The thylakoid twin arginine protein translocation (Tat) system operates by a cyclical mechanism in which precursors bind to a cpTatC-Hcf106 receptor complex, which then recruits Tha4 to form the translocase. After translocation, the translocase disassembles. Here, we fine-mapped initial interactions between precursors and the components of the receptor complex. Precursors with (Tmd)Phe substitutions in the signal peptide and early mature domain were bound to thylakoids and photo-cross-linked to components. cpTatC and Hcf106 were found to interact with different regions of the signal peptide. cpTatC cross-linked strongly to residues in the immediate vicinity of the twin arginine motif. Hcf106 cross-linked less strongly to residues in the hydrophobic core and the early mature domain. To determine whether precursors must leave their initial sites of interaction during translocation, crosslinked precursors were subjected to protein transport conditions. tOE17 cross-linked to cpTatC was efficiently translocated, indicating that the mature domain of the precursor can be translocated while the signal peptide remains anchored to the receptor complex.

Signal peptide binding modulates assembly of chloroplast Tha4 onto the twin-arginine translocase cpTatC subunit to assemble a functional protein-conducting pore.

Genes encoding proteins that exhibit similarity to the C-terminal domain of Escherichia coli colicin M were identified in the genomes of some Pseudomonas species, namely, P. aeruginosa, P. syringae, and P. fluorescens. These genes were detected only in a restricted number of strains. In P. aeruginosa, for instance, the colicin M homologue gene was located within the ExoU-containing genomic island A, a large horizontally acquired genetic element and virulence determinant. Here we report the cloning of these genes from the three Pseudomonas species and the purification and biochemical characterization of the different colicin M homologues. All of them were shown to exhibit Mg 2؉ -dependent diphosphoric diester hydrolase activity toward the two undecaprenyl phosphate-linked peptidoglycan precursors (lipids I and II) in vitro. In all cases, the site of cleavage was localized between the undecaprenyl and pyrophospho-MurNAc moieties of these precursors. These enzymes were not active on the cytoplasmic precursor UDP-MurNAc-pentapeptide or (or only very poorly) on undecaprenyl pyrophosphate. These colicin M homologues have a narrow range of antibacterial activity. The P. aeruginosa protein at low concentrations was shown to inhibit growth of sensitive P. aeruginosa strains. These proteins thus represent a new class of bacteriocins (pyocins), the first ones reported thus far in the genus Pseudomonas that target peptidoglycan metabolism.Certain Escherichia coli strains produce and release in the growth medium toxins designated colicins in order to kill competitors belonging to the same species or to related species (8,28,29). The various modes of action of colicins include formation of pores in the cytoplasmic membrane, inhibition of protein synthesis, enzymatic degradation of cellular DNA or 16S rRNA, and interference with cell envelope biosynthesis. Colicins and proteins conferring immunity to the producer are generally encoded by plasmids. Depending on the import pathway they use to enter the cells, the Tol or TonB system, the colicins have been classified in group A or B, respectively. Their lethal action occurs in three steps: binding to a specific outer membrane receptor protein, translocation through the cell envelope, and interaction with the target leading to the bactericidal effect. A specific protein domain corresponds to each of the three steps, and the different colicins display a similar three-domain structural organization (8).Colicin M (ColM) exhibits a unique mode of action, as it is the only colicin known to interfere with peptidoglycan biosynthesis and to cause cell lysis (35). This class B colicin is internalized via the TonB translocation machinery and uses the FhuA outer membrane protein as the receptor. ColM lacks peptidoglycan-degrading activity and acts synergistically with ␤-lactam antibiotics, which inhibit the last polymerization step of peptidoglycan synthesis performed by penicillin-binding proteins (34,35). Since ColM inhibited both peptidoglycan synthesis and lipopolysaccharide O-antigen sy...