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Nov 10, 1995 - Regulation of gene expression by ambient pH in Aspergillus: genes expressed at acid pH. S. Satkar**, M. X. Caddickt, E. Bignell*, J. Tilburn* ...
Biochemical Society Transactions

Regulation of gene expression by ambient pH in Aspergillus: genes expressed at acid pH 360

S. Satkar**, M. X. Caddickt, E. Bignell*, J. Tilburn* and H. N.Ant, jr.*§ *Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, Ducane Road, London W I 2 ONN, U.K., and tDepartment of Genetics and Microbiology, Donnan Laboratories, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K.

Ambient pH strongly influences the synthesis of secreted enzymes, permeases and exported metabolites by a wide range of micro-organisms. For example, numerous microbes secrete acid phosphatase in acidic environments and alkaline phosphatase in alkaline environments. Only in the ascomycete Aspergillus nidulans, however, is characterization [ 1-51 of the pH-regulatory system sufficiently advanced to have enabled construction of a comprehensive model covering both acid- and alkaline-expressed structural genes [ S ] . Briefly, the DNA-binding region of the pacC-encoded transcription factor (PacC) contains three Cys2His2zinc fingers. When converted into a functional form at alkaline ambient pH in response to the ambient pH signal transduced by the (six) pal gene product pathway, PacC activates expression of genes expressed in acidic growth conditions (such as that encoding acid phosphatase). PacC is itself an alkalineexpressed gene subject to autogenous regulation [S] . pacC' mutations, mimicking alkaline growth conditions, remove a C-terminal highly acidic region, containing an acidic glutamine repeat, responsible for negatively modulating PacC function [5,6]. These pacC' mutations obviate the need for pH signalling and therefore constitute a gain-of-function class [ 1,4-61. More severe truncation mutations, designated pacC constitute a partial loss-of-function class and mimic acid growth conditions [5,6]. A null mutant, deleted for the entire pace coding region, mimics acidic growth conditions more extremely and, in addition, leads to poor growth and conidiation [S]. Gel mobility shifts and DNase I footprinting have identified three single and one double PacCbinding sites in the promoter for the isopenicillin N synthetase (ipd)gene, enabling a consensus core-binding sequence Sr-GCCARG-3' to be identified and confirmed by cross-competition experiments involving a single base-pair change

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~ 1 . $Present address: Centro de Investigaciones Biol6gicas del CSIC, Velkzquez 144, 28006 Madrid, Spain. §To whom correspondence should be addressed.

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Proteolysis of PacC is an essential pH-sensitive step in the regulation of gene expression by pH [6]. Two forms of PacC are detectable in extracts, both forming specific retardation complexes with a PacC-binding site [6]. Under acidic growth conditions or in acidity-mimicking pal mutants, the full-length form of PacC predominates [6]. Under alkaline growth conditions or in alkalinity-mimicking pa&' mutants (which do not require the ambient pH signal) a proteolysed version containing the N-terminal 40% of PacC predominates [6]. This specifically cleaved shorter version is functional, both as an activator for alkaline-expressed genes and in repression of acid-expressed genes, but the fulllength form of PacC is inactive [6]. It should be noted that, although pacC' mutations remove the C-terminus of PacC, further proteolysis occurs to yield a functional version indistinguishable from that of the wild type except possibly where the pacC' mutation occurs in a codon very close to that encoding the C-terminal residue of the fully processed form ([6]; M. Orejas, unpublished work). Although considerable progress has been made in characterizing PacC [S, 61, there are two immediate further questions for understanding pH regulation: (a) how is ambient pH sensed and what is the mechanism by which the resulting pH signal effects proteolytic cleavage of PacC to its functional form, and (b) although it is established that PacC activates expression of alkaline-expressed genes directly [3], how does PacC prevent expression of acid-expressed genes? Although integrity of the sixpal genes is necessary for conversion of the inactive form of PacC to the functional form, n o p a l gene product directly effects this conversion [6]. Nevertheless the palB sequence indicates that it encodes a cysteine protease, the catalytic domain of which has considerable similarity to those of the calpain family [8]. PalB might catalyse an earlier and more C-terminal proteolysis of PacC than that yielding the functional form; alternatively it might proteolyse another component of the pH signal-transduction pathway [8].

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This communication addresses the second question. Does PacC function directly as a repressor? Or does it activate expression of a gene encoding a repressor? If PacC acts directly, is any other gene product required for repression but not activation? Or does PacC recognize two classes of cognate DNA sites, one for activation, one for repression? There are preliminary indications that the mechanisms for activation and repression in pH-regulated gene expression might differ substantially. Whereas the promoters of alkaline-expressed genes such as ipnA [5] , pacC [5] and the alkaline protease-encoding prtA (Figure 1) contain a number of GCCARG sites, the pH-regulated acid phosphatase-encoding p a d gene contains none in the 1311 bp upstream of the initiation codon (Figure 2). As the homology-defined three-zinc-finger-containing DNA-binding regions of the A. niger and A. nidulans conceptually translated PacC proteins are very similar (92 identities and three conservative differences in 97 residues [12]), it is also

