Label-Free Monitoring of Coculture System Dynamics: Probing Probiotic and Cancer Cell Interactions via Infrared Spectroscopic Imaging.

Evaluating the dynamic interaction of microorganisms and mammalian cells is challenging due to the lack of suitable platforms for examining interspecies interactions in biologically relevant coculture conditions. In this work, we demonstrate the interaction between probiotic bacteria (Lactococcus lactis and Escherichia coli) and A498 human cancer cells in vitro, utilizing a hydrogel-based platform in a label-free manner by infrared spectroscopy. The L. lactis strain recapitulated in the compartment system secretes polypeptide molecules such as nisin, which has been reported to trigger cell apoptosis. We propose a mid-infrared (IR) spectroscopic imaging approach to monitor the variation of biological components utilizing kidney cells (A498) as a model system cocultured with bacteria. We characterized the biochemical composition (i.e., nucleic acids, protein secondary structures, and lipid conformations) label-free using an unbiased measurement. Several IR spectral features, including unsaturated fatty acids, ß-turns in protein, and nucleic acids, were utilized to predict cellular response. These features were then applied to establish a quantitative relationship through a multivariate regression model to predict cellular dynamics in the coculture system to assess the effect of nisin on A498 kidney cancer cells cocultured with bacteria. Overall, our study sheds light on the potential of using IR spectroscopic imaging as a label-free tool to monitor complex microbe-host cell interactions in biological systems. This integration will enable mechanistic studies of interspecies interactions with insights into their underlying physiological processes.

Simulation-Guided Molecular Modeling of Nisin and Lipid II Assembly and Membrane Pore Formation.

The lantibiotic pore-forming peptide nisin is a promising candidate in the fight against multidrug-resistant bacteria due to its unique structure, which allows it to disrupt bacteria in two distinct ways─Lipid II trafficking and transmembrane pore formation. However, exactly how nisin and Lipid II assemble into oligomeric pore structures in the bacterial membrane is not known. Spontaneous peptide assembly into pores is difficult to observe in even the very long-time scale molecular dynamics (MD) simulations. In this study, we adopted an MD-guided modeling approach to investigate the nisin-nisin and nisin-Lipid II associations in the membrane environment. Through extensive microsecond-time scale all-atom MD simulations, we established that nisin monomers dimerize by forming ß-sheets in a POPE:POPG lipid bilayer and oligomerize further to form stable transmembrane channels. We determined that these nisin dimers use Lipid II as a dimer interface to incur enhanced stability. Our results provide a clearer understanding of the self-assembly of nisin monomers within the membrane and insights into the role of Lipid II in the structural integrity of oligomeric structures.

Production of nisin from Lactococcus lactis in acid-whey with nutrient supplementation.

The production of Nisin, an FDA-approved food preservative, was attempted by Lactococcus lactis subsp. lactis ATCC® 11454 using the underutilized milk industry effluent, acid-whey, as a substrate. Nisin production was further improved by studying the effect of supplementation of nutrients and non-nutritional parameters. The addition of yeast extract (6% w/v) as nitrogen source and sucrose (4% w/v) as carbon source were found to be suitable nutrients for the maximum nisin production. The changes in the medium pH due to lactic acid accumulation during batch fermentation and its influence on the production of nisin were analyzed in the optimized whey medium (OWM). The production characteristics in OWM were further compared with the nisin production in MRS media. The influence of nisin as an inducer for its own production was also studied and found that the addition of nisin at 0.22 mg/ml promote the nisin production. The analysis of consumption of various metal ions present in the OWM during the nisin production was also analyzed, and found that the copper ions are the most consumed ion. The highest nisin yield of 2.6 × 105 AU/mL was obtained with OWM.

Nisin a probiotic bacteriocin mitigates brain microbiome dysbiosis and Alzheimer's disease-like neuroinflammation triggered by periodontal disease.

