the aspergillus niger acua and acub genes correspond to the faca and facb genes in aspergillus...

The Aspergillus niger acuA and acuB genes correspond to the facAand facB genes in Aspergillus nidulans

Stella Papadopoulou, Heather M. Sealy-Lewis *Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK

Received 30 June 1999; accepted 1 July 1999

Abstract

Mutants in Aspergillus niger unable to grow on acetate as a sole carbon source were previously isolated by resistance to 1.2%propionate medium containing 0.1% glucose. AcuA mutants lacked acetyl-CoA synthetase (ACS) activity and acuB mutantslacked both ACS and isocitrate lyase activity. An acuA mutant was transformed to the acu� phenotype with a clone of ACS(facA) from Aspergillus nidulans. The acuB mutant was transformed with the A. niger facB clone which has been identified bycross-hybridisation of an A. nidulans facB clone. These results confirm that acuA in A. niger is the gene for ACS and acuB isanalogous to the A. nidulans facB regulatory gene. ß 1999 Federation of European Microbiological Societies. Published byElsevier Science B.V. All rights reserved.

Keywords: Acetyl-CoA synthetase; Regulator of acetate induction; facA ; facB ; Aspergillus nidulans ; Aspergillus niger

1. Introduction

Recently we reported the isolation of mutants in atleast three genes defective in acetate utilisation inAspergillus niger by selection for resistance to 1.2%propionate in the presence of 0.1% glucose [1]. Oneclass of mutant (acuA) lacked acetyl-CoA synthetase(ACS) activity while the most frequent class of mu-tant (acuB) lacked both ACS and isocitrate lyase. Byanalogy with mutants that had previously been de-scribed in Aspergillus nidulans we proposed that theacuA mutants were defective in the structural genefor ACS while the acuB mutants were defective in a

regulatory gene necessary for induction of ACS andenzymes of the glyoxylate bypass [2^4]). In this paperwe con¢rm the identity of these genes by demon-strating that the A. niger acuA and acuB mutantscan be transformed to an acu� phenotype by trans-formation with the A. nidulans facA (structuralgene for ACS [5]) and A. niger facB [6,7] cloned se-quences.

2. Materials and methods

2.1. Strains and genetic analysis

The wild-type strain was cspA1 ; nicA1. The acu3

strains used were 98.1(12): cspA1 ; fwA1 ; bioA1 ;lysA7 ; leuA1 ; cnxC5 ; acuB98.1 and 117.1: cspA1 ;

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 3 3 9 - 0

* Corresponding author. Tel. : +44 (1482) 465970; Fax: +44(1482) 465458; E-mail: h.m.sealy-lewis@biosci.hull.ac.uk

FEMSLE 8929 11-8-99

FEMS Microbiology Letters 178 (1999) 35^37

nicA1 ; acuA117 [1]. The markers and parasexualanalysis have been described by Bos et al. [8,9].The pyrG mutant strain was AB4.1 [10]. Mediaused for A. niger were identical to that used for A.nidulans [11^13].

2.2. Plasmids and transformation procedure

The transformation protocol was as described byTilburn et al. [14]. The co-transforming plasmid waspAB4-1 that carries the pyrG gene of A. niger [10].The A. niger facB plasmid, nFacB SK+3, was iso-lated from a genomic library of A. niger after prob-ing with the A. nidulans facB clone [6,7] and the A.nidulans facA plasmid was pRAS7 [5].

3. Results and discussion

Recombinant genotypes used as recipient strainsin the transformation were obtained after haploidisa-tion of a diploid between 98.1(12): AB4.1 to yieldHN14: cspA1 ; fwA1 ; pyrG ; leuA1 ; acuB98.1 andbetween 117.1: AB4.1 to yield SP3: cspA1 ; nicA1 ;pyrG ; acuA117.1. Care was taken to ensure that adiploid had been formed by checking the spore vol-ume and again in the case of the recombinant segre-gants that they were consistent with the haploid vol-ume.

Strain HN14 was transformed with 5 Wg pAB4-1and 5 Wg nFacB SK+3 and transformants were se-lected by their ability to grow on selective plateslacking uridine (pyr�). 35% of pyr� transformants(75 tested) were able to grow on acetate medium.None of the acu� tranformants had an inhibitedphenotype on acetate medium which was a charac-teristic of tranformants with increased copy numberof facB in A. nidulans [6]. Strain SP3 was trans-formed with 5 Wg pAB4-1 and 5 Wg of the A. nidulansfacA plasmid pRAS7. 27% of pyr� transformants(140 tested) were able to grow on acetate. Theacu� transformants had lost their ability to growon propionate+glucose medium. None of the trans-formants were able to grow on propionate alone. Incontrol transformations in which only the pyrG plas-mid was used in the transformation all the trans-formants selected were still unable to grow on ace-tate medium. These results con¢rm that the strains

selected in A. niger that were designated acuA andacuB correspond to mutants previously described inA. nidulans namely in facA, the structural gene forACS and facB, a regulatory gene responsible foracetate induction of ACS, cytosolic acetyl carnitinetransferase (facC), enzymes of the glyoxylate bypass,acetamidase and NADP-isocitrate dehydrogenase[2,3,15,16,6,7]. The availability of these mutants inA. niger, especially the acuB mutants, will aid com-parative studies on the regulation of metabolism oftwo carbon compounds in these ¢lamentous fungi.

