is known The leu Operon Can Be Activated by an Inducible tac that cytosines deaminate to uracils in ssDNA at Ͼ100ϫ the Promoter. In strain 78AL, the chromosomal leu promoter rate seen in double-stranded DNA (3, 4, 44). The first inves- (including the attenuator and ppGpp discriminator sequences) tigators to propose transcriptional activation as a mechanism has been replaced by the tac promoter (see Materials and for increasing mutation rates in specific operons were Brock Methods). Fig. 4 summarizes the effect of 1 mM IPTG on leuB (45) and Herman and Dworkin (33). Their investigations mRNA levels during growth and during 30 min of starvation Ϫ for either arginine or leucine. As might be expected in this demonstrated that recA-independent lac reversion rates of construct, IPTG enhances leuB mRNA levels maximally dur- frameshift and point mutations were higher when transcription was induced by IPTG and that the effect was specific to that ing growth (in the presence of leucine), in contrast to the Ϫ conditions found optimal for derepression in the parental gene; that is, lys reversions were not affected, and mutants strain CP78 (Fig. 2). Reversion rates in the construct also were

ϩ FIG. 3. Ribonuclease protection assay of E. coli CP78 (relA , ppGppϩ) and CP79 (relAϪ, ppGppϪ). Total RNA was recovered, and the levels of specific transcripts were determined by nuclease protec- tion assay and densitometry (see Materials and Methods). Total RNA [5 ␮g (lanes 1–4) and 10 ␮g (lanes 5–8)] was hybridized simultaneously FIG. 4. Construct 78AL was grown to log phase in the absence or with probes for leuB, pyrD, and glpK mRNA. E. coli CP78 and CP79 presence of 1 mM IPTG (log) or was grown to log phase and was cells were grown to log phase (L) and then were washed and starved washed and transferred to minimal medium without arginine (Ϫarg) for leucine for 60 min (S). RNA markers 200–500 nt in length were run or leucine (Ϫleu), in the presence or absence of IPTG. Total RNA was in the last lane. The leuB hybridized fragment is 436 nt, pyrD is 313 nt, recovered, and the level of leuB transcript was determined by nuclease and glpK is 244 nt. protection assay and densitometry (see Materials and Methods). Downloaded by guest on October 3, 2021 Evolution: Wright et al. Proc. Natl. Acad. Sci. USA 96 (1999) 5093

