Three main ele-ments are involved: a high level expression of the positively autoregulated activator AlcR; b the strong promoters of the structural genes for alcohol dehydrogenase alcA a
Trang 1Relationships between the ethanol utilization ( alc ) pathway
Michel Flipphi, Janina Kocialkowska and Be´atrice Felenbok
Institut de Ge´ne´tique et Microbiologie, CNRS UMR 8621, Universite´ Paris-Sud XI, Centre d’Orsay, Orsay, France
The ethanol utilization pathway in Aspergillus nidulans is a
model system, which has been thoroughly elucidated at the
biochemical, genetic and molecular levels Three main
ele-ments are involved: (a) high level expression of the positively
autoregulated activator AlcR; (b) the strong promoters of
the structural genes for alcohol dehydrogenase (alcA) and
aldehyde dehydrogenase (aldA); and (c) powerful
activa-tion of AlcRby the physiological inducer, acetaldehyde,
produced from growth substrates such as ethanol and
L-threonine We have previously characterized the chemical
features of direct inducers of the alc regulon These studies
allowed us to predict which type of carbonyl compounds
might induce the system In this study we have determined
that catabolism of different amino acids, such asL-valine,
L-isoleucine,L-arginine andL-proline, produces aldehydes that are either not accumulated or fail to induce the alc system On the other hand, catabolism of D-galacturonic acid and putrescine, during which aldehydes are transiently accumulated, gives rise to induction of the alc genes We show that the formation of a direct inducer from carboxylic esters does not depend on alcA-encoded alcohol dehydro-genase I or on AlcR, and suggest that a cytochrome P450 might be responsible for the initial formation of a physio-logical aldehyde inducer
Keywords: Aspergillus nidulans; activation of transcription; alcgenes; aldehydes; carboxylic esters
The saprophytic hyphal fungus Aspergillus nidulans can
utilize a wide range of organic compounds as sole sources
of carbon and nitrogen One such alternative nutrient
is ethanol The pathway-specific transcriptional activator
AlcRis essential for the ethanol-induced expression of the
two structural genes necessary for the utilization of the
alcohol, alcA and aldA (reviewed in [1]) These genes
encode, respectively, alcohol dehydrogenase I (ADHI),
which oxidizes ethanol into acetaldehyde, and aldehyde
dehydrogenase (ALDH), converting acetaldehyde into
acetate Acetate is further metabolized into acetyl-CoA by
an acetyl-CoA synthetase encoded by the facA gene [2–4],
which is not subject to ethanol-specific control
The molecular means by which transcriptional regulation
of ethanol utilization is achieved have been studied
exten-sively [1] The functional characteristics of the AlcR
binuclear zinc cluster activator, its DNA-binding specificity,
its three-dimensional structure and its nuclear localization
sequence, have been elucidated [5–15] The alcA, alcR
and aldA promoters are subject to different powerful
mechanisms of transcriptional activation [9,13] These properties have been exploited in the use of the A nidulans alc system as a strongly inducible tool for heterologous expression [1,16] When a rich carbon source such as glucose
is present, expression of the alc system is repressed by the action of CreA, the DNA-binding protein mediating carbon catabolite repression in A nidulans Several mechanisms account for the direct repression of the alcR and alcA genes while the aldA gene is subject to indirect CreA control via direct repression of alcR [7,12,13,17,18] A subtle interplay between induction and repression allows A nidulans to adapt rapidly to changing nutritional conditions in the environment
The degradation pathways of small aliphatic primary alcohols and monoamines as well as that of the amino acid
L-threonine, converge on a common catabolic intermediate,
an aliphatic aldehyde [1,13,19] The three principal alc genes, alcA, aldA and alcR, are essential for the use of ethylamine andL-threonine as sole sources of carbon (but not for their utilization as sources of nitrogen) Recently, it has been demonstrated that ethanol, ethylamine andL-threonine do not induce the alc system directly but that these growth substrates have to be converted into acetaldehyde, which is the physiological inducer of the alc system [13,19]
However, the alc system was found to be inducible by a range of carbonyl compounds and, interestingly, some of these provoke substantially higher levels of expression than the maximal level obtainable with acetaldehyde [19] Fur-thermore, transcription of three genes of yet unknown function, alcO, alcM and alcS (clustered along with the alcR and alcA genes on chromosome VII) is subject to strict AlcR control [20] Despite being coordinately expressed with the alcR, alcA and aldA genes, these three additional alc genes
Correspondence to M Flipphi, Consejo Superior de Investigaciones
Cientı´ficas (CSIC), Instituto de Agroquı´mica y Tecnologı´a de
Ali-mentos (IATA), Apartado de Correos 73, 46100 Burjassot, Valencia,
Spain Fax: + 34 96 363 63 01, Tel.: + 34 96 390 00 22,
E-mail: flipphi@iata.csic.es
Abbreviations: ADHI, alcohol dehydrogenase I; ALDH, aldehyde
dehydogenase; GABA, c-aminobutyric acid; P450, cytochrome P450.