Acknowledgements

We thank Professor Michael Hynes for providingplasmids nFacB SK+3 and pRAS7.

References

[1] Sealy-Lewis, H.M. and Fairhurst, V. (1998) Isolation of mu-tants de¢cient in acetyl-CoA synthetase and a possible regu-lator of acetate induction in Aspergillus niger. Microbiology144, 1895^1900.

[2] Apirion, D. (1965) The two way selection of mutants andrevertants in respect of acetate utilisation and resistance to£uoroacetate in Aspergillus nidulans. Genet. Res. 6, 317^329.

[3] Armitt, S., McCullough, W. and Roberts, C.R. (1976) Anal-ysis of acetate non-utilising (acu) mutants in Aspergillus nidu-lans. J. Gen. Microbiol. 92, 263^282.

[4] Sealy-Lewis, H.M. (1994) A new selection method for isolat-ing mutants defective in acetate utilisation in Aspergillus nidu-lans. Curr. Genet. 25, 47^48.

[5] Sandeman, R.A. and Hynes, M.J. (1989) Isolation of the facA(acetyl-Coenzyme A synthetase) and acuE (malate synthase)genes of Aspergillus nidulans. Mol. Gen. Genet. 218, 87^92.

[6] Katz, M.E. and Hynes, M.J. (1989) Isolation and analysis ofthe acetate regulatory gene, facB, from Aspergillus nidulans.Mol. Cell. Biol. 9, 5696^5701.

[7] Todd, R.B., Murphy, R.L., Martin, H.M., Sharp, J.A., Davis,M., Katz, M.E. and Hynes, M.J. (1997) The acetate regula-tory gene facB of Aspergillus nidulans encodes a Zn(II)2Cys6transcriptional activator. Mol. Gen. Genet. 254, 495^504.

[8] Bos, C.J., Debets, A.J.M., Swart, K., Huybers, A., Kobus, G.and Slakhorst, S.M. (1988) Genetic analysis and constructionof master strains for assignment of genes to six linkage groupsin Aspergillus niger. Curr. Genet. 14, 437^443.

[9] Bos, C.J., Slakhorst, S.M., Debets, A.J.M. and Swart, K.(1993) Linkage group analysis in Aspergillus niger. Appl. Mi-crobiol. Biotechnol. 38, 742^745.

[10] van Hartingsveldt, W., Mattern, I.E., van Zeijl, C.M.J., Pou-wels, P.H. and van den Hondel, C.A.M.J.J. (1987) Develop-

FEMSLE 8929 11-8-99

S. Papadopoulou, H.M. Sealy-Lewis / FEMS Microbiology Letters 178 (1999) 35^3736

ment of a homologous transformation system for Aspergillusniger based on the pyrG gene. Mol. Gen. Genet. 206, 7^75.

[11] Pontecorvo, G., Roper, J.A., Hemmons, L.M., MacDonald,K.D. and Bufton, A.W. (1953) The genetics of Aspergillusnidulans. Adv. Genet. 5, 141^238.

[12] McCully, K.S. and Forbes, E. (1965) The use of p-£uorophen-ylalanine with master strains of Aspergillus nidulans for assign-ing genes to linkage groups. Genet. Res. 6, 352^359.

[13] Cove, D.J. (1966) The induction and repression of nitratereductase in the fungus, Aspergillus nidulans. Biochim. Bio-phys. Acta 113, 51^56.

[14] Tilburn, J., Scazzocchio, C., Taylor, G.G., Zabicky-Zissman,J.H., Lockington, R.A. and Davies, R.W. (1983) Transforma-tion by integration in Aspergillus nidulans. Gene 26, 205^221.

[15] Kelly, J.M. and Hynes, M.J. (1977) Increased and decreasedsensitivity to carbon catabolite repression of enzymes of ace-tate metabolism in mutants of Aspergillus nidulans. Mol. Gen.Genet. 156, 87^92.

[16] Stemple, C.J., Davis, M.A. and Hynes, M.J. (1998) The facCgene of Aspergillus nidulans encodes an acetyl inducible carni-tine acetyltransferase. J. Bacteriol. 180, 6242^6251.

FEMSLE 8929 11-8-99

S. Papadopoulou, H.M. Sealy-Lewis / FEMS Microbiology Letters 178 (1999) 35^37 37