Metagenomic Analysis of Bacterial Community and Isolation of Representative Strains from Vranjska Banja Hot Spring, Serbia.

The hot spring Vranjska Banja is the hottest spring on the Balkan Peninsula with a water temperature of 63-95 °C and a pH value of 7.1, in situ. According to the physicochemical analysis, Vranjska Banja hot spring belongs to the bicarbonated and sulfated hyperthermal waters. The structures of microbial community of this geothermal spring are still largely unexplored. In order to determine and monitor the diversity of microbiota of the Vranjska Banja hot spring, a comprehensive culture-independent metagenomic analysis was conducted in parallel with a culture-dependent approach for the first time. Microbial profiling using amplicon sequencing analysis revealed the presence of phylogenetically novel taxa, ranging from species to phyla. Cultivation-based methods resulted in the isolation of 17 strains belonging to the genera Anoxybacillus, Bacillus, Geobacillus, and Hydrogenophillus. Whole-genome sequencing of five representative strains was then performed. The genomic characterization and OrthoANI analysis revealed that the Vranjska Banja hot spring harbors phylogenetically novel species of the genus Anoxybacillus, proving its uniqueness. Moreover, these isolates contain stress response genes that enable them to survive in the harsh conditions of the hot springs. The results of the in silico analysis show that most of the sequenced strains have the potential to produce thermostable enzymes (proteases, lipases, amylases, phytase, chitinase, and glucanase) and various antimicrobial molecules that can be of great importance for industrial, agricultural, and biotechnological applications. Finally, this study provides a basis for further research and understanding of the metabolic potential of these microorganisms.

Exploring the antibacterial potential of Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 by genome mining, bacteriocin gene overexpression, and chemical protein synthesis of lactolisterin BU variants.

Lactic acid bacterium Lactococcus lactis BGBU1-4 produces 43 amino acids (aa) long bacteriocin, lactolisterin BU (LBU), a 5.161 kDa peptide with potent antibacterial activity against many Gram-positive pathogens. In addition, BGBU1-4 produces an additional unknown product of 3.642 kDa with antibacterial activity. Here, we determined that the significant amount of naturally produced LBU breaks down to create a 3.642 kDa truncated form of LBU bacteriocin consisting of 31 N-terminal aa (LBU1-31) that exhibits 12.5% the antibacterial activity of the full-length LBU. We showed that chemically synthesized LBU is stable and 50% less active than native LBU, and so we used the synthetic peptides of LBU and its variants to further study their activities and antibacterial potential. Deletion analysis of LBU revealed that the 24 N-terminal aa of LBU (LBU1-24) are responsible for antibacterial activity, while downstream aa (25-43) determine the species-specific effectiveness of LBU. Although LBU1-31 contains aa 1-24, the truncation at position 31 is predicted to change the structure within aa 15-31 and might impact on antibacterial activity. Intriguingly, whole genome sequencing and genome mining established that BGBU1-4 is abundant in genes that encode potential antibacterials, but produces LBU and its breakdown product LBU1-31 exclusively.

In vitro colistin susceptibility of pandrug-resistant Ac. baumannii is restored in the presence of selenium nanoparticles.

AIMS: To investigate the synergistic activity of colistin and selenium nanoparticles (SeNPs) against pandrug-resistant (PDR) Ac. baumannii. METHODS AND RESULTS: Chequerboard and time-kill assays were employed to explore the potential synergistic interactions between colistin and SeNPs against Ac. baumannii isolates (8), previously determined as colistin-resistant (MIC range 16-256 µg ml-1 ). Also, whole-genome sequencing (WGS) and gene expression analyses were used to elucidate the mechanisms of colistin resistance. Exceptionally strong synergistic activity (FICI range 0.004-0.035) of colistin and SeNPs against colistin-resistant isolates was revealed. Colistin (0.5 or 1 µg ml-1 ) used in combination with SeNPs (0.5 µg ml-1 ) was able to reduce initial inoculum during the first 4 h of incubation, in contrast to colistin (0.5, 1 or 2 µg ml-1 ) alone. CONCLUSIONS: These findings propose colistin/SeNPs combination as a new option to fight PDR Ac. baumannii, the therapeutic possibilities of which should be proved in future in vivo studies. SIGNIFICANCE AND IMPACT OF STUDY: Here we present the first evidence of synergy between colistin and selenium compounds against bacteria in general. Also, WGS and gene expression analyses provide some new insights into Ac. baumannii colistin resistance mechanisms.

Redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides.

AIMS: The aim of this study was to construct the improved pMAL expression vector to increase the efficacy of purification of small native peptides and their clear-cut separation from MBP tag. The modifications we introduced can be applied to many expression vectors. METHODS AND RESULTS: To improve the pMAL expression vector, we introduced the His6 tag and the enterokinase cleavage site (Ek) downstream from the MBP tag and Xa cleavage site on the original vector. For cloning of a desired peptide DNA, the enterokinase site contains a unique BsaBI restriction site adjacent to the original multi-cloning site. This redesigned pMAL vector was optimized for the purification of cytoplasmic (pMALc5HisEk) and periplasmic (pMALp5HisEk) peptides. The purification of native and active peptide (P) was obtained following two-step affinity chromatography. In the first step, the entire MBP-His6 -Ek-P fusion protein is purified using the Ni-NTA agarose column. This fusion protein was cleaved with active His6 tagged enterokinase. In the second step, the further purification was performed by column containing the mixture of amylose and Ni-NTA agarose resins. This removes both the MBP-His6 and His6 -enterokinase leaving pure native protein in solution. These new vectors and the two-step purification protocol were successfully applied in purification of active native small antimicrobial peptides (AMPs), lactococcin A and human ß-defensin. CONCLUSIONS: We constructed the improved pMAL expression vectors and established the pipeline and optimal conditions for their use in efficient purification of large amounts of active native small peptides. SIGNIFICANCE AND IMPACT OF THE STUDY: Choice of expression vector impacts on the efficiency of expression and purification of desired proteins. The idea of redesigning pMAL vector was driven by the need for rapid purification of larger amounts of active native AMPs. This newly improved pMAL vector, the cloning strategy, expression conditions and two-step purification protocol represent a unique simple approach which can be applied in every laboratory.

Genomic Analysis of Multidrug-Resistant Salmonella enterica Serovar Kentucky Isolates from Humans, Turkey, and Food in the Republic of Serbia.

Owing to the emerging resistance to antimicrobials in Salmonella Kentucky isolates around the globe, the genomic comparison of all the registered multidrug-resistant Salmonella Kentucky isolates in Serbia (five from humans, one from turkey flock, and one from meat) was done. Most of the isolates were isolated from patients returning from Egypt or Tunisia or originated from imported turkey flock and turkey meat. The comparative analysis of resistance and virulence genes was done. All isolates belonged to sequence type-ST198 and were resistant to ciprofloxacin (Cip). The resistance to Cip was mediated by target mutations of the gyrA and parC genes, which encode topoisomerase I and II, respectively. Multidrug-resistant phenotype to aminoglycosides, ß-lactam antibiotics, sulfonamides, and tetracyclines was detected in five isolates. However, none of the isolates was pan-resistant to antimicrobials. The number of single nucleotide polymorphisms between isolates varied from 8 to 43 and phylogenomics revealed the genetic proximity of the human isolate 10475/11 and the turkey meat isolate 5264/14, indicating a possible meat-to-human transfer. All isolates belonged to the main Salmonella Kentucky MDR lineage, carrying the Salmonella genomic island 1 (SGI1-K) subtype. The SGI1-K of Serbian isolates showed mosaicism attributed to rapid intraclonal evolution. Many virulence factors were detected in all the isolates, including SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-9, and C63PI. Although Salmonella Kentucky has rarely been isolated from humans, food, and animals in Serbia, further surveillance is needed to diminish the risk of the spreading of resistant clones and their meat-to-human transmission.

RclS Sensor Kinase Modulates Virulence of Pseudomonas capeferrum.

