Development of novel natto using legumes produced in Europe.

Natto is a traditional Japanese fermented product consisting of cooked soybeans fermented with Bacillus subtilis var. natto. We assessed three different B. subtilis strains and investigated their impact on product quality aspects, such as microbial quality, textural quality (poly-γ-glutamate strand formation), free amino acids (FAA), and volatile organic compounds (VOCs), but also the vitamin K1, K2 and B1 content, and presence of nattokinase. Using Bayesian contrast analysis, we conclude that the quality attributes were influenced by both the substrate and strain used, without significant differences in bacterial growth between strain or substrate. Overall, all the tested European legumes, except for brown beans, are adequate substrates to produce natto, with comparable or higher qualities compared to the traditional soy. Out of all the tested legumes, red lentils were the most optimal fermentation substrate. They were fermented most consistently, with high concentrations of vitamin K2, VOCs, FAA.

The impact of processing technology on microbial community composition and functional properties of Beninese maize ogi.

Traditionally fermented maize starch, called ogi, is produced to prepare akpan, a yoghurt-like street food widely consumed in Benin. Current maize ogi production practices were compared to assess the impact of different processing technologies on the characteristics of the fermented product as a basis to determine best practices. Maize starch slurry samples were collected from processors in five municipalities in southern Benin and analysed before fermentation (starch samples) and after spontaneous fermentation (ogi samples). Four technological pathways for maize starch production were distinguished based on variations in the duration of steeping the grains, which ranged from 6 to 72 h, and whether or not kneading of the wet flour before filtration was practised. Six categories of maize ogi were derived from the four technology groups based on the duration of the fermentation, which lasted from 6 to 24 h. The average pH of maize starch varied from 3.2 to 5.3, with the lowest values for the two technology groups that also had the highest lactate concentrations (9-11.8 g/L). The six maize ogi categories had a pH ranging from 3.1 to 4.0. Viable plate counts of lactic acid bacteria were similar for maize starch samples and for ogi samples, whereas yeast counts showed clear differences. Members of the genera Limosilactobacillus, Lactobacillus, Weissella, Streptococcus and Ligilactobacillus, dominated the bacterial community in maize starch, and were also dominant in maize ogi. The members of the genera dominating the fungal community in maize starch were also dominant in maize ogi, except for Aspergillus and Stenocarpella spp., which decreased in relative abundance by fermentation. The highest total free essential amino acid concentration was 61.6 mg/L in maize starch and 98.7 mg/L in ogi. The main volatile organic compounds in maize starch samples were alcohols, esters, and carboxylic acids, which also prevailed in maize ogi samples. The results indicate that the characteristics of traditional maize ogi depend on the processing technologies used to produce the maize starch before the intentional fermentation into ogi, with no clear-cut connection with the production practices due to high variations between samples from the same technology groups. This revealed the importance of a standardized maize starch production process, which would benefit controlling the starch fermentation and the characteristics of maize ogi. Further research is needed to understand the hidden fermentation during maize starch production for determination of the best practices that support the production of quality maize ogi.

Lactococcus cell envelope proteases enable lactococcal growth in minimal growth media supplemented with high molecular weight proteins of plant and animal origin.

Lactic acid bacteria (LAB) have evolved into fastidious microorganisms that require amino acids from environmental sources. Some LAB have cell envelope proteases (CEPs) that drive the proteolysis of high molecular weight proteins like casein in milk. CEP activity is typically studied using casein as the predominant substrate, even though CEPs can hydrolyze other protein sources. Plant protein hydrolysis by LAB has rarely been connected to the activity of specific CEPs. This study aims to show the activity of individual CEPs using LAB growth in a minimal growth medium supplemented with high molecular weight casein or potato proteins. Using Lactococcus cremoris MG1363 as isogenic background to express CEPs, we demonstrate that CEP activity is directly related to growth in the protein-supplemented minimal growth media. Proteolysis is analyzed based on the amino acid release, allowing a comparison of CEP activities and analysis of amino acid utilization by L. cremoris MG1363. This approach provides a basis to analyze CEP activity on plant-based protein substrates as casein alternatives and to compare activity of CEP homologs.

