Promoter analysis of the acetate-inducible isocitrate lyase gene (acu-3) from Neurospora crassa

Bibbins, Martha; Sheffield, Peter; Gainey, L. D. S.; Mizote, Tomoko; Connerton, Ian F.

doi:10.1016/s0167-4781(98)00194-8

Cited by 8 publications

(11 citation statements)

References 24 publications

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“…The presence of C 2 compounds resulted in the upregulation of the glyoxylate cycle in these entomopathogenic fungi. The regulation of the glyoxylate cycle in M. anisopliae and B. bassiana was found to be comparable to those reported in other fungi, with respect to glucose and acetate catabolism (Bibbins et al, 1998;Chaure & Connerton, 1995;De Lucas et al, 1994). In B. bassiana, the presence of glucose with acetate did not repress gene expression of the glyoxylate cycle genes, which has been observed in the fungus Leptosphaeria maculans (Idnurm & Howlett, 2002).…”

Section: Discussionsupporting

confidence: 82%

Flexible metabolism in Metarhizium anisopliae and Beauveria bassiana: role of the glyoxylate cycle during insect pathogenesis

et al. 2011

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Insect pathogenic fungi such as Metarhizium anisopliae and Beauveria bassiana have an increasing role in the control of agricultural insect pests and vectors of human diseases. Many of the virulence factors are well studied but less is known of the metabolism of these fungi during the course of insect infection or saprobic growth. Here, we assessed enzyme activity and gene expression in the central carbon metabolic pathway, including isocitrate dehydrogenase, aconitase, citrate synthase, malate synthase (MLS) and isocitrate lyase (ICL), with particular attention to the glyoxylate cycle when M. anisopliae and B. bassiana were grown under various conditions. We observed that ICL and MLS, glyoxylate cycle intermediates, were upregulated during growth on 2-carbon compounds (acetate and ethanol) as well as in insect haemolymph. We fused the promoter of the M. anisopliae ICL gene (Ma-icl) to a marker gene (mCherry) and showed that Ma-icl was upregulated when M. anisopliae was grown in the presence of acetate. Furthermore, Ma-icl was upregulated when fungi were engulfed by insect haemocytes as well as during appressorium formation. Addition of the ICL inhibitor 3-nitroproprionate delayed conidial germination and inhibited appressorium formation. These results show that these insect pathogenic fungi have a flexible metabolism that includes the glyoxylate cycle as an integral part of germination, pathogenesis and saprobic growth.

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Section: Discussionsupporting

confidence: 82%

Flexible metabolism in Metarhizium anisopliae and Beauveria bassiana: role of the glyoxylate cycle during insect pathogenesis

et al. 2011

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“…The affinity column was constructed with a 671-bp BamHI-ApaI fragment from the promoter region of the acu-3 gene (encoding isocitrate lyase; Gainey et al 1991Gainey et al , 1992Mizote et al 1996). The promoter fragment was selected because it produces a reliable gel shift with the DEAEpurified protein, and it is required in vivo for induction of the acu-3 gene in response to acetate (Bibbins et al 1998). The restriction fragment was gel-purified and ligated to the solid matrix Easy Anchor (TakaRa).…”

Section: Protein Transfer and Detectionmentioning

confidence: 99%

“…The DEAE-purified protein (1.2 mg) was adsorbed (batchwise) to the matrix, which was then loaded into a column, and washed with 50 mM KCl. The column was then eluted with a gradient of 50-250 mM KCl and the resulting fractions were assayed for binding to promoter DNA fragments (Mizote et al 1996;Bibbins et al 1998). Fractions showing gel retardation were pooled (300 mg) and dialysed against 20 mM HEPES-KOH (pH 7.9).…”

Section: Protein Transfer and Detectionmentioning

confidence: 99%

“…FacB binding sites have been observed in the promoter regions of several acu genes from Neurospora. These sites have also been demonstrated to bind in vitro to a recombinant fusion protein carrying the FacB DNA-binding domain and a native, acetate-induced, protein extract from N. crassa (Mizote et al 1996;Bibbins et al 1998). …”

Section: The Facb Homologue In N Crassamentioning

confidence: 99%

“…The affinity column was constructed by ligating a 671-bp BamHI-ApaI restriction fragment of the acu-3 promoter to the compatible BamHI site in the oligonucleotide linker covalently bound to the Easy Anchor resin. The fragment was selected because it was the minimal upstream sequence capable of conferring acetate induction in vivo and it showed efficient binding to the DEAE-purified protein in vitro (Bibbins et al 1998). The DNA-affinity column was eluted with a gradient of 50-250 mM KCl following batch adsorption of the DEAE-purified protein.…”

Section: The Facb Homologue In N Crassamentioning

confidence: 99%

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A regulator gene for acetate utilisation from Neurospora crassa

Bibbins

et al. 2002

Mol Gen Genomics

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The Neurospora crassa homologue of the Aspergillus nidulans regulatory gene facB has been cloned. The gene encodes a putative transcriptional activator of 865 amino acids that contains a DNA-binding domain with a Zn(II)(2)Cys(6) binuclear cluster, a linker region and a leucine zipper-like heptad repeat. Two internal amino acid sequences are identical to peptide sequences determined from proteolytic fragments of a DNA-binding protein complex specific for genes involved in acetate utilisation and expressed in acetate-induced mycelia of N. crassa. Recombinant expression of the predicted DNA-binding domain demonstrates that it is capable of independent recognition of a subset of the promoter sequences that bind the protein complex from N. crassa. A duplication-induced mutation in the corresponding gene results in an acetate non-utilising phenotype that is characterised by inefficient induction of the enzymes required for acetate utilisation. The new gene does not fall into any existing complementation group and has been designated acu-15.

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Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

Gold

Duick

Redman

et al. 2001

Agriculture and Food Production

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Promoter analysis of the acetate-inducible isocitrate lyase gene (acu-3) from Neurospora crassa

Cited by 8 publications

References 24 publications

Flexible metabolism in Metarhizium anisopliae and Beauveria bassiana: role of the glyoxylate cycle during insect pathogenesis

Flexible metabolism in Metarhizium anisopliae and Beauveria bassiana: role of the glyoxylate cycle during insect pathogenesis

A regulator gene for acetate utilisation from Neurospora crassa

Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

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