Furthermore, strains carrying mutations

in the genes required for the formation of uroporphyrinogen III (UroIII) contain an additional auxotrophy for methionine because of the lack of sirohaem formation (Gollub et al., 1977). Sirohaem is a haem-like prosthetic group required for sulphite and nitrite reductases, which are essential for assimilation of sulphur and nitrogen into all life forms (Crane & Getzoff, 1996; Schubert GSK1120212 et al., 2002; Raux et al., 2003). The highly conserved haem biosynthesis pathway is also responsible for the synthesis of Vitamin B12, chlorophyll, factor F430 and sirohaem (Warren & Scott, 1990) but only sirohaem is also synthesized in fungi (Murphy & Siegel, 1973; Crane & Getzoff, 1996; Raux et al., 1999). As S. cerevisiae is unable to assimilate nitrate, lack of the sirohaem pathway has no additional effects on N-metabolism. Unlike the yeast haem-deficient mutants, growth of a 5′-aminolevulinic acid synthase (ALAS)-deficient strain of Aspergillus oryzae (ΔhemA) could not be restored by hemin supplementation (Elrod et al., 2000). Also, additional supplementation with ergosterol and/or vitamin B12 was also unsuccessful, and

additional methionine supplementation was not examined. Therefore, it remains unclear whether Aspergillus spp. are unable to use the external haem sources or that other metabolic processes are disturbed. To generate more insight into the haem biosynthesis pathway and its regulation in filamentous fungi, pathway-specific gene expression was studied, and an A. niger ΔhemA mutant strain was analysed. Our results demonstrate A. niger is capable of haem uptake from its environment Ibrutinib molecular weight and suggest a role of the sirohaem biosynthesis pathway in growth defects

observed in strains deficient in the haem biosynthesis pathway. Northern analysis furthermore suggests a limiting role for hemA and a regulatory mechanism to Glutathione peroxidase direct early intermediates either to haem or to sirohaem synthesis. Aspergillus niger N402 [cspA1 derivative of ATCC9029 (Bos et al., 1988)] and its pyrG- derivative, AB4.1 (van Hartingsveldt et al., 1987), were used during this study. Strains were grown on minimal medium (MM) (Bennet & Lasure, 1991) or complete medium (CM) consisting of MM with yeast extract (10 g L−1) and casamino acids (5 g L−1) containing sodium nitrate (70 mM) as nitrogen source. Wherever indicated, nitrate may be omitted or replaced by ammonium chloride (10 mM). Growth medium was supplemented with 10 mM uridine when required. Escherichia coli DH5α was used for the amplification of recombinant DNA as previously described (Inoue et al., 1990). Aspergillus niger transformations were performed according to Meyer et al. (2010). 50 μM ALA (Sigma-Aldrich) was supplemented in transformation experiments to generate hemA deletion mutants (ΔhemA). Chromosomal DNA of A. niger was isolated as described by Kolar et al. (1988).