Proteins interacting with CreA and CreB in the carbon catabolite repression network in Aspergillus nidulans.

In Aspergillus nidulans, carbon catabolite repression (CCR) is mediated by the global repressor protein CreA. The deubiquitinating enzyme CreB is a component of the CCR network. Genetic interaction was confirmed using a strain containing complete loss-of-function alleles of both creA and creB. No direct physical interaction was identified between tagged versions of CreA and CreB. To identify any possible protein(s) that may form a bridge between CreA and CreB, we purified both proteins from mycelia grown in media that result in repression or derepression. The purified proteins were analysed by LC/MS and identified using MaxQuant and Mascot databases. For both CreA and CreB, 47 proteins were identified in repressing and derepressing conditions. Orthologues of the co-purified proteins were identified in S. cerevisiae and humans. Gene ontology analyses of A. nidulans proteins and yeast and human orthologues were performed. Functional annotation analysis revealed that proteins that preferentially interact with CreA in repressing conditions include histones and histone transcription regulator 3 (Hir3). Proteins interacting with CreB tend to be involved in cellular transportation and organization. Similar findings were obtained using yeast and human orthologues, although the yeast background generated a number of other biological processes involving Mig1p which were not present in the A. nidulans or human background analyses. Hir3 was present in repressing conditions for CreA and in both growth conditions for CreB, suggesting that Hir3, or proteins interacting with Hir3, could be a possible target of CreB.

The CreB deubiquitinating enzyme does not directly target the CreA repressor protein in Aspergillus nidulans.

Ubiquitination/deubiquitination pathways are now recognized as key components of gene regulatory mechanisms in eukaryotes. The major transcriptional repressor for carbon catabolite repression in Aspergillus nidulans is CreA, and mutational analysis led to the suggestion that a regulatory ubiquitination/deubiquitination pathway is involved. A key unanswered question is if and how this pathway, comprising CreB (deubiquitinating enzyme) and HulA (ubiquitin ligase) and other proteins, is involved in the regulatory mechanism. Previously, missense alleles of creA and creB were analysed for genetic interactions, and here we extended this to complete loss-of-function alleles of creA and creB, and compared morphological and biochemical phenotypes, which confirmed genetic interaction between the genes. We investigated whether CreA, or a protein in a complex with it, is a direct target of the CreB deubiquitination enzyme, using co-purifications of CreA and CreB, first using strains that overexpress the proteins and then using strains that express the proteins from their native promoters. The Phos-tag system was used to show that CreA is a phosphorylated protein, but no ubiquitination was detected using anti-ubiquitin antibodies and Western analysis. These findings were confirmed using mass spectrometry, which confirmed that CreA was differentially phosphorylated but not ubiquitinated. Thus, CreA is not a direct target of CreB, and nor are proteins that form part of a stable complex with CreA a target of CreB. These results open up new questions regarding the molecular mechanism of CreA repressing activity, and how the ubiquitination pathway involving CreB interacts with this regulatory network.

The CreB deubiquitinating enzyme does not directly target the CreA repressor protein in Aspergillus nidulans.

Ubiquitination/deubiquitination pathways are now recognized as key components of gene regulatory mechanisms in eukaryotes. The major transcriptional repressor for carbon catabolite repression in Aspergillus nidulans is CreA, and mutational analysis led to the suggestion that a regulatory ubiquitination/deubiquitination pathway is involved. A key unanswered question is if and how this pathway, comprising CreB (deubiquitinating enzyme) and HulA (ubiquitin ligase) and other proteins, is involved in the regulatory mechanism. Previously, missense alleles of creA and creB were analysed for genetic interactions, and here we extended this to complete loss-of-function alleles of creA and creB, and compared morphological and biochemical phenotypes, which confirmed genetic interaction between the genes. We investigated whether CreA, or a protein in a complex with it, is a direct target of the CreB deubiquitination enzyme, using co-purifications of CreA and CreB, first using strains that overexpress the proteins and then using strains that express the proteins from their native promoters. The Phos-tag system was used to show that CreA is a phosphorylated protein, but no ubiquitination was detected using anti-ubiquitin antibodies and Western analysis. These findings were confirmed using mass spectrometry, which confirmed that CreA was differentially phosphorylated but not ubiquitinated. Thus, CreA is not a direct target of CreB, and nor are proteins that form part of a stable complex with CreA a target of CreB. These results open up new questions regarding the molecular mechanism of CreA repressing activity, and how the ubiquitination pathway involving CreB interacts with this regulatory network.

