Surface-Induced Formation and Redox-Dependent Staining of Outer Membrane Extensions in Shewanella oneidensis MR-1

Abstract

The metal-reducing bacterium Shewanella oneidensis MR-1 produces extensions of its outer membrane (OM) and periplasm that contain cytochromes responsible for extracellular electron transfer (EET) to external redox-active surfaces, including minerals and electrodes. While the role of multi-heme cytochromes in transporting electrons across the cell wall is well established, their distribution along S. oneidensis OM extensions is also thought to allow lateral electron transport along these filaments. These proposed bacterial nanowires, which can be several times the cell length, would thereby extend EET to more distant electron acceptors. However, it is still unclear why these extensions form, and to what extent they contribute to respiration in living cells. Here, we investigate physical contributors to their formation using in vivo fluorescence microscopy. While previous studies focused on the display of S. oneidensis outer membrane extensions (OMEs) as a response to oxygen limitation, we find that cell-to-surface contact is sufficient to trigger the production of OMEs, including some that reach >100 µm in length, irrespective of medium composition, agitation, or aeration. To visualize the extent of heme redox centers along OMEs, and help distinguish these structures from other extracellular filaments, we also performed histochemical redox-dependent staining with transmission electron microscopy on wild type and cytochrome-deficient strains. We demonstrate that redox-active components are limited to OMEs and not present on other extracellular appendages, such as pili and flagella. We also observed that the loss of 8 functional periplasmic and outer membrane cytochromes significantly decreased both the frequency and intensity of redox-dependent staining found widespread on OMEs. These results will improve our understanding of the environmental conditions that influence the formation of S. oneidensis OMEs, as well as the distribution and functionality of EET components along extracellular appendages.

A Prokaryotic Membrane Sculpting BAR Domain Protein

“…S. oneidensis OMEs are more commonly observed during surface attachment rather than planktonic cultures 7,29 . BAR domain proteins can directly promote tubule formation from liposomes in vitro 24 , so inducing expression of an additional copy of the bdpA gene prior to attachment could result in OME formation even during planktonic growth.…”

“…After ~6 hours at 30°C and 225 rpm, when the OD 600 was 2.4 (late log phase), 5 mL of cells were collected by centrifugation at 4226 x g for 5 min and washed twice in defined medium. The perfusion chamber, microscope, and flow medium described previously 7,29,30 were used for all perfusion flow OME statistics experiments. During each 5 hour imaging experiment, the perfusion chamber was first filled with this flow medium, then <1 mL of washed cells were slowly injected for a surface density of ~100-300 cells per 112 x 112 μm field of view on a Nikon Ti-E inverted microscope.…”

“…OMV formation is ubiquitous and has many documented functions 9 . OMEs are less commonly observed, remain attached to the cell, and various morphologies can be seen extending from single cells including Myxococcus xanthus 14,15 , flavobacterium strain Hel3_A1_48 8 , Vibrio vulnificus 10 , Francisella novicida 28 , Shewanella oneidensis 7,29–31 , and as cell-cell connections in Bacillus subtilis 32–34 and Eschericia coli 35 . Several bacterial proteins have demonstrated membrane tubule formation capabilities in vitro 16,36–40 , but despite the growing number of reports, proteins involved in shaping bacterial membranes into OMV/Es have yet to be identified.…”

“…Shewanella oneidensis is a model organism for extracellular electron transfer (EET), a mode of respiration whereby electrons traverse the inner membrane, periplasm, and outer membrane via multiheme cytochromes to reach exogenous insoluble terminal electron acceptors, such as metals and electrodes 41,42 . It is also known to produce redox-active OMVs 43 and OMEs coated with mulitheme cytochromes, particularly upon surface attachment 7,30,43 . However, little is known about their formation mechanism, control of shape or curvature, and electrochemical properties that influence EET function.…”

“… Summary Paragraph Bin/Amphiphysin/RVS (BAR) domain proteins belong to a ubiquitous superfamily of coiled-coil proteins that influence membrane curvature in eukaryotes and are associated with vesicle biogenesis, vesicle-mediated protein trafficking, and intracellular signaling 1–6 . BAR domain proteins have not been identified in bacteria, despite certain organisms displaying an array of membrane curvature phenotypes 7–16 . Here we identify a prokaryotic BAR domain protein, BdpA, from Shewanella oneidensis MR-1, an iron reducing bacterium known to produce redox active membrane vesicles and micrometer-scale membrane extensions.…”