selection, adaptation, and bacterial operons

MOLECULAR BIOLOGY

OF NATURAL SELECTION

23.5. i Selection, adaptation, and bacterial operons

BARRY G. HALL Molecular and Cell Biology, U-44, University of Connecticut, Storrs, CT 06268, U. S. A.

Symposium Editor: D. A. Hickey

HALL, B. G. 1989. Selection, adaptation, and bacterial operons. Genome, 31: 265-271. Bacteria are especially useful as systems to study the molecular basis of adaptive evolution. Selection for novel metabolic

capabilities has allowed us to study the evolutionary potential of organisms and has shown that there are three major "strate- gies" for the evolution of new metabolic functions. ( i ) Regulatory mutations may allow a gene to be expressed under unusual conditions. If the product of that gene is already active toward a novel resource, then a regulatory mutation alone may confer a new metabolic capability. ( i i ) Structural gene mutations may alter the catalytic properties of enzymes so that they can act on novel substrates. These structural gene mutations may dramatically improve catalytic capabilities, and in some cases they can confer entirely new capabilities upon enzymes. In most cases both regulatory and structural gene mutations are required for the effective evolution of new metabolic functions. (iii) Operons that are normally silent, or cryptic, may be activated by either point mutations or by the action of mobile genetic elements. When activated, these operons can provide entirely new pathways for the metabolism of novel resources. Selection can also play a role in modulating the probability that a particular adaptive mutation will occur. In this paper I present evidence that a specific adaptive mutation, reversion of the metBl mutation, occurs 60 to 80 times more frequently during prolonged selection on plates under conditions where the members of the population are not growing than it does in growing cells under nonselective conditions. This selective condition, methionine starvation, does not increase the frequency of other mutations unrelated to methionine biosynthesis. Thus, con- trary to our present notions, selection can act not only to reveal preexisting mutations but to modulate the frequency with which adaptive mutations occur.

Key words: mutation rates, molecular evolution, adaptive mutations, cryptic genes.

HALL, B. G. 1989. Selection, adaptation, and bacterial operons. Genome, 31 : 265 -271. Les bactkries sont particulikrement utiles, en tant que systkmes, pour l'ktude de l'adaptation Cvolutive sur une base molkcu-

laire. La sklection en fonction de nouvelles potentialites mCtaboliques nous a permis d'Ctudier le potentiel Cvolutif des . organismes et rCvClC l'existence de trois stratkgies majeures de dkveloppement de nouvelles fonctions mktaboliques. ( i ) Des

mutations rkgulatrices peuvent permettre l'expression d'un gkne dans des conditions inhabituelles. Si le produit de ce gkne est dkj2 actif dans la production d'une nouvelle ressource, alors la mutation rkgulatrice peut, 2 elle seule, confCrer une nouvelle capacitk mktabolique. ( i i ) Des mutations structurales de gknes peuvent altCrer les propriCtCs catalytiques des enzymes et leur permettre d'agir sur de nouveaux substrats. Ces mutations peuvent amCliorer CnormCment les capacitCs catalytiques et, dans certains cas, leur confkrer de nouvelles capacitCs sur les enzymes. Dans la plupart des cas, les mutations gkniques, tant rkgulatrices que structurales, sont requises pour 1'Cvolution opCrationnelle de nouvelles fonctions mktaboliques. (iii) Les opCrons, qui sont normalement inactifs ou cryptiques, peuvent &re activks soit par des mutations ponctuelles, soit par l'action d'k1Cments gCnCtiques mobiles. S'ils sont activCs, ils peuvent fournir des sentiers tout 2 fait nouveaux pour le mktabolisme de nouvelles ressources. La sClection peut aussi jouer un r6le en modulant la probabilitC qu'une mutation d'adaptation particu- likre puisse survenir. L'Cvidence est ici prksentke qu'une mutation spkcifique d'adaptation, soit le reversion de la mutation metB1, survient de 60 2 80 fois plus frkquemment en cours d'une sklection prolongCe sur plaque dans des conditions o i ~ les membres de la population ne sont pas en croissance, par rapport 2 celle qui survient chez les cellules en croissance dans des conditions de non-sklection. Cette condition de sklection, soit l'kpuisement en mCthionine, n'augmente pas la frkquence d'autres mutations qui ne sont pas reliCes 2 la biosynthkse de la mCthionine. Donc, 2 l'opposC des notions actuelles, la sClec- tion peut agir non seulement pour rkvCler des mutations prC-existantes, mais aussi pour moduler la frCquence avec laquelle les mutations d'adaptation peuvent survenir.

