Nemanja Mirkovic

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

ABSTRACT 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 (Trp25) and terminal alanine (Ala30). It was also shown that the distance between Trp25 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 (Tyr356) and alanine (Ala353). 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 Tyr356 and Ala353 residues of YvjB are essential for the function of this Zn-dependent membrane-located protease as a bacteriocin receptor.

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

ABSTRACT 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.

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.

Lactolisterin BU, a novel Class II broad spectrum bacteriocin from Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4

ABSTRACT 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.

Lactococcin B is inactivated by intrinsic proteinase PrtP digestion in Lactococcus lactis subsp. lactis BGMN1-501

In our previous study, we showed that PrtP is able to impair bacteriocin LcnB activity despite being produced by the same organism, and even if they were encoded by the same plasmid. However, exact cleavage site within LcnB bacteriocin, as well as the activity of the resulting peptides remained unknown. Here we further explored the interplay between these two proteins and defined, using mass spectrometry, that the hydrolysis occurs between the sixth and seventh amino acid on the N terminus of LcnB. Although it was suspected that the cleaved form of LcnB could retain some level of activity, both chemically synthesized and recombinant variant of truncated LcnB exhibited no antimicrobial activity. Wild type form of LcnB was recombinantly overexpressed using the same expression system, its antimicrobial activity was tested before and after the treatment with PrtP proteinase, and the degradation products were 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. Importance Lactococcal enzyme PrtP, considered as a growth promoting factor, is involved in casein breakdown and enabling bacteria efficient growth in amino acids-poor, but protein-rich media. However, its interaction with bacteriocins was not known until recently. Bacteriocin LcnB can also be considered as growth promoting factor, since its known physiological role mirrors in preventing competing bacteria of reaching high growth densities. In this manuscript, we define the exact peptide bond inside the bacteriocin LcnB which is recognized by PrtP. This N-terminal removal of six amino acids completely inactivates the bacteriocin. The biological function of such action remains elusive. It is unexpected that in the same strain one enzyme inactivates a protein important for survival, unless it is some type of regulation.

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.

Izolacija i karakterizacija bakteriocina i faktora promicanja agregacije u soju bakterije Lactococcus lactis ssp. lactis BGBM50

In this study, the influence of lactic acid fermentation on the quality of tomato powder was evaluated. The effect of adding fermented tomato powder to ready-to-cook minced pork meat to improve its nutritional value and sensory characteristics was also analysed. The cell growth of Lactobacillus sakei (7.53 log CFU/g) was more intense in the medium containing tomato powder, compared to the growth of Pediococcus pentosaceus (6.35 log CFU/g) during 24 h of fermentation; however, higher acidity (pH=4.1) was observed in the tomato powder samples fermented with Pediococcus pentosaceus. The spontaneous fermentation of tomato powder reduced cell growth by 38% and pH values slightly increased to 4.17, compared to the fermentation with pure LAB. The lactofermentation of tomato powder increased the average β-carotene and lycopene mass fractions by 43.9 and 50.2%, respectively, compared with the nonfermented samples. Lycopene and β-carotene contents in the ready-to-cook minced pork meat were proportional to the added tomato powder (10 and 30%). After cooking, β-carotene and lycopene contents decreased, on average, by 24.2 and 41.2%, respectively. The highest loss (up to 49.2%) of carotenoids was found in samples with 30% nonfermented tomato powder. Tomato powder fermented with 10% Lactobacillus sakei KTU05-6 can be recommended as both a colouring agent and a source of lycopene in the preparation of ready-to-cook minced pork meat.Chokeberries (Aronia melanocarpa) are rarely used in diet in Croatia but they have high content of polyphenolic compounds and one of the highest in vitro antioxidant activities among fruits. The aim of this study is to compare the quality, phenolic content and antioxidant capacity of different chokeberry products (juices, powders, fruit tea, capsules and dried berries). It can be expected that processing influences antioxidant activity and phenolic content of final products reaching consumers. Characterisation of phenolic compounds was carried out by using spectroscopic methods (Folin-Ciocalteu and pH differential methods). Antioxidant activity of chokeberry products was determined using 2,2-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) methods. The results show that the investigated products contain high amount of phenols (3002 to 6639 mg per L and 1494 to 5292 mg per 100 g of dry matter) and lower amount of total anthocyanins (150 to 1228 mg per L and 141 to 2468 mg per 100 g of dry matter). The examined juices and other chokeberry products possess high antioxidant capacity (12.09 to 40.19 mmol per L or 58.49 to 191.31 mmol per 100 g of dry matter, respectively) and reducing power (38.71 to 79.86 mmol per L or 13.50 to 68.60 mmol per 100 g of dry matter, respectively). On the basis of phenolic content and antioxidant activity, capsules and powders stand out among other products. The study indicates that there are significant differences (p<0.05) in the quality, phenolic content and antioxidant capacity among examined products.