Dextran and levan exopolysaccharides from tempeh-associated lactic acid bacteria with bioactivity against enterotoxigenic Escherichia coli (ETEC).

Soybean tempeh contains bioactive carbohydrate that can reduce the severity of diarrhea by inhibiting enterotoxigenic Escherichia coli (ETEC) adhesion to mammalian epithelial cells. Lactic acid bacteria (LAB) are known to be present abundantly in soybean tempeh. Some LAB species can produce exopolysaccharides (EPS) with anti-adhesion bioactivity against ETEC but there has been no report of anti-adhesion bioactive EPS from tempeh-associated LAB. We isolated EPS-producing LAB from tempeh-related sources, identified them, unambiguously elucidated their EPS structure and assessed the bioactivity of their EPS against ETEC. Pediococcus pentosaceus TL, Leuconostoc mesenteroides WA and L. mesenteroides WN produced both dextran (α-1,6 linked glucan; >1000 kDa) and levan (ß-2,6 linked fructan; 650-760 kDa) in varying amounts and Leuconostoc citreum TR produced gel-forming α-1,6-mixed linkage dextran (829 kDa). All four isolates produced EPS that could adhere to ETEC cells and inhibit auto-aggregation of ETEC. EPS-PpTL, EPS-LmWA and EPS-LmWN were more bioactive towards pig-associated ETEC K88 while EPS-LcTR was more bioactive against human-associated ETEC H10407. Our finding is the first to report on the bioactivity of dextran against ETEC. Tempeh is a promising source of LAB isolates that can produce bioactive EPS against ETEC adhesion and aggregation.

Impact of vitamin B12 on rhamnose metabolism, stress defense and in-vitro virulence of Listeria monocytogenes.

Listeria monocytogenes is a facultative anaerobe which can cause a severe food-borne infection known as listeriosis. L. monocytogenes is capable of utilizing various nutrient sources including rhamnose, a naturally occurring deoxy sugar abundant in foods. L. monocytogenes can degrade rhamnose into lactate, acetate and 1,2-propanediol. Our previous study showed that addition of vitamin B12 stimulated anaerobic growth of L. monocytogenes on rhamnose due to the activation of bacterial microcompartments for 1,2-propanediol utilization (pdu BMC) with concomitant production of propionate and propanol. Notably, anaerobic 1,2-propanediol metabolism has been linked to virulence of enteric pathogens including Salmonella spp. and L. monocytogenes. In this study we investigated the impact of B12 and BMC activation on i) aerobic and anerobic growth of L. monocytogenes on rhamnose and ii) the level of virulence. We observed B12-induced pdu BMC activation and growth stimulation only in anaerobically grown cells. Comparative Caco-2 virulence assays showed that these pdu BMC-induced cells have significantly higher translocation efficiency compared to non-induced cells (anaerobic growth without B12; aerobic growth with or without B12), while adhesion and invasion capacity is similar for all cells. Comparative proteome analysis showed specific and overlapping responses linked to metabolic shifts, activation of stress defense proteins and virulence factors, with RNA polymerase sigma factor SigL, teichoic acid export ATP-binding protein TagH, DNA repair and protection proteins, RadA and DPS, and glutathione synthase GshAB, previously linked to activation of virulence response in L. monocytogenes, uniquely upregulated in anaerobically rhamnose grown pdu-induced cells. Our results shed light on possible effects of B12 on L. monocytogenes competitive fitness and virulence activation when utilizing rhamnose in anaerobic conditions encountered during transmission and the human intestine.

Lytic bacteriophages affect the population dynamics of multi-strain microbial communities.

Background: Lytic bacteriophages infect and lyse bacteria and, as a by-product, may affect diversity in microbial communities through selective predation on abundant bacterial strains. We used a complex dairy starter named Ur to investigate population dynamics of Lactococcus lactis, Lactococcus cremoris and Leuconostoc mesenteroides strains in terms of constant-diversity and periodic selection models. Methods: To mimic the starter Ur, we designed blends of 24 strains representing all eight previously identified genetic lineages in the starter culture. The blends were propagated by daily transfers in milk for over 500 generations in the presence or absence of a cocktail of lytic bacteriophages. The relative abundance of genetic lineages of L. lactis, L. cremoris and Lc. mesenteroides strains present in the complex blend, as well as phage presence, were monitored. Results: Control blends without phage predation showed decreased strain diversity, leading to a stable state due to the domination of the fittest strain(s) of a particular lineage according to periodic selection dynamics. However, in phage-challenged blends, predation caused a large shift in the microbial composition by killing the fittest and sensitive strains. Conclusion: It was demonstrated that phage-challenged blends maintained their diversity at the level of genetic lineages, thus providing experimental support for the constant-diversity dynamics model in a complex microbial community.

