Protein translocation occurs across the energy‐conserving bacterial membrane at the SecYEG channel. The crystal structure of the channel has revealed a possible mechanism for gating and opening. This study evaluates the plug hypothesis using cysteine crosslink experiments in combination with various allelic forms of the Sec complex. The results demonstrate that the SecY plug domain moves away from the center of the channel toward SecE during polypeptide translocation, and further show that the translocation‐enhancing prlA3 mutation and SecG subunit change the properties of channel gating. Locking the plug in the open state preactivates the Sec complex, and a super‐active translocase can be created when combined with the prlA4 mutation located in the pore of the channel. Dimerization of the Sec complex, which is essential for translocase activity, relocates the plug toward the open position. We propose that oligomerization may result in SecYEG cooperative interactions important to prime the translocon function.

Previous studies have shown that the SecY plug is displaced from the center of the SecYEG channel during polypeptide translocation. The structural and functional consequences of the deletion of the plug are now examined. Both in vivo and in vitro observations indicate that the plug domain is not essential to the function of the translocon. In fact, deletion of the plug confers to the cell and to the membranes a Prl-like phenotype: reduced proton-motive force dependence of translocation, increased membrane insertion of SecA, diminished requirement for functional leader peptide, and weakened SecYEG subunit association. Although the plug domain does not seem essential, locking the plug in the center of the channel inactivates the translocon. Thus, the SecY plug is important to regulate the activity of the channel and to confer specificity to the translocation reaction. We propose that the plug contributes to the gating mechanism of the channel by maintaining the structure of the SecYEG complex in a compact closed state.

Bacterial toxins are important weapons of toxicogenic pathogens. Depending on their origin, structure and targets, they show diverse mechanisms of action and effects on eukaryotic cells. Exolysin is a secreted 170 kDa pore-forming toxin employed by clonal outliers of Pseudomonas aeruginosa providing to some strains a hyper-virulent behaviour. This group of strains lacks the major virulence factor used by classical strains, the Type III secretion system. Here, we review the structural features of the toxin, the mechanism of its secretion and the effects of the pore formation on eukaryotic cells.

The FemX _Wv aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring l -Ala from Ala-tRNA ^Ala to UDP-MurNAc-pentadepsipeptide. FemX _Wv is an attractive target for the development of novel antibiotics, since the side chain is essential for the last cross-linking step of peptidoglycan synthesis. Here, we show that FemX _Wv is highly specific for incorporation of l -Ala in vivo based on extensive analysis of the structure of peptidoglycan. Comparison of various natural and in vitro -transcribed tRNAs indicated that the specificity of FemX _Wv depends mainly upon the sequence of the tRNA although additional specificity determinants may include post-transcriptional modifications and recognition of the esterified amino acid. Site-directed mutagenesis identified cytosines in the G ¹ –C ⁷² and G ² –C ⁷¹ base pairs of the acceptor stem as critical for FemX _Wv activity in agreement with modeling of tRNA ^Ala in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNA ^Ala harboring nucleotide substitutions in the G ³ –U ⁷⁰ wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX _Wv . The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX _Wv could be achieved by targeting the distal portion of tRNA ^Ala for the design of substrate analogues.