Relationships between the ethanol utilization (alc) pathway and unrelated catabolic pathways in Aspergillus nidulans.

The ethanol utilization pathway in Aspergillus nidulans is a model system, which has been thoroughly elucidated at the biochemical, genetic and molecular levels. Three main elements are involved: (a) high level expression of the positively autoregulated activator AlcR; (b) the strong promoters of the structural genes for alcohol dehydrogenase (alcA) and aldehyde dehydrogenase (aldA); and (c) powerful activation of AlcR by the physiological inducer, acetaldehyde, produced from growth substrates such as ethanol and l-threonine. We have previously characterized the chemical features of direct inducers of the alc regulon. These studies allowed us to predict which type of carbonyl compounds might induce the system. In this study we have determined that catabolism of different amino acids, such as L-valine, L-isoleucine, L-arginine and L-proline, produces aldehydes that are either not accumulated or fail to induce the alc system. On the other hand, catabolism of D-galacturonic acid and putrescine, during which aldehydes are transiently accumulated, gives rise to induction of the alc genes. We show that the formation of a direct inducer from carboxylic esters does not depend on alcA-encoded alcohol dehydrogenase I or on AlcR, and suggest that a cytochrome P450 might be responsible for the initial formation of a physiological aldehyde inducer.     

Characterization of a Transcriptional Activator Controlling Trichothecene Toxin Biosynthesis

Trichothecene biosynthetic pathway genes are localized within a gene cluster in Fusarium sporotrichioides and require the zinc-finger containing protein, TRI6, for expression. We show here that TRI6 is able to bind within the promoter regions of nine different pathway genes and that TRI6 binding is involved in pathway gene activation. TRI6 binding occurs at three distinct sites in the TRI5 promoter, all of which contain the sequence TNAGGCCT. DNA fragments from the promoter regions of six other pathway genes containing this sequence are also substrates for TRI6 binding. Specific nucleotide changes in the TNAGGCCT sequence dramatically reduced TRI6 binding. Analysis of TRI6 binding within the TRI3 and TRI11 promoters and the TRI4-TRI6 intergenic region which do not contain the TNAGGCCT motif suggests that the minimum sequence required for TRI6 binding is YNAGGCC. Two potential TRI6 binding sites, T4A and T4B, were identified within the intergenic region for the divergently transcribed TRI4 and TRI6 genes. Alteration or deletion of the T4A site resulted in the loss of nearly all in vitro TRI6 binding and was correlated with the loss of promoter activity in vivo as measured by the expression of mutant TRI4(p)/GUS fusions. This establishes a physiological role for TRI6 binding and demonstrates that TRI6 is directly involved in the regulation of pathway gene expression. To determine if a predicted Cys2His2 zinc-finger motif at the C-terminus of TRI6 is involved in DNA binding, a C187A mutant was constructed in TRI6 using site-directed mutagenesis. The C187A mutant did not bind promoter DNA fragments, supporting the role of C187 in DNA binding. In addition, a TRI6 homologue in the distantly related macrocyclic trichothecene pathway of Myrothecium roridum (MRTRI6) was also shown to bind to the same TRI5 and TRI4 promoter fragments bound by TRI6. Together, these data confirm our previous proposal that TRI6 is an activator of trichothecene pathway gene expression and that DNA binding employs the C-terminal region of TRI6 containing three predicted Cys2His2 zinc fingers.     

Allosteric negative regulation of smt O/P binding of the zinc sensor,SmtB, by metal ions: a coupled equilibrium analysis

The Synechococcus PCC 7942 smt operon is responsible for cellular resistance to excess zinc and consists of two divergently transcribed genes, smtB and smtA. SmtB is the Zn(II)-sensing metal-regulated repressor of the system and binds to a 12-2-12 imperfect inverted repeat in the smtA O/P region. Using fluorescence anisotropy to monitor SmtB-smt O/P multiple equilibria, we show that four SmtB homodimers bind to a 40 bp oligonucleotide containing a single 12-2-12 inverted repeat. The binding affinities of the first two dimers are very tight (K(int) = 2.9 x 10(9) M(-1)) with the affinities of the third and fourth dimers lower by approximately 10- and approximately 30-fold, respectively. A single monomer equivalent of Zn(II), Cd(II), or Co(II) promotes disassembly of the oligomeric complex to a mixture of (P(2)).D and (P(2))(2).D SmtB dimer-DNA complexes with the intrinsic affinity of all SmtB homodimers for DNA greatly reduced by approximately 500-2000-fold. Substitution or derivatization of cysteines which comprise the alpha3N metal binding site (Cys14 and Cys61) [VanZile, M. L., et al. (2002) Biochemistry 41, 9765-9775] has no effect on allosteric negative regulation by Zn(II); in contrast, H106Q SmtB, harboring a single zinc-liganding substitution in the alpha5 metal binding site, is refractory to zinc-induced disassembly of SmtB-DNA complexes. The alpha5 metal binding sites are therefore regulatory for Zn(II) sensing in vitro and in vivo, while the high-affinity alpha3N sites play some other role. This finding for SmtB is the opposite of that previously determined for Staphylococcus aureus pI258 CadC, a Pb(II)/Cd(II)/Bi(III) sensor [Busenlehner, L. S., et al. (2002) J. Mol. Biol. 319, 685-701], thus providing insight into the origin of functional metal ion selectivity in this family of metal sensor proteins.