The effects of solid state fermentation (SSF) on physicochemical, nutritional and antioxidant properties of common bean flour were studied. SSF increased protein content (21.7%) and decreased lipids (-38.4%), carbohydrates (-3.5%) and phytic acid (-58.3%). Fermented (tempeh) flour showed higher dispersability, lower water solubility index and pH than unfermented flour. Fermentation also increased an average of 0.21 g/100 g protein, six of the essential amino acids (EAAs), including total sulfur (Met + Cys), the limiting EAAs in unfermented flour (score = 0.91); Lys and Trp decreased 0.21 and 0.09 g/100 g protein, respectively. SSF improved the in vitro protein digestibility and the calculated protein efficiency ratio. Tempeh flour had 2.2-fold more phenolics than the bean flour and exhibited antiradical activity (43%) and antioxidant activity (38%) correlated with total phenolics content. Common bean tempeh flour may be considered for the fortification of widely consumed legume-based food products and also for the prevention of pathologies associated with oxidative stress.

Awareness of the problem of antimicrobial resistance (AMR) has escalated, and drug-resistant infections are named among the most urgent issues facing clinicians today. Bacteria can acquire resistance to antibiotics by a variety of mechanisms that, at times, involve changes in their metabolic status, thus altering diverse biochemical reactions, many of them pH-dependent. In this work, we found that modulation of the cytoplasmic pH (pH i) of Escherichia coli provides a thus far unexplored strategy to support resistance. We show here that the acidification of the cytoplasmic pH is a previously unrecognized consequence of the activation of the marRAB operon. The acidification itself contributes to the full implementation of the resistance phenotype. We measured the pH i of two resistant strains, developed in our laboratory, that carry mutations in marR that activate the marRAB operon. The pH i of both strains is lower than that of the wild type strain. Inactivation of the marRAB response in both strains weakens resistance, and pH i increases back to wild type levels. Likewise, we showed that exposure of wild type cells to weak acids that caused acidification of the cytoplasm induced a resistant phenotype, independent of the marRAB response. We speculate that the decrease of the cytoplasmic pH brought about by activation of the marRAB response provides a signaling mechanism that modifies metabolic pathways and serves to cope with stress and to lower metabolic costs. Antimicrobial resistance (AMR) is a growing public health threat of broad concern, and drug-resistant infections are named among the most urgent problems facing clinicians today 1-4. Bacteria can acquire resistance to antibiotics by horizontal gene transfer, activation of regulatory loci and genetic mutations that modify the drug target, increase drug efflux, and activate the expression of drug inactivating enzymes 5-8. High-level, clinically relevant resistant strains usually result from the accumulation of several mutations that, at times, involve changes in their metabolic status, thus altering diverse biochemical reactions 9-11. Metabolic rearrangements and changes in rates of reactions necessitate the existence of mechanisms that maintain tight homeostasis of all the physicochemical parameters in the cell within a narrow range of operation, consistent with the survival of the organism. This dynamic state of equilibrium includes many variables such as pH, and solutes and ion concentrations. Because of the accumulating evidence on the metabolic adaptation of antibiotic-resistant cells, we focused our attention on the cytoplasmic pH of these cells. Protons play a crucial role in biochemical networks because changes in intracellular pH affect cell functioning at different levels as it impacts protein folding, enzyme activities, and the protonation of biological macromolecules, lipids, and other metabolites. Moreover, the proton electrochemical gradient across the cell membrane is key to the generation and conversion of cellular energy. It is, therefore, n...

Ustilago maydis is a phytopathogenic fungus responsible for corn smut disease. Although it is a very well established model organism for the study of plant-microbe interactions, its potential to produce specialized metabolites, which might contribute to this interaction, has not been studied in detail. By analyzing the U. maydis genome, we identified a biosynthetic gene cluster whose activation led to the production of a black melanin pigment. Single deletion mutants of the cluster genes revealed that five encoded enzymes are required for the accumulation of the black pigment, including three polyketide synthases (pks3, pks4 and pks5), a cytochrome P450 monooxygenase (cyp4) and a protein with similarity to versicolorin B synthase (vbs1). Metabolic profiles of deletion mutants in this gene cluster suggested that Pks3 and Pks4 act in concert as heterodimer to generate orsellinic acid (OA) which is reduced to the corresponding aldehyde by Pks5. The OA-aldehyde can then react with triacetic acid lactone (TAL) also derived from Pks3/Pks4 heterodimers to form larger molecules including novel coumarin derivatives. Our findings suggest that U. maydis synthesizes a novel type of melanin based on coumarin and pyran-2-one intermediates, while most fungal melanins are derived from 1,8-dihydroxynaphthalene (DHN) or L-3,4-dihydroxyphenylalanine (L-DOPA). Along with these observations, this work also provides an insight into the mechanisms of polyketide synthases in this filamentous fungus. IMPORTANCE The fungus Ustilago maydis represents one of the major threats for maize plants since it is responsible for corn smut disease, which generates considerable economical losses around the world. Therefore, contributing to a better understanding of the biochemistry of defense mechanisms used by U. maydis to protect itself against harsh environments, as the synthesis of melanin, could provide improved biological tools for tackling the problem and protect the crops. In addition, the fact that this fungus synthesizes melanin in an unconventional way, requiring more than one polyketide synthase for producing melanin precursors, gives a different perspective on the complexity of these multimodular enzymes and their evolution in the fungal kingdom.