Mots cle's : taux de mutations, Cvolution molkculaire, mutations adaptatives, gknes cryptiques. [Traduit par la revue]

Microorganisms were first noticed by Leeuwenhoek in 1676, and by 1780 they had become the object of serious scientific study (Clarke 1985). In 1881 Charles Darwin men- tioned a meeting at which bacteria were discussed in an evolutionary context (Freeman 1982). Aside from bacterial systematists, microorganisms managed to escape the attention of serious evolutionary biologists for roughly the next 80 years. This is somewhat understandable. Having neither fur, feathers, bones nor hooves, virtually no sex life, and more importantly, no fossil record, they were considered to offer little to evolutionists who were primarily concerned with mor- phological changes over time. The development of molecular Printed in Canada 1 Imprime au Canada

techniques in the early 1960s suddenly made bacteria amen- able to much more sophisticated phylogenetic analyses than had previously been possible, and by 1974 there was a suffi- ciently lively interest in microbial evolution that "Evolution in the Microbial World" was the topic of the 24th Symposium of the Society for the Study of General Microbiology (Carlile and Skehel 1974). At that time microbial evolution still lay well outside of the mainstream of evolutionary biology and was still very much the domain of scientists whose primary interests were microbiology, microbial physiology, and biochemistry. The proceedings of that symposium (Carlile and Skehel 1974) show that, while most of the interest was in

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266 GENOME, VOL. 31. 1989

phylogenies and in historical evolution, several papers discussed microorganisms as models for studying evolution dynamically. A decade later microorganisms had become accepted as legitimate subjects of interest for evolutionary biologists. One enormously fruitful area has been the molecular systematics of bacteria. Using primarily RNA sequence data, microbial evolutionary trees have been constructed and have been integrated with trees of the less well developed (and much less interesting) organisms known as Eukaryotes. Another area that has been particularly rewarding has been the direct exploitation of microorganisms as model systems for studying evolutionary processes dynamically, i.e., monitoring evolution as it occurs in the laboratory.

Experimental evolution One means by which an organism can dramatically improve

its fitness is to acquire the ability to exploit an ecological niche that is unavailable to other members of its species. For bacteria that exploitation usually means evolving the ability to metabolize some novel resource in the environment, a resource that is not available to the wild type population. Because bacteria reproduce rapidly and because we can manipulate the resources in bacterial environments in the laboratory so precisely, we can use the evolution of new cata- bolic capabilities in bacteria as a model for the evolution of new metabolic functions in general.

The problem then is, How do fully functional organisms that are already well adapted to their environments evolve new functions that enable them to exploit novel resources in the environment? The standard strategy for investigation of this question is to apply a strong selective pressure to a population of a well-characterized bacterial strain so that it evolves the ability to grow on the novel resource, and then to determine what genetic changes occurred to generate the new capability.

The results of over 20 years of such experiments in a number of laboratories have shown that there are three major ways by which such new functions evolve, and I shall discuss examples of each below. More detailed discussions of these and other systems can be found in the book Microorganisms as Model System for Studying Evolution edited by R. P. Mort- lock (1984).

Regulatory mutations Although enzymes have a reputation for being highly sub-

strate specific, they are not completely specific or biochemists wouldn't have such a plethora of substrate analogs with which to work. It is often the case that an organism already possesses an enzyme necessary to utilize a novel substrate, but that the novel substrate fails to induce synthesis of the enzyme. When that is the case, selection for growth on the novel substrate results in the selection of regulatory mutants in which the properties of the relevant enzyme are unchanged. The only alteration is in the circumstances under which the gene encoding the enzyme is expressed.

