Background: 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.
Results: 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.
Conclusions: 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.
Keywords: 5′ untranslated region (5′-UTR); Clostridium papyrosolvens; Electrotransformation; Promoter; xyl-doc gene cluster.