The crystal structure of the lipid II-degrading bacteriocin syringacin M suggests unexpected evolutionary relationships between colicin M-like bacteriocins

Abstract

Colicin-like bacteriocins show potential as next generation antibiotics with clinical and agricultural applications. Key to these potential applications is their high potency and species specificity that enables a single pathogenic species to be targeted with minimal disturbance of the wider microbial community. Here we present the structure and function of the colicin M-like bacteriocin, syringacin M from Pseudomonas syringae pv. tomato DC3000. Syringacin M kills susceptible cells through a highly specific phosphatase activity that targets lipid II, ultimately inhibiting peptidoglycan synthesis. Comparison of the structures of syringacin M and colicin M reveals that, in addition to the expected similarity between the homologous C-terminal catalytic domains, the receptor binding domains of these proteins, which share no discernible sequence homology, share a striking structural similarity. This indicates that the generation of the novel receptor binding and species specificities of these bacteriocins has been driven by diversifying selection rather than diversifying recombination as suggested previously. Additionally, the structure of syringacin M reveals the presence of an active site calcium ion that is coordinated by a conserved aspartic acid side chain and is essential for catalytic activity. We show that mutation of this residue to alanine inactivates syringacin M and that the metal ion is absent from the structure of the mutant protein. Consistent with the presence of Ca(2+) in the active site, we show that syringacin M activity is supported by Ca(2+), along with Mg(2+) and Mn(2+), and the protein is catalytically inactive in the absence of these ions.

FIGURE 1.

Identification of bacteriocin produced by P. syringae pv. syringae LMG1247. A, native PAGE (8%) of DEAE anion exchange purification fraction from concentrated culture medium of P. syringae pv. syringae LMG1247, Coomassie-strained and overlaid with soft agar seeded with test strain Pseudomonas syringae pv. lachrymans. LMG 5456. The band corresponding to the zone of growth clearing was excised and identified using tandem mass spectrometry. B, trypsin-derived fragments from the band from A, matching sequence from P. syringae pv. syringae 642 ZP_07266212 identified using the MASCOT server.

FIGURE 2.

Purification and characterization of wild-type syringacin M and deletion mutants. A, susceptibility of P. syringae test strains to syringacin M; B, 15% (v/v) SDS-PAGE showing purified syringacin M (SyrM) and mutant proteins generated in this study; C, light field image of 5-fold serial dilutions (37.5 μm to 1 nm; minimal inhibitory concentration of 60 nm) of syringacin M spotted onto a soft agar overlay of susceptible strain P. syringae pv. lachrymans LMG 5456; D, dark field image of interference of killing activity of syringacin M on soft agar overlay by adjacent spotting of inactive mutants (proteins spotted at 100 μm).

FIGURE 3.

Sequence and structure of syringacin M. A, full-length structure of syringacin M showing amino acids 38–276 as a surface model and the unstructured N terminus (amino acids 2–37) as a ribbon model (orange). The positive electron density from the F_o − F_c omit map calculated after omitting the N terminus residues is shown as a chicken wire model at the 3 σ level. B, schematic representation of high resolution structure of syringacin M (amino acids 39–276) showing the receptor binding domain (green), cytotoxic domain (red), and Ca²⁺ ion (yellow). C, B-factor putty model of full-length (left) and truncated (right) structures of syringacin M. Cool colors/thin ribbon, lower relative B-factors; hot colors/fat ribbon, higher relative B-factors. D, sequence alignment of syringacin M and colicin M, showing conserved (dark blue) and similar (light blue) residues, secondary structure (sheets and helices), and location of truncation for −10 and −20 deletion mutants of syringacin M generated in this study.

FIGURE 4.

