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Owing to their unique potential to ferment carbohydrates, both homo- and heterofermentative lactic acid bacteria (LAB) are widely used in the food industry. Deciphering the genetic basis that determine the LAB fermentation type, and hence carbohydrate utilization, is paramount to optimize LAB industrial processes. Deep sequencing of 24 LAB species and comparison with 32 publicly available genome sequences provided a comparative data set including five major LAB genera for further analysis. Phylogenomic reconstruction confirmed Leuconostoc and Pediococcus species as independently emerging from the Lactobacillus genus, within one of the three phylogenetic clades identified. These clades partially grouped LABs according to their fermentation types, suggesting that some metabolic capabilities were independently acquired during LAB evolution. In order to apply a genome-wide association study (GWAS) at the multigene family level, utilization of 49 carbohydrates was also profiled for these 56 LAB species. GWAS results indicated that obligately heterofermentative species lack 1-phosphofructokinase, required for d -mannose degradation in the homofermentative pathway. Heterofermentative species were found to often contain the araBAD operon, involved in l -arabinose degradation, which is important for heterofermentation. Taken together, our results provide helpful insights into the genetic determinants of LAB carbohydrate metabolism, and opens for further experimental research, aiming at validating the role of these candidate genes for industrial applications.

This study aimed to investigate the silage quality, bacterial community, and predicted functional profiles during the ensiling of whole‐crop maize (Zea mays L.). Maize was harvested at dough stage and ensiled in laboratory‐scale silos (1‐L capacity). Triplicate silos were sampled after 1, 3, 7, 15, 30, and 60 d of ensiling. The bacterial communities on Day 3 and Day 60 were assessed through high throughput sequencing technology, and 16S rRNA gene‐predicted functional profiles were analyzed according to the KEGG using Tax4Fun. After 60 d of ensiling, the maize silages had large amounts of lactic acid (∼69.0 g dry matter [DM] kg–1), and low pH (∼3.64), butyric acid (∼1.31 g DM kg–1), and ammonia N (∼64.9 g total N kg–1). Lactobacillus was the most dominant genus during the early and late stages of fermentation. After ensiling, the metabolism of amino acid, energy, cofactors and vitamins was inhibited by 17.5, 16.3, and 16.0% respectively, whereas the metabolism of nucleotide and carbohydrate was enhanced by 37.1 and 10.3%, respectively. The promotion of 1‐phosphofructokinase and pyruvate kinase (65.9 and 32.5%, respectively) may stimulate the lactic acid fermentation, and the enhancement of arginine deiminase (61.8%) could help lactic acid bacteria to tolerate the acid environment. Overall, the high throughput sequencing technology, combined with 16S rRNA gene‐predicted functional analyses, revealed differences in maize silage related to shifts in the bacterial community consistent with increased abundance of inferred enzymatic pathways. This approach could provide comprehensive insights into bacterial community and functional profiles to further improve the silage quality. Core Ideas High throughput sequencing method provides new insights into functional profiles in silage. Metabolic prediction analysis can describe the changes of different metabolic pathways. An enhanced capacity of key predicted microbial enzymes is linked to silage quality.

In this work, we performed a comparative adaptive laboratory evolution experiment of the important biotechnological platform strain Corynebacterium glutamicum ATCC 13032 and its prophage-free variant MB001 towards improved growth rates on glucose minimal medium. Both strains displayed a comparable adaptation behavior and no significant differences in genomic rearrangements and mutation frequencies. Remarkably, a significant fitness leap by about 20% was observed for both strains already after 100 generations. Isolated top clones (UBw and UBm) showed an about 26% increased growth rate on glucose minimal medium. Genome sequencing of evolved clones and populations resulted in the identification of key mutations in pyk (pyruvate kinase), fruK (1-phosphofructokinase) and corA encoding a Mg 2+ importer. The reintegration of selected pyk and fruK mutations resulted in an increased glucose consumption rate and ptsG expression causative for the accelerated growth on glucose minimal medium, whereas corA mutations improved growth under Mg 2+ limiting conditions. Overall, this study resulted in the identification of causative key mutations improving the growth of C. glutamicum on glucose. These identified mutational hot spots as well as the two evolved top strains, UBw and UBm, represent promising targets for future metabolic engineering approaches.

