Yeast
Yeast 2004; 21: 703–746.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1113
Review
A history of research on yeasts 7: enzymic adaptation
and regulation
James A. Barnett*
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK
*Correspondence to:
James A. Barnett, School of
Biological Sciences, University of
East Anglia, Norwich NR4
7TJ, UK.
E-mail: j.barnett@uea.ac.uk
Received: 18 December 2003
Accepted: 20 January 2004
Keywords: history; yeast research; enzymic adaptation; enzyme induction and
repression; galactose pathway; GAL genes; Frédéric Dienert; Solomon Spiegelman;
Luis Leloir
The phenomenon of enzymatic adaptation may be simply stated in the following terms: a
population of cells placed in contact with some substrate acquires, after a lapse of some time,
the enzymes necessary to metabolize the added substrate (Spiegelman, 1946 [248] p. 256).
Contents
Introduction: the scope of this article
Dienert’s work on adaptation of yeast to galactose
Enzymic adaptation or selection of mutants?
Galactose fermentation by yeasts
Monod’s work on lactose utilization by Escherichia coli
Gratuitous induction
‘Permeases’: transport of metabolites into the cells
Carbon catabolite repression
The operon
The galactose pathway in yeasts: work of Leloir and others
Uridine diphosphate sugars
Genetic regulation of the galactose pathway
Regulation of the GAL genes
Genetic regulation of the utilization of other substrates
Maltose utilization: the MAL genes
Sucrose utilization: invertase and the SUC genes
Conclusion
References
Appendix: sugar structures
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Introduction: the scope of this article
The present article concerns mechanisms by which yeasts regulate the amount of certain enzymes in their
cells. Such regulations enable microbes to adapt to the different nutrients available to them in changing
environments. The history of research on these regulatory processes in yeasts is the subject discussed
below.
Most of the work which has explained the general phenomenon of enzymic adaptation1 has been done
by studying the microbial utilization of two sugars, lactose and D-galactose, both of which have long been
relatively easy to obtain and purify.2 Accordingly, this article concentrates on the research done on the
utilization of these two sugars. The structures of these and other sugars and sugar derivatives discussed
herein are shown in an appendix at the end of this article.
Frédéric Dienert3 (Figure 1), who worked on galactose utilization by yeasts in 1900, gave the first clear
account of enzymic adaptation. His research eventually led to two major advances — in biochemistry
and in molecular biology. (a) In the late 1940s and early 1950s, the great Argentinian biochemist,
Luis Leloir4 (Figure 2) worked out the pathway of galactose catabolism in yeasts. He found that this
pathway involved nucleoside diphosphate sugars and demonstrated the importance of these compounds
in biosynthetic pathways. (b) Although Leloir studied mainly Kluyveromyces marxianus, his work on the
galactose pathway made it possible to establish the details of how that pathway is controlled genetically in
Saccharomyces cerevisiae. In the latter yeast, the GAL gene system, which controls galactose utilization,
became the most extensively studied mechanism of biochemical–molecular genetic regulation in any
eukaryote. In turn, this work on galactose metabolism and its control was of medical significance,
explaining the biochemistry of the severe human autosomal recessive disorder, galactosaemia.5
A great deal of the research on enzymic adaptations by yeasts was made possible by the researches
carried out at the Institut Pasteur in Paris in the 1940s and 1950s by Jacques Monod6 and his colleagues.
By studying the β-galactosidase7 activity of Escherichia coli, they elucidated the general characteristics of
enzyme induction [54,183,189]. Accordingly, some of their work is described below. In his biographical
memoir of Monod, André Lwoff8 explains:
The enzyme β-galactosidase . . . was only a tool for the understanding of the relation between
genes and enzymes . . . When the work on the induced synthesis of enzymes was started in
1941, nothing was known except the phenomenon; the concepts developed essentially from
1948 on. In the first phase, biochemical, Melvin Cohn played a determining rôle. In the second,
genetical and regulatory, François Jacob’s intervention had been essential . . . Between 1948
1 Even
today, ‘adaptation’, the action of adapting, is still sometimes written ‘adaption’ as it was by the Anglo-Irish satirist Jonathan
Swift (1667–1745) in A Tale of a Tub (1704) [212] and in 1790 by the English chemist Charles Blagden (1748–1820) ([28] p. 344).
2 When the water is evaporated from whey, which is a by-product of cheese-making, crystalline lactose (milk sugar) is deposited and this
is easily purified by recrystallization ([32] pp. 708–709; [113] p. 496). Pasteur discovered D-galactose (which he called ‘lactose’) in 1856
by hydrolysing lactose [214]. Indeed, it is usually prepared by the acid hydrolysis of lactose forming glucose and galactose, the latter
then being separated from the glucose by direct fractional crystallization. The glucose may also be removed by a non-galactose-utilizing
yeast and the galactose then crystallized ([32] pp. 602–603; [245] p. 89).
3 Frédéric Vincent Dienert (1874–1948), French biologist, is known for his public health work on purifying water. He became Inspector
General of the Paris Water Surveillance Service, honorary professor at the Institut Agronomique and, in 1946, President of the French
Academy of Agriculture [4,33,156,157].
4 Luis Federico Leloir (1906–1987), Argentinian biochemist, received the Nobel Prize for chemistry in 1970 ‘for his discovery of sugar
nucleotides and their role in the biosynthesis of carbohydrates’. He became professor at Buenos Aires University. [209,305].
5 Patients with galactosaemia are unable to metabolize galactose, because of a deficiency of one of the enzymes of the galactose pathway.
D-Galactose 1-phosphate accumulates, damaging liver, central nervous system and other parts of the body [239,240].
6 Jacques Monod (1910–1976) was French and one of the greatest of microbiologists.Working at the Institut Pasteur in Paris, he was a
founder of molecular microbiology and largely responsible for the concept of allostery [163,164].
7 β-Galactosidase is the enzyme which hydrolyses lactose to D-glucose and D-galactose.
8 André Michel Lwoff (1902–1994), French microbiologist, worked at Institut Pasteur, Paris, from 1921, was professor at the Sorbonne
from 1959 to 1968 and shared the Nobel Prize in Physiology or Medicine with Monod and Jacob. He worked on the genetics of
bacteriophages and, like Monod, was one of the originators of molecular biology [123].
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Figure 1. Frédéric Dienert. Photograph from La Technique Sanitaire et Municipale, vol 3 (1948). Reproduced by kind
permission of the Secrétaire perpétuel, Académie d’Agriculture de France
Figure 2. Luis Leloir (1970).  The Nobel Foundation
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and 1963, the main problems posed by the induced synthesis of enzymes (that is regulation)
were solved, and molecular biology was created ex nihilo ([163] p. 399).
Melvin Cohn9 writes that it was he who decided to work with β-galactosidase because ‘it was the only
enzyme that could withstand fractionation at the summer temperature of Paris. Monod was on vacation
and the Pasteur Institute had no cold rooms, a primitive centrifuge, but it did have amonium sulfate’ (M.
Cohn, personal communication).
The workers at the Institut Pasteur made very considerable advances in understanding the physiological
and molecular control of (a) enzyme induction and repression, and (b) the mechanisms by which
substrates enter microbial cells. In their studies of the regulation of metabolism, Monod and François
Jacob10 (Figure 3) established the rôle of allosteric enzymes [190]. The activity of such enzymes depends
on their conformation (or shape) which is changed by the binding (attachment or detachment) of another
molecule, an effector.
Monod and Jacob coined the expression ‘allosteric inhibition’; that is, to use Monod’s words, when
an enzyme:
. . . inhibitor is not a steric analogue of the substrate [of that enzyme]. We propose therefore
to designate this mechanism as ‘allosteric inhibition’ ([190] p. 391).
and he emphasized:
Figure 3. Jacques Monod and François Jacob (1966). Photograph from Paris-Match
9 Melvin Cohn (b. 1922), American microbial biochemist, worked at the Institut Pasteur, Paris from 1949 to 1955 and is now at the Salk
Institute for Biological Studies, La Jolla, California. He was professor at Washington University (1955–1958) and at Stanford University
(1959–1961). His more recent work has been on immunology (M. Cohn, personal communication).
10 François Jacob (b. 1920), French microbial geneticist and molecular biologist, shared the Nobel prize for physiology or medicine in
1965 with Lwoff and Monod. When the Germans occupied France in 1940, Jacob came to London and joined the Free French forces,
serving in North Africa. In 1944, he was badly wounded in Normandy and received the Croix de la Libération. In 1964, Jacob became
professor at the Collège de France ([Nobel Lectures Physiology or Medicine 1963–1970] p. 172).
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. . . the regulatory role of allosteric proteins is absolutely fundamental; it explains everything:
hormonal action, repressor function, non-Michaelian enzyme kinetics . . . (Monod, 1961;
quoted in [276] p. 167).
The binding of an effector molecule at a second (non-catalytic) site alters the enzyme’s activity and
so provides a means for its regulation [186].
Furthermore, in 1960, the Institut Pasteur researchers also published [127] the concept of the operon,
a unit of coordinated gene activity, which made it possible to understand the underlying regulation of
inducible and repressible enzymes. They commented that up to that time:
. . . the word ‘gene’ . . . [was used to designate] a DNA molecule whose specific self-replicating
structure can, through mechanisms unknown, become translated into the specific structure of
a polypeptide chain ([125] p. 318).
Monod and his remarkable colleagues were responsible for some outstanding achievements. Their
excitement and enthusiasm is abundantly clear in their publications, occasionally obscuring some
of the experimental detail! The work was, however, described in many extensive and clearly
written reviews, many by Monod himself, making the subject both well-known and well understood
[35,50,51,54,56,125,126,180,182,189,220,249,252,260,277].
Dienert’s work on adaptation of yeast to galactose
In the middle of the nineteenth century, Louis Pasteur11 was the first to report alterations in the behaviour
of yeast in response to an environmental change, when he found that switching between aerobic and
anaerobic conditions affected the rate of sugar fermentation [215,216]. Although the great chemist, Emil
Fischer, had studied the utilization of a number of sugars by various yeasts in the 1890s (see [23]), it was
not until 1900 that Dienert gave the first lucid account of the special adaptations which yeasts undergo,
enabling them to use certain sugars. In a 50-page paper on galactose fermentation, which constituted his
thesis for a doctorate, Dienert used the word ‘l’accoutumance’ (habituation) for these adaptations. With
astonishing foresight, Dienert compared the adaptation he described to mammalian antibody production,12
as did Macfarlane Burnet13 over 50 years later [34] and Monod considered Dienert’s paper to be ‘one of
the classics’ on enzymic adaptation ([180] p. 231).
Many microbial biochemists adopted Henning Karström’s14 term ‘adaptive’, which he proposed in 1938
for microbial enzymes produced in specific response to the presence of an appropriate substrate [132].
