Bacteriocin curvaticin FS47

Bacteriocins are a heterogeneous group of ribosomally synthesized peptides or proteins with antimicrobial activity, produced predominantly by lactic acid bacteria, with potential applications as biopreservatives and probiotics. We describe here a novel strategy based on a bottom-up, shotgun proteomic approach using nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) with multiple fragmentation techniques for the quantitative profiling of bacteriocins present in the probiotic preparations of Lactobacillus acidophilus. A direct LC-MS/MS analysis with alternate collision-induced dissociation, high-energy collision dissociation, and electron-transfer dissociation fragmentation following a filter-assisted size-exclusion sample prefractionation has resulted in the identification of peptides belonging to 37 bacteriocins or related proteins. Peptides from lactacin F, helveticin J, lysin, avicin A, acidocin M, curvaticin FS47, and carocin D were predominant. The process of freeze drying under vacuum was observed to affect both the diversity and abundance of bacteriocins. Data acquisition using alternating complementary peptide fragmentation modes, especially electron-transfer dissociation, has significantly enhanced the peptide sequence coverage and number of bacteriocin peptides identified. Multi-enzyme proteolytic digestion was observed to increase the sample complexity and dynamic range, lowering the chances of detection of low-abundant bacteriocin peptides by LC-MS/MS. An analytical platform integrating size exclusion prefractionation, nanoLC-MS/MS analysis with multiple fragmentation techniques, and data-dependent decision tree-driven bioinformatic data analysis is novel in bacteriocin research and suitable for the comprehensive bioanalysis of diverse, low-abundant bacteriocins in complex samples.

Curvaticin FS47, a bacteriocin produced by Lactobacillus curvatus FS47, is inhibitory to Listeria monocytogenes, as well as Lactobacillus, Pediococcus, Enterococcus, and Bacillus spp. The bacteriocin was purified by 40% ammonium sulfate precipitation, solid-phase extraction, and reversed-phase high-pressure liquid chromatography. Purified curvaticin FS47 was determined to be 4.07 kDa by mass spectrometry and was partially sequenced. Thirty-one N-terminal amino acids were identified; the curvaticin FS47 protein sequence did not show homology to the pediocin-like group of bacteriocins.

A practical system was devised for grouping bacteriocins of lactic acid bacteria (LAB) based on mode of action as determined by changes in inhibitory activity to spontaneously-acquired bacteriocin resistance (Bac(R)). Wild type Listeria monocytogenes 39-2 was sensitive to five bacteriocins produced by 3 genera of LAB: pediocin PA-1 and pediocin Bac3 (Pediococcus), lacticin FS97 and lacticin FS56 (Lactococcus), and curvaticin FS47 (Lactobacillus). A spontaneous Bac(R) derivative of L. monocytogenes 39-2 obtained by selective recovery against lacticin FS56 provided complete resistance to the bacteriocin made by Lactococcus lactis FS56. The lacticin FS56-resistant strain of L. monocyotgenes 39-2 was also cross-resistant to curvaticin FS47 and pediocin PA-1, but not to lacticin FS97 or pediocin Bac3. The same pattern of cross-resistance was also observed with Bac(R) isolates obtained with L. monocytogenes Scott A-2. A spontaneous mutation that renders a strain cross-resistant to different bacteriocins indicates that they share a common mechanism of resistance due to similar modes of action of the bacteriocins. Spontaneous resistance was acquired to other bacteriocins (in aggregate) by following the same procedure against which the Bac(R) strain was still sensitive. In subsequent challenge assays, mixtures of bacteriocins of different modes of action provided greater inhibition than mixtures of bacteriocins of the same mode of action (as determined by our screening method). This study identifies a methodical approach to classify bacteriocins into functional groups based on mechanism of resistance (i.e., mode of action) that could be used for identifying the best mixture of bacteriocins for use as biopreservatives.