INTRODUCTION: Periodontitis-related oral microbial dysbiosis is thought to contribute to Alzheimer's disease (AD) neuroinflammation and brain amyloid production. Since probiotics can modulate periodontitis/oral dysbiosis, this study examined the effects of a probiotic/lantibiotic, nisin, in modulating brain pathology triggered by periodontitis. METHODS: A polymicrobial mouse model of periodontal disease was used to evaluate the effects of this disease on brain microbiome dysbiosis, neuroinflammation, Alzheimer's-related changes, and nisin's therapeutic potential in this context. RESULTS: 16S sequencing and real-time PCR data revealed that Nisin treatment mitigated the changes in the brain microbiome composition, diversity, and community structure, and reduced the levels of periodontal pathogen DNA in the brain induced by periodontal disease. Nisin treatment significantly decreased the mRNA expression of pro-inflammatory cytokines (Interleukin-1ß/IL-1 ß, Interleukin 6/IL-6, and Tumor Necrosis Factor α/TNF-α) in the brain that were elevated by periodontal infection. In addition, the concentrations of amyloid-ß 42 (Aß42), total Tau, and Tau (pS199) (445.69 ± 120.03, 1420.85 ± 331.40, 137.20 ± 36.01) were significantly higher in the infection group compared to the control group (193.01 ± 31.82, 384.27 ± 363.93, 6.09 ± 10.85), respectively. Nisin treatment markedly reduced the Aß42 (261.80 ± 52.50), total Tau (865.37 ± 304.93), and phosphorylated Tau (82.53 ± 15.77) deposition in the brain of the infection group. DISCUSSION: Nisin abrogation of brain microbiome dysbiosis induces beneficial effects on AD-like pathogenic changes and neuroinflammation, and thereby may serve as a potential therapeutic for periodontal-dysbiosis-related AD.

A single system detects and protects the beneficial oral bacterium Streptococcus sp. A12 from a spectrum of antimicrobial peptides.

The commensal bacterium Streptococcus sp. A12 has multiple properties that may promote the stability of health-associated oral biofilms, including overt antagonism of the dental caries pathogen Streptococcus mutans. A LanFEG-type ABC transporter, PcfFEG, confers tolerance to the lantibiotic nisin and enhances the ability of A12 to compete against S. mutans. Here, we investigated the regulation of pcfFEG and adjacent genes for a two-component system, pcfRK, to better understand antimicrobial peptide resistance by A12. Induction of pcfFEG-pcfRK was the primary mechanism to respond rapidly to nisin. In addition to nisin, PcfFEG conferred tolerance by A12 to a spectrum of lantibiotic and non-lantibiotic antimicrobial peptides produced by a diverse collection of S. mutans isolates. Loss of PcfFEG resulted in the altered spatio-temporal arrangement of A12 and S. mutans in a dual-species biofilm model. Deletion of PcfFEG or PcfK resulted in constitutive activation of pcfFEG and expression of pcfFEG was inhibited by small peptides in the pcfK mutant. Transcriptional profiling of pcfR or pcfK mutants combined with functional genomics revealed peculiarities in PcfK function and a novel panel of genes responsive to nisin. Collectively, the results provide fundamental insights that strengthen the foundation for the design of microbial-based therapeutics to control oral infectious diseases.

Nisin induces apoptosis in cervical cancer cells via reactive oxygen species generation and mitochondrial membrane potential changes.

Nisin, an antimicrobial peptide produced by Lactococcus lactis, is widely used as a safe food preservative and has recently attracted the attention of researchers as a potential anticancer agent. The cytotoxicity of nisin against human cervical cancer cell lines (HeLa), human ovarian carcinoma cell lines (OVCAR-3 and SK-OV-3), and human umbilical vein endothelial cells (HUVECs) was evaluated using an MTT assay. The apoptotic effect of nisin was identified by Annexin-V/propidium iodide assay, which was further confirmed by western blotting analysis, mitochondrial membrane potential (ΔΨm) analysis, and reactive oxygen species (ROS) assay. The MTT assay showed concentration-dependent cytotoxicity of nisin towards cancer cell lines, with IC50 values of 11.5-23 µM, but less toxicity against normal endothelial cells. Furthermore, the treatment of cervical cancer cells with 12 µM nisin significantly (P < 0.05) increased the Bax/Bcl-2 ratio (4.9 fold), reduced ΔΨm (70%), and elevated ROS levels (1.7 fold). These findings indicate that nisin may have anticancer and apoptogenic activities through mitochondrial dysfunction and oxidative stress damage in cervical cancer cells.

