regulation of β-glucosidase biosynthesis in aspergillus nidulans
TRANSCRIPT
ELSEVIER FEMS Microbiology Letters I35 ( 1996) 79-83
Regulation of P-glucosidase biosynthesis in Aspergillus nidwlans
JaeHoon Lee ‘, Ki-Sun Kwon *, Yung Chil Hah Department of Microbiology College of Natuml Sciences, Seoul Nationd Unir,ersity. Seoul. South Korea
Received 11 October 1995; revised 31 October 1995; accepted 31 October 1995
Abstract
/?-Glucosidase in Aspergillus nidulans was found to be both intracellular and extracellular. The intracellular p-gluco- sidase was synthesized after the exhaustion of carbon source in the medium. The extracellular enzyme appeared with autolysis of the mycelium. Biosynthesis of P-glucosidase was not induced by various carbohydrates but repressed to varying
extents in the presence of glucose, glycerol, and 2-deoxyglucose. This repression was not relieved by addition of cAMP. The repression was relieved much more by mutations in the creA gene than by one in the creC gene. Thus, P-glucosidase synthesis in A. nidulans is subject to carbon catabolite repression.
Keywords: Aspergillus nidulans; /3-Glucosidase; Carbon catabolite repression; creA gene
1. Introduction
P-Glucosidase ( P-D-glucoside glucohydrolase, EC 3.2.1.21) in cellulolytic microbes plays a major role
in hydrolysing cellobiose and cellodextrins to glu- cose. ,&Glucanase is generally subject to end-prod-
uct inhibition by cellobiose, and the level of /3-glu- cosidase is thus important for the efficient break-
down of cellulose. The addition of /3-glucosidase results in an enhanced rate of glucose production during cellulose hydrolysis [I].
A. nidulans is a cellulolytic fungus and produces
_ Corresponding author. Present address: Korea Research Insti-
tute of Bioscience and Biotechnology, Korea Institute of Science
and Technology, P.O. Box 1 I5 Yusong, Taejon 305600, South
Korea. Tel.: 1-82 (42) 860 4143; Fax: +82 (421 860 4593;
E-mail: [email protected].
’ Present address: Korea Research Institute of Bioscience and
Biotechnology. Korea Institute of Science and Technology, P.O.
Box I IS Yusong. Taejon 305600, South Korea.
cellulolytic enzymes including P-glucosidase [2]. The regulation of endo-@ 1,4-glucanase in the fungus
has been reported [3], and P-glucosidase was puri- fied and characterized [4]. Since the genetics of A.
nidulans are well known, this study was carried out
as a part of the genetic approach to elucidate the mechanism of regulation of cellulase biosynthesis.
2. Materials and methods
2. I. Strains and media
The organism used in this study was Aspergillus
nidulans wild-type strain C62, and carbon catabolite-derepressed cre mutants, which Were gen- erous gifts of Dr. H.N. Arst (Royal Postgraduate Medical School, London) and Dr. A.J. Clutterbuck (University of Glasgow, Glasgow). The strains and their relevant genotypes are as follaws: C62 ( pabaA 1, 48 1 ( pabaA creAd- 1). C224 ( pabaA 1
0378 1097/96/S 12.00 0 1996 Federation of European Microbiological Societies. All rights reserved S&W 0378.1097(95)00433-5
creAd-2), C235 (pabaA creAd-31, C228 (biA 1
creAd-4), 1500 (biA 1 pabaA 1 creAd-25 fwA l), G244
(biA 1 creC27), and G246 ( pabaA 1 creC302 jivA 1). Minimal and complete media were prepared accord- ing to Pontecorvo et al. [5].
2.2. Culture conditions
To compare the wild-type strain (C62) with the creAd-3 mutant (C235) for j?-glucosidase production,
a conidial suspension was inoculated at a final con- centration of IO6 ml-’ into minimal broth contain-
ing 1% (w/v) glucose and grown at 37°C for 40 h with shaking. In the induction study, the conidial
suspension was inoculated into complete broth and cultured for 18 h. The mycelia were harvested and
washed with sterile saline. After starvation in saline
for 2 h, the mycelia were transferred to minimal
induction medium with different carbon sources at a final concentration of 0.05% (w/v) and cultured for
8 h. In the study of carbon catabolite repression, the washed mycelia were incubated in minimal medium
without a carbon source for 3 h. Then glucose, glycerol, or 2-deoxyglucose was added to a final concentration of 4 mg ml- I.