A case in point is the evolution of D-arabinose utilization in Klebsiella pneumoniae and in Escherichia coli. D-Arabinose is found rarely in nature (Mortlock 1984) and is utilized by neither Klebsiella pneumoniae nor Escherichia coli. Mortlock and Wood (1964) selected spontaneous mutants of Klebsiella pneumoniae that could grow on D-arabinose, and Camyre and Mortlock (1965) showed that the mutants constitutively synthesized an enzyme that could isomerize D-arabinose to

D-ribulose. They then showed that D-arabinose isomerase was, in fact, the L-fucose isomerase that is part of the normal L-fucose metabolic pathway. D-Arabinose and L-fucose are structurally very similar, and the L-fucose isomerase effi- ciently converts D-arabinose to D-ribulose. The resulting D-ribulose is metabolized further by enzymes of the ribitol pathway, in which ribulose is both an intermediate and an inducer of the genes for that pathway (Fig. 1). The mutation that permitted D-arabinose utilization was simply a mutation that led to constitutive expression of the L-fucose operon.

In Escherichia coli the situation is slightly different. Again, D-arabinose positive mutants turned out to use the L-fucose isomerase to convert D-arabinose to D-ribulose (LeBlanc and Mortlock 1971a, 1971b, 1972), but in this case the mutants did not express the L-fucose operon constitutively. Instead, as a result of a mutation in a regulatory gene, the L-fucose operon in the mutant strain was now inducible by D-arabinose. Escherichia coli does not have a ribotol operon, and thus could not use ribitol dehydrogenase to further metabolize the resulting D-ribulose. Instead, the second enzyme in the L-fucose pathway, L-fuculokinase, acted to phosphorylate the D-ribulose and permit its entry into central metabolism (Fig. 1).

These two examples illustrate how simple regulatory mutations can generate a new metabolic capability by exploiting existing enzyme activities, sometimes involving more than one pathway.

Structural gene mutations A second means for generating new metabolic capabilities

involves mutations that alter the catalytic properties of enzymes. An example of this evolutionary process is provided by the EBG system of Escherichia coli.

When the lacZ gene, encoding P-galactosidase, is deleted, selection for growth on P-galactoside sugars yields mutations in the EBG operon that is located on the opposite side of the chromosome from the LAC operon (Campbell et al. 1973; Hall and Hart1 1974, 1975). Although evolution of this operon does involve regulatory mutations in addition to structural gene mutations, the example below focuses only on those mutations that change the properties of the EBG enzyme itself, and all of the strains in this example synthesize EBG enzyme constitutively. These results are described in detail in Hall (1978, 1981) and Hall and Clarke (1977).

Wild type EBG enzyme is a poor 6-galactosidase and does not permit utilization of lactose (galactosyl-glucose), lactulose (galactosyl-fructose), galactosyl-D-arabinose, or lactobionate (galactosyl-gluconic acid). There is detectable activity toward all substrates except lactobionate, but the activity is insufficient to permit growth (Table 1).

A spontaneous mutant selected for growth on lactose, strain A2, synthesizes an altered enzyme with greatly enhanced activity toward lactose, but with activity toward the other substrates virtually unchanged (Table 1).

A spontaneous mutant of strain A2, strain A27, was selected for growth on lactulose. As a result of a second mutation in the structural gene, the enzyme was altered so that its activity toward both lactulose and galactosyl-arabinose was dramatically increased (Table 1). At this stage activity toward lactobionate was detected, but that activity was insufficient for growth.

A third round of selection generated strain A271, selected for growth on lactobionate. Again, the properties of the enzyme were altered so that activity toward lactobionate was enhanced. Notice that this improvement was at the expense of

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ribitol D-arabinose L-f ucose

metabolic

ribitol dehydrogenase

FIG. 1. Pathways for metabolism of D-arabinose in mutants of E. coli and Klebsiella. Enzymes are shown within boxes. The ribitol pathway is present only in Klebsiella, the fucose pathway is present in both organisms.

L-fucose isomerase

TABLE 1. 6-Galactosidase activity (A)* and growth rate (GR)? of four E. coli strains on 6-galactoside sugars

Galactosyl- Lactose Lactulose arabinose Lactobionate

Strain Mutations A GR A GR A GR A GR

In Klebsiella 1 1

D-ri bulose L-fuculose

1B1 None 89 0.00 33 0.00 15 0.00 n.d. 0.00 A2 One 2230 0.45 34 0.00 175 0.03 n.d. 0.00 A27 Two 1651 0.42 336 0.24 716 0.16 7 0.00 A271 Three 574 0.36 170 0.12 291 0.12 330 0.10

*Activity is expressed as nmol pure enzyme hydrolyzed . min-' . mg-' at physiological (25 mM) substrate concentration.

tGrowth rate is p in h-'.

a reduction in activity toward some of the other substrates, and that this reduction led to a decreased growth rate on those substrates.

