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Construction of a System for the Study of Protein‐Induced Membrane Tubules
Author(s) -
Rivera Alba Katiria González,
Forest Katrina T.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.621.2
Subject(s) - pilus , flagellum , microbiology and biotechnology , vesicle , bacterial outer membrane , membrane protein , biology , secretion , membrane , adhesion , chemistry , escherichia coli , biochemistry , gene , organic chemistry
Communication between cells and the environment is crucial to life, and correspondingly cells have evolved complex signaling pathways. For example, bacteria have a repertoire of cellular structures that mediate adhesion, motility, virulence, etc. Some of the well‐studied systems are pili, flagella, and multiple secretion systems. Recently, more attention has been paid to membranous structures such as secreted vesicles and membrane appendages. These structures have been shown to play roles in adhesion, cell‐to‐cell communication, and pathogenesis. Membrane appendages appear to be very diverse structures with possible distinct assembly mechanisms. For instance, membrane appendages can appear as tubular structures with a size range of 20–70 ηm in width that extend from the bacterial membrane for very long distances. These membrane appendages are sometime referred as nanotubes and in some instances they have been shown to play a role in metabolic exchange. One study showed that Delftia sp. can form nanopods as the result of the release of outer membrane vesicles that fused together1. Other membrane tubules have been shown to contain protein filaments, such as sheathed flagella and unnatural pili. In our laboratory we attempted to construct a heterologous system for bacterial pilus assembly. However, we found that under special conditions we could trick E. coli to elaborate structures that resembled nanotubes. In our initial studies we expressed different proteins from the Pseudomonas aeruginosa Type‐4 pilus system in E. coli Bl21‐AI, E. coli BL21 (DE3), or E. coli NovaBlue (DE3) and induced protein expression with IPTG and/or arabinose when necessary. Using negative staining and electron microscopy, we observed tubular structures (about 20–40 ηm wide, and longer than 1 μm). Remarkably although formation of these extensions was dependent on the major component of the pilus, PilA, it was independent of pre‐pilin cleavage by pre‐pilin peptidase; we found long membrane tubules in the presence of a non‐cleavable pilin. This system promises a means to investigate in detail how bacteria can build nanotubes and how this process is affected by the presence of transmembrane proteins. Support or Funding Information NIH 5 T32 GM008349