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NukT, a possible ABC transporter maturation and secretion (AMS) protein, may contribute to the cleavage of the leader peptide of NukA, which is the prepeptide of the lantibiotic nukacin ISK-1, and to nukacin ISK-1 transport. In this study, we reconstituted in vitro peptidase activity of the full-length NukT overexpressed in inside-out membrane vesicles of Staphylococcus carnosus TM300. We found that the presence of unusual amino acids in NukA is required for leader peptide cleavage. Furthermore, NukT peptidase activity was inhibited by phenylmethylsulfonyl fluoride, a serine/cysteine protease inhibitor; this finding strongly suggests that NukT, like other AMS proteins, is a cysteine protease. Interestingly, NukT peptidase activity depended on ATP hydrolysis. These results suggest that the N-terminal peptidase domain of NukT may cooperatively function with the C-terminal ATP-binding domain. This is the first in vitro study on lantibiotics that reports the processing mechanism of a full-length bifunctional ABC transporter.

Nukacin ISK-1 is a lantibiotic produced by Staphylococcus warneri ISK-1. Previous studies have reported that the self-protection system of the nukacin ISK-1 producer involves the cooperative function of the ABC transporter NukFEG and the lantibiotic-binding immunity protein NukH. In this study, the cooperative mechanism between NukFEG and NukH was characterized by using fluorescein-4-isothiocyanate (FITC)-labeled nukacin ISK-1 (FITC-nuk) to clarify the localization of nukacin ISK-1 in the immunity process. Lactococcus lactis recombinants expressing nukFEGH, nukFEG, or nukH showed immunity against FITC-nuk, suggesting that FITC-nuk was recognized by the self-protection system against nukacin ISK-1. Analysis of the interaction between FITC-nuk and energy-deprived cells of the L. lactis recombinants showed that FITC-nuk specifically bound to cells expressing nukH. The interaction between FITC-nuk and nukH-expressing cells was inhibited by the addition of unlabeled nukacin ISK-1 and its derivatives with deletions of the N-terminal tail region, but not by the addition of a synthesized N-terminal tail region. This suggests that the NukH protein recognizes the C-terminal ring region of nukacin ISK-1. The addition of glucose to nukFEGH-expressing cells treated with FITC-nuk resulted in a time-dependent decrease in fluorescence intensity, indicating that FITC-nuk was transported from the cell membrane by the NukFEG protein. These results revealed that after being captured by NukH in an energy-independent manner, nukacin ISK-1 was transported to the extracellular space by NukFEG in an energy-dependent manner.

Lanthionine-containing peptide antibiotics called lantibiotics are produced by a large number of Gram-positive bacteria. Nukacin ISK-1 produced by Staphylococcus warneri ISK-1 is type-A(II) lantibiotic. Ribosomally synthesized nukacin ISK-1 prepeptide (NukA) consists of an N-terminal leader peptide followed by a C-terminal propeptide moiety that undergoes several post-translational modification events including unusual amino acid formation by the modification enzyme NukM, cleavage of leader peptide and export by the dual functional ABC transporter NukT, finally yielding a biologically active peptide. Unusual amino acids in lantibiotics contribute to biological activity and also structural stability against proteases. Thus, lantibiotic-synthesizing enzymes have a high potentiality for peptide engineering by introduction of unusual amino acids into desired peptides with altering biological and physicochemical properties, e.g., activity and stability, termed lantibiotic engineering. We report the establishment of a heterologous expression of nukacin ISK-1 biosynthetic gene cluster by the nisin-controlled expression system and discuss our recent progress in understanding of the biosynthetic enzymes for nukacin ISK-1 such as localization, molecular interaction in biophysical and biochemical aspects. Substrate specificity of the lantibiotic-synthesizing enzymes was evaluated by complementation of the biosynthetic enzymes (LctM and LctT) of closely related lantibiotic lacticin 481 for nukacin ISK-1 biosynthesis. We further explored a rapid and powerful tool for introduction of unusual amino acids by co-expression of hexa-histidine-tagged NukA and NukM in Escherichia coli.