The receptor binding and cytotoxic domains of syringacin M and colicin M show structural homology. A, structural alignment of syringacin M 57–276 (light green, residues 57–126; green, residues 127–276) and colicin M 48–271 (blue, residues 48–121; sky blue, residues 122–271) based on the catalytic domain β-barrel, residues 180–190, 204–216, 220–229, and 269–275 (minus 6 for colicin M residue numbers) (RMSD 1.765 Å). B, subsection of the alignment from A, syringacin M (residues 145–276) and colicin M (residues 140–271) showing overlay of the conserved catalytic region. C, structural alignment as in A, based on receptor binding domain helices 2–4, residues 58–70 (residues 53–65), 73–92 (73–92), and 95–101 (97–103) (colicin M residues in parentheses) (RMSD 2.051 Å). D, subsection of the alignment from C, syringacin M (residues 57–126) and colicin M (residues 48–120) showing overlay of conserved core helices in wall-eyed stereo. E, alternative orientation of alignment from D showing a fit of the two main helices of the receptor binding domain. Terminal residues are labeled for syringacin M (black) and colicin M (red).

FIGURE 5.

Comparison of the syringacin M and colicin M active sites. A, superimposition of key conserved active site residues of syringacin M (green) and colicin M (blue) in stick representation (labels for colicin M are shown in red, and those for syringacin M are in black) and Ca²⁺ ion from syringacin M (yellow). B, coordination of Ca²⁺ ion in the active site of syringacin M. Residues and the ethylene glycol (EDO) involved in coordination are shown as a stick model, and the 2F_o − F_c electron density map is shown as a chicken wire model at 1.7 σ. C, surface representation of syringacin M active site showing conserved residues essential (red) and important (orange) for activity in colicin M. D, surface representation of colicin M active site showing conserved residues essential (red) and important (orange) for cytotoxicity.

FIGURE 6.

Dependence of syringacin M activity on divalent metal ions. A, thin layer chromatography visualization of reaction of syringacin M (top, wild type; bottom, D232A mutant) and lipid II in the presence and absence of various divalent cations, all reactions identical except for the labeled component (Buffer, no additional reagents added). B, TLC visualization of time course reaction of syringacin M and lipid II in the presence of CaCl₂ or MgCl₂.

FIGURE 7.

Molecular phylogeny of catalytic domains (A) and receptor binding and translocation domains (B) of colicin M homologues. The tree was constructed using nearest neighbor joining method, and values at nodes are percent bootstrap values (1000 rounds) >55%. Tip labels correspond to the following proteins. Syringacin M [Pto. DC3000], NP_790419 P. syringae pv. tomato DC3000; Syringacin M [Pss. 642], ZP_07266212 P. syringae pv. syringae 642; Pecotcin M1 [Pcc. PC1], YP_003017875 Pectobacterium cartovorum subsp. cartovorum PC1; Pectocin M2 [Pb. BPR1692], YP_004352281 Pectobacterium brassicacarum BPR 1692; Colicin M [Ec. H29], ZP_08386556 E. coli H299; PyoM [Pa. 6077], ABD94622 P. aeruginosa str. 6077; SamM [BAA_1581], EHY70087 Salmonella enterica subsp. houtenae str. ATCC BAA-1581; SyrM [Psm. M302280], EGH10510 P. syringae pv. morsprunorum str. M302280; SyrM [Psa. M302273], EGH74181 P. syringae pv. aceris str. M302273; FluorM [Pb. MF142], YP 004352281 P. brassicacearum subsp. brassicacearum NFM421; BurkM [Bo. C6786], ZP_02366967 Burkholderia oklahomensis C6786; BurkM [Ba. AMMD], YP_772218 Burkholderia ambifaria AMMD; BurkM [Ba. MC40-6], YP_001807050 B. ambifaria MC40-6; BurkM [Bu. BU], ZP_02380104 B. ubonensis Bu.

FIGURE 8.

Graphical representation of mechanisms of diversification of bacteriocins. Diversifying recombination for novel translocation (T), receptor binding (R), or cytotoxic domain (C) functions has been widely observed in colicin-like bacteriocins, and diversifying selection is the mechanism through which novel cytotoxic immunity protein (I) specificities are generated in the DNase and rRNase type colicins (13, 16). The structural similarity between the receptor binding domains of colicin M and syringacin M suggests that diversifying selection may also be important in the generation of novel receptor binding specificities.