Sedimentation analysis of <i>S. pombe</i> strain 1252 (<i>agn1</i>Δ), complemented with <i>AGN1</i> from <i>P. brasiliensis</i>.

<p>The values presented are the mean ± SD of four individual experiments.</p

Oligonucleotides used in the amplification of PCR products for the complementation of <i>S. pombe.</i>

1<p>Annealing temperature 53°C.</p>2<p>Annealing temperature for overlap extension reactions 55°C (AB, CD and AD).</p

Complementation of <i>S. pombe agn1</i>Δ with the <i>P. brasiliensis AGN1</i> gene.

<p><i>S. pombe ags1</i>Δ was complemented with pHV3, which contains the complete <i>P. brasiliensis AGN1</i> gene, including its original signal peptide coding region (<i>S. pombe</i> strain HLVSP3) (D1, D2) or pHV4, which includes a chimeric <i>P. brasiliensis AGN1</i>, whose signal peptide coding region was substituted by the <i>S. pombe agn1</i> signal peptide coding region (<i>S. pombe</i> strain HLVSP4) (C1, C3). In both cases, the plasmids restored the wild type phenotype. As positive control, plasmid pREP3X-<i>agn1⁺</i>, which includes the complete ORF from the <i>S. pombe agn1⁺</i> gene, was transformed into <i>S. pombe ags1</i>Δ (HLVSP5) (B1 and B2). Negative control consisted of <i>S. pombe ags1</i>Δ transformed with the empty vector pREP3X (HLVSP6) (A1, A2). White arrows point to the defect in separation at the tip of the daughter cells. Left panel show cells stained with calcofluor white (A1, B1, C1, and D1). Bar 20 µm.</p

SDS-PAGE, and Western analysis of <i>P.</i><i>brasiliensis</i> Agn1p.

<p>Ni-NTA-purified Agn1p from cell lysates of <i>E. coli</i> transformed with of pQE-30Xa::<i>AGN1</i> (Agn1p), and with the empty pQE-30Xa expression vector as negative control (NC) were separated by SDS-PAGE and stained with coomasie blue (A). The Ni-NTA-purified lysates were blotted on a nitrocellulose membrane and the His-tagged <i>P. brasiliensis</i> Agn1p (Agn1p) visualized using an anti RGS-His antibody (B). E stands for eluate, and NB for unbound material. MW: molecular weight marker. 6HP: 6xHis Ladder. Black arrow signals Agn1p position in both panels.</p

Sequences used for alignments and phylogenetic tree construction.

*<p>Obtained in this work.</p><p>∼From <i>Paracoccidioides brasiliensis</i> sequence data bank: <a href="http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html" target="_blank">http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html</a>.</p

<i>P.</i><i>brasiliensis</i> Agn1p is a specific endo α-1,3-glucanase.

<p>(<b>A</b>) Inhibition profile of exo-glucoamylase from <i>A. niger</i> (gray) and endo-α-1,3-glucanase from <i>P. brasiliensis</i> (black). Note that none of the indicated inhibitors reduced Agn1p-his activity significantly, even at a high concentration of 250 µM. (<b>B</b>) Agn1p substrate specificity. Purified Agn1p-his was incubated with the indicated substrates at 1 mg/ml. Reactions were carried out at optimal conditions by triplicate.</p

Thin Layer Chromatography (TLC).

<p><i>P. brasiliensis</i> Agn1p was incubated for 1 h with CMGS. Lanes: 1, glucose (G1); 2, maltose (G2); 3, maltotriose (G3); 4, Agn1p incubated with GMGS; 5, CMGS.</p

Expression analysis of <i>P. brasiliensis AGN1</i> and <i>AGS1</i>, under horse serum supplementation.

<p>Transcriptional levels were measured by qRT-PCR. Growing <i>P. brasiliensis</i> yeast phase supplemented with horse serum (HS), induces a statistically significant increase in the relative expression of <i>AGN1</i> (A) <i>and AGS1</i> (B), when compared to a control grown without HS. Yeast H.S. (-) (cultured without horse serum), Yeast H.S. (+) (cultured with horse serum). Error bars represent the standard deviation. (*) Turkey-Kramer test between Yeast H.S.(-) and Yeast H.S.(+); P-value <0.05. Experiments were done by triplicate.</p