Role of HxkC, a mitochondrial hexokinase-like protein, in fungal programmed cell death.

Apoptosis is a form of programmed cell death (PCD) that occurs during animal development and is also triggered by a variety of signals including nutrient or oxidative stress, hypoxia, DNA damage, viral infection and oncogenic transformation. Though apoptotic-like PCD also occurs in plants and fungi, genes encoding several of the key players in mammalian apoptosis (p53 and BH-domain proteins) have not been identified in these kingdoms. In this report we investigated whether HxkC, a mitochondrial hexokinase-like protein, and XprG, a putative p53-like transcription factor similar to Ndt80, play a role in programmed cell death in the filamentous fungus Aspergillus nidulans. We show that a mutant lacking HxkC is more sensitive to oxidative stress. Autolysis, a form of fungal programmed cell death triggered by carbon starvation, is accelerated in the hxkCΔ1 mutant but not the hxkCΔ1 xprGΔ1 double mutant. In the absence of nutrient stress, the hxkCΔ1 mutant displays XprG-dependent DNA fragmentation typical of apoptosis and elevated levels of intracellular protease. HxkC and XprG are required for catabolism of N-acetylglucosamine, as in Trichoderma reesei. We show that XprG is present in the nucleus. We conclude that, like mammalian mitochondrial hexokinase, HxkC has anti-apoptotic activity and the XprG transcription factor has a pro-apoptotic role in filamentous fungi.

A second component of the SltA-dependent cation tolerance pathway in Aspergillus nidulans.

The transcriptional response to alkali metal cation stress is mediated by the zinc finger transcription factor SltA in Aspergillus nidulans and probably in other fungi of the pezizomycotina subphylum. A second component of this pathway has been identified and characterized. SltB is a 1272 amino acid protein with at least two putative functional domains, a pseudo-kinase and a serine-endoprotease, involved in signaling to the transcription factor SltA. Absence of SltB activity results in nearly identical phenotypes to those observed for a null sltA mutant. Hypersensitivity to a variety of monovalent and divalent cations, and to medium alkalinization are among the phenotypes exhibited by a null sltB mutant. Calcium homeostasis is an exception and this cation improves growth of sltΔ mutants. Moreover, loss of kinase HalA in conjunction with loss-of-function sltA or sltB mutations leads to pronounced calcium auxotrophy. sltA sltB double null mutants display a cation stress sensitive phenotype indistinguishable from that of single slt mutants showing the close functional relationship between these two proteins. This functional relationship is reinforced by the fact that numerous mutations in both slt loci can be isolated as suppressors of poor colonial growth resulting from certain null vps (vacuolar protein sorting) mutations. In addition to allowing identification of sltB, our sltB missense mutations enabled prediction of functional regions in the SltB protein. Although the relationship between the Slt and Vps pathways remains enigmatic, absence of SltB, like that of SltA, leads to vacuolar hypertrophy. Importantly, the phenotypes of selected sltA and sltB mutations demonstrate that suppression of null vps mutations is not dependent on the inability to tolerate cation stress. Thus a specific role for both SltA and SltB in the VPS pathway seems likely. Finally, it is noteworthy that SltA and SltB have a similar, limited phylogenetic distribution, being restricted to the pezizomycotina subphylum. The relevance of the Slt regulatory pathway to cell structure, intracellular trafficking and cation homeostasis and its restricted phylogenetic distribution makes this pathway of general interest for future investigation and as a source of targets for antifungal drugs.

