Metabolite repression and inducer exclusion in the proline utilization gene cluster of Aspergillus nidulans.
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The clustered prnB, prnC, and prnD genes are repressed by the simultaneous presence of glucose and ammonium. A derepressed mutation inactivating a CreA-binding site acts in cis only on the permease gene (prnB) while derepression of prnD and prnC is largely the result of reversal of inducer exclusion. (+info)
Lack of host specialization in Aspergillus flavus.
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Aspergillus spp. cause disease in a broad range of organisms, but it is unknown if strains are specialized for particular hosts. We evaluated isolates of Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans for their ability to infect bean leaves, corn kernels, and insects (Galleria mellonella). Strains of A. flavus did not affect nonwounded bean leaves, corn kernels, or insects at 22 degrees C, but they killed insects following hemocoelic challenge and caused symptoms ranging from moderate to severe in corn kernels and bean leaves injured during inoculation. The pectinase P2c, implicated in aggressive colonization of cotton balls, is produced by most A. flavus isolates, but its absence did not prevent colonization of bean leaves. Proteases have been implicated in colonization of animal hosts. All A. flavus strains produced very similar patterns of protease isozymes when cultured on horse lung polymers. Quantitative differences in protease levels did not correlate with the ability to colonize insects. In contrast to A. flavus, strains of A. nidulans and A. fumigatus could not invade living insect or plant tissues or resist digestion by insect hemocytes. Our results indicate that A. flavus has parasitic attributes that are lacking in A. fumigatus and A. nidulans but that individual strains of A. flavus are not specialized to particular hosts. (+info)
Requirement of monooxygenase-mediated steps for sterigmatocystin biosynthesis by Aspergillus nidulans.
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Sterigmatocystin (ST) and aflatoxin B(1) (AFB(1)) are two polyketide-derived Aspergillus mycotoxins synthesized by functionally identical sets of enzymes. ST, the compound produced by Aspergillus nidulans, is a late intermediate in the AFB(1) pathway of A. parasiticus and A. flavus. Previous biochemical studies predicted that five oxygenase steps are required for the formation of ST. A 60-kb ST gene cluster in A. nidulans contains five genes, stcB, stcF, stcL, stcS, and stcW, encoding putative monooxygenase activities. Prior research showed that stcL and stcS mutants accumulated versicolorins B and A, respectively. We now show that strains disrupted at stcF, encoding a P-450 monooxygenase similar to A. parasiticus avnA, accumulate averantin. Disruption of either StcB (a putative P-450 monooxygenase) or StcW (a putative flavin-requiring monooxygenase) led to the accumulation of averufin as determined by radiolabeled feeding and extraction studies. (+info)
Pantothenate kinase regulation of the intracellular concentration of coenzyme A.
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Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway in bacteria and is thought to play a similar role in mammalian cells. We examined this hypothesis by identifying and characterizing two murine cDNAs that encoded PanK. The two cDNAs were predicted to arise from alternate splicing of the same gene to yield different mRNAs that encode two isoforms (mPanK1alpha and mPanK1beta) with distinct amino termini. The predicted protein sequence of mPanK1 was not related to bacterial PanK but exhibited significant similarity to Aspergillus nidulans PanK. mPanK1alpha was most highly expressed in heart and kidney, whereas mPanK1beta mRNA was detected primarily in liver and kidney. Pantothenate was the most abundant pathway component (42.8%) in normal cells providing clear evidence that pantothenate phosphorylation was a rate-controlling step in CoA biosynthesis. Enhanced mPanK1beta expression eliminated the intracellular pantothenate pool and triggered a 13-fold increase in intracellular CoA content. mPanK1beta activity in vitro was stimulated by CoA and strongly inhibited by acetyl-CoA illustrating that differential modulation of mPanK1beta activity by pathway end products also contributed to the management of CoA levels. These data support the concept that the expression and/or activity of PanK is a determining factor in the physiological regulation of the intracellular CoA concentration. (+info)
Hypomorphic bimA(APC3) alleles cause errors in chromosome metabolism that activate the DNA damage checkpoint blocking cytokinesis in Aspergillus nidulans.
