De novo engineering riboflavin production Bacillus subtilis by overexpressing the downstream genes in the purine biosynthesis pathway.

BACKGROUND: Bacillus subtilis is widely used in industrial-scale riboflavin production. Previous studies have shown that targeted mutagenesis of the ribulose 5-phosphate 3-epimerase in B. subtilis can significantly enhance riboflavin production. This modification also leads to an increase in purine intermediate concentrations in the medium. Interestingly, B. subtilis exhibits remarkable efficiency in purine nucleoside synthesis, often exceeding riboflavin yields. These observations highlight the importance of the conversion steps from inosine-5'-monophosphate (IMP) to 2,5-diamino-6-ribosylamino-4(3 H)-pyrimidinone-5'-phosphate (DARPP) in riboflavin production by B. subtilis. However, research elucidating the specific impact of these reactions on riboflavin production remains limited. RESULT: We expressed the genes encoding enzymes involved in these reactions (guaB, guaA, gmk, ndk, ribA) using a synthetic operon. Introduction of the plasmid carrying this synthetic operon led to a 3.09-fold increase in riboflavin production compared to the control strain. Exclusion of gmk from the synthetic operon resulted in a 36% decrease in riboflavin production, which was further reduced when guaB and guaA were not co-expressed. By integrating the synthetic operon into the genome and employing additional engineering strategies, we achieved riboflavin production levels of 2702 mg/L. Medium optimization further increased production to 3477 mg/L, with a yield of 0.0869 g riboflavin per g of sucrose. CONCLUSION: The conversion steps from IMP to DARPP play a critical role in riboflavin production by B. subtilis. Our overexpression strategies have demonstrated their effectiveness in overcoming these limiting factors and enhancing riboflavin production.

Deazaflavin metabolite produced by endosymbiotic bacteria controls fungal host reproduction.

The endosymbiosis between the pathogenic fungus Rhizopus microsporus and the toxin-producing bacterium Mycetohabitans rhizoxinica represents a unique example of host control by an endosymbiont. Fungal sporulation strictly depends on the presence of endosymbionts as well as bacterially produced secondary metabolites. However, an influence of primary metabolites on host control remained unexplored. Recently, we discovered that M. rhizoxinica produces FO and 3PG-F420, a derivative of the specialized redox cofactor F420. Whether FO/3PG-F420 plays a role in the symbiosis has yet to be investigated. Here, we report that FO, the precursor of 3PG-F420, is essential to the establishment of a stable symbiosis. Bioinformatic analysis revealed that the genetic inventory to produce cofactor 3PG-F420 is conserved in the genomes of eight endofungal Mycetohabitans strains. By developing a CRISPR/Cas-assisted base editing strategy for M. rhizoxinica, we generated mutant strains deficient in 3PG-F420 (M. rhizoxinica ΔcofC) and in both FO and 3PG-F420 (M. rhizoxinica ΔfbiC). Co-culture experiments demonstrated that the sporulating phenotype of apo-symbiotic R. microsporus is maintained upon reinfection with wild-type M. rhizoxinica or M. rhizoxinica ΔcofC. In contrast, R. microsporus is unable to sporulate when co-cultivated with M. rhizoxinica ΔfbiC, even though the fungus was observed by super-resolution fluorescence microscopy to be successfully colonized. Genetic and chemical complementation of the FO deficiency of M. rhizoxinica ΔfbiC led to restoration of fungal sporulation, signifying that FO is indispensable for establishing a functional symbiosis. Even though FO is known for its light-harvesting properties, our data illustrate an important role of FO in inter-kingdom communication.

Similarities and Differences in Ligand Binding to Protein and RNA Targets: The Case of Riboflavin.

