Unexpected implications from the Helicobacter pylori genome for understanding periplasmic c‐type cytochrome assembly in Gram‐negative bacteria in coexistence with disulphide bond formation

PDZ domains are named after the three eukaryotic proteins (post-synaptic density protein, disc large and zo-1 proteins) in which they were first found and are also termed DHR or GLGF domains. They span 80–100 amino acids and have now been found in several dozen eukaryotic signalling proteins, where they mediate a range of protein–protein interactions by binding to short sequences usually at the C-termini of target polypeptides (for reviews, see Fanning and Anderson, 1996, Curr Biol 6: 1385– 1388; Ponting et al., 1997, BioEssays 19: 469–479). Many proteins containing PDZ domains are associated with the plasma membrane, in which the PDZ domains mediate the assembly of multiprotein complexes that may initiate signal transduction (for a recent example, see Tsunoda et al., 1997, Nature 388: 243–249). One of us recently described the presence of PDZ domains in five groups of bacterial proteins (Ponting, 1997, Protein Sci 6: 464– 468; Ponting et al., 1997, BioEssays in press). Using the new methodology of hidden Markov models (HMM; Eddy, 1996, Curr Opin Struct Biol 6: 361–365), we have now discovered PDZ domains in an additional important family of bacterial proteins. Here, we present these novel observations and attempt to place these and previous findings in a functional context. An alignment of PDZ domains from HtrA and Prc homologues was used to construct a Hidden Markov model using the HMMer suite of programs (Eddy, 1996, Curr Opin Struct Biol 6: 361–365). This model was used to search the NCBI’s non-redundant protein database using a PAM prior weight of 2000 and a cut-off of 25 bits. Members of all five previously described bacterial PDZ protein families were detected, as well as 81 PDZ domains from animal proteins. However, members of a sixth family of bacterial proteins were also found to contain PDZ domains – the GspC proteins. This result was substantiated by an additional search using a HMM constructed from an alignment of putative PDZ domains from GspC proteins. The GspC family of proteins (members include PulC, OutC and XcpP – see Prosite entry PDOC00878) is represented in several species of Gram-negative bacteria, where they are involved in protein export from the cell via type II secretion systems (also termed the main terminal branch of the general secretory pathway – for a review, see Pugsley, 1993, Microbiol Rev 57: 50–108). We found PDZ domains in nine of 11 known members of the GspC family (see Fig. 1), including two proteins from Escherichia coli. The XcpP proteins of Pseudomonas aeruginosa and Pseudomonas putida lack PDZ domains, despite showing similarities to GspC proteins in their N-terminal regions. Proteins exported by type II secretion systems include extracellular enzymes and toxins (cholera toxin, exotoxin A of P. aeruginosa). Export involves an initial Sec-dependent translocation across the cytoplasmic membrane, followed by a second step, in which proteins are secreted across the outer membrane to be released into the extracellular milieu. This second stage involves a large complex of proteins, most of which are of unknown function. Recent evidence (Thomas et al., 1997, Microbiology 143: 713–720) suggests that the GspC proteins span the cytoplasmic membrane and that each consists of a short cytoplasmic sequence, a single membrane-spanning helix, followed by a large hydrophilic region, which protrudes into the periplasm. This periplasmic region consists of two domains, of which the PDZ domain is the most Cterminal. A recent paper (Lindeberg et al., 1996, Mol Microbiol 20: 175–190) suggested that the GspC proteins are the gatekeepers of the type II pathway, responsible for recognizing proteins destined for export. Our identification of a PDZ domain within the GspC proteins suggests a mechanism for this specificity, namely a protein–protein interaction involving recognition by the PDZ domain of short sequences internal to or at the C-terminus of exported proteins. SpoIVB of Bacillus subtilis is a 427-residue protein that contains a single PDZ domain spanning residues 100–186 (Ponting, 1997, Protein Sci 6: 464–468). It is the key signalling component of the sigma-K checkpoint in sporulating cells (for a review on sporulation in B. subtilis, see Errington, 1993, Microbiol Rev 57: 1–33), coupling sigmaG-dependent gene expression in the forespore to proteolytic activation of sigma-K in the mother cell (Gomez et al., 1995, J Bacteriol 177: 4825–4827). To do this, SpoIVB crosses at least one membrane of the forespore protoplast to interact with a multiprotein complex consisting of SpoIVFA, SpoIVFB and BofA. SpoIVFA and BofA inhibit the activity of SpoIVFB – thought to be the protease that processes sigma-K – until the arrival of SpoIVB (Lu et al., 1995, J Bacteriol 177: 108–185; Cutting et al., 1990, Cell 62: 239–250). SpoIVB also interacts with an additional forespore protein, BofC (Gomez and Cutting, 1997, Microbiology 143: 157–170). SpoIVB’s PDZ domain is likely to be involved in the interactions between SpoIVB, the multiprotein complex and/or BofC. Molecular Microbiology (1997) 26(2), 411–415