The term referred to the general and widespread phenomenon, which Dienert had studied: that an adaptive
enzyme is one synthesized by cells only when they are in contact with a substrate for the metabolizing
of which the enzyme is necessary. By contrast, those enzymes which are present, irrespective of the
availability of any substrate, were called ‘constitutive’. Later, several kinds of physiological regulatory
mechanism (Table 1) have been found to be responsible for enzymic ‘adaptivity’. At the molecular level,
11 Some accounts of the following are given in earlier articles: E. Fischer [23], H. M. Kalckar [21], C. C. Lindegren [23], P. M. Nurse
[25], L. Pasteur [20], M. Stephenson [23], H. K. A. S. von Euler-Chelpin [21], Ø. Winge [23].
12 C’est ce qui arrive également chez les animaux qui perdent de leur immunité contre une toxine, dès que la toxine cesse d’agir ([69]
p. 152).
13 Frank Macfarlane Burnet (1899–1985), Australian virologist, immunologist and Nobel Prize winner, worked on the characteristics
and replication of bacteriophages and animal viruses and generated the clonal selection theory of antibody production. The third edition
of his Natural History of Infectious Disease (1962; the last edition of which he was sole author) is still marvellous to read [87,208].
14 Henning Karström (1899–1989) was, with the Nobel prize winner Artturi Virtanen, a pioneer of Finnish biochemistry. He worked on
food chemistry and nutrition but, in the 1940s, gave up science for religion (information kindly given by Anna-Maija Pietila, Helsinki
University Library).
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Table 1. Mechanisms underlying enzymic adaptations involving changes in the amount of enzyme in the
cell (after Entian and Barnett [81])
Kind of
regulation
Physiological observation
Enzymic or molecular mechanism
Induction
Increase in enzymic activity in response to
presence of inducer (i.e. substrate or
structurally similar compound)
Inducer evokes activation of transcription
Derepression
Increased specific activity after removing
repressing substrate
De-inhibition of transcription
Inactivation
Irreversible loss of enzymic activity
Specific proteolysis of the enzyme
control can be exerted at the level of (a) transcription, affecting mRNA synthesis, or (b) translation,
affecting protein synthesis from the mRNA transcript.
Reports of apparent enzymic adaptations had appeared even before Dienert’s work was published. In
1882, Julius Wortmann15 found that ‘Bacterium termio’ did not produce amylases unless starch was in
the growth medium [300] and in 1898, Julius Katz of the Botanical Institute at Leipzig noted that the
amylase (diastase) of a strain of a Penicillium species increased when growth was in the presence of
starch [133]. There was also a report, based on inadequate experimental evidence, of yeasts adapting to
sucrose [75]. It was Dienert, however, who in 1899 and 1900, when working under Émile Duclaux,16
published the first clear evidence of enzyme induction [67,68,69]. Using washed cells of brewer’s yeasts
and also of yeasts which fermented lactose, Dienert reported adaptation to galactose in the absence of
cell division. Hence he was not selecting mutants: the yeast cells which were present were adapting to
the sugar. They fermented D-galactose after a few hours of adaptation, whereas glucose was fermented
without delay. Furthermore, he found that some yeasts which he had adapted to galactose, had also
adapted to lactose. The kind of flask Dienert used for his adaptation experiments is shown in Figure 4.
The following were Dienert’s salient findings. (a) The rate of galactose fermentation depends on which
sugars are present in the growth medium; but the rate of glucose fermentation varies very little, irrespective
of what the yeast is grown on. (b) When galactose-grown cells are re-grown in medium containing glucose
they lose their ‘galactozymase’ (that is, the enzymic mechanism by which galactose is catabolized).
(c) When yeasts are grown in medium containing galactose, lactose or melibiose, galactozymase activity
is maximal; for those grown with sucrose or maltose, the galactozymase activity is slight; yeasts grown
a
Figure 4. Flask used for adaptation experiments by Dienert [69]. Yeast accumulates in the little bulb (a) in the base of
the vessel
15 Julius Wortmann (1856–1924), German botanist, worked inter alia on Saccharomyces cerevisiae in relation to vinification at the
Geisenheim research station for viticultural problems [194].
16 Émile Duclaux (1840–1904), French chemist and bacteriologist, helped Pasteur in his work on diseases of silk-worms and succeeded
him as director of the Institut Pasteur, Paris [169].
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on glucose or fructose have no galactozymase. (d) Yeasts grown on either melibiose or galactose form
α-galactosidase (melibiase). He wrote:
Galactose fermentation is possible only when the yeast is adapted to this sugar. The time of
adaptation varies with different yeasts . . . Adapted yeasts ferment glucose about 1.6 times
faster than galactose . . . An adapted yeast loses its adaptation little by little if it is given a
sugar other than galactose, lactose or melibiose. If growth occurs, the adaptation is destroyed
after a few hours . . . Adaptation does not affect the morphological characteristics of the yeasts
. . . Certain substances prevent adaptation without preventing the fermentation of glucose (e.g.
borate and toluene).17
In 1936 Marjory Stephenson and John Yudkin18 were not as generous as Monod, when they commented:
These experiments of Dienert, though highly suggestive, are difficult to interpret owing
principally to their non-quantitative nature, rates of fermentation, quantity of yeast and change
in cell numbers not being recorded ([266] p. 506).
Table 2 gives the results of some of Dienert’s experiments and in 1908 Arthur Slator19 confirmed
those findings (Table 3). At about the same time, Edward Armstrong20 named several yeasts which did
not adapt to galactose [14] (Table 4), although all could ferment glucose, fructose and mannose without
adaptation. In 1910 Arthur Harden confirmed that ‘some yeasts can be trained to ferment galactose by
cultivation in a medium containing that sugar’ and that ‘such a trained yeast yields a juice capable of
fermenting galactose’ ([111] p. 649).
Dienert’s publications also stimulated work on enzymic adaptations in multicellular organisms. For
example, in 1906 Ernst Weinland21 reported that repeated intravenous injections of sucrose induced
invertase activity in a dog’s plasma [288].
Enzymic adaptation or selection of mutants?
Yudkin pointed out:
As would be expected, when organisms possessing a newly acquired enzyme are grown in
the absence of the substrate, enzymes arising by adaptation are readily lost whilst enzymes
arising by mutation and selection tend to be permanent (Yudkin, 1938 [301] p. 104).
Indeed, as there were often difficulties in distinguishing between enzymic adaptation and the selection
of genetic variants, many experiments were done to decide whether microbial adaptive adjustments were
17 La fermentation du galactose n’est possible que lorsque la levure s’est acclimatée à ce sucre. La durée de l’acclimatation varie avec les
levures . . . Chez les levures acclimatées, le glucose fermente environ 1,6 fois plus vite que le galactose . . . Une levure acclimatée perd
peu à peu son acclimatation si on lui offre un autre sucre que du galactose, du lactose ou du mélibiose. Si on favorise la multiplication,
la perte de l’acclimatation se produit au bout de quelques heures . . . L’effet est nul sur les propriétés morphologiques des levures . . .
Certaines substances empêchent l’acclimatation sans empêcher la fermentation du glucose (BoO3− , toluène) ([69] p. 187).
18 John Yudkin (1910–1995) English biochemist and nutritional physiologist, worked with Marjory Stephenson on enzymic adaptation in
yeast and bacteria for about 5 years and simultaneously studied medicine. From 1946, he was professor of physiology at King’s College
of Household and Social Science of London University (later Queen Elizabeth College) (M. D. Yudkin, personal communication).
19 Arthur Slator (1879–1953), English brewing specialist, published work on rates of activities of various microbes and became chief
chemist to Bass brewers at Burton-on-Trent [5,128].
20 Edward Frankland Armstrong (1878–1945), English carbohydrate chemist with wide scientific interests, obtained his doctorate at the
University of Berlin and published six papers on sugar chemistry with Emil Fischer. Armstrong had a career in industries concerned
with biscuits, soup, dyestuffs, coal and gas [94,232].
21 Ernst Weinland (1869–1932), German medical physiologist, worked on anatomy, physiology and biochemistry at Munich, Tübingen
and Leipzig. He held a chair in physiology at Erlangen from 1913 to 1932 [103].
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Table 2. Results of some of Dienert’s experiments [69]. In each flask of
the type shown in Figure 4, the yeast was grown in yeast extract to which
a sugar was added as indicated in the table. After growth, the medium was
drawn off and the yeast, which accumulated in the bulb at the base of the
flask, was washed in sterile water. The washing water was then replaced by
a galactose solution. Incubation was at 25 ◦ C; the time was noted when 1 gas
bubble per second was produced and this time was treated as the beginning
of fermentation
Yeast
Sugar added to
growth medium
Delay before beginning
of fermentation
A yeast growing on lactose
Sucrose
Galactose
Lactose
Maltose
2h
1h
0.5 h
2h
‘Frohberg’ yeast (a
bottom-fermenting beer
yeast ([105] pp. 233–234)
Glucose
Sucrose
Fructose
Melibiose
Galactose
2 days
2 days
2 days
1h
1h
Table 3. Rates of fermentation of galactose or glucose by two yeast
species when grown in either (a) brewer’s wort (an aqueous extract of
malt) or (b) a solution of hydrolysed lactose + a trace of wort. Results of
Slator in 1908 ([242] p. 224)
Relative rates
Yeast
Growth medium
Glucose
Galactose
Saccharomyces cerevisiae
Wort
Hydrolysed lactose
100
100
<1
21–77
Saccharomyces pastorianus
Wort
Hydrolysed lactose
100
100
<1
25–155
caused by selecting a minority of cells which already had the ability to use the substrate in question, or
by the action of that substrate on most of the cells present.
Such questions were asked when ‘training’ Salmonella typhi to use lactose (1907) [275] or galactitol22
(1911) [218]. Working with yeasts, several authors in the 1920s concluded that new cells had to be
formed for these adaptations to occur. Various methods were used to stop cell division, such as adding
0.5% phenol [279,284] or measuring enzymic activity at 38 ◦ C [244]. However, such experiments were
usually inconclusive, as the techniques may well have also stopped synthesis of the required enzyme.
In 1933, Stephenson and her colleagues showed that adaptive enzyme synthesis occurs without cell
reproduction in both yeasts and bacteria, for example in the presence of formate, E. coli synthesizes
‘formic hydrogen lyase’23 without cell multiplication [265]. Three years later, they also detected adaptive
formation of galactozymase in non-proliferating cells of S. cerevisiae [266]. When washed cells, deficient
in galactozymase, were suspended in galactose solution, galactozymase activity developed within an
hour, that is before appreciable cell division could occur. Cell counts confirmed that enzyme production
occurred without the numbers of cells increasing. The criterion used for assessing enzymic activity was
22 Galactitol
(also called dulcitol or dulcite) is the reduction product of galactose (Appendix).
hydrogen lyase’, catalysing formic acid → CO2 + H2 , is made up of two enzymes [217,223,286]: (a) formate dehydrogenase
[EC 1.2.1.2], which catalyses formate + NAD+ → CO2 + NADH; (b) hydrogen dehydrogenase [EC 1.12.1.2], which catalyses H2 +
NAD+ → H+ + NADH [264].
