Effects of Hexose Analogues on Fungi: Mechanisms of Inhibition and of Resistance
Author(s): D. Moore
Source: New Phytologist, Vol. 87, No. 3 (Mar., 1981), pp. 487-515
Published by: Blackwell Publishing on behalf of the New Phytologist Trust
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http://www.jstor.org
487
New Phytol.(1981) 87, 487-515
OF HEXOSE
EFFECTS
ANALOGUES
MECHANISMS
OF INHIBITION
RESISTANCE
ON FUNGI:
AND OF
BY D. MOORE
Departmentof Botany, The University,ManchesterM13 9PL, U.K.
(Accepted5 May 1980)
CONTENTS
I.
I I.
III.
IV.
487
488
489
492
SUMMARY
INTRODUCTION
HEXOSE STRUCTURE
INHIBITIONS BY SUGAR ANALOGUES
1. Sensitivityto inhibition
2. Mechanisms of inhibition: 2-deoxy-D-glucose and
related sugars
3. Mechanisms of inhibition: sorbose and
3-0-methylglucose
V.
VI.
VI I.
SUGAR ANALOGUE-RESISTANT MUTANTS
CONCLUSIONS
REFERENCES
I.
492
493
500
504
508
510
SUMMARY
Althoughtheeffectsofa numberofsugaranaloguesare discussedin thisreview,mostattention
is devotedto 2-deoxy-D-glucose(deGlc) and L-sorbose.The growthof fungiis not uniformly
inhibitedby sugaranaloguessince some speciesor strainscan metabolizesugarswhichseverely
inhibitothers.Sugar analogues usuallycause inhibitionsby being used in metabolismin place
of glucose. Both deGlc and sorbose are metabolizedby severalspecies but, in general,only a
small fractionof the analogue is utilized.
The uptake and phosphorylationof readily metabolized sugars are inhibitedby deGlc.
sourcesofcarbon,
Inhibitionsby sugaranaloguesoccuron media containingnon-carbohydrate
so inhibitionofsugaruptakeis unlikelyto be a majorcomponentofthegrowthinhibitionwhich
occurs on sugar-containingmedia. Hexokinase phosphorylatesdeGlc, and its 6-phosphate
inhibitsthe activityof especiallyphosphohexoseisomeraseand glucose 6-phosphatedehydroofdeGlc-6P
genase.Apartfroman involvementin synthesisofmorecomplexsugars,formation
is the usual limitof metabolismof this sugar analogue. Accumulationof the phosphateester
leads to a considerabledrain on phosphatepools and ATP levels decline drastically.Indeed,
deGlc rapidlyinitiatesdegradationof purinenucleotideswhichcan proceedat leastto the level
ofhypoxanthine.In Saccharomyces
cerevisiae,deGlc can be condensedto dideoxytrehalose
and,
in most fungi,polysaccharidesynthesisis affected.Preformedwall materialis eroded and
Abbreviations:
ADP, adenosinediphosphate;
AMP, adenosinemonophosphate;
ATP, adenosinetriphosphate;
cAMP,adenosine
3',5'-cyclic
monophosphate;
deGIc,2-deoxy-D-glucose;
deGlc-6P,2-deoxyAllofthesugarsandsugar
adeninedinucleotide
D-glucose
6-phosphate;
NADP, nicotinamide
phosphate.
ofsorbosewhichshouldalways
to in thispaperaretheD-isomers
withtheexception
derivatives
referred
cerevisiae.
toSaccharomyces
be understood
tomeanL-sorbose.
The unqualified
term,'yeast',is usedtorefer
ofotheryeastsarecited.
Binomials
0028-646X/81/030487
+ 29 $02.00/0
(D 1981The New Phytologist
D. MOORE
488
synthesisof new wall componentsis preventedby sequesteringof uridine and guanosine
nucleotidesthroughreactionwith deGIc. Interferencewith the structureof the wall greatly
reducesosmoticstability,and cell lysisis the major cause of inhibitionof growthby deGlc in
yeastsand filamentousfungialike.
and the biochemicalbasis of the inhibitionscaused by this
L-Sorbose is not phosphorylated,
analogue is obscure. Some enzymes involved in polysaccharidesynthesisare sensitive to
ofgrowthon thissugaris formationofan abnormally
inhibitionby sorbose,and a characteristic
thick cell wall. Filamentous fungi grown on solid medium containingsorbose assume an
abnormalgrowthformin whichcells are muchshortenedand branchingfrequencyis increased.
hyphal
affects
withcell wall structure,
There are indicationsthatsorbose,perhapsby interfering
morphogenesisand that this effectmay be magnifiedinto a macroscopic change in the
effects.
characteristics
ofthecolonyduringgrowthon solid mediumbysecondaryenvironmental
Mutants,resistantto deGlc, have alterationsin eithertransport,hexokinaseor phosphatase
functions,
thusemphasizingtheimportantofaccumulationofdeGlc-6P intheinhibitionscaused
by this analogue. Generalizationis less easy about mutantsresistantto sorbose. The majority
are defectivein transport,but othersshow defectsin phosphoglucomutaseand polysaccharide
synthasesand thereare some correlationswith morphologicalchanges which underlinethe
involvementof the sugar in the observed alterationsin cell walls. Many aspects of early
carbohydratemetabolismneed investigationand, forsuch studies,the sugar analogues have
much to offer.However, there is also particularscope for their use in studies of hyphal
morphogenesis,especiallyin relationto the connectionbetweenwall constructionand morphology. Counterpartsof the abnormalhyphalgrowthformscaused by sugar analogues can be
foundin normalhyphalcells whichcontributeto the tissuesof complexfungalstructuressuch
as the basidiomycetecarpophore. Experimentalinductionof these cell formsin vegetative
culture by the sugar analogues shows promise for study of events which contributeto the
morphogenesisof these fungalstructures.
II.
INTRODUCTION
Metabolic inhibitors are often employed to interfere experimentally with normal
processes in order to produce a recognizable abnormality so that study of the
abnormal will lead to a better appreciation of the normal. To be successful, such
an approach must employ inhibitors which cause the smallest disturbance possible
at their primary site of action. Otherwise, the very magnitude of the initial effect
and its confusion by secondary phenomena are likely to militate against
determination of the nature and consequences of the application of the inhibitor.
Such inhibitors can be analogues of normal metabolites. Frequently, though, they
have an unexpectedly far-reaching impact because of the close integration of
metabolism. Nevertheless, analogues have considerable attractions, not least of
which is the chemical variety which is often available. Irrespective of the type of
technique used to follow the effectsof any inhibitor, its metabolic influence is very
much a consequence of molecular interactions between the inhibitor and its target
functional molecule. To understand better the molecular events involved in the
inhibiting reactions, it is useful to have available a variety of chemically diverse
analogues, as these potentially enable the significance of different aspects of the
molecular structure of the inhibitor to be assessed.
The dual requirement of close similarity to a central metabolite and chemical
variety is amply satisfied by the family of hexose sugars. The metabolic significance
of hexoses, both as nutrients and as biosynthetic intermediates, is well documented
and the molecular structure of hexoses provides a considerable variety of chemical
modifications, though comparatively few sugar analogues have been examined in
detail for their effecton fungal growth. Because of its ability to impose a restricted
colonial growth habit on Neurospora (Tatum, Barratt and Cutter, 1949), L-sorbose
Hexose analogues and fungi
489
is probablythemostwidelyused hexoseanalogue,butmuchworkhas been done
and a few others.
and some with 3-0-methylglucose
with 2-deoxy-D-glucose
rangeof
Althoughonlya fewfungalspecieshavebeen studied,a comprehensive
havebeenrecognizedand,amongmutantsselectedforresistance
effects
inhibitory
a numberof distinctresponsesoccur.
to theseinhibitions,
The inhibitionscaused by sugaranalogueshave not been reviewedsince the
ofHochsterand Quastel(1963) and
treatiseson enzymeand metabolicinhibitors
toemphasizetheresponseoffungi
Webb(1966),and no reviewhaseverattempted
as generaltoolsto probe
totheseagents.Not onlydo theyhavea greatdealto offer
and function
synthesis
and polysaccharide
and energymetabolism,
carbohydrate
theyhold promiseforinvestigating
fungiin particular,
but also, forfilamentous
hyphal
componentsofthewall in determining
thepartplayedby polysaccharide
by sugaranalogues
In thisreview,data dealingwithinhibitions
morphogenesis.
mutantsare broughttogetherin thehope thatthepotentialvalue of
and resistant
workwillbe illustrated.
theseagentsin further
III.
HEXOSE
STRUCTURE
effects
becausetheyare metabolizedin
Hexose analoguesusuallyexertinhibitory
similartothatofglucose
aresufficiently
placeofglucose;theirmolecularstructures
fromthe structureof D-glucose
forerrorsto occur. However,theirdifferences
subsequentlycause metaboliclesionswhichinhibitgrowth.Appreciationof the
of
molecularstructureof hexose sugars is thus essentialto an understanding
inhibitory
and resistancemechanismsalike.Althoughthe moleculeof D-glucose
most
can assume a numberof shapes in solution,its moststable,and therefore
usual, structureis the chairformof the pyranoseringshownin Figure 1. This
introducedby Reeves (1949, 1951).
in the terminology
is the C1 conformation
of sugarshave been dealt withby Pigmanand
Details of the stereochemistry
Horton(1972), Pigmanand Anet(1972) and Angyal(1972).
ringwithan
heterocyclic
The structure
shownin Figure1 is a six-membered
carbonatomsofthesix-carbon
and fifth
oxygen(hemiacetal)linkbetweenthefirst
two of its bonds to the ring
'chain'. Each carbonatom in the ringcontributes
are
This is fairlyflat,and thetwobondsnotinvolvedin ringformation
structure.
eq
ax
4
e
eq
eq
ax
ax
H
20HOH
HO
H
\
HO
H
\
O
aH
OH
H
Fig. 1. Top: generalizedstructureoftheC 1 chairformofthepyranosering.The fivecarbonatoms
of the ringare indicatedby numberand the equatorial (eq) and axial (ax) bonds are identified.
Bottom: the structureof f-D-glucopyranose.
