Bacteria have mechanisms to export proteins for diverse purposes, including colonization of hosts and pathogenesis. A small number of archetypal bacterial secretion machines have been found in several groups of bacteria and mediate a fundamentally distinct secretion process. Perhaps erroneously, proteins called 'autotransporters' have long been thought to be one of these protein secretion systems. Mounting evidence suggests that autotransporters might be substrates to be secreted, not an autonomous transporter system. We have discovered a new translocation and assembly module (TAM) that promotes efficient secretion of autotransporters in proteobacteria. Functional analysis of the TAM in Citrobacter rodentium, Salmonella enterica and Escherichia coli showed that it consists of an Omp85-family protein, TamA, in the outer membrane and TamB in the inner membrane of diverse bacterial species. The discovery of the TAM provides a new target for the development of therapies to inhibit colonization by bacterial pathogens.

The mechanism(s) by which nuclease colicins translocate distinct cytotoxic enzymes (DNases, rRNases, and tRNases) to the cytoplasm of Escherichia coli is unknown. Previous in vitro investigations on isolated colicin nuclease domains have shown that they have a strong propensity to associate with anionic phospholipid vesicles, implying that electrostatic interactions with biological membranes play a role in their import. In the present work we set out to test this hypothesis in vivo. We show that cell killing by the DNase toxin colicin E9 of E. coli HDL11, a strain in which the level of anionic phospholipid and hence inner membrane charge is regulated by isopropyl ␤-D-thiogalactopyranoside induction, is critically dependent on the level of inducer, whereas this is not the case for pore-forming colicins that take the same basic route into the periplasm. Moreover, there is a strong correlation between the level and rate of HDL11 cell killing and the net positive charge on a colicin DNase, with similar effects seen for wild type E. coli cells, data that are consistent with a direct, electrostatically mediated interaction between colicin nucleases and the bacterial inner membrane. We next sought to identify how membrane-associated colicin nucleases might be translocated into the cell. We show that neither the Sec or Tat systems are involved in nuclease colicin uptake but that nuclease colicin toxicity is instead dependent on functional FtsH, an inner membrane AAA ؉ ATPase and protease that dislocates misfolded membrane proteins to the cytoplasm for destruction.

Escherichia coli possesses a number of specific K(+) influx and efflux systems that maintain an appropriate intracellular K(+) concentration. Although regulatory mechanisms have been identified for a number of these transport systems, the exact mechanism through which K(+) concentration is sensed in the cell remains unknown. In this work we show that Kbp (K(+) binding protein, formerly YgaU), a soluble 16-kDa cytoplasmic protein from Escherichia coli, is a highly specific K(+) binding protein and is required for normal growth in the presence of high levels of external K(+). Kbp binds a single potassium ion with high specificity over Na(+) and other metal ions found in biological systems, although, in common with K(+) transporters, it also binds Rb(+) and Cs(+). Dissection of the K(+) binding determinants of Kbp suggests a mechanism through which Kbp is able to sense changes in K(+) concentration over the relevant range of intracellular K(+) concentrations.

Article:Mosbahi, Khédidja, Lemaître, Christelle, Mobasheri, Hamid et al. (6 more authors) (2002) The cytotoxic domain of colicin E9 is a channel-forming endonuclease. Nature Structural Biology. pp. 476-484. ISSN 1545-9985 https://doi.org/10.1038/nsb797 eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. TakedownIf you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. White Rose Consortium ePrints Repositoryhttp://eprints.whiterose.ac.uk/ This is an author produced version of a paper published in Nature Structural Biology. This paper has been peer-reviewed but does not include final publisher proof-corrections or journal pagination.White Rose Repository URL for this paper: http://eprints.whiterose.ac.uk/archive/00001066/ Citation for the published paper Mosbahi, Khédidja and Lemaître, Christelle and Keeble, Anthony H. and Mobasheri, Hamid and Morel, Bertrand and James, Richard and Moore, Geoffrey R. and Lea, Edward J. A. and Kleanthous, Colin (2002) The cytotoxic domain of colicin E9 is a channel-forming endonuclease. Nature Structural Biology, 9 (6). pp. 476-484. Citation for this paperTo refer to the repository paper, the following format may be used: Mosbahi, Khédidja and Lemaître, Christelle and Keeble, Anthony H. and Mobasheri, Hamid and Morel, Bertrand and James, Richard and Moore, Geoffrey R. and Lea, Edward J. A. and Kleanthous, Colin (2002) Colicin E9 is a 60 kDa toxin that is normally released from colicinogenic bacteria in the form of a heterodimeric complex with its 9.5 kDa immunity protein, Im9 (ref. 14). The immunity protein protects the colicin-producing bacterium from the activity of its own toxin but is jettisoned on entry of the colicin into a susceptible cell 15 . Hence, the form of the toxin tested in the bilayer experiments had the immunity protein removed (see Materials andMethods section). Previous work from our laboratory has shown that this form of the toxin retains complete biological activity 14 . Immunity-free colicin E9 (2 nM) was added to the cis chamber of a bilayer apparatus in 10 mM Tris/HCl buffer at pH 7.5, containing 0.1 M NaCl and 10 mM CaCl 2 , and a potential difference (p.d.) applied across the membrane. Random, fluctuating current was observed that showed evidence of opening and closing events with conductance of the order of ~100 pS, although larger conductance states were also seen ( Fig. 1a). In order to identify the region(s) of the protein responsible for this a...

Iron is a limiting nutrient in bacterial infection putting it at the centre of an evolutionary arms race between host and pathogen. Gram-negative bacteria utilize TonB-dependent outer membrane receptors to obtain iron during infection. These receptors acquire iron either in concert with soluble iron-scavenging siderophores or through direct interaction and extraction from host proteins. Characterization of these receptors provides invaluable insight into pathogenesis. However, only a subset of virulence-related TonB-dependent receptors have been currently described. Here we report the discovery of FusA, a new class of TonB-dependent receptor, which is utilized by phytopathogenic Pectobacterium spp. to obtain iron from plant ferredoxin. Through the crystal structure of FusA we show that binding of ferredoxin occurs through specialized extracellular loops that form extensive interactions with ferredoxin. The function of FusA and the presence of homologues in clinically important pathogens suggests that small iron-containing proteins represent an iron source for bacterial pathogens.