This study aimed to characterize the genomic and metabolic properties of a novel Lb. fermentum strain AGA52 which was isolated from a lactic acid fermented beverage called “shalgam.” The genome size of AGA52 was 2,001,184 bp, which is predicted to carry 2024 genes, including 50 tRNAs, 3 rRNAs, 3 ncRNAs, 15 CRISPR repeats, 14 CRISPR spacers, and 1 CRISPR array. The genome has a GC content of 51.82% including 95 predicted pseudogenes, 56 complete or partial transposases, and 2 intact prophages. The similarity of the clusters of orthologous groups (COG) was analyzed by comparison with the other Lb. fermentum strains. The detected resistome on the genome of AGA52 was found to be intrinsic originated. Besides, it has been determined that AGA52 has an obligate heterofermentative carbohydrate metabolism due to the absence of the 1-phosphofructokinase ( pfK ) enzyme. Furthermore, the strain is found to have a better antioxidant capacity and to be tolerant to gastrointestinal simulated conditions. It was also observed that the AGA52 has antimicrobial activity against Yersinia enterocolitica ATCC9610, Bacillus cereus ATCC33019, Salmonella enterica sv. Typhimurium, Escherichia coli O157:h7 ATCC43897, Listeria monocytogenes ATCC7644, Klebsiella pneumoniae ATCC13883, and Proteus vulgaris ATCC8427. Additionally, AGA52 exhibited 42.74 ± 4.82% adherence to HT29 cells. Cholesterol assimilation (33.9 ± 0.005%) and GABA production capacities were also confirmed by “in silico” and “in vitro.” Overall, the investigation of genomic and metabolic features of the AGA52 revealed that is a potential psychobiotic and probiotic dietary supplement candidate and can bring functional benefits to the host.

BackgroundThis study aimed to evaluate the ensiling characteristics, bacterial community structure, co-occurrence networks, and their predicted functionality and pathogenic risk in high-moisture oat (Avena sativa L.) silage. The oat harvested at heading stage (224 g/kg fresh weight) was spontaneously ensiled in plastic silos (10 L scale). Triplicate silos were opened after 1, 3, 7, 15, 30 and 60 days of fermentation, respectively. The bacterial community structure on day 3 and 60 were investigated using high-throughput sequencing technology, and 16S rRNA-gene predicted functionality and phenotypes were determined by PICRUSt2 and BugBase tools, respectively.ResultsAfter 60 days, the oat silage exhibited moderate fermentation quality, as indicated by large amounts of acetic acid (~ 50.4 g/kg dry matter (DM)) and lactic acid (~ 55.4 g/kg DM), relatively high pH (~ 4.79), acceptable levels of ammonia nitrogen (~ 75.2 g/kg total nitrogen) and trace amounts of butyric acid (~ 3.36 g/kg DM). Psychrobacter was prevalent in fresh oat, and Enterobacteriaceae and Lactobacillus dominated the bacterial community on day 3 and 60. Ensilage reduced the complexity of bacterial community network at the initial stage of fermentation. The bacterial functional pathways in fresh and ensiled oat are primarily characterized by the metabolism of carbohydrate and amino acid. During ensiling, the elevated pyruvate kinase and 1-phosphofructokinase levels were correlated with the lactic acid production, and the increased levels of 6-phosphogluconate dehydrogenase and ribulose-5-phosphate 3-epimerase may be responsible for the abundant acetic acid contents. Greater (P < 0.01) proportions of “Potentially Pathogenic” were observed in the bacterial community of oat silage compared to fresh oat.ConclusionsAltogether, the findings indicated that the high-moisture oat silage exhibited moderate fermentation quality, and the potential for microbial contamination and pathogens remained after 60 days of ensiling. Therefore, some effective chemical and microbial additives are recommended to ensure the quality, hygiene, and safety in high-moisture oat silage production.

Roseburia inulinivorans is a recently identified motile representative of the Firmicutes that contributes to butyrate formation from a variety of dietary polysaccharide substrates in the human large intestine. Microarray analysis was used here to investigate substrate-driven gene-expression changes in R. inulinivorans A2-194. A cluster of fructo-oligosaccharide/inulin utilization genes induced during growth on inulin included one encoding a β-fructofuranosidase protein that was prominent in the proteome of inulin-grown cells. This cluster also included a 6-phosphofructokinase and an ABC transport system, whereas a distinct inulin-induced 1-phosphofructokinase was linked to a fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS II transport enzyme). Real-time PCR analysis showed that the β-fructofuranosidase and adjacent ABC transport protein showed greatest induction during growth on inulin, whereas the 1-phosphofructokinase enzyme and linked sugar phosphotransferase transport system were most strongly up-regulated during growth on fructose, indicating that these two clusters play distinct roles in the use of inulin. The R. inulinivorans β-fructofuranosidase was overexpressed in Escherichia coli and shown to hydrolyze fructans ranging from inulin down to sucrose, with greatest activity on fructo-oligosaccharides. Genes induced on starch included the major extracellular α-amylase and two distinct α-glucanotransferases together with a gene encoding a flagellin protein. The latter response may be concerned with improving bacterial access to insoluble starch particles.

