Floxuridine supports UPS independent of germline signaling and proteostasis regulators via involvement of detoxification in C. elegans.

The ubiquitin-proteasome system (UPS) is critical for maintaining proteostasis, influencing stress resilience, lifespan, and thermal adaptability in organisms. In Caenorhabditis elegans, specific proteasome subunits and activators, such as RPN-6, PBS-6, and PSME-3, are associated with heat resistance, survival at cold (4°C), and enhanced longevity at moderate temperatures (15°C). Previously linked to improving proteostasis, we investigated the impact of sterility-inducing floxuridine (FUdR) on UPS functionality under proteasome dysfunction and its potential to improve cold survival. Our findings reveal that FUdR significantly enhances UPS activity and resilience during proteasome inhibition or subunit deficiency, supporting worms' normal lifespan and adaptation to cold. Importantly, FUdR effect on UPS activity occurs independently of major proteostasis regulators and does not rely on the germ cells proliferation or spermatogenesis. Instead, FUdR activates a distinct detoxification pathway that supports UPS function, with GST-24 appearing to be one of the factors contributing to the enhanced activity of the UPS upon knockdown of the SKN-1-mediated proteasome surveillance pathway. Our study highlights FUdR unique role in the UPS modulation and its crucial contribution to enhancing survival under low-temperature stress, providing new insights into its mechanisms of action and potential therapeutic applications.

A novel de novo FEM1C variant is linked to neurodevelopmental disorder with absent speech, pyramidal signs and limb ataxia.

The principal component of the protein homeostasis network is the ubiquitin-proteasome system. Ubiquitination is mediated by an enzymatic cascade involving, i.e. E3 ubiquitin ligases, many of which belong to the cullin-RING ligases family. Genetic defects in the ubiquitin-proteasome system components, including cullin-RING ligases, are known causes of neurodevelopmental disorders. Using exome sequencing to diagnose a pediatric patient with developmental delay, pyramidal signs and limb ataxia, we identified a de novo missense variant c.376G>C; p.(Asp126His) in the FEM1C gene encoding a cullin-RING ligase substrate receptor. This variant alters a conserved amino acid located within a highly constrained coding region and is predicted as pathogenic by most in silico tools. In addition, a de novo FEM1C mutation of the same residue p.(Asp126Val) was associated with an undiagnosed developmental disorder, and the relevant variant (FEM1CAsp126Ala) was found to be functionally compromised in vitro. Our computational analysis showed that FEM1CAsp126His hampers protein substrate binding. To further assess its pathogenicity, we used the nematode Caenorhabditis elegans. We found that the FEM-1Asp133His animals (expressing variant homologous to the FEM1C p.(Asp126Val)) had normal muscle architecture yet impaired mobility. Mutant worms were sensitive to the acetylcholinesterase inhibitor aldicarb but not levamisole (acetylcholine receptor agonist), showing that their disabled locomotion is caused by synaptic abnormalities and not muscle dysfunction. In conclusion, we provide the first evidence from an animal model suggesting that a mutation in the evolutionarily conserved FEM1C Asp126 position causes a neurodevelopmental disorder in humans.

Mycofactocin is essential for the establishment of methylotrophy in Mycobacterium smegmatis.

Mycobacterium smegmatis possesses (N,N-dimethyl-4-nitrosoaniline)-dependent (NDMA) methanol dehydrogenase (Mno) to establish methylotrophy by utilizing methanol as the source of both carbon and energy. In this study, we show that Mno forms decamer and has NADPH as the bound cofactor. Interestingly, Mno uses NDMA and not NADP+ as an electron acceptor in in vitro reactions. We further show that the operon mftAD required for the biosynthesis of mycofactocin, a ribosomally-synthesized electron carrier, is indispensable for the growth of M. smegmatis on methanol. Our data obtained from 2,6-Dichlorophenolindophenol reduction assays also suggest that Mno uses mycofactocin as an in vivo electron acceptor for the oxidation of methanol to formaldehyde. We thus provide here biochemical evidence for mycofactocin as an electron carrier in mycobacterial physiology.

MnoSR Is a Bona Fide Two-Component System Involved in Methylotrophic Metabolism in Mycobacterium smegmatis.

