Bacteriocin detection from whole bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry - PubMed (original) (raw)

Bacteriocin detection from whole bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Thomas Hindré et al. Appl Environ Microbiol. 2003 Feb.

Abstract

Class I bacteriocins (lantibiotics) and class II bacteriocins are antimicrobial peptides secreted by gram-positive bacteria. Using two lantibiotics, lacticin 481 and nisin, and the class II bacteriocin coagulin, we showed that bacteriocins can be detected without any purification from whole producer bacteria grown on plates by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). When we compared the results of MALDI-TOF-MS performed with samples of whole cells and with samples of crude supernatants of liquid cultures, the former samples led to more efficient bacteriocin detection and required less handling. Nisin and lacticin 481 were both detected from a mixture of their producer strains, but such a mixture can yield additional signals. We used this method to determine the masses of two lacticin 481 variants, which confirmed at the peptide level the effect of mutations in the corresponding structural gene.

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Figures

FIG. 1.

FIG. 1.

Lantibiotics nisin A and lacticin 481 and the mutations leading to the lacticin 481 variants S4T and ΔS27. (A and B) Amino acid sequences and bridging patterns of nisin A and of lacticin 481 (16) and its variants. The unusual residues are 2,3-didehydroalanine (Dha), 2,3-didehydrobutyrine (Dhb), lanthionine (alanine-S-alanine), 3-methyllanthionine (aminobutyric acid-S-alanine), and aminobutyric acid (Abu). (C) Mutations created within the lacticin 481 structural gene lctA. This gene codes for a precursor peptide, the C-terminal part of which (propeptide) yields mature lacticin 481 after creation of the unusual residues and cleavage of the N-terminal part (leader peptide) (18). Only the propeptide-encoding part of lctA is shown, and the nucleotide sequence is numbered as it is in the GenBank database (accession no. U91581). The propeptide sequence is indicated above the nucleotide sequence. Lysine +1 is the N-terminal amino acid of mature lacticin 481. The asterisks indicate stop codons. The mutated bases and amino acids are indicated by boldface type and underlining, and the changes in the DNA and peptide sequences are indicated below and above the vertical arrows, respectively.

FIG. 2.

FIG. 2.

MALDI-TOF spectra of the lacticin 481-producing strain L. lactis ADRIA85LO30 (A), of L. lactis IL1403, which does not produce any bacteriocin (B), of the nisin-producing strain L. lactis NCDO1402 (C), and of a mixture of the lacticin 481- and nisin-producing strains L. lactis ADRIA85LO30 and NCDO1402 (D).

FIG. 3.

FIG. 3.

Detail of the peak cluster shown in Fig. 2A. The peaks at m/z 2,902, 2,924, and 2,940 correspond to the ions [M+H]+, [M+Na]+, and [M+K]+ of lacticin 481, respectively.

FIG. 4.

FIG. 4.

MALDI-TOF spectrum of the coagulin-producing strain B. coagulans I4. The peak at m/z 4,650 corresponds to the ion [M+K]+ of coagulin.

FIG. 5.

FIG. 5.

MALDI-TOF spectra of L. lactis IL1403(pEBΔS27) (A) and of L. lactis IL1403(pEBS4T) (B), which produce lacticin 481 mutant molecules which lack the C-terminal serine (ΔS27) or in which the serine at position 4 is replaced by a threonine (S4T).

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References

    1. Chopin, A., M. C. Chopin, A. Moillo-Batt, and P. Langella. 1984. Two plasmid-determined restriction and modification systems in Streptococcus lactis. Plasmid 11:260-263. - PubMed
    1. Claydon, M. A., S. N. Davey, V. Edwards-Jones, and D. B. Gordon. 1996. The rapid identification of intact microorganisms using mass spectrometry. Nat. Biotechnol. 14:1584-1586. - PubMed
    1. Cleveland, J., T. J. Montville, I. F. Nes, and M. L. Chikindas. 2001. Bacteriocins: safe, natural antimicrobials for food preservation. Int. J. Food Microbiol. 71:1-20. - PubMed
    1. Delves-Broughton, J., P. Blackburn, R. J. Evans, and J. Hugenholtz. 1996. Applications of the bacteriocin, nisin. Antonie Leeuwenhoek 69:193-202. - PubMed
    1. Dufour, A., D. Thuault, A. Boulliou, C. M. Bourgeois, and J.-P. Le Pennec. 1991. Plasmid-encoded determinants for bacteriocin production and immunity in a Lactococcus lactis strain and purification of the inhibitory peptide. J. Gen. Microbiol. 137:2423-2429. - PubMed

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