The physiological functions of the Cbp2D and Cbp2E proteins are important for insoluble cellulose-dependent growth in Cellvibrio japonicus

Abstract

Microbial deconstruction of plant polysaccharides is important for environmental nutrient cycling, and bacteria proficient at this process have extensive suites of polysaccharide-specific enzymes. In the gram-negative saprophyte Cellvibrio japonicus, genome annotation suggests that 17 genes are predicted to encode Carbohydrate-Active enZymes (CAZymes) with roles in cellulose degradation; however, previous work suggested that only a subset of these genes is essential. Building upon that work, here, we identify the required and minimally sufficient set of enzymes for complete degradation of cellulose using a combination of transcriptomics, gene deletion analysis, heterologous expression studies, and metabolite analysis. We identified six CAZyme-encoding genes required for cellulose deconstruction in C. japonicus, which are cel3B, cel5B, cel6A, lpmo10B, cbp2D, and cbp2E. These genes encode for a β-glucosidase, an endoglucanase, a cellobiohydrolase, a lytic polysaccharide mono-oxygenase, and two carbohydrate-binding proteins, respectively. These CAZyme-encoding genes are essential for growth using insoluble cellulose by C. japonicus and sufficient for using soluble cellulose when heterologously expressed in Escherichia coli. Moreover, during C. japonicus growth using insoluble cellulose, we detected no cellodextrins in the medium, which suggested that cello-oligosaccharide uptake is highly efficient. RNA-seq analysis corroborates these results as we observed several genes significantly upregulated during growth using cellulose that encode TonB-dependent and ABC transporters. Our revised model of cellulose utilization by C. japonicus suggests a greater importance for the Cbp2D and Cbp2E proteins than previously thought and that rapid cellodextrin uptake by C. japonicus is a mechanism to maximize the energetic return on investment for the production and secretion of CAZymes.