Signal transduction systems are the key players of bacterial adaptation and survival. The orthodox two-component signal transduction systems perceive diverse environmental stimuli and their regulatory response leads to cellular changes. Although rarely described, the unorthodox three-component systems are also implemented in the regulation of major bacterial behavior such as the virulence of clinically relevant pathogen P. aeruginosa. Previously, we described a novel three-component system in P. capeferrum WCS358 (RclSAR) where the sensor kinase RclS stimulates the intI1 transcription in stationary growth phase. In this study, using rclS knock-out mutant, we identified RclSAR regulon in P. capeferrum WCS358. The RNA sequencing revealed that activity of RclSAR signal transduction system is growth phase dependent with more pronounced regulatory potential in early stages of growth. Transcriptional analysis emphasized the role of RclSAR in global regulation and indicated the involvement of this system in regulation of diverse cellular activities such as RNA binding and metabolic and biocontrol processes. Importantly, phenotypic comparison of WCS358 wild type and ΔrclS mutant showed that RclS sensor kinase contributes to modulation of antibiotic resistance, production of AHLs and siderophore as well as host cell adherence and cytotoxicity. Finally, we proposed the improved model of interplay between RclSAR, RpoS and LasIR regulatory systems in P. capeferrum WCS358.

Natural bacterial isolates as an inexhaustible source of new bacteriocins.

Microorganisms isolated from various traditionally fermented food products prepared in households without commercial starter cultures are designated as natural isolates. In addition, this term is also used for microorganisms collected from various natural habitats or products (silage, soil, manure, plant and animal material, etc.) that do not contain any commercial starters or bacterial formulations. They are characterized by unique traits that are the result of the selective pressure of environmental conditions, as well as interactions with other organisms. The synthesis of antimicrobial molecules, including bacteriocins, is an evolutionary advantage and an adaptive feature that sets them apart from other microorganisms from a common environment. This review aims to underline the knowledge of bacteriocins produced by natural isolates, with a particular emphasis on the most common location of their genes and operons, plasmids, and the importance of the relationship between the plasmidome and the adaptive potential of the isolate. Applications of bacteriocins, ranging from natural food preservatives to supplements and drugs in pharmacology and medicine, will also be addressed. The latest challenges faced by researchers in isolating new natural isolates with desired characteristics will be discussed, as well as the production of new antimicrobials, nearly one century since the first discovery of colicins in 1925. KEY POINTS: • Natural bacterial isolates harbor unique properties shaped by diverse interactions. • Horizontal gene transfer enables constant engineering of new antimicrobials. • Fermented food products are important source of bacteriocin-producing natural isolates.

C-protein α-antigen modulates the lantibiotic thusin resistance in Streptococcus agalactiae.

Screening for producers of potent antimicrobial peptides, resulted in the isolation of Bacillus cereus BGNM1 with strong antimicrobial activity against Listeria monocytogenes. Genome sequence analysis revealed that BGNM1 contains the gene cluster associated with the production of the lantibiotic, thusin, previously identified in B. thuringiensis. Purification of the antimicrobial activity confirmed that strain BGMN1 produces thusin. Both thusin sensitive and resistant strains were detected among clinical isolates of Streptococcus agalactiae. Random mutagenesis of a thusin sensitive strain, S. agalactiae B782, was performed in an attempt to identify the receptor protein for thusin. Three independent thusin resistant mutants were selected and their complete genomes sequenced. Comparative sequence analysis of these mutants with the WT strain revealed that duplication of a region encoding a 79 amino acids repeat in a C-protein α-antigen was a common difference, suggesting it to be responsible for increased resistance to thusin. Since induced thusin resistant mutants showed higher level of resistance than the naturally resistant B761 strain, complete genome sequencing of strain B761 was performed to check the integrity of the C-protein α-antigen-encoding gene. This analysis revealed that this gene is deleted in B761, providing further evidence that this protein promotes interaction of the thusin with receptor.

Burkholderia cepacia YtnP and Y2-aiiA lactonases inhibit virulence of Pseudomonas aeruginosa via quorum quenching activity.