It is nowadays clear that RNA molecules can play active roles in several biological processes. As a result, an increasing number of RNAs are gradually being identified as potentially druggable targets. In particular, noncoding RNAs can adopt highly organized conformations that are suitable for drug binding. However, RNAs are still considered challenging targets due to their complex structural dynamics and high charge density. Thus, elucidating relevant features of drug-RNA binding is fundamental for advancing drug discovery. Here, by using Molecular Dynamics simulations, we compare key features of ligand binding to proteins with those observed in RNA. Specifically, we explore similarities and differences in terms of (i) conformational flexibility of the target, (ii) electrostatic contribution to binding free energy, and (iii) water and ligand dynamics. As a test case, we examine binding of the same ligand, namely riboflavin, to protein and RNA targets, specifically the riboflavin (RF) kinase and flavin mononucleotide (FMN) riboswitch. The FMN riboswitch exhibited enhanced fluctuations and explored a wider conformational space, compared to the protein target, underscoring the importance of RNA flexibility in ligand binding. Conversely, a similar electrostatic contribution to the binding free energy of riboflavin was found. Finally, greater stability of water molecules was observed in the FMN riboswitch compared to the RF kinase, possibly due to the different shape and polarity of the pockets.

Enhancing Extracellular Electron Transfer of a 3D-Printed Shewanella Bioanode with Riboflavin-Modified Carbon Black Bioink.

3D printing of a living bioanode holds the potential for the rapid and efficient production of bioelectrochemistry systems. However, the ink (such as sodium alginate, SA) that formed the matrix of the 3D-printed bioanode may hinder extracellular electron transfer (EET) between the microorganism and conductive materials. Here, we proposed a biomimetic design of a 3D-printed Shewanella bioanode, wherein riboflavin (RF) was modified on carbon black (CB) to serve as a redox substance for microbial EET. By introducing the medicated EET pathways, the 3D-printed bioanode obtained a maximum power density of 252 ± 12 mW/m2, which was 1.7 and 60.5 times higher than those of SA-CB (92 ± 10 mW/m2) and a bare carbon cloth anode (3.8 ± 0.4 mW/m2). Adding RF reduced the charge-transfer resistance of a 3D-printed bioanode by 75% (189.5 ± 18.7 vs 47.3 ± 7.8 Ω), indicating a significant acceleration in the EET efficiency within the bioanode. This work provided a fundamental and instrumental concept for constructing a 3D-printed bioanode.

Isolation, characterisation and description of the roseoflavin producer Streptomyces berlinensis sp. nov.

The Gram-positive bacteria Streptomyces davaonensis and Streptomyces cinnabarinus have been the only organisms known to produce roseoflavin, a riboflavin (vitamin B2) derived red antibiotic. Using a selective growth medium and a phenotypic screening, we were able to isolate a novel roseoflavin producer from a German soil sample. The isolation procedure was repeated twice, that is, the same strain could be isolated from the same location in Berlin 6 months and 12 months after its first isolation. Whole genome sequencing of the novel roseoflavin producer revealed an unusual chromosomal arrangement and the deposited genome sequence of the new isolate (G + C content of 71.47%) contains 897 genes per inverted terminal repeat, 6190 genes in the core and 107 genes located on an illegitimate terminal end. We identified the roseoflavin biosynthetic genes rosA, rosB and rosC and an unusually high number of riboflavin biosynthetic genes. Overexpression of rosA, rosB and rosC in Escherichia coli and enzyme assays confirmed their predicted functions in roseoflavin biosynthesis. A full taxonomic analysis revealed that the isolate represents a previously unknown Streptomyces species and we propose the name Streptomyces berlinensis sp. nov. for this roseoflavin producer.

Hydrolysis of riboflavins in root exudates under iron deficiency and alkaline stress.

Riboflavins are secreted under iron deficiency as a part of the iron acquisition Strategy I, mainly when the external pH is acidic. In plants growing under Fe-deficiency and alkaline conditions, riboflavins have been reported to accumulate inside the roots, with very low or negligible secretion. However, the fact that riboflavins may undergo hydrolysis under alkaline conditions has been so far disregarded. In this paper, we report the presence of riboflavin derivatives and products of their alkaline hydrolysis (lumichrome, lumiflavin and carboxymethylflavin) in nutrient solutions of Cucumis sativus plants grown under different iron regimes (soluble Fe-EDDHA in the nutrient solution, total absence of iron in the nutrient solution, or two different doses of FeSO4 supplied as a foliar spray), either cultivated in slightly acidic (pH 6) or alkaline (pH 8.8, 10 mM bicarbonate) nutrient solutions. The results show that root synthesis and exudation of riboflavins is controlled by shoot iron status, and that exuded riboflavins undergo hydrolysis, especially at alkaline pH, with lumichrome being the main product of hydrolysis.