Analysis of Cross-Functionality within LanBTC Synthetase Complexes from Different Bacterial Sources with Respect to Production of Fully Modified Lanthipeptides.

Lanthipeptides belong to a family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) containing (methyl)lanthionine residues. Commonly, class I lanthipeptides are synthesized by a gene cluster encoding a precursor peptide (LanA), biosynthetic machinery (LanBTC), a protease (LanP), a two-component regulatory system (LanRK), and an immunity system (LanI and LanFEG). Although nisin and subtilin are highly similar class I lanthipeptides, the cross-regulation by LanRK and the cross-immunity by LanI and LanFEG between the nisin and subtilin systems have been proven to be very low. Here, the possibility of the cross-functionality of LanBTC to modify and transport nisin precursor (NisA) and subtilin precursor (SpaS) was evaluated in Bacillus subtilis and Lactococcus lactis. Interestingly, we found that a promiscuous NisBC-SpaT complex is able to synthesize and export nisin precursor, as efficiently as the native nisin biosynthetic machinery NisBTC, in L. lactis but not B. subtilis. The assembly of the NisBC-SpaT complex at a single microdomain, close to the old cell pole, was observed by fluorescence microscopy in L. lactis. In contrast, such a complex was not formed in B. subtilis. Furthermore, the isolation of the NisBC-SpaT complex and its subcomplexes from the cytoplasmic membrane of L. lactis by pulldown assays was successfully conducted. Our work demonstrates that the association of LanBC with LanT is critical for the efficient biosynthesis and secretion of the lanthipeptide precursor with complete modifications and suggests a cooperative mechanism between LanBC and LanT in the modification and transport processes. IMPORTANCE A multimeric synthetase LanBTC complex has been proposed for the in vivo production of class I lanthipeptides. However, it has been demonstrated that LanB, LanC, and LanT can perform their functionality in vivo and in vitro, independently of other Lan proteins. The role of protein-protein interactions, especially between the modification complex LanBC and the transport system LanT, in the biosynthesis process of lanthipeptides is still unclear. In this study, the importance of the presence of a well-installed LanBTC complex in the cell membrane for lanthipeptide biosynthesis and transport was reinforced. In L. lactis, the recruitment of SpaT from the peripheral cell membrane to the cell poles by the NisBC complex was observed, which may explain the mechanism by which the secretion of the premature peptide is prevented.

Structure and mechanism of the tRNA-dependent lantibiotic dehydratase NisB.

Lantibiotics are a class of peptide antibiotics that contain one or more thioether bonds. The lantibiotic nisin is an antimicrobial peptide that is widely used as a food preservative to combat food-borne pathogens. Nisin contains dehydroalanine and dehydrobutyrine residues that are formed by the dehydration of Ser/Thr by the lantibiotic dehydratase NisB (ref. 2). Recent biochemical studies revealed that NisB glutamylates Ser/Thr side chains as part of the dehydration process. However, the molecular mechanism by which NisB uses glutamate to catalyse dehydration remains unresolved. Here we show that this process involves glutamyl-tRNA(Glu) to activate Ser/Thr residues. In addition, the 2.9-Å crystal structure of NisB in complex with its substrate peptide NisA reveals the presence of two separate domains that catalyse the Ser/Thr glutamylation and glutamate elimination steps. The co-crystal structure also provides insights into substrate recognition by lantibiotic dehydratases. Our findings demonstrate an unexpected role for aminoacyl-tRNA in the formation of dehydroamino acids in lantibiotics, and serve as a basis for the functional characterization of the many lantibiotic-like dehydratases involved in the biosynthesis of other classes of natural products.

