scholarly journals Engineered shell proteins confer improved encapsulated pathway behavior in a bacterial microcompartment

2017 ◽  
Author(s):  
Marilyn F. Slininger Lee ◽  
Christopher M. Jakobson ◽  
Danielle Tullman-Ercek
Keyword(s):  
Growth Enhancement ◽  
Shell Protein ◽  
Bacterial Microcompartment ◽  
Hydropathy Index ◽  
Shell Proteins ◽  
Structure Result ◽  
Pore Lining ◽  
Protein Shell ◽  
Control Diffusion ◽  
Do So

AbstractBacterial microcompartments are a class of proteinaceous organelles comprising a characteristic protein shell enclosing a set of enzymes. Compartmentalization can prevent escape of volatile or toxic intermediates, prevent off-pathway reactions, and create private cofactor pools. Encapsulation in synthetic microcompartment organelles will enhance the function of heterologous pathways, but to do so, it is critical to understand how to control diffusion in and out of the microcompartment organelle. To this end, we explored how small differences in the shell protein structure result in changes in the diffusion of metabolites through the shell. We found that the ethanolamine utilization (Eut) protein EutM properly incorporates into the 1,2-propanediol utilization (Pdu) microcompartment, altering native metabolite accumulation and the resulting growth on 1,2-propanediol as the sole carbon source. Further, we identified a single pore-lining residue mutation that confers the same phenotype as substitution of the full EutM protein, indicating that small molecule diffusion through the shell is the cause of growth enhancement. Finally, we show that the hydropathy index and charge of pore amino acids are important indicators to predict how pore mutations will affect growth on 1,2- propanediol, likely by controlling diffusion of one or more metabolites. This study highlights the success of two strategies to engineer microcompartment control over metabolite transport: altering the existing shell protein pore via mutation of the pore-lining residues, and generating chimeras using shell proteins with the desired pores.TOC Abstract Graphic

scholarly journals A Survey of Bacterial Microcompartment Distribution in the Human Microbiome

2021 ◽  
Vol 12 ◽  
Author(s):  
Kunica Asija ◽  
Markus Sutter ◽  
Cheryl A. Kerfeld
Keyword(s):  
Sequence Data ◽  
Human Microbiome ◽  
Metabolic Potential ◽  
Bacterial Phyla ◽  
Bacterial Microcompartments ◽  
Bacterial Microcompartment ◽  
Different Types ◽  
Shell Proteins ◽  
Enzymatic Pathways ◽  
Protein Shell

Bacterial microcompartments (BMCs) are protein-based organelles that expand the metabolic potential of many bacteria by sequestering segments of enzymatic pathways in a selectively permeable protein shell. Sixty-eight different types/subtypes of BMCs have been bioinformatically identified based on the encapsulated enzymes and shell proteins encoded in genomic loci. BMCs are found across bacterial phyla. The organisms that contain them, rather than strictly correlating with specific lineages, tend to reflect the metabolic landscape of the environmental niches they occupy. From our recent comprehensive bioinformatic survey of BMCs found in genome sequence data, we find many in members of the human microbiome. Here we survey the distribution of BMCs in the different biotopes of the human body. Given their amenability to be horizontally transferred and bioengineered they hold promise as metabolic modules that could be used to probiotically alter microbiomes or treat dysbiosis.

scholarly journals In Salmonella enterica, Ethanolamine Utilization Is Repressed by 1,2-Propanediol To Prevent Detrimental Mixing of Components of Two Different Bacterial Microcompartments

2015 ◽  
Vol 197 (14) ◽  
pp. 2412-2421 ◽  
Author(s):  
Ryan Sturms ◽  
Nicholas A. Streauslin ◽  
Shouqiang Cheng ◽  
Thomas A. Bobik
Keyword(s):  
Protein Interactions ◽  
Regulatory Protein ◽  
Gene Clusters ◽  
Content Type ◽  
Bacterial Microcompartments ◽  
Shell Protein ◽  
Bacterial Microcompartment ◽  
Genes Encoding ◽  
Shell Proteins ◽  
High Conservation

