Cooperative transport between NukFEG and NukH in immunity against the lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1

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.     

Enhanced production of nukacin D13E in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> NZ9000 by the additional expression of immunity genes

Nukacin D13E (D13E) is a variant of type-A(II) lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1. D13E exhibited a twofold higher specific antimicrobial activity than nukacin ISK-1 against a number of Gram-positive bacteria. We previously reported the heterologous production of D13E in Lactococcus lactis NZ9000 under the control of nisin-controlled gene expression system. In this study, we demonstrated enhanced production of D13E by the additional expression of immunity genes, nukFEG. The nukacin ISK-1 immunity, conferred by the ABC transporter complex, NukFEG, and the lantibiotic-binding protein, NukH, was not overwhelmed by D13E. The additional NukFEG resulted in a fourfold increase in the immunity level of the strain and a 5.2-fold increase in D13E production. The additional NukFEGH-expressing strain with the highest D13E immunity showed reduced level of production. Further improvement in D13E production was achieved by using pH-controlled batch fermentation.     

Description of complete DNA sequence of two plasmids from the nukacin ISK-1 producer, Staphylococcus warneri ISK-1

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.     

Binding Specificity of the Lantibiotic-Binding Immunity Protein NukH

NukH is a lantibiotic-binding immunity protein that shows strong binding activity against type A(II) lantibiotics. In this study, the binding specificity of NukH was analyzed by using derivatives of nukacin ISK-1, which is a type A(II) lantibiotic produced by Staphylococcus warneri ISK-1. Interactions between cells of Lactococcus lactis transformants expressing nukH and nukacin ISK-1 derivatives were analyzed by using a quantitative peptide-binding assay. Differences in the cell-binding rates of each nukacin ISK-1 derivative suggested that three lysine residues at positions 1 to 3 of nukacin ISK-1 contribute to the effective binding of nukacin ISK-1 to nukH-expressing cells. The binding levels of mutants with lanthionine and dehydrobutyrine substitutions (S11A nukacin_4-27 and T24A nukacin_4-27, respectively) to nukH-expressing cells were considerably lower than those of nukacin_4-27, suggesting that unusual amino acids play a decisive role in NukH recognition. Additionally, it was suggested that T9A nukacin_4-27, a mutant with a 3-methyllanthionine substitution, binds to NukH via an intermolecular disulfide bond after it is weakly recognized by NukH. We succeeded in the detection of specific type A(II) lantibiotics from the culture supernatants of various bacteriocin producers by using the binding specificity of nukH-expressing cells.     

A novel lantibiotic, nukacin ISK-1, of Staphylococcus warneri ISK-1: cloning of the structural gene and identification of the structure

Staphylococcus warneri ISK-1, which we had previously reported as Pediococcus sp. ISK-1, produces a novel bacteriocin, nukacin ISK-1. Edman degradation of the chemically reduced nukacin ISK-1 produced a sequence of 27 amino acids, 7 of which were unidentified. Using single-specific-primer-PCR product as a probe, a 3.6-kb HindIII fragment containing the nukacin ISK-1 structural gene (nukA) was cloned and sequenced. The deduced amino acid sequence of nukacin ISK-1 had 57 amino acids, including a 30-amino acid leader region. The propeptide sequence showed significant similarity to those of lacticin-481 type lantibiotics. In the region upstream of nukA, a part of a long open reading frame (ORF), designated as nukM, encoding a putative modification enzyme was oriented in the opposite direction. In the region downstream of nukA, ORF1 was found in which the sequence of the putative translational product was similar to various response regulatory proteins.     

A Novel Lantibiotic,Nukacin ISK-1, of Staphylococcus warneri ISK-1: Cloning of the Structural Gene and Identification of the Structure

Staphylococcus warneri ISK-1, which we had previously reported as Pediococcus sp. ISK-1, produces a novel bacteriocin, nukacin ISK-1. Edman degradation of the chemically reduced nukacin ISK-1 produced a sequence of 27 amino acids, 7 of which were unidentified. Using single-specific-primer-PCR product as a probe, a 3.6-kb HindIII fragment containing the nukacin ISK-1 structural gene (nukA) was cloned and sequenced. The deduced amino acid sequence of nukacin ISK-1 had 57 amino acids, including a 30-amino acid leader region. The propeptide sequence showed significant similarity to those of lacticin-481 type lantibiotics. In the region upstream of nukA, a part of a long open reading frame (ORF), designated as nukM, encoding a putative modification enzyme was oriented in the opposite direction. In the region downstream of nukA, ORF1 was found in which the sequence of the putative translational product was similar to various response regulatory proteins.     

