Introduction The story of my favorite molecule, ethanol aside, is inextricably interwoven with that of one of my favorite scientists, Professor Daniel Hartl. Dan Hartl and I met when I was an undergraduate in Allen Fox’s Drosophila lab at the University of Wisconsin, and Dan was a graduate student in Jim Crow’s population genetics laboratory next door. We finished our respective degrees, B.S. and Ph.D., in June of 1968 and went our separate ways but kept in touch. Two years later, while I was a graduate student studying E. coli genetics, I visited Dan at the University of Minnesota where he had just taken an Assistant Professorship. Over several drinks we bemoaned the state of evolutionary biology and wished that somehow the experimental power of E. coli could be brought to bear on evolutionary problems so that we could study the process of evolution dynamically. In 1972 we met, quite by chance, at a U.C.L.A. Lake Arrowhead meeting where John Campbell was presenting his finding that a strain of E. coli that was deleted for the l a c 2 (bgalactosidase) gene could give rise to spontaneous lactose-utilizing mutants. John called the gene responsible for lactose utilization ebg, standing for evolved 8-galactosidase, and it was immediately apparent that his system could be a powerful tool for studying the evolution of new metabolic functions. After a discussion that lasted from early evening until about 6:OO AM, and that involved the consumption of serious quantities of my truly favorite molecule, John, Dan and I agreed to a threeway collaboration, and the following year I transferred my post-doctoral fellowship to Dan Hartl’s lab in Minnesota. We were interested in understanding the evolutionary potential that lay within the genome of an organism, and in the processes and mechanisms by which the organism could exploit that potential to adapt to new demands and selective pressures. The general question that I wanted to answer was, ‘Exactly what information about an organism is required in order to predict the probability that it will be able to evolve a specific new function?’ At that time the lac2 gene, encoding /3galactosidase, and its regulatory gene, l a d , were still the focus of intensive study as a model for regulation of gene expression. We could therefore ask, ‘When this gene for degrading lactose is deleted, exactly how does
E. coli go about reinventing the /$galactosidase function?’, and have some hope that the answer might be of interest both to the molecular and evolutionary communities. Evolutionary biologists are usually forced to infer a historical process by examining the present day outcome of that process, a very unsatisfactory approach to understanding any dynamic process. With that in mind, several studies, primarily in Drosophila melanogaster, sought to study the adaptive process experimentally, rather than retrospectively. In general those studies involved selection for complex traits in populations that were genetically heterogeneous. Enhancement of the selected phenotype was often observed, but it was concluded that it was based on preexisting variation that was already in the population, and the complexity of the selected traits made interpretation of the results frustrating in any but the broadest terms. We hoped that by starting with a single, clonal, genetically well understood organism, and applying a specific selection pressure, under carefully defined conditions, to a biochemically well understood pathway that we would be able experimentally to demonstrate the operation of those mechanisms that have produced the enormous array of highly adapted organisms we see today. Because of the simplicity of the system we hoped to be able to look at a level of detail (biochemical, molecular, regulatory, etc.) that was not possible in more complex systems. Discovery of ebg John Campbell(’) had isolated the first ebg mutant by starting with an E. coli K12 strain that was deleted for the entire lac operon, but which carried the (constitutively expressed) lactose permease (lacY) gene on an F’ plasmid. The starting strain was allowed to form colonies on an indicator medium that permits growth at the expense of small peptides, and which included lactose (which the cells could transport, but not hydrolyze) and a color indicator for lactose fermentation. After several weeks, papillae, or outgrowths, appeared on the surface of some colonies, and the color of these papillae indicated that they were weakly fermenting lactose. Cells from one papilla were restreaked on the same medium, and allowed to form colonies which subsequently produced more papillae. After five rounds of such selection Campbell isolated the mutant that he designated ebg-5; the number ‘5’ reflected his assumption that each round of selection had produced an additional mutation which improved growth on lactose. The ebg-5 strain grew well on lactose, but not as well as a wild type lacz+ strain. Campbell el al. showed that the ebg-5 mutant constitutively synthesized a /3-galactosidase with biochemical and immunological properties that were completely distinct from those of the classical lac2 encoded pgalactosidase. A particularly interesting observation was that the ebg 8-galactosidase was incapable of converting lactose into an inducer of the lac operon. BioEssays
Vol. 12, No. 11 - November 1990
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Therefore, in later experiments, when the lacy gene was under control of the lacl encoded repressor it was necessary to include the lac operon inducer IPTG in the medium if the cells were to transport lactose. They mapped the ebg gene to a position near tolC on the opposite side of the chromosome from the lac operon. Subsequent genetic and physical mapping studies have confirmed that mapping, with the gene placed at 68.2 min on the current genetic and physical maps(23’).
Development of ebg as a Tool for Studying Evolutionary Processes When Hartl and I initiated our studies we began with a distantly related E . coli K12 strain, strain DS4680A, that was deleted for the middle 1/3 of the lacZ gene, but that was both l a d + and was lacy+. We had two immediate questions in mind: How many different genes could mutate so as to evolve pgalactosidase activity?; and, What were the properties of the intermediate-step ebg isolates, equivalent to the postulated ebg-1 through ebg-4 mutants which Campbell had not retained? The first question has been thoroughly answered over the course of the last 17 years, both in my lab and in Hartl’s lab: no genes other than ebg have ever been observed to give rise to lactose-utilizing mutants. Deletion of the ebg gene results in a complete inability to mutate to lactose utilization, thus E. coli has a very limited potential, in this sense, for evolving this particular new function. The answer to the second question emerged over the course of the next two years. In my first study(‘), using the same papillae method as that used by Campbell et al., I isolated 34 independent ebg mutants, three of which were indistinguishable from Campbell’s ebg-5 mutant. Since all of these were isolated in a single round of selection, we began to suspect that the ebg-5 mutant did not necessarily involve five separate mutations. The remaining 31 mutants grew much more slowly than the ebg-5-like mutants, and they proved to differ only in their regulation of ebg enzyme synthesis: they were inducible by lactose. The enzyme produced by the inducible mutants was indistinguishable from that synthesized by the three constitutive strains and by Campbell’s ebg-5 strain. The slower growth resulted from a lower level of induced than constitutive synthesis, a common property of regulated enzyme systems. Campbell had believed that the ebg-5 encoded enzyme was the result of structural gene mutations in a gene that was expressed constitutively in the original AZacZ strain. The finding that the majority of ebgt mutants were inducible led us to suspect that the ‘unevolved’ or ancestral gene might also be inducible by lactose. (‘Ancestral’, in this context, means the allele that was present in the AlacZ strain that we started with, not a gene that was present in an ancestor of E. coli.) We showed that in strain DS4680A, and in other
AlacZ strains, ebg enzyme activity was inducible, but that the fully induced level of activity was insufficient for growth on lactose(’). At this point, I accepted an Assistant Professorship at the Memorial University of Newfoundland, where further work showed that the gene encoding for ebg enzyme, designated ebgA, was under negative control by a repressor that was the product of the adjacent ebgR gene(6). Physical studied7), completed some 14 years later, confirmed and extended the early genetic conclusions(6,8): the EBG gene cluster consists of a repressor gene, ebgR, that is adjacent to an operon consisting of at least three structural genes; ebgA and ebgC, which encode the 118 kilodalton a subunit and the 20 kilodalton /3 subunit of ebg enzyme, and ebgB, which encodes a 67 kilodalton protein of unknown function. In 1975 enough was known about the state of the wild-type, or ‘ancestral’ ebg operon to permit the pursuit of fundamental questions concerning the evolution of lactose utilization via the ebg genes. The most fundamental question was, ‘Just how long is the pathway for the evolution of lactose utilization, or, in other words, how many steps are required?’ Campbell thought that five structural (ebg enzyme encoding) gene mutations were required for maximal growth, but the finding that 90 % of the Ebg+ (lactose utilizing) mutants were inducible suggested that the pathway might be as short as one structural mutation for slow growth, with a second mutation to ebgR- (constitutive) expression for maximal growth. From the wild-type stain I isolated an ebgR- mutant that synthesized the ancestral enzyme, designated ebg’ for ‘original’, constitutively. Although the mutant synthesized 5 % of its soluble protein as ebg enzyme, it was incapable of growth on lactose. Using that strain I measured the spontaneous mutation rate to Ebg+ as 2xlOP9 per cell division, a rate that was consistent with a single difference between unevolved (ebg’) and evolved (ebg+) enzyme(’). Since the single mutant ebg+ strains grew as well as Campbell’s ebg-5 and as my previously isolated ebg+ strains, 1 concluded that only two mutations, one structural and one regulatory, were required for maximal growth on lactose. Multiple Evolutionary Destinations and Evolutionary Pathways John Campbell appeared to have lost interest in the project, and at this point Dan Hartl concluded that a two-step evolutionary pathway was not sufficiently interesting to warrant further study. Two things, however, encouraged me to stay with the project: (1) I had a grant to study EBG, and no other bright ideas about what to do; and (2) a second mutant hunt had revealed a curious new ebg+ phenotype. One of the striking features of the EBG system had been the homogeneity of lactose growth rates exhibited by the two mutant classes, inducible and constitutive:
Table 1. Properties of ebg enzyme Property
EBG class
(Wild type)
Lactose
Lactulose
Km' Vma,2 Growth rate3 Km Vm,, Growth rate
0
Km
I1
150 620 Undetectable 22 3600 0.4 59 2400 0.19 0.82 1460 0.37 0.69 590 0.18
180 270 Undetectable 34 70 Undetectable 26 1900 0.26 8 430 0.18 6.5 215 0.1
I
Vmax
Growth rate
K,
IV
Vmax
Growth rate Km Vmax
Growth rate
v
Galactosylarabinose
64 52 Undetectable 14 185 0.03 25 360 0.02 3 740 0.13 4.9 349 0.07
Lactobionate Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable 15 105 Undetectable 3 370 0.2
'
K, in m M substrate. 'Vmaxin nmol hydrolyzed min-'mg-'. Growth rates are first order rate constant in hr-'.
