Sir, Bacteriocins produced by Escherichia coli have been grouped into two major families, namely the nuclease and pore-forming colicins. In the nuclease colicins, the colicin E2 and colicin E3 subfamilies can be distinguished based on their DNase and RNase activities respectively. Pore-forming colicins are divided into the colicin E1 subfamily, the colicin A subfamily and the colicin Ia/Ib subfamily (Braun et al., 1994, Arch Microbiol161: 199–206). The nuclease domain of the RNase colicin E5 exhibits no sequence similarity to those of the E3 subfamily colicins. Colicin E5 has recently been shown to hydrolyse transfer RNAs for Tyr, His, Asn and Asp on the 3′ side of the nucleotide containing the modified base queuine, found at the wobble position of each anticodon (Ogawa et al., 1999, Science283: 2097–2100). A colicinogenic strain acquires specific immunity towards its bacteriocin through the production of an immunity protein that usually interacts with the C-terminal domain of the bacteriocin (Kleanthous et al., 1998, Mol Microbiol28: 227–233). Both killing and immunity proteins are encoded by closely linked plasmid-encoded genes. This genetic organization is also found in the chromosomally encoded S-type pyocins produced by Pseudomonas aeruginosa. Hitherto, the genes for four S-type pyocins (S1, S2, S3 and AP41) have been characterized in different P. aeruginosa strains (Sano et al., 1993, J Bacteriol175: 2907–2916; Sano and Kageyama, 1993, Mol Gen Genet237: 161–170; Duport et al., 1995, J Biol Chem270: 8920–8927). Each of these pyocins is produced as a protein complex consisting of a killing protein with DNase activity and an immunity protein (Fig. 1). The killing proteins are organized in functional domains similar to the E2 colicins. In these colicins, the N-terminal region, involved in translocation, is followed by a receptor recognition domain and a DNase/immunity protein-binding domain. In pyocins, the order of receptor recognition and translocation domains is reversed (Fig. 1). The nuclease domains in pyocins S1, S2 and AP41 share about 50% identity with the nuclease domains of the E2 colicins (Kageyama et al., 1996, J Bacteriol178: 103–110). The C-terminal domain of pyocin S3 and its immunity protein are unrelated to the nuclease domains and cognate immunity proteins of the other pyocins. Pyocins S2, S3 and AP41 contain an additional domain of unknown function (but dispensable for killing) linking the receptor-binding and translocation domains (Sano et al., 1993, J Bacteriol175: 6179–6185). . Homology of killing and immunity proteins (grey shading) of pyocin S4 and S5 complexes with related bacteriocins. The relative positions of the genes and ORFs are indicated by block arrows. The protein sequences of the novel pyocin complexes were deduced from the nucleic acid sequences at positions 4 007 722–4 010 016 (killing protein of pyocin S4), 4 010 016–4 010 354 (immunity protein of pyocin S4), 1 005 444–1 006 940 (killing protein of pyocin S5) and 1 006 965–1 007 291 (complement; immunity protein of pyocin S5) in the Pseudomonas aeruginosa PAO1 genome database (http://www.ncbi.nlm.nih.gov/BLAST/unfinishedgenome.html, last update 14 July 1999). Domain structure is represented by different shading patterns (see inset), which do not necessarily imply significant domain homology (Sano et al., 1993, J Bacteriol 175: 6179–6185; Duport et al., 1995, J Biol Chem 270: 8920–8927). Regions in the killing proteins with high homology (> 45% identity) are linked by dashed lines. No homology was detected for the non-shaded region in pyocin S5. Only the sequence of the C-terminal part of colicin E5 has been determined (Lau and Condie, 1989, Mol Gen Genet 217: 269–277). The N-terminal extension in the immunity protein of colicin E5 identified by Ogawa et al. (1999, Science 283: 2097–2100) is included. The availability of the P. aeruginosa PAO1 genomic sequence prompted us to search for additional pyocin genes. Homology searches with the four S-type pyocins and the cognate immunity proteins confirmed the presence of the S2 pyocin gene, but also revealed a novel pyocin gene. The deduced protein sequence (764 amino acids) shows the highest level of overall homology with pyocin S3 (71% identity). Similar to pyocins AP41, S2 and S3, this new pyocin (designated pyocin S4) is composed of four domains. However, pyocin S4 has a novel composite domain structure (Fig. 1). Surprisingly, its C-terminal domain does not show homology with the corresponding regions of the known S pyocins, but with the C-terminal region of the tRNase colicin E5 (Fig. 1). The conserved operon structure of other pyocin genes was also found for pyocin S4 (Fig. 1). Consistent with the identification of a colicin E5-like tRNase domain in pyocin S4, its putative immunity protein (112 amino acids) showed significant homology with the immunity protein of colicin E5. The P. aeruginosa PAO1 genome appears to encode yet another novel type of pyocin and linked immunity protein, pyocin S5 (498 amino acids), containing only a short region with high homology to pyocin S2 (Fig. 1). The C-terminal domain is homologous to the active domain of the pore-forming colicins Ia and Ib. These colicins kill bacterial cells by the formation of voltage-gated, ion- conducting channels across the plasma membrane (Wiener et al., 1997, Nature385: 461–464). Downstream of the pyocin S5 gene, we identified an open reading frame (ORF) for the probable immunity protein (108 amino acids), showing significant homology with the colicins Ia and Ib immunity proteins (Fig. 1). The presence of three potential transmembrane helices in the pyocin S5 immunity protein (TMpred; http://www.ch.embnet.org/software/TMPRED_ form.html) is consistent with the predicted channel-forming activity of the S5 pyocin. Immunity proteins for pore-forming colicins reside in the inner membrane, enabling specific intramembrane helix–helix interactions with the toxins (Cramer et al., 1995, Annu Rev Biophys Biomol Struct24: 611–641; Espesset et al., 1996, EMBO J15: 2356–2364). A P box, a conserved regulatory sequence found in the promoter regions of the four known S pyocins (Matsui et al., 1993, J Bacteriol178: 103–110), was also identified in the promoter region of the pyocin S4 operon (positions −124 to −76 relative to the start codon of the killing gene). No such P box is present in the pyocin S5 promoter region, indicating that pyocin S5 expression is probably regulated differently from the other pyocins. In conclusion, our analysis shows that, in addition to the four known pyocins with DNase activity, a potential pore-forming pyocin and a colicin E5-like pyocin are produced by P. aeruginosa. Hitherto, the specific tRNase activity of colicin E5 was considered unique among bacteriocins. Furthermore, it indicates that domain swapping, well documented for colicins, has also played an important role in the evolution of bacteriocins in P. aeruginosa.
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Parret et al. (2000) studied this question.