Synthetic Protein Microcompartment Generation By Expressing a Propanediol Utilisation Organelle from Geobacillus thermoglucosidasius in Bacillus Subtilis

Bacterial microcompartments (BMCs) are bacterial sub-cellular proteinaceous organelles which host specific biochemical reactions in a manner which is physically separated from other cytoplasmic processes. This includes metabolosome housing the propanediol metabolic pathway where the purpose appears to be to sequester highly reactive pathway intermediates. Gaining control of assembly of BMCs and their biochemical cargo offers biotechnological opportunities, and this has yet to be achieved in thermophiles. In this study, we have established a strategy for construction of an empty organelle from Geobacillus thermoglucosidasius using components of the propanediol utilisation metabolosome and targeting of enzymes in its lumen in Bacillus subtilis, as model Gram-positive organism.

The >18kb pdu operon consisting of 19 genes was examined. The five genes, pduA, pduBB’, pduJ, pduK and pduN, encoding proteins that form the shell of BMCs were dispersed with others. These genes were amplified from the G. thermoglucosidasius chromosome and assembled in the same order as in the locus and integrated into the amyE locus of the B. subtilis 168CA chromosome under control of a IPTG inducible promoter to give the BMCG strain. Initially, the ability to express heterologous proteins was confirmed by the appearance of a green fluorescent signal after two hours induction in the strains where the superfolder GFP (sfGFP) was fused in frame to each individual genes at the C-terminus. Cytoplasmic fluorescence intensity differed between the strains indicating an abundance of shell proteins with PduBB’ and PduJ having the highest levels. Western immunoblotting of the whole-cell protein extract of fusion sfGFP to pduA and pduN, using anti-sfGFP antibody, revealed bands with molecular weights of 37 and 36kDa, respectively, in contrast to 27kDa in the control. Circular structures with clear boundaries were visualised in thin-sectioned cells of BMCG by TEM and were absent in the control strain with empty vector integration. In order to determine the protein "signal" sequences required for targetting sfGFP to the empty BMC, it was linked to the N-terminal sequences of Propanediol utilisation protein (PduP) equal to 18, 24 and 461 amino acids. The constructs were placed into the lacA genomic region of the BMCG strain under the xylose inducible promoter. Discrete fluorescent puncta were observed after co-expression of shell and both P24 and P461 sfGFP fused proteins. Separately, the P18 sfGFP fusion seemed to be unable to incorporate sfGFP in the BMCs, resulting in a diffuse fluorescent signal. Time-lapse imaging showed an ability to assemble a BMC after 20 minutes.

These results show for the first time the capacity to generate synthetic protein compartments derived from the shell proteins, PduABB’JKN, of the thermophile, G. thermoglucosidasius. Based on studies with PduP, targeting of heterologous protein to the interior of the compartment requires fusion of an N-terminal sequence of at least 24 amino acids. The current Bacillus-based system will assist in future construction of empty compartments for targeting proteins in Geobacillus spp. at high temperatures.