"It made you feel like you've still got it": experiences of people with chronic low back pain undertaking a single session of body image training in virtual reality.

INTRODUCTION: Embodying fit avatars in virtual reality (VR) is proposed as a possible treatment for cortical body representations and pain-related self-perceptions. OBJECTIVE: To explore consumer perceptions of a novel VR intervention (VR-BiT) for chronic low back pain. METHODS: Adults (n = 17, mean age(SD) = 52(14)) with chronic low back pain who had undergone a single session of VR-BiT as part of a randomized controlled trial underwent a semi-structured interview using open-ended questions. Interviews were audio-recorded, transcribed verbatim, and analyzed thematically. RESULTS: Data reduction identified four themes: clinically beneficial and beyond; helping and hindering use; desire for more; and individualized future. Participants experienced wide ranging effects, including improved physical self-efficacy, pain, ability to perform physical activity and psychological symptoms. The intervention was well tolerated, except for two reports of nausea, and a few participants indicating pain associated with unaccustomed movement. Most participants were motivated to use VR-BiT again, despite some having technical issues. Participants suggested that personalizing VR-BiT and regular use would be beneficial. CONCLUSIONS: There was strong consumer support for further use of VR-BiT. Future studies of VR-BiT effectiveness are warranted and should consider incorporating individual user preferences, including people with diverse pain presentations, and involving a multi-session design.

Disruption of Trichoderma reesei cre2, encoding an ubiquitin C-terminal hydrolase, results in increased cellulase activity.

BACKGROUND: The filamentous fungus Trichoderma reesei (Hypocrea jecorina) is an important source of cellulases for use in the textile and alternative fuel industries. To fully understand the regulation of cellulase production in T. reesei, the role of a gene known to be involved in carbon regulation in Aspergillus nidulans, but unstudied in T. reesei, was investigated. RESULTS: The T. reesei orthologue of the A. nidulans creB gene, designated cre2, was identified and shown to be functional through heterologous complementation of a creB mutation in A. nidulans. A T. reesei strain was constructed using gene disruption techniques that contained a disrupted cre2 gene. This strain, JKTR2-6, exhibited phenotypes similar to the A. nidulans creB mutant strain both in carbon catabolite repressing, and in carbon catabolite derepressing conditions. Importantly, the disruption also led to elevated cellulase levels. CONCLUSIONS: These results demonstrate that cre2 is involved in cellulase expression. Since the disruption of cre2 increases the amount of cellulase activity, without severe morphological affects, targeting creB orthologues for disruption in other industrially useful filamentous fungi, such as Aspergillus oryzae, Trichoderma harzianum or Aspergillus niger may also lead to elevated hydrolytic enzyme activity in these species.

Independent duplications of α-amylase in different strains of Aspergillus oryzae.

Aspergillus oryzae is a filamentous fungus that has arisen through the ancient domestication of Aspergillus flavus for making traditional oriental foods and beverages. In the many centuries A. oryzae has been used for fermenting the starch in rice to simple sugars, it has undergone selection for increased secretion of starch-degrading enzymes. In particular, all A. oryzae strains investigated thus far have two or more copies of a gene encoding α-amylase, whereas A. flavus has only one. Here we investigate the duplications leading to these copies in three A. oryzae strains. We find evidence of at least three separate duplications of α-amylase, an example of parallel evolution in a micro-organism under artificial selection. At least two of these duplications appear to be associated with activity of transposable elements of the Tc1/mariner class. Both involve a 9.1 kb element that terminates in inverted repeats, encodes a putative transposase and another putative protein of unknown function, and contains an unusual arrangement of four short internal imperfect repeats. Although "unusual Mariners" of this size have previously been identified in A. oryzae, Aspergillus fumigatus and Aspergillus nidulans, this is the first evidence we know of that at least some of them are active in modern times and that their activity can contribute to beneficial genetic changes.