inducible strains grew at 0.1 h-l, and constitutive strains at 0.39 h-', with no intermediate growth rates. In a second hunt, on a larger scale than the first, I isolated three mutants that grew at an intermediate rate, about 0.24 h-', but that were fully constitutive. The new mutants proved to have additional interesting features: ebg enzyme from these mutants had a much lower K, for the synthetic substrate ONPG than did ebg enzyme from previous mutants, and the new mutants were able to use both lactose (galactosyl-/3-1,4D-glucose) and lactulose (galactosyl-p-1,CD-fructose), whereas previous mutants were unable to utilize lactulose. The availability of these new mutants (designated Class II), the previously isolated constitutive mutants (Class I), and the ebgR- mutant that synthesized unevolved (i.e. wild-type or ancestral) enzyme (ebg') constitutively permitted me to purify all three classes to homogeneity and to characterize them both physically and biochemically("). At that time I failed to detect the 20 kilodalton /3 subunit of the enzyme, and from the apparent M.W. of the native protein, estimated as 720 kilodaltons, and the MW of the a subunit (then estimated as 120 kilodaltons) I concluded that native ebg enzyme was a hexamer. More recent studies (M. Sinnott, pers. commun.) suggest that the native structure is probably a&,, with a molecular weight of 551 kalodaltons. (The earlier estimate was based on assumed partial specific volume that now, based upon amino acid sequence, appears to have been incorrect .) Availability of the purified enzyme permitted me to assay its activity directly on a variety of pgalactoside sugars using a cou led assay that was not possible with crude The ancestral enzyme (ebg') was a truly wimpy pgalactosidase, with a high Km and a low V,,, for lactose (Table 1). In contrast, the Class I
enzyme had dramatically improved activity toward lactose, but showed no real improvement on lactulose; whereas Class I1 enzyme showed less increase in activity toward lactose, but it had dramatically increased activity toward lactulose. When enzyme velocities at physiological substrate concentrations were calculated, the biochemical data were entirely consistent with the observed growth rates on the respective substrates (Table 1). Although mutation rate data indicated that the evolutionary pathways were short (both Class I and Class I1 enzymes involved only a single mutation), the ancestral gene clearly had at least two distinct evolutionary destinations. With two evolutionary destinations in hand, the temptation to see if the ebg operon had additional possibilities was too great to resist(12). Starting with the constitutive wild type (ebg') strain, I selected for spontaneous mutations that would permit growth on lactulose. All that I obtained were strains (called Class 111) that were indistinguishable from Class 11. Boring.. .however, the possibility of starting with a Class I strain and selecting for lactulose utilization held some interesting possibilities. Were the two evolutionary destinations mutually exclusive, would the Class I mutation lock out the possibility of lactulose utilization? Would the Class I1 mutation dominate, so that the double mutant enzyme would be indistinguishable from Class 11, or would the double mutant be something altogether different? The latter proved to be the case.. .the double mutant strains grew nearly as well as a Class I on lactose, and nearly as well as a Class I1 on lactulose. Biochemical characterization showed that this was clearly a different class (Table l ) , and it was named Class IV. I now had a two-step evolutionary pathway, but how different was Class IV from Class I1 in evolutionary terms? Although their growth rates
were different, both could use lactose and lactulose. Perhaps Class IV was just a more tedious route to approximately the same end.
Completely Novel Activities for ebg Enzyme As it turned out, Class IV proved to provide a key step in ebg enzyme evolution. Two more Fgalactoside sugars were commercially readily available: galactosyland lactobioarabinose (galactosyl-p-1,3-D-arabinose), nic acid (galactosyl-p-1 ,QD-gluconic acid). Class IV strains, to my surprise, turned out to be able to utilize galactosyl-arabinose, whereas none of the previous classes could do so (Table 1). Biochemical analysis showed that ebg' enzyme had barely detectable activity toward galactosyl-arabinose, and that the gal-ara activity was improved in both Class I and Class I1 strains, but that in none of these cases was it sufficient for growth. Only in Class IV enzyme had activity toward galactosyl-arabinose improved enough to permit decent growth. I used all four classes (wild-type, Class I, Class 11, and Class IV) to try to select for spontaneous mutants that could use lactobionate, which were designated Class V (showing absolutely no imagination with respect to naming Classes). Only Class IV strains were capable of yielding such mutants, and I concluded that lactobionate utilization required three mutations. Class IV strains thus differed from previous classes in two critical aspects: they could utilize an additional resource, galactosyl-arabinose, and they had the potential to evolve to utilization of a fourth resource, lactobionate. As before, biochemical analysis of the Class V enzyme showed a reduction in K, and an increase in V,, for lactobionate when compared with the parental class. For all the other substrates it could be legitimately argued that all of this evolutionary tinkering had produced nothing new, it had only increased existing activity to the point where it was sufficient for growth. This was not the case, however, for lactobionate. N o activity toward lactobionate could be detected with purified ebg', Class I or Class I1 enzyme. Lactobionate hydrolysis was truly a novel activity for Class IV enzyme, and the Class V mutation improved it to the point of being useful. John Campbell(') had originally noted that ebg+ strains with functional (lacZ+) lac operon repressors required a gratuitous inducer, IPTG, in order to grow on lactose. This was because ebg enzyme was incapable of converting lactose to the true lac-operon inducer, allolactose. This feature provided a convenient way to check the authenticity of ebg+ strains: they were Lacin the absence of IPTG, Lac+ in the presence of IPTG. An undergraduate student in my lab had isolated an unusual ebg+ mutant, but upon checking it proved to be Lac+ in both the presence and absence of IPTG. Genetic analysis showed that it was not a contaminant, and that it was not simply a lacl- mutant. With some excitement we published the observation that we had a
mutant ebg enzyme that could convert lactose into a lac operon inducer(13).Not long after I discovered that the mutant strain could grow on galactosyl-arabinose, and both growth rate studies and biochemical analysis of ebg enzyme purified from that strain showed (somewhat to my sheepish dismay) that the 'unusual' mutant was a perfectly ordinary Class IV mutant, and that all Class IV eb enzyme could synthesize allolactose from lactose$")). This activity, too, proved to originate with Class IV ...none of the previous classes could convert lactose to allolactose, and I will return to this later when considering the evolution of the regulatory circuits for ebg.