Enzymes: ADHI, alcohol dehydrogenase I (EC 1.1.1.1); ALDH,
aldehyde dehydogenase (EC 1.2.1.5); P450, cytochrome P450
(EC 1.14.14.1).
(Received 15 May 2003, revised 26 June 2003, accepted 2 July 2003)
Trang 2are dispensable for growth on ethanol, ethylamine and
L-threonine (S Fillinger, M Flipphi & B Felenbok,
unpublished results) This raises the question as to whether
the alc system contributes to the nutritional versatility of
A nidulans beyond providing the fungus with the ability
to utilize ethanol, ethylamine and L-threonine as growth
substrates A whole range of structurally diverse alternative
carbon and nitrogen sources are catabolized via aldehyde
intermediates, some of which might serve as in vivo
substrates for ADHI and/or ALDH under physiologically
relevant conditions In this study, we have addressed the
possible involvement of the alc genes in the conversion of
a number of nutrients, i.e D-galacturonate, glycerol, six
amino acids, putrescine, c-aminobutyric acid (GABA) and
small carboxylic esters
Materials and methods
Strains, media and growth conditions
Aspergillus nidulansstrains used in this study are listed in
Table 1 The references refer to the mutations relevant to
this work; all other markers are in standard use [21] Media
composition, supplements and basic growth conditions at
37°C were as described by Cove [22], using diammonium
tartrate (5 mM) as nitrogen source with the carbon source
present at 1% (w/v or v/v), unless stated otherwise
Mycelia for transcript analysis and esterase expression
were grown for 20–24 h in minimal medium with lactose
(3% w/v) as the carbon source and urea (5 mM) as the
nitrogen source Figure 1 shows that this growth medium is
perfectly neutral with respect to induction by ethanol alc
gene transcript levels in lactose-grown and ethanol-induced
mycelia are essentially the same as those observed in
ethanol-grown biomass and notably 10- to 20-fold higher
than those obtained in ethanol-induced mycelia grown on
0.1% (w/v)D-fructose Regardless of the growth substrate,
the gratuitous inducer 2-butanone always elicites more
powerful induction than the convertible compound ethanol
Furthermore, the response of the acetyl-CoA synthetase
gene (facA) showed that inducing amounts of acetate had
been formed from the ethanol applied to lactose-grown
biomass during the induction period as well as in the
ethanol-grown mycelia The use of lactose as the growth
substrate allows accurate analysis of alc gene transcription
upon addition of different compounds to principally
nonrepressed and noninduced mycelia in all genetic
back-grounds, and even monitoring catabolism of certain effector compounds beyond the first aldehyde intermediate Induction was achieved by addition of the effector compounds to 50 mM (final concentration), unless stated otherwise Cultures were harvested after a further 2.5 h of incubation (inducing conditions) for Northern analysis and after 4–6 h for esterase expression Where necessary, the effector compound was added from a concentrated solution
Table 1 A nidulans strains and transformants used in this study.
Strain Genotype References for characterized mutation or strain
BF054 yA2 pabaA1
BF064 yA2 pabaA1; alc500 [13,20]
BF107 yA2 pabaA1; aldA67 [13]
BF129 yA2 pabaA1; alcR125; aldA67 [13,55] M.-F Cochet & B Felenbok (unpublished results)
H1269–12.1 yA2 biA1; (riboB2) D alcC::riboB a [43]
TgpdA::alcR yA2; (argB2); (alcR125) pantoB100 TargB/gpdA::alcR b [13]
TgpdA::aldA pabaA1; (argB2); (aldA67) TargB/gpdA::aldAb [13]
alc500 TalcR yA2 pabaA1; (argB2); alc500 TargB/alcR c This work (see Materials and methods)
Corresponding phenotype: a riboflavin prototroph; b arginine prototroph, ethanol utilizing; c arginine prototroph, ethanol nonutilizing.