pertinent that the A. niger pepF gene [13], encoding a pH-regulated acid protease, contains only one GCCARG site (at -1050 to -1045) in the 1166 bp upstream of the initiation codon and that the A. ficuum (closely related to A. niger) aphA ( p a d ) gene encoding pH-regulated acid phosphatase [lo] contains none in the 319 bp upstream of the initiation codon. In addition, the acid-expressed [14] acid-protease-encoding genes pepA of A. niger var. awamori 11.51 and pepB of A. niger var. macrosporus [16] contain respectively no GCCARG sites in the 176 bp upstream of the initiation codon and one GCCARG site (at -516 to -511) in the 613 bp upstream of the initiation codon. A further indication that the mechanism of regulation of acid-expressed (i.e. alkaline-repressible) genes might be different from that of alkaline-expressed (i.e. alkaline-activatable) genes is provided by the pacM gene. The UV-induced pacM1.52 mutation was selected as reversing hypersensitivity to neomycin toxicity in a pacC 14

Figure I Promoter region of the A. nidulans alkaline protease-encodingprtA gene Nucleotide numbering is based on the putative initiation codon The sequence beginning at -552 and extending 3’, including the putative coding region and beyond, has been published by Katz et al [9] The sequence shown has been independently determined by us At position - 196 our sequence differs from that of Katz et al [9] in having a G rather than a C The source of this difference is not known GCCARG sequences in either direction are boxed -1312 -1232 -1152 -1072 -992 -912 -832 -752 -672 -592 -512 -432 -352 -272 -192 -112 -32

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strain, and other UV-induced putative pacM mutations have been selected as increasing neomycin tolerance in a pacC63 strain. These pacM mutations, all very similar in phenotype, apparently suppress alkalinity-mimicking pacC' mutations for a number of characteristics associated with reduced expression of acid-expressed genes whilst not affecting characteristics associated with alkaline-expressed genes. Thus they restore the ability to stain for acid phosphatase activity, increase neomycin tolerance and reduce resistance to molybdate toxicity but have^ no effect on alkaline phosphatase staining. Their suppression of pacCc mutations is clearly physio-

logical rather than translational because pacM15I suppresses chain-termination alleles of the ochre (pacC'14 [ 5 ] ) , opal (pacC'5 and pacC'II [ 5 ] ) and amber (pacC'50 [6]) types, and pacMl58 and other putative pucM mutations suppress the pacC'63 (S. Sarkar, J. Tilburn and H. N. Arst, unpublished work) missense mutation. Moreover, pacM mutations are readily detectable in a pacC background (see below). An interesting physiological feature of pacM151 (and presumably other mutant pacM alleles) is that pacM double mutants with the more phenotypically extreme pacC' alleles grow very poorly under all conditions tested, perhaps reflecting deleterious

Figure 2 Nucleotide and derived amino acid sequences of the A. nidulans acid phosphatase-encoding pacA gene The A. nidulans pacA gene was cloned by cross-hybridization to its Aspergillus niger homologue [ lo, I I ] and restores acid phosphatase activity when transformed into an A. nidulans pocA- strain. Nucleotide numbering is based on the putative initiation codon. When compared with its A. niger and Aspergillus ficuurn homologues [ 101, the derived A. nidulans P a d sequence has, respectively, 74.4 and 75. I % identity over 606 residues. -1311 -1211 -1111 5 -1011 f l -911 P P -811 u -711 -611

-511

u

-411 -311 -211 -111 lcN32Kc -11 : 1 U I U N A W L A A K U K L V A V L L A L A T V E A R P T V D 9 0 w T T Y P Y T G P A V P I G D W V N P T I N G N G K G F P R L V C A 31 190 r 6 4 P A V K P R S A H P K N N V N V I S L S Y L P D G U H I H Y Q T P 290 G 9 7 F G L G Q A P S V R W G T S P A N L N K V A H G W S H T 390 9 125 Y D R T P S C A Q V K A V T Q C S 490 7 142 Q F F H E V S L P H L K P E T T Y Y Y R I P A A N G T T Q S D I L 590 1 7 5 S F K T A R A P G H K R S F T V A V L N D U G Y T N A H G T H R Q

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2 0 8 L L K A A N E G A A F A W H G G D L S Y A D D W F S G I L P C A D D 790 242 W P V C Y N G T S T Q L P G G G P I P E E Y K Q P L P Q G E T A N 890 2 7 5 Q G G P Q G G D U S V L Y E S N W D L W Q Q W U T N L T V K I P H 3 0 8 U V U P G N H E S C A A E F D G P G N P I T A Y L N E G I P N G T W 1090 342 A A E N L T Y Y S C P P S Q R N F T A F Q H R ? H U P G K E T G G 1190 3 3 7 5 V G N F W Y S F D Y G L A H F V S L D G E T D F A N S P F S T F E

