A genomic integration platform for heterologous cargo encapsulation in 1,2-propanediol utilization bacterial microcompartments

Nichols, Taylor M.; Kennedy, Nolan W.; Tullman‐Ercek, Danielle

doi:10.1016/j.bej.2020.107496

Cited by 21 publications

(23 citation statements)

References 53 publications

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“…Thus, compartment distribution throughout the cell is indicated by the presence of the green fluorescence associated with the ssD-GFP reporter encapsulated within the MCP lumen. As in previous studies, expression of Pdu MCPs in the wild type (PduNcontaining) background results in punctate fluorescence throughout the cell (Figure 2c), suggesting that well-formed compartments are distributed within the cell [33][34][35] . In contrast, when the pduN gene is knocked out, expression of the pdu operon results in lines of fluorescence, typically aligned with the long axis of the cell (Figure 2c).…”

Section: Pdun Mediates the Morphology Of Pdu Compartment Structuressupporting

confidence: 80%

“…There is great interest in repurposing MCPs for metabolic engineering applications, where they have the potential to alleviate bottlenecks such as slow pathway kinetics, toxic intermediate buildup, and cofactor competition 2,11 . While strides have been made in loading non-native cargo into these systems in a controlled fashion 35,[46][47][48][49][50][51] , the selection criteria for an MCP system in any given engineering application is lacking. This includes MCP features such as size and morphology.…”

Section: Discussionmentioning

confidence: 99%

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Vertex protein PduN tunes encapsulated pathway performance by dictating bacterial metabolosome morphology

Mills

Waltmann

Archer

et al. 2021

Preprint

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Engineering subcellular organization in microbes shows great promise in addressing bottlenecks in metabolic engineering efforts; however, rules guiding selection of an organization strategy or platform are lacking. Here, we study compartment morphology as a factor in mediating encapsulated pathway performance. Using the 1,2-propanediol utilization microcompartment (Pdu MCP) system from Salmonella enterica serovar Typhimurium LT2, we find that we can shift the morphology of this protein nanoreactor from polyhedral to tubular by removing vertex protein PduN. Analysis of the metabolic function between these Pdu microtubes (MTs) shows that they provide a diffusional barrier capable of shielding the cytosol from a toxic pathway intermediate, similar to native MCPs. However, kinetic modeling suggests that the different surface area to volume ratios of MCP and MT structures alters encapsulated pathway performance. Finally, we report a microscopy-based assay that permits rapid assessment of Pdu MT formation to enable future engineering efforts on these structures.

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Section: Pdun Mediates the Morphology Of Pdu Compartment Structuressupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Vertex protein PduN tunes encapsulated pathway performance by dictating bacterial metabolosome morphology

Mills

Waltmann

Archer

et al. 2021

Preprint

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“…6) (40). Microcompartment formation was tested using a GFP encapsulation assay, in which GFP is targeted to microcompartments using an N-terminal signal sequence sufficient for microcompartment targeting (41,42). We found that strains expressing the pduN pseudogene (ΔN::N*) exhibited aberrant microcompartment morphologies similar to those observed in the pduN knockout strain (ΔN), indicating improper microcompartment assembly due to a loss of vertex capping.…”

Section: Application: Distribution and Diversity Of Full And Partial Pdu Gene Clusters In S Entericamentioning

confidence: 98%

Density-based binning of gene clusters to infer function or evolutionary history using GeneGrouper

McFarland¹,

Kennedy²,

Mills³

et al. 2021

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Motivation: Identifying gene clusters of interest in phylogenetically proximate and distant taxa can help to infer phenotypes of interest. Conserved gene clusters may differ by only a few genes, which can be biologically meaningful, such as the formation of pseudogenes or insertions interrupting regulation. These qualities may allow for unsupervised clustering of similar gene clusters into bins that provide a population-level understanding of the genetic variation in similar gene clusters. Results: We developed GeneGrouper, a command-line tool that uses a density-based clustering method to group gene clusters into bins. GeneGrouper demonstrated high recall and precision in benchmarks for the detection of the 23-gene Salmonella enterica LT2 Pdu gene cluster and four-gene Pseudomonas aeruginosa PAO1 Mex gene cluster in 435 genomes containing mixed taxa. In a subsequent application investigating the diversity and impact of gene complete and incomplete LT2 Pdu gene clusters in 1130 S. enterica genomes, GeneGrouper identified a novel, frequently occurring pduN pseudogene. When replicated in vivo, disruption of pduN with a frameshift mutation negatively impacted microcompartment formation. We next demonstrated the versatility of GeneGrouper by clustering both distant homologous gene clusters and variable gene clusters found in integrative and conjugative elements.

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“…[15][16][17] MCP enzyme contents can be readily modified to vary catalytic activity and to include additional cargo like fluorescent proteins. [18][19][20] MCPs are therefore an ideal platform to investigate the chemotaxis of nanoscale compartments where shell structure, enzymatic activity, and fluorescent contents can be independently tuned.…”

Section: Introductionmentioning

confidence: 99%

Enzymatically-active bacterial microcompartments follow substrate gradients and are protected from aggregation in a cell-free system

Steinkühler

Abrahamson

Agudo-Canalejo

et al. 2022

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The ability to dynamically control organelle movement and position is essential for cellular function. Yet the underlying mechanisms driving this organization have not been fully resolved. Here, we draw from recent experimental observations and theoretical models of enzyme chemotaxis to demonstrate the chemotaxis of a bacterial organelle, the 1,2 propanediol (1,2-PD) utilization bacterial microcompartment (MCP) from Salmonella enterica. Upon encapsulating MCPs in a cell-like, biomimetic compartment, we observed the directed movement of MCPs along an external gradient of substrate. Our analysis shows that MCPs not only chemotax towards their substrate but also that enzymatic activity and substrate turnover protect them against large-scale aggregation. Our results provide a first experimental demonstration of organelle chemotaxis in a synthetic cellular system and support a recent theoretical model of chemotaxis. Together this work reveals a potentially significant driver of organelle organization while contributing to the construction of synthetic cell-like materials.

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A genomic integration platform for heterologous cargo encapsulation in 1,2-propanediol utilization bacterial microcompartments

Cited by 21 publications

References 53 publications

Vertex protein PduN tunes encapsulated pathway performance by dictating bacterial metabolosome morphology

Vertex protein PduN tunes encapsulated pathway performance by dictating bacterial metabolosome morphology

Density-based binning of gene clusters to infer function or evolutionary history using GeneGrouper

Enzymatically-active bacterial microcompartments follow substrate gradients and are protected from aggregation in a cell-free system

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