Mycobacterium smegmatis and several other mycobacteria are able to utilize methanol as the sole source of carbon and energy. We recently showed that N,N-dimethyl-p-nitrosoaniline (NDMA)-dependent methanol dehydrogenase (Mno) is essential for the growth of M. smegmatis on methanol. Although Mno from this bacterium shares high homology with other known methanol dehydrogenases, methanol metabolism in M. smegmatis differs significantly from that of other described methylotrophs. In this study, we dissect the regulatory mechanism involved in the methylotrophic metabolism in M. smegmatis We identify a two-component system (TCS), mnoSR, that is involved in the regulation of mno expression. We show that the MnoSR TCS is comprised of a sensor kinase (MnoS) and a response regulator (MnoR). Our results demonstrate that MnoS undergoes autophosphorylation and is able to transfer its phosphate to MnoR by means of phosphotransferase activity. Furthermore, MnoR shows specific binding to the putative mno promoter region in vitro, thus suggesting its role in the regulation of mno expression. Additionally, we find that the MnoSR system is involved in the regulation of MSMEG_6239, which codes for a putative 1,3-propanediol dehydrogenase. We further show that M. smegmatis lacking mnoSR is unable to utilize methanol and 1,3-propanediol as the sole carbon source, which confirms the role of MnoSR in the regulation of alcohol metabolism. Our data, thus, suggest that the regulation of mno expression in M. smegmatis provides new insight into the regulation of methanol metabolism, which furthers our understanding of methylotrophy in mycobacteria.IMPORTANCE Methylotrophic metabolism has gained huge attention considering its broad application in ecology, agriculture, industries, and human health. The genus Mycobacterium comprises both pathogenic and nonpathogenic species. Several members of this genus are known to utilize methanol as the sole carbon source for growth. Although various pathways underlying methanol utilization have been established, the regulation of methylotrophic metabolism is not well studied. In the present work, we explore the regulation of methanol metabolism in M. smegmatis and discover a dedicated two-component system (TCS), MnoSR, that is involved in its regulation. We show that the loss of MnoSR renders the bacterium incapable of utilizing methanol and 1,3-propanediol as the sole carbon sources. Additionally, we establish that MnoS acts as the common sensor for the alcohols in M. smegmatis.

Methylotrophy in Mycobacteria: Dissection of the Methanol Metabolism Pathway in Mycobacterium smegmatis.

The mycobacteria comprise both pathogenic and nonpathogenic bacteria. Although several features related to pathogenicity in various mycobacterial species, such as Mycobacterium tuberculosis, have been studied in great detail, methylotrophy, i.e., the ability of an organism to utilize single-carbon (C1) compounds as the sole source of carbon and energy, has remained largely unexplored in mycobacteria. Reports are available that suggest that mycobacteria, including M. tuberculosis and M. smegmatis, are capable of utilizing alternative C1 compounds to meet their carbon and energy requirements. However, physiological pathways that are functional in mycobacteria to utilize such carbon compounds are only poorly understood. Here we report the identification and characterization of the gene products required for establishing methylotrophy in M. smegmatis We present N,N-dimethyl-p-nitrosoaniline (NDMA)-dependent methanol oxidase (Mno) as the key enzyme that is essential for the growth of M. smegmatis on methanol. We show that Mno has both methanol and formaldehyde dehydrogenase activities in vitro Further, M. smegmatis is able to utilize methanol even in the absence of the major formaldehyde dehydrogenase MscR, which suggests that Mno is sufficient to dissimilate methanol and the resulting formaldehyde in vivo Finally, we show that M. smegmatis devoid of phosphoenolpyruvate carboxykinase, which has been shown to fix CO2 in M. tuberculosis, does not grow on methanol, suggesting that the final step of methanol utilization requires CO2 fixation for biomass generation. Our work here thus forms the first comprehensive report that explores methylotrophy in a mycobacterial species.IMPORTANCE Methylotrophy, the ability to utilize single-carbon (C1) compounds as the sole carbon and energy sources, is only poorly understood in mycobacteria. Both pathogenic and nonpathogenic mycobacteria, including Mycobacterium tuberculosis, are capable of utilizing C1 compounds to meet their carbon and energy requirements, although the precise pathways are not well studied. Here we present a comprehensive study of methylotrophy in Mycobacterium smegmatis With several genetic knockouts, we have dissected the entire methanol metabolism pathway in M. smegmatis We show that while methanol dissimilation in M. smegmatis differs from that in other mycobacterial species, the concluding step of CO2 fixation is similar to that in M. tuberculosis It is therefore both interesting and important to examine mycobacterial physiology in the presence of alternative carbon sources.

Bile acid fitness determinants of a Bacteroides fragilis isolate from a human pouchitis patient.

IMPORTANCE: The Gram-negative bacterium Bacteroides fragilis is a common member of the human gut microbiota that colonizes multiple host niches and can influence human physiology through a variety of mechanisms. Identification of genes that enable B. fragilis to grow across a range of host environments has been impeded in part by the relatively limited genetic tractability of this species. We have developed a high-throughput genetic resource for a B. fragilis strain isolated from a UC pouchitis patient. Bile acids limit microbial growth and are altered in abundance in UC pouches, where B. fragilis often blooms. Using this resource, we uncovered pathways and processes that impact B. fragilis fitness in bile and that may contribute to population expansions during bouts of gut inflammation.

Ms6244 is a novel Mycobacterium smegmatis TetR family transcriptional repressor that regulates cell growth and morphophysiology.