Strain diversity in Saccharomyces cerevisiae thiamine production capacity.

Vitamin B1 , also known as thiamine, is an important vitamin that, besides its role in human health, is converted to meat aromas upon exposure to high temperatures. Therefore, it is relevant for the production of vegan meat-like flavours. In this study, we investigated 48 Saccharomyces cerevisiae strains for their thiamine production capacity by measuring the intracellular and extracellular vitamins produced in the thiamine-free minimal medium after 72 h of growth. We found approximately an 8.2-fold difference in overall thiamine yield between the highest and lowest-producing strains. While the highest thiamine yield was 254.6 nmol/L, the highest thiamine-specific productivity was 160.9 nmol/g DW. To assess whether extracellular thiamine was due to leakage caused by cell damage, we monitored membrane permeabilization using propidium iodide (PI) staining and flow cytometry. We found a good correlation between the percentage of extracellular thiamine and PI-stained cells (Spearman's ρ = 0.85). Finally, we compared S. cerevisiae CEN.PK113-7D (wild type [WT]) to three strains evolved in a thiamine-free medium for their thiamine production capacity. On average, we saw an increase in the amount of thiamine produced. One of the evolved strains had a 49% increase in intracellular thiamine-specific productivity and a biomass increase of 20% compared with the WT. This led to a total increase in thiamine yield of 60% in this strain, reaching 208 nmol/L. This study demonstrated that it is possible to achieve thiamine overproduction in S. cerevisiae via strain selection and adaptive laboratory evolution.

The stressostat: A novel approach in adaptive laboratory evolution to improve end-product resistance.

End-product inhibition in pH-controlled batch cultures, is the major limiting factor for bacterial biomass formation in the starter culture industry as well as in many other biotechnological processes. Adaptive laboratory evolution (ALE) has emerged over the past decades as a powerful tool for phenotype optimization, but none of the existing ALE methods could select for improved end-product resistance. Therefore, we developed the stressostat (STress Resistance Evolution in Substrate Surplus) as a novel continuous ALE method. Stressostat cultivation applies end-product concentrations as constant evolutionary pressure on microorganisms in the presence of substrate surplus. In this study, we improved the lactate resistance of Lactococcus lactis FM03P in 35 days of stressostat cultivations. The lactate concentrations increased over time from 530 to 675 mM, indicating the successful selection for variants with improved lactate resistance. Thirty-four variants were isolated and grouped into four clusters based on their growth rates at high lactate concentrations. In the high-throughput screening without pH control, most isolated variants could grow at high lactate concentrations (870-928 mM), while the wild type was completely inhibited. The variants grew slower than wild type at low lactate media indicating possible evolutionary trade-off. However, in pH-controlled batch cultivations, most variants produced more biomass than the wild type. In conclusion, stressostat cultivation is a valuable method to obtain L. lactis variants with improved end-product resistance and further characterization is needed to elucidate underlying resistance mechanisms and potential industrial applications.

Influence of metabolic guilds on a temporal scale in an experimental fermented food derived microbial community.

The influence of community diversity, which can be measured at the level of metabolic guilds, on community function is a central question in ecology. Particularly, the long-term temporal dynamic between a community's function and its diversity remains unclear. We investigated the influence of metabolic guild diversity on associated community function by propagating natural microbial communities from a traditionally fermented milk beverage diluted to various levels. Specifically, we assessed the influence of less abundant microbial types, such as yeast, on community functionality and bacterial community compositions over repeated propagation cycles amounting to ∼100 generations. The starting richness of metabolic guilds had a repeatable effect on bacterial community compositions, metabolic profiles, and acidity. The influence of a single metabolic guild, yeast in our study, played a dramatic role on function, but interestingly not on long-term species sorting trajectories of the remaining bacterial community. Our results together suggest an unexpected niche division between yeast and bacterial communities and evidence ecological selection on the microbial communities in our system.