2.3. Enzyme preparations
Mycelia were harvested at the indicated times.
The filtrate was used to determine extracellular en-
zyme levels. The filtered mycelium was washed and resuspended in 0.05 M sodium acetate buffer, pH 5.0
and then sonicated (120 W, 3 min) in an ice bath. After centrifugation (8000 X g, 30 min, 4”C), the supematant was used to determine intracellular en-
zyme levels. The pellet was resuspended in the same buffer, and used as a mycelial-bound enzyme.
2.4. Analytical methods
,&GIucosidase activity was estimated using p-
nitrophenyl-P-Dglucopyranoside (PNPG) (Sigma) as substrate. Protein was determined by the Bradford method [6] with bovine serum albumin as a standard. One unit of enzyme activity was defined as the amount of enzyme releasing 1 pg of p-nitrophenol under assay conditions. Glucose in the spent medium
was measured according to the method of Somogyi [7]. Mycelial dry weight was obtained after drying at 80°C to a constant weight. Results are expressed as
the mean of triplicate determinations.
3. Results and discussion
3.1. The biosynthesis of intracellular and extracellu-
Jar P-glucosidase
Growth, glucose consumption, and P-glucosidase
activity were monitored during cultivation of the C62 and C235 strains in minimal medium. Both
strains showed a similar growth pattern, and autoly-
sis began at about 32 h when mycelial dry weight started to decline (Fig. la, b). In the wild-type strain (C62), the exhaustion of glucose was immediately
accompanied by the appearance of intracellular and,
subsequently, extracellular Pglucosidase after about 26 h incubation (Fig. la). In the mutant, intracellular @glucosidase was detected earlier, at about 14 h incubation, when 60% of the original glucose re-
mained in the medium. Thereafter, the activity in- creased with myceliaf growth (Fig. 1 b). However,
extracellular @glucosidase began to appear in the medium after 32 h, behaviour very similar to that of
the wild-type.
The extracellular enzyme might originate from the intracellular one by either secretion or autolysis. If secretion is involved in the appearance of extracellu-
lar enzyme, the extracellular enzyme activity should concomitantly increase with intracellular activity. However, the above results showing different time scales of intracellular and extracellular enzyme pro- duction suggest that P-glucosidase was synthesized
intracellularly and appeared in the medium as a result of autolysis. The observation that intracellular and extracellular enzymes shared the same compo- nents (Kwon, K.-S., Lee, J.H. and Hah, Y.C., unpub- lished data) could support this suggestion. Therefore, we measured the intracellular enzyme activity in the following study of regulation of P-glucosidase bio- synthesis. Previously, mycelial-bound @glucosidase was also reported to be released by autolysis in Trichoderma reesei [8].
J. Lee et al. / FEMS Microbiology Lerters 135 C 1996) 79-84 81
a
0.0
Culture time (h)
b
0 0.0 0 10 20 30 40
Culture time (h)
Fig. 1. Growth, glucose consumption, and synthesis of P-glucosidase in A. nidulans wild-type strain C62 (a) and carbon catabolite-dere-
pressed creAd-3 mutant C235 (b) during growth in minimal medium with 1% glucose. Glucose concentration (0) and dry weight (0) are
expressed as g I-’ and mg per flask, respectively. The intracellular (0) and extracellular (m) enzyme activities are expressed as units
ml-‘.
3.2. The effect of L>ariou.s carbohydrates on p-gluco-
sidase induction
Table 1 shows no induction effect by the tested carbohydrates on @-glucosidase synthesis, suggest-
ing that P-glucosidase is not an inducible enzyme in A. nidulans. In Neurospora crussa, cellobiose, gen-
tiobiose, and laminaribiose all induced p-gluco-
sidase [9]. Methyl-P-glucoside was a reported in- ducer of P-glucosidase in T. reesei [IO]. In Mucor
rucemosus and Chaetomium thermophile, however,
no induction by carbohydrates was found [ 11,121.