23 ‘Formic
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Table 4. Selected publications arranged chronologically on enzymic ‘adaptation’ of yeasts
Date
Authors
System studied
Findings
1888
Bourquelot [30]
Galactose fermentation by brewer’s
top- and bottom-fermenting yeasts
Fermentation of galactose enabled by
presence of small amounts of glucose or
fructose
1900
Dienert [69]
Galactose fermentation by brewer’s
yeasts and lactose-fermenting yeasts
Washed cells ferment galactose only after
induction period with galactose, but glucose
fermented without delay. Adaptation can
occur without cell multiplication
1905
Armstrong [14]
Galactose fermentation by several
yeasts
Some yeasts do not adapt to ferment
galactose, e.g. Hanseniaspora uvarum,
Saccharomycodes ludwigii,
Schizosaccharomyces octosporus,
Schizosaccharomyces pombe, Williopsis
saturnus
1908
Slator [242]
Galactose fermentation by
Saccharomyces pastorianus
This yeast fermented galactose when grown
on hydrolysed lactose, but not when grown
on beer wort
1910
Harden and Norris [111]
Saccharomyces pastorianus galactose
fermentation
Confirmation that yeast could be ‘trained’ to
ferment galactose and extracts of such yeast
also ferment galactose
1936
Stephenson and Yudkin
[266]
Galactose fermentation by
Saccharomyces cerevisiae
Adaptive enzymes formed without cell
division
1939
Schultz and Atkin [237]
Maltose fermentation by baker’s
yeast
Long induction period for α-glucosidase
formation
1940
Schultz, Atkin & Frey
[238]
Maltose and galactose fermentation
by baker’s yeast
Presence of oxygen shortens induction
period
1944
Spiegelman, Lindegren
and Hedgecock [253,254]
Aerobic growth of haploid and
diploid strains of S. cerevisiae on
D-galactose
When growing aerobically on D-galactose,
diploid cells adapted without cell division;
adaptation of haploid cells involved growth
1947
Spiegelman, Reiner and
Morgan [256]
Galactose fermentation by S.
cerevisiae
Extracts from adapted, but not unadapted,
yeast ferment galactose; but both extracts
ferment glucose
1951
Spiegelman, DeLorenzo
and Campbell [250]
S. cerevisiae adaptation to galactose
Perhaps the first published evidence of
regulator genes in a yeast
1956
Davies [60]
Invertase of Kluyveromyces marxianus
Glucose represses synthesis of invertase
1956
Davies [61]
β-Galactosidase of Kluyveromyces
marxianus
β-Galactosidase synthesis is repressed by
glucose and >0.2 mM galactose
1958
Duerksen and Duerksen
and Halvorson [76]
β-Glucosidase of Rhodotorula minuta
(misnamed Saccharomyces cerevisiae
[18])
Inducible β-glucosidase catalyses hydrolysis
of aryl and alkyl β-D-glucosides
1959
Duerksen and Halvorson
[77]
Specificity of inducing β-glucosidase
of Rhodotorula minuta
Methyl β-D-glucopyranoside is a particularly
strong inducer of the β-glucosidase
1960
MacQuillan, Winderman
and Halvorson [165]
Induction and glucose repression of
β-glucosidase of hybrid
Kluyveromyces marxianus ×
Kluyveromyces dobzhanskii
At least two distinct sites involved in
regulating β-glucosidase synthesis: an
induction site and a repressor site
1962
Sutton and Lampen [270]
S. cerevisiae sucrose utilization
Invertase repressed by D-glucose
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Table 4. Continued
Date
Authors
System studied
Findings
1964
Heinrich [116]
Galactokinase from Kluyveromyces
marxianus
Nineteen strains grown on galactose or
lactose give about same yield of
galactokinase; those on glucose give none
1964
Halvorson, Okada and
Gorman [109]
S. cerevisiae: methyl α-D-glucoside
carrier and two α-D-glucoside
hydrolases, using antisera specific to
precipitate each enzyme, leaving the
other active
Oligo-1,6-glucosidase (isomaltase) is
inducible. The inducer, ethyl
1-thio-α-D-glucopyranoside (α-TEG), enters
the cells by constitutive facilitated diffusion
or inducible active transport
1965
Polakis and Bartley [219]
Activities of various enzymes of S.
cerevisiae grown on different carbon
sources
Glucose represses malate synthase,
isocitrate dehydrogenase (NADP+ ) and
(NAD+ ), glutamate dehydrogenase,
aconitate hydratase
1965
Kohlhaw, Drägert and
Holzer [138]
Regulation of glutamine synthetase
(glutamate–ammonia ligase) of S.
cerevisiae
NH4 + , glutamine or asparagine repress
synthesis of glutamine synthetase
1968
Cirillo [48]
Galactose transport by haploid
strains of S. cerevisiae
Inducible galactose transport is by facilitated
diffusion and depends on GAL2
1968
Barnett [Barnett 1968]
Catabolism of alditols Candida
saitoana
Various alditol dehydrogenases inducible
1971
Ferguson and Sims [88]
Glutamine synthetase and glutamate
dehydrogenase of Candida utilis
Both enzymes subject to ammonia
repression and inactivation
1972
Gascón and Ottolenghi
[93]
Effect of D-glucose concentration on
invertase of diploid S. cerevisiae
Invertase concentration increases as
exogenous glucose concentration decreases
1975
Barnett [17]
Transport of D-ribose by Pichia pini
Pichia pini has two ribose carriers, one
inducible and the other constitutive
1979
Dickson, Dickson and
Markin [65,66]
Kluyveromyces lactis β-galactosidase
Induction of β-galactosidase by lactose
1980
Krátký and Biely [140]
Xylan degrading system of
Cryptococcus albidus
Induction of extracellular β-xylanase is
accompanied by induction of an active
transport system for methyl β-D-xyloside
and β-1,4-xylo-oligosaccharides
1981
Williamson, Young and
Ciriacy [292]
Alcohol dehydrogenase (ADH) of S.
cerevisiae
Glucose represses constitutive ADHII, which
is involved in ethanol utilization
1983
Dickson and Barr [64]
Kluyveromyces lactis lactose transport
Induction of lactose carrier by lactose or
galactose
carbon dioxide production, measured manometrically. Stephenson followed this work with a study of the
‘adaptability of galactozymase’ in E. coli [263].
In 1944, by means of a simple and elegant experiment with two strains of S. cerevisiae, Solomon
Spiegelman24 (Figure 5) and Carl Lindegren addressed the practical problem of distinguishing between
enzymic adaptation and the selection of mutants. Both strains of S. cerevisiae could adapt to ferment
galactose, but one was haploid and phenotypically heterogeneous for galactose fermentation, while the
other was diploid and homogeneous. The amount of galactose fermented by the former was directly
related to the number of cells in the culture, so this was an example of selection; while the rate of
24 Solomon Spiegelman (1914–1983), American microbiologist, was professor of microbiology at the University of Illinois, Urbana, and
in 1968 became professor at Columbia’s College of Physicians and Surgeons [287].
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713
Figure 5. Soloman Spiegelman. Photo courtesy of the University of Illinois at Urbana-Champaign Archives; record series
number 26/4/1, Alumni and Faculty File
galactose fermentation by the latter strain was almost independent of the number of cells and hence, as
there was no growth, this was a case of physiological adaptation (Figure 6) [254].
Galactose fermentation by yeasts
By the late 1940s, the fermentation of galactose by yeasts had become the most thoroughly studied
system of enzymic adaptation and the then current findings may be summarized as follows:
1. Yeasts which could ferment galactose did so only after being incubated with galactose [69,111,242,246,
266].
2. This adaptation did not depend on cell multiplication and, hence, selection was not involved
[69,246,266].
3. Cell-free extracts from adapted yeast could ferment galactose; extracts from unadapted cells did not do
so, although they fermented glucose [111,256]. These observations showed that the adaptation could
not be explained simply in terms of changes in the permeability of the cells.
In the course of his work on adaptation to galactose, Spiegelman reported a singular observation on
Schizosaccharomyces pombe, which is usually held to be galactose-negative [14,141,143,161,262]. He
found that when cultures composed of >20% asci (as meiotic products, the ascospores would be haploid)
were inoculated into a medium of 8% galactose and 2% glucose, some strains could be isolated which
utilized galactose. Consistent with Spiegelman’s finding, a more recent work on yeast systematics records
that some strains can use galactose after a delay of more than 7 days ([24] p. 678). However, the
nature of this ‘adaptation’, if indeed it occurs, does not seem to have been investigated and Paul Nurse
(personal communication, 2003) comments that if indeed Spiegelman selected mutants, such mutants
would probably be rare.
By 1950, microbiologists had examined adaptations of many other enzymes ([249] pp. 268–270), such
as those by yeasts for: (a) invertase, studied by Hans von Euler-Chelpin and his colleagues [278,280–284];
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J. A. Barnett
Figure 6. Adaptation to D-galactose without cell multiplication by a strain of S. cerevisiae: an experiment of Spiegelman
and his colleagues carried out in 1943. Cells grown on D-glucose were washed in 67 mM KH2 PO4 , then resuspended in
phosphate under nitrogen and D-galactose added. Carbon dioxide produced anaerobically was measured manometrically
for 5 h. , CO2 production (µl3 /h); , log of number of cells/cm3 ([254] Figure 2). Reproduced by permission
°
(b) α-galactosidase (‘melibiozymase’); (c) α-glucosidase (‘maltozymase’) [251,255]; and (d) also those
acting on nitrogen compounds, e.g. asparagine by Candida utilis [98].
Spiegelman followed up some earlier observations [238] and, in 1945, found that the adaptation of Saccharomyces spp. to galactose was much faster aerobically than anaerobically (Figure 7) [247]. He studied
some strains which completely failed to adapt to galactose in anaerobic conditions; these yeasts had to
adapt aerobically, then galactose could be fermented to provide energy for synthesizing more enzyme.
Monod’s work on lactose utilization by E. coli
In the early 1950s, Monod and his colleagues proposed abandoning the expression ‘enzymic adaptation’
and, instead, adopted the phrase ‘the induced biosynthesis of enzymes’.25 Indeed, in a letter to Nature,
25 Nous proposons donc d’abandonner l’expression d ‘adaption enzymatique’ [sic] pour adopter celle de ‘biosynthèse induite des enzymes’
. . . ([189] p. 68).