Technological variations, microbial diversity and quality characteristics of maize ogi used for akpan production in Benin.

Fermented maize starch, called ogi in Benin, is used for preparing akpan, a traditional yoghurt-like food that contributes to the food and nutrition security of its consumers. Current ogi processing technologies used by two socio-cultural groups of Benin, namely the Fon and the Goun, and aspects of the quality of the fermented starches were studied to assess the current state-of-the-art, explore changes in key product characteristics over time and identify priorities for follow-up research to increase product quality and shelf life. A survey on processing technologies was conducted in five municipalities in south Benin and samples of maize starch were collected, which were analysed after the fermentation required to obtain ogi. Four processing technologies were identified, two from the Goun (G1, G2) and two from the Fon (F1, F2). The main difference between the four processing technologies was the steeping procedure used for the maize grains. The pH of the ogi samples ranged between 3.1 and 4.2, with the highest values for G1 samples, which also contained relatively higher concentrations of sucrose (0.05-0.3 g/L) than F1 samples (0.02-0.08 g/L), and lower citrate and lactate concentrations (0.2-0.3 and 5.6-16.9 g/L, respectively) than F2 samples (0.4-0.5 and 14-27.7 g/L, respectively). Fon samples collected in Abomey were particularly rich in volatile organic compounds and free essential amino acids. Members of the genera Lactobacillus (8.6-69.3%), Limosilactobacillus (5.4-79.1%), Streptococcus (0.6-59.3%) and Weissella (2.6-51.2%) dominated the bacterial microbiota of ogi with a significant abundance of Lactobacillus spp. in Goun samples. Sordariomycetes (10.6-81.9%) and Saccharomycetes (6.2-81.4%) dominated the fungal microbiota. The yeast community of ogi samples mainly consisted of the genera Diutina, Pichia, Kluyveromyces, Lachancea and unclassified members of the Dipodascaceae family. Hierarchical clustering of metabolic data showed similarities between samples from different technologies at a default threshold of 0.05. No obvious trend in the composition of the samples' microbial communities reflected the clusters observed for the metabolic characteristics. The results indicate that beyond the general impact of the use of Fon or Goun technologies on fermented maize starch, the individual contribution of processing practices warrants study, under controlled conditions, to determine the drivers of difference or similarity between maize ogi samples to further contribute to improving product quality and shelf life.

Dynamic genome-scale modeling of Saccharomyces cerevisiae unravels mechanisms for ester formation during alcoholic fermentation.

Fermentation employing Saccharomyces cerevisiae has produced alcoholic beverages and bread for millennia. More recently, S. cerevisiae has been used to manufacture specific metabolites for the food, pharmaceutical, and cosmetic industries. Among the most important of these metabolites are compounds associated with desirable aromas and flavors, including higher alcohols and esters. Although the physiology of yeast has been well-studied, its metabolic modulation leading to aroma production in relevant industrial scenarios such as winemaking is still unclear. Here we ask what are the underlying metabolic mechanisms that explain the conserved and varying behavior of different yeasts regarding aroma formation under enological conditions? We employed dynamic flux balance analysis (dFBA) to answer this key question using the latest genome-scale metabolic model (GEM) of S. cerevisiae. The model revealed several conserved mechanisms among wine yeasts, for example, acetate ester formation is dependent on intracellular metabolic acetyl-CoA/CoA levels, and the formation of ethyl esters facilitates the removal of toxic fatty acids from cells using CoA. Species-specific mechanisms were also found, such as a preference for the shikimate pathway leading to more 2-phenylethanol production in the Opale strain as well as strain behavior varying notably during the carbohydrate accumulation phase and carbohydrate accumulation inducing redox restrictions during a later cell growth phase for strain Uvaferm. In conclusion, our new metabolic model of yeast under enological conditions revealed key metabolic mechanisms in wine yeasts, which will aid future research strategies to optimize their behavior in industrial settings.