Fig 1 Lactose is a neutral growth substrate with respect to induction of the alc genes by ethanol Wild type mycelia were grown on either
D -fructose or lactose (noninduced conditions, NI) and cultures were induced by addition of ethanol (E) or the gratuitous inducer 2-butanone (2B) to a final concentration of 50 m M , as detailed in Materials and method Ethanol-grown biomass is considered to be induced from the start of cultivation (E*) Culture growth conditions, RNA isolation and Northern blots were as described in Materials and methods Membranes were hybridized with 32 P-labelled probes specific for the alcA, alcR, facA, facB and c-actin (acnA) genes Hybridization with a probe specific for the 18S rRNA species provided a control of the quantity of total RNA in each lane that correlates well with the con-comitant expression of the regulatory facB gene (encodes a transacti-vator of acetate catabolism [2,29]), under the various growth conditions Note that transcription of the c-actin gene was much higher in D -fructose-grown mycelia than in biomass generated on lactose or ethanol The c-actin gene cannot therefore be used as an internal control to normalize the amounts of mRNA when comparing
D -fructose-grown mycelia with lactose- or ethanol-grown mycelia However, this gene still provided a reliable control among different cultures grown on one particular carbon source, and also for com-paring lactose- with ethanol-grown cultures.
Trang 3of pH 6.8 In those cases where the effector compound
constitutes a poor nitrogen source for the fungus mycelia
were also grown in the presence of the effector (25 mM) as
the sole nitrogen source Such cultures were supplemented
withD-biotin to enhance uptake of these compounds [23]
Chemicals were purchased either from Sigma-Aldrich or
Merck-Eurolab
Generation of a mutant strain lacking the ADHI-encoding
alcA gene
The best characterized alc deletion mutant isolated upon
selection for resistance to allyl alcohol is alc500 [24,25] This
mutant lacks all five AlcR-controlled genes comprizing the
alcgene cluster [20] (J Kocialkowska, B Felenbok & M
Flipphi, unpublished results) The unlinked aldA gene is no
longer inducible but is constitutively expressed at a low level
[13] To monitor pathway-specific induction of the aldA gene
in the absence of ADHI-activity, a single, functional copy of
the alcR gene was introduced into anL-arginine-auxotroph
argB2 alc500double mutant strain by cotransformation with
the plasmids pBSalcRSal (carrying the alcR gene [26]) and
pFB39 (carrying the A nidulans argB gene [27]), essentially
as described by Flipphi et al [13].L-Arginine-prototrophs
were screened for the presence of the alcR gene by dot-blot
analysis A single copy cotransformant was selected by
Southern blot analysis (not shown) This transformant was
back-crossed with an alc500 mutant and offspring carrying a
functional alcR gene were screened via their ability to be
crossfed by a leaky aldA15 mutant strain, which accumulates
and secretes acetaldehyde on plates containing 1% ethanol
as sole carbon source and 10 mM sodium nitrate as the
nitrogen source [13] The alcR-complemented alc500 strains
behave as alcA loss-of-function mutants In one of the
crossfed strains, alc500 TalcR, proper inducibility of alcR
and aldA was demonstrated by Northern blot analysis
Isolation of RNA and Northern blot analysis
Transcript analysis was carried out as described by Flipphi
et al [13] using 32P-labelled probes corresponding to
fragments of the cloned A nidulans genes alcA [9], alcR
[26], aldA [28], facA [4], facB [29], c-actin (acnA) [30] and
prnD(GenBankÒ AJ223459) To monitor gabA
transcrip-tion, a probe was made from gabA cDNA [31] 18S
ribosomal RNA was detected with a probe for horseradish
18S rDNA [32] Autoradiographs were exposed for various
times to avoid saturation of the film In lactose-grown
mycelia the c-actin gene is constitutively expressed and was
used as an internal control for the amounts of mRNA
loaded All induction experiments were repeated at least
once
Qualitative zymogram analysis of carboxyl-/
acetylesterase activity
For the analysis of intracellular esterase activity cell-free
extracts were prepared from about 250 mg mycelial powder,
obtained by grinding freshly harvested mycelia in liquid
nitrogen The mycelial powder was quickly suspended in
500 lL of ice-cold extraction buffer (10 mMsodium
phos-phate pH 6.5, 2 m dithiothreitol) The suspension was
subsequently centrifuged in an Eppendorf centrifuge for
5 min at 10 000 g at 4 °C and the supernatant was recovered and put on ice For the analysis of extracellular esterase activity, media samples ( pH 6.8) were taken directly from cultures After centrifugation as above, the supernatant was recovered and put on ice Protein concen-tration was determined with Bradford’s method using bovine serum albumin as the standard
Proteins were separated in native 7.5% polyacrylamide (29 : 1 acrylamide/bisacrylamide, v/v) minigels buffered with 10 mMTris, 76 mM glycine, pH 8.5 Electrophoresis was performed at 4°C For cell-free extracts, samples containing 25 lg protein were mixed with 1/5 volume of loading buffer (10 mM sodium phosphate pH 7.0, 50% (v/v) glycerol, 2 per thousand (w/v) bromophenol blue) For extracellular samples, 20 lL of culture medium (containing 3–5 lg proteinÆmL)1) were applied to gels The samples were allowed to migrate into the gel at 1 VÆcm)1, after which the gel was run at 4 VÆcm)1for 3–4 h After electrophoresis, the gel was immersed in 100 mL of assay buffer (25 mM
