ÓRGANOS DE COORDINACIÓN DOCENTE

Colicins begin their journey into an E. coli cell by initially binding with high affinity to an OM receptor (Di Masi, White et al. 1973). Most receptors used by colicins are monomeric, 22-strand -barrel proteins that habitually transport small metabolites. In addition, outer membrane porins such as OmpF often act as co-receptors for colicins and are referred to as translocator proteins. The events that take place after colicin localization to the cell surface have not been fully elucidated but presumably require the assembly of what has been described as a translocon (Kurisu, Zakharov et al. 2003). The translocon assembly is thought to be facilitated by an unstructured region of the T- domain, which recruits the OM translocator protein to deliver at least part of the colicin T-domain into the periplasm of the target E. coli cell, where it can recruit the Tol proteins, allowing the entry of the cytotoxic domain through the OM (Cao and Klebba 2002; Housden, Loftus et al. 2005). It is still yet to be demonstrated if the entire colicin molecule is translocated into the cell but recent evidence has suggested that the cytotoxic domain is cleaved through FtsH mediated catalysis before it crosses the cytoplasmic membrane (Chauleau, Mora et al. 2011). The tol-dependent translocation system consists of the proteins TolA, TolB, TolQ, TolR and Pal (James, Penfold et al. 2002), which are all important for group A colicin entry. E. coli strains containing a mutation in the tol genes are tolerant of group A colicins. The outer membrane-associated lipoprotein Pal interacts with the periplasmic TolB protein (Bouveret, Derouiche et al. 1995). A number of recent studies of the Tol-Pal proteins have provided useful information on the structure/function relationships between Tol proteins and colicin biology (Cascales, Buchanan et

al. 2007). One approach to study in vivo interactions between colicins and components of the Tol–Pal or TonB systems has been the expression and secretion of colicins and/or colicin domains to the cell periplasm followed by colicin challenge of those cells from the external mileau. The aim of this technique is to perturb the function of individual proteins of the Tol or Ton systems to determine their role in the uptake of the colicin molecule. This can be extended to include mutated colicins to investigate important residues in these processes. Also it is a useful tool to examine the diverse interaction between colicins or bacteriophage g3p protein of filamentous bacteriophages with the translocation machineries. The first observation that the periplasmic production of interacting proteins perturbed the Tol–Pal system was from strains that were infected with filamentous bacteriophages which displayed a tol phenotype. These strains behaved like tol mutants showing a tolerance to group A colicins; having leaky phenotypes and hypersensitivity to drugs and detergents (Zinder 1973; Smilowitz 1974) that they normally would be resistant too. As these bacteriophages require the TolA-Q-R proteins to infect sensitive cells, it was shown that expression of g3p and secretion into the periplasm could create leakiness of cell contents through inhibition of the normal functioning of the Tol proteins. This approach was extended to examine the role of Tol proteins on colicin biology (Cascales, Buchanan et al. 2007). Thus by analogy, expressed and secreted colicin translocation domains should interact with the cell’s translocation machinery in the periplasm, thereby disturbing the normal functioning of this cellular system and rendering the producing cells less sensitive or ’tolerant’ to colicins of the same group attacking the cells from the external environment. Moreover, proteins of the

Tol or TonB systems could be secreted to the periplasm and subsequently preventing colicin action either by directly trapping the imported colicin to non-functional Tol or by disturbing the function of the envelope systems due to the presence of non-functional protein-interactions in the periplasm, and thus preventing the translocation process. Overexpressed TonB fragments suggested that TonB interfered with the receptors interaction in E. coli and yet abolished sensitivity of cells to bacteriophage 80 and colicin M, both of which use FhuA as the receptor, these results were obtained using the periplasmic production technique (Howard, Herrmann et al. 2001). Secretion of TolAIII, colicins or g3p domains in the periplasm of wild-type cells produce a tol-associated phenotype (Henry, Pommier et al. 2004), and it has been postulated that these domains may alter outer membrane integrity by altering the stability of the Tol-Pal interactions (Bouveret, Journet et al. 2002). Moreover, sequestration of active TolA-TolQ-TolR complexes found to be due to the periplasmic overexpressed TolR molecules (Journet, Rigal et al. 1999). Using this technique allowed identification of an interaction between TolABR and ColK T-domain and for the first time an interaction with TolQ protein (Barneoud-Arnoulet, Gavioli et al. 2010). Generally cells secreting the T- domains of group A colicins become tolerant specifically to group A colicins, while cells secreting the T-domains of group B colicins become tolerant specifically to group B colicins. Cells producing Tol or TonB domains become tolerant specifically to colicins or phages that require the same translocation machinery for cell entry (Bouveret, Rigal et al. 1997; Bouveret, Journet et al. 2002).

One of the problems of studying enzymatic colicins in the past has been the lack of a rapid and quantifiable assay for the determination of cell killing that would be necessary to study the effects of periplasmic expression of Tol proteins or colicin domains. An approach for monitoring cell killing through DNA damage was developed that incorporated a SOS promoter-lux fusion reporter system. Furthermore, because the reporter assay enables a quantitative assessment of colicin activity, the effect of individual mutations on the biological activity of colicin (Vankemmelbeke, Healy et al. 2005) can be compared. The reporter assay has recently been used to show the importance of the Tol proteins in the release of immunity protein from the catalytic domains of enzymatic colicins (Vankemmelbeke, Zhang et al. 2009).

This chapter describes a method for expressing and secreting colicin domains into the cell periplasm to monitor the interaction between the Tol system and externally added colicins. Using this ‘periplasmic expression technique’ to perturb the function of the Tol system in the cell envelope, interactions of secreted colicin domains with the Tol proteins can be monitored by the patterns of sensitivity/resistance of the cells to externally added colicins. It is shown that expression and secretion of the translocation domains of colicins E9 and A protect cells from externally added ColE9 and A through interactions of those domains with Tol proteins which prevent subsequent interactions of these proteins with the translocation domains of the externally added colicins. In addition competitive inhibition of TolA has a larger effect on cell resistance than inhibition of TolB providing further insights into the role of different Tol proteins in the translocation of different colicins.