Distribution and Diversity of Nisin Producing LAB in Fermented Food.

An attempt was made, to characterize natural antibiotics or lantibiotics from unconventional sources and its antibacterial spectrum against food borne pathogens and drug resistant bacteria. Six different traditional fermented foods i.e., fermented fish, fermented soybeans, Soibum (fermented bamboo shoots), milk, idly and dosa batter were used for the isolation of bacteriocin producing Lactic acid bacteria (LAB). Among all bacterial cultures isolated from the various sources, 129 cultures have found to produce antimicrobial compounds. Nisin specific reporter bacteria was utilized as biosensor to identify the Nisin like bacteriocin, where 10 cultures found to be positive Nisin producer. Identified Nisin like bacteriocin was partially concentrated by using ammonium sulphate followed by butanol extraction. Minimum inhibitory concentration (MIC) was analyzed against food borne pathogen and drug resistant bacteria. MIC of partially purified Nisin (pp-Nisin) of all the LAB isolates against food-borne pathogens are ranged between 0.5 and 92 µg/ml respected to various Gram-positive bacteria. Similarly, the drug resistant bacteria were also inhibited by pp-Nisin (MIC ranged between 15 and 175 µg/ml). All samples of ppnisin exhibited auto induction ability. Taxonomic identification of the nisin producers was done by whole genome sequencing which reveals that cultures belongs to Lactococcus lactis ssp. lactis. Also it was found that Lactococcus lactis ssp. lactis C2d and Lactococcus lactis ssp. lactis SP2C4 harbor nisA gene and Lactococcus lactis ssp. lactis FS2 (L. lactis FS2) harbor nisQ gene. The finding of this study highlights the first case of L. lactis FS2 isolated from fermented fish harbor nisQ gene. Antibacterial activity of pp-Nisin against drug resistant LAB is also reported.

Investigation of combinations of rationally selected bioengineered nisin derivatives for their ability to inhibit Listeria in broth and model food systems.

In this study, we examined the ability of nisin A and a rationally assembled bank of 36 nisin derivative producing Lactococcus lactis strains to inhibit Listeria. A broth-based bioluminescence assay for screening single and combinations of bioengineered nisin derivatives using cell-free supernatants (CFS) from nisin derivative producing strains was developed. In this way, we screened 630 combinations of nisin derivative producing strains, identifying two (CFS from M17Q + N20P and M17Q + S29E) which exhibited enhanced anti-listerial activity when used together compared to when used alone, or to the nisin A producing strain. Minimal inhibitory concentration assays performed with purified peptides revealed than when used singly, the specific activities of M17Q, N20P and S29E (3.75-7.5 µM) against L. innocua were equal to, or less than that of nisin A (MIC of 3.75 µM). Broth-based growth curve assays using purified peptides demonstrated that use of the double peptide combinations and a triple peptide combination (M17Q + N20P + S29E) resulted in an extended lag phase of L. innocua, while kill curve assays confirmed the enhanced bactericidal activity of the combinations in comparison to the single derivative peptides or nisin A. Furthermore, the enhanced activity of the M17Q + N20P combination was maintained in a model food system (frankfurter homogenate) at both chill (4 °C) and abusive (20 °C) temperature conditions, with final cell numbers significantly less (1-2 log10 CFU/ml) than those observed with the derivative peptides alone, or nisin A. To our knowledge, this study is the first investigation that combines bioengineered bacteriocins with the aim of discovering a combination with enhanced antimicrobial activity.

Nisin J, a Novel Natural Nisin Variant, Is Produced by Staphylococcus capitis Sourced from the Human Skin Microbiota.

