C/N Ratio Drives Soil Actinobacterial Cellobiohydrolase Gene Diversity

Abstract

dCellulose accounts for approximately half of photosynthesis-fixed carbon; however, the ecology of its degradation in soil is still relatively poorly understood. The role of actinobacteria in cellulose degradation has not been extensively investigated despite their abundance in soil and known cellulose degradation capability. Here, the diversity and abundance of the actinobacterial glycoside hydrolase family 48 (cellobiohydrolase) gene in soils from three paired pasture-woodland sites were determined by using terminal restriction fragment length polymorphism (T-RFLP) analysis and clone libraries with gene-specific primers. For comparison, the diversity and abundance of general bacteria and fungi were also assessed. Phylogenetic analysis of the nucleotide sequences of 80 clones revealed significant new diversity of actinobacterial GH48 genes, and analysis of translated protein sequences showed that these enzymes are likely to represent functional cellobiohydrolases. The soil C/N ratio was the primary environmental driver of GH48 community compositions across sites and land uses, demonstrating the importance of substrate quality in their ecology. Furthermore, mid-infrared (MIR) spectrometry-predicted humic organic carbon was distinctly more important to GH48 diversity than to total bacterial and fungal diversity. This suggests a link between the actinobacterial GH48 community and soil organic carbon dynamics and highlights the potential importance of actinobacteria in the terrestrial carbon cycle. C ellulases are responsible for the degradation of cellulose, an insoluble, recalcitrant substrate which comprises approximately half of the biologically fixed CO 2 on earth (1). Cellulases are classified as glycosyl hydrolases (GHs) together with other enzymes that target the glycosidic bonds in oligo-and polysaccharides and are grouped into families that reflect their protein folding structure (2). Metagenomic studies have characterized various cellulose-rich environments, such as the bovine rumen (3-6), rabbit cecum (7), ant fungus gardens (8), compost (9, 10), earthworm casts (11), termite gut (12), and forest soil (13,14). These studies have revealed a rich new GH gene diversity not thus far observed in cultured microorganisms. However, little is known about the role of GH genes in natural environments and the enzymes which they encode.Glycoside hydrolases are a large and complex group of enzymes, with some GH families showing multiple substrate specificities (15). Horizontal gene transfer has also been documented for many GH families (15-21). The presence of multiple substrate specificities within the same GH family has precluded the design of molecular tools for in-depth investigation of their environmental role. Importantly, all 13 functionally characterized bacterial glycoside hydrolase family 48 (GH48) enzymes have been shown to target cellulose, and in most bacteria that carry the GH48 gene, it is present as a single genomic copy (19). Only in insects have the characterized GH48 enzymes been shown not to targe...

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