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The Aspergillus nidulans sepI(+) gene has been implicated in the coordination of septation with nuclear division and cell growth. We find that the temperature-sensitive (ts) sepI1 mutation represents a novel allele of bimA(APC3), which encodes a conserved component of the anaphase-promoting complex/cyclosome (APC/C). We have characterized the septation, nuclear division, cell-cycle checkpoint defects, and DNA sequence alterations of sepI1 (renamed bimA10) and two other ts lethal bimA(APC3) alleles, bimA1 and bimA9. Our observations that bimA9 and bimA10 strains had morphologically abnormal nuclei, chromosome segregation defects, synthetic phenotypes with mutations in the DNA damage checkpoint genes uvsB(MEC1/rad3) or uvsD(+), and enhanced sensitivity to hydroxyurea strongly suggest that these strains accumulate errors in DNA metabolism. We found that the aseptate phenotype of bimA9 and bimA10 strains was substantially relieved by mutations in uvsB(MEC1/rad3) or uvsD(+), suggesting that the presence of a functional DNA damage checkpoint inhibits septation in these bimA(APC3) strains. Our results demonstrate that mutations in bimA(APC3) lead to errors in DNA metabolism that indirectly block septation. (+info)
On how a transcription factor can avoid its proteolytic activation in the absence of signal transduction.
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In response to alkaline ambient pH, the Aspergillus nidulans PacC transcription factor mediating pH regulation of gene expression is activated by proteolytic removal of a negative-acting C-terminal domain. We demonstrate interactions involving the approximately 150 C-terminal PacC residues and two regions located immediately downstream of the DNA binding domain. Our data indicate two full-length PacC conformations whose relative amounts depend upon ambient pH: one 'open' and accessible for processing, the other 'closed' and inaccessible. The location of essential determinants for proteolytic processing within the two more upstream interacting regions probably explains why the interactions prevent processing, whereas the direct involvement of the C-terminal region in processing-preventing interactions explains why C-terminal truncating mutations result in alkalinity mimicry and pH-independent processing. A mutant PacC deficient in pH signal response and consequent processing behaves as though locked in the 'closed' form. Single-residue substitutions, obtained as mutations bypassing the need for pH signal transduction, identify crucial residues in each of the three interactive regions and overcome the processing deficiency in the 'permanently closed' mutant. (+info)
Molecular characterization of ubiquitin genes from Aspergillus nidulans: mRNA expression on different stress and growth conditions.
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We are interested in studying the ubiquitin (UBI) gene expression during different stress and growth conditions in the filamentous fungus Aspergillus nidulans. Here, we report the cloning of a cDNA clone that corresponds to a gene, ubi1, that encodes a carboxyl extension protein from A. nidulans. This cDNA corresponds to a gene that encodes a protein that showed high homology to other polyubiquitin and CEP-80 genes at the N- and C-terminus, respectively. We characterize the mRNA expression of the CEP and polyubiquitin genes during several growth and stress conditions. Expression of the ubi1 and ubi4 genes was correlated with cell growth in most of the carbon sources used, except maltose. Both ubi1 and ubi4 genes were induced upon heat-shock, although the levels of expression were raised quicker for ubi4 than for ubi1. The ubi1 and ubi4 genes displayed a very complex expression pattern in presence of drugs with a different mechanism of action suggesting that the regulatory processes controlling UBI gene expression discriminate between different stresses and can affect individually each UBI gene. The ubi1 gene was highly expressed in presence of hydrogen peroxide while the ubi4 mRNA level was not affected; several metals in our experimental conditions were not able to induce either ubi1 nor ubi4 genes. (+info)
A new approach for the identification and cloning of genes: the pBACwich system using Cre/lox site-specific recombination.
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With current plant transformation methods ( Agrobacterium, biolistics and protoplast fusion), insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences and multigene interactions can make gene expression experiments difficult to interpret and plant phenotypes less predictable. An alternative approach to random integration of large DNA fragments into plants is to utilize one of several site-specific recombination (SSR) systems, such as Cre/ lox. Cre has been shown in numerous instances to mediate lox site-specific recombination in animal and plant cells. By incorporating the Cre/ lox SSR system into a bacterial artificial chromosome (BAC) vector, a more precise evaluation of large DNA inserts for genetic complementation should be possible. Site-specific insertion of DNA into predefined sites in the genome may eliminate unwanted 'position effects' caused by the random integration of exogenously introduced DNA. In an effort to make the Cre/ lox system an effective tool for site-directed integration of large DNAs, we constructed and tested a new vector potentially capable of integrating large DNA inserts into plant and fungal genomes. In this study, we present the construction of a new BAC vector, pBACwich, for the system and the use of this vector to demonstrate SSR of large DNA inserts (up to 230 kb) into plant and fungal genomes. (+info)