The skin microbiota is thought to play a key role in host protection from infection. Nisin J is a novel nisin variant produced by Staphylococcus capitis APC 2923, a strain isolated from the toe web space area in a screening study performed on the human skin microbiota. Whole-genome sequencing and mass spectrometry of the purified peptide confirmed that S. capitis APC 2923 produces a 3,458-Da bacteriocin, designated nisin J, which exhibited antimicrobial activity against a range of Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and Cutibacterium acnes The gene order in the nisin J gene cluster (nsjFEGBTCJP) differs from that of other nisin variants in that it is lacking the nisin regulatory genes, nisRK, as well as the nisin immunity gene nisI Nisin J has 9 amino acid changes compared to prototypical nisin A, with 8 amino acid substitutions, 6 of which are not present in other nisin variants (Ile4Lys, Met17Gln, Gly18Thr, Asn20Phe, Met21Ala, Ile30Gly, Val33His, and Lys34Thr), and an extra amino acid close to the C terminus, rendering nisin J the only nisin variant to contain 35 amino acids. This is the first report of a nisin variant produced by a Staphylococcus species and the first nisin producer isolated from human skin.IMPORTANCE This study describes the characterization of nisin J, the first example of a natural nisin variant, produced by a human skin isolate of staphylococcal origin. Nisin J displays inhibitory activity against a wide range of bacterial targets, including MRSA. This work demonstrates the potential of human commensals as a source for novel antimicrobials that could form part of the solution to antibiotic resistance across a broad range of bacterial pathogens.

NisI Maturation and Its Influence on Nisin Resistance in Lactococcus lactis.

NisI confers immunity against nisin, with high substrate specificity to prevent a suicidal effect in nisin-producing Lactococcus lactis strains. However, the NisI maturation process as well as its influence on nisin resistance has not been characterized. Here, we report the roles of lipoprotein signal peptidase II (Lsp) and prolipoprotein diacylglyceryl transferase (Lgt) in NisI maturation and nisin resistance of L. lactis F44. We found that the resistance of nisin of an Lsp-deficient mutant remarkably decreased, while no significant differences in growth were observed. We demonstrated that Lsp could cleave signal peptide of NisI precursor in vitro Moreover, diacylglyceryl modification of NisI catalyzed by Lgt played a decisive role in attachment of NisI on the cell envelope, while it exhibited no effects on cleavage of the signal peptides of NisI precursor. The dissociation constant (KD ) for the interaction between nisin and NisI exhibited a 2.8-fold increase compared with that between nisin and pre-NisI with signal peptide by surface plasmon resonance (SPR) analysis, providing evidence that Lsp-catalyzed signal peptide cleavage was critical for the immune activity of NisI. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production.IMPORTANCE Nisin, a safe and natural antimicrobial peptide, has a long and impressive history as a food preservative and is also considered a novel candidate to alleviate the increasingly serious threat of antibiotic resistance. Nisin is produced by certain L. lactis strains. The nisin immunity protein NisI, a membrane-bound lipoprotein, is expressed by nisin producers to avoid suicidal action. Here, we report the roles of Lsp and Lgt in NisI maturation and nisin resistance of L. lactis F44. The results verified the importance of Lsp to NisI-conferred immunity and Lgt to localization. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production.

Staphylococcus aureus Virulence Affected by an Alternative Nisin A Resistance Mechanism.