D. MOORE
490
directed one in the plane of the ring (described as equatorial) and one almost at
rightangles to the plane of the ring(axial). In ,-D-glucopyranose, the formof the
sugar which predominates in aqueous solution, all of the major substituents
(hydroxylgroups on C-1, -2, -3 and -4, and a hydroxymethylgroup on C-5) are
arranged equatorially to the ring with the hydrogen atoms attached to the axial
bonds (Fig. 1). Other aldose sugars can be related to this structureby simply
indicating the manner by which they differ(Fig. 2 illustrates several). The
hydroxylgroup at C-2 is replaced by a hydrogenatom in deGlc, by an amino group
in D-glucosamine and, in glucosone, by a ketonic group. In 3-0-methylglucose,
thehydroxylat C-3 is methylated.In D-galactose,thehydroxylat C-4 is axial rather
than equatorial while, in D-mannose, the C-2 hydroxylis axial. For mannose, a
secondary consequence is that the a-anomer predominates at equilibrium in
aqueous solution as shown in Figure 2.
H
H
HO)~CH20H
0.
H~~~~~~~~~~~~~~
H
-
CH20H
HO
D-mannose
H
13~~-0-glucose
OH
H~~~~~
H~H
H
H
H
H3-0-methylglucose
1B-2-deoxy-D-glucose
OH
H
HO
OH
CH30O
OH
H
IH20H
.1
HO
HO~~~~~~~~~~~~~~~~~~~
H
H
(3~~-D-glucosamine
OH
~~~H
CH0
H
-,-
HB1-D-galactose
H
O
HO
H
H
Fig. 2. Molecular structuresof a selectionof aldose sugarsand sugar analogues.
It is not difficultto envisage the interactionbetween an analogue of an aldose
and an enzyme which normally reacts with glucose. Such comparisons often
emphasize the greatspecificitywhich can be displayed by enzymicand regulatory
systems,Thus the differentorientationof an hydroxylgroup as in the glucosegalactose pair is a sufficientdifferenceto enable discriminationbetween separate
hexose transportsystemsin both Aspergillusnidulans(Mark and Romano, 1971)
and Saccharomycescerevisiae(Kotyk, 1967).
When ketosesugarslikefructoseand sorbose are considered,itis not immediately
clear how theirmolecularconformationsrelateto enzymicand otherproteinswhich
normallyreactwithglucose. Ketoses formpyranoserings,so again theirstructures
can be compared directlywith that of f-D-glucopyranose(Fig. 3). However, ring
closure in ketopyranosesoccurs between C-2 and C-6, so this ring structurecan
only be 'fitted' to the aldopyranose after inversion and rotation; i.e. the sixth
carbon atom of the glucopyranose ring is equated, for the sake of diagrammatic
comparisononly,withC-i oftheketopyranose,C-5 = C-2, etc. (Lardy, Wiebelhaus
and Mann, 1950). The similarity between these diagrams is close and this
ketopyranoseconformationmust play a part in any systeminvolvingfructoseand
to understand
makesitdifficult
sorbose in solution.However, such an interpretation
Hexose analogues and fungi
49I
H
H
HO
0
O
HO
OH
H
H
1-D-glucopyranose
H
H
H
HO
HO
H
HO
____O\
CH0
HO
HO
H
O
H
*CH20H
OH
0O
H
OH
H
B-D-fructopyranose
and ,-D-fructose.The fructopyranose
Fig. 3. Comparisonofthepyranoseringformsof/J-D-glucose
ring is shown on the left in the conventionalorientationand, in the right-handdiagram, the
molecular structurehas been invertedand rotated to present a view which allows a closer
comparisonwith glucose. In each diagram,the C-1 carbon atom is indicatedwith a star.
H
OH
H
HO
H
H
B-D-glucopyranose
CH2O
H
H
O
H_
H
CH20H
OH
B-D-fructofuranose
HOH
H
Hr
OH
OH
CH2OH
a-L-sorbofuranose
Fig. 4. Comparisonof the pyranoseringformof glucose with the furanoseformsof
fructoseand sorbose
some reactions involving specific carbon atoms, particularly the hexokinase
reaction in which fructose6-phosphate is formed.An alternativeconformationof
ketoses is the five-memberedfuranosering. Indeed, it is in this formthat fructose
is always found when in combination in biological materials. Purich, Fromm and
Rudolph (1973) accounted forthe formationoffructose6-phosphate by hexokinase
by assuming that the enzyme acts on the furanose ring formof fructose. Figure
4 shows the comparison between fl-D-glucopyranoseand furanose ring formsof
fructoseand sorbose.
D. MOORE
492
IV.
INHIBITIONS
BY SUGAR
ANALOGUES
1. Sensitivityto inhibition
The overall effectsof sugar analogues on fungalgrowthcan be readilyassessed
by measurementsof hyphalgrowthratesof filamentousfungiand by turbidimetric
methods with yeasts. Sensitivitiesto inhibitionobserved for differentorganisms
are very variable. The basidiomycete, Coprinuscinereus(often incorrectlycalled
C. lagopus or C. macrorhizus),with which I have mainly worked is very sensitive
to inhibitionby hexose analogues and is almost restrictedto using glucose as a
carbon source (Moore, 1969a). It is intolerant of deviation from the ,l-Dglucopyranosestructure.This leads to some contrastsbetween Coprinusand other
fungi. For instance, while it is not surprisingto find that its hyphal growth is
inhibitedby deGlc withan ED50 (concentrationcausing 50 % inhibitionof growth
rate) of 0 05 mmand by sorbose (ED50 = 1 5 mM), it is unusual thathyphalgrowth
is inhibitedby galactose (ED50 = 10 mM) and glucosamine (ED50 = 0-2mM). Only
about 20 % of the species surveyedby Lilly and Barnett(1953) were unable to use
galactose, but it is generallya poorer carbon source than glucose. In some cases,
good growth occurs in mixtures of galactose with other poor carbon sources
(Steinberg, 1939). There are fewreportsof toxicityalthoughHorr (1936) reported
that,when used alone, galactose decreased ratesof spore germinationand mycelial
development, and caused abnormal hyphal growth in Aspergillus niger and
Penicilliumglaucum. Similar effectshave been reportedfor mixturesof galactose
with fructoseor glucose forPodospora anserina(Lysek and Esser, 1971). Glucosamine is similarlyvariable in effect,servingas sole source of carbon and nitrogen
in Aspergillusnidulans yet being inhibitoryto Coprinus. To some extent, such
organisms.The soil fungus
differencesmaybe relatedto the ecologyofthe different
A. nidulansgrowsin an environmentin which 5 to 40 % ofthe totalnitrogenoccurs
in the formof polymersof aminosugars (Bremner, 1967), so it mightbe expected
to make some use of these materials. On the other hand, C. cinereusgrows in
conditionswherecellulose is by farthemost importantcarbohydrate(Chang, 1967;
Chang and Hudson, 1967). However, similar variations in sensitivityhave also
been observed with unnaturalsugar analogues to which such ecological interpretations are unlikelyto apply. Sorbose, for example, is rarelyfound in nature; it
does not occur in the freeformbut is foundin the pectinofPassifloraedulis(Martin
and Reuter, 1949). Its occurrence in the fermentedjuice of Sorbus berries is a
secondaryfeatureresultingfrombacterial metabolism (Schaffer,1972). Although
the growthof many fungiis inhibitedby sorbose (Barnett and Lilly, 1951), some
are able to utilize it either as their sole carbon source or synergisticallyin the
presence of glucose or maltose (Lilly and Barnett, 1953). A similar spectrum of
effects,complete inhibition in some cases contrastingwith ability to utilize the
genera
analogue in othercases, has been observed forsorbose in studies ofdifferent
of basidiomycetes (Oddoux, 1952), differentspecies of Phyllosticta(Bilgrami,
1963) and differentmonosporidial isolates of Ustilago maydis (Matsushima and
Klug, 1958). Agaricus macrosporusand Neurospora can metabolize sorbose, the
latterapparentlyvia D-glucitol(sorbitol),but only aftera long period of adaptation
also growsslowly
(Bohus, 1967; Crocken and Tatum, 1968). Pyrenochaetaterrestris
with sorbose as sole carbon source but the finalyield of myceliumwas only 20 %
less than thatobtained with otherhexoses and the myceliumwas foundto contain
sorbitol in addition to the usually present mannitol (Wright and Le Tourneau,
1965). It is thereforeimpossible to generalize or confidentlypredict the likely
response of any isolate to exposure to sorbose. Fewer organismscan utilize deGlc
Hexose analogues and fungi
493
Chaetomium,
but Neurospora,Aspergillusspp., Glomerella,Cunninghamella,
and Xylaria all have at leastsome abilityto use thisanalogueas
Neocosmospora
poorand,in somecases,such
a sole sourceofcarbon,thoughgrowthis generally
through
of analoguehave been employedthatcross-feeding
highconcentrations
out
be
ruled
cannot
sugars
metabolized
readily
by
analogue
of
the
contamination
(Ruiz-Amil,1959; Sols, Herediaand Ruiz-Amil,1960; Atkin,Spencerand Wain,
1964; Moore, 1969b).
and relatedsugars
2-deoxy-D-glucose
ofinhibition:
2. Mechanisms
metabolismin twobroadareas;
sugaranaloguesseemto influence
Inhibitory
metabolism)and
(energy-yielding
metabolism
intheinitialstagesofcarbohydrate
by
The potentialsitesinvolvedin inhibition
synthesis.
in stagesofpolysaccharide
(iii)
and
hexokinase
of
inhibition
withsugaruptake,(ii)
deGlc are: (i) interference
isomereasebydeGlc-6P.The lattercompoundis also
ofphosphoglucose
inhibition
likelyto affectenzymesat otherstagesof glycolysisand in otherpathways,and
metabolizedalso has implicationsin termsof the
the factthatit is not further
thesegeneralheadings
depletionof pools of phosphateand ATP. Effects-under
occur in a wide range of cell types includingyeasts and filamentousfungi.
