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Bacterial microcompartments (BMCs) are protein-based organelles found across the bacterial tree of life. They consist of a shell, made of proteins that oligomerize into hexagonally and pentagonally shaped building blocks, that surrounds enzymes constituting a segment of a metabolic pathway. The proteins of the shell are unique to BMCs. They also provide selective permeability; this selectivity is dictated by the requirements of their cargo enzymes. We have recently surveyed the wealth of different BMC types and their occurrence in all available genome sequence data by analyzing and categorizing their components found in chromosomal loci using HMM (Hidden Markov Model) protein profiles. To make this a "do-it yourself" analysis for the public we have devised a webserver, BMC Caller ( https://bmc-caller.prl.msu.edu ), that compares user input sequences to our HMM profiles, creates a BMC locus visualization, and defines the functional type of BMC, if known. Shell proteins in the input sequence data are also classified according to our function-agnostic naming system and there are links to similar proteins in our database as well as an external link to a structure prediction website to easily generate structural models of the shell proteins, which facilitates understanding permeability properties of the shell. Additionally, the BMC Caller website contains a wealth of information on previously analyzed BMC loci with links to detailed data for each BMC protein and phylogenetic information on the BMC shell proteins. Our tools greatly facilitate BMC type identification to provide the user information about the associated organism's metabolism and enable discovery of new BMC types by providing a reference database of all currently known examples.

Bacterial microcompartments (BMCs) are protein-based organelles found across the bacterial tree of life. They consist of a shell, made of proteins that oligomerize into hexagonally and pentagonally shaped building blocks, that surrounds enzymes constituting a segment of a metabolic pathway. The proteins of the shell are unique to BMCs. They also provide selective permeability; this selectivity is dictated by the requirements of their cargo enzymes. We have recently surveyed the wealth of different BMC types and their occurrence in all available genome sequence data by analyzing and categorizing their components found in chromosomal loci using HMM (Hidden Markov Model) protein profiles. To make this a "do-it yourself" analysis for the public we have devised a webserver, BMC Caller ( https://bmc-caller.prl.msu.edu ), that compares user input sequences to our HMM profiles, creates a BMC locus visualization, and defines the functional type of BMC, if known. Shell proteins in the input sequence data are also classified according to our function-agnostic naming system and there are links to similar proteins in our database as well as an external link to a structure prediction website to easily generate structural models of the shell proteins, which facilitates understanding permeability properties of the shell. Additionally, the BMC Caller website contains a wealth of information on previously analyzed BMC loci with links to detailed data for each BMC protein and phylogenetic information on the BMC shell proteins. Our tools greatly facilitate BMC type identification to provide the user information about the associated organism's metabolism and enable discovery of new BMC types by providing a reference database of all currently known examples.

Bacterial microcompartments (BMCs) are proteinaceous prokaryotic organelles that enable the utilization of substrates such as 1,2-propanediol and ethanolamine. BMCs are mostly linked to the survival of particular pathogenic bacteria by providing a growth advantage through utilization of 1,2-propanediol and ethanolamine which are abundantly present in the human gut. Although a 1,2-propanediol utilization cluster was found in the probiotic bacterium Propionibacterium freudenreichii, BMC-mediated metabolism of 1,2-propanediol has not been demonstrated experimentally in P. freudenreichii. In this study we show that P. freudenreichii DSM 20271 metabolizes 1,2-propanediol in anaerobic conditions to propionate and 1-propanol. Furthermore, 1,2-propanediol induced the formation of BMCs, which were visualized by transmission electron microscopy and resembled BMCs found in other bacteria. Proteomic analysis of 1,2-propanediol grown cells compared to L-lactate grown cells showed significant upregulation of proteins involved in propanediol-utilization (pdu-cluster), DNA repair mechanisms and BMC shell proteins while proteins involved in oxidative phosphorylation were down-regulated. 1,2-Propanediol utilizing cells actively produced vitamin B12 (cobalamin) in similar amounts as cells growing on L-lactate. The ability to metabolize 1,2-propanediol may have implications for human gut colonization and modulation, and can potentially aid in delivering propionate and vitamin B12 in situ.

