Abstract
Understanding the strategies used by bacteria to degrade polysaccharides constitutes an invaluable tool for biotechnological applications. Bacteria are major mediators of polysaccharide degradation in nature; however, the complex mechanisms used to detect, degrade, and consume these substrates are not well-understood, especially for recalcitrant polysaccharides such as chitin. It has been previously shown that the model bacterial saprophyte Cellvibrio japonicus is able to catabolize chitin, but little is known about the enzymatic machinery underlying this capability. Previous analyses of the C. japonicus genome and proteome indicated the presence of four glycoside hydrolase family 18 (GH18) enzymes, and studies of the proteome indicated that all are involved in chitin utilization. Using a combination of in vitro and in vivo approaches, we have studied the roles of these four chitinases in chitin bioconversion. Genetic analyses showed that only the chi18D gene product is essential for the degradation of chitin substrates. Biochemical characterization of the four enzymes showed functional differences and synergistic effects during chitin degradation, indicating non-redundant roles in the cell. Transcriptomic studies revealed complex regulation of the chitin degradation machinery of C. japonicus and confirmed the importance of CjChi18D and CjLPMO10A, a previously characterized chitin-active enzyme. With this systems biology approach, we deciphered the physiological relevance of the glycoside hydrolase family 18 enzymes for chitin degradation in C. japonicus, and the combination of in vitro and in vivo approaches provided a comprehensive understanding of the initial stages of chitin degradation by this bacterium.
Highlights
Understanding the strategies used by bacteria to degrade polysaccharides constitutes an invaluable tool for biotechnological applications
Previous analyses of the C. japonicus genome and proteome indicated the presence of four glycoside hydrolase family 18 (GH18) enzymes, and studies of the proteome indicated that all are involved in chitin utilization
Transcriptomic studies revealed complex regulation of the chitin degradation machinery of C. japonicus and confirmed the importance of CjChi18D and CjLPMO10A, a previously characterized chitin-active enzyme. With this systems biology approach, we deciphered the physiological relevance of the glycoside hydrolase family 18 enzymes for chitin degradation in C. japonicus, and the combination of in vitro and in vivo approaches provided a comprehensive understanding of the initial stages of chitin degradation by this bacterium
Summary
According to previous sequence analysis [21], C. japonicus possesses four GH18 chitinases (CjChi18A, CjChi18B, CjChi18C, and CjChi18D). When using crab shells as the only source of carbon, the ⌬chi18D mutant was unable to grow, whereas the single mutants of ⌬chi18A, ⌬chi18B, and ⌬chi18C displayed growth similar to wildtype (Fig. 3C and Table S1B). The ⌬chi18A ⌬chi18B ⌬chi18C triple mutant recapitulated the growth defect observed in the ⌬chi18B ⌬chi18C double mutant in terms of the growth rate, lag phase, and the maximum OD (Fig. 3B and Table S1A) These results suggest that the chi18B and chi18C gene products have non-redundant. The comparison of gene expression during early stationary phase yielded similar results (Fig. S5A and Table S3B) This analysis revealed the up-regulation of 47 CAZyme genes, of which seven are implicated in chitin degradation (lpmo10A, chi18D, chi18C, chi18B, nag9A, hex20A, and chi19A). When comparing the transcriptomes of the exponential and the stationary phase during growth on chitin, none of the significant changes in expression concerned CAZyme genes (Fig. S5B)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
