In-Frame Deletions Allow Functional Characterization of Complex Cellulose Degradation Phenotypes in Cellvibrio japonicus
ABSTRACTThe depolymerization of the recalcitrant polysaccharides found in lignocellulose has become an area of intense interest due to the role of this process in global carbon cycling, human gut microbiome nutritional contributions, and bioenergy production. However, underdeveloped genetic tools have hampered study of bacterial lignocellulose degradation, especially outside model organisms. In this report, we describe an in-frame deletion strategy for the Gram-negative lignocellulose-degrading bacteriumCellvibrio japonicus. This method leverages optimized growth conditions for conjugation andsacBcounterselection for the generation of markerless in-frame deletions. This method produces mutants in as few as 8 days and allows for the ability to make multiple gene deletions per strain. It is also possible to remove large sections of the genome, as shown in this report with the deletion of the nine-gene (9.4-kb)gspoperon inC. japonicus.We applied this system to study the complex phenotypes of cellulose degradation inC. japonicus. Our data indicated that a Δcel5BΔcel6Adouble mutant is crippled for cellulose utilization, more so than by either single mutation alone. Additionally, we deleted individual genes in the two-genecbp2EDoperon and showed that both genes contribute to cellulose degradation inC. japonicus. Overall, these described techniques substantially enhance the utility ofC. japonicusas a model system to study lignocellulose degradation.