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.

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.

Rheological and textural properties of goat's milk set-type yoghurt as affected by heat treatment, transglutaminase addition and storage.

BACKGROUND: Production of goat's milk set-style yoghurt encounters challenges in achieving the texture characteristic for this type of product, primarily due to protein composition of this milk. This study evaluated the effects of using microbial transglutaminase (mTGase) concomitantly with starter culture in the production of goat's milk yoghurt - a method that has not been employed with this milk type until now- indicating the potential of the enzyme to change yoghurt's textural properties. Goat's milk set yoghurts were produced from milk heated at 72 °C/30 s and 90 °C/5 min, without (G72 and G90) and with mTGase (G72TG and G90TG) and starter culture addition. Protein profiles of goat's milks and yoghurts were also examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Yoghurts were evaluated for rheological properties, texture, microbiological and sensory profile over 2 weeks to study the influence of mTGase, pasteurization and storage. RESULTS: The enzyme caused significant increases of storage moduli at the end of fermentation: 8.32 ± 0.27 Pa (G90TG) and 2.89 ± 0.18 Pa (G72TG) vs. 6.13 ± 0.07 Pa (G90) and 1.27 ± 0.18 Pa (G72) without enzyme. Lower loss tangent values indicated the enhanced elastic character of the gels with enzyme. Enzyme increased yoghurt's firmness from 49.69 ± 2.61 g (G90) to 60.81 ± 5.29 g (G90TG) after 1 day and from 58.21 ± 0.53 g (G90) to 80.45 ± 0.59 g (G90TG) after 15 days' storage. Enzyme improved starter bacteria survivability during storage of G72TG yoghurt. CONCLUSION: mTGase can be used simultaneously with the starter culture to improve the rheological properties and texture of goat's milk yoghurt, without deteriorating effect on its flavour. © 2021 Society of Chemical Industry.

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.

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.

The Sensory Quality and Volatile Profile of Dark Chocolate Enriched with Encapsulated Probiotic Lactobacillus plantarum Bacteria.

Cocoa and dark chocolate have a wide variety of powerful antioxidants and other nutrients that can positively affect human health. Probiotic dark chocolate has the potential to be a new product in the growing number of functional foods. In this study, encapsulated potential probiotic Lactobacillus plantarum 564 and commercial probiotic Lactobacillus plantarum 299v were added in the production of dark chocolate. The results show very good survival of probiotic bacteria after production and during storage, reaching 108cfu/g in the first 60 days and over 106cfu/g up to 180 days. No statistically significant difference (p > 0.05) in chemical composition and no major differences in the volatile profiles between control and experimental chocolate samples were observed, indicating no impact of probiotic bacteria on compositional and sensory characteristics of dark chocolate. The sensory evaluation of control and both probiotic dark chocolate samples showed excellent sensory quality after 60 and 180 days of storage, demonstrating that probiotics did not affect aroma, texture and appearance of chocolate. Due to a high viability of bacterial cells and acceptable sensory properties, it can be concluded that encapsulated probiotics Lb. plantarum 564 and Lb. plantarum 299v could be successfully used in the production of probiotic dark chocolate.

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.

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.

Isolation and Characterisation of Bacteriocinand Aggregation-Promoting Factor Production inLactococcus lactis ssp. lactis BGBM50 Strain.

Lactococcus lactis ssp. lactis BGBM50, a producer of lactococcin G and aggregation-promoting factor, was isolated from selected lactic acid bacteria taken from semi-hard cheese traditionally produced in the village Zanjic, Montenegro. Strain BGBM50 harbours a number of plasmids of different sizes. Plasmid curing experiments showed that genes for bacteriocin production are located on pBM140, a plasmid 140 kb in length. PCR analysis with primers specific for lactococcin Q and G genes gave fragment of the expected size. In addition, after plasmid curing of strain BGBM50, different derivatives with altered phenotypes were obtained, among them BGBM50-34 strain, which retained bacteriocin synthesis but had enhanced aggregation ability.

Effects of Synthesis Parameters on Structure and Antimicrobial Properties of Bacterial Cellulose/Hydroxyapatite/TiO2 Polymer-Ceramic Composite Material.