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715
Figure 7. Spiegelman’s report in 1945 of the difference in rate of adaptation to D-galactose by a strain of Saccharomyces
pastorianus aerobically ( ) and anaerobically ( ). D-Glucose-grown yeast was washed free of glucose and resuspended in
67 mM KH2 PO4 . CO2 production was measured manometrically after adding galactose to the suspension, after flushing
with nitrogen in the case of the anaerobic yeast ([247] Figure 2)
°
they suggested a whole new terminology (Table 5). Martin Pollock,26 although one of the signatories
to the letter, recalls his opposition to ‘the dogmatic presentation of the opinions of a self-appointed
clique . . . When I refused to sign, . . . Jacques [Monod] resorted to (subtly flattering) blackmail by
threatening that in that case it would not be sent for publication at all’ ([221] p. 67). Another signatory,
Melvin Cohn, comments: ‘why were we so insufferably sure of ourselves is not clear to me’ ([55]
p. 80].
The continual success of Jacques Monod’s work explains why they were so sure of themselves. From the 1940s, he changed the whole course of research on enzymic adaptation and
much else besides. Monod’s doctoral thesis of 1941 [179], which had been devoted to the systematic study of the kinetics of the growth of E. coli, underpinned a remarkable proportion of
subsequent work on microbial physiology. He clarified two major experimental concepts, growth
rate and growth yield, and established the dependence of growth rate on the concentration of a
limiting carbon and energy source and also the independence of growth yield from growth rate
[181].
Lwoff writes:
Jacques Monod has told how, in December 1940, at the Institut Pasteur, he came and showed
me [a] . . . diauxic curve27 and asked: ‘What could that mean?’. I said it could have something
to do with enzymatic adaptation. The answer was: ‘Enzymatic adaptation, what is that?’ I told
Monod what was known — what I knew — and he objected that the diauxic curve showed an
inhibition of growth rather than an ‘adaptation’. We know today that repression and induction
are complementary, but I simply repeated that diauxy should be related to adaptation. . . .‘From
this very day of December 1940’, wrote Jacques Monod, ‘all my scientific activity has been
26 Martin Rivers Pollock (1914–1999), English medically-qualified microbial biochemist, became professor of biology at Edinburgh
university (1965–1976). Monod once remarked that Pollock’s election to a Royal Society fellowship ‘was a mistake’ [3,221].
27 In the thesis for his doctorate, Jacques Monod wrote, ‘I must apologise for having to invent a new term to describe this phenomenon:
that is, ‘diauxy’ (double growth)’. Je m’excuse d’avoir dû, pour désigner ce phénomène, créer un terme nouveau: celui de ‘diauxie’
(croissance double) ([179] p. 139).
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Table 5. Terminology of enzymic adaptation, proposed in 1953 by Cohn, Monod, Pollock,
Spiegelman and Stanier [57]
Term
Meaning
’Enzyme induction’
Increase in rate of synthesis of a specific apo-enzyme resulting
from exposure to a chemical substance
’Enzyme inducer’
Any substance inducing enzyme synthesis
’Inducible enzyme’
An enzyme-forming system which can be activated by an
exogenous inducer
’Induced enzyme’
Enzyme formed by induction
’Constitutive enzyme’
One formed in ‘considerable amounts’ in the absence of an
exogenous inducer. The amount is often increased by specific
induction
’Constitutivity’ and ‘inducibility’
Properties of enzyme-forming system, not of enzymes per se
’Sequential induction’
A single substance may induce a sequence of enzymes
Note: Certain substrates of induced enzymes do not induce them; and some inducers are not substrates
of the enzymes they induce
devoted to the study of enzymatic adaptation.’ Yet during the dark years, he had joined the
underground. He had even been arrested by the Gestapo, but cleverly managed to escape
([163] p. 388).
And Monod tells us:
Lwoff’s intuition was correct. The phenomenon of ‘diauxy’ that I had discovered was indeed
closely related to enzyme adaptation, as my experiments, included in the second part of my
doctoral dissertation, soon convinced me. It was actually a case of the ‘glucose effect’ . . .
today better known as ‘catabolite repression’ . . . ([184] p. 189).
Monod’s assessment is well-illustrated by the results of an experiment he published in 1946, when he
and a colleague worked with a lactose-utilizing strain of E. coli, which showed a typical diauxic curve
when grown in a medium containing a mixture of glucose and lactose (Figure 8). Here was an early, but
clear, representation of the ‘glucose effect’ or ‘carbon catabolite repression’, which is discussed below.
Suspensions of this strain grown (a) on lactose (‘adapted’) or (b) on glucose (‘non-adapted’) were tested
for the ability to respire lactose. Adapted cells respired fast; non-adapted cells respired no faster than
the negative control cells without exogenous substrate, and the adaptive enzyme systems were shown
to be determined genetically [185]. Glucose repression was clearly an economy measure: unnecessary
enzymes, such as β-galactosidase, are not synthesized when so readily catabolized a compound as glucose
is present.
Gratuitous induction
In 1947, concluding that microbial enzymes which attack exogenous substrates are usually ‘adaptive’,
Monod defined ‘enzymatic adaptation’ as ‘apo-enzyme28 formation induced by a specific substrate’ ([180]
p. 226). However, not long afterwards, certain enzymes were shown to be induced by compounds which
are not substrates of those enzymes. Spiegelman and his colleagues found that a strain of S. cerevisiae,
grown on or otherwise adapted to maltose, formed two enzymes (I and II) which had different substrate
28 Apoenzyme is the protein component of an enzyme; an enzyme with a tightly bound cofactor (prosthetic group) is a holoenzyme
[1,205].
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717
Figure 8. Diauxy in E. coli. This figure, from Monod’s thesis of 1942 [179], represents growth of a culture in a defined
medium containing 0.4 mM-D-glucose and 0.8 mM-lactose
Table 6. Two enzymes with α-D-glucosidase activities formed by
a strain of Saccharomyces cerevisiae when grown on maltose [258].
Substrate specificities of the two enzymes in Saccharomyces spp. were
established by various authors (see footnotes); for general reviews of
α-D-glucopyranoside hydrolases, see [99,100,146,271,272]
Enzyme
Substrate
Sucrose
Maltose
Phenyl α-D-glucopyranoside
Isomaltose
Methyl α-D-glucopyranoside
Furanose
α-Glucosidase
(maltase)
[EC 3.2.1.20]a
Oligo-1,6glucosidase
(isomaltase)
[EC 3.2.1.10]b
+
+
+
−
−
+
+
−
−
+
+
−
+, Sugar hydrolysed; −, sugar not hydrolysed.
a [47,107,108,110,136,144,151,162,173,200,271,272]. This enzyme hydrolyses terminal, (1→4)-linked α-D-glucopyranosyl groups, releasing α-D-glucose; oligosaccharides
are hydrolysed rapidly, and polysaccharides slowly or not at all.
b 97,136,137. This enzyme hydrolyses (1→6)-α-D-glucopyranosyl linkages in
isomaltose and in the gluco-oligosaccharides (’dextrins’) produced from starch and
glycogen by α-amylase [175].
specificities [258]. The current name for enzyme I is α-glucosidase (maltase) and that for enzyme II,
oligo-1,6-glucosidase (isomaltase); their specificities are shown in Table 6. Maltose induces isomaltase,
but is not hydrolysed by it. And, as described below, Monod used the term ‘gratuitous’ for the induction
of an enzyme by a compound which is not a substrate of that enzyme.29 Further, in 1947 Roger Stanier30
pointed out that several enzymes of a pathway may be induced by one intermediate of that pathway [259].
29 . . .
la synthèse d’un enzyme ‘gratuit’ c’est à dire n’intervenant pas dans le métabolisme . . . ([189] p. 89).
Yate Stanier (1916–1982), a Canadian microbiologist, worked at the University of California, Berkeley from 1947 to 1971. In
his sabbatical year at the Institut Pasteur, he met one of Monod’s colleagues, Germaine Cohen-Bazire, whom he married. He is known
especially for his work on the catabolism of organic compounds and on pseudomonad taxonomy. His admirable attitude to research is
reflected in the title of his memoir ‘The journey, not the arrival, matters’ [49,191,261].
30 Roger
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J. A. Barnett
The workers at the Institut Pasteur used a number of non-metabolizable synthetic glycosides in order to
study induction. Substituting sulphur for oxygen in the galactoside linkage converts an O-glycoside into
an S -glycoside [Figure 9; and cf. methyl β-D-galactopyranoside with methyl 1-thio-β-D-galactopyranoside
(TMG) in Appendix]. Although β-galactosidase does not hydrolyse thio-galactosides, it has about the
same affinity for them as for the corresponding O-galactosides.
These ‘gratuitous’ inducers, such as TMG, produced for Monod’s laboratory in the chemical institute of
Bonn University [117], made it practicable to study enzyme induction separately from enzymic activity.
Although it is not hydrolysed, TMG strongly induces the synthesis of β-galactosidase and at constant rate,
as the synthesis is not limited by metabolic dependence on the inducer itself. Thus, many experiments
on the induced synthesis of β-galactosidase were done with E. coli under conditions of gratuity: neither
inducer, nor the enzyme it induced, gave the cells any advantage. Before 1950, ‘adaptive’ enzymes had
been thought to be produced only in response to the presence of their substrates; but a systematic study
of the utilization of β-galactosides by E. coli enabled Monod and his colleagues to make the following
generalizations [188,189]:
1. Some enzymic substrates are inducers; others do not induce.
2. Some β-galactosides for which β-galactosidase has no affinity are powerful inducers of that enzyme.
3. Some inducers are not substrates of the induced enzyme, but inhibit its activity competitively.
By means of a series of elegant experiments on the kinetics of incorporation of sulphur into the βgalactosidase molecule during its induced synthesis, Monod’s group showed that induction of the enzyme
involves its synthesis de novo, rather than the activation of a single enzyme precursor [119].
‘Permeases’: transport of metabolites into the cells
In the 1950s, Monod and his colleagues isolated many mutants from E. coli. Some of these mutants
were ‘cryptics’,31 that is they could not catabolize exogenously-supplied β-galactosides, such as lactose,
despite possessing β-galactosidase. Such crypticity had already been reported for a number of yeasts.
For example: (a) intact brewer’s yeast fermented maltose at 4 ◦ C, but not methyl α-D-glucopyranoside,
although extracts fermented the latter at that temperature [152]; (ii) β-glucosidase, which hydrolyses
cellobiose to two glucose molecules, was present in a strain of Kluyveromyces marxianus,32 but intact
yeast did not ferment cellobiose [204]; (iii) pressed yeast (Preßhefe) contained α,α-trehalase, but did
not ferment α,α-trehalose [196]. Such observations indicated that the substrates were inaccessible to the
enzymes across cell membranes.
