| Abstrakt: |
ABSTRACTAn operon comprising two genes, CA_P0037and CA_P0036, that encode proteins of unknown function that were previously shown to be highly expressed in acidogenic cells and repressed in solventogenic and alcohologenic cells is located on the pSOL1 megaplasmid of Clostridium acetobutylicumupstream of adhE2. A CA_P0037::int(189/190s) mutant in which an intron was inserted at position 189/190 in the sense strand of CA_P0037was successfully generated by the Targetron technique. The resultant mutant showed significantly different metabolic flux patterns in acidogenic (producing mainly lactate, butyrate, and butanol) and alcohologenic (producing mainly butyrate, acetate, and lactate) chemostat cultures but not in solventogenic or batch cultures. Transcriptomic investigation of the CA_P0037::int(189/190s) mutant showed that inactivation of CA_P0037significantly affected the expression of more than 258 genes under acidogenic conditions. Surprisingly, genes belonging to the Fur regulon, involved in iron transport (CA_C1029-CA_C1032), or coding for the main flavodoxin (CA_C0587) were the most significantly expressed genes under all conditions, whereas fur(coding for the ferric uptake regulator) gene expression remained unchanged. Furthermore, most of the genes of the Rex regulon, such as the adhE2and ldhAgenes, and of the PerR regulon, such as rbr3A-rbr3Band dfx, were overexpressed in the mutant. In addition, the whole CA_P0037-CA_P0036operon was highly expressed under all conditions in the CA_P0037::int(189/190s) mutant, suggesting a self-regulated expression mechanism. Cap0037 was shown to bind to the CA_P0037-CA_P0036operon, soloperon, and adcpromoters, and the binding sites were determined by DNA footprinting. Finally, a putative Cap0037 regulon was generated using a bioinformatic approach.IMPORTANCEClostridium acetobutylicumis well-known for its ability to produce solvents, especially n-butanol. Understanding the regulatory network of C. acetobutylicumwill be crucial for further engineering to obtain a strain capable of producing n-butanol at high yield and selectivity. This study has discovered that the Cap0037 protein is a novel regulator of C. acetobutylicumthat drastically affects metabolism under both acidogenic and alcohologenic fermentation conditions. This is pioneering work for further determining the regulatory mechanism of Cap0037 in C. acetobutylicumand studying the role of proteins homologous to Cap0037 in other members of the phylum Firmicutes. |