However,a greatdeal of workhas been done withanimal cells because early
intumourcellsintherat(Woodward
ofmetabolism
on theinhibition
observations
agent.
carcinostatic
as a potentially
in
deGlc
interest
and Cramer,1952)generated
on mousetumours
ofD-glucosamine
fortheeffect
Oddly,thesimilarobservations
(Quastel and Cantero,1953) have not been followedup so assiduously.
step
Interactionbetweenutilizablesugarsand theiranaloguesat thetransport
of growthcaused
is indicatedby the abilityof the formerto reverseinhibitions
by deGlc. In almostall cases in whichthecomparisonhas been made,inhibition
mediathanon thosecontaining
is muchmorereadilyachievedon fructose-based
in sensitivity
difference
threefold
to
glucose. Thus, in yeast,therewas a twoamongmost
difference
a
tenfold
about
and
1964)
Sols,
and
Fuente
(Heredia,de la
wasexceptional
cinereus
fungitestedbyMoore(1969b).Coprinus
ofthefilamentous
in sensitivity
dependingon
in this latterstudyin havinga 100-folddifference
are interpreted
whetherthemediumcontainedfructoseor glucose.These effects
as beingdue to the relativeabilitiesof glucoseand fructoseto inhibituptakeof
fallintopatternswhich
sensitivities
the analogueintothe cell. The differential
sugars.In Coprinus,
mechanismsforthedifferent
oftransport
theaffinities
reflect
forglucosethanforfructose
the transportcarrierhas a 100-foldgreateraffinity
(Moore and Devadatham,1979)and,in yeast,thereis a five-to tenfolddifference
in affinity
(Kotyk,1967; Cirillo,1968). Clearly,in theseorganisms,fructosewill
the
analogues.Conversely,
be lessable thanglucoseto inhibituptakeofinhibitory
analoguesmustinhibituptakeofmetabolizablesugarsnotonlyby theirpresence
oftheir
theeffects
on theoutsideofthemembranebutalso through
as competitors
accumulationon the regulationof sugartransport(Heredia, de la
intracellular
oftenemploying
oftransport,
Fuenteand Sols, 1964).Directstudiesofmechanisms
of
inhibitions
the
demonstrate
clearly
sugaranaloguesfortechnicalconvenience,
impact
the
one
case,
in
at
least
different
and,
between
sugars
which
can
occur
uptake
has also beenexplored(H6fer
on laterstagesofmetabolism
transport
ofmetabolite
and Dahle, 1972). However, it is doubtfulwhetherthe inhibitionof uptake
contributes
greatlyto the overallinhibitionof growthsince,in mostcases, the
analoguescause considerableinhibitionsof growthon media containingnonsourcesof carbonsuch as acetateor glycerol.An exceptionseems
carbohydrate
whichappearsto inhibitoxidationof glucose
to be 6-deoxy-6-fluoro-D-glucose
D. MOORE
494
in yeast prior to phosphorylationat a step suggested to be involved in the entry
of sugar into the cell (Blakley and Boyer, 1955). The same conclusion was reached
in studies involving animal tissues in which neitheracetate nor lactate oxidation
was sensitive to inhibition by this analogue (Serif and Wick, 1958). At high
concentrations,inhibitionsby 6-deoxy-6-fluoro-D-glucosereached maxima which
differedfrom tissue to tissue but never reached 100 %, suggesting that this
analogue acts very specificallyon only some of the routes by which sugar enters
the cells (Serif et al., 1958). If thisis true,then it mightbe veryusefulforstudying
transportprocesses, particularlyin those cases where multiple uptake systems
differingin substrate specificityoccur. Generally, though, interactionsat the
transportlevel are probably more importantin relationto the abilityof the normal
metaboliteto protectagainst the toxicityof the analogue. As already indicated, if
the normal metaboliteis a sugar it will protectin proportionto the affinity
of the
transportcarrierand to its own abilityto compete withthe analogue fortransport.
An interestingexception occurs in Aspergillusnidulans,where acetyl coenzyme A
exerts a regulatoryinfluenceon hexose transport(Romano and Kornberg, 1968,
1969; Desai and Modi, 1974, 1977). A consequence is thatacetate protectsagainst
the toxicityof the sugar analogue such that, in acetate media, 50-fold higher
concentrationsof sorbose or deGlc are required to produce inhibitionsequivalent
to those produced with glycerol as carbon source (Elorza and Arst, 1971). This
mechanism does not operate in Coprinus,and the greatestdegrees of inhibitionof
growthof this organism are observed with acetate as carbon source (Moore and
Stewart, 1972).
The hexokinase of yeast, Neurospora and Aspergillusreadily phosphorylates
deGlc (Sols et al., 1958, 1960; Ruiz-Amil, 1959; Heredia et al., 1964; Biely and
Bauer, 1967). Yeast hexokinase does not phosphorylateeither4-deoxy-D-glucose
or 6-deoxy-D-glucose (Kotyk et al., 1975). Table 1 shows some data for the
Coprinus enzyme. The similarities of affinityconstants lead to considerable
competitive inhibitions between the analogues and utilizable sugars for phosphorylation. It has been claimed that, for at least one strain of Saccharomyces
cerevisiae,phosphorylationof deGlc is associated with its transportacross the cell
membrane(van Steveninck,1968). There is considerabledoubt about thegenerality
of involvementof phosphorylationin sugar transportin yeast (Jennings,1974;
Kotykand Janacek,1975) and, even in thisparticularcase, althoughvan Steveninck
(1968) showed deGlc-6P to be a precursorof intracellularfreedeGlc, intracellular
Table 1. Kinetic constantsof thehexokinaseof Coprinus cinereus
substrates
withdifferent
Substrate
Glucose
2-Deoxy-D-glucose
Glucosamine
Fructose
Km (mM)
Vmax
0-107
0 410
0 087
2-858
264
263
103
181
Phosphorylation
coefficient
1
0 26
0 49
0 03
withammoniumsulphateand assayedusing
The enzymewas partiallypurifiedby fractionalprecipitation
a coupled systemwhich detectedformationof ADP. Vmmaxis given in units of nmol productmin-' (mg
calculatedaccordingto Sols & Crane (1954). Sorbose was completely
coefficient
protein)-'.Phosphorylation
inertas a substrate.Unpublished data of S. J. Taj-Aldeen.
Hexose analogues and fungi
495
hydrolysisofthephosphatewas also demonstrated,so hexokinaseactivityremained
of great importance for the accumulation of deGlc-6P. A related analogue,
glucosone, inhibitsfermentationand growthofyeastlargelyby its abilityto inhibit
activity of hexokinase (Mitchell and Bayne, 1952; Woodward, Cramer and
Hudson, 1953; Hudson and Woodward, 1958). Sorbose is not phosphorylated,but
some unphosphorylatedsugar analogues show inhibitoryactivitieseven in vitro;
forinstance, 5-thio-D-glucoseat concentrationsof 0-25 mm and above completely
inhibitsactivityofcommercialpreparationsofmushroomtyrosinase(Prabhakaran,
1976).
However, it is clear from work with yeast and animal tissues that in vivo
inhibitionby deGlc is conditional on its phosphorylation(Heredia et al., 1964).
Phosphohexose isomerase and glucose 6-phosphatedehydrogenaseare particularly
sensitive to inhibition by deGlc-6P (Barban and Schulze, 1961). Glucosamine
exertsa similarpatternof effects;it is readilyphosphorylatedby brain hexokinase
(and see Table 1) and inhibitsphosphorylationof glucose (Harpur and Quastel,
1949), while the glucose 6-phosphate dehydrogenase of yeast is competitively
inhibited by glucosamine 6-phosphate (Glaser and Brown, 1955).
Formation of the 6-phosphate is probably the limit of analogue metabolism in
most cases but thereare otherpossibilities. 5-Thio-D-glucose and its 6-phosphate
are, respectively,substratesforhexokinaseand glucose 6-phosphatedehydrogenase
of yeast, though the rate with the latteris only 5 % thatwith glucose 6-phosphate
(Burton and Wells, 1977). The glucose oxidase of Aspergillusnigeroxidizes deGlc
at about 20 % of the rate observed with glucose; surprisingly,mannose was
oxidized at only 1 % and galactose at only 0-5% of the glucose rate (Pazur and
Kleppe, 1964). Other glucose oxidases behave similarly(Sols and de la Fuente,
1957). Cell-free extractsof the polypore, Trametessanguinea, oxidized sorbose
although the sugar was not assimilated for growth.The purifiedenzyme was an
L-sorbose oxidase which used substrate sorbose to form the products 5-ketoD-fructose and hydrogen peroxide. The metabolic function of the enzyme is
unclear; fructosewas inactiveas a substratealthoughglucose, galactose,xyloseand
maltose were oxidized (Yamada et al., 1966, 1967). There is evidence forsorbose
metabolismin otherfungialthough,as will be discussed later,thereis no evidence
for the formationof sorbose phosphates. Galpin, Jenningsand Thornton (1977)
reported that mycelia of Dendryphiellasalina exposed to randomly isotopically
labelled sorbose for 48 h converted about 9 % of the available 14C to 14CO2. In
Neurospora,Crocken and Tatum (1968) claim thatsorbose is eventuallyconverted
to glucose withsorbitolas an intermediate,sorbose being utilized forgrowthwhen
the medium also contains sucrose (Trinci and Collinge, 1973). Elorza and Arst
(1971) have also demonstratedthe formationof sorbitolfromsorbose in Aspergillus
nidulans; however,in thiscase, the evidence showed that14C introducedas sorbose
eventuallyappeared as fructose.Conversion of sorbose to sorbitol is also evident
in Pyrenochaeta terrestris(Wright and Le Tourneau, 1965). Although these
reactionsprovide a means wherebysorbose can be detoxified,only a small fraction
of the applied sorbose is usually metabolized in this way; the bulk of it remains
unaltered.
An inducible coenzyme-dependentglucose dehydrogenaseable to act on deGlc
has been reported in Aspergillus oryzae (Ruiz-Amil, 1959) and two similar
dehydrogenaseshave been found in Pseudomonasaeruginosa(Williams and Eagon,
1959). One of these latterenzymes could not be separated fromglucose dehydrogenase while the other oxidized deGlc but not glucose and may have been an
17
ANP 87
496
D. MOORE
expression of activityof a deoxyribose dehydrogenase. In neither case was the
deoxygluconate formed metabolized further. In Leuconostoc mesenteroides,an
enzymeable to oxidize deGlc-6P using NADP as coenzymehas been demonstrated.
This enzyme activitywas separable fromglucose 6-phosphate dehydrogenasebut
2-deoxy-D-gluconate 6-phosphate was not furthermetabolized (deMoss and
Happel, 1955). No such activitycould be found in Neurosporaor A. oryzae (Sols
et al., 1960; Ruiz-Amil, 1959).