A consistent observation throughout this study was that if even a small amount of activity toward a substrate could be detected in the purified enzyme, then it was possible to select a mutant in which that activity had been improved to a point where it permitted growth on that substrate.

Activation of cryptic genes

The third major means by which a microorganism can acquire a new metabolic capability is by the activation of a normally silent, or cryptic, gene.

Cryptic genes are defined as genes that are normally silent

in wild type members of the population, but which can be activated by genetic events that may include point mutations, genetic rearrangements, or the transposition of mobile genetic elements (Hall et al. 1983). How do we know when selection for a novel function has resulted in the activation of a cryptic gene? When biochemical and genetic analyses show that the novel function actually requires the expression of more than one gene (i.e., a transport and a hydrolysis function, or two enzymes that catalyze sequential steps in a pathway), the possibility of cryptic gene activation should be considered. If further investigation shows that expression of the required genes can not be induced in the wild type strain, either by the novel substrate or by chemically related compounds that are normally metabolized by the organisms, then it is highly likely that a cryptic operon has been activated. Even when careful

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268 GENOME, VOL. 31, 1989

investigation fails to detect any conditions under which the gene is expressed in the wild type strain, the possibility that it can be induced in natural environments can rarely be completely excluded. In some cases, however, the nature of the activating mutations as determined by the DNA sequence level make it appear highly unlikely that the gene is ever expressed at a physiologically meaningful level in the absence of a mutation (Hall et al. 1983).

The problem that is posed by the existence of cryptic genes is that, being silent, they make no positive contribution to the fitness of the organism, thus mutations in these genes are expected to be neutral. Since neutral mutations accumulate linearly with time, the structural genes in a cryptic operon are expected to accumulate mutations that will eventually lead to their irreversible inactivation. If these is a gene that is silent in the great majority of members of a population, it is expected that it would be impossible to reactivate the gene by spontaneous mutations in most members of that population. In contrast to this expectation, a survey of natural E. coli isolates has shown that cryptic genes for (3-glucoside utilization can be activated by spontaneous mutations in over 90 % of the isolates tested (Hall and Betts 1987). How do silent genes avoid random mutational inactivation? We have proposed that cryptic genes are maintained in bacterial populations by alternation between "normal" environments, in which there is selection against expression of the gene, and rarely encountered alternative environments in which there is selection favoring expression of the gene (Hall et al. 1983). According to this model, when the population is in the normal environment random mutations do accumulate in silent genes, and in some members the genes are irreversibly damaged. When the environment changes to one favoring expression, then there is selection for the rare mutants in which undamaged genes are activated, and those members that are unable to activate the gene are dis- placed from the population. When the environment returns to normal there is selection for mutations that silence the gene, and the cycle begins again. This model has been strongly supported by a theoretical study conducted by Li (1984).

The cryptic cellobiose utilization operon of E. coli More than 99 % of wild type E. coli isolated from nature are

unable to utilize cellobiose as a carbon and energy source (Hall and Faunce 1987), and the laboratory strain E. coli K12 is typical in this respect. We have isolated over 100 spontaneous mutants of E. coli K12 that utilize cellobiose and have investigated the structure and functions of the genes responsible for this new capability. The genes responsible for cellobiose utilization are organized as an operon, the cel operon, located at 38 min on the E. coli K12 map (Kricker and Hall 1984, 1987). Expression of the cel operon is undetectable in wild type strains, either by activity of the gene products (Kricker and Hall 1987) or by detection of cel operon mRNA (Hall et al. 1986). When activated the operon encodes a cellobiose transport gene (celB) whose product both transports and phos- phorylates cellobiose, and a hydrolase gene (celF) whose product hydrolyzes phosphorylated cellobiose (Kricker and Hall 1987). Cloning and sequencing of the cel operon has permitted unambiguous identification of these genes and, in addition, two open reading frames whose functions have not yet been determined (L. Parker and B. G . Hall, unpublished results).

The sequencing studies have also shown that the operon can be activated by two different kinds of spontaneous mutations.

(i) The mobile genetic elements IS 1, IS2, or IS5 can insert into a region about 200 bp upstream of the operon. In none of these cases does the insertion sequence provide an outward reading promoter in the orientation in which it activates the cel operon. (ii) The operon can be activated by "point" mutations. The sites of these mutations have not yet been determined, but they do not involve DNA rearrangements that can be detected by restriction analysis (L. Parker and B. G . Hall, unpublished results).

