Joseph Strauss

Chromatin-level regulation of biosynthetic gene clusters

Loss-of-function Aspergillus nidulans CclA, a Bre2 ortholog involved in histone H3 lysine 4 methylation, activated the expression of cryptic secondary metabolite clusters in A. nidulans. One new cluster generated monodictyphenone, emodin and emodin derivatives, whereas a second encoded two anti-osteoporosis polyketides, F9775A and F9775B. Modification of the chromatin landscape in fungal secondary metabolite clusters allows for a simple technological means to express silent fungal secondary metabolite gene clusters.

Crel, the carbon catabolite repressor protein from Trichoderma reesei

In order to investigate the mechanism of carbon catabolite repression in the industrially important fungus Trichoderma reesei, degenerated PCR‐primers were designed to amplify a 0.7‐bp fragment of the crel gene, which was used to clone the entire gene. It encodes a 402‐amino acid protein with a calculated M r, of 43.6 kDa. Its aa‐sequence shows 55.6% and 54.7% overall similarity to the corresponding genes of Aspergillus nidulans and A. niger, respectively. Similarity was restricted to the aa‐region containing the C2H2 zinc finger and several aa‐regions rich in proline and basic amino acids, which may be involved in the interaction with other proteins. Another aa‐region rich in the SPXX‐motif that has been considered analogous to a region of yeast RGR1p, was instead identified as a domain occurring in several eucaryotic transcription factors. The presence of the crel translation product was demonstrated with polyclonal antibodies against Cre1, which identified a protein of 43 (±2) kDa in cell‐free extracts from T. reesei. A Cre1 protein fragment from the two zinc fingers to the region similar to the aa‐sequence of eucaryotic transcription factors, was expressed in Escherichia coli as a fusion protein with glutathione S‐transferase. EMSA and in vitro footprinting revealed binding of the fusion protein to the sequence 5′‐GCGGAG‐3′, which matches well with the A. nidulans consensus sequence for CreA binding (5′‐SYGGRG‐3′). Cell‐free extracts of T. reesei formed different complexes with DNA‐fragments carrying this binding sites, and the presence of Cre1 and additional proteins in these complexes was demonstrated. We conclude that T. reesei Cre1 is the functional homologue of Aspergillus CreA and that it binds to its target sequence probably as a protein complex.

Bacteria-induced natural product formation in the fungus Aspergillus nidulans requires Saga/Ada-mediated histone acetylation

Sequence analyses of fungal genomes have revealed that the potential of fungi to produce secondary metabolites is greatly underestimated. In fact, most gene clusters coding for the biosynthesis of antibiotics, toxins, or pigments are silent under standard laboratory conditions. Hence, it is one of the major challenges in microbiology to uncover the mechanisms required for pathway activation. Recently, we discovered that intimate physical interaction of the important model fungus Aspergillus nidulans with the soil-dwelling bacterium Streptomyces rapamycinicus specifically activated silent fungal secondary metabolism genes, resulting in the production of the archetypal polyketide orsellinic acid and its derivatives. Here, we report that the streptomycete triggers modification of fungal histones. Deletion analysis of 36 of 40 acetyltransferases, including histone acetyltransferases (HATs) of A. nidulans, demonstrated that the Saga/Ada complex containing the HAT GcnE and the AdaB protein is required for induction of the orsellinic acid gene cluster by the bacterium. We also showed that Saga/Ada plays a major role for specific induction of other biosynthesis gene clusters, such as sterigmatocystin, terrequinone, and penicillin. Chromatin immunoprecipitation showed that the Saga/Ada-dependent increase of histone 3 acetylation at lysine 9 and 14 occurs during interaction of fungus and bacterium. Furthermore, the production of secondary metabolites in A. nidulans is accompanied by a global increase in H3K14 acetylation. Increased H3K9 acetylation, however, was only found within gene clusters. This report provides previously undescribed evidence of Saga/Ada dependent histone acetylation triggered by prokaryotes.

Heterochromatic marks are associated with the repression of secondary metabolism clusters in Aspergillus nidulans.

