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Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous PDIM-negative mutants that have attenuated virulence and increased cell wall permeability, thus impacting the relevance of experimental findings. PDIM loss can also reduce the efficacy of the BCG Pasteur vaccine. Here we show that vancomycin susceptibility can rapidly screen for M. tuberculosis PDIM production. We find that metabolic deficiency of methylmalonyl-CoA impedes the growth of PDIM-producing bacilli, selecting for PDIM-negative variants. Supplementation with odd-chain fatty acids, cholesterol or vitamin B 12 restores PDIM-positive bacterial growth. Specifically, we show that propionate supplementation enhances PDIM-producing bacterial growth and selects against PDIM-negative mutants, analogous to in vivo conditions. Our study provides a simple approach to screen for and maintain PDIM production, and reveals how discrepancies between the host and in vitro nutrient environments can attenuate bacterial pathogenicity., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

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 heterodimers 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 insight into the mechanisms of polyketide synthases in this filamentous fungus. IMPORTANCE The fungus Ustilago maydis represents one of the major threats to 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, such 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 multidomain enzymes and their evolution in the fungal kingdom., (Copyright © 2021 American Society for Microbiology.)

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.

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.