Screening the 72 natural isolates that constitute the E. coli Reference Collection (ECOR collection) (Ochman and Selander 1984) showed that none of the strains could utilize cellobiose, but that over 50% could spontaneously mutate to cellobiose utilization (Hall and Betts 1987). Some, but not all, of the cellobiose-positive mutants of that collection expressed the cel operon as judged by mRNA hybridization, demonstrating that there are at least two sets of cryptic cellobiose utilization genes extant within the E. coli population.

A deliberate screening for cellobiose-positive E. coli showed that 0.1 -0.5 % of natural isolates are cellobiose positive, but that none of these expressed the cel operon (Hall and Faunce 1987).

A central issue is whether cryptic genes really are silent under natural conditions, or whether they are instead expressed under some circumstances and thus contribute to the fitness of the organism. If the genes are truly cryptic they are expected to accumulate mutations, whereas if the genes are actually expressed then mutations are expected to be removed by purifying selection. Many mutations that do not completely inactivate a gene will nonetheless result in the product being less thermostable, thus rendering the function temperature sensitive. Thus, if the genes are actually cryptic it is to be expected that, on average, the decryptified cellobiose-positive mutants would be more temperature sensitive for growth on cellobiose than would strain that are cellobiose positive when isolated from nature, and thus expected to be subject to purifying selection.

The growth rates of six "decryptified" cellobiose-positive mutants and eight "naturally functional" cellobiose-positive mutants were determined at 30, 37, and 42°C on cellobiose and, as a control, on glucose. None of the strains exhibited any temperature sensitivity for growth on glucose. On cellobiose, all of the decryptified strains grew slower at 37 than at 30°C, and none was able to grow at 42°C. In contrast, all of the "naturally functional" strains grew well at all three tempera- tures (Hall and Faunce 1987). This provides direct evidence that strongly supports the notion that the cellobiose utilization genes are indeed cryptic, and not under selection, in the majority of E. coli isolates.

A critical part of the model for retention of cryptic genes is the hypothesis that there are conditions that select against the functional (expressed) allele and thus favor cryptic and nonfunctional (irreversibly inactivated) alleles. We have demon- strated that such conditions exist with respect to the cel operon. In the process of characterizing cel operon clones, we observed that plasmids which bore insertionally inactivated celF (cellobiose-PO4 hydrolase) genes rendered the host strain sensitive to cellobiose as a growth inhibitor. We reasoned that, in the absence of the hydrolase activity, the cells accumulated cellobiose phosphate to toxic levels. If cellobiose phosphate could act as an inhibitor, we wondered whether it might also inhibit cellobiose positive strains. The growth rates of five independent cellobiose-positive mutants of E. coli K12 were

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HALL 269

determined by glycerol, on cellobiose, and on a mixture of cellobiose and glycerol. In every case the Cel+ mutants grew slower on the mixture than they did on either carbon source alone. The presence of cellobiose reduced the growth rate on glycerol by as much as 16-fold. In contrast, the wild type (cellobiose-negative) strain grew as fast on glycerol + cellobiose as it did on glycerol alone.

In a mixed resource environment (glycerol + cellobiose) cryptic and nonfunctional cel alleles are thus at a strong selective advantage and would outcompete decryptified Cel+ mutants that arose. This would be comparable to normal environmental conditions in which mixed resources would be the rule. Under the unusual conditions in which cellobiose was the only (or perhaps just the best) carbon resource in the environment, decryptified Cel+ mutants would, in contrast, be at a strong selective advantage. Thus this critical aspect of the model for cryptic gene retention appears to be realistic.

Selection and mutation Whichever strategy is used, the first event in the acquisition

of a new metabolic function is the occurrence of a mutation. One of the basic assumptions underlying our understanding of evolutionary processes is the assumption that spontaneous mutations arise randomly and continuously, and that the only role played by selection is to increase or decrease the frequency of the mutation in subsequent generations. While there is abundant evidence that mutations do arise in the absence of selection (Luria and Delbriick 1943); Cairns et al. (1988) have recently pointed out that none of the classical experiments ruled out the possibility that the alternative also occurs, namely that the selective conditions cause some mutations to arise. Shapiro (1984), Cairns et al. (1988), and Hall (1988) have provided examples of transposon-mediated mutations that occur only when they are advantageous, and do not occur at detectable levels in growing cultures of E. coli. I have now investigated the mutation rate for reversion of a classical mutation, the metBl mutation, under selective and nonselective conditions. The results suggest that the mutation rate from metBl to metB+ is increased 50-fold under strong selective conditions.

