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We report the complete genomes of Geobacillus stearothermophilus strains EF60045 and SJEF4-2 from Korean hot springs, with 3,769 and 3,625 thermophilic genes, respectively. G. stearothermophilus EF60045 shows four methylation patterns. G. stearothermophilus SJEF4-2 harbors three plasmids. These findings enhance understanding of Geobacillus strains, aiding in their development as microbial platform hosts.
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Cathodes made of LiFePO4 (LFP) offer numerous benefits including being non-toxic, eco-friendly, and affordable. The distinctive olivine structure of LFP cathodes contributes to their electrochemical stability. Nonetheless, this structure is also the cause of their low ionic and electronic conductivity. To enhance these limitations, an uncomplicated approach has been effectively employed. A straightforward solid-state synthesis technique is used to apply a coating of biomass from potato peels to the LFP cathode, boosting its electrochemical capabilities. Potato peels contain pyridinic and pyrrolic nitrogen, which are conducive to ionic and electronic movement and facilitate pathways for lithium-ion and electron transfer, thus elevating electrochemical performance. When coated with nitrogen-doped carbon derived from potato peel biomass (PPNC@LFP), the LFP cathode demonstrates an improved discharge capacity of 150.39 mAh g-1 at a 0.1 C-rate and 112.83 mAh g-1 at a 1.0 C-rate, in contrast to the uncoated LFP which shows capacities of 141.34 mAh g-1 and 97.72 mAh g-1 at the same rates, respectively.
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The evolution of the bacterial phosphotransferase system (PTS) linked to glycolysis is dependent on the availability of naturally occurring sugars. Although bacteria exhibit sugar specificities based on carbon catabolite repression, the acquisition and evolvability of the cellular sugar preference under conditions that are suboptimal for growth (e.g., environments rich in a rare sugar) are poorly understood. Here, we generated Escherichia coli mutants via a retro-aldol reaction to obtain progeny that can utilize the rare sugar d-tagatose. We detected a minimal set of adaptive mutations in the d-fructose-specific PTS to render E. coli capable of d-tagatose utilization. These E. coli mutant strains lost the tight regulation of both the d-fructose and N-acetyl-galactosamine PTS following deletions in the binding site of the catabolite repressor/activator protein (Cra) upstream from the fruBKA operon and in the agaR gene, encoding the N-acetylgalactosamine (GalNAc) repressor, respectively. Acquired d-tagatose catabolic pathways then underwent fine-tuned adaptation via an additional mutation in 1-phosphofructose kinase to adjust metabolic fluxes. We determined the evolutionary trajectory at the molecular level, providing insights into the mechanism by which enteric bacteria evolved a substrate preference for the rare sugar d-tagatose. Furthermore, the engineered E. coli mutant strain could serve as an in vivo high-throughput screening platform for engineering non-phosphosugar isomerases to produce rare sugars. IMPORTANCE Microorganisms generate energy through glycolysis, which might have preceded a rapid burst of evolution, including the evolution of cellular respiration in the primordial biosphere. However, little is known about the evolvability of cellular sugar preferences. Here, we generated Escherichia coli mutants via a retro-aldol reaction to obtain progeny that can utilize the rare sugar d-tagatose. Consequently, we identified mutational hot spots and determined the evolutionary trajectory at the molecular level. This provided insights into the mechanism by which enteric bacteria evolved substrate preferences for various sugars, accounting for the widespread occurrence of these taxa. Furthermore, the adaptive laboratory evolution-induced cellular chassis could serve as an in vivo high-throughput screening platform for engineering tailor-made non-phosphorylated sugar isomerases to produce low-calorigenic rare sugars showing antidiabetic, antihyperglycemic, and antitumor activities.
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Excavating the molecular details of many diverse enzymes from metagenomes remains challenging in agriculture, food, health, and environmental fields. We present a versatile method that accelerates metabolic enzyme discovery for highly selective gene capture in metagenomes using next-generation sequencing. Culture-independent enzyme mining of environmental DNA is based on a set of short identifying degenerate sequences specific for a wide range of enzyme superfamilies, followed by multiplexed DNA barcode sequencing. A strategy of 'focused identification of next-generation sequencing-based definitive enzyme research' enabled us to generate targeted enzyme datasets from metagenomes, resulting in minimal hands-on obtention of high-throughput biological diversity and potential function profiles, without being time-consuming. This method also provided a targeted inventory of predicted proteins and molecular features of metabolic activities from several metagenomic samples. We suggest that the efficiency and sensitivity of this method will accelerate the decryption of microbial diversity and the signature of proteins and their metabolism from environmental samples.
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Código de Barras de DNA Taxonômico , Enzimas/genética , Sequenciamento de Nucleotídeos em Larga Escala , MetagenômicaRESUMO
Pyrrolidone carboxypeptidases (Pcps) (E.C. 3.4.19.3) can cleave the peptide bond adjacent to pyro-glutamic acid (pGlu), an N-terminal modification observed in some proteins that provides protection against common proteases. Pcp derived from extremely thermophilic Fervidobacterium islandicum AW-1 (FiPcp), that belongs to the cysteine protease family, is involved in keratin utilization under stress conditions. Although an irreversible oxidative modification of active cysteine to its sulfonic acid derivative (Cys-SO3H) renders the enzyme inactive, the molecular details for the sulfonic acid modification in inactive Pcp remain unclear. Here, we determined the crystal structure of FiPcp at 1.85 Å, revealing the oxidized form of cysteine sulfonic acid (C156-SO3H) in the catalytic triad (His-Cys-Glu), which participates in the hydrolysis of pGlu residue containing peptide bond. The three oxygen atoms of cysteine sulfonic acid were stabilized by hydrogen bonds with H180, carbonyl backbone of Q83, and water molecules, resulting in inactivation of FiPcp. Furthermore, FiPcp demonstrated a unique 139KKKK142 motif involved in inter-subunit electrostatic interactions whose mutation significantly affects the thermostability of tetrameric FiPcp. Thus, our high-resolution structure of the first inactive FiPcp with irreversible oxidative modification of active cysteine provides not only the molecular basis of the redox-dependent catalysis of Pcp, but also the structural features of its thermostability.
