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.     

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.     

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.     

Characterization of modification enzyme NukM and engineering of a novel thioether bridge in lantibiotic nukacin ISK-1

The lantibiotic nukacin ISK-1 is an antimicrobial peptide containing unusual amino acids such as lanthionine and dehydrobutyrine. The nukacin ISK-1 prepeptide (NukA) undergoes posttranslational modifications, such as the dehydration and cyclization reactions required to form the unusual amino acids by the modification enzyme NukM. We have previously constructed a system for the introduction of unusual amino acids into NukA by coexpression of NukM in Escherichia coli. Using this system, we describe the substrate specificity of NukM by the coexpression of a series of NukA mutants. Our results revealed the following characteristics of NukM: (1) its dehydration activity is not coupled to its cyclization activity; (2) its dehydration activity is site-specific; (3) the length of the substrate is important for its dehydration activity. Furthermore, we succeeded in introducing a novel thioether bridge in NukA by replacing an unmodified Ser at position 27 with a Cys residue.     

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.

     

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.     

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.     

Detection, purification and efficacy of warnerin produced by Staphylococcus warneri

Summary Three strains of Staphylococcus warneri (FM10, FM20 and FM30) isolated from meat samples were investigated for their ability to synthesize bacteriocin. All the tested strains produced warnerin, a new peptide bacteriocin; which inhibits the growth of a large number of Gram-positive and Gram-negative bacteria. The inhibitory effect of warnerin produced by the FM20 isolate was high when compared to the other isolates. The results on the effect of carbon sources, nitrogen sources, pH, temperature, incubation time and surfactant (tween 80) inferred that the bacteriocin production was high in medium supplemented with 1% glucose (12,800 AU/ml), 1% urea (6800 AU/ml), and 0.5% Tween 80 (25,600 AU/ml). The higher productivity of bacteriocin was registered during 12 h of incubation in the medium pH 6.5 at 37 °C temperature. Among the various indicator strains tested, Staphylococcus aureus was more sensitive to the bacteriocin activity. Partially purified warnerin exhibited a single band on SDS-PAGE with an apparent molecular weight of 2500 Da. Warnerin, the antibacterial compound was determined as a proteinaceous substance, since it lost its activity when pepsin was added.     

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.     

Identification of a novel two-peptide lantibiotic, haloduracin, produced by the alkaliphile Bacillus halodurans C-125

Complete genome sequencing of the alkaliphilic bacterium Bacillus halodurans C-125 revealed the presence of several genes homologous to those involved in the production of lantibiotic peptides. Additional bioinformatic analysis identified a total of eleven genes, spanning a 15 kbp region, potentially involved in the production, modification, immunity and transport of a two-peptide lantibiotic. Having established that strain C-125 exhibited antimicrobial activity against a wide range of Gram-positive bacteria, it was demonstrated through peptide purification, MS and site-directed mutagenesis that this activity was indeed attributable to the production of a lantibiotic encoded by these genes. This antimicrobial has been designated haloduracin and represents the first occasion wherein production of two-peptide lantibiotic has been associated with a Bacillus sp. It is also the first example of a lantibiotic of any kind to be produced by an alkaliphilic species.     

Gene cluster for biosynthesis of thermophilin 1277--a lantibiotic produced by Streptococcus thermophilus SBT1277, and heterologous expression of TepI, a novel immunity peptide

Aims: To identify genes cluster for thermophilin 1277 produced by Streptococcus thermophilus SBT1277. Methods and Results: To identify genes for thermophilin 1277 production, the chromosomal DNA region surrounding the structural gene, tepA, was sequenced using a primer‐walking method. The thermophilin 1277 biosynthesis gene locus (tep) is a 9·9‐kb region, which consists of at least ten open reading frames (ORFs) in the following order: tepAMTFEGKRI and ORF4. Homology analysis showed high similarity to genes involved in bovicin HJ50 production by Streptococcus bovis HJ50. tepI encodes a novel, small, positively charged hydrophobic peptide of 52 amino acids, which contains a putative transmembrane segment. By heterologous expression in Lactococcus lactis ssp. cremoris MG1363, the TepI‐expressing strain exhibited at least 1·3 times higher resistance to thermophilin 1277. Conclusions: Thermophilin 1277 biosynthesis genes were encoded by a 9·9‐kbp region containing at least ten ORFs. TepI is a novel immunity peptide, which protected Strep. thermophilus SBT1277 against thermophilin 1277 in addition to TepFEG, a putative ABC transporter. Significance and Impact of the Study: This is the first report regarding a lantibiotic gene cluster produced by Strep. thermophilus strain.     