Carbon Catabolite Repression Governs Diverse Physiological Processes and Development in Aspergillus nidulans.

Carbon catabolite repression (CCR) is a common phenomenon of microorganisms that enable efficient utilization of carbon nutrients, critical for the fitness of microorganisms in the wild and for pathogenic species to cause infection. In most filamentous fungal species, the conserved transcription factor CreA/Cre1 mediates CCR. Previous studies demonstrated a primary function for CreA/Cre1 in carbon metabolism; however, the phenotype of creA/cre1 mutants indicated broader roles. The global function and regulatory mechanism of this wide-domain transcription factor has remained elusive. Here, we applied two powerful genomics methods (transcriptome sequencing and chromatin immunoprecipitation sequencing) to delineate the direct and indirect roles of Aspergillus nidulans CreA across diverse physiological processes, including secondary metabolism, iron homeostasis, oxidative stress response, development, N-glycan biosynthesis, unfolded protein response, and nutrient and ion transport. The results indicate intricate connections between the regulation of carbon metabolism and diverse cellular functions. Moreover, our work also provides key mechanistic insights into CreA regulation and identifies CreA as a master regulator controlling many transcription factors of different regulatory networks. The discoveries for this highly conserved transcriptional regulator in a model fungus have important implications for CCR in related pathogenic and industrial species. IMPORTANCE The ability to scavenge and use a wide range of nutrients for growth is crucial for microorganisms' survival in the wild. Carbon catabolite repression (CCR) is a transcriptional regulatory phenomenon of both bacteria and fungi to coordinate the expression of genes required for preferential utilization of carbon sources. Since carbon metabolism is essential for growth, CCR is central to the fitness of microorganisms. In filamentous fungi, CCR is mediated by the conserved transcription factor CreA/Cre1, whose function in carbon metabolism has been well established. However, the global roles and regulatory mechanism of CreA/Cre1 are poorly defined. This study uncovers the direct and indirect functions of CreA in the model organism Aspergillus nidulans over diverse physiological processes and development and provides mechanistic insights into how CreA controls different regulatory networks. The work also reveals an interesting functional divergence between filamentous fungal and yeast CreA/Cre1 orthologues.

Mutations in genes encoding sorting nexins alter production of intracellular and extracellular proteases in Aspergillus nidulans.

XprG, a putative p53-like transcriptional activator, regulates production of extracellular proteases in response to nutrient limitation and may also have a role in programmed cell death. To identify genes that may be involved in the XprG regulatory pathway, xprG2 revertants were isolated and shown to carry mutations in genes which we have named sogA-C (suppressors of xprG). The translocation breakpoint in the sogA1 mutant was localized to a homolog of Saccharomyces cerevisiae VPS5 and mapping data indicated that sogB was tightly linked to a VPS17 homolog. Complementation of the sogA1 and sogB1 mutations and identification of nonsense mutations in the sogA2 and sogB1 alleles confirmed the identification. Vps17p and Vps5p are part of a complex involved in sorting of vacuolar proteins in yeast and regulation of cell-surface receptors in mammals. Protease zymograms indicate that mutations in sogA-C permit secretion of intracellular proteases, as in S. cerevisiae vps5 and vps17 mutants. In contrast to S. cerevisiae, the production of intracellular protease was much higher in the mutants. Analysis of serine protease gene expression suggests that an XprG-independent mechanism for regulation of extracellular protease gene expression in response to carbon starvation exists and is activated in the pseudorevertants.

Can in vitro metabolism-dependent covalent binding data distinguish hepatotoxic from nonhepatotoxic drugs? An analysis using human hepatocytes and liver S-9 fraction.