ABSTRACTBacterial microcompartments (MCPs) are a diverse family of protein-based organelles composed of metabolic enzymes encapsulated within a protein shell. The function of bacterial MCPs is to optimize metabolic pathways by confining toxic and/or volatile metabolic intermediates. About 20% of bacteria produce MCPs, and there are at least seven different types. Different MCPs vary in their encapsulated enzymes, but all have outer shells composed of highly conserved proteins containing bacterial microcompartment domains. Many organisms have genes encoding more than one type of MCP, but given the high homology among shell proteins, it is uncertain whether multiple MCPs can be functionally expressed in the same cell at the same time. In these studies, we examine the regulation of the 1,2-propanediol (1,2-PD) utilization (Pdu) and ethanolamine utilization (Eut) MCPs inSalmonella. Studies showed that 1,2-PD (shown to induce the Pdu MCP) represses transcription of the Eut MCP and that the PocR regulatory protein is required. The results indicate that repression of the Eut MCP by 1,2-PD is needed to prevent detrimental mixing of shell proteins from the Eut and Pdu MCPs. Coexpression of both MCPs impaired the function of the Pdu MCP and resulted in the formation of hybrid MCPs composed of Eut and Pdu MCP components. We also show that plasmid-based expression of individual shell proteins from the Eut MCP or the β-carboxysome impaired the function of Pdu MCP. Thus, the high conservation among bacterial microcompartment (BMC) domain shell proteins is problematic for coexpression of the Eut and Pdu MCPs and perhaps other MCPs as well.IMPORTANCEBacterial MCPs are encoded by nearly 20% of bacterial genomes, and almost 40% of those genomes contain multiple MCP gene clusters. In this study, we examine how the regulation of two different MCP systems (Eut and Pdu) is integrated inSalmonella. Our findings indicate that 1,2-PD (shown to induce the Pdu MCP) represses the Eut MCP to prevent detrimental mixing of Eut and Pdu shell proteins. These findings suggest that numerous organisms which produce more than one type of MCP likely need some mechanism to prevent aberrant shell protein interactions.

scholarly journals Selective molecular transport through the protein shell of a bacterial microcompartment organelle

2015 ◽  
Vol 112 (10) ◽  
pp. 2990-2995 ◽  
Author(s):  
Chiranjit Chowdhury ◽  
Sunny Chun ◽  
Allan Pang ◽  
Michael R. Sawaya ◽  
Sharmistha Sinha ◽  
...  
Keyword(s):  
Small Molecules ◽  
Carbon Fixation ◽  
Molecular Diffusion ◽  
Molecular Transport ◽  
Phenotypic Data ◽  
Bacterial Microcompartments ◽  
Shell Protein ◽  
Bacterial Microcompartment ◽  
Selective Permeability ◽  
Protein Shell

Bacterial microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for microcompartment function propose that their outer protein shell is selectively permeable to small molecules, but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. Here, biochemical and physiological studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu microcompartment) while restricting the efflux of propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the observed biochemical and phenotypic data in terms of molecular diffusion across the shell. Overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins.

scholarly journals Crystallographic Insights into the Pore Structures and Mechanisms of the EutL and EutM Shell Proteins of the Ethanolamine-Utilizing Microcompartment of Escherichia coli

2010 ◽  
Vol 192 (22) ◽  
pp. 6056-6063 ◽  
Author(s):  
Mihoko Takenoya ◽  
Kiel Nikolakakis ◽  
Martin Sagermann
Keyword(s):  
Escherichia Coli ◽  
Two Dimensional ◽  
Structural Investigations ◽  
Shell Protein ◽  
Bacterial Microcompartment ◽  
Pore Structures ◽  
Gating Mechanism ◽  
Protein Membrane ◽  
Shell Proteins ◽  
Small Channels

