C terminus of NisI provides specificity to nisin

Abstract

Nisin-producing Lactococcus lactis protects its own cell membrane against the bacteriocin with the ABC transporter NisFEG, and the immunity lipoprotein NisI. In this study, in order to localize a site for specific nisin interaction in NisI, a C-terminal deletion series of NisI was constructed, and the C-terminally truncated NisI proteins were expressed in L. lactis. The shortest deletion (5 aa) decreased the nisin immunity capacity considerably in the nisin-negative strain MG1614, resulting in approximately 78 % loss of immunity function compared with native NisI. A deletion of 21 aa decreased the immunity level even more, but longer deletions, up to 74 aa, provided the same level of nisin immunity as the 21 aa deletion, i.e. approximately 14 % of the immunity provided by native NisI. Similar to native NisI, all the C-terminally truncated NisI proteins provided higher immunity to nisin in the NisFEG-expressing strain NZ9840 than in MG1614, i.e. approximately 40-50 % of the immunity capacity of native NisI. Then, it was determined whether the NisI Cterminal 21 aa fragment could protect cells against nisin. To target the 21 aa fragment to its natural location, 21 C-terminal amino acids from the subtilin-specific immunity lipoprotein SpaI were replaced by 21 C-terminal amino acids from NisI. The expression of the SpaI9-9NisI fusion in L. lactis strains significantly increased their nisin immunity. This is the first time the immunity function of a lantibiotic immunity protein has been transferred to another protein. However, unlike native NisI, and the C-terminally truncated NisI fragments, the increase in nisin immunity conferred by the SpaI9-9NisI fusion was the same in both the NisFEG strain NZ9840 and MG1614. In conclusion, the SpaI9-9NisI fusion could not enhance nisin immunity by interacting with NisFEG, whereas the Cterminally truncated NisI fragments and native NisI were able to enhance nisin immunity, probably by co-operation with NisFEG. The results made it evident that the C terminus of NisI is involved in specific interaction with nisin, and that it confers specificity for the NisI immunity lipoprotein.

Genes Involved in Immunity to and Secretion of Aureocin A53, an Atypical Class II Bacteriocin Produced by Staphylococcus aureus A53

“…For example, NukH, a protein involved in immunity to nukacin ISK-1, is a membrane protein with three transmembrane domains, and its N terminus is located on the cytoplasmic side (31). On the other hand, NisI is a lipoprotein anchored to the extracellular side of the membrane by its N-terminal lipid (39).…”

Isolation of a Variant of Subtilosin A with Hemolytic Activity

“…NisI, which is negatively charged (the pI of pre-NisI is 5.2, and the pI of mature NisI is 5.04), interacts with cationic nisin to protect the producing cells. This interaction was proposed to involve both charge-charge interactions and sequence-specific interactions (30). In the case of anionic subtilosin A, the putative immunity peptide AlbB is likely membrane anchored and positively charged (the pI is 10.8); thus, AlbB might interact with subtilosin A, partly based on the opposite charge of the two peptides.…”

Binding Specificity of the Lantibiotic-Binding Immunity Protein NukH

“…These results suggest that NisI intercepts nisin before the latter attacks the cell membrane, thereby preventing pore formation. In addition, experiments using C-terminally truncated NisI mutants and a hybrid protein containing an immunity lipoprotein for subtilin (SpaI) have shown that the C terminus of NisI confers protection specifically against nisin (32). However, the details of the substrate-binding mechanism of NisI have not yet been explained.…”

“…LanFEG is an ATP-binding cassette (ABC) transporter, which mediates the transport of lantibiotics from the cell membrane to the extracellular space (1,4,6,10,22,23,25,28,29). On the other hand, LanI is an immunity lipoprotein anchored to the membrane surface via a lipidmodified N-terminal cysteine residue (4,11,12,16,(27)(28)(29)32). One of the well-studied LanI proteins is NisI, which is involved in nisin immunity.…”