Fungal ABC Transporter Deletion and Localization Analysis

Abstract

Fungal cells are highly complex as their metabolism is compartmentalized harboring various types of subcellular organelles that are bordered by one or more membranes. Knowledge about the intracellular localization of transporter proteins is often required for the understanding of their biological function. Among different approaches available, the localization analysis based on the expression of GFP fusions is commonly used as a relatively fast and cost-efficient method that allows visualization of proteins of interest in both live and fixed cells. In addition, inactivation of transporter genes is an important tool to resolve their specific function. Here we provide a detailed protocol for the deletion and localization analysis of ABC transporters in the filamentous fungus Penicillium chrysogenum. It includes construction of expression plasmids, their transformation into fungal strains, cultivation of transformants, microscopy analysis, as well as additional protocols on staining of fungal cells with organelle-specific dyes like Hoechst 33342, MitoTracker DeepRed, and FM4-64.

The amino‐terminal tail of Hxt11 confers membrane stability to the Hxt2 sugar transporter and improves xylose fermentation in the presence of acetic acid

“…The GFP gene in plasmid pDONR‐eGFP‐AT (Kovalchuk et al, 2012) was amplified by PCR with the primers GFP‐F and GFP‐R, and the product was cloned into the XmaI and ClaI restriction sites of pRS313‐P7T7. The resulting plasmid and PCR products of the HXT2 and chimeric HXT11/2 were digested with XbaI and Cfr9I, and ligated with the GFP cassette.…”

A Non-Canonical NRPS Is Involved in the Synthesis of Fungisporin and Related Hydrophobic Cyclic Tetrapeptides in Penicillium chrysogenum

“…Escherichia coli DH5α (F– Φ80 lacZ ΔM15 Δ( lacZYA-argF ) U169 recA 1 endA 1 hsdR 17 (rK–, mK+) phoA supE 44 λ– thi -1 gyrA 96 relA 1) was used as host strain for high frequency transformation and plasmid DNA amplification [21]. All the strains were grown on yeast nitrogen base-glucose-yeast extract (YGG)-medium for protoplasts formation and transformation [22]. Both mutant and host strains of P. chrysogenum were grown on secondary metabolite production medium as described previously [18].…”

“…The deletion plasmid pDes R4-R3p PcHcpA was transformed to the protoplasts of P. chrysogenum DS54555 [23] yielding the Δ hcpA derivative of strain DS54555 The phleomycin resistance gene was used as selection marker for the deletion of the HcpA gene [22], [24].…”

A Branched Biosynthetic Pathway Is Involved in Production of Roquefortine and Related Compounds in Penicillium chrysogenum

“…All the strains were grown on YGG-medium [22] for protoplasts formation and transformation. For analysis, cells were grown on SMP medium (glucose, 5.0 g/l; lactose, 75 g/l; urea, 4.0 g/l; Na 2 SO 4 , 4.0 g/l; CH 3 COONH 4 , 5.0 g/l; K 2 HPO 4 , 2.12 g/l; KH 2 PO 4 , 5.1 g/l) for secondary metabolites production using a shaking incubator at 200 rpm for 168 hours at 25°C.…”

“…The phleomycin resistance gene was used as selection marker for the deletion of roqA, roqT and roqD while acetamidase gene ( amdS ) was used as selection marker for the deletion of roqM, roqO, roqN and roqR using acetamide as the only nitrogen source for selection [22], [25].…”