Functional domains of S-type pyocins deduced from chimeric molecules

Abstract

Functional domain structures of pyocins AP41, Si, and S2 were assigned by examining the functions of chimeric pyocins and deletion derivatives. Pyocins AP41, S1, and S2 are essentially composed of three domains, the receptor-binding domain, the translocation domain, and the DNase domain, in that order from the N terminus to the C terminus. The alignment of these domains is distinct from that in E2-group colicins with functions similar to those of these pyocins. Pyocins AP41 and S2 have a fourth domain between the receptor-binding and the translocation domains, which is dispensable for their killing functions.Pyocins Si, S2, and AP41 are the protease-sensitive bacteriocins most frequently found among Pseudomonas aeruginosa strains (19). They are distinguished by their different receptor specificities. Recently we have cloned and sequenced the genetic determinants for pyocins AP41, S1, and S2 (18,20). Each determinant for these three pyocins constitutes an operon encoding two proteins of different sizes, one responsible for killing (the killing protein) and the other conferring immunity to its own pyocin (the immunity protein). In the 5' upstream region of each operon, a characteristic sequence (a P box), a possible regulatory element for the induced pyocin production, is conserved (10,20). The molecular weights of the killing proteins are different for the pyocins (84,000, 65,600, and 74,000 for pyocins AP41, S1, and S2, respectively), whereas the immunity proteins are of similar sizes, around 10,000. As described previously (20), the amino acid sequences of the C-terminal halves of the killing proteins of pyocins AP41, Si, and S2 are highly conserved. Those of pyocins Si and S2 are identical except for one amino acid deletion (Si) or addition (S2) in this region. Furthermore, the distal ca. 130 amino acids show striking homology to the C-terminal sequences of E2-group colicins and possess DNase activity. The less highly conserved N-terminal halves have been suggested to be receptor-binding domains because these pyocins show different receptor specificities. In addition to causing breakdown of the chromosomal DNA, pyocins Si and S2, but not pyocin AP41, inhibit lipid synthesis in the susceptible bacteria, although their susceptible strains are not the same (16,18,20).In the study described here, we constructed various chimeric pyocins and domain-deleted pyocins on the basis of their sequence conservation and attempted to determine the functions of each domain by examining their functions in vivo and in vitro.JM109 (26), and MV1304 (25) were used as host strains. For preparation of pyocins and chimeric proteins, E. coli C600 carrying the appropriate plasmids was employed. P. aeruginosa PML1516d (SiS S2') (11) and its derivatives PML1567 (S1l S2r) (13) and PML1570 (Slr S2') (this study) were used as indicators for pyocins S1 and S2, and PA03092 (17) was used as an indicator for pyocin AP41. NIH3 (6) and its derivatives NIH3S1r, NIH3S2r, and NIH3AP41r (this study)were also used to determine the receptor specificity....

The Crystal Structure of the Lipid II-degrading Bacteriocin Syringacin M Suggests Unexpected Evolutionary Relationships between Colicin M-like Bacteriocins

“…Similarly, bacteriocins from distantly related species frequently share homologous cytotoxic domains but unrelated translocation and receptor binding domains. For example, colicin E9 and pyocin S2 share sequence homology within their C-terminal cytotoxic domains, but sequences of the translocation and receptor binding domains appear to be unrelated (17). A variation on this mechanism of bacteriocin evolution is illustrated by the recently described pectocins M1 and M2.…”

Pore-forming pyocin S5 utilizes the FptA ferripyochelin receptor to kill Pseudomonas aeruginosa

“…Similar to the organization of colicins genes, the pyocin loci encode two protein components: a larger protein bearing the killing activity and a smaller protein conferring immunity towards the producing bacteria (Duport et al, 1995;Elfarash et al, 2012;Ling et al, 2010;Michel-Briand & Baysse, 2002;Sano & Kageyama, 1984;Sano et al, 1993). All S-type pyocins are composed of three or four domains.…”

Microbial defense systems in foods and feeds

“…The killing domain and the immunity region, form part of the remaining colicin protein which is a short sequence and takes part in immunity protein binding. Pyocins produced by Pseudomonas aeruginosa are also similar to colicin structure but the order of translocation and receptor recognition domains are switched (Sano et al, 1993).…”

“…Studies by Pugsley, (1984a) revealed that colicins have a mass of 29 to 90 KDa with receptors for specificity, translocation, or killing activity. Sano et al (1993) later found that pyocins secreted by P. aeruginosa have similar domain constructs. Bacteriocins produced by gram-positive bacteria appear supposed to have been formed initially as prepeptides and later separated from the main peptide to form the biologically active molecule.…”