Bacterial microcompartments (BMCs) are proteinaceous organelles that optimize specific metabolic pathways referred to as metabolosomes involving transient production of toxic volatile metabolites such as aldehydes. Previous bioinformatics analysis predicted the presence of BMCs in 23 bacterial phyla including foodborne pathogens and a link with gene clusters for the utilization of host-derived substrates such as 1,2-propanediol utilization, i.e., the Pdu cluster. Although, transcriptional regulation of the Pdu cluster and its role in Listeria monocytogenes virulence in animal models have recently been reported, the experimental identification and the physiological role of BMCs in L. monocytogenes is still unexplored. Here, we ask whether BMCs could enable utilization of 1,2-propanediol (Pd) in L. monocytogenes under anaerobic conditions. Using L. monocytogenes EGDe as a model strain, we could demonstrate efficient utilization of Pd with concomitant production of 1-propanol and propionate after 24 h of anaerobic growth, while the utilization was significantly reduced in aerobic conditions. In line with this, expression of genes encoding predicted shell proteins and the signature enzyme propanediol dehydratase is upregulated more than 20-fold in cells anaerobically grown in Pdu-induced versus non-induced control conditions. Additional proteomics analysis confirmed the presence of BMC shell proteins and Pdu enzymes in cells that show active degradation of Pd. Furthermore, using transmission electron microscopy, BMC structures have been detected in these cells linking gene expression, protein composition, and BMCs to activation of the Pdu cluster in anaerobic growth of L. monocytogenes. Studies in defined minimal medium with Pd as an energy source showed a significant increase in cell numbers, indicating that Pdu and the predicted generation of ATP in the conversion of propionyl-phosphate to the end product propionate can support anaerobic growth of L. monocytogenes. Ou

The use of bacterial production could improve commercial feasibility for biochemicals such as pharmaceuticals and natural products. However, many non-native reactions in bacterial hosts have unacceptably low yields due to pathway toxicity or competition with other cellular processes. Compartmentalization is a potential solution to these problems, but compartmentalized organelles were long thought to be found only in eukaryotic cells. Remarkably, bacterial microcompartments (MCPs) are protein-based organelles that perform this sequestration function. We aim to apply these MCPs for metabolic engineering and the enhancement of biosynthesis pathways that do not function optimally in the cytosol. MCPs may also serve as a scaffold for application outside of the cell. To achieve this goal, we characterized the stability of MCPs when exposed to a range of pH and temperature to define the limitations of this complex structure. We next demonstrated that it is possible to form chimeric MCPs by combining shell proteins from different systems in nature. Importantly, we found that these changes to shell protein structure can enhance the function of the encapsulated pathway. We discovered that pathway performance can also be improved by a single point mutation to the pore of a shell protein, likely altering small molecule diffusion across the shell. We are investigating the role of shell protein pore chemistry in tuning small molecule diffusion. Finally, we test signal sequence peptides at the C-terminus of proteins and find that variation of signal peptide sequence results in altered expression level and loading within MCPs. In total, these tools improve our ability to design custom bacterial organelles to control the partitioning of chemical reactions within cells.

The use of bacterial production could improve commercial feasibility for biochemicals such as pharmaceuticals and natural products. However, many non-native reactions in bacterial hosts have unacceptably low yields due to pathway toxicity or competition with other cellular processes. Compartmentalization is a potential solution to these problems, but compartmentalized organelles were long thought to be found only in eukaryotic cells. Remarkably, bacterial microcompartments (MCPs) are protein-based organelles that perform this sequestration function. We aim to apply these MCPs for metabolic engineering and the enhancement of biosynthesis pathways that do not function optimally in the cytosol. MCPs may also serve as a scaffold for application outside of the cell. To achieve this goal, we characterized the stability of MCPs when exposed to a range of pH and temperature to define the limitations of this complex structure. We next demonstrated that it is possible to form chimeric MCPs by combining shell proteins from different systems in nature. Importantly, we found that these changes to shell protein structure can enhance the function of the encapsulated pathway. We discovered that pathway performance can also be improved by a single point mutation to the pore of a shell protein, likely altering small molecule diffusion across the shell. We are investigating the role of shell protein pore chemistry in tuning small molecule diffusion. Finally, we test signal sequence peptides at the C-terminus of proteins and find that variation of signal peptide sequence results in altered expression level and loading within MCPs. In total, these tools improve our ability to design custom bacterial organelles to control the partitioning of chemical reactions within cells.