Lantibiotic engineering: molecular characterization and exploitation of lantibiotic-synthesizing enzymes for peptide engineering

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.     

ATPase activity regulation by leader peptide processing of ABC transporter maturation and secretion protein,NukT, for lantibiotic nukacin ISK-1

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.

     

Evaluation of essential and variable residues of nukacin ISK-1 by NNK scanning

Nukacin ISK-1, a type-A(II) lantibiotic, comprises 27 amino acids with a distinct linear N-terminal and a globular C-terminal region. To identify the positional importance or redundancy of individual residues responsible for nukacin ISK-1 antimicrobial activity, we replaced the native codons of the parent peptide with NNK triplet oligonucleotides in order to generate a bank of nukacin ISK-1 variants. The bioactivity of each peptide variant was evaluated by colony overlay assay, and hence we identified three Lys residues (Lys1, Lys2 and Lys3) that provided electrostatic interactions with the target membrane and were significantly variable. The ring structure of nukacin ISK-1 was found to be crucially important as replacing the ring-forming residues caused a complete loss of bioactivity. In addition to the ring-forming residues, Gly5, His12, Asp13, Met16, Asn17 and Gln20 residues were found to be essential for antimicrobial activity; Val6, Ile7, Val10, Phe19, Phe21, Val22, Phe23 and Thr24 were relatively variable; and Ser4, Pro8, His15 and Ser27 were extensively variable relative to their positions. We obtained two variants, Asp13Glu and Val22Ile, which exhibited a twofold higher specific activity compared with the wild-type and are the first reported type-A(II) lantibiotic mutant peptides with increased potency.     

Localization and interaction of the biosynthetic proteins for the lantibiotic, Nukacin ISK-1

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.     

Lysine-oriented charges trigger the membrane binding and activity of nukacin ISK-1

The antibacterial activities and membrane binding of nukacin ISK-1 and its fragments and mutants were evaluated to delineate the determinants governing structure-function relationships. The tail region (nukacin(1-7)) and ring region (nukacin(7-27)) were shown to have no antibacterial activity and also had no synergistic effect on each other or even on nukacin ISK-1. Both a fragment with three lysines in the N terminus deleted (nukacin(4-27)) and a mutant with three lysines in the N terminus replaced with alanine (K1-3A nukacin ISK-1) imparted very low activity (32-fold lower than nukacin ISK-1) and also exhibited a similar antagonistic effect on nukacin ISK-1. Addition of two lysine residues at the N terminus (+2K nukacin ISK-1) provided no further increased antibacterial activity. Surface plasmon resonance sensorgrams and kinetic rate constants determined by a BIAcore biosensor revealed that nukacin ISK-1 has remarkably higher binding affinity to anionic model membrane than to zwitterionic model membrane. Similar trends of strong binding responses and kinetics were indicated by the high affinities of nukacin ISK-1 and +2K nukacin ISK-1, but there was no binding of tail region, ring region, nukacin(4-27), and K1-3A nukacin ISK-1 to the anionic model membrane. Our findings therefore suggest that the complete structure of nukacin ISK-1 is necessary for its full activity, in which the N-terminus three lysine residues play a crucial role in electrostatic binding to the target membrane and therefore nukacin ISK-1's ability to exert its potent antibacterial activity.     

Identification of the nukacin KQU-131, a new type-A(II) lantibiotic produced by Staphylococcus hominis KQU-131 isolated from Thai fermented fish product (Pla-ra)

Staphylococcus hominis KQU-131, isolated from Thai fermented marine fish, produces a heat stable bacteriocin. Structural and genetic analysis indicated that the bacteriocin is a variant of nukacin ISK-1, a type-A(II) lantibiotic, and we termed the bacteriocin nukacin KQU-131. There were three different amino acid residues between nukacin ISK-1 and nukacin KQU-131, one residue in the leader peptide and the other two in the mature peptide.     

