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Fermentative processes by lactic acid bacteria can produce metabolites of interest to the health and food industries. Two examples are the production of B-group vitamins, and of prebiotic and immunomodulatory dextran-type exopolysaccharides. In this study, three riboflavin- and dextran-producing Weissella cibaria strains (BAL3C-5, BAL3C-7 and BAL3C-22) were used to develop a new method for selection and isolation of spontaneous riboflavin-overproducing W. cibaria mutants. This method was based on the selection of strains resistant to roseoflavin. The DNA sequencing of the FMN riboswitch of bacterial cell populations treated with various roseoflavin concentrations, revealed the existence of at least 10 spontaneous and random point mutations at this location. Folding and analysis of the mutated FMN riboswitches with the RNA fold program predicted that these mutations could result in a deregulation of the rib operon expression. When the roseoflavin-treated cultures were plated on medium supporting dextran synthesis, the most promising mutants were identified by the yellow color of their mucous colonies, exhibiting a ropy phenotype. After their isolation and recovery in liquid medium, the evaluation of their riboflavin production revealed that the mutant strains synthesized a wide range of riboflavin levels (from 0.80 to 6.50 mg/L) above the wild-type level (0.15 mg/L). Thus, this was a reliable method to select spontaneous riboflavin-overproducing and dextran-producing strains of W. cibaria. This species has not yet been used as a starter or adjunct culture, but this study reinforces the potential that it has for the food and health industry for the production of functional foods or as a probiotic. Furthermore, analysis of the influence of FMN present in the growth medium, on rib mRNA and riboflavin levels, revealed which mutant strains produce riboflavin without flavin regulation. Moreover, the BAL3C-5 C120T mutant was identified as the highest riboflavin-overproducer. Determination of its chromosomal DNA sequence and that of BAL3C-5, revealed a total identity between the 2 strains except for the C120T mutation at the FMN riboswitch. To our knowledge, this work is the first demonstration that only a single alteration in the genome of a lactic acid bacteria is required for a riboflavin-overproducing phenotype., 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 © 2023 Diez-Ozaeta, Martín-Loarte, Mohedano, Tamame, Ruiz-Masó, del Solar, Dueñas and López.)

DNA replication is essential to all living organisms as it ensures the fidelity of genetic material for the next generation of dividing cells. One of the simplest replication initiation mechanisms is the rolling circle replication. In the streptococcal plasmid pMV158, which confers antibiotic resistance to tetracycline, replication initiation is catalysed by RepB protein. The RepB N-terminal domain or origin binding domain binds to the recognition sequence (bind locus) of the double-strand origin of replication and cleaves one DNA strand at a specific site within the nic locus. Using biochemical and crystallographic analyses, here we show how the origin binding domain recognises and binds to the bind locus using structural elements removed from the active site, namely the recognition α helix, and a β-strand that organises upon binding. A new hexameric structure of full-length RepB that highlights the great flexibility of this protein is presented, which could account for its ability to perform different tasks, namely bind to two distinct loci and cleave one strand of DNA at the plasmid origin., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

This work describes a method for deriving riboflavin overproducing strains of Weissella cibaria by exposing three strains (BAL3C-5, BAL3C-7, and BAL3C-22) isolated from dough to increasing concentrations of roseoflavin. By this procedure, we selected one mutant overproducing strain from each parental strain (BAL3C-5 B2, BAL3C-7 B2, and BAL3C-22 B2, respectively). Quantification of dextran and riboflavin produced by the parental and mutant strains in a defined medium lacking riboflavin and polysaccharides confirmed that riboflavin was only overproduced by the mutant strains, whereas dextran production was similar in both mutant and parental strains. The molecular basis of the riboflavin overproduction by the mutants was determined by nucleotide sequencing of their rib operons, which encode the enzymes of the riboflavin biosynthetic pathway. We detected a unique mutation in each of the overproducing strains. These mutations, which map in the sensor domain (aptamer) of a regulatory element (the so-called FMN riboswitch) present in the 5' untranslated region of the rib operon mRNA, appear to be responsible for the riboflavin-overproducing phenotype of the BAL3C-5 B2, BAL3C-7 B2, and BAL3C-22 B2 mutant strains. Furthermore, the molecular basis of dextran production by the six W. cibaria strains has been characterized by ( i ) the sequencing of their dsr genes encoding dextransucrases, which synthesize dextran using sucrose as substrate, and ( ii ) the detection of active Dsr proteins by zymograms. Finally, the parental and mutant strains were analyzed for in situ production of riboflavin and dextran during experimental bread making. The results indicate that the mutant strains were able to produce experimental wheat breads biofortified with both riboflavin and dextran and, therefore, may be useful for the manufacture of functional commercial breads., 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 Hernández-Alcántara, Chiva, Mohedano, Russo, Ruiz-Masó, del Solar, Spano, Tamame and López.)

Manufacturing of probiotics and functional foods using lactic acid bacteria (LAB) that overproduce vitamin B 2 has gained growing interest due to ariboflavinosis problems affecting populations of both developing and affluent countries. Two isogenic Lactiplantibacillus plantarum strains, namely a riboflavin-producing parental strain (UFG9) and a roseoflavin-resistant strain (B2) that carries a mutation in the FMN-aptamer of the potential rib operon riboswitch, were analysed for production and intra- and extracellular accumulation of flavins, as well as for regulation of the rib operon expression. Strain B2 accumulated in the medium one of the highest levels of riboflavin+FMN ever reported for LAB, exceeding by ~ 25 times those accumulated by UFG9. Inside the cells, concentration of FAD was similar in both strains, while that of riboflavin+FMN was ~ 8-fold higher in B2. Mutation B2 could decrease the stability of the aptamer's regulatory P1 helix even in the presence of the effector, thus promoting the antiterminator structure of the riboswitch ON state. Although the B2-mutant riboswitch showed an impaired regulatory activity, it retained partial functionality being still sensitive to the effector. The extraordinary capacity of strain B2 to produce riboflavin, together with its metabolic versatility and probiotic properties, can be exploited for manufacturing multifunctional foods., (© 2021 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)