Optimal method for efficiently removing extracellular nanofilaments from Shewanella oneidensis MR-1

Area of Science:

• Microbiology and Microbial Ecology
• Biophysics and Nanotechnology
• Protein Biochemistry

Background:

• Bacterial extracellular nanofilaments, particularly in Shewanella oneidensis MR-1, are crucial for electron conductivity.
• Many research applications necessitate the removal of these nanofilaments from bacterial cells.
• Effective nanofilament removal must preserve the integrity of the bacterial cell body for subsequent analyses.

Purpose of the Study:

• To evaluate and compare various methods for detaching extracellular nanofilaments from S. oneidensis MR-1.
• To determine the optimal nanofilament shearing technique that maximizes yield of extracellular proteins.
• To ensure minimal contamination from membrane and intracellular proteins after nanofilament removal.

Main Methods:

• Qualitative assessment using Atomic Force Microscopy (AFM) to visualize nanofilament detachment.
• Quantitative analysis via Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC/MS-MS) to measure protein content.
• Comparison of multiple nanofilament shearing techniques on washed S. oneidensis MR-1 cultures.

Main Results:

• Vortexing a washed cell culture for 10 minutes was identified as the most effective method.
• This method significantly maximized the recovery of extracellular filamentous proteins.
• Contamination by membrane and intracellular proteins was minimized using the optimized vortexing procedure.

Conclusions:

• Optimized vortexing provides an efficient and non-destructive method for removing bacterial extracellular nanofilaments.
• This technique is suitable for preparing S. oneidensis MR-1 for downstream proteomic and functional studies.
• The findings contribute to advancing research on microbial electron transport and extracellular structures.