Metabolic and physiological adaptations of microalgal growth-promoting bacterium Azospirillum brasilense growing under biogas atmosphere: a microarray-based transcriptome analysis.

Using microalgal growth-promoting bacteria (MGPB) to improve the cultured microalga metabolism during biotechnological processes is one of the most promising strategies to enhance their benefits. Nonetheless, the culture condition effect used during the biotechnological process on MGPB growth and metabolism is key to ensure the expected positive bacterium growth and metabolism of microalgae. In this sense, the present research study investigated the effect of the synthetic biogas atmosphere (75% CH4-25% CO2) on metabolic and physiological adaptations of the MGPB Azospirillum brasilense by a microarray-based transcriptome approach. A total of 394 A. brasilense differentially expressed genes (DEGs) were found: 201 DEGs (34 upregulated and 167 downregulated) at 24 h and 193 DEGs (140 upregulated and 53 downregulated) under the same conditions at 72 h. The results showed a series of A. brasilense genes regulating processes that could be essential for its adaptation to the early stressful condition generated by biogas. Evidence of energy production is shown by nitrate/nitrite reduction and activation of the hypothetical first steps of hydrogenotrophic methanogenesis; signal molecule modulation is observed: indole-3-acetic acid (IAA), riboflavin, and vitamin B6, activation of Type VI secretion system responding to IAA exposure, as well as polyhydroxybutyrate (PHB) biosynthesis and accumulation. Moreover, an overexpression of ipdC, ribB, and phaC genes, encoding the key enzymes for the production of the signal molecule IAA, vitamin riboflavin, and PHB production of 2, 1.5 and 11 folds, respectively, was observed at the first 24 h of incubation under biogas atmosphere Overall, the ability of A. brasilense to metabolically adapt to a biogas atmosphere is demonstrated, which allows its implementation for generating biogas with high calorific values and the use of renewable energies through microalga biotechnologies.

Mathematical kinetic modelling followed by in vitro and in vivo assays reveal the bifunctional rice GTPCHII/DHBPS enzymes and demonstrate the key roles of OsRibA proteins in the vitamin B2 pathway.

BACKGROUND: Riboflavin is the precursor of several cofactors essential for normal physical and cognitive development, but only plants and some microorganisms can produce it. Humans thus rely on their dietary intake, which at a global level is mainly constituted by cereals (> 50%). Understanding the riboflavin biosynthesis players is key for advancing our knowledge on this essential pathway and can hold promise for biofortification strategies in major crop species. In some bacteria and in Arabidopsis, it is known that RibA1 is a bifunctional protein with distinct GTP cyclohydrolase II (GTPCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) domains. Arabidopsis harbors three RibA isoforms, but only one retained its bifunctionality. In rice, however, the identification and characterization of RibA has not yet been described. RESULTS: Through mathematical kinetic modeling, we identified RibA as the rate-limiting step of riboflavin pathway and by bioinformatic analysis we confirmed that rice RibA proteins carry both domains, DHBPS and GTPCHII. Phylogenetic analysis revealed that OsRibA isoforms 1 and 2 are similar to Arabidopsis bifunctional RibA1. Heterologous expression of OsRibA1 completely restored the growth of the rib3∆ yeast mutant, lacking DHBPS expression, while causing a 60% growth improvement of the rib1∆ mutant, lacking GTPCHII activity. Regarding OsRibA2, its heterologous expression fully complemented GTPCHII activity, and improved rib3∆ growth by 30%. In vitro activity assays confirmed that both OsRibA1 and OsRibA2 proteins carry GTPCHII/DHBPS activities, but that OsRibA1 has higher DHBPS activity. The overexpression of OsRibA1 in rice callus resulted in a 28% increase in riboflavin content. CONCLUSIONS: Our study elucidates the critical role of RibA in rice riboflavin biosynthesis pathway, establishing it as the rate-limiting step in the pathway. By identifying and characterizing OsRibA1 and OsRibA2, showcasing their GTPCHII and DHBPS activities, we have advanced the understanding of riboflavin biosynthesis in this staple crop. We further demonstrated that OsRibA1 overexpression in rice callus increases its riboflavin content, providing supporting information for bioengineering efforts.