ABSTRACT The ethanolamine-utilizing bacterial microcompartment (Eut-BMC) of Escherichia coli is a polyhedral organelle that harbors specific enzymes for the catabolic degradation of ethanolamine. The compartment is composed of a proteinaceous shell structure that maintains a highly specialized environment for the biochemical reactions inside. Recent structural investigations have revealed hexagonal assemblies of shell proteins that form a tightly packed two-dimensional lattice that is likely to function as a selectively permeable protein membrane, wherein small channels are thought to permit controlled exchange of specific solutes. Here, we show with two nonisomorphous crystal structures that EutM also forms a two-dimensional protein membrane. As its architecture is highly similar to the membrane structure of EutL, it is likely that the structure represents a physiologically relevant form. Thus far, of all Eut proteins, only EutM and EutL have been shown to form such proteinaceous membranes. Despite their similar architectures, however, both proteins exhibit dramatically different pore structures. In contrast to EutL, the pore of EutM appears to be positively charged, indicating specificity for different solutes. Furthermore, we also show that the central pore structure of the EutL shell protein can be triggered to open specifically upon exposure to zinc ions, suggesting a specific gating mechanism.

Structure of a trimeric bacterial microcompartment shell protein, EtuB, associated with ethanol utilization in Clostridium kluyveri

2009 ◽  
Vol 423 (2) ◽  
pp. 199-207 ◽  
Author(s):  
Dana Heldt ◽  
Stefanie Frank ◽  
Arefeh Seyedarabi ◽  
Dimitrios Ladikis ◽  
Joshua B. Parsons ◽  
...  
Keyword(s):  
Rotational Symmetry ◽  
Protein A ◽  
Ethanol Metabolism ◽  
Hexagonal Symmetry ◽  
Precise Control ◽  
Shell Protein ◽  
Bacterial Microcompartment ◽  
Evolutionary Advantage ◽  
Shell Proteins ◽  
Clostridium Kluyveri

It has been suggested that ethanol metabolism in the strict anaerobe Clostridium kluyveri occurs within a metabolosome, a subcellular proteinaceous bacterial microcompartment. Two bacterial microcompartment shell proteins [EtuA (ethanol utilization shell protein A) and EtuB] are found encoded on the genome clustered with the genes for ethanol utilization. The function of the bacterial microcompartment is to facilitate fermentation by sequestering the enzymes, substrates and intermediates. Recent structural studies of bacterial microcompartment proteins have revealed both hexamers and pentamers that assemble to generate the pseudo-icosahedral bacterial microcompartment shell. Some of these shell proteins have pores on their symmetry axes. Here we report the structure of the trimeric bacterial microcompartment protein EtuB, which has a tandem structural repeat within the subunit and pseudo-hexagonal symmetry. The pores in the EtuB trimer are within the subunits rather than between symmetry related subunits. We suggest that the evolutionary advantage of this is that it releases the pore from the rotational symmetry constraint allowing more precise control of the fluxes of asymmetric molecules, such as ethanol, across the pore. We also model EtuA and demonstrate that the two proteins have the potential to interact to generate the casing for a metabolosome.

Substrate channels revealed in the trimericLactobacillus reuteribacterial microcompartment shell protein PduB

2012 ◽  
Vol 68 (12) ◽  
pp. 1642-1652 ◽  
Author(s):  
Allan Pang ◽  
Mingzhi Liang ◽  
Michael B. Prentice ◽  
Richard W. Pickersgill
Keyword(s):  
Tandem Repeats ◽  
Lactobacillus Reuteri ◽  
Protein Complexes ◽  
Protein Subunits ◽  
Shell Protein ◽  
Domain Structures ◽  
Bacterial Microcompartment ◽  
Major Shell ◽  
Carbon Molecules ◽  
Trimeric Structure

Lactobacillus reuterimetabolizes two similar three-carbon molecules, 1,2-propanediol and glycerol, within closed polyhedral subcellular bacterial organelles called bacterial microcompartments (metabolosomes). The outer shell of the propanediol-utilization (Pdu) metabolosome is composed of hundreds of mainly hexagonal protein complexes made from six types of protein subunits that share similar domain structures. The structure of the bacterial microcompartment protein PduB has a tandem structural repeat within the subunit and assembles into a trimer with pseudo-hexagonal symmetry. This trimeric structure forms sheets in the crystal lattice and is able to fit within a polymeric sheet of the major shell component PduA to assemble a facet of the polyhedron. There are three pores within the trimer and these are formed between the tandem repeats within the subunits. The structure shows that each of these pores contains three glycerol molecules that interact with conserved residues, strongly suggesting that these subunit pores channel glycerol substrate into the metabolosome. In addition to the observation of glycerol occupying the subunit channels, the presence of glycerol on the molecular threefold symmetry axis suggests a role in locking closed the central region.