By the mid-1950s, some studies had been published on the passage of various substrates across
the plasma membrane of yeasts. An example is the work on the uptake of sugars by yeasts [233].
In addition, there was evidence for the active transport of monosaccharides across the membranes of
Figure 9. Exchanging a sulphur atom for the oxygen of the bridge converts an O-glycoside to a thioglycoside. From Barnett,
1981 [19]
31 The state of ‘crypticity’: certain cells are unable to metabolize a given substrate which is supplied exogenously, although they possess
the relevant enzyme system [51].
32 This work and much of the later work on galactose metabolism was done with ‘Saccharomyces fragilis’, here called Kluyveromyces
marxianus (see [24]).
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719
primate erythrocytes by means of ‘a temporary complex formed between the sugars and a cell-surface
component during the transfer’ ([150] p. 135).
Georges Cohen33 and Monod argued that this entry of organic substrates into microbial cells
is ‘mediated by more or less selective permeation systems’ ([51] p. 169), which they proceeded
to characterize. In 1956, they published their seminal findings, which led them to the concept of
‘permeases’.34 Monod and his colleagues wrote:
In this report we describe a system characterized by the property of accumulating exogenous
galactosides in the cells of Escherichia coli. The discovery of this inducible system, distinct
from β-galactosidase, but which controls in vivo the activity of this enzyme as well as its
induction, gives a solution to numerous problems posed by the metabolism of galactosides and
by the induction of β-galactosidase in E. coli, and provides experimental confirmation of the
hypothesis, often discussed, that stereospecific and functionally specialized catalytic systems,
distinct from metabolic enzymes themselves, govern the penetration of certain substances into
microbial cells.35
Two observations provided their primary evidence: (a) cells may be cryptic towards one sugar, yet quite
normal towards others; and (b) some cells have the capacity to accumulate certain substrates internally.
Accordingly, they thought it necessary to explain such observations in terms of a number ‘of specific
permeation systems for which no positive evidence existed, and towards which no direct experimental
approach seemed open’ ([51] p. 171). The alternative interpretation, that specific crypticity was due to
inactivity of the intracellular enzyme, soon had to be abandoned.
The workers in Monod’s laboratory made use of various synthetic glycosides (Table 7) for their
induction experiments, notably o-nitrophenyl β-D-galactopyranoside36 (ONPG) as well as the thiogalactoside, TMG (Appendix). Joshua Lederberg37 had already introduced ONPG in 1950 for studying
β-galactosidase activity [148]; this enzyme acts on ONPG, for which the enzyme has a high affinity,38
and liberates o-nitrophenol which is easy to measure spectrophotometrically:39
β-galactosidase
o-nitrophenyl β-D-galactopyranoside (ONPG) −−→ D-galactose + o-nitrophenol
In a momentous and much quoted experiment, using ONPG, Monod and his disciples compared the
β-galactosidase activity of intact (a) wild-type cells and (b) cells of a cryptic mutant of E. coli [51,118]
(Figure 10). They showed that uptake of a non-hydrolysed thio-β-galactoside had enzyme-like saturation
kinetics [134]. This observation was consistent with the observed rates of hydrolysis by intact wild-type
33 Georges N. Cohen (b. 1920), French biochemist and molecular geneticist, Directeur de Recherche Émérite, Centre National de la
Recherche Scientifique and Professeur Honoraire à l’Institut Pasteur [7].
34 ‘Permeases’, also called ‘carriers’ or ‘transportases’ (the present writer favours ‘carriers’), are associated physically with the plasma
membrane. They bind the specific solute to be transported and undergo a series of conformational changes, thereby transferring the
bound substrate across the membrane.
35 Nous décrivons, dans ce mémoire, un système caractérisé par la propriété d’accumuler, dans les cellules d’Escherichia coli, les
galactosides exogènes. La découverte de ce système inductible, distinct de la β-galactosidase, mais qui commande in vivo l’activité
de cet enzyme ainsi que son induction, donne une solution à de nombreux problèmes que posaient le métabolisme des galactosides
et l’induction de la β-galactosidase chez E. coli, et apporte une confirmation expérimentale à l’hypothèse, souvent envisagée, que
des systèmes catalytiques stériquement spécifiques et fonctionellement spécialisés, distincts des enzymes métaboliques proprement dits,
gouvernent la pénétration de certains substrats dans les cellules microbiennes ([226] p. 829).
36 ONPG was prepared for Lederberg by chemists at Wisconsin [241].
37 Joshua Lederberg (b. 1925), American microbial geneticist was, with Edward Tatum (1909–1975), the first to demonstrate sexual
reproduction in bacteria [149]. Lederberg, who shared the Nobel Prize for physiology or medicine in 1958 with Tatum and George
Beadle (1903–1989), was professor of genetics in the School of Medicine at Stanford University, California, 1959–1978 [Who’s Who
in America, 2003].
38 Dissociation constants of E. coli β-galactosidase: 1.4 × 10−3 M-lactose and 1.4 × 10−4 M-ONPG ([148] p. 388).
39 ONPG has a λ
max of 420 nm at pH 10.2.
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J. A. Barnett
Table 7. Inducers of the β-galactosidase of Escherichia coli. Findings of Monod and his
colleagues published in 1951 [188]
Enzymic activity in vitro
Glycoside
D-Galactose
Activity induced by
mM glycoside
(nmol/min/mg)
Relative
affinity
Hydrolysis
420
30
ONPG
o-Nitrophenyl-β-D-galactopyranoside
1060
1000
+
Lactose
4-O-β-D-Galactopyranosyl-D-glucopyranose
2500
100
+
Melibiose
6-O-α-D-Galactopyranosyl-D-glucopyranose
2400
0
−
>100 000
300
−
TMG
Methyl-1-thio-β-D-galactopyranosidea
a
Quantities from [226].
Figure 10. Rates of hydrolysis of o-nitrophenyl β-D-galactopyranoside (ONPG) by living cells of E. coli. Upper plot,
wild-type; lower plot, cryptic (‘permeaseless’) mutant. Ordinates on left apply to upper plot; those on right apply to lower
plot. Results of Cohen, Monod and L. A. Herzenberg. Reproduced by permission from [51]
cells with varying external concentrations of ONPG. By contrast, the rate of hydrolysis by cells of the
cryptic mutant was not only much slower than for the wild-type but, moreover, was a linear function
of the exogenous galactoside concentration [51]. Hence ONPG entered (a) normal cells by a process
involving enzyme-like saturation kinetics and (b) cryptic cells by simple diffusion.
Carbon catabolite repression
Carbon catabolite repression has not been investigated as extensively in yeasts as in E. coli ; nonetheless,
a number of distinct regulatory mechanisms have been described (for review, see [90]). In 1942, Helen
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721
Epps and Ernest Gale40 described the ‘glucose effect’: ‘the presence of glucose in the medium during the
growth of E. coli suppresses the formation of certain enzymes’ [85]. Monod confirmed this observation
in 1947 for several enzymes responsible for the breakdown of other sugars and their derivatives. In 1960,
Harlyn Halvorson41 explained that ‘The mechanism of these ‘glucose effects’ is little understood’ ([106]
p. 121) and a year later Boris Magasanik42 renamed Gale’s ‘glucose effect’ as ‘catabolite repression’
[166].
In 1956, A. Davies43 showed that glucose represses the production of constitutive invertase44 by
Kluyveromyces marxianus. He detected the greatest activity of this enzyme in yeast which had its growth
rate much restricted, that is when it was grown in <60 µM-glucose [60]. In the same yeast, invertase
formation was shown to be inhibited too by its own substrate, sucrose, unless the sucrose concentration
was low: in this case, repression probably occurs because hydrolysis of the sucrose produces excess
hexose. In addition, Davies observed the glucose repression of β-galactosidase in K. marxianus [61].
The first unequivocal evidence that the ‘glucose effect’ involved the specific inhibition of enzyme
synthesis was discovered only in 1953 [56,187]. Furthermore, Monod and his colleagues soon realized
that, although repression and induction produced opposite effects, they were strikingly similar and with
similar kinetics. Both are highly specific and control the rate of enzyme synthesis, although unrelated
to substrate-specificity. Functionally associated enzymes were often found to be co-induced or corepressed and, hence, induction and repression were thought to have similar underlying mechanisms
[125]. Moreover, since specificity of induction or repression of an enzyme is not associated with its
structural specificity, the structural genes could not also be responsible for regulation. In the words of
Monod and Jacob:
Since the specificity of induction or repression is not related to the structural specificity of the
controlled enzymes, and since the rate of synthesis of different enzymes appears to be governed
by a common element, this element is presumably not controlled . . . by the structural genes
themselves. This inference . . . is confirmed by the study of certain mutations which convert
inducible or repressible systems into constitutive systems ([125] p. 328).
Juana Maria Gancedo’s excellent review of the extensive research on carbon catabolite repression
in S. cerevisiae describes its complex regulatory system and also considers some of the work on a
few other species [90]. Another valuable review of the subject is that of Karl-Dieter Entian and HansJoachim Schüller [83]. Unlike in E. coli [29], catabolite repression in yeasts is not associated with low
concentrations of cAMP [86]. Gancedo points out that ‘Catabolite repression can be exerted not only by
the three related sugars glucose, mannose and fructose, but also by other types of sugars like galactose
[219] or maltose’ [86].
Many of the components of the repression mechanisms were identified by obtaining mutants in
which catabolite repression had been abolished. Zimmermann and Scheel, of the Technische Hochschule
Darmstadt, made an ingenious technical advance with the selection system they introduced in 1977
[303]. They plated S. cerevisiae, which was growing exponentially on glucose as carbon source, on
media containing 2-deoxy-D-glucose (2-deoxy-D-arabino-hexose) plus raffinose. Selection depends on
two particular characteristics of this sugar which, unlike D-glucose, is not used for growth but, like
D-glucose, represses the synthesis of a number of catabolic enzymes [299]. S. cerevisiae hydrolyses
40 Ernest
Frederick Gale (b. 1914), English microbial biochemist, was professor at Cambridge University 1960–1981 [12].
Odell Halvorson (b. 1925), American microbial biochemist, was professor at the University of Wisconsin (1956–1971) and at
Brandeis University since 1971 [8].
42 Boris Magasanik (b. 1919), American microbiologist, emigrated from the USSR to Austria in 1921 and thence to America in 1938,
becoming professor at Massachusetts Institute of Technology from 1960 [9,11,168].
43 A. Davies (1931–?1994) was a student in the Biochemistry Department of Cambridge University. His thesis (1955) was entitled ‘The
Effect of Environment on the Enzyme Constitution of Yeast’ and subsequently he worked for Imperial Chemical Industries (information
kindly supplied by Elizabeth Stratton, Archivist, Selwyn College, Cambridge).
