Distinct colicin M-like bacteriocin-immunity pairs in Burkholderia

Abstract

The Escherichia coli bacteriocin colicin M (ColM) acts via degradation of the cell wall precursor lipid II in target cells. ColM producers avoid self-inhibition by a periplasmic immunity protein anchored in the inner membrane. In this study, we identified colM-like bacteriocin genes in genomes of several β-proteobacterial strains belonging to the Burkholderia cepacia complex (Bcc) and the Burkholderia pseudomallei group. Two selected Burkholderia ambifaria proteins, designated burkhocins M1 and M2, were produced recombinantly and showed antagonistic activity against Bcc strains. In their considerably sequence-diverged catalytic domain, a conserved aspartate residue equally proved pivotal for cytotoxicity. Immunity to M-type burkhocins is conferred upon susceptible strains by heterologous expression of a cognate gene located either upstream or downstream of the toxin gene. These genes lack homology with currently known ColM immunity genes and encode inner membrane-associated proteins of two distinct types, differing in predicted transmembrane topology and moiety exposed to the periplasm. The addition of burkhocins to the bacteriocin complement of Burkholderia reveals a wider phylogenetic distribution of ColM-like bacteriotoxins, beyond the γ-proteobacterial genera Escherichia, Pectobacterium and Pseudomonas, and illuminates the diversified nature of immunity-providing proteins.

A Colicin M-Type Bacteriocin from Pseudomonas aeruginosa Targeting the HxuC Heme Receptor Requires a Novel Immunity Partner

“…The catalytic motif in the ColM domain of PaeM4 can be recognized as DxYD(x 5 )QR, slightly deviating from the equivalent motif from colicin M and PaeM, DxYD(x 5 )HR (27,31,34). However, it was found previously that limited variation in this sequence motif does not necessarily affect the bacteriocin function of ColM-type toxins (29). Furthermore, the presence of a proline-rich TonB box at the amino terminus of PaeM4 suggests that this pyocin likely targets a TonBdependent receptor (27,35), as was demonstrated previously for PaeM (32) and several other modular S-type pyocins (6).…”

“…3A) (36). By genome analysis, a putative ColM immunity partner, PmiB, with a periplasmic domain was previously identified in a small set of pseudomonads; however, no pmiB immunity genes can be retrieved from P. aeruginosa genomes (27,29) (Fig. 1).…”

“…The paeM4 gene (locus tag Q057_04089, from P. aeruginosa BL03) was cloned in expression vector pET28a(ϩ). Previously, it was found that a coexpressed immunity gene protecting cells from colicin M toxin function is not needed when such bacteriocins are expressed in the cytoplasm of E. coli (24,28,29); therefore, the candidate immunity gene pmiC was not included in the construct. The bacteriocin gene was cloned to encode a carboxy-terminal His- tagged protein, since an amino-terminal fusion product would interfere with the import-related function of the amino-terminal translocation segment of the pyocin (15,34,35).…”

“…A subset of these modular bacteriocins in pseudomonads are those equipped with a ColM domain (24), a toxin module that was first identified in colicin M of E. coli (25,26) but actually occurs in a wide variety of proteobacterial genera (27), including other gammaproteobacteria (Pectobacterium) (28), as well as betaproteobacteria (Burkholderia) (29). The ColM domain acts in the periplasm and provokes cellular killing through degradation of lipid II (30,31).…”

“…The cell surface target of Pseudomonas ColM␤ bacteriocins remains unknown at this point, and such bacteriocin genes are lacking in P. aeruginosa genomes. Immunity to ColM␤-type bacteriocins was demonstrated in Burkholderia but not yet in Pseudomonas and is provided by an integral membrane protein with 3 TMHs (Burkholderia) or a periplasmic module anchored in the inner membrane (Burkholderia and Pseudomonas) (PmiB) (29). More recently, a phylogenetically distinct P. aeruginosa ColM bacteriocin, PaeM4, was functionally identified (10).…”