Identifying RmdA Protein Interactions in Streptomyces Using a Bacterial Two-Hybrid System

Streptomyces is a genus of the phylum actinobacteria most commonly found as soil bacteria and used as a major source of antibiotics. RmdA and RmdB are phosphodiesterases that break down the ubiquitous second messenger cyclic-di-GMP which determines colony morphology and development of Streptomyces. The objective of this research is to identify whether RmdA will have interactions with itself using the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) System. Each gene was fused into one of two BACTH vectors that encode a different domain of a single protein (T18 and T25) and then cotransformed into the BACTH indicator strain. The transformants were plated on the indicator plates, LB-X-Gal and MacConkey-maltose, and incubated to qualitatively show their possible interactions. If the proteins interact, they will bring the separated T18 and T25 domains in close proximity to produce beta-galactosidase on LB-X-Gal or ferment maltose on MacConkey-maltose which will be seen as blue or red colonies respectively. The plasmids containing T18-RmdA and T25-RmdA were successfully created and cotransformed to determine whether RmdA interacts with itself. When testing RmdA fused to T18 with an open C-terminus (T18-RmdA) and RmdA fused to T25 with an open C-terminus (T25-RmdA), no interactions were detected. This could be due to the T18 or T25 fragments blocking or preventing the function of the N-terminus of RmdA which could be required for protein interactions. Further testing is being conducted to determine if the N-terminus is needed to interact