44 Davies’s ‘invertase’ may well have been inulinase (EC 3.2.1.7), not β-fructofuranosidase (EC 3.2.1.26) [243] (discussed in [18]
pp. 187–188).
41 Harlyn
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J. A. Barnett
raffinose with invertase [296,297], so those cells which grew had high invertase activity, even when
glucose was the carbon source. In the 1980s and 1990s, a number of genes specifically involved in
glucose repression and derepression were identified, using Zimmermann and Scheel’s technique, and the
mutants obtained are listed in Table 8. Studies of these mutants have thrown much light on the regulatory
system involved in glucose repression in yeasts; an exceedingly complex subject which has been well
reviewed [41,80,81,90].
The operon
In 1956, Monod and his entourage predicted that mutants affecting regulation would not be allelic
to the structural genes. The prediction was confirmed by studying (a) mutants which do not synthesize β-galactosidase and (b) constitutive mutants which synthesize β-galactosidase without induction
[182,213,226]. From these researches came the concept of the ‘operon’, that is a ‘group of genes, their
expression coordinated by an operator’.45 For lactose utilization by E. coli, enzymic synthesis was found
to involve two genes with quite separate and distinct functions: the structural gene (z) is responsible for
the enzyme structure and the regulator gene controls the expression of z [124]. The regulator genes which
Monod and his colleagues identified had a coordinated pleiotropic46 effect: each controlled the expression
Table 8. Some mutant genes of Saccharomyces cerevisiae involved in glucose repression and derepression (after Entian and
Barnett [81])
Mutant
(alternative
names in
parentheses)
Enzymes affected
Mutants affecting glucose repression
hxk2 (hex1, glr1)
α-Glucosidase, invertase, enzymes of galactose
pathway and tricarboxylic cycle enzymes
Physiological rôle of wild-type gene
Structural gene for hexokinase PII
References
[84]
hex2 (reg1)
α-Glucosidase, invertase and enzymes of
galactose pathway
?Negative regulation
[84,206]
cat80 (grr1)
α-Glucosidase, invertase, enzymes of galactose
pathway and tricarboxylic cycle enzymes
Protein–protein interactions
[84,89]
cid1
α-Glucosidase, invertase and enzymes of
galactose pathway
?
[203]
Mutants affecting derepression
cat1 (ccr1, snf1)
α-Glucosidase, invertase; enzymes of galactose
pathway, tricarboxylic and glyoxylate cycles and
of gluconeogenesis
Protein kinase: required for transcription
of several glucose-repressed genes,
when glucose is limiting
[42,112]
α-Glucosidase, invertase; enzymes of galactose
pathway, tricarboxylic and glyoxylate cycles and
of gluconeogenesis
CAT3 gene is required for expressing
glucose-repressible genes in response to
glucose deprivation
[43]
?
[236]
Strong derepression
[236]
cat3 (snf4)
Mutants epistatica to mutants cat1 and cat3
cat2
α-Glucosidase, invertase; enzymes of galactose
pathway, tricarboxylic and glyoxylate cycles and
of gluconeogenesis
cat4 (mg1)
a
α-Glucosidase, invertase; enzymes of galactose
pathway
A gene which prevents the expression of another is said to be epistatic to it.
45 L’opéron:
46 A
groupe de gènes à expression coordonnée par un opérateur [127].
pleiotropic gene has more than one phenotypic effect.
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723
of several structural genes, which were closely associated with each other and encoded enzymes of the
same biochemical sequence.47 Jacob and his fellow-authors explained the mechanism of this effect in
terms of a new genetic entity, the operator [127]. This operator, adjacent to a group of genes, (a) would
control their activity and (b) would be responsive to the repressor produced by a particular regulator
gene (Figure 11). This hypothesis of how enzymic activity is regulated formed the basis of valid later
explanations, including that of the molecular regulation of the galactose pathway in yeasts.
The galactose pathway in yeasts: work of Leloir and others
Working in Buenos Aires between 1948 and 1952, Leloir and his colleagues elucidated the pathway of
D-galactose catabolism in Kluyveromyces marxianus. In this pathway (Figure 12), the isomerization of
D-galactose 1-phosphate to D-glucose 1-phosphate involves uridine diphosphate (UDP) intermediates, and
not the direct isomerization of the galactose phosphate molecule (Figure 13).
As long ago as 1935, glucose and fructose phosphates had been found to accumulate during galactose
fermentation by top and bottom brewing yeasts [101]. Furthermore, in 1943 Hans Kosterlitz48 had
suggested that D-galactose 1-phosphate49 had a rôle in galactose fermentation as it is fermented by
extracts from a galactose-adapted brewing yeast [139]. He made the suggestion, which later proved to be
true, that adaptation to galactose involved the formation of two new enzymes catalysing the following
reactions:
(i)
(ii)
D-galactose
D-galactose
+ ATP −−→ D-galactose 1-phosphate + ADP
−−
→
1-phosphate −
←
− D-glucose 1-phosphate
Although in his 1946 review, Spiegelman was still writing of the adaptation of ‘galactozymase’
[248], the next year he and his colleagues found that the mechanism by which galactose is
catabolized by S. cerevisiae involves more than one (unspecified) enzyme [256]. Then in 1949 John
Wilkinson,50 working in Cambridge, showed that Dutch top yeast (S. cerevisiae) produces galactokinase
Figure 11. Diagram of the operon and the mechanisms for synthesis and regulation of enzymes, suggested in 1961 by
Jacob and Monod ([125] p. 344). Copyright 1961, reproduced with permission from Elsevier
47 Les gènes régulateurs identifiés jusqu’à ce jour présentent la propriété remarquable d’exercer un effet pléiotrope coordonné, chacun
gouvernant l’expression de plusieurs gènes de structure, étroitement liés entre eux, et correspondant à des protéines-enzymes appartenant
à une même séquence biochimique ([127] p. 1727).
48 Hans Walter Kosterlitz (1903–1996), German-born British pharmacologist, professor at Aberdeen University (1968–1973), Director
of Unit for Research on Addictive Drugs from 1973; discovered (1975), with John Hughes, enkephalins, two potent naturally-occurring
opiates in the brain [6,10].
49 Not galactose 6-phosphate, as might have been expected by analogy with glucose fermentation.
50 John Frome Wilkinson (b. 1925) received his doctorate at Cambridge in 1950, one of his examiners having been Jaques Monod.
Wilkinson worked at Edinburgh University from 1949 to 1991, where he became professor of microbiology (personal communications
from B. E. B. Moseley and J. F. Wilkinson).
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Figure 12. The galactose pathway (Barnett, 1976 [18]). NB phosphoglucomutase is now EC 5.4.2.7 [286]
Figure 13. The change in configuration of the C-4 of D-galactose 1-phosphate to D-glucose 1-phosphate achieved in the
Leloir pathway
adaptively. Extracts of galactose-adapted yeast formed ‘an easily hydrolysable phosphoric ester from
galactose and adenosinetriphosphate’. Some evidence was also presented ‘that galactose-1-phosphate is
further transformed into glucose-6-phosphate’ [291]. Wilkinson named the adaptively-produced enzyme
‘galactokinase’, as had Leloir and his colleagues a year earlier for an enzyme which they detected in
lactose-grown Kluyveromyces marxianus (‘Saccharomyces fragilis’) [36]. Clearly, these were two quite
independent observations, since the Biochemical Journal received Wilkinson’s script in July 1948, so he
would not have seen the earlier publication in Enzymologia before submitting his paper.
What led Leloir, working in Buenos Aires in the 1940s, to study galactose utilization by K. marxianus?
He relates:
. . . it was known . . . that glycogen could be formed from glucose-1-phosphate . . . and it had
been shown . . . that sucrose could be formed from glucose-1-phosphate and fructose with an
enzyme of bacterial origin . . . Consequently the idea that lactose originated from glucose-1phosphate and galactose was floating in the air . . . we started studies with a lactose-utilizing
yeast (Saccharomyces fragilis) which grew on whey in large milk cans . . . This led us to the
study of galactose utilization ([155] p. 28).
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725
His laboratory was not well equipped:
The most sophisticated piece [of equipment] was a Beckman DU spectrophotometer, run by
dry cells which were very difficult to get. We also had a Klett–Summerson photocolorimeter, a
microscope and a home-made Warburg respirometer. No refrigerated centrifuge was available
for a long time and of course all the initial basic discoveries on sugar nucleotides were made
without the help of radioactive materials ([155] p. 27).
And further:
. . . we used to have around the lab all kinds of fancy bottles, that were originally containers
of perfumes, shampoos or prescriptions. Many came from Dr Leloir’s home and he insisted in
storing reagents in them with the idea that the non-uniformity of the shapes and colors helped
to avoid mistakes ([155] p. 34).
In 1949 Leloir and his colleagues found that K. marxianus converts D-glucose 1-phosphate to D-glucose
6-phosphate by the action of phosphoglucomutase51 [39], so they now had evidence that ‘galactozymase’
involves the following series of reactions:
D-galactose
+ ATP
↓ galactokinase
D-galactose
1-phosphate + ADP
↓ thermostable factor
D-glucose
1-phosphate
↓ phosphoglucomutase
D-glucose
6-phosphate
Uridine diphosphate sugars
A year later, Leloir and his colleagues announced a finding of major significance when they described
‘uridine diphosphate glucose: the coenzyme of the galactose–glucose phosphate isomerization’ [37,40].
Still working with Kluyveromyces marxianus, they detected a thermostable factor necessary for converting
D-galactose 1-phosphate to D-glucose 1-phosphate. They determined the activity of this ‘coenzyme’ by
using galactose 1-phosphate as substrate with excess phosphoglucomutase and determining the glucose
6-phosphate formed:
‘coenzyme’
phosphoglucomutase
galactose 1-phosphate −−→ glucose 1-phosphate −−→ glucose 6-phosphate
Uridine diphosphate (UDP) was extracted with ethanol from baker’s yeast, fractionally precipitated with
mercuric acetate, absorbed on charcoal, eluted with ethanol and treated with a cation-exchange resin [37].
The authors comment that ‘The mechanism by which uridine diphosphate accelerates the conversion of
galactose into glucose will require further investigation’ ([40] p. 192). This remarkable reaction involves
51 The Coris, in whose laboratory Leloir worked in the 1940s, discovered phosphoglucomutase in 1938 [58,59], which was purified from
muscle in 1948 by Victor Assad Najjar [197]. This enzyme converts glucose 1-phosphate to glucose 6-phosphate. The catalytic site of the
mutase includes a phosphorylated serine residue. This phosphoryl group transfers to the C-6 hydroxyl group of glucose 1-phosphate to
form glucose 1,6-bisphosphate, the C-1 phosphoryl group of which attaches to the same serine residue, so forming glucose 6-phosphate
and regenerating the mutase [198,224,225].
