Two Components of a velvet-Like Complex Control Hyphal Morphogenesis, Conidiophore Development, and Penicillin Biosynthesis in Penicillium chrysogenum

Microarray analysis of selected Pcku70- and PcvelA-dependent genes. Heat maps of developmental genes (A) and genes of secondary and amino sugar metabolisms (B) are shown. Red, upregulation in the ΔPcvelA mutant compared to the ΔPcku70 strain; green, downregulation in the ΔPcvelA mutant compared to the ΔPcku70 strain. The color of each square represents the log₂-fold change in expression at the given time point.

Penicillin biosynthesis of knockout mutants. Transcript levels of pcbC and penDE genes in the ΔPclaeA (A) and ΔPcvelA (B) deletion strains and their corresponding complemented strains, which are indicated by “T.” Quantification of autoradiograms as shown was done with Scion Image. The gpdA transcript level served as a reference for all transcripts from individual strains, and the level in the ΔPcku70 strain was set to 100%. (C) HPLC analysis for the quantification of the penicillin V titer in recipient and knockout strains after 72 h of growth in submerged-liquid shaking cultures. Mean values and standard deviations are derived from three independent experiments. (D) Penicillin bioassays with S. aureus as a Gram-positive indicator bacterium.

Morphogenesis of the ΔPcvelA and ΔPclaeA strains as well as the corresponding recipients and complemented strains. (A) Quantification of light- and dark-dependent conidial formation in strains as indicated. All strains were grown for 168 h under constant light or dark conditions. The experiment was performed in triplicate; bars show standard deviations. (B) Scanning electron microscopy of conidiophores from the wild-type P2niaD18 and the ΔPclaeA mutant strains. While P2niaD18 produces long chains of conidia (indicated by arrows), the ΔPclaeA strain consistently forms only a single conidium from a terminal vesicle of each phialide (indicated by arrows). Scale bars are valid for either both top or three bottom figures. (C) PcvelA-dependent hyphal morphology after 72 h of cultivation in surface liquid culture. White arrows indicate dichotomous hyphal branching sites. (D) Calcofluor white staining of hyphae from strains as indicated. Hyphae were stained after 48, 72, and 96 h of growth in surface-liquid cultures. (E) PcvelA-dependent pellet formation after 72, 120, and 168 h in submerged-liquid shaking cultures. (F) PclaeA-dependent hyphal morphology after 72 h of cultivation in surface-liquid culture. White arrows indicate dichotomous hyphal branching sites.

Bimolecular fluorescence complementation studies. (A) Control to confirm the nuclear localization of two interacting velvet-like complex components by DAPI staining and demonstration that a strain producing only the two split EYFPs lacks any fluorescence. (B) Protein-protein interactions of components of the velvet-like protein complex in P. chrysogenum as indicated. The fusions of each gene pair with fragments encoding the N or C terminus of EYFP are given above each image set. Micrographs illustrate DIC microscopy, DAPI, or EYFP fluorescence and the merged image, as indicated.