It seems that the most usual pathway for metabolism of deGlc leads, through
hexokinase, to the formation of the 6-phosphate which, as it is not further
metabolized, accumulates and inhibitsearly glycolyticenzymes. Among enzymes
which deGlc-6P inhibitsare phosphohexose isomerase, phosphofructokinaseand
aldolase (evidence mostlyfromanimal cells; Webb, 1966). Both phosphoglucose
isomerase and phosphomannose isomerase of yeast are sensitiveto deGlc-6P, and
inhibition of their activities in vivo causes drastic alteration to the balance of
carbohydratemetabolism (Kuo and Lampen, 1972). Inhibition of enzymes by
deGlc-6P is evidentlyan importantaspect of the overall growthinhibitioncaused
by this analogue but it must also be significantthat formationof the 6-phosphate
both depletes cellular supplies of ATP and impairs the metabolic ability to
replenishthose supplies. This aspect can have wide-rangingeffects,since any part
of metabolism which is regulated by the energy charge or is dependent on
nucleotide pools will be influenced by changing levels of adenine nucleotides.
Much of the evidence that such changes can be extremelydramatic again comes
fromwork with animal tissues (Webb, 1966); one example is worth description.
Within 12 min of firstexposure to deGlc, Krebs ascites cells lose 65 % of their
adenine nucleotides; the loss proceeds throughATP, ADP and AMP, but the latter
is then deaminated and the resultinginosine monophosphatedephosphorylatedso
thatinosine appears within1 min and, by the 12thmin, inosine is the predominant
250 to 260 nm absorbing compound in the cell extracts(McComb and Yushok,
1964a, b). No fungalcell typehas been examined in as greatdetail as have animal
cells, but this drasticeffecton purine nucleotide metabolismis probably a general
one. Accumulation of deGlc-6P in Saccharomyces cerevisiae [and 2-deoxy-Dgalactose 1-phosphate in Kluyveromycesmarxianus(= S. fragilis)] was balanced
quantitativelyby simultaneous decreases in levels of ATP, orthophosphateand
polyphosphate(van Steveninck,1968; Jaspersand van Steveninck,1976). In yeast
exposed to 33 mm deGlc for 6 h, the concentrationof inosine (not detectable in
glucose-growncells) increased until it representedabout 60 % of the total purines
in the cell extract. There was a concomintant drop in the level of adenine
nucleotides, ATP in particularbeing reduced to a level only 13 % of that in cells
exposed to glucose. Indications of accumulationof hypoxanthinein the yeastwhen
incubationwithdeGlc was prolonged suggestthatthe cells embarkupon full-scale
purine degradation(Oppenheim and Avigad, 1965). Anotherstudyhas illustrated
the rapidity with which deGlc affectsphosphate and purine nucleotide status
(Maitra and Estabrook, 1962). Addition of ethanol to starved yeast leads to an
immediate increase in ATP levels which is mirroredby reduction in amounts of
both ADP and inorganic phosphate. Addition of 20 mm deGlc causes a rapid
decline in both ATP and inorganic phosphate. Within 1 min of the addition of
deGlc, the phosphate level is reduced to about 30 % of its initial value and ATP
to about 50 % of its startingconcentration.In the same period, ADP levels more
than double. Under these conditions sufficientphosphate can be trapped in
deGlc-6P to make respirationlimitedby phosphate availabilityeven thoughADP
Hexose analogues and fungi
497
levels remain high (Maitra and Estabrook, 1967). Six minutes afteraddition of
deGic, both inorganic phosphate and ATP declined to between 10 and 15 % of
the levels existingat the time the analogue was firstadministered,ADP levels also
began to decline as, presumably,nucleotide degradationwas initiated(Maitra and
Estabrook, 1962). Anaerobic fermentationis stimulated in yeast. In dialysed
extracts, a fermentationof fructose 1,6-bisphosphate which is independent of
adenosine phosphates is stimulated by deGlc. In intact cells under anaerobic
conditions,production of CO2 was enhanced by deGlc by a factorof about four,
endogenous glycogenstoresbeing fermentedto alcohol and CO2. In the suggested
pathway, hexose phosphates take the place of adenine nucleotides as phosphate
acceptors (Scharff,Moffittand Montgomery, 1965; Scharffand Montgomery,
1965, 1966). A transientstimulationof oxygen consumption occurs in both yeast
and tumour cells very soon afterfirstexposure to deGlc which is presumably a
response to the rapid change in energycharge resultingfromphosphorylationof
the sugar (Feldheim, Augustin and Hofmann, 1966; Coe, 1968). Most metabolic
processes, however, are inhibited. In ascites cells, contentsof nucleotide triphosphates other than ATP also decline followingtreatmentwith deGlc (Letnansky,
1964) and, in consequence, ratesof synthesisofboth DNA and RNA can be greatly
reduced (Klenow, 1963). Inhibitionsof oxidation of Pyruvate,lactate,acetate and
palmitate in yeast have been ascribed to depletion of ATP by deGlc (Feldheim
et al., 1966; Sauermann, 1968). On the otherhand, inhibitionof ethanol oxidation
is thoughtto be due more to direct enzyme inhibitionby deGlc-6P than to lack
of ADP or phosphate (Feldheim et al., 1966).
Recent work with baker's yeast has revealed what may be considered to be a
detoxificationreaction for deGlc-6P. After60 min incubation with isotopically
labelled deGlc, the bulk of the intracellular radioactivity is associated with
2,2'-dideoxy-a,a'-trehalose rather than with deGlc or its 6-phosphate (Farkas,
Bauer and Zemek, 1969; Meredith and Romano, 1977). A trehalose-likederivative
has also been found within cells of Kluyveromycesmarxianus exposed to 2deoxy-D-galactose (Jaspers and van Steveninck, 1976). Large-scale synthesisof
trehalose is a normal accompaniment of sugar uptake in yeast (Kotyk and
Michaljanicova', 1974) so the synthesisof dideoxytrehaloseon exposure to deGlc
is another instance of the analogue being used metabolicallyin place of glucose;
disaccharide synthesisin itselfis not a pathological response to exposure to deGlc.
Synthesisofdisaccharideanalogues is not restrictedto yeast./3-D-Fructofuranosyl2-deoxy-D-glucosehas been isolated fromleaves of several plant species following
2 days incubation with 50 mM deGlc (Barber, 1959). The same disaccharide was
obtained in vitrowhen leaf homogenates of Impatienssultaniwere incubated with
deGlc and sucrose in conditionsfavouringinvertaseactivity.In this disaccharide,
the glycosidic bond occurred at eitherC-5 or C-6 of the deGlc moiety,so this was
not a sucrose analogue (Barber, 1959). However, in-vitrosynthesisof 'deoxysucrose' has been demonstratedwith a sucrose synthetasepreparationfrompea
seedlings (Farkas, Biely and Bauer, 1968). The only reportof deGlc in nature is
in the formof a glycoside in Erysimum(Kowalewski, Schindler and Reichstein,
1960). The dideoxytrehalosein yeast representedthe major portion of partially
metabolized intracellulardeGlc, but energy was still expended continuously to
maintain an intracellular pool of deGlc-6P (Meredith and Romano, 1977).
Diversion of deGlc intothe disaccharidewill provide some alleviationofphosphate
sequestration but continued utilizationof ATP will be damaging.
In theirreviewofthe effectofmannose and itsanalogues on greenplants,Herold
17-2
498
D. MOORE
and Lewis (1977) argue 'that the primarycause of almost all of the inhibitory
effectsof mannose, deoxyglucose and glucosamine in green plants is the synthesis
and limitedfurthermetabolismoftheirphosphate esters'. The rapid effectexerted
by deGlc on phosphate and purine nucleotide balances must make this a central
featureof any growthinhibitioncaused by this sugar. However, it is unlikelythat
the inhibition
all of the inhibitoryeffectsin fungiare due to thiscause since, firstly,
of growthis generallymuch greaterthan any inhibitionof respirationor glycolysis
that can be measured and, secondly, there are some sugar analogues which are
potentinhibitorsof growthin filamentousfungibut which are not phosphorylated.
Fermentationin Saccharomycescerevisiaeis inhibitedless than 50 % by a 1: 0 3
molar ratio of glucose: deGlc, whereas growthis inhibited more than 50 % at a
1:0-05 ratio (Heredia et al., 1964). These authors concluded that inhibition of
growthby deGlc was not a direct consequence of the demonstratedinhibitionof
glycolysis, because quite high growth rates were obtained when disaccharide
fermentationwas considerablyinhibitedby otherunfavourableconditionssuch as
adverse pH values and concentrationsof substrate. Similar results have been
obtained with Coprinuscinereus.In thisorganism,mixturesof glucose + deGlc and
fructose+ deGlc which caused at least 50 % inhibitionof hyphal growthhad very
little effecton oxygen uptake. Even in media containing 5 mm deGlc as sole
carbon+ energy source, oxygen uptake was only 75 % inhibited although linear
growth did not occur (Moore, 1968). Clearly, factors other than inhibition of
energy-yieldingmetabolismmust contributeto inhibitionofcell growth,and there
is now convincingevidence thatmuch of the inhibitoryactivityof deGlc concerns
synthesisof polysaccharide.
It is well establishedthatdeGlc adverselyaffectscell wall synthesisin yeast.This
is undoubtedlythe basis of the fungicidalaction thatthis compound has on yeasts,
since sensitivitywas proportionalto the mean rate of extension of the individual
yeast cells (Johnson and Rupert, 1967); only growing cells were killed (Heredia
et al., 1964) and fragmentsof cell wall were found in the treated suspensions
(Megnet, 1965). Lysis occurred at points coinciding with the regions of growth
of the glucan layersofthe yeastcell wall (Johnson,1968a). The major consequence
to growth in deGlc is the osmotic fragilityresultingfromweakening of the cell
walls. Erosion of preformed glucan seems to be a likely component of the
weakeningprocess. Johnson(1 968b) showed thatthe amounts ofglucan in cultures
of Schizosaccharomycespombe decreased after about 1 h exposure to deGlc.