Nisin A is a bacteriocin produced by Lactococcus lactis and is widely used as a food preservative. Staphylococcus aureus has the BraRS-VraDE system that provides resistance against low concentrations of nisin A. BraRS is a two-component system that induces the expression of the ABC transporter VraDE. Previously, we isolated a highly nisin A-resistant strain with increased VraDE expression due to a mutation in braRS In this study, we isolated S. aureus MW2 mutants with BraRS-VraDE-independent nisin A resistance. These mutants, designated SAN2 ( S.aureusnisin resistant) and SAN469, had a mutation in pmtR, which encodes a transcriptional regulator responsible for the expression of the pmtABCD operon. As a result, these mutants exhibited increased expression of PmtABCD, a transporter responsible for the export of phenol-soluble modulin (PSM). Characterization of the mutants revealed that they have decreased susceptibility to human ß-defensin-3 (hBD3) and LL37, which are innate immune factors. Additionally, these mutants showed higher hemolytic activity than the original MW2 strain. Furthermore, in a mouse bacteremia model, the SAN2 strain exhibited a lower survival rate than the original MW2 strain. These results indicate that the increased expression of pmtABCD due to a pmtR mutation is an alternative nisin A resistance mechanism that also affects virulence in S. aureusIMPORTANCE Recently, the emergence of antibiotic-resistant bacteria has resulted in serious problems for chemotherapy. In addition, many antibacterial agents, such as disinfectants and food additives, are widely used. Therefore, there is a possibility that bacteria are becoming resistant to some antibacterial agents. In this study, we investigated whether Staphylococcus aureus can become resistant to nisin A, one of the bacteriocins applied as a food additive. We isolated a highly nisin A-resistant strain designated SAN2 that displayed increased expression of Pmt proteins, which are involved in the secretion of virulence factors called phenol-soluble modulins (PSMs). This strain also showed decreased susceptibility to human antimicrobial peptides and increased hemolytic activity. In addition, SAN2 showed increased lethal activity in a mouse bacteremia model. Our study provides new insights into the possibility that the acquisition of resistance against food preservatives may modulate virulence in S. aureus, suggesting that we need to pay more attention to the use of food preservatives together with antibiotics.

No more cleaning up - Efficient lactic acid bacteria cell catalysts as a cost-efficient alternative to purified lactase enzymes.

ß-galactosidases, commonly referred to as lactases, are used for producing lactose-free dairy products. Lactases are usually purified from microbial sources, which is a costly process. Here, we explored the potential that lies in using whole cells of a food-grade dairy lactic acid bacterium, Streptococcus thermophilus, as a substitute for purified lactase. We found that S. thermophilus cells, when treated with the antimicrobial peptide nisin, were able to hydrolyze lactose efficiently. The rate of hydrolysis increased with temperature; however, above 50 °C, stability was compromised. Different S. thermophilus strains were tested, and the best candidate was able to hydrolyze 80% of the lactose in a 50 g/L solution in 4 h at 50 °C, using only 0.1 g/L cells (dry weight basis). We demonstrated that it was possible to grow the cell catalyst on dairy waste, and furthermore, that a cell-free supernatant of a culture of a nisin-producing Lactococcus lactis strain could be used instead of purified nisin, which reduced cost of use significantly. Finally, we tested the cell catalysts in milk, where lactose also was efficiently hydrolyzed. The method presented is natural and low-cost, and allows for production of clean-label and lactose-free dairy products without using commercial enzymes from recombinant microorganisms. KEY POINTS: • Nisin-permeabilized Streptococcus thermophilus cells can hydrolyze lactose efficiently. • A low-cost and more sustainable alternative to purified lactase enzymes. • Reduction of overall sugar content. • Clean-label production of lactose-free dairy products.

The ability of Lactococcus lactis subsp. lactis bv. diacetylactis strains in producing nisin.

Lactococcus lactis subsp. lactis bv. diacetylactis is a relevant microorganism for the dairy industry because of its role in the production of aromatic compounds. Despite this technological property, the identification of bacteriocinogenic potential of obtained strains can offer the additional positive aspect of biosafety. A panel of 15 L. lactis subsp. lactis bv. diacetylactis strains was characterised for the presence and expression of bacteriocin related genes, and further investigated regarding the nisin operon. Eight strains were positive only for nisA, and one strain (SBR4) presented a full nisin operon, with sequencing that was shown to be similar to nisin Z. Only SBR4 presented inhibitory activity against 16 microbial target strains. The growth curves of selected targets strains confirmed the inhibitory activity of SBR4 and consequently the nisin production. This research has demonstrated the inhibitory potential of L. lactis subsp. lactis bv. diacetylactis strain, SBR4, due to its ability to produce nisin Z. This biopreservative potential, associated to previously characterised technological properties, allow the indication of this strain as a promising candidate to be used by the dairy industry as a starter or adjunct culture.

