Molecular analyses of β-glucosidase diversity and function in soil

Abstract

The incongruence of genetic potential and apparent functions associated with enzyme production has attracted research attempts to elucidate the environmental influences on the molecular underpinnings of soil biogeochemistry. β-glucosidase is a phylogenetically conserved enzyme that plays an important role in carbon cycling; and its involvement in the final stages of cellulose degradation suggests an important role for soil prokaryotes in moderating enzymatic diversity and function in environmentally dynamic niches. Therefore, detailed assessment of the molecular diversity of this enzyme in soils is critical for refining models of terrestrial carbon fluxes. Here, we tested the hypothesis that the availability of specific carbon substrates for β-glucosidase constraint the diversity of phylogenetic groups that are enzymatically active in soils. To test this hypothesis, we constructed microcosms consisting of vegetation covered and bare soils amended either with cellobiose or glucose. We then proceeded to directly assess the responses of genomic and proteomic contexts of β-glucosidase diversity over an incubation period. We monitored the relative population densities of bacteria capable of degrading cellulose, and we successfully designed and used a set of degenerate primers for real-time PCR to quantify genetic determinants of β-glucosidase in composite bacterial DNA extracts. We also developed a complementary proteomic strategy for electrophoretically resolving β-glucosidase activities in-gel. A pure culture of Pseudomonas putida capable of degrading cellobiose that we isolated from the natural soil was used as a positive control throughout the culture-independent experiments. We demonstrated that this organism contains three distinct proteins of molecular sizes 120, 300, and 669 kDa exhibiting β-glucosidase activity, and we detected proteins in these size ranges in direct protein extracts from soil. Our results also show that there is a narrow range of bacteria capable of processing cellobiose in soil, but there is a broader versatility of enzyme action in the dominant organisms advantageously selected by amendment of soil with specific carbon sources. This study contributes to the strategies for molecular-level understanding of soil enzyme diversity and function in composite systems.