Efficient nitrate and Cr(VI) removal by denitrifier: The mechanism of S. oneidensis MR-1 promoting electron production, transportation and consumption.

When Cr(VI) and nitrate coexist, the efficiency of both bio-denitrification and Cr(VI) bio-reduction is poor because chromate hinders bacterial normal functions (i.e., electron production, transportation and consumption). Moreover, under anaerobic condition, the method about efficient nitrate and Cr(VI) removal remained unclear. In this paper, the addition of Shewanella oneidensis MR-1 to promote the electron production, transportation and consumption of denitrifier and cause an increase in the removal of nitrate and Cr(VI). The efficiency of nitrate and Cr(VI) removal accomplished by P. denitrificans as a used model denitrifier increased respectively from 51.3% to 96.1% and 34.3% to 99.8% after S. oneidensis MR-1 addition. The mechanism investigations revealed that P. denitrificans provided S. oneidensis MR-1 with lactate, which was utilized to secreted riboflavin and phenazine by S. oneidensis MR-1. The riboflavin served as coenzymes of cellular reductants (i.e., thioredoxin and glutathione) in P. denitrificans, which created favorable intracellular microenvironment conditions for electron generation. Meanwhile, phenazine promoted biofilm formation, which increased the adsorption of Cr(VI) on the cell surface and accelerated the Cr(VI) reduction by membrane bound chromate reductases thereby reducing damage to other enzymes respectively. Overall, this strategy reduced the negative effect of chromate, thus improved the generation, transportation, and consumption of electrons. SYNOPSIS: The presence of S. oneidensis MR-1 facilitated nitrate and Cr(VI) removal by P. denitrificans through decreasing the negative effect of chromate due to the metabolites' secretion.

Iron-mediated DAMO-anammox process: Revealing the mechanism of electron transfer.

The nitrate denitrifying anaerobic methane oxidation-anaerobic ammonia oxidation (DAMO-anammox) can accomplish nitrogen removal and methane (CH4) reduction. This process greatly contributes to carbon emission mitigation and carbon neutrality. In this study, we investigated the electron transfer process of functional microorganisms in the iron-mediated DAMO-anammox system. Fe3+ could be bound to several functional groups (-CH3, COO-, -CH) in extracellular polymeric substance (EPS), and the functional groups bound were different at different iron concentration. Fe3+ underwent reduction reactions to produce Fe2+. Most Fe3+ and Fe2+ react with microorganisms and formed chelates with EPS. Three-dimensional fluorescence spectra showed that Fe3+ affected the secretion of tyrosine and tryptophan, which were essential for cytochrome synthesis. The presence of Fe3+ accelerated c-type cytochrome-mediated extracellular electron transfer (EET), and when more Fe3+ existed, the more cytochrome C expressed. DAMO archaea (M. nitroreducens) in the system exhibited a high positive correlation with the functional genes (resa and ccda) for cytochrome c synthesis. Some denitrifying microorganisms showed positive correlations with the abundance of riboflavin. This finding showed that riboflavin secreted by functional microorganisms acted as an electron shuttle. In addition, DAMO archaea were positively correlated with the hair synthesis gene pily1, which indicated that direct interspecies electron transfer (DIET) may exist in the iron-mediated DAMO-anammox system.

Effect of riboflavin on redox balance, osmolyte accumulation, methylglyoxal generation and nutrient acquisition in indian squash (Praecitrullus fistulosus L.) under chromium toxicity.