This study aimed to assess the fermentation characteristics, bacterial community structure, co-occurrence networks, and their predicted functionality and pathogenic risk in high-moisture Italian ryegrass (IR; Lolium multiflorum Lam.) silage. The IR harvested at heading stage (208 g dry matter (DM)/kg fresh weight) was spontaneously ensiled in plastic silos (10 L scale). Triplicated silos were opened after 1, 3, 7, 15, 30, and 60 days of fermentation, respectively. The bacterial community structure on days 3 and 60 were investigated using high-throughput sequencing technology, and 16S rRNA-gene predicted functionality and phenotypes were determined by PICRUSt2 and BugBase tools, respectively. After 60 days, the IR silage exhibited good ensiling characteristics indicated by large amounts of acetic acid (~58.7 g/kg DM) and lactic acid (~91.5 g/kg DM), relatively low pH (~4.20), acceptable levels of ammonia nitrogen (~87.0 g/kg total nitrogen), and trace amounts of butyric acid (~1.59 g/kg DM). Psychrobacter was prevalent in fresh IR, and Lactobacillus became the most predominant genus after 3 and 60 days. The ensilage process reduced the complexity of the bacterial community networks in IR silage. The bacterial functional pathways in fresh and ensilaged IR are primarily characterized by the metabolism of carbohydrate and amino acid. The pyruvate kinase and 1-phosphofructokinase were critical in promoting lactic acid fermentation. A greater (p < 0.01) abundance of the “potentially pathogenic” label was noticed in the bacterial communities of ensiled IR than fresh IR. Altogether, the findings indicated that the high-moisture IR silage exhibited good ensiling characteristics, but the potential for microbial contamination and pathogens still remained after ensiling.

Objective: This study aimed to investigate the fermentation profiles, bacterial community and predicted metabolic characteristics of Sudangrass (Sorghum sudanense Stapf.) during ensiling.Methods: First-cutting Sudangrass was harvested at the vegetative stage and ensiled in laboratory-scale silos (1 L capacity). Triplicate silos were sampled after 1, 3, 7, 15, 30, and 60 days of ensiling, respectively. The bacterial communities on day 3 and 60 were assessed through high-throughput sequencing technology, and 16S rRNA-gene predicted functional profiles were analyzed according to the Kyoto encyclopedia of genes and genomes using Tax4Fun.Results: The Sudangrass silages showed good fermentation quality, indicated by higher lactic acid contents, and lower pH, butyric acid and ammonia nitrogen contents. The dominant genus Lactococcus on day 3 was replaced by Lactobacillus on day 60. The metabolism of amino acid, energy, cofactors and vitamins was restricted, and metabolism of nucleotide and carbohydrate was promoted after ensiling. The 1-phosphofructokinase and pyruvate kinase of bacterial community seemed to play important roles in stimulating the lactic acid fermentation, and the promotion of arginine deiminase could help lactic acid bacteria to tolerate the acidic environment. Conclusion: High-throughput sequencing technology combined with 16S rRNA genepredicted functional analyses revealed the differences during the early and late stages ofSudangrass ensiling not only for distinct bacterial community but also for specific functional metabolites. The results could provide a comprehensive insight into bacterial community and metabolic characteristics to further improve the silage quality.

Fructose utilization in Corynebacterium glutamicum starts with its uptake and concomitant phosphorylation via the phosphotransferase system (PTS) to yield intracellular fructose 1-phosphate, which enters glycolysis upon ATP-dependent phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux on fructose (∼10%) compared to glucose (∼60%). Consequently, the biosynthesis of NADPH demanding products, e.g., L -lysine, by C. glutamicum is largely decreased when fructose is the only carbon source. Previous works reported that fructose is partially utilized via the glucose-specific PTS presumably generating fructose 6-phosphate. This closer proximity to the entry point of the oxPPP might increase oxPPP flux and, consequently, NADPH availability. Here, we generated deletion strains lacking either the fructose-specific PTS or 1-phosphofructokinase activity. We used these strains in short-term evolution experiments on fructose minimal medium and isolated mutant strains, which regained the ability of fast growth on fructose as a sole carbon source. In these fructose mutants, the deletion of the glucose-specific PTS as well as the 6-phosphofructokinase gene, abolished growth, unequivocally showing fructose phosphorylation via glucose-specific PTS to fructose 6-phosphate. Gene sequencing revealed three independent amino acid substitutions in PtsG (M260V, M260T, and P318S). These three PtsG variants mediated faster fructose uptake and utilization compared to native PtsG. In-depth analysis of the effects of fructose utilization via these PtsG variants revealed significantly increased ODs, reduced side-product accumulation, and increased L -lysine production by 50%.

Silver ions act as a powerful, broad-spectrum antimicrobial agent and are known to kill over 650 different kinds of pathogens. We investigated the protein expression pattern and identity after silver ion treatment in Escherichia coli and Staphylococcus aureus, which are primarily responsible for the majority of bovine mastitis cases using proteomics. Two-dimensional electrophoresis showed that silver ion treatment significantly reduced 5 spot’s density in E. coli and S. aureus, respectively. We identified 10 proteins (alkyl hydroperoxide reductase C22 subunit, phosphoglucomutase, fructose-1-phosphate kinase, putative carbamoyl transferase, alpha-galactosidase, carbamate kinase, ornithine transcarbamoylase, fumarate hydratase class II, alcohol dehydrogenase, and conserved hypothetical protein) by matrix-assisted laser desorption ionization time of flight (MALDI-TOF). These results demonstrated that silver ions have bactericidal effects through energy deprivation, inhibition of DNA replication, and accumulation of oxidants in bovine mastitis pathogens and suggested that silver ions can be applied for the treatment of bovine mastitis.