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Insights into the Utility of the Focal Adhesion Scaffolding Proteins in the Anaerobic Fungus Orpinomyces sp. C1A.

ABSTRACT: Focal adhesions (FAs) are large eukaryotic multiprotein complexes that are present in all metazoan cells and function as stable sites of tight adhesion between the extracellular matrix (ECM) and the cell's cytoskeleton. FAs consist of anchor membrane protein (integrins), scaffolding proteins (e.g. α-actinin, talin, paxillin, and vinculin), signaling proteins of the IPP complex (e.g. integrin-linked kinase, α-parvin, and PINCH), and signaling kinases (e.g. focal adhesion kinase (FAK) and Src kinase). While genes encoding complete focal adhesion machineries are present in genomes of all multicellular Metazoa; incomplete machineries were identified in the genomes of multiple non-metazoan unicellular Holozoa, basal fungal lineages, and amoebozoan representatives. Since a complete FA machinery is required for functioning, the putative role, if any, of these incomplete FA machineries is currently unclear. We sought to examine the expression patterns of FA-associated genes in the anaerobic basal fungal isolate Orpinomyces sp. strain C1A under different growth conditions and at different developmental stages. Strain C1A lacks clear homologues of integrin, and the two signaling kinases FAK and Src, but encodes for all scaffolding proteins, and the IPP complex proteins. We developed a protocol for synchronizing growth of C1A cultures, allowing for the collection and mRNA extraction from flagellated spores, encysted germinating spores, active zoosporangia, and late inactive sporangia of strain C1A. We demonstrate that the genes encoding the FA scaffolding proteins α-actinin, talin, paxillin, and vinculin are indeed transcribed under all growth conditions, and at all developmental stages of growth. Further, analysis of the observed transcriptional patterns suggests the putative involvement of these components in alternative non-adhesion-specific functions, such as hyphal tip growth during germination and flagellar assembly during zoosporogenesis. Based on these results, we propose putative alternative functions for such proteins in the anaerobic gut fungi. Our results highlight the presumed diverse functionalities of FA scaffolding proteins in basal fungi.

SUBMITTER: Calkins S

PROVIDER: S-EPMC5042518 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background. The anaerobic gut fungi (phylum Neocallimastigomycota) represent a promising source of novel lignocellulolytic enzymes. Here, we report on the cloning, expression, and characterization of a glycoside hydrolase family 39 (GH39) enzyme (Bgxg1) that is highly transcribed by the anaerobic fungus Orpinomycessp. strain C1A under different growth conditions. This represents the first study of a GH39-family enzyme from the anaerobic fungi. Methods. Using enzyme activity assays, we performed a biochemical characterization of Bgxg1 on a variety of substrates over a wide range of pH and temperature values to identify the optimal enzyme conditions and the specificity of the enzyme. In addition, substrate competition studies and comparative modeling efforts were completed. Results. Contrary to the narrow range of activities (β-xylosidase or α-L-iduronidase) observed in previously characterized GH39 enzymes, Bgxg1 is unique in that it is multifunctional, exhibiting strong β-xylosidase, β-glucosidase, β-galactosidase activities (11.5 ± 1.2, 73.4 ± 7.15, and 54.6 ± 2.26 U/mg, respectively) and a weak xylanase activity (10.8 ± 1.25 U/mg), as compared to previously characterized enzymes. Further, Bgxg1 possesses extremely high affinity (as evident by the lowest K m values), compared to all previously characterized β-glucosidases, β-galactosidases, and xylanases. Physiological characterization revealed that Bgxg1 is active over a wide range of pH (3-8, optimum 6) and temperatures (25-60 °C, optimum 39 °C), and possesses excellent temperature and thermal stability. Substrate competition assays suggest that all observed activities occur at a single active site. Using comparative modeling and bioinformatics approaches, we putatively identified ten amino acid differences between Bgxg1 and previously biochemically characterized GH39 β-xylosidases that we speculate could impact active site architecture, size, charge, and/or polarity. Discussion. Collectively, the unique capabilities and multi-functionality of Bgxg1 render it an excellent candidate for inclusion in enzyme cocktails mediating cellulose and hemicellulose saccharification from lignocellulosic biomass.

Project description:BACKGROUND:Anaerobic fungi reside in the rumen and alimentary tract of herbivores where they play an important role in the digestion of ingested plant biomass. The anaerobic fungal isolate Orpinomyces sp. strain C1A is an efficient biomass degrader, capable of simultaneous saccharification and fermentation of the cellulosic and hemicellulosic fractions in multiple types of lignocellulosic biomass. To understand the mechanistic and regulatory basis of biomass deconstruction in anaerobic fungi, we analyzed the transcriptomic profiles of C1A when grown on four different types of lignocellulosic biomass (alfalfa, energy cane, corn stover, and sorghum) versus a soluble sugar monomer (glucose). RESULTS:A total of 468.2 million reads (70.2 Gb) were generated and assembled into 27,506 distinct transcripts. CAZyme transcripts identified included 385, 246, and 44 transcripts belonging to 44, 13, and 8 different glycoside hydrolases (GH), carbohydrate esterases, and polysaccharide lyases families, respectively. Examination of CAZyme transcriptional patterns indicates that strain C1A constitutively transcribes a high baseline level of CAZyme transcripts on glucose. Although growth on lignocellulosic biomass substrates was associated with a significant increase in transcriptional levels in few GH families, including the highly transcribed GH1 ?-glucosidase, GH6 cellobiohydrolase, and GH9 endoglucanase, the transcriptional levels of the majority of CAZyme families and transcripts were not significantly altered in glucose-grown versus lignocellulosic biomass-grown cultures. Further, strain C1A co-transcribes multiple functionally redundant enzymes for cellulose and hemicellulose saccharification that are mechanistically and structurally distinct. Analysis of fungal dockerin domain-containing transcripts strongly suggests that anaerobic fungal cellulosomes represent distinct catalytic units capable of independently attacking and converting intact plant fibers to sugar monomers. CONCLUSIONS:Collectively, these results demonstrate that strain C1A achieves fast, effective biomass degradation by the simultaneous employment of a wide array of constitutively-transcribed cellulosome-bound and free enzymes with considerable functional overlap. We argue that the utilization of this indiscriminate strategy could be justified by the evolutionary history of anaerobic fungi, as well as their functional role within their natural habitat in the herbivorous gut.

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Insights into the Utility of the Focal Adhesion Scaffolding Proteins in the Anaerobic Fungus Orpinomyces sp. C1A.

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