Mutation rates are classically determined using the Luria- Delbriick fluctuation test method (Luria and Delbriick 1943). In such experiments a large number of cultures are each started from a few cells and allowed to grow up to a known final cell density. The entirety of each culture is then plated under selective conditions, and the number of mutant cells in each culture is determined from the number of colonies on the selective plate. The number of mutants per culture is greater than the number of mutations that occurred during the growth of that culture because mutants that arose prior to the last generation of growth were able to divide one or more times. The average number of mutations per culture (m) is best estimated from the fraction of cultures in which no mutants were present (Po) as m = -In (Po), and the mutation rate is expressed as p = m ln2lnumber of cells per culture and is expressed as mutations per cell division. This method mea- sures the mutation rate in growing cultures.

The metBl mutation, which causes a requirement for methionine, was one of the earliest mutations isolated in the study of E. coli genetics (Gray and Tatum 1944; Bachmann 1987b). It is widely known as a particularly stable mutation, its product produces no detectable enzyme activity (Johnson

et al. 1977), and because it was induced by X-ray mutagenesis it is thought by many to be a deletion. To the best of my knowledge there is only one report in the literature of a reversion of the metBl mutation (Garen and Levinthal 1960), and the conditions under which that reversion was obtained are not described.

To measure the reversion rate of metBl 110 independent cultures of strain ~ 3 4 2 L D (HfrC, metB1, AlacZ4680) were grown to an average density of 5.9 x lo9 cells per culture, and each culture was plated onto glucose mineral salts medium without methionine. Of the 1 10 plates, 96 had no colonies and the mutation rate during growth is thus estimated as 1.6 x 10- per cell division. This is roughly an order of magnitude lower than the average mutation rate per base.

To measure the mutation rate under selective conditions cells were inoculated onto the surface of medium containing a very low (5 pM) concentration of methionine. That concentration of methionine limits growth to 3 x lo7 cells per colony. Instead of casting the plates in traditional Petri dishes, the medium was distributed to the wells of micro-titer plates at 1 mL per well, and each well was inoculated so that it contained a single colony. This permitted me to resuspend a number of individual colonies in order to estimate the number of cells per colony, as well as to keep exact track of colonies in which a mutation to metB+ occurred.

These small colonies were observed daily under a dissecting microscope, and metB+ revertants were detected as outgrowths on the surface of the colonies. In each case where a metB+ revertant was detected, it completely overgrew the original colony within 3 days.

Although no further growth of the original colonies occurred after the 2nd day, the first metB+ revertant was not detected until 6 days after inoculation. During the subsequent 5 days an additional five revertants were detected, after which no additional revertants were detected for the remainder of the experiment, another 10 days.

The data from the above experiment can be treated exactly as for a fluctuation test. Of ,the 331 colonies containing 3 x lo7 cells per colony, six produced revertants. The mutation rate that is thus estimated is 8 x 10-lo per cell.

In order to be sure that the revertants were not con- taminants, all of the metB+ revertants that were detected were isolated, purified, and tested for the presence of the AlacZ and for the ability to act as donors in genetic crosses (HfrC). All proved to have the expected genotypes.

Considering the origin of the metBl mutation (X-ray mutagenesis) and the rarity of reversion, it was necessary to con- sider two possible origins for the revertants that had been obtained. The revertants could either have been true metB+ revertants, or they could have been the result of activation of a cryptic gene. To distinguish these possibilities, I chose five of the revertants that had been isolated as outgrowths from methionine limited colonies, and compared them with strain XMT1, an isogenic metB+ strain that had been constructed by transducing the wild type allele into strain ~342LD. Growth rates of all six strains were measured in glucose minimal medium without methionine, and, on average, the revertants grew 93% as fast as did the wild type strain. Next, all six strains were mated with strain AB356 (argH, metB1, his, rpsL). argH is tightly linked to metB (Bachmann 1987a), and when argH+ rpsL recombinants were selected the wild type strain XMT 1 showed 12.6 % recombination between argH and metB. The five revertants showed 13.3 + 2%

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270 GENOME, VOL. 31, 1989

recombination between argH and the gene that conferred methionine prototrophy, showing that indeed the revertants were metB+. Taken together these data indicate that methionine prototrophy resulted from reversion of the metBl mutation, and not from activation of a cryptic gene.