Quantitative Physiology and Proteome Adaptations of Bifidobacterium breve NRBB57 at Near-Zero Growth Rates.

In natural environments, nutrients are usually scarce, causing microorganisms to grow slowly while staying metabolically active. These natural conditions can be simulated using retentostat cultivations. The present study describes the physiological and proteome adaptations of the probiotic Bifidobacterium breve NRBB57 from high (0.4 h-1) to near-zero growth rates. Lactose-limited retentostat cultivations were carried out for 21 days in which the bacterial growth rate progressively reduced to 0.00092 h-1, leading to a 3.4-fold reduction of the maintenance energy requirement. Lactose was mainly converted into acetate, formate, and ethanol at high growth rates, while in the retentostat, lactate production increased. Interestingly, the consumption of several amino acids (serine, aspartic acid, and glutamine/arginine) and glycerol increased over time in the retentostat. Morphological changes and viable but nonculturable cells were also observed in the retentostat. Proteomes were compared for all growth rates, revealing a downregulation of ribosomal proteins at near-zero growth rates and an upregulation of proteins involved in the catabolism of alternative energy sources. Finally, we observed induction of the stringent response and stress defense systems. Retentostat cultivations were proven useful to study the physiology of B. breve, mimicking the nutrient scarcity of its complex habitat, the human gut. IMPORTANCE In natural environments, nutrients are usually scarce, causing microorganisms to grow slowly while staying metabolically active. In this study we used retentostat cultivation to investigate how the probiotic Bifidobacterium breve adapts its physiology and proteome under severe nutrient limitation resulting in near-zero growth rates (<0.001 h-1). We showed that the nutrient limitation induced a multifaceted response including stress defense and stringent response, metabolic shifts, and the activation of novel alternative energy-producing pathways.

Species dynamics in natural bacterial communities over multiple rounds of propagation.

Our experimental work illustrates how microbial ecosystems can be shaped by selective pressures over long-term ecological time scales. Natural microbial ecosystems generally consist of various co-existing species, where community composition describes the frequency at which species or types are present. Overall functionality of the system is achieved by interacting species. Upon short-term selection, for instance by transfer to a novel environment, community composition and functionality may change in a process referred to as species sorting. Various factors, such as initial community composition and selective pressures from the environment, may influence this change. Mabisi is a traditional fermented food from Zambia that naturally contains a bacterial community of around twenty unique bacterial types. We used six comparable but different natural bacterial Mabisi communities, each split into five identical replicates, for 16 propagation cycles in a novel, common laboratory environment. Composition of the bacterial communities changed upon propagation. The influence of four main factors on community composition, i.e. initial composition (history), impact of the environment (adaptation), changes due to interaction between species and random processes (chance) in species dynamics, was tested using maximum likelihood ratios. Initial community composition seemed to determine the change in community composition, followed by random processes. Interestingly, we observed convergence at the level of ecosystem functionality, which was measured as profiles of metabolic output. This suggests different combinations of species or types can achieve similar eco-system functionality.

Microaerobic metabolism of lactate and propionate enhances vitamin B12 production in Propionibacterium freudenreichii.

BACKGROUND: Propionibacterium freudenreichii is used in biotechnological applications to produce vitamin B12. Although cultured mainly in anaerobic conditions, microaerobic conditions can greatly enhance biomass formation in P. freudenreichii. Since B12 yields may be coupled to biomass formation, microaerobic conditions show great potential for increasing B12 yields in P. freudenreichii. RESULTS: Here we show biomass formation increases 2.7 times for P. freudenreichii grown in microaerobic conditions on lactate versus anaerobic conditions (1.87 g/L vs 0.70 g/L). Consumption of lactate in microaerobic conditions resulted first in production of pyruvate, propionate and acetate. When lactate was depleted, pyruvate and propionate were oxidised with a concomitant sixfold increase in the B12 titer compared to anaerobic conditions, showing potential for propionate and pyruvate as carbon sources for B12 production. Consequently, a fed-batch reactor with anaerobically precultured lactate-grown cells was fed propionate in microaerobic conditions resulting in biomass increase and production of B12. Vitamin yields increased from 0.3 [Formula: see text] B12 per mmol lactate in anaerobic conditions to 2.4 [Formula: see text] B12 per mmol lactate and 8.4 [Formula: see text] B12 per mmol propionate in microaerobic conditions. Yield per cell dry weight (CDW) increased from 41 [Formula: see text] per g CDW in anaerobic conditions on lactate to 92 [Formula: see text] per g CDW on lactate and 184 [Formula: see text] per g CDW on propionate in microaerobic conditions. CONCLUSIONS: Here we have shown both B12 yield per substrate and per CDW were highest on cells oxidising propionate in microaerobic conditions, showing the potential of propionate for biotechnological production of vitamin B12 by P. freudenreichii.