Ustilago maydis is a phytopathogenic fungus responsible for corn smut disease. Although it is a very well established model organism for the study of plant-microbe interactions, its biosynthetic potential has not been totally explored. By analyzing U. maydis genome, we identified a biosynthetic gene cluster whose activation led to the production of a black melanin pigment. Single deletion mutants of the cluster genes revealed that five encoded enzymes are required for the accumulation of the black pigment, including three polyketide synthases (pks3, pks4 and pks5), a cytochrome P450 monooxygenase (cyp4) and a protein with similarity to versicolorin B-synthase (vbs1). Moreover, metabolic profiles of the mutants defective for pks3 and pks4 indicated that the products of these genes catalyze together the first step in the melanin biosynthetic pathway since none of the mutants accumulated any melanin or intermediate products. Mutants deleted for pks5 produced orsellinic acid (OA) and triacetic acid lactone (TAL), suggesting that both products are produced by Pks3 and Pks4. It might thus demonstrate that Pks5 plays a role in a reaction downstream of that catalyzed by Pks3 and Pks4. OA and TAL were also found in extracts of a cyp4 deletion mutant along with several heterodimers of TAL and Pks5-derived orsellinic aldehyde compounds. According to their phenotypes and the intermediate products isolated from these strains, Cyp4 and Vbs1 seem to be involved in reactions downstream of Pks5. Our findings suggest that U. maydis synthesizes a new melanin based on coumarin and pyran-2-one intermediates, while most fungal melanins are derived from 1,8-dihydroxynaphthalene (DHN) or L-3,4dihydroxyphenylalanine (L-DOPA). Along with these observations, this work also provides an insight into the mechanisms of polyketide synthases in this filamentous fungus. IMPORTANCEUstilago maydis represents one of the major threats for maize plants since it is responsible for corn smut disease, which generates considerable economical losses around the world. Therefore, contributing to a better understanding of the biochemistry of defense mechanisms used by U. maydis to protect itself against harsh environments, as the synthesis of melanin, could provide improved biological tools for tackling the problem and protect the crops. In addition, the fact that this fungus synthesizes melanin in a very unique way, requiring more than one polyketide synthase for producing this secondary metabolite, gives a different perspective on the complexity of these multimodular enzymes and their evolution in the fungal kingdom.

Awareness of the problem of antimicrobial resistance (AMR) has escalated, and drugresistant infections are named among the most urgent issues facing clinicians today. Bacteria can acquire resistance to antibiotics by a variety of mechanisms that, at times, involve changes in their metabolic status, thus altering diverse biochemical reactions, many of them pH-dependent. In this work, we found that modulation of the cytoplasmic pH (pHi) of Escherichia coli provides a thus far unexplored strategy to support resistance. We show here that the acidification of the cytoplasmic pH is a previously unrecognized consequence of the activation of the marRAB operon. The acidification itself contributes to the full implementation of the resistance phenotype. We measured the pHi of two resistant strains, developed in our laboratory, that carry mutations in marR that activate the marRAB operon.The pHi of both strains is lower than that of the wild type strain. Inactivation of the marRAB response in both strains weakens resistance, and pHi increases back to wild type levels.Likewise, we showed that exposure of wild type cells to weak acids that caused acidification of the cytoplasm induced a resistant phenotype, independent of the marRAB response. We speculate that the decrease of the cytoplasmic pH brought about by activation of the marRAB response provides a signaling mechanism that modifies metabolic pathways and serves to cope with stress and to lower metabolic costs. SIGNIFICANCEThe decreasing effectiveness of antibiotics in treating common infections has quickened in recent years, and resistance has spread worldwide. There is an urgent need to understand the mechanisms underlying acquisition and maintenance of resistance, and here we identify a novel element in the chain of events leading to a full-fledged clinically relevant state.The Escherichia coli multiple antibiotic resistance (mar) regulon is induced by a variety of signals and modulates the activity of dozens of target genes involved in resistance to antibiotics. We report here a thus far unidentified result of this activation: acidification of the cytoplasmic pH. Manipulation of the cytoplasmic pH with weak acids and bases, independently of the mar response, shows that the acidification significantly increases resistance.