Table 2. Comparisons between leuBϪ reversion rates and leuB pausing of RNA polymerase (21), which could increase the mRNA levels in CP78, CP79, and 78AL concentration of ssDNA and mutation rates at specific pause Cells Condition leuB mRNA* MR ϫ 10Ϫ9† sites. It is therefore interesting that an analysis of predicted Ϫ Ϯ mRNA folding (Mfold, GC Gene, University of Wisconsin) CP79 relA log 29 5 Not indicates that both the leuB and pyrD mutations occurred at applicable possible pause sites (data not shown). Somatic hypermutation CP79 relAϪ Leu-starved 17 Ϯ 5 0.22 Ϯ 0.1 ϩ Ϯ in mature B lymphocytes depends on transcription, and RNA CP78 relA log 30 5 Not polymerase pausing is thought to be a mechanism that could applicable ϩ Ϯ Ϯ determine the site of mutagenesis (50). CP78 relA Leu-starved 269 60 1.5 0.5 How Are Mutations Expressed in Nongrowing Cells? When 78AL‡ log 213 Ϯ 4 1.0 Ϯ 0.1 ϩ Ϯ Ϯ mutations occur at the onset of starvation, reversions on the 78AL log IPTG 1,275 175 2.7 0.5 transcribed strand would immediately be immortalized by the 78AL SP Ϫ Leu 63 Ϯ 4 0.9 Ϯ 0.1 Ϫ ϩ Ϯ Ϯ synthesis of functional enzyme and leucine, allowing cell 78AL SP Leu IPTG 333 32 1.6 0.5 division. If most mutations occur on the nontranscribed strand SP, stationary phase. and are never expressed, then the actual mutation rates are *pg leuB mRNA͞␮g total RNA. higher than calculated. Mutations on the nontranscribed Ϫ Ϫ †leuB mutation rates (MR) ϫ10 9 (see Materials and Methods). strand could be expressed, for example, by the stimulation of ‡ Lacks attenuation and ppGpp-binding sequences. DNA repair activities that attempt to repair the complemen- Ϫ ϩ tary position on the transcribed strand or by the induction of associated with uninducible regulatory systems (i o ) were ‘‘cross-strand deamination’’ (51). It is also possible that a not differentially influenced by IPTG. Transcription- damaged base on the nontranscribed strand will cause the enhanced mutations also have been shown for reversions of a Ϫ complementary base to become altered at a higher frequency lys frameshift allele in yeast (46); reversion rates of an than paired bases (52). Mutants defective in a variety of DNA uninducible lys allele and mutations to canavanine resistance repair functions should provide further insights into the mech- at an unrelated locus were not affected. The mutability of the anism of transcription-enhanced mutations. nontranscribed strand also was demonstrated (47) in a plasmid Are Stringent Response Mutations Specifically Enhanced? system in which a 4-fold increase in the frequency of transitions The extent to which starvation regimen-dependent mutation occurred selectively in the nontranscribed strand when tran- rates are exclusively increased by derepression and ppGpp scription was induced; for a number of reasons, this strand bias activation of a targeted operon appears to justify use of the could not be ascribed to transcription-coupled repair (48). In word ‘‘specific’’. The effect of leucine starvation in up- our view, these studies demonstrating enhanced mutation rates regulating leu operon transcription is specific in that starvation as a result of artificially induced transcription are all examples for other amino acids is not effective; in the arg operon, of specifically induced mutations. However, they have not been starvation for arginine but not histidine increases argH mRNA recognized as such, nor have they been related to transcription abundance (S. McAllister, personal communication). Genes induced by starvation (derepression), which is directly relevant down-regulated or not regulated during the stringent response to environmental stress and evolution. include pyrD and glpK (Fig. 3), ‘‘housekeeping’’ genes (53), and Up-Regulation of the leu Operon Requires ppGpp Activa- starvation regimen-independent genes involved in DNA, tion. When bacteria are starved for an amino acid, the rRNA, tRNA, nucleotide, phospholipid, and cell wall synthesis biosynthetic operon is derepressed; that is, it is no longer (17). Whether hypermutation specific to the leu operon is down-regulated by attenuation (thr, his, ilv, and leu), repression described as being induced, enhanced, or directed by leucine (arg), or both (trp and phe). However, in addition to derepres- starvation is arbitrary. The specificity of stringent response sion, our data indicate that the operon is always activated by mutations and their dependence on derepression (relA) re- ppGpp. At 15 min starvation, leu operon expression apparently cently has been observed by others in a Bacillus subtilus system depends on ppGpp activation because leucine starvation of the (54). Mutation rates in amino acid biosynthetic genes, but not ϩ Ϫ ϩ relA strain, but not the relA strain, enhanced leuB mRNA drug resistance genes, were enhanced in relA compared with levels (Fig. 2). Although leuB mRNA levels increased some- relAϪ strains under conditions that evoke the stringent re- what after 60 min of starvation in the ppGpp-deficient strain Ϫ sponse. (Fig. 3), this may be related to the fact that the relA allele is In our investigations, an increase in reversion rate of leuBϪ somewhat ‘‘leaky’’ (17). is taken as evidence of increased mutation rates also in the leu Operon Activation by IPTG in the Construct Is Less other (normal) genes of that operon. The reversion of an Effective than ppGpp Activation in the Parental Strain. Table auxotroph to prototrophy represents the restoration of a 2 compares the construct 78AL and the parental strains with Ϫ previously existing ability and therefore only serves as a model respect to leuB mRNA levels and leuB reversion rates. The for the analysis of mechanisms underlying ‘‘forward’’ muta- high basal transcription level of the uninduced tac promoter in tions required in the evolution of complex metabolic networks 78AL log cells is consistent with higher mRNA levels com- (55, 56). We suggest that the stringent response played, and pared with log cells of the parental strains. In growing cells, a continues to play, a role in optimizing functional genes in 6-fold increase in mRNA level induced by IPTG is apparently organisms confronted with novel stress conditions and in ‘‘required’’ for a 2- to 3-fold increase in mutation rate in 78AL. derepressing and mobilizing genes to serve in new biochemical During leucine starvation, IPTG also stimulates leuB mRNA pathways. Genetic derepression frequently is a prerequisite for levels. However, this is not sufficient to have a significant affect further mutations that modify existing genes, enabling them to on reversion rates under these conditions. IPTG induction use new substrates during acquisitive evolution (57). Ϫ previously has been observed to enhance lac reversion rates Unaware of the evidence supporting his hypothesis, Fitch in growing (33) but not starving cells (49). With respect to (58) speculated that transcribing genes might have higher affecting mutation rates, the normal starvation mechanism is mutation rates than other genes. The growth-dependent, ar- apparently more efficient than IPTG induction in the con- tificially induced, specifically enhanced mutations discussed struct (which lacks the ppGpp discriminator sequence). This earlier occurred under nonselective conditions and are there- suggests that ppGpp may be affecting the concentration of fore not relevant to environmental stress and evolution. How- ssDNA by some mechanism related to and in addition to its ever, stringent response mutations occur in response to im- effect on transcription. Work in progress concerns leuB mRNA pending starvation. In nature, bacteria likely starve simulta- turnover, as well as investigations into the effect of ppGpp on neously for many required metabolites; nutritional stress, gene Downloaded by guest on October 3, 2021 5094 Evolution: Wright et al. Proc. Natl. Acad. Sci. USA 96 (1999)

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