Bacterial microcompartments (MCPs) are protein-based organelles that encapsulate metabolic pathways. Metabolic engineers have recently sought to repurpose MCPs to encapsulate heterologous pathways to increase flux through pathways of interest. As MCP engineering becomes more common, standardized methods for analyzing changes to MCPs and interpreting results across studies will become increasingly important. In this study, we demonstrate that different imaging techniques yield variations in the apparent size of purified MCPs from Salmonella enterica serovar Typhimurium LT2, likely due to variations in sample preparation methods. We provide guidelines for preparing samples for MCP imaging and outline expected variations in apparent size and morphology between methods. With this report we aim to establish an aid for comparing results across studies.

Metabolic engineers seek to produce high-value products from inexpensive starting materials in a sustainable and cost-effective manner by using microbes as cellular factories. However, pathway development and optimization can be arduous tasks, complicated by pathway bottlenecks and toxicity. Pathway organization has emerged as a potential solution to these issues, and the use of protein- or DNA-based scaffolds has successfully increased the production of several industrially relevant compounds. These efforts demonstrate the usefulness of pathway colocalization and spatial organization for metabolic engineering applications. In particular, scaffolding within an enclosed, subcellular compartment shows great promise for pathway optimization, offering benefits such as increased local enzyme and substrate concentrations, sequestration of toxic or volatile intermediates, and alleviation of cofactor and resource competition with the host. Here, we describe the 1,2-propanediol utilization (Pdu) bacterial microcompartment (MCP) as an enclosed scaffold for pathway sequestration and organization. We first describe methods for controlling Pdu MCP formation, expressing and encapsulating heterologous cargo, and tuning cargo loading levels. We further describe assays for analyzing Pdu MCPs and assessing encapsulation levels. These methods will enable the repurposing of MCPs as tunable nanobioreactors for heterologous pathway encapsulation.

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A microcrystalline collagen hemostat (MCH) widely used in general surgery was tested in the control of bleeding from experimentally produced gastric ulcers. Five dogs had a gastrotomy and were given heparin. Using the standard "ulcer maker," three sets of three ulcers were made in the gastric mucosa of each animal. Blood from each ulcer was collected for a 5-min period to allow for stabilization of bleeding. MCH powder or slurry or no MCH was placed directly on one ulcer of each set in random order. The bleeding rate for the next 10 min was measured. Mean decrements in the bleeding rate for slurry MCH and dry MCH-treated ulcers were 87% and 81%, respectively, compared with 51% for controls, P less than 0.05. Twelve MCH-treated ulcers, but no control ulcer, stopped bleeding completely, P less than 0.01. Preliminary observations show that MCH slurry can be applied through an endoscope and may be hemostatically effective in man. MCH may have a role in the endoscopic control of gastrointestinal bleeding.

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 ...