The Colicin E1 TolC Box: Identification of a Domain Required for Colicin E1 Cytotoxicity and TolC Binding

Abstract

Colicins are protein toxins made by Escherichia coli to kill related bacteria that compete for scarce resources. All colicins must cross the target cell outer membrane in order to reach their intracellular targets. Normally, the first step in the intoxication process is the tight binding of the colicin to an outer membrane receptor protein via its central receptor-binding domain. It is shown here that for one colicin, E1, that step, although it greatly increases the efficiency of killing, is not absolutely necessary. For colicin E1, the second step, translocation, relies on the outer membrane/transperiplasmic protein TolC. The normal role of TolC in bacteria is as an essential component of a family of tripartite drug and toxin exporters, but for colicin E1, it is essential for its import. Colicin E1 and some N-terminal translocation domain peptides had been shown previously to bind in vitro to TolC and occlude channels made by TolC in planar lipid bilayer membranes. Here, a set of increasingly shorter colicin E1 translocation domain peptides was shown to bind to Escherichia coli in vivo and protect them from subsequent challenge by colicin E1. A segment of only 21 residues, the "TolC box," was thereby defined; that segment is essential for colicin E1 cytotoxicity and for binding of translocation domain peptides to TolC. IMPORTANCEThe Escherichia coli outer membrane/transperiplasmic protein TolC is normally an essential component of the bacterium's tripartite drug and toxin export machinery. The protein toxin colicin E1 instead uses TolC for its import into the cells that it kills, thereby subverting its normal role. Increasingly shorter constructs of the colicin's N-terminal translocation domain were used to define an essential 21-residue segment that is required for both colicin cytotoxicity and for binding of the colicin's translocation domain to bacteria, in order to protect them from subsequent challenge by active colicin E1. Thus, an essential TolC binding sequence of colicin E1 was identified and may ultimately lead to the development of drugs to block the bacterial drug export pathway.KEYWORDS TolC, colicins, drug efflux, membrane translocation, toxins E scherichia coli competes for scarce resources by making plasmid-encoded protein toxins called colicins, which efficiently kill closely related bacteria. All of the few dozen or so colicins that have been identified kill their targets by one of a few basic mechanisms: (i) making an ion-permeable channel in the inner membrane of the target cell, which depolarizes and kills the cell (1); (ii) enzymatically cleaving its rRNA, tRNA, or DNA in the cytoplasm (2, 3); or (iii) degrading peptidoglycan precursors in the periplasm (4, 5). Regardless of their ultimate killing mechanism, all colicins must cross at least the outer membrane in order to reach their targets. The way one particular colicin, E1, makes that transit is the subject of this study.Colicins have a well-defined domain structure, with the killing (catalytic or channelforming) domain at the ...

Colicin E1 opens its hinge to plug TolC

“…3 A ). This observation is notable in light of the fact that, in previous studies, similar peptides were shown to bind TolC (18, 20) and to occlude the channel, as reflected in diminished conductance (18, 19). These studies, however, did not investigate the effects of colicin E1 fragments on the efflux function of the pump.…”

“…In particular, peptides including residues 100-120 of colicin E1 (termed the ‘TolC box’), with additional amino acids at the C-terminus of the ‘box’, have been shown to co-elute with TolC (18) and to disrupt channel conductance (19). A subsequent study confirmed the identity of the minimal T-domain segment that binds TolC, showing that E. coli exposure to peptides comprising residues 100–120 is sufficient to prevent subsequent binding of full-length colicin E1 (as reflected in the fact that the bacteria were protected from the colicin’s cytotoxic effects (20)). Notably, none of these studies investigated the effects of exposure to colicin E1 fragments on TolC’s efflux activity.…”

Dead cells release a ‘necrosignal’ that activates antibiotic survival pathways in bacterial swarms

“…Another curious finding is the structural homology of AcrA to colicin E3 (Fig. 7a-c, RMSD = 0.85 Å), which interacts with BtuB (vitamin B12 receptor), a β-barrel OM protein similar to TolC 52,53 . We have used the colicin homology to suggest possible modes of AcrA binding to TolC (Fig.…”

Rapid production and characterization of antimicrobial colicins using Escherichia coli-based cell-free protein synthesis

“…Over 20 colicins have been studied and the functional diversity of colicins offers unique cell-killing mechanisms ( 1 ). For example, colicins Ia ( 5 ) and E1 ( 6 ) form pores on the inner membrane, colicin M inhibits cell-wall biosynthesis by degrading an undecaprenyl phosphate-linked peptidoglycan precursor ( 7 ), colicin E2 degrades DNA templates ( 8 ) and colicin E3 degrades 16S ribosomal RNA resulting in slow translation ( 9 ). Therefore, colicins can target specific microbes by penetrating cells and disrupting cellular components.…”