In vitro covalent binding studies in which xenobiotics are shown to undergo metabolism-dependent covalent binding to macromolecules have been commonly used to shed light on the biochemical mechanisms of xenobiotic-induced toxicity. In this paper, 18 drugs (nine hepatotoxins and nine nonhepatotoxins) were tested for their proclivity to demonstrate metabolism-dependent covalent binding to macromolecules in human liver S-9 fraction (9000 g supernatant) or human hepatocytes, as an extension to previous work that used human liver microsomes published in this journal [ Obach et al. ( 2008 ) Chem. Res. Toxicol. 21 , 1814 -1822 ]. In the S-9 fraction, seven out of the nine drugs in each category demonstrated some level of metabolism-dependent covalent binding. Inclusion of reduced glutathione, cofactors needed by conjugating enzymes, and other parameters (total daily dose and fraction of total intrinsic clearance comprised by covalent binding) improved the ability of the system to separate hepatotoxins from nonhepatotoxins to a limited extent. Covalent binding in human hepatocytes showed that six out of the nine hepatotoxins and four out of eight nonhepatotoxins demonstrated covalent binding. Taking into account estimates of total daily body burden of covalent binding from the hepatocyte data showed an improvement over other in vitro systems for distinguishing hepatotoxins from nonhepatotoxins; however, this metabolism system still displayed some false positives. Combined with the previous study using liver microsomes, these findings identify the limitations of in vitro covalent binding data for prospective prediction of hepatotoxicity for new drug candidates and highlight the need for a better understanding of the link between drug bioactivation, covalent adduct formation, and toxicity outcomes. Directly relating covalent binding to hepatotoxicity is likely an oversimplification of the process whereby adduct formation ultimately leads to toxicity. Understanding underlying complexities (e.g., which macromolecules are important covalent binding targets, interindividual differences in susceptibility, etc.) will be essential to any understanding of the problem of metabolism-dependent hepatotoxicity and predicting toxicity from in vitro experiments.

CreA-mediated repression in Aspergillus nidulans does not require transcriptional auto-regulation, regulated intracellular localisation or degradation of CreA.

The major regulatory protein in carbon repression in Aspergillus nidulans is CreA. Strains constitutively over-expressing creA show normal responses to carbon repression, indicating that auto-regulation of creA is not essential for CreA-mediated regulation. In these strains, high levels of CreA are present whether cells are grown in repressing or derepressing conditions, indicating large-scale degradation of CreA does not play a key role. CreA is located in the nucleus and cytoplasm in cells when grown in either repressing or derepressing conditions, and absence of CreB, CreD or AcrB does not affect either the localisation or amount of CreA. Therefore, CreA must require some modification or interaction to act as a repressor. Deletion analysis indicates that a region of CreA thought to be important for repression in Trichoderma reesei and Sclerotina sclerotiorum CreA homologues is not critical for function in Aspergillus nidulans.

Production of L(+)-lactic acid using acid-adapted precultures of Rhizopus arrhizus in a stirred tank reactor.

Cultivations of filamentous fungi in stirred tank reactors (STRs) to produce metabolites are often limited by insufficient mixing and mass transfer because of the formation of mycelial clumps inside the reactors. This study developed an acid-adapted preculture approach to control the morphology of filamentous Rhizopus arrhizus in a STR, consequently to enhance the production yield and productivity of L(+)-lactic acid efficiently using waste potato starch as substrate. Using the acid-adapted precultures as inoculum, the morphology of R. arrhizus was maintained as large clumps, coalesced loose small pellets, and freely dispersed small pellets. The highest lactic acid concentration of 85.7 g/L with a yield of 86% was obtained in association with the formation of coalesced loose small pellets. The results indicate that the use of the acid-adapted precultures as inoculum is a promising approach for lactic acid production in STRs.

Physiotherapist-delivered Stress Inoculation Training for acute whiplash-associated disorders: A qualitative study of perceptions and experiences.

