Exploring the Regulation of a Bacillus pumilus Bioplastic Degrading Protein

Biodegradable plastics are being adopted to reduce the environmental impacts associated with petroleum-based plastics, including long residence times, and reliance on fossil fuels as a feedstock. Microbial degradation of bioplastics is still poorly understood, which leads to inconsistent degradation outcomes and challenges for implementation. Bacillus pumilus B12 degrades the biodegradable bioplastic polylactic acid (PLA) via the protease AprE, but the regulatory mechanisms controlling aprE expression in B. pumilus B12 are largely unknown. To explore this question, we established developed a conjugation- based method to introduce DNA into B. pumilus B12 and diverse Bacillus species that is applicable to environmental and industrially relevant species.

We are leveraging this conjugation technique to begin exploring aprE regulation in B. pumilus B12. Despite belonging to the same genus there is a considerable difference in the aprE upstream regulatory region sequence of B. pumilus B12 and B. subtilis. Thus, we wanted to begin defining subregions of the upstream regulatory region that influence expression, especially transcription factor binding sites. We generated a series of transcriptional reporters to GFP with varying lengths of the upstream regulatory region. These reporters provide indications of repressor and activator binding sites, including for Spo0A. Additionally, we used site directed mutagenesis (SDM) to mutate the bases corresponding to putative transcription factor DNA binding motifs. We observe that SinR acts as a repressor of aprE but ScoC has no effect.t

We then show that expression of aprE is repressed by the addition of inosine, adenine and adenosine, which supports our previous work. In other Bacillus species, AprE is a highly regulated secreted protein expressed during stationary phase, but the mechanism for this nucleotide- mediated repression remains unknown, as no one transcription factor abrogated the repressive effect of inosine addition. We hypothesize that purine addition disrupts the activity of a pleiotropic regulator, which is reflected in the repression of additional genes. Overall, we show that aprE expression is controlled over 500 bases upstream of the predicted open reading frame and that there may be more regulatory elements than described in B. subtilis. Further, we describe aprE repression by inosine for the first time.