These two experiments show that the mutation rate for metBl to metB+ is 80-fold higher in aged colonies on medium depleted of methionine than it is in growing cultures. Does this mean that these selective conditions are generally mutagenic? To determine this I measured the mutation rate to an unrelated phenotype, valine resistance, under the same two conditions.

Growth of E. coli K12 is inhibited by valine which acts as a false feedback inhibitor of isoleucine biosynthesis, and mutations at several loci can overcome this inhibition. In growing cultures of strain ~ 3 4 2 L D the mutation rate to valine resistance is 2.1 x per cell division as determined from a Luria-Delbriick fluctuation test (Hall 1988).

To measure the mutation rate in aged colonies, I repeated the experiment in which colonies were grown on methionine limited medium. In this experiment five metB+ revertants were obtained from 235 colonies that contained an average of 3.4 x lo7 cells per colony, yielding an estimated reversion rate of 6 x 10-lo. Again, the first revertant appeared when the colonies were 6 days old. Eleven days after inoculation, colonies from 60 wells were resuspended and plated onto medium containing both methionine and a high concentration of valine. At that point the average number of cells per colony was 1.4 x lo7. Four of the plates contained no valine resis- tant mutants, leading to an estimated mutation rate of 1.3 x

a value that is indistinguishable from that obtained in growing cells.

.These results indicate that the selective conditions were not generally mutagenic. The mutation rate was increased 60- to 80-fold only with respect to a mutation that would be strongly advantageous under the conditions employed.

We now have a sufficient number of examples (Shapiro 1984; Cairns et al. 1988; Hall 1988; these results) in which the probability of a specifically advantageous mutation was modulated by the selective conditions that we must begin to seriously question the paradigm that mutations arise randomly. The assumption of randomness underlies almost all evolutionary thought. For instance, mutational distance between two sequences is taken as a measure of the time since the sequences diverged. If the probabilities of particular mutations occurring are subject to this sort of environmental modulation, then the number of observed differences between two sequences may be completely unrelated to the time since they diverged, and may instead simply reflect the variability in environmental conditions encountered by the species from which the sequences were obtained. Although it is very diffi- cult to imagine mechanisms that might permit selective conditions to modulate the frequencies of specific adaptive mutations, it is clear that such a feature would be strongly advantageous. While we can not yet rule out the possibility that these diverse observations are the result of some consistent artifact, the issues raised by the studies warrant thorough and systematic investigation.

Acknowledgement This study was supported by U.S. Public Health Service

grant GM37 1 10.

BACHMANN, B. J. 1987a. Linkage map of Escherichia coli K 12. 7th ed. In Escherichia coli and Salmonella typhimurium. Cellular and molecular biology. Edited by F. C. Neidhardt. American Society for Microbiology, Washington, D.C. pp. 807 - 876.

1987b. Derivations and genotypes of some mutant deriva- tives of Eschericia coli K 12. In Escherichia coli and Salmonella typhimurium. Cellular and molecular biology. Edited by F. C . Neidhart. American Society for Microbiology, Washington, D .C . pp. 1190- 1219.

CAIRNS, J., OVERBAUGH, J., and MILLER, S. 1988. The origin of mutants. Nature (London), 335: 142 - 145.

CAMPBELL, J., LENGYEL, J., and LANGRIDGE, J. 1973. Evolution of a second gene for 0-galactosidase in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 70: 1841 - 1845.

CAMYRE, K. P., and MORTLOCK, R. P. 1965. Growth of Aerobacter aerogenes on D-arabinose and L-xylose. J. Bacteriol. 90: 1157- 1158.

CARLILE, M. J., and SKEHEL, J. J. (Editors). 1974. Evolution in the microbial world. Cambridge University Press, London.

CLARKE, P. H. 1985. The scientific study of bacteria. In Bacteria in nature. Vol. 1 . Edited by E. R. Leadbetter and J. S. Poindexter. Plenum Press, New York. pp. 1770- 1980.

FREEMAN, R. B. 1982. Darwin and Gower Street. Exhibition, 19 April 1982, Catalogue item 14, University College, London.

