The effects of time, temperature, and pH on the stability of PDU bacterial microcompartments

Kim, Edward Y.; Slininger, Marilyn F.; Tullman‐Ercek, Danielle

doi:10.1002/pro.2527

Cited by 16 publications

(20 citation statements)

References 36 publications

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“…No βGal activity was detected for temperatures above 58°C. Our observations are in line with the results by Kim et al who observed the onset of BMC-shell denaturation starting at temperatures of 53°C [29]. Association of the enzyme with BMCs thus could not increase the thermostability of the bGal-activity, an expected result as heat-unlike pH-is a physical stressor.…”

Section: The Bmc-shell Proteins Protect β-Galactotsidase From Ph But supporting

confidence: 93%

Engineering bacterial microcompartments with heterologous enzyme cargos

Wagner

Capitain

Richter

et al. 2016

Engineering in Life Sciences

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Bacterial microcompartments (BMCs) are intracellular proteinaceous organelles devoid of a lipid membrane that encapsulates enzymes of metabolic pathways. Salmonella enterica synthesizes propanediol‐utilization BMCs containing enzymes involved in the degradation of 1,2‐propanediol. BMCs can be designed to enclose heterologous proteins, paving the way to engineered catalytic microreactors. Here, we investigate broader applicability of this design principle by directing three different enzymes to the BMC. We demonstrate that β‐galactosidase, esterase Est5, and cofactor‐dependent glycerol dehydrogenase can be directed to the BMC and copurified with the microcompartment shell in a catalytically active form. We show that the BMC shell protects enzymes from pH‐dependent but not from temperature stress. Moreover, we provide evidence that the heterologously expressed BMCs act as a moderately selective diffusion barrier for lipophilic small molecules.

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Section: The Bmc-shell Proteins Protect β-Galactotsidase From Ph But supporting

confidence: 93%

Engineering bacterial microcompartments with heterologous enzyme cargos

Wagner

Capitain

Richter

et al. 2016

Engineering in Life Sciences

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“…The protein concentration has also been documented as a critical factor for driving shell formation [41,43]. In addition, in vitro solubility studies have illustrated that pH and ionic strength in solution could influence the structural stability of BMCs [17,27] as well as the assembly behaviors of BMC shell proteins in the formation of two-dimensional sheets [37,41], nanotubes [13,17], and nanocages [28], reminiscent of their impact on virus capsid assembly [44,45]. We also found protein precipitation and no patches formed when pH > 10 and < 3 or the salt concentration < 10 mM or > 600 mM (unpublished data).…”

Section: Discussionmentioning

confidence: 99%

“…However, some key issues remain to be tackled in BMC bioengineering, for example, how stable the BMC structures are and how to manipulate and assess effectively the self-assembly and formation of BMC protein aggregates. Investigations of the structures and assembly of BMC shells and entire BMCs have been carried out using X-ray crystallography, electron microscopy (EM), fluorescence microscopy, and dynamic light scattering (DSL) [10,11,16,22,[27][28][29][30][31]. Recently, we have exploited high-speed AFM (HS-AFM) to conduct the first visualization of the dynamic self-assembly process of BMC-H proteins [12].…”

Section: Introductionmentioning

confidence: 99%

Self-Assembly Stability and Variability of Bacterial Microcompartment Shell Proteins in Response to the Environmental Change

et al. 2019

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Bacterial microcompartments (BMCs) are proteinaceous self-assembling organelles that are widespread among the prokaryotic kingdom. By segmenting key metabolic enzymes and pathways using a polyhedral shell, BMCs play essential roles in carbon assimilation, pathogenesis, and microbial ecology. The BMC shell is composed of multiple protein homologs that self-assemble to form the defined architecture. There is tremendous interest in engineering BMCs to develop new nanobioreactors and molecular scaffolds. Here, we report the quantitative characterization of the formation and self-assembly dynamics of BMC shell proteins under varying pH and salt conditions using high-speed atomic force microscopy (HS-AFM). We show that 400-mM salt concentration is prone to result in larger single-layered shell patches formed by shell hexamers, and a higher dynamic rate of hexamer self-assembly was observed at neutral pH. We also visualize the variability of shell proteins from hexameric assemblies to fiber-like arrays. This study advances our knowledge about the stability and variability of BMC protein self-assemblies in response to microenvironmental changes, which will inform rational design and construction of synthetic BMC structures with the capacity of remodeling their self-assembly and structural robustness. It also offers a powerful toolbox for quantitatively assessing the self-assembly and formation of BMC-based nanostructures in biotechnology applications. Electronic supplementary material The online version of this article (10.1186/s11671-019-2884-3) contains supplementary material, which is available to authorized users.

