Bacterial flagella are potent immunogens and aromatic-dependent (aro) Salmonella as live vaccines evoke humoral and cellular immune responses. Such strains expressing epitopes of protective antigens as inserts in flagellin would provide a novel way to vaccinate against diseases caused by unrelated pathogens. A synthetic oligonucleotide specifying an epitope of cholera toxin subunit B was inserted in a Salmonella flagellin gene. The chimeric flagellin functioned normally and the epitope was expressed at the flagellar surface. Parenteral administration to mice of an aro A flagellin-negative strain of S. dublin expressing the chimeric flagellin gene evoked antibody to cholera toxin.

SummaryThe siderophore ferric enterobactin enters Escherichia coli through the outer membrane (OM) porin FepA, which contains an aqueous transmembrane channel that is normally occluded by other parts of the protein.After binding the siderophore at a site within the surface loops, FepA undergoes conformational changes that promote ligand internalization. We assessed the participation of different loops in ligand recognition and uptake by creating and analysing a series of deletions. We genetically engineered 26 mutations that removed 9-75 amino acids from nine loops and two buried regions of the OM protein. The mutations had various effects on the uptake reaction, which we discerned by comparing the substrate concentrations of half-maximal binding (K d ) and uptake (K m ): every loop deletion affected siderophore transport kinetics, decreasing or eliminating binding affinity and transport efficiency. We classified the mutations in three groups on the basis of their slight, strong or complete inhibition of the rate of ferric enterobactin transport across the OM. Finally, characterization of the FepA mutants revealed that prior experiments underestimated the affinity of FepA for ferric enterobactin: the interaction between the protein and the ferric siderophore is so avid (K d < 0.2 nM) that FepA tolerated the large reductions in affinity that some loop deletions caused without loss of uptake functionality. That is, like other porins, many of the loops of FepA are superficially dispensable: ferric enterobactin transport occurred without them, at levels that allowed bacterial growth.

The Escherichia coli iron transporter, FepA, has a globular N terminus that resides within a transmembrane ␤-barrel formed by its C terminus. We engineered 25 cysteine substitution mutations at different locations in FepA and modified their sulfhydryl side chains with fluorescein maleimide in live cells. The reactivity of the Cys residues changed, sometimes dramatically, during the transport of ferric enterobactin, the natural ligand of FepA. Patterns of Cys susceptibility reflected energy-and TonB-dependent motion in the receptor protein. During transport, a residue on the normally buried surface of the N-domain was labeled by fluorescein maleimide in the periplasm, providing evidence that the transport process involves expulsion of the globular domain from the ␤-barrel. Porin deficiency much reduced the fluoresceination of this site, confirming the periplasmic labeling route. These data support the previously proposed, but never demonstrated, ball-and-chain theory of membrane transport. Functional complementation between a separately expressed N terminus and C-terminal ␤-barrel domain confirmed the feasibility of this mechanism.FepA is a Gram-negative bacterial outer membrane (OM) 6 protein that transports ferric enterobactin (FeEnt) (1-3). The crystal structures of FepA (4) and other bacterial metal transporters (FhuA, BtuB,57)), contain a C-terminal, 22-stranded ␤-barrel, placing them in the porin superfamily (5). Their ϳ150-residue globular N termini (N-domain; see Fig. 1) reside within their ␤-barrels. This architecture is potentially consistent with the "ball-and-chain" mechanism of membrane transport, whereby the globule controls solute (ligand) uptake by moving in and out of the channel. This process was postulated for nervous system channels (6), but no demonstrated examples of ball-and-chain transport are known.FepA and its relatives are unlike other porins (7, 8), because they selectively adsorb metal chelates with high affinity (3, 9 -14). Ligand binding causes small conformational changes that activate them to transport competency (15-17), hence their designation "ligand-gated porin" (LGP). The requirements for metabolic energy (18 -20) and another cell envelope protein, TonB (21-24), in LGP mediated transport are well known but unaccounted for: the OM has no source of energy and cannot sustain an ion gradient because of its open porin channels (7); TonB is a minor cell envelope protein whose functions are not yet understood.In live cells, FepA binds and transports FeEnt via sub-reactions with different dependences on energy and TonB. (i) In the absence of ligand the receptor opens, and its flexible surface loops extend outward (25). (ii) FeEnt binds to FepA in a biphasic reaction (26) that begins with adsorption to aromatic amino acids in the loop extremities (27, 28). Multiple determinants in multiple loops, including L7 (25), converge on the iron complex, creating a closed conformation that associates the negatively charged (Ϫ3), catecholate iron center with basic and aromatic residues in the r...

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SummaryListeria monocytogenes is a Gram-positive bacterium that causes severe opportunistic infections in humans and animals. We biochemically characterized, for the first time, the iron uptake processes of this facultative intracellular pathogen, and identified the genetic loci encoding two of its membrane iron transporters. Strain EGD-e used iron complexes of hydroxamates (ferrichrome and ferrichrome A, ferrioxamine B), catecholates (ferric enterobactin, ferric corynebactin) and eukaryotic binding proteins (transferrin, lactoferrin, ferritin, haemoglobin). Quantitative determinations showed 10-100-fold lower affinity for ferric siderophores ( K m ª 1-10 nM) than Gramnegative bacteria, and generally lower uptake rates.

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Sortases are transamidases that covalently link proteins to the peptidoglycan of gram-positive bacteria. The genome of the pathogenic bacterium Listeria monocytogenes encodes two sortases genes, srtA and srtB. The srtA gene product anchors internalin and some other LPXTG-containing proteins to the listerial surface. Here, we focus on the role of the second sortase, SrtB. Whereas SrtA acts on most of the proteins in the peptidoglycan fraction, SrtB appears to target minor amounts of surface polypeptides. We identified one of the SrtB-anchored proteins as the virulence factor SvpA, a surface-exposed protein which does not contain the LPXTG motif. Therefore, as in Staphylococcus aureus, the listerial SrtB represents a second class of sortase in L. monocytogenes, involved in the attachment of a subset of proteins to the cell wall, most likely by recognizing an NXZTN sorting motif. The ⌬srtB mutant strain does not have defects in bacterial entry, growth, or motility in tissue-cultured cells and does not show attenuated virulence in mice. SrtB-mediated anchoring could therefore be required to anchor surface proteins involved in the adaptation of this microorganism to different environmental conditions.

Ligand-gated membrane channels selectively facilitate the entry of iron into prokaryotic cells. The essential role of iron in metabolism makes its acquisition a determinant of bacterial pathogenesis and a target for therapeutic strategies. In Gram-negative bacteria, TonB-dependent outer membrane proteins form energized, gated pores that bind iron chelates (siderophores) and internalize them. The time-resolved operation of the Escherichia coli ferric enterobactin receptor FepA was observed in vivo with electron spin resonance spectroscopy by monitoring the mobility of covalently bound nitroxide spin labels. A ligand-binding surface loop of FepA, which normally closes its transmembrane channel, exhibited energy-dependent structural changes during iron and toxin (colicin) transport. These changes were not merely associated with ligand binding, but occurred during ligand uptake through the outer membrane bilayer. The results demonstrate by a physical method that gated-porin channels open and close during membrane transport in vivo.