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

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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

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