Evolution by Recombination Within a Gene I had demonstrated a three step pathway in the lab (ebgo+Class I-Class IV+Class V, and see Fig. 1) that ultimately resulted in the ability to use four new substrates that were unavailable to the ancestral strain. But what of poor Class II? Was it an evolutionary dead end, or did it too have additional potential? I couldn't select for lactulose utilization, as was done from Class I , because it already used lactulose; and experiments had shown that it could not produce spontaneous lactobionate-utilizing mutants. Despite the exorbitant cost of galactosyl-arabinose, I decided to see if I could select mutants that could use it from Class I1 strains. It proved relatively easy to do so, but the resulting gal-ara utilizing strains proved t o be, both by growth and biochemical criteria, ordinary Class IV strains. Boring? Not this time! From two different starting points (Classes I and 11) I had obtained apparently the same end point! Genetic analysis confirmed the intuitively obvious: a Class IV was simply a Class I mutation and a Class I1 mutation in the same gene("*")). The evidence was that a Class IV strain could be obtained by a cross between a Class I and a Class I1 strain. The two mutations were required to be in cis, the Class IV activity could not be achieved by complementation. This was the first direct demonstration that a set of new enzymatic functions (gal-ara utilization, lactobionate hydrolysis, allolactose synthesis) could be evolved by recombination within a gene. Ebg had a branchedconverging evolutionary pathway that led to lactobionate utilization (Fig. l)! Evolution of Regulation The studies on the evolution of multiple new functions for ebg enzyme had all been done in constitutive (ebgR-) strains because most of the p-galactoside sugars were poor inducers of the ebg operon(I6).While convenient, this was not the most realistic model for the evolution of new metabolic functions because such evolution requires not only novel enzyme activities, but integration of those activities into the regulatory network of the cell. The availability of purified enzymes allowed me to determine the level of expression in
Select on lactose
Select on galactosyl-arabinose
Select on lactobionate
EiEZl Fig. 1. Evolutionary pathway for utilization of p-galactoside sugars by mutations in ebgA.
regulated strains simply by measuring enzyme activity in crude extracts. The wild-type repressor, encoded by ebgR+, permitted a basal (uninduced) level of synthesis equivalent to 3-5 molecules of ebg enzyme per cell, or about one transcription per generation. The maximum level of expression, defined by ebgR- mutants, was about 2000-fold higher, and produced about 5 % of the soluble protein of the cell as ebg enzyme(16). For the wild-type repressor, lactose was the best inducer, allowing enzyme synthesis at 100-fold the basal level, but this was still poor induction compared with the maximum level permitted by the ebg promoter. Lactobionate was a non-inducer, as were thiogalactosides such as IPTG. Both lactulose and galactosylarabinose were very weak inducers, allowing only 10fold induction. The ancestral (ebgR’ ebgA’) strain thus had at least three major barriers to growth on the array of @galactoside sugars: first, the ancestral enzyme was virtually inactive toward all substrates; second, none of the substrates was sufficiently effective as an inducer to permit growth even with the most active enzyme being expressed; third, in the absence of the artificial inducer IPTG, the lacy encoded permease could not be expressed so no lactose could even enter the cell. Was it possible for this metabolic cripple to evolve into a superstrain that could utilize a variety of @galactosides without depending on artificial inducers, and could still regulate expression of all the genes involved? In an early experiment I had streaked the ancestral ALacZ strain, DS4680A, onto MacConkey lactose medium to obtain Ebg+ papillae. One papilla was only faintly pink, instead of exhibiting the deep red color
characteristic of ebg+ mutants. Upon testing it turned out to be unable to grow on lactose. Biochemical analysis showed that it synthesized Class I1 enzyme under the control of the wild-type repressor. This was an important strain, from an evolutionary point of view because it was unable to use lactose. Those of us who work with bacteria in laboratories are accustomed to such strong selection, and such clear phenotypes, that we tend to think of fitness only in terms of good growth. This mutant, strain 5A1, had formed a papilla, an outgrowth, on the surface of a Lac- colony. It was clearly an advantageous mutation, but it was not sufficient for growth on lactose as a sole carbon source. Evolution often proceeds by such relatively small increases in fitness, but those small increases are not often observed in the lab. I next plated strain 5A1 onto lactulose minimal medium to obtain lactulose-utilizing mutants. Because the Class I1 enzyme of strain 5A1 could already hydrolyze lactulose, I expected to obtain regulatory mutants, and indeed the great majority of the few thousand mutants were ebgR- constitutives, but nine isolates turned out to be inducible by lactulose(”). The nine inducible mutants turned out to have altered repressor genes, designated ebgRfL alleles, whose products had become sensitive to lactulose as inducers. Interestingly, the mutant expressed fully functional repressors with an unaltered basal level of ebg enzyme synthesis in the absence of inducer. Not only were the mutant repressors now sensitive to lactulose as an inducer, they had become 5-8 fold more sensitive to lactose (depending upon the specific allele) and had become 30-200 fold more sensitive to galactosyl-arabinose as inducers. This double mutant strain (Class I1 ebg enzyme, and evolved repressor) could now grow on both lactulose and lactose while completely regulating expression of the ebg operon. The final step was to select, from that double mutant, a strain that could synthesize Class IV enzyme by selecting a galactosyl-arabinose utilizing mutant. The resulting triple mutant strain now synthesized Class IV enzyme in response to an appropriate environmental signal, the presence of lactose in the environment. Because Class IV enzyme converts lactose into allolactose, the triple mutant growing on lactose required no additional inducer, such as IPTG, to turn on the lacy permease gene. Thus, in the absence of lactose, neither the ebg nor the Lac operon was expressed. In the presence of lactose the ebg operon was induced, the resulting Class IV ebg enzyme converted lactose to allolactose, the lac operon was induced, lactose was rapidly transported into the cell and was hydrolyzed to permit growth. Three mutations, two in an enzyme structural gene and one in a regulatory gene, had resulted in the evolution of the ability to utilize lactose, lactulose, and galactosylarabinose and in complete integration of these capabilities into the regulatory network of the cell.
Molecular Characterization of the ebg Operon The eb A and ebgR genes were sequenced several years ago(”,‘), and recently sequencing of several different alleles was completed(7). The Class I mutations all involved a G+A at bp 1566 in ebgA, resulting in A ~ p ~ ~ - + A the s n ;Class I1 mutations all had G - + Tat bp Interestingly, ys. in 4223 in ebgA, resulting in T r ~ ~ ~ ~ + C addition, some Class I1 mutations also involved simultaneous substitutions in the adjacent codon at bp 4227 in ebgA and at bp 4749 some 522 bp downstream, in ebgC. Since there is no detectable enzymatic difference between the Class I1 alleles with only the single substitution in ebgA and those that have 3 substitutions, it is concluded that the additional mutations have no direct effect on fitness. Only one Class V allele was sequenced, and the mutation was G+A in ebgA at bp 1569 resulting in G l ~ ~ ~ + L None ys. of these mutations lie within active site eptides that were identified by substrate labeling(28, thus they probably lie in portions of the peptide chain that form part of the active site, but that do not directly participate as intermediates in catalysis. The sites of all nine ebgR+= mutations were identified as falling into three groups that corresponded exactly to the phenotypes of the repressors with respect to inducibility by various galactosides. The allele most sensitive to lactose, lactulose and gal-ara as inducers involved a G+A at bp 708 in ebgR, the next most inducible alleles were T+G at bp 712, and the least inducible alleles involved T+G at bp 695. The ebg operon is almost exactly across the chromosome from the lac operon. Comparisons between the ebgR and lacl amino acid sequences, and the ebgAC and lacZ amino acid sequences showed that these operons clearly descended from a common ancestral gene. The divergence time must, however, be very ancient since there is about half as much homology between ebg and lac Pgalactosidases of E. coli as there is between the lac enzymes of Klebsiella pneumonia and E. coli. Similarly, the ebg repressor amino acid sequence has, respectively, 23.3 YO and 20.1 YO sequence identity with the E. coli gal repressor and the K. pneumoniae lac repressor genes. Clearly neither ebgR nor ebgAC are pseudogenes.. .they all produce functional products with detectable activities in the unevolved strain. Why have they been retained by E. coli? There are two reasonable possibilities. The ebg operon may, in natural environments, play a role in metabolizing substrates, presumably /3-galactosides, that have not been tested in the lab. Considerable effort (Hall, unpublished data) has failed to reveal any such role, but the possibility is certainly not excluded. Alternatively, ebg may be a ‘cryptic’ operon that can be called upon, via mutation if it is needed. E. coli is replete with cryptic enes that require mutations in order to be expressedT2’-”), but in those cases there is not an active gene, such as the lac operon, that is already capable of producing the desired function at a
very high level. The position of ebg at about 180”from lac provides supporting evidence for the intriguing that the chromosome of the Enterobacteriuce, the bacterial family that includes E. coli may have evolved by genome doubling.