Short communication: An inducible CRISPR/dCas9 gene repression system in Lactococcus lactis.

Lactococcus lactis, one of the most important probiotic lactic acid bacteria (LAB), is widely used in the dairy industry as a cell factory for recombinant protein production. Currently, a nisin-controlled inducible expression system is used for this purpose and represents the only commercial expression system in LAB. However, the available genetic modification methods are rather limited for modulating gene expression in L. lactis. Here, we developed a 2-plasmid system for gene transcription repression in L. lactis NZ9000 that uses inducible clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9. An inducible promoter Pnisin was used to drive the expression of dCas9 from Streptococcus pyogenes, whereas a strong constitutive promoter P44 drove single guide RNA expression for single or multiple target genes. dCas9 enabled CRISPR interference-mediated silencing of single or multiple target genes with significant reduction of gene expression, up to 99%. In addition, LLNZ_07335, a putative penicillin acylase, was identified as bile salt hydrolase for bile salt resistance in NZ9000 using this system. To our knowledge, this report is the first for a functional gene for bile salt tolerance in L. lactis. Overall, our work introduces a new gene repression tool for various applications in L. lactis or other LAB.

LanI-Mediated Lantibiotic Immunity in Bacillus subtilis: Functional Analysis.

Lantibiotics subtilin and nisin are produced by Bacillus subtilis and Lactococcus lactis, respectively. To prevent toxicity of their own lantibiotic, both bacteria express specific immunity proteins, called SpaI and NisI. In addition, ABC transporters SpaFEG and NisFEG prevent lantibiotic toxicity by transporting the respective peptides to the extracellular space. Although the three-dimensional structures of SpaI and NisI have been solved, very little is known about the molecular function of either lipoprotein. Using laser-induced liquid bead ion desorption (LILBID)-mass spectrometry, we show here that subtilin interacts with SpaI monomers. The expression of either SpaI or NisI in a subtilin-nonproducing B. subtilis strain resulted in the respective strain being more resistant against either subtilin or nisin. Furthermore, pore formation provided by subtilin and nisin was prevented specifically upon the expression of either SpaI or NisI. As shown with a nisin-subtilin hybrid molecule, the C-terminal part of subtilin but not any particular lanthionine ring was needed for SpaI-mediated immunity. With respect to growth, SpaI provided less immunity against subtilin than is provided by the ABC transporter SpaFEG. However, SpaI prevented pore formation much more efficiently than SpaFEG. Taken together, our data show the physiological function of SpaI as a fast immune response to protect the cellular membrane.IMPORTANCE The two lantibiotics nisin and subtilin are produced by Lactococcus lactis and Bacillus subtilis, respectively. Both peptides have strong antimicrobial activity against Gram-positive bacteria, and therefore, appropriate protection mechanisms are required for the producing strains. To prevent toxicity of their own lantibiotic, both bacteria express immunity proteins, called SpaI and NisI, and in addition, ABC transporters SpaFEG and NisFEG. Whereas it has been shown that the ABC transporters protect the producing strains by transporting the toxic peptides to the extracellular space, the exact mode of action and the physiological function of the lipoproteins during immunity are still unknown. Understanding the exact role of lantibiotic immunity proteins is of major importance for improving production rates and for the design of newly engineered peptide antibiotics. Here, we show (i) the specificity of each lipoprotein for its own lantibiotic, (ii) the specific physical interaction of subtilin with its lipoprotein SpaI, (iii) the physiological function of SpaI in protecting the cellular membrane, and (iv) the importance of the C-terminal part of subtilin for its interaction with SpaI.

