SrbA interacts with a transcriptional enhancer within the 34 <i>mer</i>.

<p>(A) Alignment of TR34, TR46.1, TR46.2 and TR53. The 34 <i>mer</i> is duplicated in all TR variants (adapted from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005775#ppat.1005775.ref030" target="_blank">30</a>]). bold, 34 <i>mer</i>; underlined, duplicated sequence. (B) To further analyze the <i>in vivo</i> binding data described by Chung <i>et al</i>. [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005775#ppat.1005775.ref027" target="_blank">27</a>], we measured binding of recombinant SrbA to this section using real-time SPR analysis. The putative binding sites identified by comparison to the consensus sequence are shown as SRE1 and SRE2. DNA duplexes containing either SRE1, SRE2 or both were used for SPR analysis. Sensorgrams of 200, 100, 50, 25, 12.5, 6.25, and 3.13 nM SrbA161-267 binding injected in duplicate (black lines) are shown overlaid with the best fit derived from a 1:1 interaction model including a mass transport term (red lines).</p

The CBC plays a crucial role in the growth of <i>A</i>. <i>fumigatus</i> and is required for pathogenicity in a pulmonary (non-leucopenic) and systemic (leucopenic) model of aspergillosis.

<p>(A) Radial growth of strains was determined under different nutrient supply. Strains were grown on AMM, RPMI as well as SAB solid medium; (B) Survival of cortisone acetate immunosuppressed OF-1 mice challenged intranasally with 1x10⁵ CFU/animal of <i>A</i>. <i>fumigatus</i>. P < 0.05 in comparison to wt and <i>hapC</i>^<i>REC</i>. PM, pulmonary model; (C) Survival proportions of cyclophosphamide immunosuppressed OF-1 mice challenged intravenously with 3x10⁴ CFU/animal of <i>A</i>. <i>fumigatus</i>. P < 0.05 in comparison to wt and <i>hapC</i>^<i>REC</i>. SM, systemic model.</p

CBC mutants show increased production of sterols.

<p>Sterol levels of transcription factor mutants incubated in AMM for 24 h at 37 C, 200 rpm have been determined by GC-MS. Δ<i>cyp51A</i> served as control for the double deletion mutant Δ<i>hapC</i>Δ<i>cyp51A</i>. Sterol content has been normalised to that of wt. Samples were assessed in biological triplicates. p-values were calculated by Student’s T-test (reference: wt): *, <0.05; ** <0.01. TS, total sterols; bold, >1.5 fold increase.</p

<i>hapC</i> deletion leads to derepression of ergosterol biosynthetic genes and confers resistance to non-azole ergosterol biosynthesis inhibitors.

<p>(A) Transcript levels of six ergosterol biosynthetic genes have been monitored in Δ<i>hapC</i> using RT-qPCR (<i>gpdA</i> was used as reference gene). Strains were grown in AMM for 18h at 37°C, 200 rpm. Results represent the mean of biological triplicates and error bars illustrate the standard deviation. p-values were calculated by Student’s T-test (reference: wt): *, <0.05; ** <0.01. (B) MIC levels of simvastatin and terbinafine, both blocking enzymes upstream Cyp51. (C) Schematic of ergosterol biosynthetic pathway including targets for inhibitors.</p

Azole sensitivity of strains generated in this study.

<p>MIC testing has been carried out using the EUCAST broth microdilution reference method [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005775#ppat.1005775.ref026" target="_blank">26</a>].</p

The 34 <i>mer</i> is essential for activation of <i>cyp51A</i> gene expression and azole resistance.

<p>(A) <i>cyp51A</i> transcript levels in strains lacking the 34 <i>mer</i> (<i>cyp51A</i>^Δ<i>34</i>) and strains carrying TR34 (<i>cyp51A</i>^<i>TR34</i>) have been measured using RT-qPCR (<i>gpdA</i> was used as reference gene). Strains for the expression analysis have been grown in AMM for 18h at 37°C, 200 rpm. Samples have been assessed in biological duplicates. Error bars indicate the standard deviation of respective samples. p-values were calculated by Student’s T-test (reference: <i>cyp51A</i>^<i>REC</i>): *, <0.05; ** <0.01; (B) MIC levels of itraconazole (ITRA), voriconazole (VORI) and posaconazole (POSA); (C) Proposed model highlighting the mechanistic basis of the 34 <i>mer</i> associated azole resistance in the promoter of <i>cyp51A</i>. In susceptible isolates (wt) SrbA homodimers bind to SRE1 and SRE2 thereby activating expression of <i>cyp51A</i>. The CBC competes with SrbA binding to the 34 <i>mer</i>. In resistant isolates, carrying the duplicated 34 <i>mer</i> (TR34), both SREs are duplicated whereas only the CGAAT motif located at the 3’ end of the 34 <i>mer</i> is effectively bound by the CBC. Upregulation of <i>cyp51A</i> in resistant isolates expressing mutated <i>hapE</i> (<i>hapE</i>^<i>P88L</i>) is caused by decreased binding affinity of the mutated complex. Hence, CBC-based competition with SrbA is perturbed enhancing binding of SrbA to the 34 <i>mer</i>.</p