The immunity to a lantibiotic, nukacin ISK-1, is conferred by NukFEG (ABC transporter) and NukH (lantibiotic-binding protein) cooperatively. The present study identifies the functional domains of NukH. The topological analysis indicated that NukH possesses two external loops and three transmembrane helices. Deletion of N or C terminus of NukH did not affect the function. Amino acids substitutions in the respective loops abolished the function. Deletion of the third transmembrane helix resulted in loss of immunity but did not affect the binding activity. These findings suggested that the whole structure of NukH, except for N and C termini, is essential for its full immunity function, and that NukH inactivates nukacin ISK-1 after binding.

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 NukFEG. 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.

Nukacin ISK-1 is a type-A(II) lantibiotic produced by Staphylococcus warneri ISK-1. In this study, we characterized NukM and NukT, which are predicted to be involved in modification of prepeptide (NukA) and cleavage of leader peptide and subsequent secretion respectively. Localization analysis of NukM and NukT in the wild-type strain indicated that both proteins were located at the cytoplasm membrane. Interestingly, NukM expressed heterologously in St. carnosus TM300 was also located at the cytoplasm membrane even in the absence of NukT. Yeast two-hybrid assay showed that a complex of at least two each of NukM and NukT was associated with NukA. In vitro interaction analysis by surface plasmon resonance biosensor further suggested that membrane-located NukM interacted with NukA. These results indicate that NukM and NukT form a membrane-located multimeric protein complex and that post-translational modification of nukacin ISK-1 would occur at the cytoplasm membrane.

We report the whole DNA sequence of two plasmids, pPI-1 (30.2 kb) and pPI-2 (2.8 kb). These plasmids are from Staphylococcus warneri ISK-1, which produces a lantibiotic, nukacin ISK-1. Curing of pPI-1 resulted in a loss of bactericidal activity in the culture supernatant and the host's immunity to nukacin ISK-1, suggesting that the biosynthetic genes of the bacteriocin are encoded by pPI-1. Based on the results of a homology search of each open reading flame, pPI-1 is comprised of the following four distinct regions: (1) the nukacin ISK-1 biosynthesis and immunity gene cluster, (2) the thioredoxin gene cluster, (3) the replication region, and (4) a region of Staphylococcus epidermidis ATCC 12228, highly homologous to pSE-12228-05. Gene organization in the nukacin ISK-1 biosynthesis and immunity gene cluster is different from that in other lacticin-481 type gene clusters. The features of the replication protein encoded in the replicating region are somewhat different from other staphylococcus theta-replicating plasmids. pPI-2 comprised a disinfectant resistant gene, qacC, and the whole DNA sequence showed significant similarity to those of other qacC plasmids such as pSK108, suggesting that pPI-2 belongs to the qacC plasmid group.

We characterized a gene cluster in a plasmid designated pPI-1 of Staphylococcus warneri ISK-1 encoding the biosynthesis of and immunity to the lacticin-481 type lantibiotic, nukacin ISK-1. The DNA sequence suggested that the nukacin ISK-1 gene cluster consists of at least six genes, nukA (a structural gene), -M, -T, -F, -E, -G, and two open reading frames, ORF1 and ORF7. NukM and NukT were predicted to be involved in post-translational modification and secretion of nukacin ISK-1 respectively. NukF, -E, and -G were predicted to form a membrane complex which contributes to self-protection from nukacin ISK-1. Transcriptional analyses revealed that nukM through ORF7 comprises an operon, and that ORF1 is transcribed independently from downstream of nukA. The transcriptional levels of the nukA and nukM genes were enhanced by osmotic stress. The expression level of the nukA transcript was scarcely enhanced by nukacin ISK-1, suggesting that expression is not under the control of the autoregulatory circuit.

