Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic

Abstract

Bacteriocin producers normally possess dedicated immunity systems to protect themselves from their own bacteriocins.Lactococcus lactis strains LMG2081 and BGBM50 are known as lactococcin G producers. However, BGBM50 was sensitive to LMG2081, which indicated that LMG2081 might produce additional bacteriocins that are not present in BGBM50. Therefore, whole-genome sequencing of the two strains was performed, and a lantibiotic operon (called lctLMG) was identified in LMG2081 but not in BGBM50. The lctLMG operon contains six open reading frames; the first three genes,lmgA ,lmgM, and lmgT, are involved in the biosynthesis and export of bacteriocin, while the other three genes,lmgF,lmgE, and lmgG, are involved in lantibiotic immunity. Mutational analysis confirmed that the lctLMG operon is responsible for the additional antimicrobial activity. Specifically, site-directed mutation within this operon rendered LMG2081 inactive toward BGBM50. Subsequent purification and electrospray ionization-time of flight mass spectrometric analysis confirmed that the lantibiotic bacteriocin called lacticin LMG is exported as a 25-amino-acid peptide. Lacticin LMG is highly similar to the lacticin 481 group. It is interesting that a bacteriocin producer produces two different classes of bacteriocins, whose operons are located in the chromosome and a plasmid.

FIG 1

Analysis of the bacteriocin activity of L. lactis LMG2081 and its mutants. (A) LMG2081 against BGMN1-596. (B) LMG2081 against BGBM50 (lactococcin G producer). (C) LMG2081/pG⁺host9lcnG (ΔlcnG) against BGMN1-596. (D) LMG2081/pG⁺host9lctLMG (ΔlcnLMG) against BGBM50. (E) LMG2081/pG⁺host9lctLMG against BGMN1-596.

FIG 2

Linear gene map of the region carrying the 8,000-bp lctLMG operon in L. lactis LMG2981. The positions of relevant restriction sites are indicated. Arrows, size and orientation of predicted ORFs.

FIG 3

Alignment of the deduced peptide sequence for lacticin LMG and homologous type A(II) lantibiotics, including nukacin ISK-1 from Staphylococcus warneri (GenBank accession no. Q9KWM4), lacticin 481 from Lactococcus lactis (GenBank accession no. WP_032489363), variacin from Kocuria varians (GenBank accession no. CAA63706), butyrivibriocin OR79 from Butyrivibrio fibrisolvens (GenBank accession no. AAC19355), lacticin LmgA from Lactococcus lactis (GenBank accession no. CUH82811.1), and salivaricin G32 from Streptococcus salivarius (GenBank accession no. AFB74480). Underlined amino acids, leader peptide; x, undefined residues. Gray shading and asterisks indicate identical amino acids, periods indicate amino acids belonging to similar groups, and colons indicate amino acids belonging to the same group.

FIG 4

PFGE profiles of Lactococcus lactis LMG2081 and lacticin LMG and lactococcin G mutants (A) and Southern blot hybridization with a lmgM probe (B). Total DNA from Lactococcus lactis LMG2081 (lanes 1, 5, 8, and 12), LMG2081/pG⁺host9lctLMG (lanes 2, 6, 9, and 13), or LMG2081/pG⁺host9lcnG (lanes 3, 7, 10, and 14) was digested with NotI (lanes 1 to 3) or SmaI (lanes 5 to 7), not digested (lanes 8 to 10), or treated with S1 nuclease (lanes 12 to 14). Lanes 4 and 11, λ concatemers. White arrows, linearized plasmids carrying the lctLMG operon; black arrows, differences in chromosomal fragments between the WT strain and the lactococcin G mutant.

FIG 5

Bioassay for the production of lacticin LMG in L. lactis LMG2081 and mass spectrometric analysis. (A) Reverse-phase HPLC. The active fraction was spotted on GM17 soft agar that had been inoculated with the indicator strain L. lactis subsp. lactis BGBM50. (B) Calculated molecular mass of lacticin LMG, determined by ESI-TOF mass spectrometry.