potassium phosphate pH 7.0, 1 mMEDTA, 1 per thousand (v/v) 2-mercaptoethanol) at room temperature for 2 min 4-Methylumbelliferyl acetate was gradually added from a
1Msolution in dimethylsulfoxide to a final concentration of
500 lM The fluorogenic ester is a substrate for both carboxyl- and acetylesterases (EC 3.1.1.1/3.1.1.6) Forma-tion of 4-methylumbelliferone could be detected within minutes upon illumination with UV light (312 nm)
Results and discussion
D-Galacturonic acid provokes an induction response but glycerol does not
Induction of the alc system in lactose-grown mycelia by
D-galacturonic acid is very strong at 50 mM (Fig 2A, left panel) but hardly significant at 10 mM (not shown)
D-Galacturonic acid is a good growth substrate for A nidu-lansthat is catabolized into pyruvate andD-glyceraldehyde [33], and the observed induction should be attributable to the latter compound D-Glyceraldehyde appears to be a relatively weak inducer when added to pregrown mycelia (Fig 2B) but this could be due to inefficient uptake
D-Glyceraldehyde is converted into the glycolytic inter-mediate D-glyceraldehyde-3-phosphate via glycerol and glycerol-3-phosphate [34] Glycerol is noninducing (Fig 2B) even in the strongly derepressed mutant creAd30 or in a strain carrying a derepressed alcA gene in addition to a constitutively expressed alcR gene (results not shown) Dihydroxyacetone, a noninducing double hydroxyl-substituted a-ketone [19], is also catabolized via glycerol
In A nidulans, reduction ofD-glyceraldehyde is achieved by NADPH-dependent reductases, one of which is constitu-tively expressed but substrate-specific [35] while the second
is specifically induced onD-galacturonate [36] It is therefore not surprising that mutants in alc grow normally on both
D-galacturonic acid and glycerol [34] The alc gene induction observed upon addition of the higher concentration of the uronic acid is probably due to a transient accumulation of
D-glyceraldehyde, which ceases upon full expression of the inducible reductase In mycelia grown on D-galacturonic acid, expression of the alc genes is repressed (Fig 2A, right
Trang 4panel) However, induction does not cease in mycelia that
are grown on ethanol, or on lactose with either ethylamine
orL-threonine as the nitrogen source (results not shown)
Catabolism ofL-proline andL-arginine does not
lead to induction of thealc genes
L-Proline and L-arginine can serve as sole carbon and
nitrogen sources for A nidulans and both are catabolized
viaL-D1-pyrroline-5-carboxylate, the internal Schiff base of
L-glutamic semialdehyde with which it is in continuous
equilibrium [37,38] The Schiff base intermediate can be
reduced by PrnD (L-D1-pyrroline-5-carboxylate reductase/
L-proline oxidase EC 1.5.1.2) to yieldL-proline, and
oxi-dized by PrnC (L-D1-pyrroline-5-carboxylate dehydrogenase
EC 1.5.1.12) to produce L-glutamate These three amino
acids do not induce the alc genes while, as expected,
L-proline andL-arginine do induce the prnD gene This was
also the case in a strain constitutively overexpressing the
alcRgene, TgpdA::alcR (Fig 3) In this genetic background,
alcA transcription is more sensitive to minor inducing
signals because the expression of its activator, AlcR, is no
longer limiting [13] Thus, and in agreement with previous
predictions [19], the semialdehyde does not induce the alc
genes as it carries a carboxy substituent Loss-of-function aldAmutants are reported to grow more slowly onL-proline than wild type strains [37] This could be a pleiotropic effect due to accumulation of acetaldehyde [13] in addition to that
ofL-glutamic semialdehyde in these mutants However, we were unable to detect a phenotype characteristic for the aldA67mutation on plates withL-proline orL-arginine as sole carbon and nitrogen sources, distinguishing it from alcmutations (results not shown)
Catabolism ofL-valine,L-isoleucine andL-tryptophan does not induce thealc system
The branched-chain aliphatic amino acids L-valine,
L-leucine and L-isoleucine can serve as (poor) growth substrates for A nidulans [39,40] The first catabolic step is
a transamination to yield the corresponding 2-oxo acids In Saccharomyces cerevisiaethese latter compounds enter the Ehrlich pathway where they undergo decarboxylation to form the corresponding aldehydes, which are subsequently detoxified by constitutive alcohol dehydrogenase activity [41]
2-Methylbutyraldehyde emerged previously as the most effective alc gene inducer [19] In A nidulans, this aldehyde could be formed as a physiological intermediate when
L-isoleucine is catabolized via the Ehrlich pathway However, transcript analyses in wild type (Fig 4) and an alcA-deletion strain (not shown) grown on either of the branched-chain aliphatic amino acids as sole nitrogen source showed that the alc system was not induced Therefore, the expected aldehyde intermediates are either not accumulated or not formed at all This result can be anticipated forL-leucine as this amino acid is not catabo-lized via the Ehrlich pathway in A nidulans [40] Even if
L-isoleucine andL-valine are degraded in A nidulans as they are in yeast, then the formation and conversion of the
Fig 2 D -Galacturonic acid provokes induction of the alc genes.