The presence of high chromium (Cr) levels induces the buildup of reactive oxygen species (ROS), resulting in hindered plant development. Riboflavin (vitamin B2) is produced by plants, fungi, and microbes. It serves as a precursor to the coenzymes flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which play a crucial role in cellular metabolism. The objective of this work was to clarify the underlying mechanisms by which riboflavin alleviates Cr stress in Praecitrullus fistulosus L. Further, the role of riboflavin in growth, ions homeostasis, methylglyoxal detoxification, and antioxidant defense mechanism are not well documented in plants under Cr toxicity. We found greater biomass and minimal production of ROS in plants pretreated with riboflavin under Cr stress. Results manifested a clear abridge in growth, chlorophyll content, and nutrient uptake in Indian squash plants exposed to Cr stress. Findings displayed that Cr stress visibly enhanced oxidative injury reflected as higher malondialdehyde (MDA), hydrogen peroxide (H2O2), superoxide radical (O2•‒), methylglyoxal (MG) levels alongside vivid lipoxygenase activity. Riboflavin strengthened antioxidant system, enhanced osmolyte production and improved membrane integrity. Riboflavin diminished Cr accumulation in aerial parts that led to improved nutrient acquisition. Taken together, riboflavin abridged Cr phytotoxic effects by improving redox balance because plants treated with riboflavin had strong antioxidant system that carried out effective ROS detoxification. Riboflavin protected membrane integrity that, in turn, improved nutrient uptake in plants.

DIET-like and MIET-like mutualism of S. oneidensis MR-1 and metal-reducing function microflora boosts Cr(VI) reduction.

Microbial treatment of Cr(VI) is an environmentally friendly and low-cost approach. However, the mechanism of mutualism and the role of interspecies electron transfer in Cr(VI) reducing microflora are unclear. Herein, we constructed an intersymbiotic microbial association flora to augment interspecies electron transfer via functionalizing electroactive Shewanella oneidensis MR-1 with metal-reducing microflora, and thus the efficiency of Cr(VI) reduction. The findings suggest that the metal-reducing active microflora could converts glucose into lactic acid and riboflavin for S. oneidensis MR-1 to act as a carbon source and electron mediator. Thus, when adding initial 25 mg/L Cr (VI), this microflora exhibited an outstanding Cr (VI) removal efficiency (100%) at 12 h and elevated Cr (III) immobilization efficiency (80%) at 60 h with the assistance of 25 mg/L Cu(II). A series of electrochemical experiments proved this remarkable removal efficiency were ascribed to the improved interspecies electron transfer efficiency through direct interspecies electron transfer and riboflavin through mediated interspecies electron transfer. Furthermore, the metagenomic analysis revealed the expression level of the electron transport pathway was promoted. Intriguing high abundance of genes participating in the bio-reduction and biotransformation of Cr(VI) was also observed in functional microflora. These outcomes give a novel strategy for enhancing the reduction and fixation of harmful heavy metals by coculturing function microflora with electrogenic microorganisms.

A comparative study on riboflavin responsive multiple acyl-CoA dehydrogenation deficiency due to variants in FLAD1 and ETFDH gene.

Lipid storage myopathy (LSM) is a heterogeneous group of lipid metabolism disorders predominantly affecting skeletal muscle by triglyceride accumulation in muscle fibers. Riboflavin therapy has been shown to ameliorate symptoms in some LSM patients who are essentially concerned with multiple acyl-CoA dehydrogenation deficiency (MADD). It is proved that riboflavin responsive LSM caused by MADD is mainly due to ETFDH gene variant (ETFDH-RRMADD). We described here a case with riboflavin responsive LSM and MADD resulting from FLAD1 gene variants (c.1588 C > T p.Arg530Cys and c.1589 G > C p.Arg530Pro, FLAD1-RRMADD). And we compared our patient together with 9 FLAD1-RRMADD cases from literature to 106 ETFDH-RRMADD cases in our neuromuscular center on clinical history, laboratory investigations and pathological features. Furthermore, the transcriptomics study on FLAD1-RRMADD and ETFDH-RRMADD were carried out. On muscle pathology, both FLAD1-RRMADD and ETFDH-RRMADD were proved with lipid storage myopathy in which atypical ragged red fibers were more frequent in ETFDH-RRMADD, while fibers with faint COX staining were more common in FLAD1-RRMADD. Molecular study revealed that the expression of GDF15 gene in muscle and GDF15 protein in both serum and muscle was significantly increased in FLAD1-RRMADD and ETFDH-RRMADD groups. Our data revealed that FLAD1-RRMADD (p.Arg530) has similar clinical, biochemical, and fatty acid metabolism changes to ETFDH-RRMADD except for muscle pathological features.

