Novel Immunity Proteins Associated with Colicin M-like Bacteriocins Exhibit Promiscuous Protection in Pseudomonas

Abstract

Bacteriocins related to colicin M, acting via cleavage of the cell wall precursor lipid II, have been characterized in γ- and β-proteobacteria. Depending on the species, immunity is provided by either an inner membrane-anchored periplasmic protein or by an integral membrane protein. In Pseudomonas however, the immunity partner of colicin M-like bacteriocins remains unknown. Based on an in silico analysis in pseudomonad genomes, we here identify a gene encoding a putative immunity partner that represents a novel type of integral membrane protein (PmiA, Pseudomonas colicin M-like immunity type A). By heterologous expression of pmiA genes in susceptible strains, we show that immunity to colicin M-like bacteriocins is indeed provided by the cognate PmiA. Sequence homology among PmiA proteins is essentially absent, except for a short motif with a conserved periplasm-exposed aspartate residue. However, PmiA's protective function is not abolished by changing this acidic residue to the uncharged alanine. Immunity by PmiAs appears promiscuous to the extent that PmiA homologs from a clade sharing <40% pairwise amino acid identity, equally provide protection against the bacteriocin linked to the original PmiA. This study shows that multiple immunity factors have evolved independently to silence lipid II-targeting enzymatic bacteriocins. Their relaxed bacteriocin immunization capacity contrasts to the strict specificity of immunity proteins shielding the enzymatic domain of nuclease bacteriocins. The nature of associated immune functions needs consideration when using such natural protein antibiotics or designing novel variants.

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

“…4C). Conversely, pmiC could not immunize cells against the PaeM function in P. aeruginosa PA7 (15) (Fig. 4D to F).…”

“…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). After transformation into E. coli BL21(DE3), pyocin gene expression was induced, cells were harvested, soluble proteins were isolated, and the recombinant protein was purified (Fig.…”

“…To circumvent suicidal expression in producer strains, immunity genes are expressed concomitantly with the bacteriocin. The encoded immunity partners temporarily form a complex with the bacteriocin's toxin module (14) or are inserted in the inner membrane to enable bacteriocin secretion without causing harm to the producer cell (15,16). Cognate immunity genes are usually located downstream of the bacteriocin genes, allowing efficient coexpression.…”

“…Pectocins of Pectobacterium also belong to the ColM␣ subtype but target FusA, a TonB-dependent receptor mediating the uptake of ironbound plant ferredoxin (33). Immunity to Pseudomonas ColM␣ bacteriocins is provided by integral membrane proteins named PmiA (for Pseudomonas ColM-type immunity, type A), consisting of 4 transmembrane helices (TMHs) (15). The actual mechanism by which these proteins provide immunity remains unclear.…”

LlpB represents a second subclass of lectin‐like bacteriocins

“…If so, llpB genes are organized in tandem, whereas llpA genes in strains carrying two representatives usually appear at distant loci (Ghequire and De Mot, ). As noted for other (mid‐sized) bacteriocins (Ghequire et al ., , ,; Dingemans et al ., ; Sharp et al ., ), llpB genes are typified by a lower GC content than the genomic average (~47% versus ~60%), pointing towards foreign origin. Yet another similarity with llpA genes is that some of these llpB genes arise in prophage/tailocin clusters (Ghequire et al ., ), for example in a Rp3 tailocin gene cluster of Pseudomonas libanensis DSM 17149.…”