Conclusions This study was begun with several questions in mind, many of which were answered. Does this mean that the ebg project has been a success? At one level the answer is, ‘yes’. Direct evidence for the operation of evolutionary processes whose existence had previously been inferred, but not experimentally demonstrated was obtained. These include specific evolutionary pathways involving sequential mutations, and evolution of novel functions by intragenic recombination. At another, and very important level, the answer is clearly, ‘No!’. The project was begun primarily to answer one very specific question: ‘What do we have to know about an organism in order to predict the best way to direct the evolution of a new metabolic function and to evaluate the probability of success?’ We are apparently no closer to answering that question today, in 1990, than we were in that room at Lake Arrowhead with John Campbell and Dan Hart1 in 1972. An awful lot of time, effort, and public funds were put into the acquisition of a lot of data that proved to be consistent, interpretable, and interesting, but that failed to provide the hoped for general answers...the ‘rules’ of the evolutionary game. The closest we might come to a rule is: ‘If you wish to evolve a new metabolic function, such as lactose utilization, determine whether the organism has a biochemically related function, such as O-nitrophenyl/3-galactoside (ONPG) hydrolysis’. Even that rule, however, is pretty pathetic. Just what do we mean by ‘biochemically related’? Deletion of the lac operons from Klebsiella reveals the presence of weak ONPG hydrolyzing activity(30). Selection of lactose utilizing mutants from that deletion strain, however, does not result in enhancement of that activity toward lactose(”). Instead, a different new activity is revealed, one that is related to utilization of cellobiose, a /?glucoside sugar. Recent studies have shown that cellobiose utilization proteins in E. coli are related to lactose utilization genes in Staphylococcus aureus(26). The connection is therefore no longer as puzzling, but this gives little comfort with respect to predicting evolutionary outcomes. Lest this sounds overly pessimistic, remember that evolutionary biology is the only branch of biology that deals almost exclusively with the past. It has generally been limited to making up plausible stories about past processes in order to explain existing organisms. It is the only branch of biology that is not expected to test its hypotheses by verifying predictions with data of a different nature than that which generated the hypotheses. Partly as a result, evolutionary biology lacks credibility with biologists from the ‘harder’, more
molecular, branches. Cell biologists now have realistic hopes of understanding the details of cellular differentiation and organization; geneticists have a realistic hope of understanding the details of recombination and chromosome assortment. Until evolutionary biologists develop both the physical and intellectual tools that will provide similarly detailed and rigorous insights into evolutionary processes we can not even enter into the arena of modern biology. The ebg system, as much fun as it was, did not provide the tools and insights that had been hoped for when the project was conceived. In other senses, however, the project has been a success. While the ebg system may not have served my evolutionary interests as well as I had hoped, it has served other peoples interest by creating tools for their utilization. The ebg system has been used by Professor Michael Sinnott, a physical-organic chemist who is one of the world's experts on enzyme catalytic mechanisms, to study the catal tic consequences of experimental evolution in detailgz3"), and recently another young scientist has taken up the ebg system to study the molecular details of the process by which evolution gradually improves and fine-tunes existing enzymatic functions (M. Riley, pers. commun.). Similarly, the ebg system has served my evolutionary interests in ways that were not anticipated at its inception. Investigation of the ebg system was what gradually led me into the systems and problems that I work with today. My investi ations of the molecular evolution of cryptic were stimulated by trying to test the generality of my findings from the ebg system and finding that, lo and behold, E. coli carried full blown, perfectly functional, silent operons for other catabolic functions and that it had no need to tinker with the catalytic machinery for those particular functions. Likewise, my current interest in the origins of spontaneous mutations, particularly those that occur more often when they are advantageous than they do when they are n e ~ t r a l ( ~ ~ -came ~ ' ) , directly from the observation that the ebg mutants were all spontaneous double mutants and that those double mutations arose some 10' more frequently than was expected under our present understanding of mutation processes. It isn't surprising that the goals of a naive young postdoc weren't attained by the exploitation of a single system such as ebg, and now, some 16 years older, I no longer expect that I will ever develop the level of understanding necessary to be able to really predict specific evolutionary outcomes. (At this stage I take some comfort from the fact that biophysicists still can't predict complete three dimensional structures of proteins from amino acid sequences, either.) I am very optimistic, however, that the general goal of understanding evolution as a dynamic process, and understanding the details of its important mechanisms, will be obtained in the foreseeable future. There is a new generation of molecular evolutionary geneticists upon us. Trained in the 1980s, they combine expertise in molecular biology with a thorough grounding in
gene^('^".^^-^')
population and evolutionary theory. The combination of the rigor of molecular genetics with an appreciation of both the history of evolutionary biology and the important issues that confront evolution today is allowing these young scientists to have an enormous impact on the field of Evolution. It is not unreasonable to predict that by the end of this century they will have generated the physical and intellectual tools to put Evolutionary Biology in a position comparable to that enjoyed by Developmental Biology today.
References 1 CAMPBELL, J . . LENGYEL, J. AND LANGRIDGE. J . (1973). Evolution of a second gene for /3-galactosidase in Escherichia coli. Proc. Nafl Acad. Sci. USA 70. 1841-1845. 2 BACHMANN, B. J . (1990). Linkage map of Escherichia coli K-12. Edition 8. Microbiol. Rev. 54, 130-197. 3 KOHARA, Y . . AKIYAMA. K . AND ISONO. K. (1987). The physical map of the whole E . coli chromosome: Application of a new strategy for rapid analysis and sorting of a large genomic library. Cell 50. 495-508. 4 HALL. B. G . AND HARTL,D. L. (1974). Regulation of newly cvolved enzymes. I . Selection of a novel lactase regulated by lactose in Eseherichia coli. Genetics 76. 391-400. 5 HARTL.D. L. A N D HALL,B. G. (1974). A second naturally occurring /igalactosidase in E . coli. Nature 248, 152-153. 6 HALL, B. G. AND HARTL,D. L. (1975). Regulation of newly evolved enzymes. 11. The ebg repressor. Genetics 81. 427-435. 7 HALL.B. G . . BETTS,P. W. AND WOOTTON, J. C. (1989). D N A sequence analysis of artificially evolved ebg enzyme and ebg repressor genes. Genetics 123. 635-648. 8 HALL.B. G . A N D ZUZEL,T. (1980). The ebg operon consists of at least two genes. J . Bacteriol. 144, 1208-1211. 9 HALL.B. G. (1977). The number of mutations required to evolve a new lactase function in Escherichia coli. J. Bacreriol. 129, 540-543. 1 0 HAT-I., B. G. (1976). Experimental evolution of a new enzymatic function. Kinetic analysis of the ancestral (ebgu) and evolved ( e b g + ) enzymes. J . M o l . Biol. 107. 71-84. 11 HALL,B. G. (1981). Changes in the substrate specificities of a n enzymc during directed evolution of new functions. Biochern. 20. 4042-4049. 1 2 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. 1 3 ROLSETH.S . J., FRIED,V. A . AND HALL.B. G. (1980). A mutant ehg enzyme that converts lactose into an inducer of the lac operon. J . Bacteriol. 142. 1036-1039. 1 4 HALL,B . G . (1982). Transgalactosylation activity of ebg /3-galactosidase synthesizes allelactose from lactose. J . Bacteriol. 150, 132-140. 1 5 HALL,B. G. AND ZUZEL,T. (1980). Evolution of a new enzymatic function by recombination within a gene. Proc. Natl Acad. Sci. U . S . A . 7 7 . 3529-3533. 1 6 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, 193-201. 1 7 H A I . I ,B. G. (1978). Regulation of newly evolved enzymes. IV. Directed evolution of the ebg repressor. Genetics 90. 673-691. 18 STOKES. H . W., B E ~ s P. , W. AND HALL.B. G. (1985). Sequence of the ebgA gene of Escherichia coli: Comparison with the lacZ gene. Mol. Biol. E d . 2, 469-477. 1 9 STOKES, H. W. AND HALL,B. G. (1985). Sequence of the ehgR gene of Escherichia coli: Evidence that the EBG and LAC operons are descended from a common ancestor. Mol. B i d . Evol. 2, 478-483. 2 0 FOWLER, A . V. AND SMITH, P. J. (1983). The active site regions of lacZ and ebg Pgalactosidases are homologous. J . Biol. Chern. 258, 10204-10207. 21 HALL,B. G. (1982). A chromosomal mutation for citrate utilization by Escherichia coli K12. J. Bacferiol. 152, 269-273. 22 HALL,B . G., YOKOYAMA, S . AND CALHOUN. D. (1983). Role of cryptic genes in microbial evolution. Mol. Biol & Evol. 1, 109-124. 2 3 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. 24 HALL. B. G. AND BETTS,P. W. (1987). Cryptic genes for cellobiose utilization in natural isolates of Escherichia cofi. Generics 115, 431-439. 25 PARKER, L. L. AND HALL,B. G. (1988). A fourth E. coli gene system with the potential to evolve &glucoside utilization. Genetics 119, 485-490. 26 PARKER, L. L. AND HALL,B. G . (1990). Characterization and nucleotide sequence of the cryptic cel operon of E. coli K12. Genetics 124, 455-471.