GAREN, A., and LEVINTHAL, C. 1960. A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli. I. Purification and characterization of alkaline phosphatase. Biochim. Biophys. Acta, 38: 470-483.

GRAY, C. H., and TATUM, E. L. 1944. X-ray induced growth factor requirements in bacteria. Proc. Natl. Acad. Sci. U.S.A. 30: 404-410.

HALL, B. G. 1978. Experimental evolution of a new enzymatic function. 11. Evolution of multiple functions for EBG enzyme in E. coli. Genetics, 89: 453 -465.

1981. Changes in the substrate specificities of an enzyme during directed evolution of new functions. Biochemistry, 20: 4042 -4049.

1988. Adaptive evolution that requires multiple spontaneous mutations. I. Mutations mediated by insertion sequences. Genetics, 120: 887 - 897.

HALL, B. G., and BETTS, P. W. 1987. Cryptic genes for cellobiose utilization in natural isolates of Escherichia coli. Genetics, 115: 431 -439.

HALL, B. G., and CLARKE, N. D. 1977. Regulation of newly evolved enzymes. 111. Evolution of the ebg repressor during selection for enhanced lactase activity. Genetics, 85: 193 - 20 1 .

HALL, B. G., and FAUNCE 111, W. 1987. Functional genes for cellobiose utilization in natural isolates of Escherichia coli. j. Bacteriol. 169: 2713-2717.

HALL, B. G., and HARTL, D. L. 1974. Regulation of newly evolved enzymes. I. Selection of a novel lactase regulated by lactose in Escherichia coli. Genetics, 76: 391 -400.

1975. Regulation of newly evolved enzymes. 11. The ebg repressor. Genetics, 81: 427 -435.

HALL, B. G., YOKOYAMA, S., and CALHOUN, D. H. 1983. Role of cryptic genes in microbial evolution. Mol. Biol. Evol. 1: 109- 124.

HALL, B. G., BETTS, P. W., and KRICKER, M. 1986. Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state. Mol. Biol. Evol. 3: 389-402.

JOHNSON, J. R., GREENE, R. C., and KRUEGE, J. H. 1977. Isolation and characterization of a specialized lambda transducing bacterio- phage carrying the metBJF methionine gene cluster. J. Bacteriol. 131: 795 -800.

KRICKER, M., and HALL, B. G. 1984. Directed evolution of cellobiose utilization in E. coli K- 12. Mol. Biol. Evol. 1 : 17 1 - 182.

1987. Biochemical genetics of the cryptic gene system for cellobiose utilization in Escherichia coli K- 12. Genetics, 115: 419-429.

Gen

ome

Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

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com

by

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IV O

F N

OR

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14Fo

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HALL 27 1

LEBLANC, D. J., and MORTLOCK, R. P. 1971a. Metabolism of D-arabinose: Origin of a D-ribulokinase activity in Escherichia coli. J. Bacteriol. 106: 82 -89.

1971b. Metabolism of D-arabinose: a new pathway in Escherichia coli. J . Bacteriol. 106: 90 - 96.

1972. Metabolism of D-arabinose: alternate kinases for the phosphorylation of D-ribulose in Escherichia coli and in Aerobac- ter aerogenes. Arch. Biochem. Biophys. 156: 390 - 396.

LI, W.-H. 1984. Retention of cryptic genes in microbial populations. Mol. Biol. Evol. 1: 212-218.

LURIA, S. E., and DELBR~~CK, M. 1943. Mutations of bacteria from virus sensitivity to virus resistance. Genetics, 28: 491 -5 1 1 .

MORTLOCK, R. P. (Editor). 1984. Microorganisms as model systems for studying evolution. Plenum Press, New York.

MORTLOCK, R. P., and WOOD, W. A. 1964. Metabolism of pentoses and pentitols by Aerobacter aerogenes. I. Demonstration of pen- tose isomerase, pentulokinase, and pnetitol dehydrogenase enzyme families. J. Bacteriol. 88: 838 - 844.

OCHMAN, H., and SELANDER, R. K. 1984. Standard reference strains of Escherichia coli from natural populations. J. Bacteriol. 157: 690 - 693.

SHAPIRO, J. A. 1984. Observations on the formation of clones containing araB-lac2 cistron fusions. Mol. Gen. Genet. 194: 79 - 90.

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selection, adaptation, and bacterial operons

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