(A) Northern blot analysis of induction upon addition of D
-galact-uronic acid ( D -GAA) to lactose-grown wildtype mycelia (left panel)
and upon growth on D -galacturonic acid ( D -GAA*) (right panel).
(B) Northern blot analysis of the response of the alc genes to addition
of D -glyceraldehyde and glycerol Ethanol served as the reference for
induction Induction was achieved by adding effector compounds
to uninduced cultures to 50 m M (final concentration) except for
D -glyceraldehyde (*), which was added to 4 m M When the effector
compound served as the carbon source for growth, its initial
concen-tration in culture was 50 m M Experimental details and abbreviations
were as described in the legend to Fig 1.
Fig 3 The amino acids L -proline and L -arginine do not induce the alc system Northern blot analysis was done in gpdA::alcR, a strain con-stitutively overexpressing the alcR gene (alcR*) The prnD ( L -proline oxidase) and alcA genes were used to monitor the responses to the two amino acids Experimental details and abbreviations were as described
in the legends to Figs 1 and 2.
Trang 5aldehyde intermediates clearly does not require activation of
the ethanol utilization pathway Interestingly bothL
-isoleu-cine andL-valine modestly induce the alc genes in a strain
deleted for another alcohol dehydrogenase, ADHIII,
pre-sumably due to some aldehyde accumulation, while
L-leucine does not (results not shown) Expression of the
ADHIII-encoding alcC gene is not under the control of
AlcRand is irrelevant for ethanol catabolism [1,42,43]
L-Tryptophan can also serve as a nitrogen source for
A nidulans [39] Multiple routes are known in bacteria,
fungi and plants to convert this amino acid into
indole-3-acetic acid, an important plant hormone (in bacteria,
reviewed by [44]; in fungi [45,46]) The pathways via
indole-3-pyruvate and tryptamine produce indole-3-acetaldehyde
as an intermediate, which is further oxidized by an
alcohol-inducible ALDH in the fungus Ustilago maydis [45] In
A nidulansit has been observed that tryptamine is inducing
for the alc genes at relatively low (nontoxic) concentrations
(M Flipphi, J Kocialkowska & B Felenbok, unpublished
results), probably by conversion into indole-3-acetaldehyde
This reaction would be similar to that of the deamination of
unbranched aliphatic monoamines; these latter compounds
have been shown previously to be inducers of the alc system
[19] However,L-tryptophan itself is noninducing even in
mycelia grown on lactose with this amino acid as sole
nitrogen source (results not shown) Therefore
indole-3-acetaldehyde is apparently not accumulated uponL
-trypto-phan degradation in A nidulans
Putrescine provokes an induction of thealc system
but GABA does not
A nidulans is capable of using putrescine
(1,4-diamino-butane) as a sole source of nitrogen but not as a carbon
source [47] Breakdown of this diamine into the tricarboxylic acid-cycle intermediate succinate (Fig 5) involves two different aldehyde intermediates, c-aminobutyraldehyde and succinic semialdehyde [37,48–50] Both have a back-bone of four carbon atoms, the optimal length for the aliphatic tail of an alc-inducing aldehyde Succinic semi-aldehyde is predicted to be noninducing due to its terminal carboxy group [19] but c-aminobutyraldehyde permits analysis of the effect of an amino substituent on the inductive capacity of a physiologically relevant aldehyde Transcript analysis shows that putrescine is indeed able to moderately induce the alc genes when added to pregrown mycelia (Fig 6A) As expected, no induction is observed upon addition of c-aminobutyric acid (GABA) The forma-tion of GABA from putrescine can be monitored with the expression of the GABA permease gene (gabA) [31,49] Using the leaky punA11 mutation, which partially impairs the utilization of putrescine [47], we could confirm that the diamine needs to be converted into the corresponding aldehyde to induce the alc genes; putrescine does not induce the alc system in this background, while the gabA gene is only very modestly expressed (Fig 6B) Moreover, induction
by putrescine is titratable in vivo with semicarbazide, an aldehyde scavenger (see References [13,19]; results not shown) Thus, it can be concluded that putrescine breakdown results in the accumulation of a weakly inducing compound, c-aminobutyraldehyde, and that an amino substituent strongly limits the inducing capacity of an aldehyde
As is the case for D-galacturonic acid, no alc gene induction could be observed in mycelia grown on lactose/ putrescine although the gabA gene is expressed under these growth conditions (results not shown) Expression of the alc gene is thus likely to respond to a transient accumulation of c-aminobutyraldehyde when putrescine is added to a non-induced culture Interestingly, GABA is also produced from putrescine in the stringent loss-of-function aldA67 mutant as well as in an aldA67 alcR125 double mutant, which is unable
to induce the alc genes (Fig 6A) This indicates that
Fig 4 The branched-chain aliphatic amino acids L -valine and L
-isoleu-cine do not induce the alc system Transcript analysis was performed in
a wild-type strain Induced mycelia were grown in the presence of
either amino acid (25 m M ) as the sole nitrogen source instead of urea.