27 REYNOLDS, A. E., FLLTON. J . AND WRIGHT. A. (1981). Insertion of DNA activates the cryptic bgl operon of E . coli. Nature 203, 625-629. 28 RILEY,M..SOLOMON, L. AND Z I P K A S . D. (1978). Relationship between gene function and gene location in Escherichia coli. J. Mol. Evol. 11, 47-56. 29 RILEY,M. A N D ANILLONIS. A. (1978). Evolution of the bacterial genome. Annu. Rev. Microbiol. 32. 519-560. 30 HALL.B . G. AND REEVE.E. C. R. (1977). A third Pgalactosidase in a strain of Klcbsiella which possesses two Lac genes. J. Bacteriol. 132, 219-223. 31 H A I L .B. G . , IMAI,K. AND ROMANO, C. (1982). Genetics of the lac-PTS system of Klebsiella. Genet. RES.39. 287-302. 32 BURTON.J . AND SINNOTT. M. (1983). Catalytic Consequenccs of Experimental Evolution. Part 1 . Catalysis by the Wild-type Second bGalactosidase (ebg") of Escherichia coli: a Comparison with the lacZ Enzyme. J. Chem. Soc. Perkin Trans. I I . 359-364. 33 LI, B. F . . OSBORNE, S. A N D SINNOIT,M. (1983). Catalytic Consequences of Experimental Evolution. Part 2. Rate-limiting Degalactosylation in the Hydrolysis of Aryl PD-Galactopyranosides by the Experimental Evolvants ebg' and ebg". J. Chem. SOC. Perkin Trans. I t . . pp. 365-369. 34 HALL,B. G. MURRAY, M..OSBORNE, S. AND SINNOIT.M. L. (1983). The
catalytic consequences of experimental evolution. Part 111. Construction of reaction profiles for hydrolysis of lactose by ebg", ebg", and ebg" enzymes via measurements of the enzyme-catalyzed exchange of galactose-l-180 by 13C NMR spectroscopy. J. Chem. SOC. Perkm Trans. It, 1595-1598. 35 KRICKER, M. AND HALL,B. G. (1984). Directed evolution of cellobiose utilization in Escherichia coli. Mol. B i d . & Evol. 1. 171-182. 36 HALL.B . G. (1988). Adaptive evolution that requires multiple spontaneous mutations. I . Mutations involving and inscrtion sequence. Generia 120. 887-897. 37 HALL. B. G. (1989). Selection, adaptation, and bacterial operons. Genome 31, 265-271. 38 HALL,B. G. (1990). Spontaneous point mutations that occur more often when they are advantageous than when they arc neutral. Genetics 126. 5-16.
Barry G. Hall is at the Department of Biology, Hutchison Hall, University of Rochester, Rochester, NY 14627, USA.
'1
"Stop complaining, Munshaw. You knew you'd be working with a computer when we hired you. 'I
Reprinted courtesy OMNl Magazine @ 1985
E. coli go about reinventing the /$galactosidase function?’, and have some hope that the answer might be of interest both to the molecular and evolutionary communities. Evolutionary biologists are usually forced to infer a historical process by examining the present day outcome of that process, a very unsatisfactory approach to understanding any dynamic process. With that in mind, several studies, primarily in Drosophila melanogaster, sought to study the adaptive process experimentally, rather than retrospectively. In general those studies involved selection for complex traits in populations that were genetically heterogeneous. Enhancement of the selected phenotype was often observed, but it was concluded that it was based on preexisting variation that was already in the population, and the complexity of the selected traits made interpretation of the results frustrating in any but the broadest terms. We hoped that by starting with a single, clonal, genetically well understood organism, and applying a specific selection pressure, under carefully defined conditions, to a biochemically well understood pathway that we would be able experimentally to demonstrate the operation of those mechanisms that have produced the enormous array of highly adapted organisms we see today. Because of the simplicity of the system we hoped to be able to look at a level of detail (biochemical, molecular, regulatory, etc.) that was not possible in more complex systems. Discovery of ebg John Campbell(’) had isolated the first ebg mutant by starting with an E. coli K12 strain that was deleted for the entire lac operon, but which carried the (constitutively expressed) lactose permease (lacY) gene on an F’ plasmid. The starting strain was allowed to form colonies on an indicator medium that permits growth at the expense of small peptides, and which included lactose (which the cells could transport, but not hydrolyze) and a color indicator for lactose fermentation. After several weeks, papillae, or outgrowths, appeared on the surface of some colonies, and the color of these papillae indicated that they were weakly fermenting lactose. Cells from one papilla were restreaked on the same medium, and allowed to form colonies which subsequently produced more papillae. After five rounds of such selection Campbell isolated the mutant that he designated ebg-5; the number ‘5’ reflected his assumption that each round of selection had produced an additional mutation which improved growth on lactose. The ebg-5 strain grew well on lactose, but not as well as a wild type lacz+ strain. Campbell el al. showed that the ebg-5 mutant constitutively synthesized a /3-galactosidase with biochemical and immunological properties that were completely distinct from those of the classical lac2 encoded pgalactosidase. A particularly interesting observation was that the ebg 8-galactosidase was incapable of converting lactose into an inducer of the lac operon. BioEssays
Vol. 12, No. 11 - November 1990
551
Therefore, in later experiments, when the lacy gene was under control of the lacl encoded repressor it was necessary to include the lac operon inducer IPTG in the medium if the cells were to transport lactose. They mapped the ebg gene to a position near tolC on the opposite side of the chromosome from the lac operon. Subsequent genetic and physical mapping studies have confirmed that mapping, with the gene placed at 68.2 min on the current genetic and physical maps(23’).
Development of ebg as a Tool for Studying Evolutionary Processes When Hartl and I initiated our studies we began with a distantly related E . coli K12 strain, strain DS4680A, that was deleted for the middle 1/3 of the lacZ gene, but that was both l a d + and was lacy+. We had two immediate questions in mind: How many different genes could mutate so as to evolve pgalactosidase activity?; and, What were the properties of the intermediate-step ebg isolates, equivalent to the postulated ebg-1 through ebg-4 mutants which Campbell had not retained? The first question has been thoroughly answered over the course of the last 17 years, both in my lab and in Hartl’s lab: no genes other than ebg have ever been observed to give rise to lactose-utilizing mutants. Deletion of the ebg gene results in a complete inability to mutate to lactose utilization, thus E. coli has a very limited potential, in this sense, for evolving this particular new function. The answer to the second question emerged over the course of the next two years. In my first study(‘), using the same papillae method as that used by Campbell et al., I isolated 34 independent ebg mutants, three of which were indistinguishable from Campbell’s ebg-5 mutant. Since all of these were isolated in a single round of selection, we began to suspect that the ebg-5 mutant did not necessarily involve five separate mutations. The remaining 31 mutants grew much more slowly than the ebg-5-like mutants, and they proved to differ only in their regulation of ebg enzyme synthesis: they were inducible by lactose. The enzyme produced by the inducible mutants was indistinguishable from that synthesized by the three constitutive strains and by Campbell’s ebg-5 strain. The slower growth resulted from a lower level of induced than constitutive synthesis, a common property of regulated enzyme systems. Campbell had believed that the ebg-5 encoded enzyme was the result of structural gene mutations in a gene that was expressed constitutively in the original AZacZ strain. The finding that the majority of ebgt mutants were inducible led us to suspect that the ‘unevolved’ or ancestral gene might also be inducible by lactose. (‘Ancestral’, in this context, means the allele that was present in the AlacZ strain that we started with, not a gene that was present in an ancestor of E. coli.) We showed that in strain DS4680A, and in other