Svoboda and Smith (1972) distinguishedearlylysis,caused by degradationof wall
material at the growing sites, from late lysis caused by deposition of aberrant
material. However, the latterauthors showed that deGlc was not incorporated
into the walls of the fissionyeast, although incorporationhas been demonstrated
into wall glucan of Saccharomycescerevisiae(Biely et al., 1971; Kraitky,Biely and
Bauer, 1975). Rather than being due to inclusion of the analogue, general
weakening of the wall is thoughtto be largelythe consequence of interferenceby
phosphorylatedmetabolites of deGlc with metabolic processes involved in the
incorporationofglucose and mannose intostructuralwall polysaccharides(Heredia
et al., 1964; Farkas, Svoboda et al., 1969; Biely et al., 1971). The phosphorylated
metabolitesinclude both uridine and guanosine nucleotide derivatives(Biely and
Bauer, 1966, 1968; Jaspersand van Steveninck, 1976). It is generallyconsidered
that,by trappingthese nucleotides into a metabolicallyunusable form,deGlc (and
2-deoxy-D-galactose) is able to block synthesis of striictural polysaccharides
(Heredia et al., 1964; Biely and Bauer, 1968). Much the same seems to be true
Hexose analogues and fungi
499
of 2-deoxy-2-fluoro-D-glucose,although this compound acts more specificallyas
an analogue of glucose (deGlc is an analogue of both glucose and mannose) and
in cosequence glucan synthesisis the most sensitiveprocess (Biely, Kovarik and
Bauer, 1973). Although glucono-8-lactone also inhibits glucanase and glucan
formationin the yeast, Pichia polymorpha,as well as causing inhibitionsof RNA
and protein synthesis,there was only a small effecton the growthrate. After12
h incubation,up to 20 % of the cell population were aberrant- abnormallylarge,
sphericaland lackingbuds (Villa et al., 1976). The authorsconclude thatthisagent
seriously affectscell wall development.
Such detailed analyses have not been attemptedwithany filamentousfungusbut
it seems likelythatsimilarbiochemical eventsoccur aftertheirexposure to deGlc.
It inhibited glucan formationin Aspergillusnidulans(Zonneveld, 1973) and lysis
ofhyphaltip cells occurredin activelygrowinghyphaeof Coprinuscinereusexposed
to deGlc (Moore and Stewart, 1972). Although glucosamine did not cause lysis,
thetip cells ofCoprinushyphaeswelled enormously,probablyowingto interference
with wall synthesis.A characteristicand structurallyimportantcomponent of the
fungal wall is chitin, a (1 -4)-f3-homopolymerof N-acetylglucosamine. At low
substrateconcentrations,glucosamine is an allostericactivatorof chitin synthase
in Coprinuswhile glucosamine 6-phosphate is an inhibitor(de Rousset-Hall and
Gooday, 1975). Wall growthis thoughtto depend on a balance between lysis of
existing chitin fibrilsby chitinase and synthesisof new chitin by the synthase
(Gooday, 1975, 1977; Gooday, de Rousset-Hall and Hunsley, 1976; Gooday and
Trinci, 1980) and it is possible that the swelling observed in Coprinus hyphae
exposed to glucosamine results from disturbance of the lysis/synthesisbalance.
That this may well be the case is indicated by work with a temperature-sensitive
mutantof Aspergillusnidulansin which the hyphal tips were osmoticallyunstable
at 42 ?C (Cohen, Katz and Rosenberger,1969). The osmotic instabilitywas caused
by a lack of chitin in the cell wall, but this defect could be reversed by
supplementing the growth medium with glucosamine (Katz and Rosenberger,
1970). Rather similar results have been reported for a mutant of Saccharomyces
cerevisiaedefectivein glucosamine 6-phosphate ketol-isomerase.In this case, the
lesion in chitin synthesisresulted in defectiveseptation so that,in the absence of
supplementation with glucosamine, vegetativedaughter cells failed to separate.
Ascospore walls were also abnormal, lacking a glucanase-resistanthydrophobic
surface layer. Viability of vegetative cells decreased after 3 to 5 h incubation
without glucosamine because of lysis in the highly disorganized septum region
(Ballou et al., 1977). Such observations emphasize the direct involvement of
glucosamine in chitinsynthesisand indicatethe inabilityof otheraspects of hyphal
metabolism to compensate for imbalance in supply of the amino sugar. Similar
work with an A. nidulansmutantin which the wall defectwas relieved by growth
on mannose (Valentine and Bainbridge, 1978) shows that synthesisof other wall
polysaccharidesis equally sensitiveto the level of supply oftheirconstituentsugars
and again indicates that, in constructionof the total wall, there is no ability to
compensate for defectsin any one of the wall components.
As faras non-wall polysaccharidesare concerned,glucosamine and its N-acetyl
derivativeboth inhibitedconversionof glucose or fructoseto glycogenby rat liver
slices, probably via inhibition of glucose phosphorylation (Spiro, 1958). The
reduced net accumulation of glycogen may result fromacceleration of glycogen
breakdown ratherthan a true inhibitionof synthesis(Webb, 1966). Synthesisof
glycogen in hepatoma cells is also inhibited by deGlc (Nigam, 1966) and it is
500
D. MOORE
certainlythe case that this analogue stimulates degradation of glycogen in yeast
(Farkas, Svoboda & Bauer, 1969). In this latter case, though, deGlc was not
incorporatedinto glycogenin vivo despite the factsthatan in vitroenzyme system
can incorporatedeGlc intosome outerchains ofthe glycogenprimer(Biely, Farkas
and Bauer, 1967) and incorporationinto glycogen in vivo had been demonstrated
in hepatoma cells (Nigam, 1967).
There is also firmevidence that deGlc inhibits the synthesis of exocellular
enzymes,particularlyinvertaseand acid phosphatase, in Saccharomycescerevisiae
and Pichia polymorpha(Kraitkyet al., 1975; Kuo and Lampen, 1972; Villa et al.,
1975). There is a distinctdifferenceof opinion about the basis of this inhibition
in S. cerevisiae.Kuo and Lampen (1972) showed that the synthesisof invertase
and acid phosphatase is inhibited by deGlc under conditions which cause only
slightdecreases in general proteinsynthesis.They suggestthat deGlc-6P inhibits
or restrictssynthesisof the carbohydratecomponent of these glycoproteinsand
therebyreduces formationof the active extracellularenzyme. On the other hand,
Kraitkyet al. (1975) could find no correlation between inhibitions of mannan
synthesisand the appearance of invertaseand-acid phosphatase. They suggestthat
the effectof deGlc on the secretionof exoenzymes is complex but is related more
to inhibitionof synthesisof the proteincomponent than to effectsexerted on the
carbohydratemoiety.
3. Mechanismsof inhibition:sorboseand 3-0-methylglucose
Sorbose causes what is probably the best known and most widely employed
morphological change in the growth of susceptible filamentousX
fungi. The
extension growthrate is reduced, cells are much shortened,branchingfrequency
is increased withthe enhanced occurrenceof branched tips. Although sorbose has
been much used to impose colonial growth morphology on rapidly growing
filamentousfungi,very little is known about the enzymological aspects of these
effects.
Hexokinase and aldolase of beef brain are inhibitedby sorbose 1-phosphatebut
not by sorbose and its 6-phosphate (Lardy et al., 1950). Sorbose 1-phosphateand
the 1,6-bisphosphatebehaved as competitiveinhibitorsof yeast aldolase, with K1
values of the same order of magnitude as the Km for fructose 1,6-bisphosphate
(Richards and Rutter, 1961) but sorbose 1,6-bisphosphate was not used as a
substrate.However, no evidence has been obtained forphosphorylationof sorbose
in fungialthoughformationof sorbose 1-phosphateby ketohexosekinaseshas been
detected in mammalian systems(Hers, 1952; Cadenas and Sols, 1960).
In baker's yeast, sorbose is inertboth as substrateand inhibitorof hexokinase
(Sols et al., 1958). The same is true for brain hexokinase under conditions able
to detect phosphorylationrates less than 10-4 that of glucose (Sols and Crane,
1954). In Aspergillusparasiticus, the rate of phosphorylation of sorbose by
hexokinasewas no more than 2 % thatof glucose (Davidson, 1960). These studies
leave open thepossibilitythatketokinasesmayexist. Indeed, as sorbose 1-phosphate
formationhas been observed in vitro by the action of crystallinerabbit muscle
aldolase on hexose bisphosphate and L-glyceraldehyde (Lardy et al., 1950),
alternativemodes of synthesisof sorbose phosphates may be real possibilities. It
is particularlyimportant,therefore,to note thatseveralunsuccessfulattemptshave
been made to extract sorbose phosphates from the cell contents. Most of the
relevantwork with Neurospora concerned sugar transportin that organism and,
althoughthereare manycontradictionswithregardto thesortsoftransportsystems
Hexose analogues and fungi
50I
involved and their kinetic characters,there is unanimitythat no phosphorylated
formsof sorbose can be found in cell extracts(Klingmiiller, 1967a, b; Crocken
and Tatum, 1968; Scarborough, 1970). The same is true forAspergillusnidulans
(Elorza and Arst, 1971) and Coprinuscinereus(S. J. Taj-Aldeen, unpublished). In
sharp contrast to deGlc, sorbose evidently exerts its inhibitoryactions while
chemically unchanged.
Although Crocken and Tatum (1967) claimed thatsorbose was not accumulated
against a concentrationgradientin Neurospora,Klingmiiller(1 967a) reportedits
accumulation in this organismto more than 600 times the externalconcentration,
and Scarborough (1970) showed thatthe internalconcentrationof sorbose reached
300 mm after 30 min exposure of germinatedconidia of Neurospora to 10 mM
sorbose. It may be thatthe strainused by Crocken and Tatum (1967) was in some
way defectivein its sugar transportapparatus, since the Km, Vmaxand mode of
transportreportedby these authorsdifferconsiderablyfromany hexose transport
process reported subsequently for Neurospora (Klingmiuller and Huh, 1972).
Sorbose is accumulated by an active uptake systemin Aspergillusnidulans(Elorza
and Arst, 1971) and Coprinuscinereus(Moore and Devadatham, 1979) and, in the
latterorganism,accumulationcan proceed untilthe internalconcentrationis more
than three orders of magnitude higher than the external. The problem remains,
however, of identifyingthe point of action of this inhibitor. Sorbose does not
inhibit phosphorylationof fructosein Neurospora (Klingmuller, 1967a) and has
no effecton either in vivo or in vitroactivities of hexokinase, glucosephosphate
isomerase, phosphoglucomutase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenaseor NADP-linked isocitratedehydrogenase(Mishra
and Tatum, 1972). Althoughparticularenzymeinhibitionshave notbeen identified
by Neurospora mycelia in
in primarymetabolism,glucose is used less efficiently
the presence of sorbose. Mycelial dry wt yield is lessened, more CO2 is produced
and more 02 is absorbed per unit glucose used. Because of the many similarities
between the effectsof sorbose and of compounds known to uncouple oxidative
phosphorylation and respiration. Crocken and Tatum (1968) suggested that
sorbose causes such uncoupling.
