The mechanism by which enzymatic E colicins such as colicin E3 (ColE3) and ColE9 cross the outer membrane, periplasm, and cytoplasmic membrane to reach the cytoplasm and thus kill Escherichia coli cells is unique in prokaryotic biology but is poorly understood. This requires an interaction between TolB in the periplasm and three essential residues, D35, S37, and W39, of a pentapeptide sequence called the TolB box located in the N-terminal translocation domain of the enzymatic E colicins. Here we used site-directed mutagenesis to demonstrate that the TolB box sequence in ColE9 is actually larger than the pentapeptide and extends from residues 34 to 46. The affinity of the TolB box mutants for TolB was determined by surface plasmon resonance to confirm that the loss of biological activity in all except one (N44A) of the extended TolB box mutants correlates with a reduced affinity of binding to TolB. We used a PCR mutagenesis protocol to isolate residues that restored activity to the inactive ColE9 D35A, S37A, and W39A mutants. A serine residue at position 35, a threonine residue at position 37, and phenylalanine or tyrosine residues at position 39 restored biological activity of the mutant ColE9. The average area predicted to be buried upon folding (AABUF) was correlated with the activity of the variants at positions 35, 37, and 39 of the TolB box. All active variants had AABUF profiles that were similar to the wild-type residues at those positions and provided information on the size, stereochemistry, and potential folding pattern of the residues of the TolB Box.

Enzymatic colicins such as colicin E9 (ColE9) bind to BtuB on the cell surface of Escherichia coli and rapidly recruit a second coreceptor, either OmpF or OmpC, through which the N-terminal natively disordered region (NDR) of their translocation domain gains entry into the cell periplasm and interacts with TolB. Previously, we constructed an inactive disulfide-locked mutant ColE9 (ColE9 s-s ) that binds to BtuB and can be reduced with dithiothreitol (DTT) to synchronize cell killing. By introducing unique enterokinase (EK) cleavage sites in ColE9 s-s , we showed that the first 61 residues of the NDR were inaccessible to cleavage when bound to BtuB, whereas an EK cleavage site inserted at residue 82 of the NDR remained accessible. This suggests that most of the NDR is occluded by OmpF shortly after binding to BtuB, whereas the extreme distal region of the NDR is surface exposed before unfolding of the receptor-binding domain occurs. EK cleavage of unique cleavage sites located in the ordered region of the translocation domain or in the distal region of the receptor-binding domain confirmed that these regions of ColE9 remained accessible at the E. coli cell surface. Lack of EK cleavage of the DNase domain of the cell-bound, oxidized ColE9/Im9 complex, and the rapid detection of Alexa Fluor 594-labeled Im9 (Im9 AF ) in the cell supernatant following treatment of cells with DTT, suggested that immunity release occurred immediately after unfolding of the colicin and was not driven by binding to BtuB.During times of nutrient stress, many bacteria synthesize and release a range of bacteriocins that target and kill related organisms. Presumably, this gives them greater access to the limited food supply and improves their chances of survival over "unarmed" neighbors occupying the same ecological niche (36). Escherichia coli is no exception and produces a range of toxic bacteriocins, called colicins, that target other enterobacteriaceae (9). Colicins are bactericidal antibiotics that (i) bind to one or more ␤-barrel-shaped outer membrane receptors of target cells (that are principally involved with the uptake of small metabolite growth factors), (ii) translocate across the cell envelope via protein-protein interactions with host periplasmic proteins, and (iii) kill the host bacterium by either depolarizing the cytoplasmic membrane, inhibiting murein biosynthesis or degrading the host's nucleic acids.A combination of deletion mapping and structural studies (12,21,31,32,39,42) have shown that each step in the colicin killing process is accomplished by one of three separate domains of the colicin that tend to be separated by a short stretch of residues that act as a linker. Receptor binding occurs via a centrally located receptor-binding domain (i.e., the R domain). Translocation is via a N-terminal natively disordered region (NDR) joined to an ordered region making up the translocation domain (i.e., the T domain), while cell killing is achieved by a C-terminally located cytotoxic domain. Enzymatic colicins differ from pore-...

Bacteriocins are proteins produced by bacteria to destroy other bacteria occupying their ecological niche. Photorhabdus luminescens is an insect pathogenic bacterium carried by an entomopathogenic nematode and occupies several different niches in its life cycle. The nematode enters the insect and releases a single strain of P. luminescens. The bacteria then kill the host and the bacteria and nematodes replicate within the cadaver. Strikingly, at the end of the infection the cadaver is still occupied by a single strain of bacterium, suggesting that P. luminescens can destroy other bacteria entering, or present within, the insect. Here we describe four loci encoding 'lumicins' in P. luminescens subsp. akhurstii strain W14. The lumicins are novel bacteriocins capable of killing other strains of Photorhabdus and Escherichia coli. These loci predict killer proteins and multiple dual type immunity proteins with domains similar to pyocins and colicins. The killer proteins are chimeric in nature with multiple domains, one of which is similar to the uropathogenic-specific protein (USP) described from uropathogenic E. coli. The implications of these novel bacteriocins for the lifestyle of Photorhabdus and the potential role of USP as a bacteriocin in E. coli are discussed.