Fungal secondary metabolites are important bioactive compounds but the conditions leading to expression of most of the putative secondary metabolism (SM) genes predicted by fungal genomics are unknown. Here we describe a novel mechanism involved in SM‐gene regulation based on the finding that, in Aspergillus nidulans, mutants lacking components involved in heterochromatin formation show de‐repression of genes involved in biosynthesis of sterigmatocystin (ST), penicillin and terrequinone A. During the active growth phase, the silent ST gene cluster is marked by histone H3 lysine 9 trimethylation and contains high levels of the heterochromatin protein‐1 (HepA). Upon growth arrest and activation of SM, HepA and trimethylated H3K9 levels decrease concomitantly with increasing levels of acetylated histone H3. SM‐specific chromatin modifications are restricted to genes located inside the ST cluster, and constitutive heterochromatic marks persist at loci immediately outside the cluster. LaeA, a global activator of SM clusters in fungi, counteracts the establishment of heterochromatic marks. Thus, one level of regulation of the A. nidulans ST cluster employs epigenetic control by H3K9 methylation and HepA binding to establish a repressive chromatin structure and LaeA is involved in reversal of this heterochromatic signature inside the cluster, but not in that of flanking genes.

Carbon catabolite repression of xylanase I (xyn1) gene expression in Trichoderma reesei

The filamentous fungus Trichoderma reesei forms two specific, xylan‐inducible xylanases encoded by xyn1 and xyn2 to degrade the β‐1,4‐d‐xylan backbone of hemicelluloses. This enzyme system is formed in the presence of xylan, but not glucose. The molecular basis of the absence of xylanase I formation on glucose was the purpose of this study. Northern blotting of the xyn1 transcript as well as the use of the Escherichia coli hygromycin B phosphotransferase‐encoding gene (hph) as a reporter consistently showed that the basal expression of xyn1 was affected by glucose, whereas its induction by xylan remained uninfluenced. The repression of basal xyn1 transcription is mediated by the carbon catabolite repressor protein Cre1, which in vivo binds to two of four consensus sites (5‐SYG‐GRG‐3) in the xyn1 promoter, which occurred in the form of an inverted repeat. T. reesei strains, bearing a xynlv. hph reporter construct, in which four nucleotides from the middle of the inverted repeat had been removed, expressed hph on glucose at a level comparable to that observed during growth on a carbon catabolite derepressing carbon source. Northern analysis of xynl expression in a T. reesei mutant strain (RUT C‐30), which contains a truncated, non‐functional crel gene, also confirmed basal transcription of xyn1. In this strain, xyn1 transcription was still inducible by xylose or xylan to an even higher degree than in the wild‐type strain, suggesting that induction overcomes glucose repression at the level of xynl expression. Based on these data, we postulate that basal transcription of xyn1 is repressed by glucose and mediated by an inverted repeat of the consensus motif for Cre1‐mediated carbon catabolite repression.

The effect of resource quantity and resource stoichiometry on microbial carbon‐use‐efficiency

The carbon-use-efficiency (CUE) of microorganisms is an important parameter in determining ecosystem-level carbon (C) cycling; however, little is known about how variance in resources affects microbial CUE. To elucidate how resource quantity and resource stoichiometry affect microbial CUE, we cultured four microorganisms - two fungi (Aspergillus nidulans and Trichoderma harzianum) and two bacteria (Pectobacterium carotovorum and Verrucomicrobium spinosum) - under 12 unique C, nitrogen (N) and phosphorus (P) ratios. Whereas the CUE of A. nidulans was strongly affected by C, bacterial CUE was more strongly affected by mineral nutrients (N and P). Specifically, CUE in P. carotovorum was positively correlated with P, while CUE of V. spinosum primarily depended on N. This resulted in a positive relationship between fungal CUE and resource C : nutrient stoichiometry and a negative relationship between bacterial CUE and resource C : nutrient stoichiometry. The difference in the direction of the relationship between CUE and C : nutrient for fungi vs. bacteria was consistent with differences in biomass stoichiometry and suggested that fungi have a higher C demand than bacteria. These results suggest that the links between biomass stoichiometry, resource demand and CUE may provide a mechanism for commonly observed temporal and spatial patterns in microbial community structure and function in natural habitats.