The aldehyde that was presumed to be formed upon L -isoleucine
turnover, 2-methylbutyraldehyde (2MB), served as an additional
control of induction and was added to urea-grown mycelia at 2 m M
[19] Further experimental details and abbreviations were as described
in the legends to Figs 1 and 2 Similar results were obtained for
L -leucine (results not shown).
Fig 5 Catabolism of putrescine and GABA in A nidulans Putrescine
is first deaminated to form c-aminobutyraldehyde, which is further oxidized by an unknown ALDH to yield c-amino butyric acid (GABA) The second amino group is liberated by GABA amino transferase (gatA gene [48]) to produce succinic semialdehyde, which is finally converted into succinate by a substrate-specific ALDH specified
by the ssuA locus [37,49,50].
Trang 6c-aminobutyraldehyde is oxidized by another ALDH
enzyme not dependent upon AlcRfor its expression In
mammals, a specialized ALDH is involved in the conversion
of this aminoaldehyde [51] However, its expression does not
seem to be as rapid as that of the alc genes Interestingly,
constitutive ALDH overexpression in the TgpdA::aldA
transformant not only decreases alc gene induction by
putrescine but also results in reduced gabA expression
(Fig 6A) Overall, the data suggest that
c-aminobutyralde-hyde can serve as a (poor) in vivo substrate of both
aldA-encoded ALDH and alcA-aldA-encoded ADHI Moreover, it
appears that expression of the putrescine pathway-specific
ALDH is inducible by its aminoaldehyde substrate
Interestingly, the level of pseudo-constitutive expression
of the alc genes in the loss-of-function aldA67 mutant [13]
is reduced upon addition of either putrescine or GABA (Fig 6A) This could be explained on the basis that the accumulated aldehyde is partly neutralized as an inducer in the presence of these primary amines, suggesting that Schiff base interactions between aldehydes and primary amines occur in vivo The formation of a Schiff base is a possible mechanism by which the DNA-binding regulator AlcRis activated by direct binding of the inducer compound [19]
An alternative hypothesis is that the aldehydes formed from putrescine and GABA (c-aminobutyraldehyde and succinic semialdehyde, respectively) are able to compete in some way with the aldehyde accumulated from general metabolism in aldA67, leading to decreased pseudo-constitutive alc gene expression This second hypothesis implies that a non-inducing aldehyde, succinic semialdehyde, competes with the inducer for binding to AlcR, although it is unable to effect activation
Some small carboxylic esters induce thealc system but lactones do not
Carboxylic esters are interesting compounds with respect to alcgene induction They are structurally related to ketones, one class of direct inducers of the alc system, and their hydrolysis results in alcohols that upon oxidation yield aldehydes, a second class of direct inducers We have shown previously that the small methyl ketones 2-butanone and 2-pentanone induce the alc genes [19] Circular ketones such
as cyclopentanone and cyclohexanone also induce but the smallest linear b-ketone, 3-pentanone, does not We have therefore investigated whether or not the esters and lactones (intramolecular esters) corresponding to the above ketones induce the alc genes
Transcript analyses show that whereas the ester ethyl-acetate (which corresponds to the inducer 2-pentanone) indeed provokes a strong induction of the alc genes, methylpropionate (which corresponds to the inert ketone 3-pentanone) fails to induce (Fig 7A) By contrast, none of the lactones tested (c-butyrolactone, d-valerolactone and e-caprolactone) provoked induction (Fig 7B) Moreover, although 2-butanone is the most powerful of all the ketone inducers, its structurally related ester methylacetate elicits only a marginal induction of alcA while alcR and aldA expression do not exceed their basal, noninduced levels (results not shown)
These data thus show that there is no correlation between the inducing capabilities of a ketone and the structurally related ester or lactone It rather appears that esters induce the alc genes when their degradation results in the produc-tion of an inducing aldehyde derived from the ester’s alcohol moiety Acetaldehyde would therefore be responsible for the induction observed on ethylacetate Methylpropionate does not induce as formaldehyde is inert while hydrolysis of the lactones tested would principally yield noninducing carb-oxy-substituted alcohols and aldehydes, in agreement with previous predictions [19]
We have verified these deductions using the unbranched aliphatic esters propylacetate and ethylpropionate Upon hydrolysis, ethylpropionate would yield acetaldehyde while propylacetate would yield propionaldehyde, both direct
Fig 6 Putrescine catabolism provokes induction of the alc system.
