Fig. 1. Genetic organization of the P.aeruginosa tat gene cluster. The genetic organization of the P.aeruginosa tat genes is compared with that of E.coli. The percentage identity between the corresponding homologous Tat proteins is indicated. The tatD and tatE genes are not clustered with the other tat genes of P.aeruginosa and E.coli, respectively. In P.aeruginosa, the intergenic region between the tatA and tatB genes is 14 bp long, whereas the start codon of tatC overlaps with the stop codon of tatB. The stop codon of the gene (hisE) encoding a 111 amino acid protein homologous to the A.chroococcum phosphoribosyl ATP pyrophosphatase is located 26 bp upstream from the tatA start codon. The start codon of the gene encoding a 235 amino acid protein homologous to a hypothetical protein from A.haemolyticus overlaps with the stop codon of tatC. The deduced size in amino acids of the gene products is indicated above or under each gene for E.coli and P.aeruginosa, respectively.

Fig. 2. (A) Schematic representation of the amino acid sequence at the junction point of the hybrid RR–pfColA. The twin-arginine signal peptide is represented in bold letters, with the RR residues of the twin-arginine motif in a larger font size. The pfColA amino acid sequence is in italics. The added linker region in the hybrid proteins is indicated in lower case letters. The leader peptidase cleavage site is indicated by an arrow and is followed by the first three residues of the mature TorA protein (ATD). N and C indicate the N- and C-termini of the protein, respectively. (B) Tat-dependent processing of RR–pfColA. The presence (+) or absence (–) of the tat, cai and RR–pfcolA genes for each cell sample is indicated. Whole-cell extracts of E.coli wild-type strains containing pImTc and pRR-pfColA or the vector pMMB67EH, and the E.coli tat mutant (tatABCDE) containing pRR-pfColA were loaded on an 11% acrylamide gel containing SDS, and proteins were separated by electrophoresis and blotted onto nitrocellulose. The immunoblot was revealed using antibodies directed against pfColA (diluted 1:1000). The arrow indicates the position of the precursor (RR–pfColA) and mature (pfColA) proteins. The deduced mol. wts of 26.6 and 22.8 kDa are in agreement with the expected size of RR–pfColA and pfColA, respectively. The asterisk indicates the position of a putative degradation product. Molecular weight markers are indicated on the left.

Fig. 3. N-terminal signal sequences of PlcH and PlcN, protein profiles of the supernatants and haemolytic activities of the wild type and the tatC mutant (PAK-TC). (A) The twin-arginine motif R-R-x-F-(I/L)-(K/R) is represented in bold and upper case letters. The number of residues (aa) in each signal peptide is indicated. The signal peptide cleavage site is indicated by an arrow. (B) Proteins from culture supernatants of PAK and PAK-TC strains equivalent to 1 absorption unit of cells were separated on a denaturing SDS–gel containing 11% acrylamide and stained with Coomassie Blue (left panel), or immunoblotted and revealed by using monoclonal antibodies directed against PlcH (right panel). The expected migration position of PlcH and PlcN is indicated by an arrow. The bands analysed by mass spectrometry and missing in the tatC mutant are boxed (PlcN) or indicated with an asterisk (GlpQ). The positions of molecular weight markers (kDa) are indicated on the left. (C) Haemolytic activity on blood agar plates of PAK or PAO1, and the derivative xcpR-Z or plcHR and xcpZ-Q (D40ZQ) mutant strains, respectively.

Fig. 4. Mosaic model of protein secretion via the type II pathway. The Xcp-dependent exoproteins exotoxin A (ETA), containing a Sec-dependent signal peptide (SP), or the phospholipases C (Plc’s), bearing a twin-arginine (RR) signal peptide, are exported across the inner membrane through the Sec and the Tat pathway, respectively. After cleavage of the signal peptides, the exoproteins are recognized in the periplasm by the Xcp system, directed to the secretin XcpQ, and released into the external medium. C = cytoplasm; IM = inner membrane; P = periplasm; OM = outer membrane. Different shades in the Sec and Tat translocons correspond to different subunits.