AlacZ strains, ebg enzyme activity was inducible, but that the fully induced level of activity was insufficient for growth on lactose(’). At this point, I accepted an Assistant Professorship at the Memorial University of Newfoundland, where further work showed that the gene encoding for ebg enzyme, designated ebgA, was under negative control by a repressor that was the product of the adjacent ebgR gene(6). Physical studied7), completed some 14 years later, confirmed and extended the early genetic conclusions(6,8): the EBG gene cluster consists of a repressor gene, ebgR, that is adjacent to an operon consisting of at least three structural genes; ebgA and ebgC, which encode the 118 kilodalton a subunit and the 20 kilodalton /3 subunit of ebg enzyme, and ebgB, which encodes a 67 kilodalton protein of unknown function. In 1975 enough was known about the state of the wild-type, or ‘ancestral’ ebg operon to permit the pursuit of fundamental questions concerning the evolution of lactose utilization via the ebg genes. The most fundamental question was, ‘Just how long is the pathway for the evolution of lactose utilization, or, in other words, how many steps are required?’ Campbell thought that five structural (ebg enzyme encoding) gene mutations were required for maximal growth, but the finding that 90 % of the Ebg+ (lactose utilizing) mutants were inducible suggested that the pathway might be as short as one structural mutation for slow growth, with a second mutation to ebgR- (constitutive) expression for maximal growth. From the wild-type stain I isolated an ebgR- mutant that synthesized the ancestral enzyme, designated ebg’ for ‘original’, constitutively. Although the mutant synthesized 5 % of its soluble protein as ebg enzyme, it was incapable of growth on lactose. Using that strain I measured the spontaneous mutation rate to Ebg+ as 2xlOP9 per cell division, a rate that was consistent with a single difference between unevolved (ebg’) and evolved (ebg+) enzyme(’). Since the single mutant ebg+ strains grew as well as Campbell’s ebg-5 and as my previously isolated ebg+ strains, 1 concluded that only two mutations, one structural and one regulatory, were required for maximal growth on lactose. Multiple Evolutionary Destinations and Evolutionary Pathways John Campbell appeared to have lost interest in the project, and at this point Dan Hartl concluded that a two-step evolutionary pathway was not sufficiently interesting to warrant further study. Two things, however, encouraged me to stay with the project: (1) I had a grant to study EBG, and no other bright ideas about what to do; and (2) a second mutant hunt had revealed a curious new ebg+ phenotype. One of the striking features of the EBG system had been the homogeneity of lactose growth rates exhibited by the two mutant classes, inducible and constitutive:
Table 1. Properties of ebg enzyme Property
EBG class
(Wild type)
Lactose
Lactulose
Km' Vma,2 Growth rate3 Km Vm,, Growth rate
0
Km
I1
150 620 Undetectable 22 3600 0.4 59 2400 0.19 0.82 1460 0.37 0.69 590 0.18
180 270 Undetectable 34 70 Undetectable 26 1900 0.26 8 430 0.18 6.5 215 0.1
I
Vmax
Growth rate
K,
IV
Vmax
Growth rate Km Vmax
Growth rate
v
Galactosylarabinose
64 52 Undetectable 14 185 0.03 25 360 0.02 3 740 0.13 4.9 349 0.07
Lactobionate Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable Undetectable 15 105 Undetectable 3 370 0.2
'
K, in m M substrate. 'Vmaxin nmol hydrolyzed min-'mg-'. Growth rates are first order rate constant in hr-'.
inducible strains grew at 0.1 h-l, and constitutive strains at 0.39 h-', with no intermediate growth rates. In a second hunt, on a larger scale than the first, I isolated three mutants that grew at an intermediate rate, about 0.24 h-', but that were fully constitutive. The new mutants proved to have additional interesting features: ebg enzyme from these mutants had a much lower K, for the synthetic substrate ONPG than did ebg enzyme from previous mutants, and the new mutants were able to use both lactose (galactosyl-/3-1,4D-glucose) and lactulose (galactosyl-p-1,CD-fructose), whereas previous mutants were unable to utilize lactulose. The availability of these new mutants (designated Class II), the previously isolated constitutive mutants (Class I), and the ebgR- mutant that synthesized unevolved (i.e. wild-type or ancestral) enzyme (ebg') constitutively permitted me to purify all three classes to homogeneity and to characterize them both physically and biochemically("). At that time I failed to detect the 20 kilodalton /3 subunit of the enzyme, and from the apparent M.W. of the native protein, estimated as 720 kilodaltons, and the MW of the a subunit (then estimated as 120 kilodaltons) I concluded that native ebg enzyme was a hexamer. More recent studies (M. Sinnott, pers. commun.) suggest that the native structure is probably a&,, with a molecular weight of 551 kalodaltons. (The earlier estimate was based on assumed partial specific volume that now, based upon amino acid sequence, appears to have been incorrect .) Availability of the purified enzyme permitted me to assay its activity directly on a variety of pgalactoside sugars using a cou led assay that was not possible with crude The ancestral enzyme (ebg') was a truly wimpy pgalactosidase, with a high Km and a low V,,, for lactose (Table 1). In contrast, the Class I
enzyme had dramatically improved activity toward lactose, but showed no real improvement on lactulose; whereas Class I1 enzyme showed less increase in activity toward lactose, but it had dramatically increased activity toward lactulose. When enzyme velocities at physiological substrate concentrations were calculated, the biochemical data were entirely consistent with the observed growth rates on the respective substrates (Table 1). Although mutation rate data indicated that the evolutionary pathways were short (both Class I and Class I1 enzymes involved only a single mutation), the ancestral gene clearly had at least two distinct evolutionary destinations. With two evolutionary destinations in hand, the temptation to see if the ebg operon had additional possibilities was too great to resist(12). Starting with the constitutive wild type (ebg') strain, I selected for spontaneous mutations that would permit growth on lactulose. All that I obtained were strains (called Class 111) that were indistinguishable from Class 11. Boring.. .however, the possibility of starting with a Class I strain and selecting for lactulose utilization held some interesting possibilities. Were the two evolutionary destinations mutually exclusive, would the Class I mutation lock out the possibility of lactulose utilization? Would the Class I1 mutation dominate, so that the double mutant enzyme would be indistinguishable from Class 11, or would the double mutant be something altogether different? The latter proved to be the case.. .the double mutant strains grew nearly as well as a Class I on lactose, and nearly as well as a Class I1 on lactulose. Biochemical characterization showed that this was clearly a different class (Table l ) , and it was named Class IV. I now had a two-step evolutionary pathway, but how different was Class IV from Class I1 in evolutionary terms? Although their growth rates
were different, both could use lactose and lactulose. Perhaps Class IV was just a more tedious route to approximately the same end.
Completely Novel Activities for ebg Enzyme As it turned out, Class IV proved to provide a key step in ebg enzyme evolution. Two more Fgalactoside sugars were commercially readily available: galactosyland lactobioarabinose (galactosyl-p-1,3-D-arabinose), nic acid (galactosyl-p-1 ,QD-gluconic acid). Class IV strains, to my surprise, turned out to be able to utilize galactosyl-arabinose, whereas none of the previous classes could do so (Table 1). Biochemical analysis showed that ebg' enzyme had barely detectable activity toward galactosyl-arabinose, and that the gal-ara activity was improved in both Class I and Class I1 strains, but that in none of these cases was it sufficient for growth. Only in Class IV enzyme had activity toward galactosyl-arabinose improved enough to permit decent growth. I used all four classes (wild-type, Class I, Class 11, and Class IV) to try to select for spontaneous mutants that could use lactobionate, which were designated Class V (showing absolutely no imagination with respect to naming Classes). Only Class IV strains were capable of yielding such mutants, and I concluded that lactobionate utilization required three mutations. Class IV strains thus differed from previous classes in two critical aspects: they could utilize an additional resource, galactosyl-arabinose, and they had the potential to evolve to utilization of a fourth resource, lactobionate. As before, biochemical analysis of the Class V enzyme showed a reduction in K, and an increase in V,, for lactobionate when compared with the parental class. For all the other substrates it could be legitimately argued that all of this evolutionary tinkering had produced nothing new, it had only increased existing activity to the point where it was sufficient for growth. This was not the case, however, for lactobionate. N o activity toward lactobionate could be detected with purified ebg', Class I or Class I1 enzyme. Lactobionate hydrolysis was truly a novel activity for Class IV enzyme, and the Class V mutation improved it to the point of being useful. John Campbell(') had originally noted that ebg+ strains with functional (lacZ+) lac operon repressors required a gratuitous inducer, IPTG, in order to grow on lactose. This was because ebg enzyme was incapable of converting lactose to the true lac-operon inducer, allolactose. This feature provided a convenient way to check the authenticity of ebg+ strains: they were Lacin the absence of IPTG, Lac+ in the presence of IPTG. An undergraduate student in my lab had isolated an unusual ebg+ mutant, but upon checking it proved to be Lac+ in both the presence and absence of IPTG. Genetic analysis showed that it was not a contaminant, and that it was not simply a lacl- mutant. With some excitement we published the observation that we had a
mutant ebg enzyme that could convert lactose into a lac operon inducer(13).Not long after I discovered that the mutant strain could grow on galactosyl-arabinose, and both growth rate studies and biochemical analysis of ebg enzyme purified from that strain showed (somewhat to my sheepish dismay) that the 'unusual' mutant was a perfectly ordinary Class IV mutant, and that all Class IV eb enzyme could synthesize allolactose from lactose$")). This activity, too, proved to originate with Class IV ...none of the previous classes could convert lactose to allolactose, and I will return to this later when considering the evolution of the regulatory circuits for ebg.