The morphological changes which affectNeurospora hyphae grown on agar
media containingsorbose are accompanied by changes in the chemical composition
of the hyphal walls. The ratioof glucosamine to glucose in wall hydrolysatesmore
than doubles (de Terra and Tatum, 1961, 1963) and there is a marked decrease
in the concentrationof cell wall fl-1,3-glucanin sorbose-grownmycelia (Mahadevan and Tatum, 1965). The obvious inference from these data is that the
paramorphic change induced by sorbose results froma considerable alterationin
the balance of importantpolymericconstituents(probably chitin and glucan) of
the cell wall. A verysimilarglucosamine to glucose ratiowas found in hydrolysates
of cell walls fromcolonial mutantsgrown on sucrose. These are mutantswhich,
on sucrose media, have a morphologyvery similar to the paramorphicgrowthof
the wild type on sorbose+sucrose media (de Terra and Tatum, 1963). Sorbose
inhibitedthe in vitroand in vivo activitiesofglycogensynthetasesand ,3-1,3-glucan
synthetasesof Neurospora(Mishra and Tatum, 1972). The latterenzymewas found
only in cell-wall preparations,a feature which might account for the apparent
localization of the sorbose effectto the Neurospora hyphal surface for,although
theNeurosporacolonyhas a characteristicmorphologyimposed upon itby sorbose,
not all of the hyphae are equally affected.Aerial hyphae do not show the abnormal
growth form of submerged and surface hyphae (de Terra and Tatum, 1961).
502
D. MOORE
Moreover,hyphaeinjectedwithsorbosedidnotdeveloptheabnormalmorphology
was muchgreaterthanthatusually
despitethefactthattheinjectedconcentration
foundin paramorphichyphae(Wilkins,unpublished,quoted in de Terra and
Tatum, 1961). Thus it seems thatthe sorboseeffectin Neurosporamightbe
localizedat the cell surface.If the enzymesystemis locatedin the wall,thenan
is evident.Trinciand Collinge(1973)
sensitivity
explanationforthe differential
effectof sorboseon hyphaein
for
the
differential
have providedan explanation
colonywhichdoes notdependon localizationof
partsoftheNeurospora
different
thatsorbosehad littleor no effect
enzymesin thehyphalwall.They demonstrated
strainsgrownin liquidcultures.On solidmedia
ofNeurospora
on themorphology
gellingagents),radialgrowthratesofcoloniesweregreatly
(usingthreedifferent
reducedalthoughsorbosehad littleeffecton themaximumspecificgrowthrate.
salina(Galpinetal., 1977).Trinciand Collinge
The sameis trueforDendryphiella
of sorbosein solidmediais itsability
effect
(1973) stressthatthemostsignificant
altersthespatialdistribution
this
radically
because
branching,
to induceprofuse
of hyphae,producingdense colonies in which adverse conditions(nutrient
ofstalingproducts,pH change,etc.)areestablishedmuch
accumulation
limitation,
closertothecolonymarginthanis normal.In liquidculture,andforaerialhyphae,
no such localizationof adversitycan occur. Trinci and Collinge (1973) thus
ofsorboseas beingsecondaryand
macroscopiceffects
thecharacteristic
interpret
dependenton growthon solid media.The primaryeffectof sorboseobservedin
culturesgrownin liquid and on solid media seems to be an increasein wall
withhyphal
presumablyso interferes
thickness.This alterationin wall structure
on solidmedia
patterns
(Moore and Stewart,1972)thatbranching
morphogenesis
change and precipitatethe secondaryconsequencesreferredto by Trinci and
of
Collinge(1973). Apartfromnotingthatthereis no large-scaleincorporation
effect
sorboseintothehyphalwall(de Terra and Tatum,1961)and theinhibitory
to (Mishraand Tatum,1972),the,
alreadyreferred
ofsorboseon glucansynthetase
is unknown.Neither
mannerbywhichsorboseleadstoan increaseinwallthickness
wall structureand
between
of
relation
the
understanding
yet
is thereany clear
hyphalmorphogenesis.
partsofthe
betweendifferent
sensitivity
As well as accountingfordifferential
of Trinciand Collinge(1973) can accountfor
fungalcolony,thisinterpretation
to sorbose,whichdepended
in theresponseofNeurospora
thereportedvariability
on the natureand mannerof preparationof the growthmedium(de Serres,
Kolmarkand Brockman,1962; Brockmanand de Serres,1963). It canalso account
forthe absenceofgrowthinhibitionin yeasts(Woodward,Cramerand Hudson,
to
1953) forthese have been testedin liquid culture;it would be interesting
the effectof sorboseon yeastgrowthon solid media.A paramorphic
investigate
of sorboseon Saccharomyces
spp. has been reported.Lewis (1971) showed
effect
strainswhich
that sorbose restorednormalmorphologyto inositol-requiring
otherwisegrew as pseudomyceliain mediumcontainingmaltoseor c-methyl
glucoside.
The factthatno paramorphicchangesoccur in liquid mediumshows that
morphologicalchanges induced by sorbose do not resultfromany osmotic
imbalance.It is worthnoting,though,thatsimilarparamorphicchangescan be
induced by osmotic shocks (Robertson, 1958, 1959). Sorbose and 3-0cause extensiveburstingof hyphaltips in the marinefungus
methylglucose
1976) and sorbosecauses
salina (Thornton,Galpin and Jennings,
Dendryphiella
in a sporecolourmutantofNeurospora
(Rizviand Robertson,
apicaldisintegration
Hexose analogues and fungi
503
1965). The glucose analogue, 3-O-methylglucose, is not phosphorylated by
hexokinase (Sols and Crane, 1954; Sols et al., 1958) but is readily accumulated
by manycell types; accumulated materialin Neurosporawas completelyextractable
in unalteredform(Scarborough, 1970). In D. salina, 3-0-methylglucosepromotes
loss of mannitolinto the medium and conversionof this hexitolto polysaccharide.
The decline in mannitollevel cannot be compensated because 3-O-methylglucose
inhibitshydrolysisof glycogen.These events contributeto loss of osmotic control
by the hyphae (McDermott and Jennings,1976).
Galpin et al. (1977) have concluded that sorbose and 3-O-methylglucoseexert
their effectsby influencingthe level of intracelluarcAMP. Although there is no
directevidence, this conclusion was reached because of the similaritybetween the
effectof the sugar analogues and of theophylline [an inhibitor of the cAMPdependent phosphodiesterasein Neurosporacrassa (Scott and Solomon, 1973)] on
colony diameter and hyphal branching in Dendryphiella,and on the synergism
existing between these agents. The interpretationis essentiallythat the glucose
analogues so reduce carbohydratemetabolism that cAMP levels are affectedand
processes regulatedbythenucleotidebalance are consequentlydisturbed.Although
speculative, this interpretationfocuses attentionon the purine nucleotides, the
metabolism of which is also severely affectedby deGlc and glucosamine (see
above). However, simple comparison between morphological or other effectsis a
very weak foundationfor any interpretationand it should be rememberedthat,
on the same basis, Crocken and Tatum (1968) concluded that sorbose might act
as an uncoupler of oxidative phosphorylation.Furthermore,although change in
the concentrationof cAMP is clearly implicated in a number of morphological
alterationsin Neurospora,the data are too contradictoryforuseful generalization.
Thus Mishra (1976) showed thathigh concentrationsof cAMP in the medium (up
to. 6 mg ml.') induced colonial growth in both wild-type N. crassa and the
sorbose-resistantmutantpatch. Conversely,the morphologicalabnormalityof the
crisp-1mutant,which is correlatedwith a deficiencyof intracellularcAMP, is at
least partiallyrepaired by growthin medium containingcAMP (10 to 30 mM) or
some cAMP analogues (at 1 mM) (Rosenberg and Pall, 1979); however, thefrost
mutant,whichlacks adenylatecyclaseactivity,does notrespondto supplementation
with cAMP. Drugs like theophylline,atropine and histamine, which adversely
affectcAMP metabolism, induce wild-type N. crassa to grow with colonial or
semi-colonial growth habit in liquid medium (Scott and Solomon, 1975). The
morphological change is correlated with an approximate 75 % decrease in endogenous cAMP concentration. On the other hand, drugs which uncouple
oxidativephosphorylationcause 3- to 20-foldincreasesin endogenous cAMP levels
in N. crassa, yeast and Mucor racemosus(Trevillyan and Pall, 1979) and, although
transportfluxesofsugars(includingdeGlc and 3-O-methylglucose)cause transient
increases in cAMP level in N. crassa, the same response is obtained when the cells
are mechanicallystressed by vigorous shaking (Pall, 1977).
It remains extremelydifficultto understand the primary metabolic effectof
sorbose. The absence of the characteristic'sorbose growth' morphologyin liquid
culturesofNeurospora(Trinci and Collinge, 1973) is a keyobservationwhichneeds
to be verifiedforother fungiand which offersthe promise of a technique which
might distinguishprimaryfromsecondary effects.
504
D. MOORE
V.
SUGAR
ANALOGUE-RESISTANT
MUTANTS
Mutants resistant to the inhibitions of growth imposed by a variety of sugar
analogues have been isolated froma number of organisms. Although mutational
alterations in wall and cell structurehave been reported, most of the mutants
obtained have proved to have defectsin early metabolic stages, specificallythose
which influencethe accumulation of sugar analogue and/or analogue phosphate
withinthe cell. Judgingby the mutantswhich have been described,thereare three
main ways in which the last effectmay be achieved; uptake of the analogue may
be altered,a defectin phosphorylationmay arise or the phosphorylatedsugar may
be hydrolysedby mutant phosphatases ratherthan accumulated.
In Neurosporacrassa, mutationsin any one of six chromosomal genes can give
riseto resistanceto sorbose (Klingmiuller,1967c, d, e). All of the resistantmutants
were recessive to wild type,but only fourof the genes were involved in transport.