Induction of Oxidative Stress in Sirtuin Gene-Disrupted Ashbya gossypii Mutants Overproducing Riboflavin.

AgHST1 and AgHST3 genes encode sirtuins that are NAD+-dependent protein deacetylases. According to previous reports, their disruption leads to the overproduction of riboflavin in Ashbya gossypii. In this study, we investigated the potential causes of riboflavin overproduction in the AgHST1Δ and AgHST3Δ mutant strains of A. gossypii. The generation of reactive oxygen species was increasd in the mutants compared to in WT. Additionally, membrane potential was lower in the mutants than in WT. The NAD+/NADH ratio in AgHST1Δ mutant strain was lower than that in WT; however, the NAD+/NADH ratio in AgHST3Δ was slightly higher than that in WT. AgHST1Δ mutant strain was more sensitive to high temperatures and hydroxyurea treatment than WT or AgHST3Δ. Expression of the AgGLR1 gene, encoding glutathione reductase, was substantially decreased in AgHST1Δ and AgHST3Δ mutant strains. The addition of N-acetyl-L-cysteine, an antioxidant, suppressed the riboflavin production in the mutants, indicating that it was induced by oxidative stress. Therefore, high oxidative stress resulting from the disruption of sirtuin genes induces riboflavin overproduction in AgHST1Δ and AgHST3Δ mutant strains. This study established that oxidative stress is an important trigger for riboflavin overproduction in sirtuin gene-disrupted mutant strains of A. gossypii and helped to elucidate the mechanism of riboflavin production in A. gossypii.

Tetraacetyl riboflavin derivative mediates caspase 3/7 activation via MAPK in A431 cells upon blue light influence.

An acetylated riboflavin derivative, 3-methyl-tetraacetyl riboflavin (3MeTARF), is a compound with high photostability and photophysical properties similar to riboflavin, including the ability to photogenerate singlet oxygen. In the present study, we compared the effects of irradiation on A431 cancer cells with blue LED light (438 nm) in the presence of 3MeTARF and riboflavin on MAPK phosphorylation, apoptosis, caspase 3/7 activation and PARP cleavage. We observed that photogenerated oxidative stress in this reaction activates MAPK by increasing phosphorylation of p38 and JNK proteins. Preincubation of cells with inhibitors specific for phosphorylation of p38 and JNK proteins (SB203580, SP600125), respectively, results in decreased caspase 3/7 activation and PARP cleavage. We showed that the tetraacetyl derivative more effectively activates MAPK and skin cancer cell death compared to riboflavin. These data, together with results of our previous study, support the hypothesis that 3MeTARF, of riboflavin, might be more useful and desirable as a compound for use in photodynamic oxidation processes, including its therapeutic potential.

New Perspective on the Interaction Behavior Between Riboflavin and ß Lactoglobulin-ß Casein Complex by Biophysical Techniques.

Riboflavin (RF) is a vitamin that only exists in plants and microorganisms and must be procured externally by humans. On the other hand, there are two major allergic factors in cow's milk, including ß-lactoglobulin (ßLG) and ß-casein (ßCN), while their allergic properties can be eliminated by binding to micronutrients. In this regard, we examined the binding process of RF to ßLG and ßCN in the binary and ternary systems by different spectroscopies such as zeta potential, electric conductivity, and molecular modeling. According to the result of the fluorescence spectrum regarding the interaction of RF with ßLG and ßCN in binary and ternary systems, an increase in RF concentration declined the fluorescence intensity of three systems and also caused the quenching of proteins. Static quenching plays a pivotal role in the formation of stable interactions. The obtained thermodynamic parameters by Van't Hoff equation ascertained the predominance of hydrogen bonds and van der Waals interaction in all the systems. Considering how the negative value of ΔH0 resulted in the negative value of ΔG0, the systems were assumed to be enthalpy driven. The outcomes of circular dichroism (CD) disclosed that the attachment of RF to the targets of systems increased their a-helix content, which particularly included the binding of RF to ßLG that led to the conversion of ß-sheet to α-helix content. As indicated by the results of zeta potential, the low concentration of RF contained the dominance of hydrophobic forces in the interactions, whereas the enlargement of this concentration prevailed electrostatic forces. Moreover, conductometry measurements showed an extension in the rate of ionizable groups due to the addition of RF to the systems, which may increase the probability of an interaction between RF, ßCN, and ßLG in binary and ternary systems. In consistency with the outcomes of molecular dynamics simulation, the data of molecular docking approved the capability of RF in forming strong and stable interactions with ßCN and ßLG.