Evolution by Recombination Within a Gene I had demonstrated a three step pathway in the lab (ebgo+Class I-Class IV+Class V, and see Fig. 1) that ultimately resulted in the ability to use four new substrates that were unavailable to the ancestral strain. But what of poor Class II? Was it an evolutionary dead end, or did it too have additional potential? I couldn't select for lactulose utilization, as was done from Class I , because it already used lactulose; and experiments had shown that it could not produce spontaneous lactobionate-utilizing mutants. Despite the exorbitant cost of galactosyl-arabinose, I decided to see if I could select mutants that could use it from Class I1 strains. It proved relatively easy to do so, but the resulting gal-ara utilizing strains proved t o be, both by growth and biochemical criteria, ordinary Class IV strains. Boring? Not this time! From two different starting points (Classes I and 11) I had obtained apparently the same end point! Genetic analysis confirmed the intuitively obvious: a Class IV was simply a Class I mutation and a Class I1 mutation in the same gene("*")). The evidence was that a Class IV strain could be obtained by a cross between a Class I and a Class I1 strain. The two mutations were required to be in cis, the Class IV activity could not be achieved by complementation. This was the first direct demonstration that a set of new enzymatic functions (gal-ara utilization, lactobionate hydrolysis, allolactose synthesis) could be evolved by recombination within a gene. Ebg had a branchedconverging evolutionary pathway that led to lactobionate utilization (Fig. l)! Evolution of Regulation The studies on the evolution of multiple new functions for ebg enzyme had all been done in constitutive (ebgR-) strains because most of the p-galactoside sugars were poor inducers of the ebg operon(I6).While convenient, this was not the most realistic model for the evolution of new metabolic functions because such evolution requires not only novel enzyme activities, but integration of those activities into the regulatory network of the cell. The availability of purified enzymes allowed me to determine the level of expression in
Select on lactose
Select on galactosyl-arabinose
Select on lactobionate
EiEZl Fig. 1. Evolutionary pathway for utilization of p-galactoside sugars by mutations in ebgA.
regulated strains simply by measuring enzyme activity in crude extracts. The wild-type repressor, encoded by ebgR+, permitted a basal (uninduced) level of synthesis equivalent to 3-5 molecules of ebg enzyme per cell, or about one transcription per generation. The maximum level of expression, defined by ebgR- mutants, was about 2000-fold higher, and produced about 5 % of the soluble protein of the cell as ebg enzyme(16). For the wild-type repressor, lactose was the best inducer, allowing enzyme synthesis at 100-fold the basal level, but this was still poor induction compared with the maximum level permitted by the ebg promoter. Lactobionate was a non-inducer, as were thiogalactosides such as IPTG. Both lactulose and galactosylarabinose were very weak inducers, allowing only 10fold induction. The ancestral (ebgR’ ebgA’) strain thus had at least three major barriers to growth on the array of @galactoside sugars: first, the ancestral enzyme was virtually inactive toward all substrates; second, none of the substrates was sufficiently effective as an inducer to permit growth even with the most active enzyme being expressed; third, in the absence of the artificial inducer IPTG, the lacy encoded permease could not be expressed so no lactose could even enter the cell. Was it possible for this metabolic cripple to evolve into a superstrain that could utilize a variety of @galactosides without depending on artificial inducers, and could still regulate expression of all the genes involved? In an early experiment I had streaked the ancestral ALacZ strain, DS4680A, onto MacConkey lactose medium to obtain Ebg+ papillae. One papilla was only faintly pink, instead of exhibiting the deep red color
characteristic of ebg+ mutants. Upon testing it turned out to be unable to grow on lactose. Biochemical analysis showed that it synthesized Class I1 enzyme under the control of the wild-type repressor. This was an important strain, from an evolutionary point of view because it was unable to use lactose. Those of us who work with bacteria in laboratories are accustomed to such strong selection, and such clear phenotypes, that we tend to think of fitness only in terms of good growth. This mutant, strain 5A1, had formed a papilla, an outgrowth, on the surface of a Lac- colony. It was clearly an advantageous mutation, but it was not sufficient for growth on lactose as a sole carbon source. Evolution often proceeds by such relatively small increases in fitness, but those small increases are not often observed in the lab. I next plated strain 5A1 onto lactulose minimal medium to obtain lactulose-utilizing mutants. Because the Class I1 enzyme of strain 5A1 could already hydrolyze lactulose, I expected to obtain regulatory mutants, and indeed the great majority of the few thousand mutants were ebgR- constitutives, but nine isolates turned out to be inducible by lactulose(”). The nine inducible mutants turned out to have altered repressor genes, designated ebgRfL alleles, whose products had become sensitive to lactulose as inducers. Interestingly, the mutant expressed fully functional repressors with an unaltered basal level of ebg enzyme synthesis in the absence of inducer. Not only were the mutant repressors now sensitive to lactulose as an inducer, they had become 5-8 fold more sensitive to lactose (depending upon the specific allele) and had become 30-200 fold more sensitive to galactosyl-arabinose as inducers. This double mutant strain (Class I1 ebg enzyme, and evolved repressor) could now grow on both lactulose and lactose while completely regulating expression of the ebg operon. The final step was to select, from that double mutant, a strain that could synthesize Class IV enzyme by selecting a galactosyl-arabinose utilizing mutant. The resulting triple mutant strain now synthesized Class IV enzyme in response to an appropriate environmental signal, the presence of lactose in the environment. Because Class IV enzyme converts lactose into allolactose, the triple mutant growing on lactose required no additional inducer, such as IPTG, to turn on the lacy permease gene. Thus, in the absence of lactose, neither the ebg nor the Lac operon was expressed. In the presence of lactose the ebg operon was induced, the resulting Class IV ebg enzyme converted lactose to allolactose, the lac operon was induced, lactose was rapidly transported into the cell and was hydrolyzed to permit growth. Three mutations, two in an enzyme structural gene and one in a regulatory gene, had resulted in the evolution of the ability to utilize lactose, lactulose, and galactosylarabinose and in complete integration of these capabilities into the regulatory network of the cell.
Molecular Characterization of the ebg Operon The eb A and ebgR genes were sequenced several years ago(”,‘), and recently sequencing of several different alleles was completed(7). The Class I mutations all involved a G+A at bp 1566 in ebgA, resulting in A ~ p ~ ~ - + A the s n ;Class I1 mutations all had G - + Tat bp Interestingly, ys. in 4223 in ebgA, resulting in T r ~ ~ ~ ~ + C addition, some Class I1 mutations also involved simultaneous substitutions in the adjacent codon at bp 4227 in ebgA and at bp 4749 some 522 bp downstream, in ebgC. Since there is no detectable enzymatic difference between the Class I1 alleles with only the single substitution in ebgA and those that have 3 substitutions, it is concluded that the additional mutations have no direct effect on fitness. Only one Class V allele was sequenced, and the mutation was G+A in ebgA at bp 1569 resulting in G l ~ ~ ~ + L None ys. of these mutations lie within active site eptides that were identified by substrate labeling(28, thus they probably lie in portions of the peptide chain that form part of the active site, but that do not directly participate as intermediates in catalysis. The sites of all nine ebgR+= mutations were identified as falling into three groups that corresponded exactly to the phenotypes of the repressors with respect to inducibility by various galactosides. The allele most sensitive to lactose, lactulose and gal-ara as inducers involved a G+A at bp 708 in ebgR, the next most inducible alleles were T+G at bp 712, and the least inducible alleles involved T+G at bp 695. The ebg operon is almost exactly across the chromosome from the lac operon. Comparisons between the ebgR and lacl amino acid sequences, and the ebgAC and lacZ amino acid sequences showed that these operons clearly descended from a common ancestral gene. The divergence time must, however, be very ancient since there is about half as much homology between ebg and lac Pgalactosidases of E. coli as there is between the lac enzymes of Klebsiella pneumonia and E. coli. Similarly, the ebg repressor amino acid sequence has, respectively, 23.3 YO and 20.1 YO sequence identity with the E. coli gal repressor and the K. pneumoniae lac repressor genes. Clearly neither ebgR nor ebgAC are pseudogenes.. .they all produce functional products with detectable activities in the unevolved strain. Why have they been retained by E. coli? There are two reasonable possibilities. The ebg operon may, in natural environments, play a role in metabolizing substrates, presumably /3-galactosides, that have not been tested in the lab. Considerable effort (Hall, unpublished data) has failed to reveal any such role, but the possibility is certainly not excluded. Alternatively, ebg may be a ‘cryptic’ operon that can be called upon, via mutation if it is needed. E. coli is replete with cryptic enes that require mutations in order to be expressedT2’-”), but in those cases there is not an active gene, such as the lac operon, that is already capable of producing the desired function at a
very high level. The position of ebg at about 180”from lac provides supporting evidence for the intriguing that the chromosome of the Enterobacteriuce, the bacterial family that includes E. coli may have evolved by genome doubling.