The GATA factor AreA is essential for chromatin remodelling in a eukaryotic bidirectional promoter

The linked niiA and niaD genes of Aspergillus nidulans are transcribed divergently. The expression of these genes is subject to a dual control system. They are induced by nitrate and repressed by ammonium. AreA mediates derepression in the absence of ammonium and NirA supposedly mediates nitrate induction. Out of 10 GATA sites, a central cluster (sites 5–8) is responsible for ∼80% of the transcriptional activity of the promoter on both genes. We show occupancy in vivo of site 5 by the AreA protein, even under conditions of repression. Sites 5–8 are situated in a pre‐set nucleosome‐free region. Under conditions of expression, a drastic nucleosomal rearrangement takes place and the positioning of at least five nucleosomes flanking the central region is lost. Remodelling is strictly dependent on the presence of an active areA gene product, and independent from the NirA‐specific and essential transcription factor. Thus, nucleosome remodelling is independent from the transcriptional activation of the niiA–niaD promoter. The results presented cast doubts on the role of NirA as the unique transducer of the nitrate induction signal. We demonstrate, for the first time in vivo, that a GATA factor is involved directly in chromatin remodelling.

nirA, the pathway-specific regulatory gene of nitrate assimilation in Aspergillus nidulans, encodes a putative GAL4-type zinc finger protein and contains four introns in highly conserved regions.

The nucleotide sequence of nirA, mediating nitrate induction in Aspergillus nidulans, has been determined. Alignment of the cDNA and the genomic DNA sequence indicates that the gene contains four introns and encodes a protein of 892 amino acids. The deduced NIRA protein displays all characteristics of a transcriptional activator. A putative double-stranded DNA-binding domain in the amino-terminal part comprises six cysteine residues, characteristic for the GAL4 family of zinc finger proteins. An amino-terminal highly acidic region and two proline-rich regions are also present. The nucleotide sequences of two mutations were determined after they were mapped by transformation with overlapping DNA fragments, amplified by the polymerase chain reaction. nirA87, a mutation conferring noninducibility by nitrate and nitrite, has a -1 frameshift at triplet 340, which eliminates 549 C-terminal amino acids from the polypeptide. Under the assumption that the truncated polypeptide is stable, it comprises the zinc finger domain and the acidic region, which seem not sufficient for transcriptional activation. nirAd-106, an allele conferring nitrogen metabolite derepression of nitrate and nitrite reductase activity, includes two transitions, changing a glutamic acid to a lysine and a valine to an alanine, situated between a basic and a proline-rich region of the protein. Northern (RNA) analysis of the wild type and of constitutive (nirAc) and derepressed (nirAd) mutants show that the nirA transcript does not vary between these strains, being in all cases constitutively expressed. On the other hand, transcript levels of structural genes (niaD and niiA) do vary, being highly inducible in the wild type but constitutively expressed in the nirAc mutant. The nirAd mutant appears phenotypically derepressed, because the niaD and niiA transcript levels are overinduced in the presence of nitrate but are still partially repressed in the presence of ammonium.

Culturable bacteria from Zn- and Cd-accumulating Salix caprea with differential effects on plant growth and heavy metal availability.

Aims:  To characterize bacteria associated with Zn/Cd‐accumulating Salix caprea regarding their potential to support heavy metal phytoextraction.

The function of CreA, the carbon catabolite repressor of Aspergillus nidulans, is regulated at the transcriptional and post‐transcriptional level

The creA gene of A. nidulans encodes a wide‐domain regulatory protein mediating carbon catabolite repression. Northern blot analysis of creA mRNA revealed a complex expression profile: the addition of monosaccharides to a carbon‐starved culture of A. nidulans provoked a strong transient stimulation of creA transcript formation within a few minutes. In the case of repressing carbon sources, creA mRNA levels were subsequently downregulated, whereas the high creA mRNA levels were maintained in a creA mutant strain and in the presence of derepressing monosaccharides. A high creA transcript level is essential to achieve carbon catabolite repression and is dependent on glucose transport and, at least partially, on the creB gene product. Subsequent downregulation of creA mRNA levels, on the other hand, is typical of carbon catabolite repression and requires a functional CreA recognition site in the creA promoter (and thus involves autoregulation) and formation of glucose‐6‐phosphate. Despite the presence of continuing high transcript levels of creA in the presence of derepressing carbohydrates, EMSA demonstrated the presence of only low levels of a CreA–DNA complex in respective cell‐free extracts. Upon transfer of carbon catabolite derepressed mycelia to catabolite‐repressing conditions, a CreA–DNA complex is formed, and this process is dependent on de novo protein synthesis.