A Novel Type of Immunity Protein, NukH, for the Lantibiotic Nukacin ISK-1 Produced byStaphylococcus warneriISK-1

Abstract

Staphylococcus warneri ISK-1 produces a lantibiotic, nukacin ISK-1. The nukacin ISK-1 gene cluster consists of at least six genes, nukA, -M, -T, -F, -E, and -G, and two open reading frames, ORF1 and ORF7 (designated nukH). Sequence comparisons suggested that NukF, -E, -G, and -H contribute to immunity to nukacin ISK-1. We investigated the immunity levels of recombinant Lactococcus lactis expressing nukFEG and nukH against nukacin ISK-1. The co-expression of nukFEG and nukH resulted in a high degree of immunity. The expression of either nukFEG or nukH conferred partial immunity against nukacin ISK-1. These results suggest that NukH contributes cooperatively to self-protection with Nuk-FEG. The nukacin ISK-1 immunity system might function against another lantibiotic, lacticin 481. Western blot analysis showed that NukH expressed in Staphylococcus carnosus was localized in the membrane. Peptide release/bind assays indicated that the recombinant L. lactis expressing nukH interacted with nukacin ISK-1 and lacticin 481 but not with nisin A. These findings suggest that NukH contributes cooperatively to host immunity as a novel type of lantibiotic-binding immunity protein with NukFEG.

Cooperative Transport between NukFEG and NukH in Immunity against the Lantibiotic Nukacin ISK-1 Produced by Staphylococcus warneri ISK-1

“…Plasmid pNZ8048 was used for the expression of nukF, nukE, nukG, and nukH. The methods of constructing pNZFEGH, pNZFEG, and pNZH, which are pNZ8048 derivatives containing nukFEGH, nukFEG, and nukH, respectively, have been described previously (3). The nukF gene in pNZFEGH was inactivated by digestion with the restriction enzymes BstZ17I and BsaI.…”

“…The structure of nukacin ISK-1 is believed to be formed by the N-terminal tail region (positions 1 to 7) and the C-terminal ring region, containing unusual amino acids (positions 7 to 27) (2). The self-protection system against nukacin ISK-1 is conferred by the LanFEG-type protein NukFEG and a novel type of lantibiotic-binding immunity protein, NukH (3,4). Previously, we demonstrated that expression of nukFEG and nukH increases the immunity level of a nukacin ISK-1-sensitive Lactococcus lactis strain and that expression of both is required for full immunity (3).…”

“…The self-protection systems of lantibiotic producers comprise two major mechanisms. One is a lantibiotic transport mechanism mediated by the ATP-binding cassette (ABC) transporter LanFEG (1,3,6,14,24,25,26,30,31). ABC transporters form one of the largest protein families and are found in all species.…”

“…Sequence similarities and hydrophobicity profiles suggest that LanFEG consists of two transmembrane subunits forming a heterodimer (LanEG) and two nucleotide-binding subunits forming a homodimer (LanF) (5,25). The expression of lanFEG decreases the amount of cell-associated lantibiotics compared with that in non-lanFEG-expressing cells, suggesting that Lan-FEG transports cell-associated lantibiotics from the cell membrane to the extracellular space (3,14,24,30,31). The other self-protection mechanism is a lantibiotic-binding mechanism mediated by LanI (5,15,17,19,29,30,31).…”

“…The self-protection system against nukacin ISK-1 is conferred by the LanFEG-type protein NukFEG and a novel type of lantibiotic-binding immunity protein, NukH (3,4). Previously, we demonstrated that expression of nukFEG and nukH increases the immunity level of a nukacin ISK-1-sensitive Lactococcus lactis strain and that expression of both is required for full immunity (3). In addition, an in vitro peptide release assay indicated that NukFEG transports cell-associated nukacin ISK-1 and that NukH has binding activity not only against nukacin ISK-1 but also against lacticin 481 (3).…”