(A) Transcript analysis of the effect of putrescine (Putr) addition to
pregrown mycelia of a wildtype strain (wt) and three strains mutant in
ethanol catabolism: aldA67, gpdA::aldA and aldA67 alcR125 The gabA
gene encodes GABA permease and is inducible by x-amino acids [31].
The aldA67 mutant exhibits pseudo-constitutive expression of the alcA
and alcR genes (NI *) [13] (B) Putrescine-induced transcription of the
alcA and gabA genes in the leaky putrescine utilization mutant punA11
compared to that in a wild type strain Putrescine (1,4-diaminobutane)
was added to uninduced cultures to 25 m M Further experimental
de-tails and abbreviations were as described in the legends to Figs 1 and 2.
Trang 7inducers of the alc system Figure 7A clearly shows that both these esters are inducing despite the fact that the carbonyl function in ethylpropionate resides in the b-position, as is the case for the inert compounds methyl-propionate and 3-pentanone That the induction provoked
by ethylacetate, apparently the most effective ester, is considerably less in a strain constitutively overexpressing ALDH, TgpdA::aldA (data not shown), is in accord with the formation of acetaldehyde from this ester Furthermore, induction of the acetyl-CoA synthetase gene (facA) upon addition of ethylacetate, propylacetate and ethylpropionate (Fig 7A), actually suggests that all esters are hydrolyzed into acetate during the induction period In this regard it is worth noting that wild type facA transcription responds to acetate and its precursor ethanol (see Fig 1) but not to propionate or n-propanol (M Flipphi & B Felenbok, unpublished results) explaining why this gene is not induced
in the presence of methylpropionate
In conclusion, the results obtained are in agreement with our previous data and show that aliphatic and cyclic ketones directly induce the alc genes There is no correlation between the inductive capacities of ketones and their structurally related ester counterparts In addition, small aliphatic carboxylic esters induce the alc genes indirectly via the aldehyde formed from their ethanol or n-propanol moieties
Acetylester breakdown yields inducing compounds independently of thealc system
The induction of the facA gene by all esters tested except methylpropionate suggests that carboxylic esters are degraded during the induction period of 2.5 h Strong indications that the hydrolysis of esters is a catalyzed process come from zymogram analyses of cell-free extracts and culture medium samples, which evidenced the presence
of multiple intra- and extracellular carboxyl-/acetylesterase activities As shown for the intracellular activity in Fig 8A, these esterase activities are constitutively produced by the fungus when grown on lactose and urea Interestingly, the esterase spectrum did not change upon addition of typical inducers of the alc gene system, i.e L-threonine and 2-butanone, nor by the supply of the inducing ester ethylacetate Only addition of 2-methylbutyraldehyde to a lactose-grown culture resulted in induction of an intracel-lular esterase However, even this inducible activity does not depend on AlcRfor its expression as the esterase spectrum
in the alc500 deletion mutant was identical to that in a wild type strain (Fig 8A)
In line with our previous studies [19], it is to be expected that the alcohol resulting from ester hydrolysis is oxidized to
an inducing aldehyde Transcript analysis in a strain completely lacking the ADHI-encoding alcA gene (alc500-TalcR) showed that the two AlcR-controlled genes present
in this background, alcR and aldA, are induced by ethylacetate and ethylpropionate (Fig 8B) These genes are also induced in the presence of ethanol despite the fact that this strain cannot use the latter as a growth substrate The acetyl-CoA synthetase gene facA is not ethanol-inducible in the deletion mutant (M Flipphi & B Felenbok, unpublished results), also indicating that not much acetate is formed from ethanol These observations suggest that inducing amounts of the physiological aldehyde inducer are
Fig 7 Analysis of the induction of the alc genes by carboxylic esters
and lactones (A) Transcript analysis of the effect of four carboxylic
esters on the expression of the alcA, alcR and facA genes in a wild type
strain The inducing ketone 2-pentanone, which is structurally related
to ethylacetate, and the inert ketone 3-pentanone, structurally related
to methylpropionate, provided the controls The structures of these
latter four compounds are shown below (B) Northern analysis of wild
type mycelia supplemented with three different lactones The inducing
ketone cyclohexanone served as the control for induction The
struc-tures of the circular ketone and its lactone counterpart,
d-valero-lactone, are shown below Experimental details and abbreviations were
as described in the legends to Figs 1 and 2.
