Apparent size and morphology of bacterial microcompartments varies with technique

Abstract

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.

Fig 1. TEM of purified MCPs.

(A) Schematic representation and transmission electron micrograph of negatively stained purified MCPs. Note that MCPs appear collapsed as evidenced by the pooled stain near the center of MCPs. (B) Schematic representation and transmission electron micrograph of negatively stained purified MCPs that were first dehydrated in ethanol and a high vapor-pressure solvent (HMDS). Note that MCPs appear less collapsed than in (A). Scale bar (white) is 100 nm.

Fig 2. Apparent size of MCPs analyzed with different imaging techniques.

Box-and-whisker plot of the size distribution of MCPs analyzed with various techniques. Note that apparent size and distribution varies widely with each technique. A single-factor ANOVA test revealed that populations differed significantly (p < 0.001). The only populations that are not significantly different (as defined by a p-value greater than .001 in a two-tailed t-test) are TEM of purified MCPs vs thin section TEM and SEM of purified MCPs vs TEM of dehydrated samples (p = .12 and .26, respectively). N = 300 for all, where 100 measurements were made for each of three biological replicates (three different MCP growths and purifications). Abbreviations: transmission electron microscopy (TEM), transmission electron microscopy with samples dehydrated in hexamethyldisilazane (TEM + HMDS), scanning electron microscopy (SEM), cryo transmission electron microscopy (Cryo TEM), ultra-thin section transmission electron microscopy (UTS TEM).

Fig 3. SEM of purified MCPs.

(A) Schematic representation of MCPs imaged by SEM. (B) SEM of MCPs with >6 nm of gold staining. (C) SEM of MCPs with the minimal 6 nm gold coat thickness. Note that MCPs appear more inflated than in Fig 1A and surface features are apparent. Scale bars (white) are 100 nm.

Fig 4. Cryo TEM of purified MCPs.

(A) Schematic representation of cryo TEM of MCPs. Note that MCPs retain their native shape and are frozen in a layer of vitreous ice. (B) Micrographs of purified MCPs visualized using cryo TEM. Scale bars (white) are 100 nm.

Fig 5. TEM of ultra-thin sections of purified MCPs.

(A) Schematic representation of an MCP undergoing ultra-thin sectioning. Note that due to the irregular shape of MCPs, thin sectioning will lead to a wide range of apparent diameters. (B) Micrographs of purified and ultra-thin sectioned MCPs. Scale bar (white) is 250 nm.

Fig 6. Higher-throughput sizing of purified MCPs using DLS.

Sizing MCPs in solution via light scattering techniques. (A) Particle size distributions measured via Nanosight, and (B) Zeta Sizer. (C) Comparison of average diameters measured via NTA and DLS. Error bars represent the standard deviation of the three measured samples.