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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 (Δ menF ), (ii) a presumed MK-1 producer (Δ ispA ), and (iii) an MK-3 producer (Δ llmg_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 ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Charamis, Boeren, Blok, Lewis, Smid and Abee.)

Vitamin K2 is an important vitamin for human health. Vitamin K2 enrichment in the human diet is possible by using vitamin K2-producing bacteria such as Lactococcus lactis in food fermentations. Based on previous observations that aerated cultivation conditions improved vitamin K2 content in L. lactis , we performed laboratory evolution on L. lactis MG1363 by cultivating this strain in a shake flask in a sequential propagation regime with transfers to a fresh medium every 72h. After 100 generations of propagation, we selected three evolved strains that showed improved stationary phase survival in oxygenated conditions. In comparison to the original strain MG1363, the evolved strains showed 50-110% increased vitamin K2 content and exhibited high resistance against hydrogen peroxide-induced oxidative stress. Genome sequencing of the evolved strains revealed common mutations in the genes ldh and gapB . Proteomics analysis revealed overproduction of glyceraldehyde 3-phosphate dehydrogenase (GapA), universal stress protein A2 (UspA2), and formamidopyrimidine-DNA glycosylase (MutM) under aerated conditions in evolved strains, proteins with putative functions in redox reactions, universal stress response, and DNA damage repair, all of which could contribute to the enhanced oxidative stress resistance. The mechanisms underlying elevated vitamin K2 content in the evolved strains remain to be elucidated. Two out of the three evolved strains performed similar to the original strain MG1363 in terms of growth and acidification of culture media. In conclusion, this study demonstrated a natural selection approach without genetic manipulations to obtain vitamin K2 overproducers that are highly relevant for food applications and contributed to the understanding of oxidative stress resistance in L. lactis ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liu, de Groot, Boeren, Abee and Smid.)

Bacterial microcompartments (BMCs) are proteinaceous prokaryotic organelles that enable the utilization of substrates such as 1,2-propanediol and ethanolamine. BMCs are mostly linked to the survival of particular pathogenic bacteria by providing a growth advantage through utilization of 1,2-propanediol and ethanolamine which are abundantly present in the human gut. Although a 1,2-propanediol utilization cluster was found in the probiotic bacterium Propionibacterium freudenreichii , BMC-mediated metabolism of 1,2-propanediol has not been demonstrated experimentally in P. freudenreichii . In this study we show that P. freudenreichii DSM 20271 metabolizes 1,2-propanediol in anaerobic conditions to propionate and 1-propanol. Furthermore, 1,2-propanediol induced the formation of BMCs, which were visualized by transmission electron microscopy and resembled BMCs found in other bacteria. Proteomic analysis of 1,2-propanediol grown cells compared to L-lactate grown cells showed significant upregulation of proteins involved in propanediol-utilization ( pdu -cluster), DNA repair mechanisms and BMC shell proteins while proteins involved in oxidative phosphorylation were down-regulated. 1,2-Propanediol utilizing cells actively produced vitamin B 12 (cobalamin) in similar amounts as cells growing on L-lactate. The ability to metabolize 1,2-propanediol may have implications for human gut colonization and modulation, and can potentially aid in delivering propionate and vitamin B 12 in situ ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dank, Zeng, Boeren, Notebaart, Smid and Abee.)

Many traditionally fermented milk products such as mabisi involve spontaneous fermentation, which can result in bacterial community composition variation due to selection pressure. The aim of this study was to determine the composition of bacterial communities in the different types of mabisi produced across Zambia and identify the factors that influence their composition. Samples of mabisi were collected across the country, and analyzed for pH and bacterial communities using 16S rRNA amplicon sequencing. We found that the bacterial community composition was dominated by members of two phyla, i.e., Firmicutes and Proteobacteria, from which the top 10 most abundant genera were Lactococcus, Lactobacillus, Streptococcus, Enterobacter, Citrobacter, Klebsiella, Kluyvera, Buttiauxella , Aeromonas , and Acinetobacter . The most dominant genus was Lactococcus , which was present in all types of mabisi produced from all regions. The mabisi products from traditional mabisi production regions (TMPRs) were dominated by lactic acid bacteria (LAB) whereas products from non-TMPRs were dominated by non-LAB species. Tonga mabisi, the most popular type of mabisi produced in non-TMPRs, had the most complex and diverse bacterial community composition compared to the other types, which included barotse, backslopping, creamy, and thick-tonga mabisi. Other factors that influenced bacterial community composition were geographical location, fermentation duration and pH while the type of fermentation container and producer did not. This study provides new insights that can be applied in starter culture development as well as microbial functionality studies., (Copyright © 2020 Moonga, Schoustra, van den Heuvel, Linnemann, Samad, Shindano and Smid.)