Burkholderia cepacia is well known as the causative agent of infections in humans where often shares niche with other pathogens, like Pseudomonas aeruginosa. Clinical isolate Burkholderia sp. BCC4135 was selected due to its strong quorum quenching (QQ) activity. Whole genome sequencing unveiled this isolate as B. cepacia with unique sequence type ST1485 and a myriad of genes belonging to resistome and virulome. Two QQ lactonases YtnP and Y2-aiiA originated from B. cepacia BCC4135 were cloned, expressed, and functionally characterized. They were active against a broad substrate spectrum of the N-acyl-homoserine lactones (AHLs). The YtnP lactonase was inactive, while Y2-aiiA was active against N-tetradecanoyl-dl-homoserine lactone (C14-HSL) which could imply the difference in their biological roles from the aspect of its quorum sensing (QS) autoregulation and interference with the QS systems of bacteria residing within the same niche. Both YtnP and Y2-aiiA were able to attenuate virulence potential of P. aeruginosa MMA83 clinical isolate declining its biofilm formation and virulence factors production. B. cepacia BCC4135 lactonases interfered with the las, rhl, and even pqs QS circuit of P. aeruginosa MMA83 transcription and the effect of combined enzymes was even more prominent. B. cepacia BCC4135 also employs the CepI/R QS system for governing its own virulence traits and possibly self-regulates the QQ/QS network through the different expression and activity of YtnP and/or Y2-aiiA. Our findings pointed out that BCC4135 lactonases could be exploited as an effective antivirulence drugs against P. aeruginosa and gave us a new insight into B. cepacia QQ/QS machinery.

Shotgun metagenomics reveals differences in antibiotic resistance genes among bacterial communities in Western Balkans glacial lakes sediments.

Long-term overuse of antibiotics has driven the propagation and spreading of antibiotic resistance genes (ARGs) such as efflux pumps in the environment, which can be transferred to clinically relevant pathogens. This study explored the abundance and diversity of ARGs and mobile genetic elements within bacterial communities from sediments of three Western Balkans glacial lakes: Plav Lake (high impact of human population), Black Lake (medium impact of human population) and Donje Bare Lake (remote lake, minimal impact of human population) via shotgun metagenomics. Assembled metagenomic sequences revealed that Resistance-Nodulation-Division (RND) efflux pumps genes were most abundant in metagenome from the Plav Lake. The Integron Finder bioinformatics tool detected 38 clusters of attC sites lacking integron-integrases (CALIN) elements: 20 from Plav Lake, four from Black Lake and 14 from Donje Bare Lake. A complete integron sequence was recovered only from the assembled metagenome from Plav Lake. Plasmid contents within the metagenomes were similar, with proportions of contigs being plasmid-related: 1.73% for Plav Lake, 1.59% for Black Lake and 1.64% for Donje Bare Lake. The investigation showed that RNDs and mobile genetic elements content correlated with human population impact.

Benzo[4,5]thieno[2,3-d]pyrimidine phthalimide derivative, one of the rare noncompetitive inhibitors of dipeptidyl peptidase-4.

A small library of benzo[4,5]thieno[2,3-d]pyrimidine phthalimide and amine derivatives was evaluated for inhibitory activity against dipeptidyl peptidase-4 (DPP-4). The phthalimide derivatives exhibited better activity than the amine precursors, with 2-(2-(3-chlorobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)isoindoline-1,3-dione (compound 14) as the most effective inhibitor (IC50 = 34.17 ± 5.11 µM). The five most potent selected inhibitors did not show cytotoxicity to a greater extent on Caco-2 cells, even at a concentration of 250 µM. Compound 14 is considered as a novel representative of the rare noncompetitive DPP-4 inhibitors. Molecular docking and dynamics simulation indicated the importance of the Tyr547, Lys554, and Trp629 residues of DPP-4 in the formation of the enzyme-inhibitor complex. These observations could be potentially utilized for the rational design and optimization of novel (structurally similar, with phthalimide moiety, or different) noncompetitive DPP-4 inhibitors, which are anyway rare, but favorable in terms of the saturation of substrate competition.

PsrA Regulator Connects Cell Physiology and Class 1 Integron Integrase Gene Expression Through the Regulation of lexA Gene Expression in Pseudomonas spp.

