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, 703, 134-144

Molecular and Bioinformatics Analyses Reveal Two Differentially Expressed Intracellular GH1 β-Glucosidases From the Rare Alkalophilic Fungus Stachybotrys Microspora

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Molecular and Bioinformatics Analyses Reveal Two Differentially Expressed Intracellular GH1 β-Glucosidases From the Rare Alkalophilic Fungus Stachybotrys Microspora

Salma Abdeljalil et al. Gene.

Abstract

The present study reports the isolation and analysis of two novel GH1 β-glucosidases from the alkalophilic fungus Stachybotrys microspora, using PCR and Nested-PCR. Three major gene fragments were obtained by PCR: the first two are very similar and constitute a novel gene, which was named Smbgl1A, and the third PCR fragment is part of a different gene, named Smbgl1B. The truncated gene sequences were completely filled using the recent partial whole genome sequencing data of S. microspora (data not yet published). Moreover, we investigated the relative effects of glucose in comparison to cellulose rather than evaluate their absolute effects. In fact, RT-PCR analysis showed that while Smbgl1A was expressed when the fungus was grown in the presence of cellulose but not when grown with glucose, Smbgl1B was equally expressed under both conditions. The putative catalytic residues and the conserved glycone binding sites were identified. Zymogram analysis showed the intracellular production of β-glucosidases in S. microspora. The predicted secondary structure exhibited a classical (β/α)8 barrel fold, showing that both SmBGL1A and SmBGL1B belong to the GH1 family. Phylogenetic studies showed that SmBGL1A and SmBGL1B belong to the same branch as β-glucosidases from Stachybotrys chlorohalonata and Stachybotrys chartarum. However, SmBGL1A and SmBGL1B form two distinct clades.

Keywords: Differential expression; Family 1 β-glucosidases; Secondary structure; Stachybotrys microspora.

Figures

Fig. 1
Fig. 1
(a) Position of Smbgl1A and Smbgl1B genes with consensus primers according to the bgl2 gene from T. reesei. (b) Nested-PCR analysis of Family 1 β-glucosidase genes. Lane M: 1 kb DNA Ladder; Lane 1: fragment F1–1 amplified with primers P12 and P14; Lane 2: fragment F1–2 amplified with primers P12 and P16; Lane 3: fragment F1–3 amplified with primers P15 and P17.
Fig. 2
Fig. 2
(a) RT-PCR analysis of Family 1 β-glucosidase mRNAs using 25, 35, and 45 cycles, with primers P12 and P16. (b) RT-PCR analysis of Family 1 β-glucosidase mRNAs using 40 cycles and primers P12 and P16 for Smbgl1A and P12 and P14 for Smbgl1B. mRNAs were extracted from an A19 culture grown in the presence of glucose or cellulose. The actin transcript was used as an internal control. RT-PCR products were analyzed by electrophoresis on 1% agarose gel.
Fig. 3
Fig. 3
(a) ClustalW alignment of SmBGL1A (GenBank acc. no.: MH036528) and SmBGL1B (MH036529) from Stachybotrys microspora with known fungal β-glucosidase amino acid sequences: Stachybotrys chlorohalonata (KFA66942.1 and KFA68795.1), S. chartarum (KFA49121.1 and KEY71876.1), Aspergillus calidoustus (CEL09739.1), and Humicola grisea (BAA74958.1). The highly conserved catalytic residues are boxed. The residues involved in glycone binding sites are indicated by blue triangles. (b) Zymogram analysis of intracellular β-glucosidases under non-denaturing conditions. G: glucose-containing media; C: cellulose-containing media. The image on the left is a blue Coomassie-stained gel ran under the same conditions. M: Protein Molecular Weight Marker. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
Structural modeling of SmBGL1A (a) and SmBGL1B (b) using the templates 5jbo.1A and 4mdo.1A, respectively, generated by the SwissModel automated mode and 3D-visualization with Swiss-Pdb Viewer 4.0. The two catalytic glutamate residues are indicated in the two constructed models.
Fig. 5
Fig. 5
Phylogenetic tree constructed with the phylogeny.fr platform, using PhyML for tree building based on the amino acid sequences of fungal β-glucosidases. Branch-support values calculated by bootstrapping procedure are indicated in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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