Function analysis of 5′-UTR of the cellulosomal xyl-doc cluster in Clostridium papyrosolvens

Anaerobic, mesophilic, and cellulolytic Clostridium papyrosolvens produces an efficient cellulolytic extracellular complex named cellulosome that hydrolyzes plant cell wall polysaccharides into simple sugars. Its genome harbors two long cellulosomal clusters: cip-cel operon encoding major cellulosome components (including scaffolding) and xyl-doc gene cluster encoding hemicellulases. Compared with works on cip-cel operon, there are much fewer studies on xyl-doc mainly due to its rare location in cellulolytic clostridia. Sequence analysis of xyl-doc revealed that it harbors a 5′ untranslated region (5′-UTR) which potentially plays a role in the regulation of downstream gene expression. Here, we analyzed the function of 5′-UTR of xyl-doc cluster in C. papyrosolvens in vivo via transformation technology developed in this study. In this study, we firstly developed an electrotransformation method for C. papyrosolvens DSM 2782 before the analysis of 5′-UTR of xyl-doc cluster. In the optimized condition, a field with an intensity of 7.5–9.0 kV/cm was applied to a cuvette (0.2 cm gap) containing a mixture of plasmid and late cell suspended in exponential phase to form a 5 ms pulse in a sucrose-containing buffer. Afterwards, the putative promoter and the 5′-UTR of xyl-doc cluster were determined by sequence alignment. It is indicated that xyl-doc possesses a long conservative 5′-UTR with a complex secondary structure encompassing at least two perfect stem-loops which are potential candidates for controlling the transcriptional termination. In the last step, we employed an oxygen-independent flavin-based fluorescent protein (FbFP) as a quantitative reporter to analyze promoter activity and 5′-UTR function in vivo. It revealed that 5′-UTR significantly blocked transcription of downstream genes, but corn stover can relieve its suppression. In the present study, our results demonstrated that 5′-UTR of the cellulosomal xyl-doc cluster blocks the transcriptional activity of promoter. However, some substrates, such as corn stover, can relieve the effect of depression of 5′-UTR. Thus, it is speculated that 5′-UTR of xyl-doc was a putative riboswitch to regulate the expression of downstream cellulosomal genes, which is helpful to understand the complex regulation of cellulosome.