Bacterial microcompartments (BMCs) are proteinaceous organelles that optimize specific metabolic pathways referred to as metabolosomes involving transient production of toxic volatile metabolites such as aldehydes. Previous bioinformatics analysis predicted the presence of BMCs in 23 bacterial phyla including foodborne pathogens and a link with gene clusters for the utilization of host-derived substrates such as 1,2-propanediol utilization, i.e., the Pdu cluster. Although, transcriptional regulation of the Pdu cluster and its role in Listeria monocytogenes virulence in animal models have recently been reported, the experimental identification and the physiological role of BMCs in L. monocytogenes is still unexplored. Here, we ask whether BMCs could enable utilization of 1,2-propanediol (Pd) in L. monocytogenes under anaerobic conditions. Using L. monocytogenes EGDe as a model strain, we could demonstrate efficient utilization of Pd with concomitant production of 1-propanol and propionate after 24 h of anaerobic growth, while the utilization was significantly reduced in aerobic conditions. In line with this, expression of genes encoding predicted shell proteins and the signature enzyme propanediol dehydratase is upregulated more than 20-fold in cells anaerobically grown in Pdu-induced versus non-induced control conditions. Additional proteomics analysis confirmed the presence of BMC shell proteins and Pdu enzymes in cells that show active degradation of Pd. Furthermore, using transmission electron microscopy, BMC structures have been detected in these cells linking gene expression, protein composition, and BMCs to activation of the Pdu cluster in anaerobic growth of L. monocytogenes . Studies in defined minimal medium with Pd as an energy source showed a significant increase in cell numbers, indicating that Pdu and the predicted generation of ATP in the conversion of propionyl-phosphate to the end product propionate can support anaerobic growth of L. monocytogenes . Our findings may suggest a role for BMC-dependent utilization of Pd in L. monocytogenes growth, transmission, and interaction with the human host., (Copyright © 2019 Zeng, Smid, Boeren, Notebaart and Abee.)

Esters are essential for the flavor and aroma of fermented products, and are mainly produced by alcohol acyl transferases (AATs). A recently discovered AAT family named Eat (Ethanol acetyltransferase) contributes to ethyl acetate synthesis in yeast. However, its effect on the synthesis of other esters is unknown. In this study, the role of the Eat family in ester synthesis was compared to that of other Saccharomyces cerevisiae AATs (Atf1p, Atf2p, Eht1p, and Eeb1p) in silico and in vivo . A genomic study in a collection of industrial S. cerevisiae strains showed that variation of the primary sequence of the AATs did not correlate with ester production. Fifteen members of the EAT family from nine yeast species were overexpressed in S. cerevisiae CEN.PK2-1D and were able to increase the production of acetate and propanoate esters. The role of Eat1p was then studied in more detail in S. cerevisiae CEN.PK2-1D by deleting EAT1 in various combinations with other known S. cerevisiae AATs. Between 6 and 11 esters were produced under three cultivation conditions. Contrary to our expectations, a strain where all known AATs were disrupted could still produce, e.g., ethyl acetate and isoamyl acetate. This study has expanded our understanding of ester synthesis in yeast but also showed that some unknown ester-producing mechanisms still exist.

During recent years it has become increasingly clear that the release of extracellular vesicles (EVs) is a feature inherent to all cellular life forms. These lipid bilayer-enclosed particles are secreted by members of all domains of life: Eukarya, Bacteria and Archaea, being similar in size, general composition, and potency as a functional entity. Noticeably, the recent discovery of EVs derived from bacteria belonging to the Gram-positive phyla Actinobacteria and Firmicutes has added a new layer of complexity to our understanding of bacterial physiology, host interactions, and pathogenesis. Being nano-sized structures, Gram-positive EVs carry a large diversity of cargo compounds, including nucleic acids, viral particles, enzymes, and effector proteins. The diversity in cargo molecules may point to roles of EVs in bacterial competition, survival, material exchange, host immune evasion and modulation, as well as infection and invasion. Consequently, the impact of Gram-positive EVs on health and disease are being revealed gradually. These findings have opened up new leads for the development of medical advances, including strategies for vaccination and anti-bacterial treatment. The rapidly advancing research into Gram-positive EVs is currently in a crucial phase, therefore this review aims to give an overview of the groundwork that has been laid at present and to discuss implications and future challenges of this new research field.