Genes of sterigmatocystin biosynthesis pathway form a 60 kb gene cluster (stc cluster) on
Figure 2: Regulation of sterigmatocystin production in A. nidulans (adopted from Fox and Howlett, 2008).
The schematic figure explains the regulation process of sterigmatocystin production through activation of many regulators such as MpkB, velvet family proteins (VelB, VeA), LaeA and master regulator AflR.
Histone deacetylase HdaA represses LaeA functions and inghibits sterigmatocystin production. PkaA:
Protein kinase A, a member of the PkaA signal transduction pathway that upon activation inhibits asexual sporulation. Arrows represent gene activation, while blocked arrows represent repression. At the bottom the schematic representation of the sterigmatocystin biosynthesis stc gene cluster is shown.
The expression of aflR is regulated during the early stationary phase of the life cycle and depends on LaeA and VeA functions (Yu et al., 1996). AflR expression and thus the sterigmatocystin biosynthesis is regulated at chromatin level by the methyltransferase LaeA (Figure 2) (Bok and Keller, 2004). LaeA, is a global regulator of secondary metabolism, which contains a classical NLS region located at the N-terminus of the protein that results in its nuclear localization. On the other hand, LaeA is part of the velvet complex that control development and thereby LaeA plays role in coupling developmental processes and secondary metabolite production (Sarikaya Bayram et al., 2010). The A. nidulans putative mitogen-activated protein kinase, encoded by mpkB, is necessary for normal expression of laeA to regulate cluster genes for sterigmatocystin production (Atoui et al., 2008) (Figure 2).
Literature:
Atoui A, Bao D, Kaur N, Grayburn WS. and Calvo AM (2008) Aspergillus nidulans natural product
Bayram O, Braus GH, Fischer R, Rodriguez-Romero J (2010) Spotlight on Aspergillus nidulans photosensory systems. Fungal Genet. Biol. 47(11): 900–908.
Bok JW and Keller NP (2004). LaeA, a regulator of secondary metabolism in Aspergillus spp.
Eukaryot. Cell. 3: 527–535.
Brown DW, Yu JH, Kelkar HS, Fernandes M, Nesbitt TC, Keller NP, Adams TH, Leonard TJ (1996) Twenty-five coregulated transcripts define a sterigmatocystin gene cluster in Aspergillus nidulans. Proc Natl Acad Sci U S A. 93(4): 1418-1422.
Fox EM and Howlett BJ (2008). Secondary metabolism: regulation and role in fungal biology.
Current Opinion in Microbiology, 11(6): 481–487.
Kim H, Han K, Kim K, Han D, Jahng K & Chae K (2002) The veA gene activates sexual development in Aspergillus nidulans. Fungal Genet Biol. 37(1): 72-80.
Sarikaya Bayram O, Bayram O, Valerius O, Park HS, Irniger S, Gerke J, Ni M, Han KH, Yu JH, Braus GH (2010) LaeA control of velvet family regulatory proteins for light-dependent
development and fungal cell-type specificity. PLoS Genet. 6(12):e1001226.
Yu J H, Butchko R A, Fernandes M, Keller N P, Leonard T J, Adams T H (1996). Conservation of structure and function of the aflatoxin regulatory gene aflR from Aspergillus nidulans and A. flavus. Curr Genet. May; 29(6):549–555.
II. Overview of the course
1. Inoculation of A. nidulans strain HZS.450 (veA+) and HZS.145 (veA1)
2. Comparison of growth properties of the veA+ and veA1 strains. Extraction of sterigmatocystin and visualisation of sterigmatocystine content in the samples by using thin layer chromatograph.
III. Practice 16 – from week 1 to week 2
Inoculation of a veA+ and a veA1 strain for the purpose of detection of sterigmatocystin production.
Used A. nidulans strains: veA1 mutant HZS.145: veA1; veA+ control HZS.450: riboB2.
Compounds for the preparation of 100 ml minimal medium with nitrate nitrogen-source and glucose carbon-source (glucose, sodium nitrate, 50 salt solution stock, 100 vitamin solution stock, agar); sterile Petri dish (4 pieces); 3 days old cultures of HZS.145 and HZS.450; sterile tooth ticks; glass container; pipette set; sterile tips for pipettes; sterile eppendorf tubes; sterile 0.01%
Tween-80 solution; alufolie, sterile 15 ml Falcon tubes (4 pieces); 1 piece of 10 cm long glass test tube with aluminium cap.
Prepare 100 ml liquid minimal medium with nitrate nitrogen-source (5 mM) and glucose carbon-source (1 %) supplemented with vitamins (1). Separate 90 ml medium in an infusion glass bottle supplemented with agar (2.5%) and 10 ml medium in a without agar. After sterilization by using high pressure cooker, supplement the media with vitamins and prepare 4 plates from the solid
1
stweek
Goal
Required materials
Tasks
Extraction of sterigmatocystine and comparative analysis of produced sterigmatocystine in veA+ and a veA1 strains grown under different light conditions by using thin layer chromatography (TLC).
10 ml chloroform; Kieselgel 60 TLC silica gel; pipette set; tips for pipettes; 1 % AlCl3 dissolved in methanol; 40 ml 4:4:1 TEF (toluol:ethylacetate:formic acid) solution; glass chamber for developing the TLC plates; hair dryer machine, 15x15 cm sized glass tray; UV lamp; pincers; glass beads (with 0.2-0.5 mm diameter)
1. Compare the conidiospore productivity of HZS.145 and HZS.450 incubated in dark and light!
Describe your observations in a few sentences and discuss your result with those results that were expected on the basis of literature.
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Tasks
2
ndweek
Goal
Required materials
2. Place 1 ml chloroform in each 15 ml Falcon tube containing the 6 days old cultures and add a few glass beads to the samples. Vortex the samples for 5 minutes. Collect the organic phase into an eppendorf tube by using a pipette. Place 45 µl sterigmatocystin containing chloroform drops of the samples side by side and add a drop from sterigmatocystin standard. Develop the chromatogram in 5:4:1 TEF solution in a glass chamber with covered top. When the solvent reaches the top part of the silica gel plate, take out the plate and let it dry under a hood. Pour out the 1% AlCl3 solution into a glass tray and submerge the plate in it for a moment. Take out the plate and dry it immediately by using a hair dryer machine. Place the silica gel plate under UV light and document the chromatogram.
Please, answer the following questions!
Which kind of color is characteristics for the sterigmatocystin compound developed according to the above executed protocol?
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Do you detect difference in the sterigmatocystine content of HZS.450 and HZS.145 incubated in dark and light?
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Discuss your results with those results that were expected on the basis of literature!
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