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“…The total protein concentration of MCP samples was measured using the Pierce™ BCA Protein Assay Kit (Thermo Scientific) per the manufacturer’s instructions, and concentrations were normalized as necessary for each analysis method. All MCP samples were stored at 4°C until use and were prepared for analysis within 5 days of purification to avoid MCP aggregation and degradation [40].…”

Section: Methodsmentioning

confidence: 99%

Apparent size and morphology of bacterial microcompartments varies with technique

Kennedy¹,

Hershewe²,

Nichols³

et al. 2019

Preprint

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27Bacterial microcompartments (MCPs) are protein-based organelles which encapsulate 28 metabolic pathways. Metabolic engineers have recently sought to repurpose MCPs to encapsulate 29 heterologous pathways to increase flux through pathways of interest. As MCP engineering 30 becomes more common, standardized methods for analyzing changes to MCPs and interpreting 31 results across studies will become increasingly important. In this study, we demonstrate that 32 different imaging techniques yield variations in the apparent size of purified MCPs from 33 Salmonella enterica serovar Typhimurium LT2, likely due to variations in sample preparation 34 methods. We provide guidelines for preparing samples for MCP imaging and outline expected 35 variations in apparent size and morphology between methods. With this report we aim to establish 36 an aid for comparing results across studies. 37Bacterial microcompartments (MCPs) are protein-based organelles found in diverse 48 species of bacteria [4][5][6]. These were originally identified in cyanobacteria and were hypothesized 49 to be viruses based on their appearance [7,8]. However, these structures were later determined to 50 be important for the carbon concentrating mechanism for certain species of autotrophic microbes 51 [9][10][11][12]. Since then, numerous diverse types of MCPs have been identified in species ranging from 52 cyanobacteria and halophilic ocean-dwelling bacteria, to enteric pathogens and soil-dwelling 53 microbes [13,14]. In addition to the cyanobacterial compartments used for carbon fixation, many 54 MCPs are used by enteric pathogens for the metabolism of unique carbon sources that move 55 through toxic or volatile intermediates, imparting a competitive advantage [15-18]. 56 The flagship archetype for metabolic MCPs is the 1,2-propanediol utilization (Pdu) MCP 57 found in Salmonella enterica. The Pdu MCP encapsulates the enzymatic machinery necessary for 58 metabolism of 1,2-propanediol (1,2-PD), a carbon source found in the gut of Salmonella hosts 59 [15]. The 1,2-PD metabolic enzymes are surrounded by a protein shell composed of multiple types 60 of trimeric, pentameric, and hexameric shell proteins. The reported size of these irregularly-shaped 61protein organelles varies widely from 77-220 nm in diameter (S1 Table), and rigorous methods 62 for size quantification are sparse [8,13,15,[19][20][21][22][23]. 63The Pdu MCP has been studied in-depth since the early 1990s, but it has recently increased 64 in popularity due to its potential utility in metabolic engineering [24][25][26]. Metabolic engineers 65 have sought to increase flux through target pathways of interest by increasing local concentrations 66 of enzymes and their substrates [27]. MCPs can accomplish this task and offer the potential added 67 benefit of sequestering toxic or volatile intermediates from damaging or escaping the cell [28,29]. 68They also have the potential to reduce unwanted side reactions and provide private cofactor pools 69 separate from central metabolism [30]. 70Recent ...

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The effects of time, temperature, and pH on the stability of PDU bacterial microcompartments

Cited by 16 publications

References 36 publications

Engineering bacterial microcompartments with heterologous enzyme cargos

Engineering bacterial microcompartments with heterologous enzyme cargos

Self-Assembly Stability and Variability of Bacterial Microcompartment Shell Proteins in Response to the Environmental Change

Apparent size and morphology of bacterial microcompartments varies with technique

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