Lantibiotic nukacin ISK-1 is produced by Staphylococcus warneri ISK-1. The dual functional transporter NukT, an ABC transporter maturation and secretion protein, contributes to cleavage of the leader peptide from the prepeptide (modified NukA) and the final transport of nukacin ISK-1. NukT consists of an N-terminal peptidase domain (PEP), a C-terminal nucleotide-binding domain (NBD), and a transmembrane domain (TMD). In this study, NukT and its peptidase-inactive mutant were expressed, purified, and reconstituted into liposomes for analysis of their peptidase and ATPase activities. The ATPase activity of the NBD region was shown to be required for the peptidase activity of the PEP region. Furthermore, we demonstrated for the first time that leader peptide cleavage by the PEP region significantly enhanced the ATPase activity of the NBD region. Taken together, the presented results offer new insights into the processing mechanism of lantibiotic transporters and the necessity of interdomain cooperation. [ABSTRACT FROM AUTHOR]

The article presents a study that identifies a variant of nukacin ISK-1, a type -A (II) lantibiotic, in Staphylococcus hominis KQU-131 that was isolated from Thai fermented marine fish. It discusses about bacteriocins which are grouped in three major classes, Thailand's various fermented foods such as fermented fish pork and vegetables, as well as lactic and halophilic bacteria found in these products. Details on the findings that shows the amino acid and lysine residue are also offered.

Lantibiotics are antibacterial peptides containing unique thioether cross-links termed lanthionine and methyllanthionine. NukM, the modifying enzyme of nukacin ISK-1, which is produced by Staphylococcus warneri ISK-1, catalyzes the dehydration of specific Ser/Thr residues in a precursor peptide, followed by conjugative addition of intramolecular Cys to dehydrated residues to generate a cyclic structure. By contrast, the precursor peptide of nisin is modified by 2 enzymes, NisB and NisC, which mediate dehydration and cyclization, respectively. While the C-terminal domain of NukM is homologous to NisC, the N-terminal domain has no homology with other known proteins. We expressed and characterized the N- and C-terminal domains of NukM, NukM N , and NukM C , separately. In vitro reconstitution revealed that full-length NukM fully modified the substrate peptide NukA. NukM N partially phosphorylated, dehydrated, and cyclized NukA. By contrast, NukM C did not catalyze dehydration, phosphorylation, or cyclization reactions. Interaction studies using surface plasmon resonance analysis indicated that NukM and NukM N can bind NukA with high affinity, whereas NukM C has low substrate-recognition activity. These results suggest that NukM N is mainly responsible for substrate recognition and dehydration and that the whole NukM structure, including the C-terminal domain, is required for the complete modification of NukA. To the best of our knowledge, this is the first report providing insights into the in vitro catalytic activity of individual domains of a LanM-type modification enzyme. [ABSTRACT FROM AUTHOR]

Nukacin ISK-1 is a lantibiotic produced by Staphylococcus warneri ISK-1. The gene cluster of nukacin ISK-1 consists of at least nukAMTFEG, ORF1 and ORF7. In this study, we demonstrated the heterologous production of nukacin ISK-1 in Lactococcus lactis by the artificial polycistronic expression of nukAMTFEG-ORF7 under the control of the nisin-controlled expression (NICE) system. Consequently, the recombinant L. lactis showed antimicrobial activity. Mass analysis clarified the presence of nukacin ISK-1 produced in the culture supernatant. These results suggested that the recombinant L. lactis produced nukacin ISK-1 heterologously. Inactivation of nukA, -M or -T resulted in the complete loss of the nukacin ISK-1 production phenotype. This finding suggested that nukAMT are indispensably associated with the biosynthesis of nukacin ISK-1. To our knowledge, this is the first report of the heterologous production of lantibiotic using the NICE system. [Copyright &y& Elsevier]

Gelatinase biosynthesis-activating pheromone (GBAP) is an autoinducing peptide involved in Enterococcus faecalis fsr quorum sensing, and its 11-amino-acid sequence has been identified in the C-terminal region of the 242-residue deduced fsrB product (J. Nakayama et al., Mol. Microbiol. 41:145-154, 2001). In this study, however, we demonstrated the existence of fsrD, encoding the GBAP propeptide, which is in frame with fsrB but is translated independently of fsrB. It was also demonstrated that FsrB', an FsrD segment-truncated FsrB, functions as a cysteine protease-like processing enzyme to generate GBAP from FsrD. This revised model is consistent with the staphylococcal agr system. [ABSTRACT FROM AUTHOR]