Deletion of <i>pex3</i> in <i>P. chrysogenum</i> results in cells completely devoid of peroxisomes.

<p><i>P. chrysogenum</i> Δ<i>pex3</i> cells, producing GFP.SKL were grown for 40 h in PPM medium and analyzed by fluorescence (<b>A</b>) and electron (<b>B</b>) microscopy. Scale bars represent 5 µm in A and 1 µm in B; M-mitochondrion, N – nucleus.</p

Pex16 is not essential for peroxisome biogenesis.

<p>Fluorescence microscopy analysis of <i>P. chrysogenum</i> Δ<i>pex16</i> GFP.SKL (<b>A</b>) and Δ<i>pex11</i> Δ<i>pex11B</i> Δ<i>pex11C</i> Δ<i>pex16</i> GFP.SKL (<b>B</b>) cells. Cells were grown for 40 h in PPM. For corresponding WT see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035490#pone-0035490-g003" target="_blank">Fig. 3A</a>. Scale bars represent 5 µm. <b>C</b>. Cells devoid of <i>pex16</i> are sporulation deficient. Colonies of WT and both Δ<i>pex16</i> GFP.SKL and Δ<i>pex11</i> Δ<i>pex11B</i> Δ<i>pex11C</i> Δ<i>pex16</i> GFP.SKL strains were grown for 7 days on sporulation inducing R-agar plates. <b>D</b>. Δ<i>pex16</i> cells are characterized by decreased levels of Pex11. Western blots of WT and Δ<i>pex16</i> GFP.SKL cell extracts were prepared and decorated with α-Pex11 antibodies; translation elongation factor 1- (eEF1A) was used as a loading control. <b>E</b>. <i>P. chrysogenum</i> cells lacking <i>pex16</i> are able to grow on oleic acid although at decreased rates. WT, Δ<i>pex16</i> GFP.SKL and Δ<i>pex11</i> Δ<i>pex11B</i> Δ<i>pex11C</i> Δ<i>pex16</i> GFP.SKL strains were grown for 10 days on mineral medium containing 0.5% glucose or 0.1% oleic acid as a sole carbon source. Electron micrographs of Δ<i>pex16</i> GFP.SKL (<b>F</b>) and Δ<i>pex11</i> Δ<i>pex11B</i> Δ<i>pex11C</i> Δ<i>pex16</i> GFP.SKL (<b>G</b>) cells grown for 40 h in PPM; P – peroxisome; M – mitochondrion; V – vacuole; arrows indicate protein dense inclusions. Scale bars represent 1 µm. Electron micrographs representing α-IAT immunolabelling of sections of Δ<i>pex16</i> GFP.SKL (<b>H</b>) and Δ<i>pex11</i> Δ<i>pex11B</i> Δ<i>pex11C</i> Δ<i>pex16</i> GFP.SKL (<b>I</b>) cells. Scale bars represent 1 µm.</p

<i>P. chrysogenum</i> cells devoid of all Pex11 family members still contain peroxisomes.

<p>Double and triple deletion mutants of <i>pex11</i> family genes were prepared and analyzed by CLSM after growth for 40 h in PPM: WT (<b>A</b>), <i>Δpex11</i> (<b>B</b>), <i>Δpex11</i> Δ<i>pex11B</i> GFP.SKL (<b>C</b>), Δ<i>pex11</i> Δ<i>pex11C</i> GFP.SKL (<b>D</b>), Δ<i>pex11B</i> Δ<i>pex11C</i> GFP.SKL (<b>E</b>), Δ<i>pex11</i> Δ<i>pex11B</i> Δ<i>pex11C</i> GFP.SKL (<b>F</b>). Scale bars represent 5 µm.</p

The effect of overproduction of Pex11 family members on peroxisome proliferation.

<p>Representative CLSM images of <i>P. chrysogenum</i> GFP.SKL cells: WT (<b>A</b>); overproducing Pex11 (<b>B</b>), Pex11B (<b>C</b>) or Pex11C (<b>D</b>). Cells were grown for 40 h in PPM. Scale bars represent 5 µm. <b>E</b>. Western blot showing overproduction of Pex11 (top), Pex11B, (center) or Pex11C (bottom). Crude extracts of DS17690 and strains overproducing Pex11 proteins were used for SDS-PAGE and western blotting and probed with specific antisera. Equal amounts of protein were loaded per lane. <b>F</b>. Electron micrograph of <i>P. chrysogenum</i> cells overproducing Pex11B. P-peroxisome; M-mitochondrion. Scale bar represents 1 µm.</p

Impact of deletion of <i>pex11</i> family genes, <i>pex16</i> or <i>pex3</i> on penicillin production.

<p><b>A.</b> Analysis of the production of antibacterial compounds by selected strains using plate bioassays with <i>M. luteus</i> as an indicator strain. Clarified culture supernatants were diluted 3200 times before analysis. During each experiment a corresponding WT supernatant at the same dilution was tested on the same plate as the supernatants of the analyzed mutants. <b>B and C.</b> Western blot analysis of the levels of penicillin biosynthetic enzymes IPNS and IAT in strains with manipulated levels of Pex11 family proteins (<b>B</b>), Pex3 or Pex16 (<b>C</b>). eEF1A was used as a loading control.</p

Subcellular localization of Pex11 family members.

<p><i>P. chrysogenum</i> cells producing C-terminal mGFP fusions of Pex11 (<b>A</b>), Pex11C (<b>B</b>) or Pex11B (<b>C</b>) with DsRed.SKL as peroxisome marker were grown for 40 h in PPM and analyzed by CLSM. <b>D</b>. CLSM analysis of <i>P. chrysogenum</i> hyphae producing Pex11B.mGFP and Sec63.mCherry as marker of the ER, grown for 40 h in PPM. Scale bars represent 5 µm. Arrowheads (in <b>C</b>) indicate the sites of overlap between Pex11B.mGFP and Sec63.mCherry fluorescence.</p

<i>P. chrysogenum</i> Pex16 is a peroxisomal membrane protein.

<p><i>P. chrysogenum</i> cells producing Pex16.mGFP and either DsRed.SKL (<b>A</b>) or Sec63.mCherry (<b>B</b>) were grown for 40 h in PPM and analyzed by fluorescence microscopy. Scale bars represent 5 µm.</p