Pseudomonas aeruginosa, which is a clinically important representative of Pseudomonas spp., has been recognized as causative agent of severe nosocomial infections worldwide. An increase in antibiotic resistance of P. aeruginosa clinical strains could be attributed to their capacity to acquire resistance through mobile genetic elements such as mobile integrons that are present in one-half of multidrug-resistant P. aeruginosa strains. Mobile class 1 integrons are recognized as genetic elements involved in the rapid dissemination of multiple genes encoding for antibiotic resistance. The LexA protein is a major repressor of integrase transcription, but differences in transcription regulation among bacterial species have also been noted. In this study, the promoter activity of class 1 integron integrase gene (intI1) and its variant lacking the LexA binding site in Pseudomonas putida WCS358 wild type, ΔrpoS and ΔpsrA was analysed. The results show that the activity of the intI1 gene promoter decreased in the rpoS and psrA mutants in the stationary phase of growth compared to the wild type, which indicates the role of RpoS and PsrA proteins in the positive regulation of integrase transcription. Additionally, it was determined that the activity of the lexA gene promoter decreased in ΔrpoS and ΔpsrA, and thus, we propose that PsrA indirectly regulates the intI1 gene promoter activity through regulation of lexA gene expression in co-operation with some additional regulators. In this study, intI1 gene expression was shown to be controlled by two major stress response (SOS and RpoS) regulons, which indicates that integrase has evolved to use both systems to sense the cell status.

Binding activity to intestinal cells and transient colonization in mice of two Lactobacillus paracasei subsp. paracasei strains with high aggregation potential.

Surface properties like hydrophobicity, aggregation ability, adhesion to mucosal surfaces and epithelial cells and transit time are key features for the characterization of probiotic strains. In this study, we used two Lactobacillus paracasei subsp. paracasei strains (BGNJ1-64 and BGSJ2-8) strains which were previously described with very strong aggregation capacity. The aggregation promoting factor (AggLb) expressed in these strains showed high level of binding to collagen and fibronectin, components of extracellular matrix. The working hypothesis was that strains able to aggregate have an advantage to resist in intestinal tract. So, we assessed whether these strains and their derivatives (without aggLb gene) are able to bind or not to intestinal components and we compared the transit time of each strains in mice. In that purpose parental strains (BGNJ1-64 and BGSJ2-8) and their aggregation negative derivatives (BGNJ1-641 and BGSJ2-83) were marked with double antibiotic resistance in order to be tracked in in vivo experiments in mice. Comparative analysis of binding ability of WT and aggregation negative strains to different human intestinal cell lines and mucin revealed no significant difference among them, excluding involvement of AggLb in interaction with surface of intestinal cells and mucin. In vivo experiments showed that surviving and transit time of marked strains in mice did not drastically depend on the presence of the AggLb aggregation factor.

AggLr, a novel aggregation factor in Lactococcus raffinolactis BGTRK10-1: its role in surface adhesion.

The ability of lactic acid bacteria to form multi-cellular aggregates via self-aggregation is regarded as an important mechanism for stress tolerance, adhesion, colonization and genetic material exchange. The novel aggLr gene encoding for the auto-aggregation promoting protein (AggLr) of Lactococcus raffinolactis BGTRK10-1 was cloned. Heterologous expression of AggLr enabled auto-aggregation, higher hydrophobicity and collagen and fibronectin binding of the carrier strains. Domain analysis and the type of aggregates formed by cells expressing AggLr confirmed that this aggregation factor belongs to the family of high molecular weight proteins that the authors propose to be called Snow-flake Forming Collagen Binding Aggregation Factors (SFCBAF). An additional feature of SFCBAF is that they are rich in threonine and lysine and are free of cysteine in all of the aggregation factors described so far. In contrast to previously discovered SFCBAF, the gene encoding for AggLr is located on the chromosome in the strain BGTRK10-1.

Molecular Epidemiology of Colistin-Resistant, Carbapenemase-Producing Klebsiella pneumoniae in Serbia from 2013 to 2016.

Twenty-seven colistin-resistant, carbapenemase-producing Klebsiella pneumoniae isolates were identified from hospitals in Serbia. All isolates were blaCTX-M-15 positive; ST101, ST888, ST437, ST336, and ST307 were blaOXA-48 positive; and ST340 was blaNDM-1 positive. ST307 had an insertion, and ST336 had a premature stop codon in the mgrB gene. Amino acid substitutions were detected in PmrAB of isolates ST101, ST888, ST336, and ST307. The mcr-1 and mcr-2 were not detected. An increase in phoP, phoQ, and pmrK gene transcription was detected for all sequence types.