High-level nitrofurantoin resistance in a clinical isolate of Klebsiella pneumoniae: a comparative genomics and metabolomics analysis.

Nitrofurantoin is a commonly used chemotherapeutic agent in the treatment of uncomplicated urinary tract infections caused by the problematic multidrug resistant Gram-negative pathogen Klebsiella pneumoniae. The present study aims to elucidate the mechanism of nitrofurantoin action and high-level resistance in K. pneumoniae using whole-genome sequencing (WGS), qPCR analysis, mutation structural modeling and untargeted metabolomic analysis. WGS profiling of evolved highly resistant mutants (nitrofurantoin minimum inhibitory concentrations > 256 mg/L) revealed modified expression of several genes related to membrane transport (porin ompK36 and efflux pump regulator oqxR) and nitroreductase activity (ribC and nfsB, involved in nitrofurantoin reduction). Untargeted metabolomics analysis of total metabolites extracted at 1 and 4 h post-nitrofurantoin treatment revealed that exposure to the drug caused a delayed effect on the metabolome which was most pronounced after 4 h. Pathway enrichment analysis illustrated that several complex interrelated metabolic pathways related to nitrofurantoin bacterial killing (aminoacyl-tRNA biosynthesis, purine metabolism, central carbohydrate metabolism, and pantothenate and CoA biosynthesis) and the development of nitrofurantoin resistance (riboflavin metabolism) were significantly perturbed. This study highlights for the first time the key role of efflux pump regulator oqxR in nitrofurantoin resistance and reveals global metabolome perturbations in response to nitrofurantoin, in K. pneumoniae.IMPORTANCEA quest for novel antibiotics and revitalizing older ones (such as nitrofurantoin) for treatment of difficult-to-treat Gram-negative bacterial infections has become increasingly popular. The precise antibacterial activity of nitrofurantoin is still not fully understood. Furthermore, although the prevalence of nitrofurantoin resistance remains low currently, the drug's fast-growing consumption worldwide highlights the need to comprehend the emerging resistance mechanisms. Here, we used multidisciplinary techniques to discern the exact mechanism of nitrofurantoin action and high-level resistance in Klebsiella pneumoniae, a common cause of urinary tract infections for which nitrofurantoin is the recommended treatment. We found that the expression of multiple genes related to membrane transport (including active efflux and passive diffusion of drug molecules) and nitroreductase activity was modified in nitrofurantoin-resistant strains, including oqxR, the transcriptional regulator of the oqxAB efflux pump. Furthermore, complex interconnected metabolic pathways that potentially govern the nitrofurantoin-killing mechanisms (e.g., aminoacyl-tRNA biosynthesis) and nitrofurantoin resistance (riboflavin metabolism) were significantly inhibited following nitrofurantoin treatment. Our study could help inform the improvement of nitrofuran derivatives, the development of new pharmacophores, or drug combinations to support the resurgence of nitrofurantoin in the management of multidrug resistant K. pneumouniae infection.

Exogenous riboflavin (vitamin B2) application enhances salinity tolerance through the activation of its biosynthesis in rice seedlings under salinity stress.