The phenotypesof the mutantshave not yetbeen well characterized;the functions
of the two genes not involved in transporthave not been identifiedat all. However,
fromanalysis of double mutants,it has been concluded thatthe different
transport
mutantscontributeneitherto a compound permease nor to componentsof a single
uptake pathway. Instead, it is thoughtthatthe product of each gene catalyzes the
same process so that,ratherlike isoenzymes,theyconstitutea set of isopermeases
(Klingmiiller, 1967f). Presumably, when mutation occurs in one of these genes,
it causes a defectin sorbose uptake which cannot be compensated by the normal
functioning of the remaining unmutated genes. Resistance conferred by the
differentgenes is additive at least to the extent that double mutants are more
resistantto sorbose than the originalsingle mutants.Neurosporamutantsselected
forresistanceto inhibitionof growthby sorbose do not appear to have been tested
forcross-resistanceto other sugar analogues. However, in both Coprinuscinereus
and Aspergillusnidulans,mutantsselected forresistanceto sorbose, and identified
as having transportdefects, are cross-resistantto deGlc. The situation in C.
cinereus has been the most extensively analyzed. Here, mutants resistant to
inhibitions of growth caused by deGlc, glucosamine and sorbose have been
separatelyselected. All are cross-resistantto the two analogues not used in their
selection. Furthermore,all ofthe mutantswere latershown to be able to grow only
poorly with fructoseas sole carbon source, whereas the wild type grows equally
well with eitherglucose or fructose(Moore and Stewart, 1971; Moore, 1973). In
N. crassa, Klingmiiller (1967d) isolated only 19 sorbose-resistantmutants yet
found that they represented six differentgenes. In sharp contrast, over 450
analogue-resistantmutantshave been reportedin C. cinereus,but theyall appear
to be allelic mutationsof a single geneticlocus (Moore and Stewart,1971; Moore,
1973). The definitionof allelism has been based both on complementationtests
in which the complementing dikaryon should be unable to grow on medium
containingthe inhibitor(Klingmiullerand Kaudewitz, 1967; Moore and Stewart,
1971) and on studies of allelic recombination (Moore and Devadatham, 1975).
These Coprinusmutantshad apparentlynormal abilities to metabolize sugars but
were imparedin the accumulationof sugars fromthemedium. They were therefore
assumed to be transportdefectiveand, because fructosewas the only utilizable
sugar with which the transport defect was apparent, the gene was called the
fructose-transport
(ftr)cistron(Moore and Stewart, 1971). Analysis of transport
of hexose in the C. cinereuswild type (Moore and Devadatham, 1979) showed that
a single negativelyco-operative uptake systemwas responsible for accumulation
Hexose analogues and fungi
505
of glucose, fructose and the sugar analogues. It is presumed that the ftr gene
product is the carrier (or permease) component of this system. Utilization of
of the
glucose may not be seriously impaired in the mutantsbecause the affinity
carrier for glucose is so high in the wild type (Km = 27 JLM)that a mutational
even if it causes a drasticalteration,will still
alterationwhich changes the affinity,
leave sufficient
residual functionto supportgood mycelialgrowthon normalmedia.
of the carrierforfructose(Km = 2-3
As possible support forthis view, the affinity
mM) is about 100 timesless thanthatforglucose in the wild type,yetthe mycelium
grows equally well on these two sugars. However, the system for transportof
glucose in the wild type does exhibit substrate-dependentmodificationsof its
activity,and it may be that understandingof the behaviour of the mutated gene
product is complicated by the dual role (modulating ligand and substrate) of the
[= C. radiatus
glucose molecule. A sorbose-resistantstrainof Coprinusfimetarius
sensuGuerdoux (1974)] has been reported(Prevost, 1958). The mutant,called sorr,
was isolated following u.v.-mutagenesis. It was markedly differentfrom the
C. cinereusftrmutantsin thatsorrwas dominantand showed a pronounced dosage
effectin homozygous dikaryons.All ftralleles are-recessive.The sorrmutantwas
used to study nuclear migrationbut no attempthas been made to establish the
biochemical basis of the resistantphenotype.
Elorza and Arst (1971) described sorbose-resistant mutants of Aspergillus
nidulans which conferredcross-resistanceto deGlc. The locus to which these
recessive mutations belonged was designated sorA. The mutants were defective
in accumulation of sorbose but utilization of fructose,glucose, glucosamine and
other sugars as carbon sources was unaffectedand uptake of glucose only slightly
depressed. It was concluded that the sorA gene product contributedto an uptake
systemwhich accounted forthe greaterpartof sorbose transportbut was relatively
unimportantin glucose transport(Elorza and Arst, 1971). If this interpretation
to understandhow cross-resistanceto deGlc can arise. The
is correct,it is difficult
systemfortransportingglucose into A. nidulanshas been characterizedby use of
deGlc as a non-metabolizedanalogue (Mark and Romano, 1971). Sorbose did not
inhibituptake of deGlc or fructoseby the strainused in thislatterstudy,suggesting
that, in Aspergillus,the normal route of uptake of sorbose is independent of the
separate permeases forglucose and fructose.At present,it is impossible to identify
the normal functionto which sorA corresponds. This will only be possible when
the normal process of sorbose uptake in A. nidulansis properlydescribed.
Interestin selection of mutantsresistantto sugar analogues in otherorganisms
has concentratedon selection forresistanceto deGlc. The mutantsobtained have
proved to have eitheraltered phosphorylationor phosphatase activities.Resistant
mutantsof Schizosaccharomyces
pombeobtained by selection on media containing
deGlc were unable to utilize glucose, fructoseor mannose and were deficientin
hexokinase (Megnet, 1965). Maitra (1970) isolated a mutant of Saccharomyces
cerevisiaelacking hexokinaseby screeningcells forresistancein galactose medium
containingincreasingconcentrations(1, 10 and 50 mM) of deGlc. S. cerevisiaehas
three enzymes able to phosphorylateglucose: hexokinase P 1, hexokinase P 2 and
glucokinase(Lobo and Maitra, 1977a). Defects in each ofthese activitieshave been
selected by exposing strains carryingonly one of the three enzymes to selection
for resistanceto deGlc (Lobo and Maitra, 1977b).
Anothertype of resistanceto deGlc in S. cerevisiaeresultedfromthe occurence
ofan intracellularneutralphosphatase which was able to hydrolysedeGlc-6P fairly
specifically(Heredia and Sols, 1964). This mutant was also obtained by serial
506
D. MOORE
(0 1 %, 1 %
cultivationin (fructose)media containingincreasingconcentrations
onlydeGlc-6P and
and 2 %) of the inhibitor.The purifiedenzymehydrolysed
fructose1-phosphate.It was presentin wild-typecellsbutonlyat about5 to 10%
function
oftheenzyme
ofthelevelsfoundinthemutant.The normalphysiological
is unknown(Martinand Heredia,1977). A somewhatsimilarsituationoccursin
HeLa humantumourcellswherea resistantcell line accumulateddeGlc-6P at a
much slowerratethanthe sensitiveparentalline (Barban, 1962). The resistant
ofalkalinephosphatasethantheoriginalcells.
strainhad4- to9-foldgreateractivity
Subsequently,Barban (1966) demonstrateda correlationbetween induced
resistanceto deGlc and alkalinephosphataseactivityby treatingcell populations
withhormonesknownto cause increasesin activityof this enzyme.A similar
correlationwas observedby Morrow and de Carli (1967). Unlike the yeast
phosphatasemutant,the phosphatasevariantof HeLa cells had an impaired
glucosemetabolism.The phosphataseactedas a net inhibitorof the hexokinase
the product,glucose6-phosphate,and the
reaction,presumablyby hydrolysing
growthof the resistantline was muchimprovedby additionof pyruvateto the
typeof resistancehas been observedin
medium(Barban,1962). An interesting
pig kidneycells (Bailey and Harris, 1968). Here, too, resistantmutantswere
isolated by serial cultivationin the presence of deGlc. The resistantcells
of hexosewerehigher
accumulatedless deGlc-6P,but ratesof phosphorylation
in extractsof resistantmutantsand alkalinephosphatasewas not detectablein
extractsof eithersensitiveor resistantcells. There was some increasein acid
phosphataselevels in resistantcells but this enzymewas unable to hydrolyse
deGlc-6P or glucose 6-phosphatein vitro.Resistantcells grewbetterthanthe
and it was concludedthatthebasis
sensitiveones at low glucoseconcentrations,
of the resistancewas that resistantcells were able to proliferateat glucose
too low to supportgrowthof sensitivecells (Bailey and Harris,
concentrations
1968). This typeof resistancehas notbeen observedin fungi.
inSaccharomyces
resistance
has revealedsome
cerevisiae
Analysisofglucosamine
mutantswithalteredresponseto cataboliterepressionresultingperhapsfroma
(Elliottand Ball, 1973). When excessglucose
changein membranepermeability
withglycerolas carbonsource,growthis first
is addedtoyeastgrowingaerobically
is initiated.Respiration
as fermentation
repressedbutsoonrecoversandaccelerates
is also repressedand, because of cataboliterepression,remainsso. Glucosamine
and completely
inhibitgrowth.Elliottand
and deGlc act as gratuitous
repressors
Ball (1973) isolatedresistantmutantsby selectingon mediumcontaining3 %
therewereat least
Among34 mutantsrecovered,
glyceroland 0 05 % glucosamine.
sevencomplementation
groups.Allbutoneofthemutantswererecessiveandthree
to deGlc(Elliottand Ball,
werecytoplasmic.
Some,butnotall,werecross-resistant
on the
1973; Ball,-Wong and Elliott,1976). Furtheranalysishas concentrated
all of whichshowedalteredcolonyand cell morphology.
mutations,
cytoplasmic
cells rather
The resistant
mutantsproducedrosettesofelongatedpseudomycelial
than the singlebuddingcells of the wild type. This alteredmorphologywas
maintainedin the presenceand absenceof glucosamine.One of the cytoplasmic
yeast)was not
mutations(designatedcry1-r forcatabolite-repression-resistant
to drugswhichinhibitmitochondriogenesis
and was assumedto be
cross-resistant
mutation(Elliottand Ball, 1975). However,in a laterstudy,these
a mitochondrial
resistantmutationswere crossedagainststrainscarryingknownmitochondrial
mutationswere
markersand the resultsindicatedthattheglucosamine-resistant
a
DNA. Whetherthesemutationshave identified
not locatedon mitochondrial
Hexose analogues and fungi
507
distinctyeastplasmidis notyetknownbut theirresistantphenotypeapparently
inpermeability
to sugars(Kunz and Ball,1977)and
arisesthroughsomealteration
membranearchitecture
theirepisomeis thoughtto havesomerolein determining
or function(Guerineau,Slonimskiand Avner,1974).
mutationswhichare eitherless
There remaina numberof analogue-resistant
well understoodor less well characterized.In the formercategoryis the sorB
nidulans(Elorza and Arst,1971). Like sorA,thismutation
mutationin Aspergillus
to sorbosebut,unlikeit,thesorBmutationdidnotconfer
was selectedforresistant
was altered,themycelium
todeGlc. However,colonymorphology
cross-resistance
onvirtually
allcarbonsources.Although
compactmorphology
withtightly
growing
levelsof hexokinase,phosphoglucoseisomerase,and glucose6-phosphatedehydrogenasewere normal,the sorB mutantshad considerablyreducedactivityof
It is obscurewhyreductionin thisenzymeactivityshould
phosphoglucomutase.