4 0 8 R D L T G N E T H P R P E E T E T T D S G P F G T I D G D R Y D D N 1390 442 T A Y A Q Y Q W L K D L A S V D R T K T P W V F V ~ ~ H R P U Y S 1490 9 4 7 5 S A Y S S Y Q N H V R N A F E N L L L Q Y G V D A Y L S G H I H W 1590 9 5 0 8 Y L R U F P U T A N G T I D E S S I A D N Q Q P N T T N S G K S U T 1690 542 H I I N G U G G N I 6 S H S W F D E G E G L T E I T A K L D R T H 1790 5 7 S ? G F S K L T V V N E T V V N W E F V K G D D G S T G D W L T L V 1890 6 0 8 K G E T C T I N V S G 1990 2090 21w

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Figure 3 Meiotic map position of pacM on chromosome VI The gene order and linkage distances in centiMorgans (fI standard deviation) are based on analysis of 250 progeny from a cross of relevant partial genotype bwA I x p c M I5 I gabA2 tornA2OO. Gene symbols are given in [ I 71.

pacM

M A

gabA

1

I

I

c16.8k2.4-18.8k2.5-

tarnA I

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consequences of the simultaneous overexpression of both ‘acidic’ and ‘alkaline’ genes. T h e most striking phenotypic characteristic of pacM mutations in a pacC‘ background is that they allow considerable acid phosphatase staining on solid low-phosphate growth media buffered at pH 8, a feature they share with acidity-mimicking mutations such as partial loss-of-function alleles of pacC and mutations in the six pal genes of the pH signal-transduction pathway. T h e pacC null allele prevents growth on such medium while wild-type strains, although growing readily, show virtually no acid phosphatase activity. Figure 3 gives the map position of pacM, which, although also on chromosome VI, is not closely linked to

pacC. It is therefore possible that pacM encodes a repressor for acid-expressed genes and that pacC activates expression of pacM or that the pacM product interacts directly or indirectly with PacC to repress acid-expressed genes. In any case, the key to understanding how acidic but not alkaline growth conditions elicit expression of genes expressed at acidic ambient pH would appear to lie in the molecular characterization of pa&. We are grateful to the BBSRC for support through a research studentshipt to S.S. and Chemicals and Pharmaceuticals Directorate grant GWH87247 to H.N.A. and to the Commission of the European Communities for support through BIOTECH contract BI02-CT93-0174 to H.N.A.

1 Caddick, M. X., Brownlee, A. G. and Arst, H. N., Jr. (1986) Mol. Gen. Genet. 203, 346-353 2 Shah, A. J., Tilburn, J., Adlard, M. W. and Arst, H. N., Jr. (1991) FEMS Microbiol. Lett. 77, 209212 3 Espeso, E. A., Tilburn, J., Arst, H. N., Jr. and Peiialva, M. A. (1993) EMBO J. 12, 3947-3956 4 Arst, H. N., Jr., Bignell, E. and Tilburn, J. (1994) Mol. Gen. Genet. 245, 787-790 5 Tilburn, J., Sarkar, S., Widdick, D. A., Espeso, E. A., Orejas, M., Mungroo, J., Peiialva, M. A. and Arst, H. N., Jr. (1995) EMBO J. 14, 779-790 6 Orejas, M., Espeso, E. A., Tilburn, J., Sarkar, S., Arst, H. N., Jr. and Peiialva, M. A. (1995) Genes Dev. 9, 1622- 1632 7 Reference deleted 8 Denison, S., Orejas, M. and Arst, H. N., Jr. (1995) J. Biol. Chem. 270,28519-28522 9 Katz, M. E., Rice, R. N. and Cheetham, B. F. (1994) Gene 150,287-292 10 Mullaney, E. J., Daly, C. B., Ehrlich, K. C. and Ullah, A. H. J. (1995) Gene 162, 117-121 1 1 MacRae, W. D., Buxton, F. P., Sibley, S., Garven, S., Gwynne, D. I., Davies, R. W. and Arst, H. N., Jr. (1988) Gene 71, 339-348 12 MacCabe, A. P., van den Hombergh, J. P. T. W., ‘Tilburn, J., Arst, H. N., Jr. and Visser, J. (1996) Mol. Gen. Genet. 250, 367-374 13 van den Hombergh, J. P. T. W., Jarai, G., Buxton, F. P. and Visser, J. (1994) Gene 151, 73-79 14 Jarai, G. and Buxton, F. (1994) Curr. Genet. 26, 238-244 15 Berka, R. M., Ward, M., Wilson, L. J., Hayenga, K. J., Kodama, K. H., Carlomagno, L. P. and Thompson, S. A. (1990) Gene 86, 153-162 16 Inoue, H., Kimura, T., Makabe, 0. and Takahashi, K. (1991) J. Biol. Chem. 266, 19484-19489 17 Clutterbuck, A. J. (1993) in Genetic Maps. Locus Maps of Complex Genomes, 6th edn. (O’Brien, S. J., ed.), vol. 3, pp. 3.71-3.84, Cold Spring Harbor Laboratory Press, Cold Spring Harbor

Received 10 November 1995

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