Role of the novel protein TmcR in regulating the expression of the cel9-cel48 operon from Myxobacter sp. AL-1.

Northern-blot analysis revealed that cel9 and cel48, which encode family 9 and 48 glycosyl hydrolases, respectively, were expressed as a bicistronic mRNA in the soil bacterium Myxobacter sp. AL-1. The two cistrons of the cel9-cel48 mRNA as well as their encoded products were detected in stationary phase cultures of Myxobacter sp. AL-1, suggesting that a mechanism delayed the transcription of cel9-cel48 until this growth phase. Interestingly, in the same strand and orientation as cel48 a different reading frame was found fully embedded within another ORF encoding a novel DNA-binding protein termed TmcR (Temporal cellulase regulator). Results of Western-blot analysis revealed that although TmcR occurred in growing cells, its concentration decreased during the late stationary growth phase. A possible regulatory role of TmcR during cel9-cel48 expression was studied in E. coli. Results showed that in comparison with E. coli cells expressing cel9-cel48 cloned in pBR322, deletion of tmcR from this plasmid increased not only the cellulase activity but also the amount of Cel9 secreted to the culture medium. Moreover, both, the cellulase activity and Cel9 production decreased in E. coli cells when tmcR was cloned back in the plasmid lacking tmcR. These results suggest that TmcR has the properties required to repress the expression of the cel9-cel48 cluster from Myxobacter sp. AL-1 and suggest the existence of a mechanism involved in regulating the expression of cellulase genes in soil bacteria.