Bacterial cellulose (BC) is a highly pure polysaccharide biopolymer that can be produced by various bacterial genera. Even though BC lacks functional properties, its porosity, three-dimensional network, and high specific surface area make it a suitable carrier for functional composite materials. In the present study, BC-producing bacteria were isolated from kombucha beverage and identified using a molecular method. Two sets of the BC hydrogels were produced in static conditions after four and seven days. Afterwards, two different synthesis pathways were applied for BC functionalization. The first method implied the incorporation of previously synthesized HAp/TiO2 nanocomposite using an immersion technique, while the second method included the functionalization of BC during the synthesis of HAp/TiO2 nanocomposite in the reaction mixture. The primary goal was to find the best method to obtain the functionalized material. Physicochemical and microstructural properties were analyzed by SEM, EDS, FTIR, and XRD methods. Further properties were examined by tensile test and thermogravimetric analysis, and antimicrobial activity was assessed by a total plate count assay. The results showed that HAp/TiO2 was successfully incorporated into the produced BC hydrogels using both methods. The applied methods of incorporation influenced the differences in morphology, phase distribution, mechanical and thermal properties, and antimicrobial activity against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Proteus mirabilis (ATCC 12453), and Candida albicans (ATCC 10231). Composite material can be recommended for further development and application in environments that are suitable for diseases spreading.

Autochthonous Enterococcus durans PFMI565 and Lactococcus lactis subsp. lactis BGBU1-4 in Bio-Control of Listeria monocytogenes in Ultrafiltered Cheese.

Nowadays, consumers are interested in cheese produced without chemical additives or high-temperature treatments, among which, protective lactic acid bacteria (LAB) cultures could play a major role. In this study, the aims were to isolate, identify and characterize antilisterial LAB from traditionally produced cheese, and utilize suitable LAB in cheese production. Among 200 isolated LAB colonies, isolate PFMI565, with the strongest antilisterial activity, was identified as Enterococcus durans. E. durans PFMI565 was sensitive to clinically important antibiotics (erytromicin, tetracycline, kanamycin, penicillin, vancomycin) and had low acidifying activity in milk. E. durans PFMI565 and the previously isolated bacteriocin producer, Lactococcus lactis subsp. lactis BGBU1-4, were tested for their capability to control Listeria monocytogenes in experimentally contaminated ultrafiltered (UF) cheeses during 35 days of storage at 4 °C. The greatest reductions of L. monocytogenes numbers were achieved in UF cheese made with L. lactis subsp. lactis BGBU1-4 or with the combination of L. lactis subsp. lactis BGBU1-4 and E. durans PFMI565. This study underlines the potential application of E. durans PFMI565 and L. lactis subsp. lactis BGBU1-4 in bio-control of L. monocytogenes in UF cheese.

Characterization of potential probiotic strain, L. reuteri B2, and its microencapsulation using alginate-based biopolymers.

In this study, Lactobacillus reuteri B2 was isolated from the feces of C57BL/6 mice and assessed on probiotic activity. L. reuteri B2 was identified by 16S rDNA sequencing, which the cell viability in acidic conditions at pH 2.0 was 64% after 2 h, and in the presents of 0.30% of the bile salts, after 6 h, was 37%. Antimicrobial assay with L. reuteri B2 showed maximum diameters against Klebsiela oxytoca J7 (12.5 ± 0.71 mm). We further hypothesized if L. reuteri B2 strain in the free form can survive all conditions in the gastrointestinal tract (GIT) then the utilization of the appropriate biomaterials would ameliorate its stability and viability in GIT. L. reuteri B2 was microencapsulated into sodium alginate-(Na-alg) and different content of Na-alg and sodium maleate (SM) beads. Characterization materials enveloped their thermal characteristics (TGA/DTA analysis) and structure using: scanning electron microscopy (SEM), FTIR, and particle size distribution. The high survival rate of L. reuteri B2 at low pH from 2.0 to 4.0 and in the presence of the bile salts, at concentrations up to 0.30%, was obtained. L. reuteri B2 showed strong antimicrobial activity and the best protection microencapsulated with Na-alg + SM in simulated gastric juices (SGJ).

Trypsin activity and freeze-thaw stability in the presence of ions and non-ionic surfactants.

Trypsin is a serine protease with important applications such as protein sequencing and tissue dissociation. Preserving protein structure and its activity during freeze-thawing and prolonging its shelf life is one of the most interesting tasks in biochemistry. In the present study, trypsin cryoprotection was achieved by altering buffer composition. Sodium phosphate buffer at pH 8.0 led to pH shift-induced destabilization of trypsin and formation of a molten globule, followed by significant activity loss (about 70%). Potassium phosphate and ammonium bicarbonate buffers at pH 8.0 were used with up to 90% activity recovery rate after 7 freeze-thaw cycles. The addition of non-ionic surfactants Tween 20 and Tween 80 led to up to 99% activity recovery rate. Amide I region changes, corresponding to specific secondary structures in the Fourier transform infrared (FTIR) spectrum, were modest in the case of Tween 20 and Tween 80. On the other hand, the addition of Triton X-100 led to the destabilization of α-helicoidal segments of trypsin structure after 7 freeze-thaw cycles but also increased protein substrate availability.