A simple, sensitive, and specific method for the extraction and determination of thiamine and thiamine phosphate esters in fresh yeast biomass.

Thiamine is an essential vitamin for most living organisms, of which yeasts are a rich nutritional source. In this study we developed a thiamine extraction and determination method to detect thiamine in fresh yeast biomass. The thiamine determination method combines the derivatization of thiamine to a highly fluorescent product, with chromatographic separation (HPLC) and fluorescence detection. The method specifically detects free thiamine (T), thiamine phosphate (TP), and thiamine pyrophosphate (TPP). It has a high sensitivity of 2 ng/ml for TPP and TP, and 1 ng/ml for T, excellent instrumental repeatability, and low day-to-day variation in retention time of the different phosphate forms. We demonstrated the robustness of the method by proving that the fluorescence signals of the derivatised samples are stable for at least 82 h after derivatization, and by showing that the final pH of the samples does not influence the fluorescent response. In addition, we developed and validated a thiamine extraction method consisting of beads beating the fresh yeast biomass in 0.1 M HCl using a lysing matrix composed of 0.1 mm silica spheres. The performance of this method was compared to extraction via heat treatment at 95 °C for 30 min, and a combination of beads beating and heat treatment carried out in different order. We demonstrated that thiamine extraction via beads beating is the only method that prevents the biologically active form thiamine pyrophosphate to be degraded to thiamine phosphate, therefore, the extraction method developed and described in this study is preferred when the different thiamine vitamers need to be detected in their actual proportions. The combination of the extraction via beads beating, the conversion of all vitamers to the thiochrome derivatives, and the separation of these compounds on the reversed phase HPLC with a fluorescence detector, yielded a sensitive, specific, repeatable, and robust method for extraction and determination of vitamin B1 in fresh yeast biomass.

The growth-survival trade-off is hard-wired in the Lactococcus lactis gene regulation network.

Most microbes reside in oligotrophic environments for extended periods of time, requiring survival strategies that maintain proliferative capacity. We demonstrate that the non-spore-forming Lactococcus lactis KF147 progressively activates the expression of stress-associated functions in response to the declining growth rate elicited by prolonged retentostat cultivation, which coincides with up to 104 -fold increased stress tolerance. Our findings provide a quantified view of the transcription and stress-tolerance adaptations underlying the growth-survival trade-off in L. lactis, and exemplify the hard-wiring of this trade-off in the lactococcal gene regulation network.

Physiological Roles of Short-Chain and Long-Chain Menaquinones (Vitamin K2) in Lactococcus cremoris.

Lactococcus cremoris and L. lactis are well known for their occurrence and applications in dairy fermentations, but their niche extends to a range of natural and food production environments. L. cremoris and L. lactis produce MKs (vitamin K2), mainly as the long-chain forms represented by MK-9 and MK-8, and a detectable number of short-chain forms represented by MK-3. The physiological significance of the different MK forms in the lifestyle of these bacterial species has not been investigated extensively. In this study, we used L. cremoris MG1363 to construct mutants producing different MK profiles by deletion of genes encoding (i) a menaquinone-specific isochorismate synthase, (ii) a geranyltranstransferase, and (iii) a prenyl diphosphate synthase. These gene deletions resulted in (i) a non-MK producer (ΔmenF), (ii) a presumed MK-1 producer (ΔispA), and (iii) an MK-3 producer (Δllmg_0196), respectively. By examining the phenotypes of the MG1363 wildtype strain and respective mutants, including biomass accumulation, stationary phase survival, oxygen consumption, primary metabolites, azo dye/copper reduction, and proteomes, under aerobic, anaerobic, and respiration-permissive conditions, we could infer that short-chain MKs like MK-1 and MK-3 are preferred to mediate extracellular electron transfer and reaction with extracellular oxygen, while the long-chain MKs like MK-9 and MK-8 are more efficient in aerobic respiratory electron transport chain. The different electron transfer routes mediated by short-chain and long-chain MKs likely support growth and survival of L. cremoris in a range of (transiently) anaerobic and aerobic niches including food fermentations, highlighting the physiological significance of diverse MKs in L. cremoris.