BACKGROUND: Formally trained and accredited physiotherapists delivered Stress Inoculation Training (SIT) integrated with guideline-based physiotherapy management to individuals with acute whiplash associated disorders (WAD) as part of a randomised controlled trial. The delivery of SIT by physiotherapists is new. OBJECTIVES: To investigate physiotherapists' perspectives on delivering SIT as part of the trial and in routine practice. DESIGN: Qualitative descriptive. METHOD: Physiotherapists (n = 11) participated in semi-structured interviews. Interviews were audio-recorded, transcribed verbatim, and analysed thematically. Findings were triangulated against an audit of physiotherapists' adherence to the SIT protocol. RESULTS: Three themes were identified: perceived value; capacity to deliver; and adaptation and implementation. Physiotherapists' saw value in SIT in that they perceived the program to have improved patient outcomes, enhanced their therapeutic alliance, and provided new skills to manage psychological contributors to WAD. Physiotherapists' capacity to deliver the program was facilitated through the development of confidence in their ability to deliver sessions, viewing SIT as falling within their current professional identity, and having confidence in their ability to manage mismatches in patients' expectations of care. All physiotherapists reported having used SIT to some extent in routine practice, by selectively delivering sessions and/or integrating the content with other management. Physiotherapists were able to deliver SIT as was intended (94.6% adherence). CONCLUSIONS: Physiotherapists' supported adding SIT to usual management of individuals with acute WAD. Education on SIT principles is recommended during pre-professional training to facilitate future implementation.

A eukaryotic nicotinate-inducible gene cluster: convergent evolution in fungi and bacteria.

Nicotinate degradation has hitherto been elucidated only in bacteria. In the ascomycete Aspergillus nidulans, six loci, hxnS/AN9178 encoding the molybdenum cofactor-containing nicotinate hydroxylase, AN11197 encoding a Cys2/His2 zinc finger regulator HxnR, together with AN11196/hxnZ, AN11188/hxnY, AN11189/hxnP and AN9177/hxnT, are clustered and stringently co-induced by a nicotinate derivative and subject to nitrogen metabolite repression mediated by the GATA factor AreA. These genes are strictly co-regulated by HxnR. Within the hxnR gene, constitutive mutations map in two discrete regions. Aspergillus nidulans is capable of using nicotinate and its oxidation products 6-hydroxynicotinic acid and 2,5-dihydroxypyridine as sole nitrogen sources in an HxnR-dependent way. HxnS is highly similar to HxA, the canonical xanthine dehydrogenase (XDH), and has originated by gene duplication, preceding the origin of the Pezizomycotina. This cluster is conserved with some variations throughout the Aspergillaceae. Our results imply that a fungal pathway has arisen independently from bacterial ones. Significantly, the neo-functionalization of XDH into nicotinate hydroxylase has occurred independently from analogous events in bacteria. This work describes for the first time a gene cluster involved in nicotinate catabolism in a eukaryote and has relevance for the formation and evolution of co-regulated primary metabolic gene clusters and the microbial degradation of N-heterocyclic compounds.

Molecular characterization and analysis of the acrB gene of Aspergillus nidulans: a gene identified by genetic interaction as a component of the regulatory network that includes the CreB deubiquitination enzyme.

Mutations in the acrB gene, which were originally selected through their resistance to acriflavine, also result in reduced growth on a range of sole carbon sources, including fructose, cellobiose, raffinose, and starch, and reduced utilization of omega-amino acids, including GABA and beta-alanine, as sole carbon and nitrogen sources. The acrB2 mutation suppresses the phenotypic effects of mutations in the creB gene that encodes a regulatory deubiquitinating enzyme, and in the creC gene that encodes a WD40-repeat-containing protein. Thus AcrB interacts with a regulatory network controlling carbon source utilization that involves ubiquitination and deubiquitination. The acrB gene was cloned and physically analyzed, and it encodes a novel protein that contains three putative transmembrane domains and a coiled-coil region. AcrB may play a role in the ubiquitination aspect of this regulatory network.

Physiological characterisation of recombinant Aspergillus nidulans strains with different creA genotypes expressing A. oryzae alpha-amylase.