Broad range of substrate specificities in papain and fig latex enzymes preparations improve enumeration of Listeria monocytogenes.

Numerous applications of proteolytic enzymes include dissociation of fermented meat products for the enumeration of `foodborne pathogenic bacteria. The use of trypsin for this cause is abandoned due to the high concentration of the enzyme affecting released bacteria. Papain, as a suggested replacement, and fig latex preparation with high extent of papain-like enzymes have the potential to be applied for bacteria enumeration. Both enzymatic preparations, originating from papaya and fig, showed a broader range of substrate specificities including gelatinolytic activity, especially prominent in the case of ficin and attributed to both, cysteine protease ficin and serine protease by the analysis of 2D zymography with specific inhibitors. The activity towards native collagen, mild in the case of papain, and extensive in the case of fig latex was proved by structural analysis of digested collagen by infrared spectroscopy. Further exploration of their potential for dissociation of fermented meat products showed that both papain and fig latex enzymes are stable in the presence of detergents Tween 20 and Triton X-100 and effective in the enumeration of Listeria monocytogenes. Gelatenolytic activity, and at least partial collagenolytic activity and stability in procedure conditions make papaya and fig latex proteases potent for this application in significantly lower concentrations than previously used enzymes. As a mixture of proteolytic enzymes with divergent characteristics, fig latex preparation shows higher efficiency in Listeria monocytogenes release than papain, conserved even in the presence of stronger non-ionic detergent Triton X-100.

Lactococcin B Is Inactivated by Intrinsic Proteinase PrtP Digestion in Lactococcus lactis subsp. lactis BGMN1-501.

In our previous study we demonstrated that proteinase PrtP is able to impair bacteriocin LcnB activity, despite being produced by the same organism and encoded by the same plasmid. However, precise mechanism of this action, i.e., the exact cleavage site within LcnB bacteriocin, as well as its effect on antimicrobial activity of the resulting peptide remained vague. Here we further explored the interplay between these two proteins and defined, using mass spectrometry, that this unusual hydrolysis indeed occurs in vivo, between the sixth and seventh amino acid on the N terminus of LcnB. To address whether the cleaved form of LcnB retains any level of activity, both recombinant and chemically synthesized variant of truncated LcnB were engineered and produced, but demonstrated no antimicrobial activity. When LcnB was recombinantly overexpressed and subjected to PrtP digestion, the change in its antimicrobial activity was monitored and the degradation products analyzed with reverse-phase high-pressure liquid chromatography. The results confirmed the inactivity of the truncated LcnB and additionally corroborated the PrtP cleavage site in LcnB bacteriocin. In addition, it was demonstrated that, once truncated, LcnB is not able to bind its receptor and is susceptible to additional hydrolysis. This is the first report on proteolytic inactivation of bacteriocins inside the same bacterial host.

Brevibacillus laterosporus strains BGSP7, BGSP9 and BGSP11 isolated from silage produce broad spectrum multi-antimicrobials.

Bacteria active against multi-drug resistant pathogens, isolated by direct selection of colonies from clover silage samples, produce zones of inhibition against two Gram-negative (Klebsiella pneumoniae Ni9 and Pseudomonas aeruginosa MMA83) and two Gram-positive (Staphylococcus aureus ATCC25923 and Listeria monocytogenes ATCC19111) pathogens. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 produced the largest zones of inhibition against all four pathogens when grown in LB broth with aeration at 37°C. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 were identified as Brevibacillus laterosporus and pulsed field gel electrophoresis and extracellular protein profiles showed that three different strains (BGSP7, BGSP9 and BGSP11) were isolated. A semi-native SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) gel overlay assay showed that BGSP7 and BGSP9 produce small antimicrobial molecules of about 1.5 kDa, while BGSP11 produces antimicrobial molecules of 1.5 and 6 kDa active against S. aureus ATCC25923. Amino acid analysis of two antimicrobial molecules (1583.73 Da; from BGSP7 and 1556.31 Da; from BGSP11) revealed that they have a similar composition and differ only by virtue of the presence of a methionine which is present only in BGSP11 molecule. Genome sequencing of the three isolates revealed the presence of gene clusters associated with the production of non-ribosomally synthesized peptides (brevibacillin, bogorol, gramicidin S, plipastatin and tyrocin) and bacteriocins (laterosporulin, a lactococcin 972-like bacteriocin, as well as putative linocin M18, sactipeptide, UviB and lantipeptide-like molecules). Ultimately, the purification of a number of antimicrobial molecules from each isolate suggests that they can be considered as potent biocontrol strains that produce an arsenal of antimicrobial molecules active against Gram-positive and Gram-negative multi-resistant pathogens, fungi and insects.