Conclusions This study was begun with several questions in mind, many of which were answered. Does this mean that the ebg project has been a success? At one level the answer is, ‘yes’. Direct evidence for the operation of evolutionary processes whose existence had previously been inferred, but not experimentally demonstrated was obtained. These include specific evolutionary pathways involving sequential mutations, and evolution of novel functions by intragenic recombination. At another, and very important level, the answer is clearly, ‘No!’. The project was begun primarily to answer one very specific question: ‘What do we have to know about an organism in order to predict the best way to direct the evolution of a new metabolic function and to evaluate the probability of success?’ We are apparently no closer to answering that question today, in 1990, than we were in that room at Lake Arrowhead with John Campbell and Dan Hart1 in 1972. An awful lot of time, effort, and public funds were put into the acquisition of a lot of data that proved to be consistent, interpretable, and interesting, but that failed to provide the hoped for general answers...the ‘rules’ of the evolutionary game. The closest we might come to a rule is: ‘If you wish to evolve a new metabolic function, such as lactose utilization, determine whether the organism has a biochemically related function, such as O-nitrophenyl/3-galactoside (ONPG) hydrolysis’. Even that rule, however, is pretty pathetic. Just what do we mean by ‘biochemically related’? Deletion of the lac operons from Klebsiella reveals the presence of weak ONPG hydrolyzing activity(30). Selection of lactose utilizing mutants from that deletion strain, however, does not result in enhancement of that activity toward lactose(”). Instead, a different new activity is revealed, one that is related to utilization of cellobiose, a /?glucoside sugar. Recent studies have shown that cellobiose utilization proteins in E. coli are related to lactose utilization genes in Staphylococcus aureus(26). The connection is therefore no longer as puzzling, but this gives little comfort with respect to predicting evolutionary outcomes. Lest this sounds overly pessimistic, remember that evolutionary biology is the only branch of biology that deals almost exclusively with the past. It has generally been limited to making up plausible stories about past processes in order to explain existing organisms. It is the only branch of biology that is not expected to test its hypotheses by verifying predictions with data of a different nature than that which generated the hypotheses. Partly as a result, evolutionary biology lacks credibility with biologists from the ‘harder’, more
molecular, branches. Cell biologists now have realistic hopes of understanding the details of cellular differentiation and organization; geneticists have a realistic hope of understanding the details of recombination and chromosome assortment. Until evolutionary biologists develop both the physical and intellectual tools that will provide similarly detailed and rigorous insights into evolutionary processes we can not even enter into the arena of modern biology. The ebg system, as much fun as it was, did not provide the tools and insights that had been hoped for when the project was conceived. In other senses, however, the project has been a success. While the ebg system may not have served my evolutionary interests as well as I had hoped, it has served other peoples interest by creating tools for their utilization. The ebg system has been used by Professor Michael Sinnott, a physical-organic chemist who is one of the world's experts on enzyme catalytic mechanisms, to study the catal tic consequences of experimental evolution in detailgz3"), and recently another young scientist has taken up the ebg system to study the molecular details of the process by which evolution gradually improves and fine-tunes existing enzymatic functions (M. Riley, pers. commun.). Similarly, the ebg system has served my evolutionary interests in ways that were not anticipated at its inception. Investigation of the ebg system was what gradually led me into the systems and problems that I work with today. My investi ations of the molecular evolution of cryptic were stimulated by trying to test the generality of my findings from the ebg system and finding that, lo and behold, E. coli carried full blown, perfectly functional, silent operons for other catabolic functions and that it had no need to tinker with the catalytic machinery for those particular functions. Likewise, my current interest in the origins of spontaneous mutations, particularly those that occur more often when they are advantageous than they do when they are n e ~ t r a l ( ~ ~ -came ~ ' ) , directly from the observation that the ebg mutants were all spontaneous double mutants and that those double mutations arose some 10' more frequently than was expected under our present understanding of mutation processes. It isn't surprising that the goals of a naive young postdoc weren't attained by the exploitation of a single system such as ebg, and now, some 16 years older, I no longer expect that I will ever develop the level of understanding necessary to be able to really predict specific evolutionary outcomes. (At this stage I take some comfort from the fact that biophysicists still can't predict complete three dimensional structures of proteins from amino acid sequences, either.) I am very optimistic, however, that the general goal of understanding evolution as a dynamic process, and understanding the details of its important mechanisms, will be obtained in the foreseeable future. There is a new generation of molecular evolutionary geneticists upon us. Trained in the 1980s, they combine expertise in molecular biology with a thorough grounding in
gene^('^".^^-^')
population and evolutionary theory. The combination of the rigor of molecular genetics with an appreciation of both the history of evolutionary biology and the important issues that confront evolution today is allowing these young scientists to have an enormous impact on the field of Evolution. It is not unreasonable to predict that by the end of this century they will have generated the physical and intellectual tools to put Evolutionary Biology in a position comparable to that enjoyed by Developmental Biology today.
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27 REYNOLDS, A. E., FLLTON. J . AND WRIGHT. A. (1981). Insertion of DNA activates the cryptic bgl operon of E . coli. Nature 203, 625-629. 28 RILEY,M..SOLOMON, L. AND Z I P K A S . D. (1978). Relationship between gene function and gene location in Escherichia coli. J. Mol. Evol. 11, 47-56. 29 RILEY,M. A N D ANILLONIS. A. (1978). Evolution of the bacterial genome. Annu. Rev. Microbiol. 32. 519-560. 30 HALL.B . G. AND REEVE.E. C. R. (1977). A third Pgalactosidase in a strain of Klcbsiella which possesses two Lac genes. J. Bacteriol. 132, 219-223. 31 H A I L .B. G . , IMAI,K. AND ROMANO, C. (1982). Genetics of the lac-PTS system of Klebsiella. Genet. RES.39. 287-302. 32 BURTON.J . AND SINNOTT. M. (1983). Catalytic Consequenccs of Experimental Evolution. Part 1 . Catalysis by the Wild-type Second bGalactosidase (ebg") of Escherichia coli: a Comparison with the lacZ Enzyme. J. Chem. Soc. Perkin Trans. I I . 359-364. 33 LI, B. F . . OSBORNE, S. A N D SINNOIT,M. (1983). Catalytic Consequences of Experimental Evolution. Part 2. Rate-limiting Degalactosylation in the Hydrolysis of Aryl PD-Galactopyranosides by the Experimental Evolvants ebg' and ebg". J. Chem. SOC. Perkin Trans. I t . . pp. 365-369. 34 HALL,B. G. MURRAY, M..OSBORNE, S. AND SINNOIT.M. L. (1983). The
catalytic consequences of experimental evolution. Part 111. Construction of reaction profiles for hydrolysis of lactose by ebg", ebg", and ebg" enzymes via measurements of the enzyme-catalyzed exchange of galactose-l-180 by 13C NMR spectroscopy. J. Chem. SOC. Perkm Trans. It, 1595-1598. 35 KRICKER, M. AND HALL,B. G. (1984). Directed evolution of cellobiose utilization in Escherichia coli. Mol. B i d . & Evol. 1. 171-182. 36 HALL.B . G. (1988). Adaptive evolution that requires multiple spontaneous mutations. I . Mutations involving and inscrtion sequence. Generia 120. 887-897. 37 HALL. B. G. (1989). Selection, adaptation, and bacterial operons. Genome 31, 265-271. 38 HALL,B. G. (1990). Spontaneous point mutations that occur more often when they are advantageous than when they arc neutral. Genetics 126. 5-16.
Barry G. Hall is at the Department of Biology, Hutchison Hall, University of Rochester, Rochester, NY 14627, USA.
'1
"Stop complaining, Munshaw. You knew you'd be working with a computer when we hired you. 'I
Reprinted courtesy OMNl Magazine @ 1985
