It is more than 80 years since Gratia first described 'a remarkable antagonism between two strains of Escherichia coli'. Shown subsequently to be due to the action of proteins (or peptides) produced by one bacterium to kill closely related species with which it might be cohabiting, such bacteriocins have since been shown to be commonplace in the internecine warfare between bacteria. Bacteriocins have been studied primarily from the twin perspectives of how they shape microbial communities and how they penetrate bacteria to kill them. Here, we review the modes of action of a family of bacteriocins that cleave nucleic acid substrates in E. coli, known collectively as nuclease colicins, and the specific immunity (inhibitor) proteins that colicin-producing organisms make in order to avoid committing suicide. In a process akin to targeting in mitochondria, nuclease colicins engage in a variety of cellular associations in order to translocate their cytotoxic domains through the cell envelope to the cytoplasm. As well as informing on the process itself, the study of nuclease colicin import has also illuminated functional aspects of the host proteins they parasitize. We also review recent studies where nuclease colicins and their immunity proteins have been used as model systems for addressing fundamental problems in protein folding and protein-protein interactions, areas of biophysics that are intimately linked to the role of colicins in bacterial competition and to the import process itself.
Ester hydrolysis is one of the most ubiquitous reactions in biochemistry. Many of these reactions rely on metal ions for various mechanistic steps. A large number of metal dependent nucleases have been crystallized with two metal ions in their active sites. In spite of an ongoing discussion about the roles of these metal ions in nucleic acid hydrolysis, there are very few studies which examine this issue using the native cofactor Mg(II) and global fitting of reaction progress curves. As part of a comprehensive study of the representative homodimeric PvuII endonuclease, we have collected single turnover DNA cleavage data as a function of Mg(II) concentration and globally fit these data to a number of models which test various aspects of metallonuclease mechanism. DNA association rate constants are about 100-fold higher in the presence of the catalytically nonsupportive Ca(II) vs. the native cofactor Mg(II), highlighting an interesting cofactor difference. A pathway in which metal ions bind prior to DNA is kinetically favored. The data fit well to a model in which both one and two metal ions per active site (EM2S and EM4S) support cleavage. Interestingly, the cleavage rate for EM2S is about 100-fold slower than that displayed by EM4S. Collectively, these data indicate that for the PvuII system, catalysis involving one metal ion per active site can indeed occur, but that a more efficient two metal ion mechanism can be operative under saturating metal ion (in vitro) conditions.
Background: TolR forms part of the proton-motive force (pmf)-linked stator of the Tol-Pal complex in bacteria.Results: Structural and biophysical analysis shows the periplasmic domain of TolR to be a strand-swapped dimer.Conclusion: Strand swapping blocks peptidoglycan binding by TolR.Significance: Pmf-linked stator proteins must disassemble strand-swapped dimer structures to traverse the periplasm and bind peptidoglycan.
Gram-negative bacteria depend on energised protein complexes that connect the two membranes of the cell envelope. However, β-barrel outer-membrane proteins (OMPs) and α-helical inner-membrane proteins (IMPs) display quite different organisation. OMPs cluster into islands that restrict their lateral mobility, while IMPs generally diffuse throughout the cell. Here, using live cell imaging of Escherichia coli, we demonstrate that when transient, energy-dependent transmembrane connections are formed, IMPs become subjugated by the inherent organisation of OMPs and that such connections impact IMP function. We show that while establishing a translocon for import, the colicin ColE9 sequesters the IMPs of the proton motive force (PMF)-linked Tol-Pal complex into islands mirroring those of colicin-bound OMPs. Through this imposed organisation, the bacteriocin subverts the outer-membrane stabilising role of Tol-Pal, blocking its recruitment to cell division sites and slowing membrane constriction. The ordering of IMPs by OMPs via an energised inter-membrane bridge represents an emerging functional paradigm in cell envelope biology.
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