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a change in configuration at C-4 of the galactose molecule to form glucose 1-phosphate (Figure 13).
The enzyme responsible for the inversion was called ‘galactowaldenase’52 in order to avoid the word
‘isomerase’, which had been used for many other enzymes [131,154]. The enzyme producing the inversion
is now called uridyl transferase (Table 9).
In 1950, Leloir and his colleagues isolated from baker’s yeast ‘the coenzyme of the galactose-1phosphate → glucose-1-phosphate transformation’. They wrote: ‘The substance contains uridine, two
phosphate groups and glucose, and has therefore been named uridine-diphosphate-glucose’ (UDPGlc)
([37] p. 349). Leloir tells us that ‘this was rather exciting, because at that time uridine was known only
as a nucleic acid constituent’ ([155] p. 30). In the following year, he described this compound (see
Figure 14) as a ‘glucose-1-phosphate molecule attached to uridine 5 -phosphate forming a pyrophosphate
link’ [153].
Leloir and his colleagues measured the molecular mass of uridine diphosphate glucose using a
Kuhlmann microbalance (Figure 15), which weighs to about 1 µg [79,222]. This remarkably sensitive
instrument has a double case to minimize the effects of room temperature changes and air movements;
but it is not easy to use. Like all research balances made before the mid-1930s, it is not air-damped, so the
Table 9. Enzymes of the galactose pathway
Enzyme and Enzyme Commission number
Reaction
Galactokinase 2.7.1.6
D-galactose
UTP-hexose-1-phosphate uridylyltransferase 2.7.7.10
(galactose-1-phosphate uridylyltransferase)
α-D-galactose-1-phosphate UDP-galactose + pyrophosphate
UDP-glucose-hexose-1-phosphate uridylyltransferase 2.7.7.12
(uridyl transferase)
UDP-glucose + α-D-galactose-1-phosphate α-D-glucose-1-phosphate + UDP-galactose
+ ATP → D-galactose-1-phosphate + ADP
UDP-glucose-4-epimerase 5.1.3.2
UDP-glucose UDP-galactose
UTP-glucose-1-phosphate uridylyltransferase 2.7.7.9
α-D-glucose-1-phosphate UDP-glucose + pyrophosphate
Phosphoglucomutase 5.4.2.2
α-D-glucose-1-phosphate α-D-glucose-6-phosphate
Figure 14. Uridine 5 -diphospho-α-D-glucopyranose (UDP-glucose) and uridine 5 -diphospho-α-D-galactopyranose
(UDP-galactose)
52 In 1950, Leloir and his colleagues had written that galactose 1-phosphate → glucose 1-phosphate consists in a Walden inversion of C-4
and is catalysed ‘by an enzyme which is currently called “galactowaldenase” in this laboratory’ ([37] p. 333). The ‘Walden inversion’,
i.e. the conversion of one optical isomer into a derivative of the other, was discovered in 1895 by the Latvian chemist, Paul Walden
(1863–1957) [285].
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727
Figure 15. A Kuhlmann microbalance. From Pregl [222]
balance does not remain at rest, but continues oscillating like a pendulum for a long time. Consequently,
it is necessary to observe and take a mean of the extreme positions of the pointer53 and estimating the
amplitude of the oscillations is a time-consuming occupation ([155] p. 31).
Treatment of UDPGlc with an extract of K. marxianus produced a galactose-containing compound,
uridine diphosphate galactose (UDPGal). Hence, Leloir was able to suggest two sequential steps to explain
the conversion of D-galactose 1-phosphate into D-glucose 1-phosphate:
−−
→
(i) UDPGlc + D-galactose 1-phosphate −
←
− UDPGal + D-glucose 1-phosphate
−
−
→
(ii) UDPGal ←−− UDPGlc
So by the early 1950s, the old ‘galactozymase’, by which galactose is converted to glucose, had been
partly unravelled [131,153,192] as follows:
galactokinase
(1) D-galactose + ATP −−→ D-galactose 1-phosphate + ATP
‘galactowaldenase’
(2) D-galactose 1-phosphate + UDPGlc −−→ UDPGal + D-glucose 1-phosphate
In addition, Herman Kalckar and his colleagues reported [131] two other reactions effected by extracts
of K. marxianus, viz. a reaction between uridine triphosphate (UTP) and D-galactose 1-phosphate:
−−
→
(3) UTP + D-galactose 1-phosphate −
←
− UDPGal + D-glucose 1-phosphate
and another by which UDPGlc is converted to D-glucose 1-phosphate and UTP:
(4) UDPGlc + pyrophosphate −−→ D-glucose 1-phosphate + UTP [192]
The enzyme responsible for reaction (3) was later named UTP-hexose-1-phosphate uridylyltransferase
[EC 2.7.7.10] and that for reaction (4) UTP-glucose-1-phosphate uridylyltransferase [EC 2.7.7.9]. The
authors’ illustration of these reactions with UDPGlc, published in 1953, is reproduced in Figure 16.
53 The method of weighing using the swinging movement of non air-damped balances is described, for example, on page 8 of reference
[273].
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J. A. Barnett
Figure 16. The points of enzymic attack on uridine diphosphoglucose, as illustrated by Kalckar and his colleagues in 1953.
Reprinted with permission from Nature [192]. Copyright (1953) Macmillan Magazines Limited
Figure 17. The reactions of the galactose pathway as represented in 1958 by Huguette de Robichon-Szulmajster [62]
Writing about the important contributions of Kalckar and his colleagues to understanding the galactose
pathway, Leloir says:
Although our laboratories worked on similar lines there was never any rivalry or resentment
among us as often happens. We exchanged information quite freely ([155] p. 33).
In 1958, Huguette de Robichon-Szulmajster54 summarized the galactose pathway (formerly known
simply as ‘galactozymase’) [62] as comprising the reactions shown in Figure 17. Reaction 4 of this
pathway shows the initial formation of a catalytic amount of UDPGlc which is necessary to start reaction
(2). Working with galactokinase-negative haploid mutants, she found that, as well as galactokinase, two
other enzymes of the galactose pathway in S. cerevisiae are ‘adaptive’. These are galactose-1-phosphate
uridyltransferase and UDP-glucose 4-epimerase, which are formed when growth is on galactose but not
when on glucose; the same is true for the epimerase in K. marxianus [63]. Her findings (Table 10) were
not consistent with Stanier’s concept of sequential adaptation, mentioned above, since the free galactose
appeared to be a ‘multi-inducer’. She and a colleague purified UDP-glucose 4-epimerase in 1960 [174].
Table 9 lists the enzymes of the galactose pathway.
54 Huguette de Robichon-Szulmajster worked at the Laboratoire d’Enzymologie, Centre national de la Recherche scientifique, Gif-surYvette, France.
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729
Table 10. Activities of enzymes of the galactose pathway in yeasts grown on
D-galactose. Results of de Robichon-Szulmajster [62,63] (after Barnett [18])
Galactokinaseless strain of
Saccharomyces cerevisiae
D-glucose
or
Kluyveromyces
marxianus
Carbon source in growth medium
Enzyme
D-Glucose
D-Galactose
Galactokinase
0
0
UDP-glucose-hexose-1-phosphate
uridylyltransferase
0.01
8.96
UDP-glucose-4-epimerase
0
UTP-glucose-1-phosphate
uridylyltransferase
9.17
D-Glucose
D-Galactose
0
26 000
10
3680
5.70
0
333 000
12.50
1770
2220
Specific activities, expressed in (nmoles of substrate reacted) min−1 (mg of protein)−1 .
Genetic regulation of the galactose pathway
The GAL genes55
In the yeast Saccharomyces cerevisiae, the interplay between Gal3p, Gal80p and Gal4p
determines the transcriptional status of the genes needed for galactose utilization [27].
In the 1940s and 1950s, several authors reported on their studies of the genetics of the ability of S.
cerevisiae to ferment galactose [95,114,115,158,160,193,230,231,250,257,293]. The Lindegrens were the
first to describe the genetic control of galactose fermentation in 1947 [160]. When they crossed haploid
fermenters with non-fermenters, each ascus of the diploid hybrids produced two fermenters and two
non-fermenters. Their conclusion that galactose fermentation was determined by a single pair of alleles
was confirmed 4 years later by Herschel Roman56 and his colleagues [230]. The non-fermenting haploids
were found to differ from each other genetically, being recessive for the galactose fermenting genes at
different loci. One of them, G2 (later called GAL2 ), was described as probably ‘involved in the transport
of galactose into the cell’ [70], which was confirmed by Vincent Cirillo57 in 1968 [48].
Donald Hawthorne58 found three dominant genes to be necessary for fermenting galactose rapidly
and designated the recessive alleles59 as g-1, g-2 and g-3, a slow fermenter of galactose having genes
G-1G-1 G-2G-2 g-3 g-3. However, Lindegren drew attention to the difficulties in being certain of the
distinction between slow-fermenters and non-fermenters [159].
Øjvind Winge had already reported in 1948 that the ‘long-term adaptation’ of S. cerevisiae to galactose
is associated with presence of gene gs [293], and this was re-named60 ga-3 in 1963 [74] by Howard
55 The
ways in which the genes were written changed over the years, at any one time varying from one author to another.
Lewis Roman (1914–1989), Polish-born American yeast geneticist, was professor in the Department of Genetics at the
University of Washington, Seattle [91]. He wrote, in 1947, that Carl Lindegren’s ‘unorthodox interpretations presented a challenge that
was largely responsible for my choosing yeast as an experimental organism’ ([229] p. 3).
57 Vincent P. Cirillo worked at the State University of New York.
58 Donald C. Hawthorne (1926–2003), American yeast geneticist, worked at the Department of Genetics of Washington University from
1950 onwards, first as a student, later becoming a professor. Apart from his work on the genetics of galactose utilization, he is known
especially for his work on chromosome mapping and on various suppressors. Bonny Brewer writes that he never had a telephone at home
nor used a computer, that he walked to work and ‘he foraged for food . . . harvesting watercress from Ravenna Creek and collecting
fennel and mushrooms from where he planted them in his walks around the city’ [304].
59 Alleles or allelomorphs are different forms of a gene which are alternative to one another at the same locus. The term ‘allelomorph’
′
(other); µορφη′ (form).
was coined by Bateson and Saunders in 1902 ([26] p. 126) from the Greek: αλλος
60 ‘Hawthorne writes that g-3 is “allelic with” g , but from his text it appears that he means ‘identical with’ ([298] p. 136).
s
56 Herschel
\
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J. A. Barnett
Douglas, who made major contributions to the understanding of the genetic regulation of galactose
metabolism. In 1976, he showed this gal3 mutation to be pleiotropic, also impairing the utilization of
melibiose and maltose [135].
