Part:BBa_K554009

TolC

TolC

This part was used to [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Results#Device_3_testing.2C_Protein_Secretion_System Device 3 testing, Protein Secretion System]

TolC is part of the hemolysin secretion system ([http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Project#Device_3:_Secretion_system Device 3, Protein Secretion System]), very important to export the proteins produced inside bacteria and that must act in targets outside (such as IL-12 and IL-10). TolC is a specific outer membrane protein, which forms a long channel throughout the outer membrane and the periplasm, largely open towards the extracellular medium.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 395
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 247
  

Usage and Biology

You can see a representation of this device acting in the schema below: HlyB gene and product are shown as a symbolic cilinder in orange.

Representation of device 3, the protein secretion system, in a Jedi bacteria that contains Device 1 (Adrenaline sensor/IL-12 producer). To export a protein, the bacteria must have the HlyD, HlyB and TolC proteins and the target protein must have a signal sequence (HlyA tail). In this case, the target protein to be secreted is IL-12.

A more realistic schema of ABC transport system is shown below:

Three-dimensional structure representation

You can find below a tridimensional structure of Crystal structure of the open state of tolc outer membrane protein from Escherichia coli (retrieved from PDB [http://www.pdb.org/pdb/explore/explore.do?structureId=2vdd 2VDD] (Bavro et al. 2008)) solved by crystallography and X-ray diffraction at 3.3 A resolution. This is a jmol applet, in which you can interactively see the protein structure of TolC:

MIT MAHE 2020

Usage and Biology

TolC is a specific outer membrane protein (OMP) and part of the hemolysin secretion system, very important to export the proteins produced inside bacteria and that must act in targets outside. TolC (55 kDa) is a multifunctional E. coli OMP that can serve as an outer membrane component for several processes such as protein export via a type I pathway and drug export via RND (resistance, nodulation, division) type systems. This means it is likely to be connected to members of the MFP/adaptor class, which are found both in the ABC exporters and in the RND exporters (P. Delepelaire, 2004).

TolC presents a new kind of OMP architecture. It is a trimeric protein, anchored in the outer membrane by beta-strands and with a very long periplasmic part made almost uniquely of alpha-helices extending about 100 Å into the periplasm. The molecule forms a tapering tube that is almost closed at the periplasmic end and wide open at the outer membrane surface, with almost no loops at the extracellular surface, as for porin or siderophore receptors. Its internal diameter of about 30 to 35 Å stays constant throughout the molecule and delimitates a large water-filled cavity (40,000 Å3) open to the extracellular medium. This cavity may be the exit tunnel for secreted proteins and is large enough to accommodate secondary structure elements or even small folded polypeptide chains (V. Koronakis et al., 2000).

It is studied that TolC operates together with the AcrAB proteins in the inner membrane to constitute an efflux pump driven by the proton motive force. It is also somehow involved in the maintenance of outer membrane integrity and the import of certain colicins and serves as a phage receptor.

In a study to evaluate the tolerance of TolC in an acidic environment using the acid resistance assay, overnight cultures grown at pH 7 were adjusted to McFarland 0.5, inoculated into fresh TSB at pH 3, and then incubated at 37°C with shaking at 180 rpm for 30 min. Cultures designed for adaptation were cultivated at pH 5 for 16–20 h before challenge with pH 3 followed by incubation. After the acid challenge, the samples were immediately diluted with saline (0.85% NaCl) and plated onto TSA. Surviving bacteria were investigated by determining the number of colony-forming units (CFU) before the acid challenge and after 24 h of incubation. Percent survival was calculated by comparing the CFU before and after the challenge. The results indicated that tolC is required for survival in extremely acidic environments, regardless of whether it has been adapted to mild acid conditions. Based on their RT-PCR results, acid exposed tolC mutant cells showed less protection against oxidative stress, indicating that TolC is involved in coping with excess protons (Jen-Jie Lee et al., 2016).

References

Barbara D. Tzschaschel, Carlos A. Guzmán,, Kenneth N. Timmis and Victor de Lorenzo. An Escherichia coli hemolysin transport system-based vector for the export of polypeptides: Export of shiga-like toxin IIeB subunit by Salmonella typhimurium aroA. Nature Biotechnology 14, 765 - 769 (1996) [http://www.nature.com/nbt/journal/v14/n6/abs/nbt0696-765.html Article link]

P. Delepelaire. Type I secretion in Gram-negative bacteria. Biochimia et Biophysica Actca 1694, 149-161 (2004) [http://www.ncbi.nlm.nih.gov/pubmed/15546664 Link to PubMed]

Ivaylo Gentschev, Guido Dietrich and Werner Goebel. The E. coli α-hemolysin secretion system and its use in vaccine development. Trends in Microbiology 10, 39-45 (2002) [http://www.ncbi.nlm.nih.gov/pubmed/11755084 Link to PubMed]

Bavro, V.N., Pietras, Z., Furnham, N., Perez-Cano, L., Fernandez-Recio, J., Pei, X.Y., Truer, R., Misra, R., Luisi, B.Assembly and channel opening in a bacterial drug efflux machine.Journal: (2008) Mol.Cell 30: 114 [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=18406332 Link to PubMed]

Koronakis, V., Sharff, A., Koronakis, E., Luisi, B., & Hughes, C. (2000). Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature, 405(6789), 914–919. https://doi.org/10.1038/35016007

Lee, J. J., Wu, Y. C., Kuo, C. J., Hsuan, S. L., & Chen, T. H. (2016). TolC is important for bacterial survival and oxidative stress response in Salmonella enterica serovar Choleraesuis in an acidic environment. Veterinary microbiology, 193, 42–48. https://doi.org/10.1016/j.vetmic.2016.08.006