Transcriptional factors such as the TetR family of transcriptional regulators (TFTRs) are widely found amongst bacteria, including mycobacteria, and are accountable for their survival. Here, we characterized a novel TFTR, Ms6244, from Mycobacterium smegmatis that negatively autoregulates its expression and represses its neighbouring gene, Ms6243. We also report the binding of Ms6244 to the inverted repeats in the intergenic region of Ms6244 and Ms6243. Further, an Ms6244-deleted strain shows various morpho-physiological differences compared to the wild type. We further confirmed that the deletion of Ms6244 itself and not the resultant Ms6243 overexpression is the cause of the altered physiology. Our data thus suggest that Ms6244 is an essential regulator, having far-reaching effects on M. smegmatis physiology.

Bile acid fitness determinants of a Bacteroides fragilis isolate from a human pouchitis patient.

Bacteroides fragilis comprises 1-5% of the gut microbiota in healthy humans but can expand to >50% of the population in ulcerative colitis (UC) patients experiencing inflammation. The mechanisms underlying such microbial blooms are poorly understood, but the gut of UC patients has physicochemical features that differ from healthy patients and likely impact microbial physiology. For example, levels of the secondary bile acid deoxycholate (DC) are highly reduced in the ileoanal J-pouch of UC colectomy patients. We isolated a B. fragilis strain from a UC patient with pouch inflammation (i.e. pouchitis) and developed it as a genetic model system to identify genes and pathways that are regulated by DC and that impact B. fragilis fitness in DC and crude bile. Treatment of B. fragilis with a physiologically relevant concentration of DC reduced cell growth and remodeled transcription of one-quarter of the genome. DC strongly induced expression of chaperones and select transcriptional regulators and efflux systems and downregulated protein synthesis genes. Using a barcoded collection of ≈50,000 unique insertional mutants, we further defined B. fragilis genes that contribute to fitness in media containing DC or crude bile. Genes impacting cell envelope functions including cardiolipin synthesis, cell surface glycosylation, and systems implicated in sodium-dependent bioenergetics were major bile acid fitness factors. As expected, there was limited overlap between transcriptionally regulated genes and genes that impacted fitness in bile when disrupted. Our study provides a genome-scale view of a B. fragilis bile response and genetic determinants of its fitness in DC and crude bile.

Ferritin-mediated iron detoxification promotes hypothermia survival in Caenorhabditis elegans and murine neurons.

How animals rewire cellular programs to survive cold is a fascinating problem with potential biomedical implications, ranging from emergency medicine to space travel. Studying a hibernation-like response in the free-living nematode Caenorhabditis elegans, we uncovered a regulatory axis that enhances the natural resistance of nematodes to severe cold. This axis involves conserved transcription factors, DAF-16/FoxO and PQM-1, which jointly promote cold survival by upregulating FTN-1, a protein related to mammalian ferritin heavy chain (FTH1). Moreover, we show that inducing expression of FTH1 also promotes cold survival of mammalian neurons, a cell type particularly sensitive to deterioration in hypothermia. Our findings in both animals and cells suggest that FTN-1/FTH1 facilitates cold survival by detoxifying ROS-generating iron species. We finally show that mimicking the effects of FTN-1/FTH1 with drugs protects neurons from cold-induced degeneration, opening a potential avenue to improved treatments of hypothermia.

Heterologous Production of Polyunsaturated Fatty Acids in E. coli Using Δ5-Desaturase Gene from Microalga Isochrysis Sp.

Eicosapentaenoic acid (EPA) and arachidonic acid (ARA) are long-chain polyunsaturated fatty acids (PUFAs) that play a significant role in human growth and development, which deficiency can trigger several metabolic-related diseases. Since the availability of PUFA sources is limited, there arises a need to explore alternative sources. Therefore, the present study aimed to investigate whether an Escherichia coli which are engineered with Δ5Des-Iso gene isolated from Isochrysis sp. could be utilized to synthesize PUFAs. Full-length gene Δ5Des-Iso (1149 bp) was isolated from Isochrysis sp. that encodes 382 amino acids and identified as Δ5-desatruase gene using different bioinformatic analysis. Heterologous gene expression was carried out in E. coli having Δ5Des-Iso with precursor fatty acids. The Δ5Des-Iso produced novel fatty acids of EPA (ω-3) and ARA (ω-6) as respective products were identified by GC-MS. Gene expression and PUFA synthesis in E. coli were optimized by temperature, time, and concentrations of precursor fatty acid substrates. Δ5Des-Iso RNA transcript level was inversely proportional to the time and fatty acid synthesis. And, the significant production of EPA (4.1 mg/g) and ARA (8.3 mg/g) in total fatty acids was observed in E. coli grown at 37 °C for 24 h with 25 µM of external fatty acid substrate as an optimum growth conditions. E. coli could be used as alternative organism to synthesis PUFAs and widely applicable in many nutraceuticals and pharmaceuticals industry for human use.