Integration of proteome and metabolic control to show regulation of sulfur and C1 (folate) metabolic fluxes at the protein (enzyme) level

Selected proteins with levels consistently upregulated (red) or downregulated (green) with growth independently of culture conditions are shown. Sulfur, C1 metabolism, methyl cycle, methionine and -adenosylmethionine (SAM) fluxes towards methylation of proteins, rRNAs and tRNAs, and protein biosynthesis are shown here. Metabolic pathways and enzymes are from [42,82, 103-105] and the diagram is drawn with Cell Designer [136] and Adobe Illustrator [137]. Reverse methionine biosynthetic pathways [83] have been omitted for clarity. Metabolite abbreviations: THF, tetrahydrofolate; METTHF, 5,10-methylenetetrahydrofolate; MTHPTGLUT, 5-methyltetrahydropteroyltriglutamate (donor of the terminal methyl group in methionine biosynthesis); GT, glutathione; CYS, cysteine; CT, cystathionine; OAHS, -acetylhomoserine; HCYS, homocysteine; MET, methionine; SAM, -adenosylmethionine; SAH, -adenosylhomocysteine; D-SAM, decarboxylated -adenosylmethionine; MTA, methylthioadenosine. Metabolic steps (genes/enzymes): Met10p, sulfite reductase alpha subunit; Ecm17p, sulfite reductase beta subunit; , folylpolyglutamate synthetase (Met7p not detected; the relevance of polyglutamylation in the C1 metabolism branch was demonstrated at the transcriptional level (see text)); Met13p, methylenetetrahydrofolate reductase isozyme; Met6p, methionine synthase; Mes1p, methionyl-tRNA synthetase; Sam1p, S-adenosylmethionine synthetase isozyme; Sam2p, S-adenosylmethionine synthetase isozyme. Sah1p, S-adenosyl-L-homocysteine hydrolase; Ado1p, adenosine kinase.<p><b>Copyright information:</b></p><p>Taken from "Growth control of the eukaryote cell: a systems biology study in yeast"</p><p>http://jbiol.com/content/6/2/4</p><p>Journal of Biology 2007;6(2):4-4.</p><p>Published online 30 Apr 2007</p><p>PMCID:PMC2373899.</p><p></p

Integration of proteome and metabolic control to show regulation of carbon and nitrogen metabolic fluxes at the protein (enzyme) level

Shown here are the coupling of carbon and nitrogen fluxes at the level of glutamate dehydrogenase (Gdh1p, Gdh2p) and glutamine synthetase (Gln1p), the regulation of arginine biosynthesis at the carbamoyl phosphate synthetase (Cpa1p, Cpa2p) level and amino-acid biosynthesis, and amino-acid sensing by TOR. Selected proteins with levels consistently upregulated (red) with growth independently of culture conditions are shown. Enzymes responsible for the cytosolic 2-oxoglutarate pool: Aco1p and Aco2p, aconitase and putative aconitase isoenzyme; Odc1p and Odc2p, mitochondrial 2-oxoglutarate transporters; Idp2p, NADP-specific isocitrate dehydrogenase. Enzyme subunits coupling the oxidation of succinate to the transfer of electrons to ubiquinone: Sdh1p and Sdh2p, succinate dehydrogenase, flavoprotein, and iron-sulfur protein subunits, respectively. Metabolic diagram from [42, 91, 92] and drawn using Cell Designer [136] and Adobe Illustrator [137].<p><b>Copyright information:</b></p><p>Taken from "Growth control of the eukaryote cell: a systems biology study in yeast"</p><p>http://jbiol.com/content/6/2/4</p><p>Journal of Biology 2007;6(2):4-4.</p><p>Published online 30 Apr 2007</p><p>PMCID:PMC2373899.</p><p></p