LlpB represents a second subclass of lectin-like bacteriocins

Abstract

Bacteriocins are secreted bacterial proteins that selectively kill related strains. Lectin-like bacteriocins are atypical bacteriocins not requiring a cognate immunity factor and have been primarily studied in Pseudomonas. These so-called LlpAs are composed of a tandem of B-lectin domains. One domain interacts with d-rhamnose residues in the common polysaccharide antigen of Pseudomonas lipopolysaccharide (LPS). The other lectin domain is crucial for interference with the outer membrane protein assembly machinery by interacting with surface-exposed loops of its central component BamA. Via genome mining, we identified a second subclass of Pseudomonas lectin-like proteins, termed LlpB, consisting of a single B-lectin domain. We show that these proteins also display bactericidal activity. Among LlpB-resistant transposon mutants of an LlpB-susceptible Pseudomonas strain, a major subset was hit in an acyltransferase gene, predicted to be involved in LPS core modification, hereby suggesting that LlpBs equally attach to LPS for surface anchoring. This indicates that LPS binding and target strain specificity are condensed in a single B-lectin domain. The identification of this second subclass of lectin-like bacteriocins further expands the toolbox of antibacterial warfare deployed by bacteria and holds potential for their integration in biotechnological applications.

Figure 1

Maximum likelihood phylogenetic tree of B‐lectin domains from LlpA and LlpB proteins in Pseudomonas, characterized LlpA and B‐lectin domain‐containing proteins retrieved in other bacteria, and select B‐lectin mono‐domain proteins in other bacteria. The domain architecture is specified by a schematic representation, and domains are coloured according to function (see colour legend in box). Amino‐terminal and carboxy‐terminal lectin domains from LlpAs and lectin domains from LlpBs are shown on a yellow, red and blue background, respectively. B‐lectin domains from LlpBs cluster with the amino‐terminal domain of LlpAs. In the case of LlpAs, the B‐lectin domain shown in the respective cluster is highlighted by a glowing background. Highly similar sequences (> 75% pairwise amino acid sequence identity for full length LlpA/LlpB proteins) are represented by one sequence only. Previously characterized proteins with a B‐lectin domain are labelled in blue, and LlpBs characterized in this study in red. Multiple LlpA/LlpB bacteriocins in a particular strain are specified by extensions (a) and (b). Phylogenetic analysis was performed with PhyML, using the JTT substitution model. Bootstrap values (percentages of 1000 replicates) higher than 50 are shown at the branches. The tree is rooted to the amino‐terminal B‐lectin domain of the LlpA from Burkholderia ambifaria MEX‐5. Scale bar represents 0.5 substitutions per site. Bamb, Burkholderia ambifaria; Bcer, Bacillus cereus; Bsp, Burkholderia sp.; Cvac, Chromobacterium vaccinii; Ksp, Kitasatospora sp.; Msme, Mycobacterium smegmatis; Paer, Pseudomonas aeruginosa; Pasp, Paraburkholderia sp.; Pcon, Pseudomonas congelans; Pelg, Paenibacillus elgii; Pflo, Pseudomonas floridensis; Pflu, Pseudomonas fluorescens; Pfre, Pseudomonas frederiksbergensis; Pgra, Pseudomonas graminis; Pkor, Pseudomonas koreensis; Plib, Pseudomonas libanensis; Pmos, Pseudomonas mosselii; Pory, Pseudomonas oryzihabitans; Ppro, Pseudomonas protegens; Ppsy, Pseudomonas psychrophila; Pput, Pseudomonas putida; Psp, Pseudomonas sp.; Psyr (syr), Pseudomonas syringae (pathovar syringae); Pvir, Pseudomonas viridiflava; Ralb, Ruminococcus albus; Rsp, Rathayibacter sp.; Sanm, Streptacidiphilus anmyonensis; Spsp, Sphingobium sp.; Ssp, Streptomyces sp.; Tavi, Tumebacillus avium; Xcit, Xanthomonas citri.

Figure 2

SDS‐PAGE electrophoresis of purified recombinant LlpB proteins from strains P. fluorescens A506 and Pseudomonas sp. UW4. Lane 1, Precision Plus Dual Xtra size marker (kDa); lane 2, LlpB_{P fluA506} (~19 kDa, predicted size 20.6 kDa); lane 3, LlpB_{P sp UW 4} (~21 kDa, predicted size 21.4 kDa).

Figure 3

Schematic gene representation of two genomic regions in Pseudomonas fluorescens LMG 1794^T (NCTC10038^T) susceptible to bacteriocin LlpB_{P fluA506}. Genes are shown as arrows and insert locations of transposon pRL27 are indicated with black triangles. Gene synteny of the locus of oatA, target of the large majority of the LlpB‐resistant mutants, with the corresponding region in reference strain P. aeruginosa PAO1 is shown by grey shading. Dotted lines indicate the lack of an equivalent region.