Lactolisterin BU, a Novel Class II Broad-Spectrum Bacteriocin from Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4.

Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 produces a novel bacteriocin, lactolisterin BU, with strong antimicrobial activity against many species of Gram-positive bacteria, including important food spoilage and foodborne pathogens, such as Listeria monocytogenes, Staphylococcus aureus, Bacillus spp., and streptococci. Lactolisterin BU was extracted from the cell surface of BGBU1-4 by 2-propanol and purified to homogeneity by C18 solid-phase extraction and reversed-phase high-performance liquid chromatography. The molecular mass of the purified lactolisterin BU was 5,160.94 Da, and an internal fragment, AVSWAWQH, as determined by N-terminal sequencing, showed low-level similarity to existing antimicrobial peptides. Curing and transformation experiments revealed the presence of a corresponding bacteriocin operon on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4. Analysis of the bacteriocin operon revealed a leaderless bacteriocin of 43 amino acids that exhibited similarity to bacteriocin BHT-B (63%) from Streptococcus ratti, a bacteriocin with analogy to aureocin A.IMPORTANCE Lactolisterin BU, a broad-spectrum leaderless bacteriocin produced by L. lactis subsp. lactis bv. diacetylactis BGBU1-4, expresses strong antimicrobial activity against food spoilage and foodborne pathogens, such as Listeria monocytogenes, Staphylococcus aureus, Bacillus spp., and streptococci. Lactolisterin BU showed the highest similarity to aureocin-like bacteriocins produced by different bacteria. The operon for synthesis is located on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4, indicating possible horizontal transfer among producers.

Temperature, pH and Trimethoprim-Sulfamethoxazole Are Potent Inhibitors of Biofilm Formation by Stenotrophomonas maltophilia Clinical Isolates.

Stenotrophomonas maltophilia, an opportunistic pathogen usually connected with healthcare-associated infections, is an environmental bacterium. Intrinsic resistance to multiple antibiotics, with different virulence determinants in the last decade classified this bacterium in the group of global multiple drug resistant (MDR) organism. S. maltophilia clinical isolates, were collected from tertiary care pediatric hospital in Belgrade, Serbia to investigate influence of different factors on biofilm formation, kinetics of biofilm formation for strong biofilm producers and effect of trimethoprim-sulfamethoxazole (TMP/SMX) on formed biofilm. Most of the isolates (89.8%) were able to form a biofilm. Analysis of biofilm formation in different growth conditions showed that changing of temeperature and pH had the stronggest effect on biofilm formation almost equally in group of cystic fibrosis (CF) and non-CF strains. TMP/SMX in concentration of 50 µg/ml reduced completely 24 h old biofilms while concentration of 25 µg/ml effects formed biofilms in a strain dependent manner. Among strains able to form strong biofilm CF isolates formed biofilm slower than non-CF isolates, while shaking conditions did not affect biofilm formation. Swimming motility was detected in both CF and non-CF isolates, however more motile strain formed stronger biofilms. This study suggests that temperature, pH and TMP/SMX had the strongest influence on biofilm formation in analyzed collection of S. maltophilia. A positive correlation between motility and strength of formed biofilm was demonstrated.

Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic.

Bacteriocin producers normally possess dedicated immunity systems to protect themselves from their own bacteriocins.Lactococcus lactis strains LMG2081 and BGBM50 are known as lactococcin G producers. However, BGBM50 was sensitive to LMG2081, which indicated that LMG2081 might produce additional bacteriocins that are not present in BGBM50. Therefore, whole-genome sequencing of the two strains was performed, and a lantibiotic operon (called lctLMG) was identified in LMG2081 but not in BGBM50. The lctLMG operon contains six open reading frames; the first three genes,lmgA ,lmgM, and lmgT, are involved in the biosynthesis and export of bacteriocin, while the other three genes,lmgF,lmgE, and lmgG, are involved in lantibiotic immunity. Mutational analysis confirmed that the lctLMG operon is responsible for the additional antimicrobial activity. Specifically, site-directed mutation within this operon rendered LMG2081 inactive toward BGBM50. Subsequent purification and electrospray ionization-time of flight mass spectrometric analysis confirmed that the lantibiotic bacteriocin called lacticin LMG is exported as a 25-amino-acid peptide. Lacticin LMG is highly similar to the lacticin 481 group. It is interesting that a bacteriocin producer produces two different classes of bacteriocins, whose operons are located in the chromosome and a plasmid.