Trang 8produced from ethanol as well as from ethylesters by other
enzymes in alcA loss-of-function mutants However, the
limited conversion capacity is clearly not sufficient to
support growth on ethanol It was surprising to find that
ethylacetate can serve as a sole source of carbon for alc
loss-of-function mutants (not shown) Despite being highly
induced in its presence, the alc genes are dispensable for
growth on this acetylester, strongly suggesting that the
acetate moiety from the ester is preferentially consumed
From the above analysis it can be questioned whether the limited, AlcR-independent ethanol conversion capacity in the alcA deletion mutant is sufficient to produce and maintain an inducing quantity of acetaldehyde in the presence of ethylacetate Induction of the alc system in an alcA-independent manner could be explained presuming that acetaldehyde is produced directly from the ethylester by
an AlcR-independent mechanism (Fig 9) Some mamma-lian cytochrome P450 isozymes produce carbonyl com-pounds (aldehydes or ketones) from carboxylic esters
in vitro by oxidative cleavage [52,53] Interestingly, the ethanol- and acetone-inducible P450 2E1 from mammals is also capable of catalyzing conversion of ethanol into acetate via two subsequent oxidation reactions [54] Although the main product is acetate, acetaldehyde is also produced by this enzyme because the intermediate product/second substrate is only loosely bound to the catalytic site Preliminary results indicate that one putative P450-encoding gene is weakly but constitutively transcribed in A nidulans (M Flipphi &
B Felenbok, unpublished results) We therefore propose that the fungus constitutively produces at least one (but possibly more) P450 oxidase able to form the physiological aldehyde inducer from ethanol and other alcohols as well as from inducing carboxylic esters Initial, AlcR-independent production of acetaldehyde by P450 could play a crucial role
in triggering ethanol catabolism in A nidulans
Acknowledgements
We thank John Clutterbuck and Heather Sealy-Lewis for providing us with mutant A nidulans strains, Michael Hynes for plasmids harboring the facA and facB genes, Irene Garcia for the prnD probe and Joan Tilburn for the gabA cDNA clone We are grateful to Sabine Fillinger
Fig 8 Formation of the physiological aldehyde inducer from acetyl
esters does not depend on the alc system (A) Zymogram analysis of
intracellular carboxyl-/acetylesterase activity in wild type compared to
that in an alc deletion mutant, alc500 Cell-free extracts from mycelia
subjected to noninduced or induced growth conditions for the alc
genes were prepared as decribed in Materials and methods Induction
was achieved by adding L -threonine ( L -Thr) (to 50 m M ) or
2-methyl-butyraldehyde (2MB) (to 2 m M ) to lactose-grown mycelia Protein was
separated in a native polyacrylamide gel at pH 8.5
Carboxyl-/acetyl-esterase activity was resolved directly in the gel as described in
Mate-rials and methods The zymograms are presented as the negatives.
(B) Northern analysis of the induction of aldA and alcR in the
pres-ence of ethanol or acetylesters in alc500 TalcR, an absolute alcA
deletion mutant (D alcA) (see Materials and methods) Further
experimental details and abbreviations were as described in the legends
to Figs 1 and 2.
Fig 9 A role for cytochrome P450 in the initial formation of physio-logical aldehyde inducers from alcohols and carboxylic esters in
A nidulans Constitutively expressed cytochrome P450 isozymes could oxidize ethanol and ethylesters to yield directly acetaldehyde, trigger-ing a cascade reaction of coupled expression of alcA-encoded ADHI and accelerated formation of the physiological inducer Esterases are responsible for the energetically more favorable hydrolysis of carb-oxylic esters to alcohols and carbcarb-oxylic acids.
Trang 9who selected the initial alcR transformant in the alc500 deletion mutant
background, Andrew MacCabe for correcting the English, Miguel
Pen˜alva for helpful suggestions and Herb Arst for critical reading of the
manuscript This work was supported by the Centre National de la
Recherche Scientifique, UMR 8621, the Universite´ Paris-Sud XI and
the European Community, grant QLK3-CT99-00729.
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