3.3. Carbon catabolite repression
Glucose and 2-deoxyglucose fully blocked and
glycerol reduced enzyme production (Fig. 2). Since
glucose is a more rapidly metabolizable carbon source than glycerol, stronger repression by glucose was expected. Thus we conclude that the biosynthesis of
P-glucosidase in this fungus is regulated by carbon catabolite repression. It has been reported that a
readily metabolizable carbon source, such as glu- cose, is involved in repression of many enzymes in
carbon metabolism [ 131. Catabolite repression by glucose was not reversed
by adding CAMP (20 mM) (Fig. 2). Increasing the concentration of CAMP had no effect (data not
shown). In Saccharomyces cerel;isiae, CAMP re-
Table 1 Effect of various carbohydrates on P-glucosidase synthesis
Carbohydrate ’ Glycoside P-Glucosidase
bond activity (c/c) h
None _ too.o+ 5.2
Cellobiose P-l.4 104.1 f 7.9
Gentiobiose p-l.6 103.3+ 9.9
Lactose P-1,4 91.9+ 11.8
Sophorose p-1.2 80.5 + 10.0
Melibiose o-1.6 81.0+ 9.1
Trehalose cu-I.1 82.9* 6.4
Amygdalin P 95. I * 13.3
Esculin 99.8 f 12.4
Salicin ; 122.6+ 9.1
Methyl-P-glucoside P 106.2+ 12.0
A Washed mycelia were incubated for 8 h with various carbo-
hydrates at 0.05% (w/v). h Enzyme activity was expressed as a percentage (+S.D.) of the
control lacking a carbon source.
3 I
0 3 6 9
Culture time (h)
Fig. 2. Effects of glycerol, 2-deoxyglucose, and glucose on the
synthesis of intracellular P-glucosidase, and effect of CAMP on
catabolite repression of P-glucosidase synthesis by glucose. After
3 h incubation of washed wild-type C62 strain mycelium in
minimal medium without carbon source, 0.4% glycerol (0).
2-deoxyglucose ( A ), glucose ( q ), or glucose plus 20 mM CAMP
(D) was added to the medium. As a control, no addition was
made to the medium (0).
versed repression of P-glucosidase synthesis by glu-
cose [14]. In M. rucemosus, CAMP inactivated /3- glucosidase and repressed its synthesis [ 111. Thus the relationship between intracellular CAMP level and repression is not yet established in fungi.
3.4. /3-Glucosidase synthesis in carbon catabolite
derepressed mutants
In A. nidulans, creA, creB, and creC genes are related to carbon catabolite repression [ 15,161. To identify the role of these genes in P-glucosidase repression, experiments were carried out with the wild-type and the different cre mutant strains. After cultivation in minimal medium containing 1% glu- cose for 14 h, the intracellular enzyme activity was measured. Table 2 showed that both creAd and creC mutants had higher P-glucosidase activity than the wild-type. There is a difference in the degree of derepression between the mutants. While the creC
J. Lee et al. / FEMS Microbiology Letter.? 135 (19%) 79-84 x3
Table 2
Derepression of /3-glucosidase in various cre mutants
Genotype Specific activity ’
(units (mg protein)-’ )
0.12+0.02
1.15+0.14
1.59i0.1
I .Y4 + 0.20
1.47*0.13
1.19+0.27
0.35 ,0.02
0.34 f 0.05
” Intracellular enzyme activity ( + S.D.) was measured after 14 h
growth in minimal medium containing I% (w/v) glucose.
mutants showed a three-fold higher activity than the
wild-type, cl-eAd mutants showed a more than ten-
fold higher activity. These results indicated that the creA gene plays a
more important role in carbon catabolite repression
of fi-glucosidase synthesis in A. nidulans than creC.
This is consistent with the fact that creA gene nega- tively regulates the synthesis of various enzymes and
permeases which are controlled by carbon catabolite
repression [ 131. While creB and creC genes are also involved in carbon catabolite repression, the roles of
these genes are unclear to date but they are likely to
act indirectly 1171. Recently, various enzymes in utilization of arabinan were found to be under the
control of creA, although mutations in creB and in creC showed modest regulatory effects [18]. This is
quite similar to our case. The creA gene has been cloned and found to encode a zinc finger DNA-bind-
ing protein [19,20]. The negative-acting nature of this regulatory protein was confirmed at the molecu- lar level [21].
In fungi, the biosynthesis of enzyme in nitrogen
and carbon metabolism is known to be regulated by catabolite repression and specific induction. Thus the
regulatory genes were termed as wide domain and pathway-specific control genes. respectively [22]. The biosynthesis of P-glucosidase in A. nidulans was non-inducible but was regulated by carbon catabolite repression mediated by creA. Thus biosynthesis of P-glucosidase may be exclusively controlled by a wide domain control gene of carbon metabolism.
Acknowledgements
The authors thank Dr. J.T. Mullins for the critical
reading of the manuscript.
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