, 2004) Therefore, the role of

norA is most easily exami

, 2004). Therefore, the role of

norA is most easily examined in A. flavus. We now provide evidence that A. flavus, lacking a functional copy of norA, accumulates a new metabolite, deoxyAFB1, a shunt metabolite that most likely is formed by dehydration of aflatoxicol (AFOH) Omipalisib in the acidic culture medium. A vector for insertional inactivation of norA in A. flavus was constructed by PCR with the oligonucleotide primers P1, 5′-acgactacaagaatagcggtgacat and P2, 5′-tattctagagacgcagactcttggtatgg (GenBank accession #AY510451; 47574–48188) and P3, 5′-tattctagagtactgggccgcggtcagtt and P4, 5′-aatggtacctcgagtccgcgacaactaggctcattttg (48516–49107) to amplify 5′- and 3′-portions of AF13ΔnorA, respectively (Fig. 2a). The resulting 614- and 591-bp PCR fragments were cloned into the SphI/XbaI and XbaI/KpnI sites of pUC18, respectively. An XbaI fragment from the niaD-containing plasmid, pSL82 (Chang et al., 1996), was then inserted into the internal XbaI site of the norA fragments in pUC18 to create the knockout vector. Transformation of A. flavus AF13ΔniaD protoplasts was performed

as described previously using the PEG procedure (Ehrlich et al., 2004) with 10 μg of the XhoI/SphI-linearized plasmid. Confirmation that norA was insertionally inactivated (double crossover event) in the resulting transformants was carried out by PCR using the outer oligonucleotide primers (P1 and P4, Fig. 2b) with DNA from the putative transformants or from pSL82-transformed AF13 as the control. Fungal cultures grown from spores at 30 °C for 3 days on potato dextrose agar (PDA, Difco, Voigt Global Distribution, Depsipeptide Lawrence, KS) were extracted with acetone and chloroform as described previously (Ehrlich et al., 2004). Aliquots of the extract were analyzed by TLC on 250-μm silica gel plates (J.T. Baker, Phillipsburg, NJ) developed with toluene : ethyl acetate : acetic acid (8 : 1 : 1). MycoClean Mycoplasma Removal Kit A prominent blue-fluorescent compound from ΔnorA cultures was partially purified by preparative TLC. The unpurified extract, the TLC-purified metabolite, and authentic standards (AFB1, synthetic aflatoxicol, synthetic deoxyAFB1, OMST, and synthetic HOMST) were analyzed

in the positive ion mode by LC/MS. The materials were dissolved in methanol, injected on a Luna C18 100 × 4.6 mm column (5 μm, 100 Å, Phenomenex) equilibrated in 10% acetonitrile/0.1% formic acid and 90% aqueous formic acid (0.1%), and eluted with a gradient to 100% acetonitrile/0.1% formic acid over 30 min. Metabolites were monitored by both diode array UV-visible spectrophotometry and quadrupole MS (Agilent 6130). Aflatoxicol (AFOH) and deoxyAFB1 were prepared by zinc borohydride reduction of AFB1 (Sigma, St. Louis, MO) (Hsia & Chu, 1977). Aflatoxicol (AFOH) was partially purified from the reaction mix by preparative TLC. Synthetic AFOH was dissolved in 200 μL dimethyl sulfoxide and added to 3-day mycelial cultures of A.

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