LsbB Bacteriocin Interacts with the Third Transmembrane Domain of the YvjB Receptor.

The Zn-dependent membrane-located protease YvjB has previously been shown to serve as a target receptor for LsbB, a class II leaderless lactococcal bacteriocin. Although yvjB is highly conserved in the genus Lactococcus, the bacteriocin appears to be active only against the subspecies L. lactis subsp. lactis Comparative analysis of the YvjB proteins of a sensitive strain (YvjBMN) and a resistant strain (YvjBMG) showed that they differ from each other in 31 positions. In this study, we applied site-directed mutagenesis and performed directed binding studies to provide biochemical evidence that LsbB interacts with the third transmembrane helix of YvjB in susceptible cells. The site-directed mutagenesis of LsbB and YvjB proteins showed that certain amino acids and the length of LsbB are responsible for the bacteriocin activity, most probably through adequate interaction of these two proteins; the essential amino acids in LsbB responsible for the activity are tryptophan (Trp(25)) and terminal alanine (Ala(30)). It was also shown that the distance between Trp(25) and terminal alanine is crucial for LsbB activity. The crucial region in YvjB for the interaction with LsbB is the beginning of the third transmembrane helix, particularly amino acids tyrosine (Tyr(356)) and alanine (Ala(353)). In vitro experiments showed that LsbB could interact with both YvjBMN and YvjBMG, but the strength of interaction is significantly less with YvjBMG In vivo experiments with immunofluorescently labeled antibody demonstrated that LsbB specifically interacts only with cells carrying YvjBMN IMPORTANCE: The antimicrobial activity of LsbB bacteriocin depends on the correct interaction with the corresponding receptor in the bacterial membrane of sensitive cells. Membrane-located bacteriocin receptors have essential primary functions, such as cell wall synthesis or sugar transport, and it seems that interaction with bacteriocins is suicidal for cells. This study showed that the C-terminal part of LsbB is crucial for the bacteriocin activity, most probably through adequate interaction with the third transmembrane domain of the YvjB receptor. The conserved Tyr(356) and Ala(353) residues of YvjB are essential for the function of this Zn-dependent membrane-located protease as a bacteriocin receptor.

Defining the structure and receptor binding domain of the leaderless bacteriocin LsbB.

LsbB is a class II leaderless lactococcal bacteriocin of 30 amino acids. In the present work, the structure and function relationship of LsbB was assessed. Structure determination by NMR spectroscopy showed that LsbB has an N-terminal α-helix, whereas the C-terminal of the molecule remains unstructured. To define the receptor binding domain of LsbB, a competition assay was performed in which a systematic collection of truncated peptides of various lengths covering different parts of LsbB was used to inhibit the antimicrobial activity of LsbB. The results indicate that the outmost eight-amino acid sequence at the C-terminal end is likely to contain the receptor binding domain because only truncated fragments from this region could antagonize the antimicrobial activity of LsbB. Furthermore, alanine substitution revealed that the tryptophan in position 25 (Trp(25)) is crucial for the blocking activity of the truncated peptides, as well as for the antimicrobial activity of the full-length bacteriocin. LsbB shares significant sequence homology with five other leaderless bacteriocins, especially at their C-terminal halves where all contain a conserved KXXXGXXPWE motif, suggesting that they might recognize the same receptor as LsbB. This notion was supported by the fact that truncated peptides with sequences derived from the C-terminal regions of two LsbB-related bacteriocins inhibited the activity of LsbB, in the same manner as found with the truncated version of LsbB. Taken together, these structure-function studies provide strong evidence that the receptor-binding parts of LsbB and sequence-related bacteriocins are located in their C-terminal halves.