A novel lantibiotic acting on bacterial cell wall synthesis produced by the uncommon actinomycete Planomonospora sp

Important classes of antibiotics acting on bacterial cell wall biosynthesis, such as beta-lactams and glycopeptides, are used extensively in therapy and are now faced with a challenge because of the progressive spread of resistant pathogens. A discovery program was devised to target novel peptidoglycan biosynthesis inhibitors capable of overcoming these resistance mechanisms. The microbial products were first screened according to their differential activity against Staphylococcus aureus and its L-form. Then, activities insensitive to the addition of a beta-lactamase cocktail or d-alanyl-d-alanine affinity resin were selected. Thirty-five lantibiotics were identified from a library of broth extracts produced by 40,000 uncommon actinomycetes. Five of them showed structural characteristics that did not match with any known microbial metabolite. In this study, we report on the production, structure determination, and biological activity of one of these novel lantibiotics, namely, planosporicin, which is produced by the uncommon actinomycete Planomonospora sp. Planosporicin is a 2194 Da polypeptide originating from 24 proteinogenic amino acids. It contains lanthionine and methyllanthionine amino acids generating five intramolecular thioether bridges. Planosporicin selectively blocks peptidoglycan biosynthesis and causes accumulation of UDP-linked peptidoglycan precursors in growing bacterial cells. On the basis of its mode of action and globular structure, planosporicin can be assigned to the mersacidin (20 amino acids, 1825 Da) and the actagardine (19 amino acids, 1890 Da) subgroup of type B lantibiotics. Considering its spectrum of activity against Gram-positive pathogens of medical importance, including multi-resistant clinical isolates, and its efficacy in vivo, planosporicin represents a potentially new antibiotic to treat emerging pathogens.     

Isolation and characterization of enterocin W, a novel two-peptide lantibiotic produced by Enterococcus faecalis NKR-4-1

Enterococcus faecalis NKR-4-1 isolated from pla-ra produces a novel two-peptide lantibiotic, termed enterocin W, comprising Wα and Wβ. The structure of enterocin W exhibited similarity with that of plantaricin W. The two peptides acted synergistically, and their order of binding to the cell membrane was important for their inhibitory activity.     

Genes involved in immunity to the lantibiotic nisin produced by Lactococcus lactis 6F3.

The lantibiotic nisin is produced by several strains of Lactococcus lactis. The complete gene cluster for nisin biosynthesis in L. lactis 6F3 comprises 15 kb of DNA. As described previously, the structural gene nisA is followed by the genes nisB, nisT, nisC, nisI, nisP, nisR, and nisK. Further analysis revealed three additional open reading frames, nisF, nisE, and nisG, adjacent to nisK. Approximately 1 kb downstream of the nisG gene, three open reading frames in the opposite orientation have been identified. One of the reading frames, sacR, belongs to the sucrose operon, indicating that all genes belonging to the nisin gene cluster of L. lactis 6F3 have now been identified. Proteins NisF and NisE show strong homology to members of the family of ATP-binding cassette (ABC) transporters, and nisG encodes a hydrophobic protein which might act similarly to the immunity proteins described for several colicins. Gene disruption mutants carrying mutations in the genes nisF, nisE, and nisG were still able to produce nisin. However, in comparison with the wild-type strain, these mutants were more sensitive to nisin. This indicates that besides nisI the newly identified genes are also involved in immunity to nisin. The NisF-NisE ABC transporter is homologous to an ABC transporter of Bacillus subtilis and the MbcF-MbcE transporter of Escherichia coli, which are involved in immunity to subtilin and microcin B17, respectively.     

A novel secretory protein produced by rat spongiotrophoblast

The placenta secretes various factors in stage- and cell-specific manners. We have identified a cDNA encoding a novel protein with 124 amino acids, which was named spongiotrophoblast specific protein (SSP). SSP is highly homologous to mouse 4311, showing 81% and 59% similarity at the nucleotide and amino acid levels, respectively. Northern blot analysis showed that SSP mRNA was first detected on Day 14 of pregnancy, peaked on Day 16, and remained elevated until term. In situ hybridization analysis showed that SSP mRNA was specifically expressed in spongiotrophoblast cells of Day 20 placenta but not in Day 12 placenta. No expression was detected from the differentiated or undifferentiated rat choriocarcinoma Rcho-1 cell line. Native SSP was detected as a 19-kDa molecule by Western blotting in cell extracts prepared from the junctional zone. SSP was predicted to be a secretory protein, because 1) a hydropathy test revealed that SSP contained an N-terminal hydrophobic region and 2) native SSP was also detected in the cultured media of junctional zone explants. To further investigate a potential signal peptide of this protein, sets of recombinant SSP were generated using a COS7 transfection system. The N-terminal amino acid sequence of secreted recombinant SSP confirmed that the N-terminal 17 amino acids had been cleaved to produce a secretory protein. Thus, we have identified and cloned a novel secretory protein, SSP, which is specifically expressed by rat spongiotrophoblast cells during the latter half of pregnancy.