Functional Characterization of the Lactolisterin BU Gene Cluster of Lactococcus lactis subsp. lactis BGBU1-4.

The gene cluster responsible for the production of the aureocin A53-like bacteriocin, lactolisterin BU, is located on plasmid pBU6 in Lactococcus lactis subsp. lactis BGBU1-4. Heterologous expression of pBU6 confirmed that production and limited immunity to lactolisterin BU were provided by the plasmid. Comparative analysis of aureocin A53-like operons revealed that the structural genes shared a low level of identity, while other genes were without homology, indicating a different origin. Subcloning and expression of genes located downstream of the structural gene, lliBU, revealed that the lactolisterin BU cluster consists of four genes: the structural gene lliBU, the abcT gene encoding an ABC transporter, the accL gene encoding an accessory protein and the immL gene which provides limited immunity to lactolisterin BU. Reverse transcription analysis revealed that all genes were transcribed as one polycistronic mRNA. Attempts to split the lactolisterin BU operon, even when both parts were under control of the PlliBU promoter, were unsuccessful indicating that expression of lactolisterin BU is probably precisely regulated at the translational level by translational coupling and is possible only when all genes of the operon are in cis constellation. Two ρ-independent transcription terminators were detected in the lactolisterin BU operon: the first in the intergenic region of the lliBU and abcT genes and the second at the end of operon. Deletion of the second transcription terminator did not influence production of the bacteriocin in lactococci.

Survival of spray-dried and free-cells of potential probiotic Lactobacillus plantarum 564 in soft goat cheese.

A high viability of probiotics in food product, with a living cells threshold of 107 /cfu/g (colony-forming units/g) is a challenge to achieve in food production. Spray drying is an efficient and economic industrial method for probiotic bacterial preservation and its application in food products. In this study, the survival of free and spray-dried cells of potential probiotic strain Lactobacillus plantarum 564 after production and during 8 weeks of storage of soft acid coagulated goat cheese was investigated, as well as compositional and sensory quality of cheese. Total bacterial count of spray-dried Lb. plantarum 564 cells were maintained at the high level of 8.82 log/cfu/g in cheese after 8 weeks of storage, while free-cell number decreased to 6.9 log/cfu/g. However, the chemical composition, pH values and sensory evaluation between control cheese (C1 sample made with commercial starter culture) and treated cheese samples (C2 and C3, made with the same starter, with the addition of free and spray-dried Lb. plantarum 564 cells, respectively) did not significantly differ. High viability of potential probiotic bacteria and acceptable sensory properties indicate that spray-dried Lb. plantarum 564 strain could be successfully used in the production of soft acid coagulated goat cheeses.

Proteinase PrtP impairs lactococcin LcnB activity in Lactococcus lactis BGMN1-501: new insights into bacteriocin regulation.

Proteinases and bacteriocins are of great importance to the dairy industry, but their interactions have not been studied so far. Lactococcus lactis subsp. lactis BGMN1-5 is a natural isolate from homemade semi-hard cheese which produces two bacteriocins (Lactococcin B and LsbB), as well as proteinase PrtP. A medium-dependent increase in the bacteriocin LcnB activity of L. lactis BGMN1-501, a derivate of L. lactis subsp. lactis BGMN1-5, was shown to be accompanied by a decrease in its promoter activity. A similar effect of media components on gene expression was reported for proteinase PrtP, whose gene is co-localized on the same plasmid as the lcnB gene. Thus, the PrtP-LcnB interplay was investigated. Single gene knockout mutants were constructed with disrupted prtP or lcnB genes. PrtP(-) mutants showed higher bacteriocin activity that had lost its growth medium dependence, which was in contrast to the original strain. When LcnB from this mutant was combined with proteinase from the LcnB(-) mutant in vitro, its activity was rendered to the original level, suggesting that proteinase reduces bacteriocin activity. We propose a new model of medium dependent expression of these genes with regard to the effects of their interaction in vivo.