by sorbose.Elorza and Arst(1971)
to inhibition
resultin acquisitionofresistance
intheA. nidulanssorB
abnormality
discusstheparallelbetweenthemorphological
mutationand morphologicalabnormalitieswhich are associatedwith loss of
in Neurosporaspp. (Brodyand Tatum, 1967; Mishra and
phosphoglucomutase
Tatum, 1970). They also showthatthe cell-wallcompositionof sorB mycelium
fromthatofthewildtype.It seems
drastically
grownon glucosemediumdiffers
that this strainavoids inhibitionby sorbosebecause the mutationalterswall
alteration
in sucha waythatit is no longersensitiveto sorbose-induced
structure
mayresultsecondabnormality
in wall composition.The colonialmorphological
arilyfromthis change in wall structurefor,on solid medium,the sorB wall
thesorts
alterationcould imposechangesin branchingpatternswhichprecipitate
of modification
to themediumwhichTrinciand Collinge(1973) have suggested
changes.A relevantcomparisonhereis
paramorphic
giveriseto sorbose-induced
mutantof A. nidulanswhichgrewnormallyonly
withthe temperature-sensitive
when mannosewas suppliedas sole carbonsource (Valentineand Bainbridge,
thermolabile
1978). This mutant,designatedmnrA455,producedan abnormally
in theabsenceofmannose,the
temperatures
Atrestrictive
phosphomannomutase.
mutantproducedextensiveareasofswollenhyphaeand cell wallscontainedonly
about30 % ofthenormalamountofmannose.The parallelwithsorBis quiteclose,
defectwas evidentduringgrowth
withtheexceptionthatthesorBmorphological
on normalcarbon sources,whereasthe mnrA455defectcould be relievedby
Nevertheless,these two mutationsemphasizehow
mannosesupplementation.
andhowdependentthelatter
is on cellwallstructure
dependentfungalmorphology
exampleofa correlation
hexosesugars.A different
is on theabilityto interconvert
is providedby the
betweensorboseresistanceand morphologicalabnormality
Neurosporamutant,patch.This mutant,isolatedby its unusualcyclicalgrowth
pattern(Stadler, 1959), is resistantto inhibitionscaused by sorbose,and the
activitiesof its glycogensynthetaseand glucan synthetaseare not affectedby
sorboseeitherinvivoor invitro(Mishraand Tatum,1972).A transport-defective
mutantarose as one of the progenyfroma crossbetweenpatchand an acetate
to
alteredcolonymorphology
non-utilizer
whichshoweda similarrhythmically
the patch parentalstrain(Halaban, 1975). In contrastto the strictlycircadian
rhythmof patch,the new mutationhad a period of about 50 h on medium
containing5 % glucoseand theperioddecreasedwithdecreasingglucoseconcensystembut,although
glucosetransport
trations.The mutantlacksthelow-affinity
to inhibitions
by
sensitivity
substrate,
was used as a transport
3-O-methylglucose
glucose analogueswas not assessed. However,as ratesof sugartransportwere
508
D. MOORE
some eighttimeslowerthanin thewild type,it is highlylikelythatthisstrainwould
prove to be resistantto some degree at least. It is worthrecallingthat,in Podospora
anserina,galactose is inhibitoryto growthof the wild type,and that it so changes
the branching habit of the hyphae that it induces the wild type to take on the
morphologyofa morphologicalmutant,zonata, whichexhibitsa rhythmicmycelial
growthpatternon normal media (Lysek and Esser, 1971). These authors stress
that their data indicate that Podospora strainswith rhythmicgrowthare altered
in carbohydratemetabolismsuch thatsugar degradationis enhanced and substrates
are wasted. Rememberingthat,in the presence of sorbose, Neurosporauses more
than the normal amount of oxygen and produces more CO2 per unit glucose used
(Crocken and Tatum, 1968), it may be that the association of abnormalityof
rhythmicgrowth with enhanced degradation of sugar arises because, on solid
medium, abnormallyrapid carbon metabolism periodically gives rise to the type
of adverse environmentalcondition induced by growth on sorbose (Trinci and
Collinge, 1973).
Cell wall defectsalso occurred in mutants of Saccharomycescerevisiaeselected
forresistanceto 2-deoxy-2-fluoro-D-glucose(Biely et al., 1973). In fluoroglucosemedia, the resistantmutantsgrew in agglomeratesconsistingof large number of
small cells. Cell division was defectivein its final stages; in particular the septa
were anomalously wide so that wide junctions occurred between mothercell and
bud, ratherthan a bud neck. The biochemical basis of the resistantphenotype is
unknown.
VI.
CONCLUSIONS
I hope that this review will have indicated both the value of sugar analogues in
dissectingmetabolic functionand the areas which remainto be explored. The fact
that most of the resistantmutants which have been described somehow limit
accumulation of eitherthe sugar analogue itselfor its phosphate emphasizes that
the initial inhibitionsdo not depend on extensivemetabolism. These compounds
are thereforeideally suited to the studyof the veryearlieststages of carbohydrate
metabolism, specificallytransportand the production and processing of sugar
phosphates. There is scope too forinvestigationof regulatoryaspects both in terms
of the directeffectsof accumulation of sugar phosphates and of the consequential
changes in nucleotide levels and phosphate availability.Transport mechanisms in
Neurosporaare inadequately understood to make a proper characterizationof the
resistant mutants already isolated, and much the same is true for Aspergillus
nidulans,in which sorbose transportin particularmust be analyzed before even
the currentlyavailable mutants can be fullyexplained. Work with yeasts shows
that mutants involving transportand kinase and phosphatase enzymes can be
selected by exposure to sugar analogues, but in no filamentousfungus has any
attempt been made to obtain a comprehensive set of resistant mutants. The
relationshipsbetween phosphohexomutase mutationsand hyphal morphologyin
the few instances discussed above suggest that several avenues exist for the
investigationof the involvementof carbohydratemetabolism in hyphal morphogenesis. Sugar analogues induce morphological changes which are described as
being abnormal. In the context of vegetative hyphal growth, the paramorphic
changes observed certainlyare abnormalbut thisis not to say thatthe paramorphic
growth form is without relevance to other aspects of growth of the fungus. As
illustration,consider the basidiomycete Coprinuscinereus.As in other organisms,
sorbose induces compact colonial growth;instead of being well separated fromone
Hexose analogues and fungi
509
another,themarginalhyphaeare denselypacked,havemuchshortenedcellsand
(Moore and Stewart,1972). Althoughthisis a totallyabnormal
branchprofusely
colony,it is preciselythissortofgrowthwhich
growthpatternforthevegetative
duringtheearlieststagesof
ofthegillhymenium
is responsiblefortheformation
morphogenesisof the carpophore(Moore, Elhiti and Butler,1979). Further,
of glucosaminecauses the tips of
treatmentwith inhibitoryconcentrations
Coprinus
hyphaeto balloonintomuchexpandedspheroids(Moore and Stewart,
in normaldevelopmentin that,
1972). This behaviouralso has its counterpart
ofthecarpophore,paraphysealcellsofthehymenium
become
duringmaturation
enormouslyinflatedthoughtheyremainconnectedto unchanged,hypha-like,
derived
areultimately
subhymenial
cells(Mooreetal., 1979).All fungalstructures
ofthebasichyphal
fromthehypha,thedifferent
celltypesarisingbymodification
analoguesto induce
studies,usingbothinhibitory
Many different
compartment.
paramorphicchangesand geneticalterationswhich impose morphologicalabnormalities,have contributedto the generalizationthatmorphologicalchange
occurs when the balances betweenwall components,precursorsor synthetic
formulated,theessentialbalancewas seen
processesare disturbed.As originally
andofrigidification
(Robertson,
as thatexistingbetweentheratesofwallformation
to the
1958, 1959). Biochemicalknowledgeaboutfactorswhichmightcontribute
toemerge(SietsmaandWessels,1979; Gooday
latterprocessis onlyjustbeginning
and Trinci, 1980) but there is now general agreementthat growthof the
polysaccharidecomponentsof the wall is itselfa balance betweenlysisof the
existingpolymerand insertionof new sugar units. This concept has been
welldevelopedrecently
forchitinsynthesis
(Gooday,1977)butitwas
particularly
originallyadvancedas a descriptionof the growthof glucanfibresin orderto
of deGlc on yeastcells (Johnson,1968a).
accountfortheeffects
changein cell
It is implicitin thisconceptthat,duringnormalmorphogenesis,
shape resultsfromcontrolledshiftsin these balances which are directedby
to
involvesattempting
events.Studyof morphogenesis
endogenousregulatory
understandthesechangesin wall synthesisand associatedmetabolismand the
them.Sugar analoguesofferone way of approachingthis
regulationunderlying
thesamemetaboliceventswhichoccur
typeofstudy.Eveniftheydo notprecipitate
observed
theiruse can at leastindicatewaysin whichthenormally
endogenously,
eventscould arise.Thus, thefactthatglucosamineinducesCoprinushyphaltips
to swell in a mannersimilarto that normallyobservedin maturationof the
doesnotnecessarily
implythatthesameprocesses
paraphysesofthegillhymenium
are operativein each case but, at least,the responseto glucosamineprovidesa
technique for the experimentalstudy of cell inflation.The indicationthat
paramorphicchangesinduced by sorbose arise largelythroughconsequential
ofinvestenvironmental
effects
(Trinciand Collinge,1973) opensthepossibility
It should
plasticity.
factorson morphogenetic
igatingtheimpactofenvironmental
to assessthevalidity
be possibleto controlmixingofthemediumexperimentally
of Trinci and Collinge (1973). If, by this means, the
of the interpretation
characteristics
of thephenomenoncan be established,it maythenbe possibleto
devisewaysinwhichdevelopingtissues,likethegillsofbasidiomycete
carpophores,
in whichsimilarmorphological
patternsare observedcan be manipulatedto see
iftheirmorphologies
ariseforsimilarreasons.
whichoccurduringthedevelopment
offungaltissues
The hyphalmorphologies
inducedbyinhibitory
arecloselysimilarto theparamorphologies
sugaranalogues
offeredby thesecompoundsforinvestigation
of morphoand the opportunities
genesisare considerable.
D. MOORE
5IO
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