Salinity stress triggers the accumulation of reactive oxygen species (ROS), leading to impaired plant growth. Riboflavin (RIB; vitamin B2) is synthesized by plants, fungi, and microorganisms and is a precursor of the coenzymes, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which are important for cellular metabolism. In this study, we aimed to elucidate the mechanistic basis of the RIB-mediated alleviation of salinity stress in rice. We observed higher biomass accumulation and lower concentrations of malondialdehyde (MDA) and hydrogen peroxide (H2O2) in RIB-pretreated seedlings under salinity stress. In vitro assays showed that H2O2 was scavenged as the RIB concentration increased, implying that RIB may function as a non-enzymatic antioxidant in ROS detoxification. RIB-pretreated seedlings accumulated more Na+ in the roots than in the leaf blades because of the contributions of OsHKT2;1, OsNHX1, and OsHKT1;4 in the roots and leaf sheaths, respectively. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis revealed increased RIB concentration in roots and shoots and upregulation of key genes (OsRIBA1, OsGCHI, OsLS, and OsRS) involved in RIB biosynthesis in the roots of RIB-pretreated seedlings. Taken together, our findings suggest that RIB pretreatment ameliorates salinity stress in rice by improving (1) oxidative stress tolerance, as increased RIB concentration may function as a non-enzymatic antioxidant, and (2) ionic stress tolerance, as RIB pretreatment limits Na+ accumulation in the leaf blades and maintains a favorable Na+/K+ balance.

Mediating the performance of anaerobic granular sludge by exogenous flavin supplementation: Flavin binding capacity and behavior, interspecies electron transfer, and methane production.

Although flavins are known as effective electron mediators, the binding capacity of exogenous flavins by anaerobic granular sludge (AGS) and their role in interspecies electron transfer (IET) remains unknown. In this study, AGS was mediated by using three exogenous flavins of riboflavin (RF), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). Results showed that the total amounts of flavins associated with extracellular polymeric substance (EPS) of AGS increased by 2.03-2.42 and 3.83-4.94 folds, after exposure to 50 and 200 µM of exogenous flavins, respectively. A large portion of FMN and FAD was transformed into RF by AGS. Exogenous flavin mediation also stimulated the production of EPS and cytochrome c (c-Cyts) as well as cytochrome-bound flavins. The increased abundance of these electron mediators led to a reduced electrochemical impedance of EPS and improved extracellular electron transfer capacity. The methane production of AGS after mediation with exogenous RF, FMN, and FAD increased by 19.03-31.71%, 22.86-26.04%, and 28.51-33.44%, respectively. This study sheds new light on the role of exogenous flavins in promoting the IET process of a complex microbial aggregate of AGS.

Riboflavin bio-enrichment of soy beverage by selected roseoflavin-resistant and engineered lactic acid bacteria.

Some lactic acid bacteria (LAB) have the ability to synthesize riboflavin, a trait linked to the presence of ribG, ribB, ribA and ribH genes located in the rib operon. Previous screening of riboflavin producers identified several LAB strains belonging to different species with this ability, but none of them surpassed 0.25 mg/L production of the vitamin. In this study, we explored two strategies to obtain riboflavin-overproducing strains: by roseoflavin selection of mutants, and by the transformation of selected strains with plasmids pNZ:TuR.rib or pNZ:TuB.rib containing the genes ribG, ribB, ribA and ribH from Lactococcus cremoris MG1363. The resulting riboflavin-overproducing strains were able to produce yields between 0.5 and 6 mg/L in culture media and several of them were selected for the fermentation of soy beverages. Riboflavin in bio-enriched soy beverages was evaluated by direct fluorescence measurement and high-performance liquid chromatography-fluorescence analysis. Soy beverages fermented with the recombinant strains Lactococcus cremoris ESI 277 pNZ:TuB.rib and Lactococcus lactis INIA 12 pNZ:TuR.rib showed the highest riboflavin yields (>5 mg/L) after 24 h fermentation. On the other hand, roseoflavin-resistant mutant Limosilactobacillus fermentum INIA P143R2 was able to enrich fermented soy beverages with 1.5 mg/L riboflavin. Riboflavin-overproducing LAB strains constitute a good option for riboflavin enrichment of soy beverages by fermentation and the commercialization of such beverages could be very useful to prevent riboflavin deficiency.