New perspectives on Mega plasmid sequence (poh1) in Bacillus thuringiensis ATCC 10792 harbouring antimicrobial, insecticidal and antibiotic resistance genes.

Bacillus thuringiensis promotes the growth of numerous economically important crops. The present study presents the complete genome sequence for a mega plasmid present in the type strain of B. thuringiensis ATCC 10792, a typical spore-forming Gram-positive bacterium with insecticidal activity, and investigates its genetic characteristics. The genome was sequenced and assembled de novo using Pac-Bio sequencers and the Hierarchical Genome Assembly Process, respectively. Further genome annotation was performed, and a total of 489 proteins and a novel mega-plasmid (poh1) with 584,623 bps were identified. The organization of poh1 revealed the genes involved in the insecticidal toxin pathway. The genes responsible for antimicrobial, insecticidal and antibiotic activities were well conserved in poh1, indicating an intimate association with plant hosts. The poh1 plasmid contains the gene encoding a novel crystal protein kinase responsible for production of zeta toxin, which poisons insects and other Gram-negative bacteria through the global inhibition of peptidoglycan synthesis. Lantibiotics are a group of bacteriocins that include the biologically active antimicrobial peptide Paenibacillin. Further, poh1 also contains the genes that encode the gramicidin S prototypical antibiotic peptide and tetracycline resistance protein. In conclusion, the strain-specific genes of B. thuringiensis strain ATCC 10792 were identified through complete genome sequencing and bioinformatics data based on major pathogenic factors that contribute to further studies of the pathogenic mechanism and phenotype analyses.

A Chemical Biology Approach to Understanding Molecular Recognition of Lipid†II by Nisin(1-12): Synthesis and NMR Ensemble Analysis of Nisin(1-12) and Analogues.

Natural products that target lipid II, such as the lantibiotic nisin, are strategically important in the development of new antibacterial agents to combat the rise of antimicrobial resistance. Understanding the structural factors that govern the highly selective molecular recognition of lipid II by the N-terminal region of nisin, nisin(1-12), is a crucial step in exploiting the potential of such compounds. In order to elucidate the relationships between amino acid sequence and conformation of this bicyclic peptide fragment, we have used solid-phase peptide synthesis to prepare two novel analogues of nisin(1-12) in which the dehydro residues have been replaced. We have carried out an NMR ensemble analysis of one of these analogues and of the wild-type nisin(1-12) peptide in order to compare the conformations of these two bicyclic peptides. Our analysis has shown the effects of residue mutation on ring conformation. We have also demonstrated that the individual rings of nisin(1-12) are pre-organised to an extent for binding to the pyrophosphate group of lipid II, with a high degree of flexibility exhibited in the central amide bond joining the two rings.

Heterologous expression of Class IIb bacteriocin Plantaricin JK in Lactococcus Lactis.

Plantaricin JK (PlnJK) is a Class IIb LAB bacteriocin that includes two peptides; i.e., PlnJ and PlnK, which can synergistically halt many types of gram-positive bacteria, including food spoilage organisms. Purification of these peptides from natural lactic acid bacteria is difficult therefore, their application remains limited. To overproduce this two-peptide bacteriocin, the food-grade nisin-controlled expression (NICE) system was firstly used to heterologous expression of PlnJK. We constructed recombinant plasmids pNZ8124-plnJ and pNZ8124-plnK, and expression of PlnJ and PlnK was achieved in Lactococcus lactis NZ9000. A combination technique of XAD-2 macroporous resin, strong cation column, and reversed-phase high performance liquid chromatography were used to obtain recombinant proteins. Their molecular mass was quantified by ESI-MS and the results were 2929.32 Da and 3502.89 Da, respectively. An antimicrobial activity assay indicated that PlnJK had significant antimicrobial activities toward strains of Staphylococcus and the two peptides acted synergistically. Fluorescence leakage analysis indicated that PlnJK induced the increase of membrane permeabilization, which resulted in intracellular ion leakage, electrolytes efflux and ultimately cell death.