Functional Significance of the E Loop,a Novel Motif Conserved in the Lantibiotic Immunity ATP-Binding Cassette Transport Systems

Lantibiotics are peptide-derived antibacterial substances produced by some Gram-positive bacteria and characterized by the presence of unusual amino acids, like lanthionines and dehydrated amino acids. Because lantibiotic producers may be attacked by self-produced lantibiotics, they express immunity proteins on the cytoplasmic membrane. An ATP-binding cassette (ABC) transport system mediated by the LanFEG protein complex is a major system in lantibiotic immunity. Multiple-sequence alignment analysis revealed that LanF proteins contain the E loop, a variant of the Q loop, which is a well-conserved motif in the nucleotide-binding domains (NBDs) of general ABC transporters. To elucidate E loop function, we introduced a mutation in the NukF protein, which is involved in the nukacin-ISK-1 immunity system. Amino acid replacement of glutamic acid in the E loop with glutamine (E85Q) resulted in slight decreases in the immunity level and transport activity. Additionally, the E85A mutation severely impaired the immunity level and transport activity. On the other hand, ATPase activities of purified E85Q and E85A mutants were almost similar to that of the wild type. These results suggested that the E loop found in ABC transporters involved in lantibiotic immunity plays a significant role in the function of these transporters, especially in the structural change of transmembrane domains.Lantibiotics are antibacterial peptides produced by some Gram-positive bacteria and are characterized by the presence of unusual amino acids, such as lanthionine and dehydrated amino acid residues (4, 9, 20). The unusual amino acids are introduced after translation by a modification enzyme(s), and their subsequent processing and secretion are carried out by a leader peptidase and transporter, respectively. Since the secreted mature lantibiotics have the potential to attack producer cells, lantibiotic-producer cells express self-protection systems against their own lantibiotics. These self-protection systems have 2 major mechanisms: a lantibiotic transport mechanism mediated by an ATP-binding cassette (ABC) transporter (LanFEG) and a lantibiotic-binding mechanism mediated by a lipoprotein (LanI) or a membrane protein (LanH) (2, 8, 26, 33, 34).Transport by LanFEG is a common and major mechanism in the lantibiotic immunity systems. LanFEG and LanI are needed for full immunity against nisin and subtilin, which are type A(I) lantibiotics (33, 34). However, the immunity level conferred by LanFEG is much higher than that conferred by LanI. LanFEG and LanH are expressed against nukacin ISK-1, which is a type A(II) lantibiotic produced by Staphylococcus warneri ISK-1 (2). As in the case of nisin and subtilin, LanFEG plays a major role in the level of immunity against nukacin ISK-1. Moreover, against lacticin 481, which is also a type A(II) lantibiotic, only LanFEG is expressed and it confers full immunity (9).ABC transporters function as molecular pumps and transport various substrates, such as nutrients, lipids, and antibiotics coupled to ATP hydrolysis (10, 31). Bacterial ABC transporters consist of 2 transmembrane domains (TMDs) and 2 nucleotide-binding domains (NBDs). They utilize ATP hydrolysis as a source of energy for the transport. The NBD of an ABC transporter has several conserved motifs, such as Walker A, Walker B, Q loop, Signature, and H loop, in its amino acid sequence, and these motifs are involved in the functions of ABC transporters (31). Although the detailed substrate-binding mechanism is still unknown, the dimerization of NBDs concomitant with ATP binding leads to the conformational change of 2 TMDs, resulting in transport of the substrate (31). Sequence similarities and hydrophobicity profiles suggest that LanFEG consists of 2 heterodimeric subunits containing TMDs (LanEG) and 2 homodimeric subunits containing NBDs (LanF) (4, 27).In general, ABC transporters that had been identified together with their substrates mediate the transport of the substrate across the membrane. An exception reported previously is the Lol system, which releases lipoproteins from the inner membrane to the outer membrane in Gram-negative bacteria (40). However, LanFEG proteins are believed to scavenge lantibiotics present on the membrane. This hypothesis is strongly supported by the mode of action of lantibiotics: many lantibiotics, especially type A(I) lantibiotics, show pore-forming activity against model membranes (4). Taken together, the transport mechanism of LanFEG seems to be different from that of general ABC transporters.The immunity mechanism against nukacin ISK-1 mediated by NukFEG and NukH has been investigated before (2, 21-23, 39). On the basis of our analysis, we suggested that NukFEG transports both nukacin ISK-1 on the membrane and nukacin ISK-1 captured by NukH (2, 22). Since the transport reaction depended on the metabolic energy of the cells, we presumed that ATP hydrolysis by NukF is a driving force for the transport (22).Using multiple sequence alignment analysis, we have found that all the LanF proteins have the E loop as a variant of the Q loop in general ABC transporters. Therefore, in this study, we investigated the function of the E loop existing in NukF by using site-directed mutagenesis. A bioassay using nukacin ISK-1 and recombinant Lactococcus lactis expressing nukF and its mutants showed that the E loop is important for immunity. Additionally, a transport assay with fluorescein isothiocyanate (FITC)-labeled nukacin ISK-1 indicated that the E loop is involved in transport activity. Since purified NukF and E loop mutants showed similar ATPase activity, we proposed that the E loop has an important role in the function of LanFEG, especially in coupled movement with the transmembrane subunit NukEG.     

