Article Figures & Data
Figures
- FIG. 1.
Sequence alignment of the different colicin M homologues. (A) Sequences of E. coli colicin M (UniProtKB/Swiss-Prot accession number P05820) (Ecoli) and its homologues from P. aeruginosa (UniProtKB/TrEMBL accession number Q1W548) (Paeru), P. fluorescens (GenBank accession number EU982300; Dmitri Mavrodi, personal communication) (Pfluo), and P. syringae (UniProtKB/TrEMBL accession number Q88A25) (Psyri) were aligned using the ClustalW program. Amino acids that are invariant in the four proteins are indicated by the one-letter abbreviation for the amino acid below the sequences, and amino acids that are conserved in three or two of the proteins are indicated by asterisks and colons below the sequences, respectively. Gaps introduced to maximize alignment are indicated by dashes. (B) Percentages of identity of residues in the N- and C-terminal regions (N-ter and C-ter, respectively) of the ColM homologues are indicated for pair-wise comparisons. The black triangle in panel A indicates the limit of these regions that was chosen for this comparison.
- FIG. 2.
Purification of ColM homologues from Pseudomonas species. The different ColM homologues were overproduced in His6-tagged form and were purified by affinity chromatography on Ni2+-NTA agarose. The lanes contain ColM from E. coli (Ecoli) (12) and purified preparations of its three homologues from P. aeruginosa (Paeru), P. fluorescens (Pfluo), and P. syringae (Psyri). Lane M contains molecular mass standards, namely, bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), and soybean trypsin inhibitor (20 kDa). Staining was performed with Coomassie brilliant blue R250.
- FIG. 3.
In vitro degradation of the peptidoglycan lipid II intermediate by ColM-like proteins from Pseudomonas species. The activity of ColM and its different homologues was tested under standard assay conditions using 14C-lipid II labeled in the GlcNAc moiety as the substrate. Conditions that result in partial (∼20 to 50%) conversion of lipid II into 1-pyrophospho-MurNAc(-pentapeptide)-GlcNAc product were used. The radiolabeled substrate and product were separated by TLC (R f values were 0.7 and 0.3, respectively,) and spots were visualized with the PhosphorImager. Lane A, lipid II incubated in the absence of ColM; lanes B to E, lipid II incubated in the presence of E. coli ColM (0.4 μg) or one of the ColM homologues from P. aeruginosa (2 ng), P. fluorescens (28 μg), and P. syringae (0.1 μg), respectively.
- FIG. 4.
Identification of lipid I degradation product by HPLC. l-[14C]alanine-labeled lipid I was digested to completion by colicin M and the three homologues from Pseudomonas species, and the reaction mixtures were analyzed by HPLC as described in Materials and Methods. The solid line (detection at 215 nm) depicts analysis of a standard mixture of 1-pyrophospho-MurNAc-pentapeptide (peak 1), 1-phospho-MurNAc-pentapeptide (peak 2), and MurNAc-pentapeptide (peaks 3 and 4; anomeric forms β and α, respectively). The broken line (detection of radioactivity) depicts analysis of the reaction mixture following incubation of 14C-labeled lipid I with the P. aeruginosa enzyme, showing the formation of a single radiolabeled product comigrating with authentic 1-pyrophospho-MurNAc-pentapeptide. The same results were obtained with ColM and the P. fluorescens and P. syringae homologue proteins, and no radiolabeled product was observed in the absence of enzyme (data not shown).
- FIG. 5.
Effect of the ColM homologue from Pseudomonas aeruginosa on the growth of P. aeruginosa DET08. (A) Two microliters of the purified bacteriocin (4 μg) were loaded in duplicate at the surface of a 2YT agar plate that was previously inoculated with the indicator strain P. aeruginosa DET08. Diffusion of the enzyme inhibited growth, resulting in darker zones. (B) P. aeruginosa DET08 was grown at 37°C in 2YT medium, and the purified bacteriocin was added at various concentrations (mg/ml) as indicated by the arrow. OD, optical density.
Tables
- TABLE 1.
Oligonucleotide primers used in this study
Primer Sequencea PaeO1 5′-GTGATCCATGGTTATGGATCTTGGAACTACCACTATAGTC-3′ (NcoI) PaeO2 5′-TGTTAAGATCTACCAGAAATATTTACAGGGATAGCTC-3′ (BglII) PflO1 5′-AGGAGGATCC ATGGAATTCGAGCTTCCAGCTACTTATGTATATC-3′ (BamHI) PflO2 5′-GCCGAAGCTTCACCTATCGGGCGTAGCTAATAGGGAGCTC-3′ (HindIII) PsyO1 5′-GGTAGGATCC ATGCCTATTGAGCTTCCTCCGACATACATCACCC-3′ (BamHI) PsyO2 5′-CTGGGAGCTCCACTGCTCAGGCGCTACCTGTCATGCCTTTGAC-3′ (SacI) ↵ a Restriction sites (in boldface type) introduced in oligonucleotide primers are indicated in parentheses. The initiation codons of the genes are underlined.
- TABLE 2.
Kinetic parameters of E. coli ColM and homologues from Pseudomonas species
ColM homologue Km (μM) for lipid II kcat (min−1) kcat/Km (μM−1·min−1) (103) E. coli 44 ± 4 0.055 ± 0.002 1.2 ± 0.1 P. aeruginosa 42 ± 6 32 ± 1 760 ± 110 P. fluorescens 150 ± 20 0.012 ± 0.001 0.080 ± 0.013 P. syringae 200 ± 30 0.52 ± 0.06 2.6 ± 0.5 - TABLE 3.
Spectrum of activity of the purified bacteriocins against Pseudomonas strains
Speciesa No. of strains tested No. of strains susceptible to the indicated bacteriocin ColM from E. coli Pseudomonas ColM homologue from: P. aeruginosa P. fluorescens P. syringae P. aeruginosa 14 0 2b 1b 0 P. fluorescens 4 0 0 0 0 P. syringae 24 0 0 0 0 ↵ a The strain collection tested comprised strains from three Pseudomonas spp.: P. aeruginosa strain ATCC 1045 and clinical isolates NCK001, -002, -003, -004, -005, -006, -007, -008, and -009 (Hôpital Necker); clinical isolates CLE08 and DET08 (Hôpital Européen Georges Pompidou); PA14 (CHU de Besançon); and PAO1 (LRMA); P. fluorescens CFBP strains 2392, 5755, 5756, and 5759 (Collection Française de Bactéries Phytopathogènes [CFBP], INRA); and P. syringae CFBP strains 1067, 1573, 1620, 1634, 1657, 1674, 1908, 2104, 2212, 2215, 2216, 2346, 2898, 2899, 3205, 3226, 3228, 8486, 10971, 11005, 11007, 11033, 11040, and 11056.
↵ b Strain NCK007 was inhibited by the enzyme from P. aeruginosa, and strain DET08 was inhibited by the enzymes from P. aeruginosa and P. fluorescens.