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Bactérias/enzimologia , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Queratinas/metabolismo , Pirrolidinonas/metabolismo , Motivos de Aminoácidos , Bactérias/classificação , Domínio Catalítico , Cristalografia por Raios X , Cisteína/análogos & derivados , Cisteína/química , Cisteína/metabolismo , Estabilidade Enzimática , Ligação de Hidrogênio , Hidrólise , Modelos Moleculares , Oxirredução , Oxigênio/metabolismo , Eletricidade Estática , Água/metabolismoRESUMO
Most extremophilic anaerobes possess a sulfur formation (Suf) system for Fe-S cluster biogenesis. In addition to its essential role in redox chemistry and stress responses of Fe-S cluster proteins, the Suf system may play an important role in keratin degradation by Fervidobacterium islandicum AW-1. Comparative genomics of the order Thermotogales revealed that the feather-degrading F. islandicum AW-1 has a complete Suf-like machinery (SufCBDSU) that is highly expressed in cells grown on native feathers in the absence of elemental sulfur (S0 ). On the other hand, F. islandicum AW-1 exhibited a significant retardation in the Suf system-mediated keratin degradation in the presence of S0 . Detailed differential expression analysis of sulfur assimilation machineries unveiled the mechanism by which an efficient sulfur delivery from persulfurated SufS to SufU is achieved during keratinolysis under sulfur starvation. Indeed, addition of SufS-SufU to cell extracts containing keratinolytic proteases accelerated keratin decomposition in vitro under reducing conditions. Remarkably, mass spectrometric analysis of extracellular and intracellular levels of amino acids suggested that redox homeostasis within cells coupled to extracellular cysteine and cystine recycling might be a prerequisite for keratinolysis. Taken together, these results suggest that the Suf-like machinery including the SufS-SufU complex may contribute to sulfur availability for an extracellular reducing environment as well as intracellular redox homeostasis through cysteine released from keratin hydrolysate under starvation conditions.
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Cisteína , Queratinas , Animais , Bactérias , Cistina , EnxofreRESUMO
Keratin degradation is of great interest for converting agro-industrial waste into bioactive peptides and is directly relevant for understanding the pathogenesis of superficial infections caused by dermatophytes. However, the mechanism of this process remains unclear. Here, we obtained the complete genome sequence of a feather-degrading, extremely thermophilic bacterium, Fervidobacterium islandicum AW-1 and performed bioinformatics-based functional annotation. Reverse transcription PCR revealed that 57 putative protease-encoding genes were differentially expressed in substrate-dependent manners. Consequently, 16 candidate genes were highly expressed under starvation conditions, when keratin degradation begun. Subsequently, the dynamic expression profiles of these 16 selected genes in response to feathers, as determined via quantitative real-time PCR, suggested that they included four metalloproteases and two peptidases including an ATP-dependent serine protease, all of which might act as key players in feather decomposition. Furthermore, in vitro keratinolytic assays supported the notion that recombinant enzymes enhanced the decomposition of feathers in the presence of cell extracts. Therefore, our genome-based systematic and dynamic expression profiling demonstrated that these identified metalloproteases together with two additional peptidases might be primarily associated with the decomposition of native feathers, suggesting that keratin degradation can be achieved via non-canonical catalysis of several membrane-associated metalloproteases in cooperation with cytosolic proteases.
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Plumas , Peptídeo Hidrolases , Animais , Bactérias , Perfilação da Expressão Gênica , Peptídeo Hidrolases/genéticaRESUMO
Infestation of phosphine (PH3) resistant insects threatens global grain reserves. PH3 fumigation controls rice weevil (Sitophilus oryzae) but not highly resistant insect pests. Here, we investigated naturally occurring strains of S. oryzae that were moderately resistant (MR), strongly resistant (SR), or susceptible (wild-type; WT) to PH3 using global proteome analysis and mitochondrial DNA sequencing. Both PH3 resistant (PH3-R) strains exhibited higher susceptibility to ethyl formate-mediated inhibition of cytochrome c oxidase than the WT strain, whereas the disinfectant PH3 concentration time of the SR strain was much longer than that of the MR strain. Unlike the MR strain, which showed altered expression levels of genes encoding metabolic enzymes involved in catabolic pathways that minimize metabolic burden, the SR strain showed changes in the mitochondrial respiratory chain. Our results suggest that the acquisition of strong PH3 resistance necessitates the avoidance of oxidative phosphorylation through the accumulation of a few non-synonymous mutations in mitochondrial genes encoding complex I subunits as well as nuclear genes encoding dihydrolipoamide dehydrogenase, concomitant with metabolic reprogramming, a recognized hallmark of cancer metabolism. Taken together, our data suggest that reprogrammed metabolism represents a survival strategy of SR insect pests for the compensation of minimized energy transduction under anoxic conditions. Therefore, understanding the resistance mechanism of PH3-R strains will support the development of new strategies to control insect pests.