Distribution of the NisI immunity protein and enhancement of nisin activity by the lipid-free NisI

Koponen, Olli; Takala, Timo M.; Saarela, Ulla; Qiao, Mingqiang; Saris, Per E. J.

doi:10.1016/s0378-1097(03)00934-0

Cited by 32 publications

(30 citation statements)

References 25 publications

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“…Attached to the outside of the cytoplasmic membrane, it can protect the membrane from subtilin by sequestering subtilin and thus prevent a high local density of subtilin molecules and subsequent pore formation. In L. lactis it has been estimated that nisin molecules dominate the cell-associated lipoprotein NisI by factors of 6 to 20 (22). A similar molar ratio between subtilin and SpaI can be assumed.…”

Section: Discussionmentioning

confidence: 99%

Expression and Functional Analysis of the Subtilin Immunity Genes spaIFEG in the Subtilin-Sensitive Host Bacillus subtilis MO1099

et al. 2005

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Bacillus subtilis ATCC 6633 produces the cationic pore-forming lantibiotic subtilin, which preferentially acts on gram-positive microorganisms; self protection of the producer cells is mediated by the four genes spaIFEG. To elucidate the mechanism of subtilin autoimmunity, we transferred different combinations of subtilin immunity genes under the control of an inducible promoter into the genome of subtilin-sensitive host strain B. subtilis MO1099. Recipient cells acquired subtilin tolerance through expression of either spaI or spaFEG, which shows that subtilin immunity is based on two independently acting systems. Cells coordinately expressing all four immunity genes acquired the strongest subtilin protection level. Quantitative in vivo peptide release assays demonstrated that SpaFEG diminished the quantity of cell-associated subtilin, suggesting that SpaFEG transports subtilin molecules from the membrane into the extracellular space. Homology and secondary structure analyses define SpaFEG as a prototype of lantibiotic immunity transporters that fall into the ABC-2 subfamily of multidrug resistance proteins. Membrane localization of the lipoprotein SpaI and specific interaction of SpaI with the cognate lantibiotic subtilin suggest a function of SpaI as a subtilin-intercepting protein. This interpretation was supported by hexahistidine-mediated 0-Å cross-linking between hexahistidinetagged SpaI and subtilin.Bacillus subtilis strain ATCC 6633 produces the cationic peptide antibiotic (lantibiotic) subtilin. Lantibiotics contain unusual thioether amino acids, such as meso-lanthionine and 3-methyl-lanthionine (17), which are incorporated into prepeptides through extensive posttranslational modifications (25,32,41). The subtilin and the closely related ericin gene clusters (35) encompass genes for posttranslational modification (18), transport (18), immunity (20), and regulation (19). Extracellular B. subtilis serine proteases are involved in the final processing step (7, 37). Subtilin biosynthesis and immunity are under the control of the two-component regulatory system SpaK/ SpaR (histidine kinase and response regulator, respectively) and the alternative sigma factor H (36, 38).Lantibiotics act against a wide range of gram-positive bacteria. The antimicrobial action of nisin produced by Lactococcus lactis, a structurally close relative of subtilin, is based on voltage-dependent pore formation that affects the efflux of small molecules and finally the collapse of the proton motive force (for a review see reference 4). The Bacto prenol-bound peptidoglycan precursor lipid II appears to be both a docking molecule assisting membrane targeting (5) and an integral constituent of the lethal pore itself (14). Gram-positive lantibiotic-producing strains need efficient countermeasures to obviate the lethal action of their own products (31). The nisin self protection (immunity) system is composed of ABC transporter homologue NisFEG and lipoprotein NisI (39).In the present study we report on the establishment of subtilin immunit...

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Section: Discussionmentioning

confidence: 99%

Expression and Functional Analysis of the Subtilin Immunity Genes spaIFEG in the Subtilin-Sensitive Host Bacillus subtilis MO1099

et al. 2005

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“…However, unlike NisI, NukH inactivates the bacteriocin after binding . Therefore, this LanFEG co-operative similarity does not throw much light upon the exact function of NisI in the nisin immunity mechanism, since NisI does not inactivate nisin (Stein et al, 2003;Koponen et al, 2004). Here, similar to native NisI, the truncated NisI proteins increased the nisin immunity to a greater extent in NisFEGexpressing NZ9840 than in MG1614.…”

mentioning

confidence: 93%

C terminus of NisI provides specificity to nisin

Takala

Saris

2006

Microbiology

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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.

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“…They are target molecule-specific and are highly efficient in their action. NisI, SpaI, and PepI are anchored to the cytoplasmic membrane, and unusually, NisI has also been found as a lipid-free form, presumably scavenging for exogenous nisin (21,28,48,49). In contrast, it was previously recognized that LtnI was very likely to traverse the membrane (34) in a manner similar to that predicted for CylI (6) and established for NukH (40).…”

Section: Discussionmentioning

confidence: 88%

Insights into Lantibiotic Immunity Provided by Bioengineering of LtnI

Draper

Deegan

Hill

et al. 2012

Antimicrob Agents Chemother

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The lantibiotic lacticin 3147 has been the focus of much research due to its broad spectrum of activity against many microbial targets, including drug-resistant pathogens. In order to protect itself, a lacticin 3147 producer must possess a cognate immunity mechanism. Lacticin 3147 immunity is provided by an ABC transporter, LtnFE, and a dedicated immunity protein, LtnI, both of which are capable of independently providing a degree of protection. In the study described here, we carried out an in-depth investigation of LtnI structure-function relationships through the creation of a series of fusion proteins and LtnI determinants that have been the subject of random and site-directed mutagenesis. We establish that LtnI is a transmembrane protein that contains a number of individual residues and regions, such as those between amino acids 20 and 27 and amino acids 76 and 83, which are essential for LtnI function. Finally, as a consequence of the screening of a bank of 28,000 strains producing different LtnI derivatives, we identified one variant (LtnI I81V) that provides enhanced protection. To our knowledge, this is the first report of a lantibiotic immunity protein with enhanced functionality.

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Distribution of the NisI immunity protein and enhancement of nisin activity by the lipid-free NisI

Cited by 32 publications

References 25 publications

Expression and Functional Analysis of the Subtilin Immunity Genes spaIFEG in the Subtilin-Sensitive Host Bacillus subtilis MO1099

Expression and Functional Analysis of the Subtilin Immunity Genes spaIFEG in the Subtilin-Sensitive Host Bacillus subtilis MO1099

C terminus of NisI provides specificity to nisin

Insights into Lantibiotic Immunity Provided by Bioengineering of LtnI

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