Engineering Bacterial Microcompartment Shells: Chimeric Shell Proteins and Chimeric Carboxysome Shells

2014 ◽  
Vol 4 (4) ◽  
pp. 444-453 ◽  
Author(s):  
Fei Cai ◽  
Markus Sutter ◽  
Susan L. Bernstein ◽  
James N. Kinney ◽  
Cheryl A. Kerfeld
Keyword(s):  
Bacterial Microcompartment ◽  
Shell Proteins

scholarly journals Linking the Salmonella enterica 1,2-propanediol utilization bacterial microcompartment shell to the enzymatic core via the shell protein PduB

2021 ◽  
Author(s):  
Nolan W Kennedy ◽  
Carolyn E Mills ◽  
Charlotte H Abrahamson ◽  
Andre Archer ◽  
Michael C Jewett ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Carbon Sources ◽  
Enzyme Concentration ◽  
Fluorescent Reporters ◽  
Shell Protein ◽  
Bacterial Microcompartment ◽  
Enzyme Encapsulation ◽  
Transmission Electron ◽  
Serovar Typhimurium ◽  
Enzymatic Machinery

Bacterial microcompartments (MCPs) are protein-based organelles that house the enzymatic machinery for metabolism of niche carbon sources, allowing enteric pathogens to outcompete native microbiota during host colonization. While much progress has been made toward understanding MCP biogenesis, questions still remain regarding the mechanism by which core MCP enzymes are enveloped within the MCP protein shell. Here we explore the hypothesis that the shell protein PduB is responsible for linking the shell of the 1,2-propanediol utilization (Pdu) MCP from Salmonella enterica serovar Typhimurium LT2 to its enzymatic core. Using fluorescent reporters, we demonstrate that all members of the Pdu enzymatic core are encapsulated in Pdu MCPs. We also demonstrate that PduB is the sole protein responsible for linking the entire Pdu enzyme core to the MCP shell. Using MCP purifications, transmission electron microscopy, and fluorescence microscopy we find that shell assembly can be decoupled from the enzymatic core, as apparently empty MCPs are formed in Salmonella strains lacking PduB. Mutagenesis studies also reveal that PduB is incorporated into the Pdu MCP shell via a conserved, lysine-mediated hydrogen bonding mechanism. Finally, growth assays and systems-level pathway modeling reveal that unencapsulated pathway performance is strongly impacted by enzyme concentration, highlighting the importance of minimizing polar effects when conducting these functional assays. Together, these results provide insight into the mechanism of enzyme encapsulation within Pdu MCPs and demonstrate that the process of enzyme encapsulation and shell assembly are separate processes in this system, a finding that will aid future efforts to understand MCP biogenesis.

scholarly journals Functionalization of Bacterial Microcompartment Shell Proteins With Covalently Attached Heme

2020 ◽  
Vol 7 ◽  
Author(s):  
Jingcheng Huang ◽  
Bryan H. Ferlez ◽  
Eric J. Young ◽  
Cheryl A. Kerfeld ◽  
David M. Kramer ◽  
...  
Keyword(s):  
Bacterial Microcompartment ◽  
Shell Proteins

Redox Characterization of Electrode-Immobilized Bacterial Microcompartment Shell Proteins Engineered To Bind Metal Centers

2019 ◽  
Vol 3 (1) ◽  
pp. 685-692 ◽  
Author(s):  
Jefferson S. Plegaria ◽  
Matthew D. Yates ◽  
Sarah M. Glaven ◽  
Cheryl A. Kerfeld
Keyword(s):  
Metal Centers ◽  
Bacterial Microcompartment ◽  
Shell Proteins
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