The physiology of three strains of Aspergillus nidulans was examined--a creA deletion strain, a wild type creA genotype and a strain containing extra copies of the creA gene, all producing Aspergillus oryzae alpha-amylase. The strains were cultured in batch and continuous cultivations and the biomass formation and alpha-amylase production was characterised. Overexpression of the creA gene resulted in a lower maximum specific growth rate and a slightly higher repression of the alpha-amylase production during conditions with high glucose concentration. No expression of creA also resulted in a decreased maximum specific growth rate, but also in drastic changes in morphology. Furthermore, the expression of alpha-amylase was completely derepressed and creA thus seems to be the only regulatory protein responsible for glucose repression of alpha-amylase expression. The effect of different carbon sources on the alpha-amylase production in the creA deletion strain was investigated and it was found that starch was the best inducer. The degree of induction by starch increased almost linearly with the concentration of starch in starch/glucose mixtures. High-density batch cultivation was performed with the creA deletion strain and a final titre of 6.0 g l(-1) of alpha-amylase was reached after 162 h of cultivation.

The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex in Aspergillus nidulans.

A mutation screen in Aspergillus nidulans uncovered mutations in the acdX gene that led to altered repression by acetate, but not by glucose. AcdX of A. nidulans is highly conserved with Spt8p of Saccharomyces cerevisiae, and since Spt8p is a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex, the SAGA complex may have a role in acetate repression in A. nidulans. We used a bioinformatic approach to identify genes encoding most members of the SAGA complex in A. nidulans, and a proteomic analysis to confirm that most protein components identified indeed exist as a complex in A. nidulans. No apparent compositional differences were detected in mycelia cultured in acetate compared to glucose medium. The methods used revealed apparent differences between Yeast and A. nidulans in the deubiquitination (DUB) module of the complex, which in S. cerevisiae consists of Sgf11p, Sus1p, and Ubp8p. Although a convincing homologue of S. cerevisiae Ubp8p was identified in the A. nidulans genome, there were no apparent homologues for Sus1p and Sgf11p. In addition, when the SAGA complex was purified from A. nidulans, members of the DUB module were not co-purified with the complex, indicating that functional homologues of Sus1p and Sgf11p were not part of the complex. Thus, deubiquitination of H2B-Ub in stress conditions is likely to be regulated differently in A. nidulans compared to S. cerevisiae.

SAGA complex components and acetate repression in Aspergillus nidulans.

Alongside the well-established carbon catabolite repression by glucose and other sugars, acetate causes repression in Aspergillus nidulans. Mutations in creA, encoding the transcriptional repressor involved in glucose repression, also affect acetate repression, but mutations in creB or creC, encoding components of a deubiquitination system, do not. To understand the effects of acetate, we used a mutational screen that was similar to screens that uncovered mutations in creA, creB, and creC, except that glucose was replaced by acetate to identify mutations that were affected for repression by acetate but not by glucose. We uncovered mutations in acdX, homologous to the yeast SAGA component gene SPT8, which in growth tests showed derepression for acetate repression but not for glucose repression. We also made mutations in sptC, homologous to the yeast SAGA component gene SPT3, which showed a similar phenotype. We found that acetate repression is complex, and analysis of facA mutations (lacking acetyl CoA synthetase) indicates that acetate metabolism is required for repression of some systems (proline metabolism) but not for others (acetamide metabolism). Although plate tests indicated that acdX- and sptC-null mutations led to derepressed alcohol dehydrogenase activity, reverse-transcription quantitative real-time polymerase chain reaction showed no derepression of alcA or aldA but rather elevated induced levels. Our results indicate that acetate repression is due to repression via CreA together with metabolic changes rather than due to an independent regulatory control mechanism.

Enhancement of l(+)-lactic acid production using acid-adapted precultures of Rhizopus arrhizus in a bubble column reactor.

88 g/L lactic acid was produced from waste potato starch (equivalent to 100 g/L glucose) in a bubble column reactor using appropriate acid-adapted precultures of Rhizopus arrhizus. Further experiment showed that repeated dilution of cultures caused the decrease of lactic acid concentration and productivity due to formation of large fungal pellets.