In 1968, Vincent Cirillo found that galactose enters galactokinaseless haploid cells of S. cerevisiae by
facilitated diffusion61 [48]. He showed the GAL2 gene (Roman’s G2 ) to be responsible for this (inducible)
transport of which, for some strains, D-fucose and L-arabinose are gratuitous inducers. Mutants altering
the inducibility of galactose pathway enzymes also affected the inducibility of galactose transport.
Regulation of the GAL genes
When D-galactose is the sole source of carbon, S. cerevisiae activates the GAL genes which encode the
enzymes of the Leloir pathway. The main features of the regulation of the GAL genes were worked out by
Douglas, Hawthorne and their colleagues in the 1960s [70–74] (Figure 18 and Table 11). They showed
that the closely linked genes GAL1, GAL7 and GAL10 encode galactokinase, galactose-1-phosphate
uridylyltransferase and UDP-glucose 4-epimerase, respectively. They also found that the mutation gal4
blocks the synthesis of these enzymes, since GAL4 is necessary for expressing GAL1, GAL7 and GAL10.
[71,72]. In 1966, these two authors suggested that the regulatory mechanism of the galactose pathway
conforms with concept of the operon of Jacob and Monod [72,126]. Thus, for example, the GAL80 gene
forms a repressor which represses the expression of the GAL4 gene when galactose is absent by affecting
the GAL81 site which, much like Monod’s lac operator, is the site of repressor recognition, controlling
transcription of the contiguous structural gene [170].
Subsequent work refined the Douglas–Hawthorne scheme and, by the 1990s, the mechanism of the
regulation of the galactose pathway in S. cerevisiae had been shown to be as represented in Figure 19.
The Gal4 protein activates GAL genes when galactose is in the medium. When it is absent, induction is
prevented by the regulatory protein Gal80 [120] (Figure 18). Regions of yeast DNA have been isolated
containing genes, the expression of which depends on the nature of the exogenous carbon source
[267], and one of these regions contains DNA sequences encoding three galactose-inducible RNAs,
transcription of which is galactose-specific and depends on GAL4. The region encoding these RNAs
is the GAL7–GAL10–GAL1 gene cluster [268]. D-Galactose activates the Gal3 protein, galactokinase,
which interacts with the Gal80p–Gal4p complex, alleviating repression by Gal80p, hence allowing
Gal4p to activate transcription [15]. Glucose repression was found to be mediated by GAL4, which
Figure 18. Regulation of the galactose pathway in S. cerevisiae. Symbols:
, inhibits activity; ∗, repressed by D-glucose
, gene encodes;
, stimulates activity;
61 Facilitated diffusion is carrier-mediated movement across a membrane which depends on a concentration gradient and not expenditure
of metabolic energy (for review, see [78]).
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731
Table 11. Galactose pathway genes of Saccharomyces cerevisiae (after
Johnston and Carlson [130])
Gene
Encodes/function
References
GAL1
GAL2
GAL3
GAL4
Galactokinase
Galactose carrier
Inducer
Transcriptional activator,
mediating glucose repression
Phosphoglucomutase
Uridyl transferase
UDP-glucose-4-epimerase
Inhibition of Gal4 protein
Part of Gal4 protein
Glucose repression
Glucose repression
[268]
[48,70,202,274]
[15,31]
[102,147,170]
GAL5
GAL7
GAL10
GAL80
GAL81
GAL82
GAL83
[210]
[268]
[268]
[122]
[170]
[171,172]
[171,172]
Figure 19. The enzymes of the galactose pathway in S. cerevisiae and the genes encoding them (after Johnston [129]). The
enzymes are galactokinase (encoded by GAL1), UDP-glucose–hexose-1-phosphate uridylyltransferase (encoded by GAL7),
UDP-glucose-4-epimerase (encoded by GAL10), phosphoglucomutase (encoded by GAL5)
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J. A. Barnett
encodes the transcriptional activator of GAL genes [102], although the mechanisms appear to be complex
[145].
In summary: (a) the protein encoded by the GAL4 gene activates the transcription of each regulated
GAL gene; (b) the Gal80 protein binds to the Gal4 protein, preventing its activating activity; and
(c) galactokinase, the Gal3 protein, prevents the inhibitory action of the Gal80 protein (Figure 18).
In the 1980s, some of the studies on the GAL system were extended to another yeast, Kluyveromyces
lactis, which also utilizes galactose by inducing enzymes of the galactose pathway. The GAL4 of S.
cerevisiae was shown to activate the lactose–galactose operon (or regulon62 ) of K. lactis [228], which
has similar GAL1, GAL4, GAL7, GAL10 coding to that of S. cerevisiae [177,178,227,234]. Induction
and repression of the galactose pathway enzymes in K. lactis were studied further in the 1990s, some
mechanisms involving Gal4p [142,302]. The regulation of lactose and galactose metabolism by K. lactis
was reviewed in 1996 [289].
Genetic regulation of the utilization of other substrates
Adaptations to other kinds of substrate have been studied, such as that to nitrogen compounds (for review,
see [167]). Here, it seems apposite to write briefly about some of the work which has been published on
the genetic regulation of two additional substrates, maltose and sucrose.
Genetic regulation of maltose utilization: the MAL genes
Brewers require S. cerevisiae to ferment maltose and maltotriose. Hence, along with galactose and sucrose,
maltose was among the first markers to be used for genetic studies of yeasts. Indeed, in the 1940s and
1950s, the pioneer yeast geneticist at the Carlsberg Laboratory in Copenhagen, Øjvind Winge, published
work on the genetics of α-glucosidase activity in brewing yeasts [293–295]. He identified four genes
(MAL1–MAL4 ) for maltose fermentation in S. cerevisiae and a single gene (MAL6 ) in Saccharomyces
pastorianus (formerly S. carlsbergensis), any of which would encode α-glucosidase (Winge crossed the
two species). The structural genes for maltose utilization are only expressed after induction by maltose
and are also subject to glucose repression (for reviews, see [82,83]).
Maltose utilization requires the functional presence of both a maltose carrier and a cytosolic αglucosidase. In 1976, Gennadi Naumov reported that MAL loci contained both regulatory and structural
genes, as well as three genes encoding the maltose carrier [199]. In addition, a transcriptional activator
protein was described in the 1980s [44,52,53,201]. Maltose is generally taken into the cells of S. cerevisiae
by proton symport (for review, see [78]) and each molecule is hydrolysed in the cytosol by α-glucosidase
to give two molecules of D-glucose. The maltose carrier is encoded by the MAL61 gene [45], with the
first indication of any MAL gene encoding this carrier coming in 1983 from the identification of a
MAL1-linked temperature-sensitive maltose transport mutation [96].
Qi Cheng and Corinne Michels explain the nomenclature of the MAL genes:
Each [MAL] locus is a complex locus containing three genes required for maltose fermentation
. . . We have established a two digit numbering system in order to distinguish the GENE 1, 2,
and 3 functions mapping to the different MAL loci. The first digit indicates the locus position
and the second the GENE function . . . Thus the MAL61 gene is the GENE 1 function mapping
to the MAL6 locus ([45] p. 477).
Two years later, those authors reported that the inducible, high-affinity maltose carrier of S. cerevisiae
is encoded by genes MAL11 and/or MAL61 [46]. Entian and Schüller review some of the intricacies of
the genetics of the regulation of maltose utilization by S. cerevisiae [82].
62 A regulon is a system in which the coordinated regulation of two or more structural genes or operons, each with its own promoter, is
achieved by a common regulator molecule.
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733
Genetic regulation of sucrose utilization: invertase and the SUC genes
Invertase (β-fructofuranosidase) catalyses the hydrolysis of sugars, such as sucrose and raffinose, which
have a terminal, unsubstituted β-D-fructofuranosyl group [195] (Appendix). The enzyme is located outside
the plasma membrane and is associated physically with the cell wall (for review, see [19]). In 1967,
Santiago Gascón and Paul Ottolenghi found that the invertase concentration varies by a factor of about
1000 with the concentration of exogenous D-glucose [92] and, consistent with this finding, derepression
of this enzyme at <6 mM D-glucose was reported some 7 years later [176].
From the 1950s to the 1970s genetic analysis demonstrated that at least five genes, designated
SUC1–SUC5, encode invertase, any one of which is sufficient for invertase synthesis and, hence, the
utilization of sucrose or raffinose [95,104,211,295]. Invertase molecular genetics was studied extensively
in the 1980s and 1990s and regulation was found to be at transcription [235]. Mark Johnston and Marian Carlson have reviewed this subject, as well as the regulation of the utilization of other organic
compounds [130].
Conclusion
The small beginning in 1900 of studying the adaptation of yeasts to D-galactose was followed up through
the twentieth century, eventually to generate considerable understanding of the complexities of the
molecular regulation of enzyme synthesis. The enormous volume of work published on the regulation of
enzymes and transport carriers, and on the molecular control of both, for S. cerevisiae and also for E.
coli, using increasingly refined techniques, has made it impracticable to give here more than an outline
of how this study developed. Only mechanisms underlying the regulation of the amount of enzymes in
the cells have been discussed; other important systems of enzymic regulation, such as interconversion
by covalent modification and systems affecting enzymic activity, allosteric activation and deactivation,
have been ignored. The genetic regulatory mechanism of S. cerevisiae, acting on the GAL genes which
encode the enzymes of galactose utilization, has been the most intensively studied and has become the
best understood genetic regulatory mechanism in any eukaryote.
Kevin Struhl tells us:
. . . classical and molecular yeast genetics has permitted the discovery and functional
characterization of transcriptional regulatory proteins that were not identified in biochemical
studies. Thus, genetic analysis in yeast has often generated information complementary to that
obtained from biochemical studies of transcription in vitro, and it has provided unique insights
into mechanisms of eukaryotic transcriptional regulation ([269] p. 651).
And work on this mechanism continues today [2].
An account of the history of research on aspects of more general metabolic regulation in yeasts, such
as the Pasteur and Kluyver effects, the regulation of glycolysis and aerobic metabolism, will be given in
a later article.
Acknowledgements
The errors are entirely mine; but I thank the following most warmly for all the help they have given me: B. J.
Brewer, Melvin Cohn, H. B. F. Dixon, K.-D. Entian, Peter Gray, Robert Hauer, Alexandre Herlea, M. C. KiellandBrandt, B. E. B. Moseley, Andrea Munsterberg, P. M. Nurse, Georges Pédro, N. M. Temperley, J. F. Wilkinson, M. D.
Yudkin, F. K. Zimmermann. I am also very much indebted to L. K. Barnett for extensive criticisms of the text and much
help with the figures, as well as to the Royal Society for a research grant.
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Appendix. The structures of some sugars and sugar-derivatives
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