H-092. Fusobacterium mortiferum MalR, a Putative Transcriptional Regulator, Is a DNA-Binding Protein

P. K. Boyd, C. L. Bouma;
West Texas A&M Univ., Canyon, TX.

Background: Fusobacterium mortiferum is a Gram-negative, anaerobic bacillus that resides in the human intestinal tract. This organism is unique among fusobacteria for its ability to metabolize numerous carbohydrates. The mal operon in F. mortiferum encodes components of a phosphotransferase system (PTS) for maltose and alpha-glucosides. One gene, malR, encodes a putative transcriptional regulator. We are investigating the role of MalR in regulating mal transcription. As there currently are no genetic methods for constructing gene fusions or deletions in fusobacteria, we used gel-shift assays to determine if MalR binds to mal promoter regions. Methods: Gel-shift assays were used to determine whether MalR binds to two promoters in the mal operon. MalR was amplified with PCR and cloned into pET24, which added a C-terminal 6x-His tag. MalR-6xHis was expressed in E. coli and purified by metal chelate affinity chromatography. A single protein of 13 kDa was observed on SDS-PAGE and protein blotting confirmed presence of the His tag. Two possible regulatory sites in the mal operon were amplified with PCR: a 143-bp region upstream of malR and a 248-bp region upstream of malB. The targets were purified and labeled with digoxygenin. Gel-shift assays were conducted on 6% PAGE with purified MalR-6xHis and digoxygenin-labeled targets. The gels were visualized with alkaline phosphatase-coupled anti-digoxygenin antibody and CSPD chemiluminescent substrate. Results: Purified MalR-6xHis (20 ng) retarded the mobility of a PCR product amplified from the upstream region of malR. 80 ng of MalR-6xHis completely bound 15 fmol of this target. In contrast, we observed no change in mobility of the malB target when incubated with MalR-6xHis. Conclusion: F. mortiferum MalR is a DNA-binding protein which binds to the malR promoter. This evidence supports a role for MalR in regulating transcription of mal genes. Future studies will be concerned with further defining the binding site and demonstrating whether F. mortiferum MalR is a repressor or activator of transcription.