Structure of the atypical bacteriocin pectocin M2 implies a novel mechanism of protein uptake

Abstract

The colicin-like bacteriocins are potent protein antibiotics that have evolved to efficiently cross the outer membrane of Gram-negative bacteria by parasitizing nutrient uptake systems. We have structurally characterized the colicin M-like bacteriocin, pectocin M2, which is active against strains of Pectobacterium spp. This unusual bacteriocin lacks the intrinsically unstructured translocation domain that usually mediates translocation of these bacteriocins across the outer membrane, containing only a single globular ferredoxin domain connected to its cytotoxic domain by a flexible α-helix, which allows it to adopt two distinct conformations in solution. The ferredoxin domain of pectocin M2 is homologous to plant ferredoxins and allows pectocin M2 to parasitize a system utilized by Pectobacterium to obtain iron during infection of plants. Furthermore, we identify a novel ferredoxin-containing bacteriocin pectocin P, which possesses a cytotoxic domain homologous to lysozyme, illustrating that the ferredoxin domain acts as a generic delivery module for cytotoxic domains in Pectobacterium.

Pectocin M1 (PcaM1) Inhibits Escherichia coli Cell Growth and Peptidoglycan Biosynthesis through Periplasmic Expression

“…The fast migrating species could represent a more compact and, thus, a more SDS-resistant form of the protein. It is tempting to link this observation to the two protein populations (compact and extended forms) that have been previously revealed by SAXS experiments on pectocin M2 by Grinter and collaborators [15]. MALDI-TOF mass spectrometry analyses of the wild-type PcaM1 revealed only one major peak at m/z 30,266 Da for the [M + H] + ion, which is in agreement with the theoretical mass of 30,401 Da calculated for the His 6 -tagged protein and with the loss of the N-terminal methionine residue (Supplementary Materials Figure S2).…”

“…During the last few years, several ColM orthologues have been identified, by sequence alignments of their C-terminal domains, in some other bacterial genera, such as Pseudomonas , Burkholderia and Pectobacterium species, and several of them have been both biochemically and structurally characterized [11,12,13,14,15]. All of the purified ColM orthologues displayed the same enzymatic activity of cleavage of lipid II and a bacteriolytic (or at least bacteriostatic) activity [11,12].…”

“…On the other hand, the structure of pectocin M2 from Pectobacterium carotovorum and probably also that of pectocin M1, since it has been strongly suggested that both pectocins M1 and M2 used the same outer membrane receptor on Pectobacterium spp. [14], presented an atypical translocation domain, where the helical receptor-domain and unstructured N-terminus were replaced by a single globular plant-like [2Fe-2S] ferredoxin domain, directly connected to the cytotoxic one through a small α-helix linker [15]. Moreover, while numerous receptors for colicins and closely-related pyocins were Tol- or TonB-dependent, these ferredoxin-containing pectocins presumably used a bacterial ferredoxin uptake mechanism to cross the outer membrane, without any evidence for Tol or TonB complex requirements [15].…”

Structure of the bacterial plant-ferredoxin receptor FusA

“…Whether the intact ferredoxin is then imported by FusA or somehow the iron sulphur cluster is liberated at the bacterial cell surface remains to be proven. However, the fact that the ferredoxin-containing pectocins are able to cross the outer membrane and enter the periplasm, through interaction with FusA suggests that importation of the intact ferredoxin is plausible26. Importation of the ferredoxin by FusA is also reasonable given then interior dimensions of the FusA barrel (Fig.…”

“…Pectocins M1 and M2, which are produced by phytopathogenic Pectobacterium spp. for intra-species competition, contain a cytotoxic domain that is active against the cell wall precursor lipid II in the periplasm, fused to an iron-containing plant-like ferredoxin that acts as a receptor-binding domain13. During our characterization of the pectocins, it became apparent that in addition to being susceptible to a ferredoxin-containing bacteriocin, Pectobacterium spp.…”