Lysine-Oriented Charges Trigger the Membrane Binding and Activity of Nukacin ISK-1

The antibacterial activities and membrane binding of nukacin ISK-1 and its fragments and mutants were evaluated to delineate the determinants governing structure-function relationships. The tail region (nukacin_1-7) and ring region (nukacin_7-27) were shown to have no antibacterial activity and also had no synergistic effect on each other or even on nukacin ISK-1. Both a fragment with three lysines in the N terminus deleted (nukacin_4-27) and a mutant with three lysines in the N terminus replaced with alanine (K1-3A nukacin ISK-1) imparted very low activity (32-fold lower than nukacin ISK-1) and also exhibited a similar antagonistic effect on nukacin ISK-1. Addition of two lysine residues at the N terminus (+2K nukacin ISK-1) provided no further increased antibacterial activity. Surface plasmon resonance sensorgrams and kinetic rate constants determined by a BIAcore biosensor revealed that nukacin ISK-1 has remarkably higher binding affinity to anionic model membrane than to zwitterionic model membrane. Similar trends of strong binding responses and kinetics were indicated by the high affinities of nukacin ISK-1 and +2K nukacin ISK-1, but there was no binding of tail region, ring region, nukacin_4-27, and K1-3A nukacin ISK-1 to the anionic model membrane. Our findings therefore suggest that the complete structure of nukacin ISK-1 is necessary for its full activity, in which the N-terminus three lysine residues play a crucial role in electrostatic binding to the target membrane and therefore nukacin ISK-1's ability to exert its potent antibacterial activity.     

Bactericidal activity of nukacin ISK-1: an alternative mode of action

We previously reported bacteriostatic action of nukacin ISK-1 against Bacillus subtilis JCM 1465^T. Here, we found its bactericidal activity against Micrococcus luteus DSM 1790 and Staphylococcus simulans 22, showing decrease in cell viability, cell lysis, and dissipation of the membrane potential. Moreover, leakage of small molecules such as K⁺, suggested the formation of small-sized or specific K⁺-conducting-pores by nukacin ISK-1.     

Characterization of the lacticin 481 operon: the Lactococcus lactis genes lctF, lctE, and lctG encode a putative ABC transporter involved in bacteriocin immunity.

The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis strains. The genetic determinants of lacticin 481 production are organized as an operon encoded by a 70-kb plasmid. We previously reported the first three genes of this operon, lctA, lctM, and lctT, which are involved in the bacteriocin biosynthesis and export (A. Rincé, A. Dufour, S. Le Pogam, D. Thuault, C. M. Bourgeois, and J.-P. Le Pennec, Appl. Environ. Microbiol. 60:1652-1657, 1994). The operon contains three additional open reading frames: lctF, lctE, and lctG. The hydrophobicity profiles and sequence similarities strongly suggest that the three gene products associate to form an ABC transporter. When the three genes were coexpressed into a lacticin 481-sensitive L. lactis strain, the strain became resistant to the bacteriocin. This protection could not be obtained when any of the three genes was deleted, confirming that lctF, lctE, and lctG are all necessary to provide immunity to lacticin 481. The quantification of the levels of immunity showed that lctF, lctE, and lctG could account for at least 6% and up to 100% of the immunity of the wild-type lacticin 481 producer strain, depending on the gene expression regulation. The lacticin 481 biosynthesis and immunity systems are discussed and compared to other lantibiotic systems.     

Identification of the Nukacin KQU-131, a New Type-A(II) Lantibiotic Produced by Staphylococcus hominis KQU-131 Isolated from Thai Fermented Fish Product (Pla-ra)

Staphylococcus hominis KQU-131, isolated from Thai fermented marine fish, produces a heat stable bacteriocin. Structural and genetic analysis indicated that the bacteriocin is a variant of nukacin ISK-1, a type-A(II) lantibiotic, and we termed the bacteriocin nukacin KQU-131. There were three different amino acid residues between nukacin ISK-1 and nukacin KQU-131, one residue in the leader peptide and the other two in the mature peptide.