Extracellular vesicle formation in Lactococcus lactis is stimulated by prophage-encoded holin-lysin system.

Gram-positive bacterial extracellular membrane vesicles (EVs) have been drawing more attention in recent years. However, mechanistic insights are still lacking on how EVs are released through the cell walls in Gram-positive bacteria. In this study, we characterized underlying mechanisms of EV production and provide evidence for a role of prophage activation in EV release using the Gram-positive bacterium Lactococcus lactis as a model. By applying a standard EV isolation procedure, we observed the presence of EVs in the culture supernatant of a lysogenic L. lactis strain FM-YL11, for which the prophage-inducing condition led to an over 10-fold increase in EV production in comparison with the non-inducing condition. In contrast, the prophage-encoded holin-lysin knockout mutant YL11ΔHLH and the prophage-cured mutant FM-YL12 produced constantly low levels of EVs. Under the prophage-inducing condition, FM-YL11 did not show massive cell lysis. Defective phage particles were found to be released in and associated with holin-lysin-induced EVs from FM-YL11, as demonstrated by transmission electron microscopic images, flow cytometry and proteomics analysis. Findings from this study further generalized the EV-producing phenotype to Gram-positive L. lactis, and provide additional insights into the EV production mechanism involving prophage-encoded holin-lysin system. The knowledge on bacterial EV production can be applied to all Gram-positive bacteria and other lactic acid bacteria with important roles in fermentations and probiotic formulations, to enable desired release and delivery of cellular components with nutritional values or probiotic effects.

Chronic Release of Tailless Phage Particles from Lactococcus lactis.

Lactococcus lactis strains residing in the microbial community of a complex dairy starter culture named "Ur" are hosts to prophages belonging to the family Siphoviridae. L. lactis strains (TIFN1 to TIFN7) showed detectable spontaneous phage production and release (109 to 1010 phage particles/ml) and up to 10-fold increases upon prophage induction, while in both cases we observed no obvious cell lysis typically described for the lytic life cycle of Siphoviridae phages. Intrigued by this phenomenon, we investigated the host-phage interaction using strain TIFN1 (harboring prophage proPhi1) as a representative. We confirmed that during the massive phage release, all bacterial cells remain viable. Further, by monitoring phage replication in vivo, using a green fluorescence protein reporter combined with flow cytometry, we demonstrated that the majority of the bacterial population (over 80%) is actively producing phage particles when induced with mitomycin C. The released tailless phage particles were found to be engulfed in lipid membranes, as evidenced by electron microscopy and lipid staining combined with chemical lipid analysis. Based on the collective observations, we propose a model of phage-host interaction in L. lactis TIFN1 where the phage particles are engulfed in membranes upon release, thereby leaving the producing host intact. Moreover, we discuss possible mechanisms of chronic, or nonlytic, release of LAB Siphoviridae phages and its impact on the bacterial host. IMPORTANCE In complex microbial consortia such as fermentation starters, bacteriophages can alter the dynamics and diversity of microbial communities. Bacteriophages infecting Lactococcus lactis are mostly studied for their detrimental impact on industrial dairy fermentation processes. In this study, we describe a novel form of phage-bacterium interaction in an L. lactis strain isolated from a complex dairy starter culture: when the prophages harbored in the L. lactis genome are activated, the phage particles are engulfed in lipid membranes upon release, leaving the producing host intact. Findings from this study provide additional insights into the diverse manners of phage-bacterium interactions and coevolution, which are essential for understanding the population dynamics in complex microbial communities like fermentation starters.