Laboratory evolution of E. coli with a natural vitamin B ₁₂ analog reveals roles for cobamide uptake and adenosylation in methionine synthase-dependent growth

Journal of Bacteriology, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 28, 2025

ABSTRACT Bacteria encounter chemically similar nutrients in their environment, which impact growth distinct ways. Among such are cobamides, the structurally diverse family of cofactors related to vitamin B 12 (cobalamin), function as for metabolic processes. Given that different environments contain varying abundances bacteria likely cobamides enable them grow robustly and also those do not efficiently metabolism. To gain insights into how might respond under latter conditions, we performed a laboratory evolution cobamide-dependent strain Escherichia coli with pseudocobalamin (pCbl), cobamide E. uses less effectively than cobalamin MetH-dependent methionine synthesis. Propagation sequencing nine independent lines identified potential genetic adaptations cobamide-related genes improved less-preferred cobamides. We constructed targeted mutants validate these findings found increasing expression outer membrane transporter BtuB is beneficial during cobamide-limiting conditions. Unexpectedly, overexpression adenosyltransferase BtuR confers specific advantage pCbl. Characterization phenotype revealed adenosylated contribute optimal growth. Together, improve our understanding expand potential. IMPORTANCE In nature, commonly experience fluctuations availability required nutrients. Thus, environment often contains insufficient quantity or poorly Cobamides, cofactors, ideal investigating influence nutrient quality on bacterial experiment examine whether can uptake modification key shared metabolites microbial communities, results reveal interactions competition

Language: Английский

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The inorganic chemistry of the cobalt corrinoids – an update

Journal of Inorganic Biochemistry, Journal Year: 2023, Volume and Issue: 242, P. 112154 - 112154

Published: Feb. 3, 2023

Language: Английский

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Microbes display broad diversity in cobamide preferences

mSystems, Journal Year: 2025, Volume and Issue: unknown

Published: March 21, 2025

ABSTRACT Cobamides, the vitamin B 12 (cobalamin) family of cofactors, are used by most organisms but produced only a fraction prokaryotes, and thus considered key shared nutrients among microbes. Cobamides structurally diverse, with multiple different cobamides found in microbial communities. The ability to use has been tested for several bacteria microalgae, nearly all show preferences certain cobamides. This approach is limited commercial unavailability other than cobalamin. Here, we have extracted purified seven commercially unavailable characterize bacterial cobamide based on growth specific cobamide-dependent conditions. include engineered strains Escherichia coli , Sinorhizobium meliloti Bacillus subtilis expressing native or heterologous enzymes, cultured under conditions that functionally isolate processes such as methionine synthesis. Comparison these results those previous studies diverse microalgae revealed broad diversity exists not across also between metabolic pathways within same organism. microbes differed support efficiently, do growth, minimum concentrations required growth. latter differ up four orders magnitude from environments 20-fold enzymes Given shared, substrates, essential central metabolism organisms, likely impact community structure function. IMPORTANCE Nearly communities tens thousands species. Molecular interactions cooperation competition factors underlying assembly structure. enzyme one class nutrients, minority prokaryotes A unique aspect their diversity, 20 structural forms identified nature. Importantly, this impacts forms. We measured model compared analyses preference. vary widely bacteria, showing importance characterizing aspects biology understand

Language: Английский

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Expanding the Riboglow-FLIM Toolbox with Different Fluorescence Lifetime-Producing RNA Tags

Biochemistry, Journal Year: 2025, Volume and Issue: unknown

Published: May 15, 2025

RNAs are essential elements of biology with subcellular localizations critical for function. Genetically tagging fluorescent RNA reporters to an interest allows investigating spatiotemporal dynamics. We previously developed a fluorescence lifetime imaging microscopy (FLIM)-based RNA-tagging platform, Riboglow-FLIM. Here, genetically encoded tag binds probe, causing increase in both intensity and lifetime. Importantly, the Riboglow platform is derived from bacterial riboswitch family different sequences nature may build basis multiplexing capabilities. observed differences two tags vitro live mammalian cells as proof-of-concept demonstration. As in-depth expansion capabilities, here we evaluate performance systematically expanding sequence space use methods varying genetic literature-guided rational design approach. The approach includes indirect direct evidence probe binding. For this, phylogenetic tree riboswitch-derived binding results was constructed, members branches were characterized. also designed mutations rationally based on insights established tags. Together, nine yielded wide range lifetimes Riboglow-FLIM building foundation track several simultaneously. These findings will serve achieving multiplexed using sensor.

Language: Английский

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Fluorescent riboswitch-controlled biosensors for the genome scale analysis of metabolic pathways

Scientific Reports, Journal Year: 2024, Volume and Issue: 14(1)

Published: May 31, 2024

Abstract Fluorescent detection in cells has been tremendously developed over the years and now benefits from a large array of reporters that can provide sensitive specific real time. However, intracellular monitoring metabolite levels still poses great challenges due to often complex nature detected metabolites. Here, we systematic analysis thiamin pyrophosphate (TPP) metabolism Escherichia coli by using TPP-sensing riboswitch controls expression fluorescent gfp reporter. By comparing different combinations reporter fusions riboswitches, determine key elements are associated with strong TPP-dependent sensing. Furthermore, Keio collection as proxy for growth conditions differing TPP levels, perform high-throughput screen high-density solid agar plates. Our study reveals several genes whose deletion leads increased or decreased levels. The approach here could be applicable other riboswitches genes, thus representing framework onto which further development lead highly sophisticated platforms allowing metabolic screens identification orphan riboswitches.

Language: Английский

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Methylations in vitamin B12 biosynthesis and catalysis

Current Opinion in Structural Biology, Journal Year: 2022, Volume and Issue: 77, P. 102490 - 102490

Published: Nov. 10, 2022

Language: Английский

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Corrins and porphyrins: two of nature’s pigments of life

Journal of Coordination Chemistry, Journal Year: 2024, Volume and Issue: 77(11), P. 1161 - 1210

Published: April 22, 2024

Given the relative scarcity of cobalt in earth's crust, its retention biological systems, principally (but not exclusively) corrinoids or derivatives vitamin B12, may be surprising. The chemistry these compounds and much more widely utilized iron porphyrins, is compared contrasted an attempt made to explain biology.

Language: Английский

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Bacterial Metallostasis: Metal Sensing, Metalloproteome Remodeling, and Metal Trafficking

Chemical Reviews, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 10, 2024

Transition metals function as structural and catalytic cofactors for a large diversity of proteins enzymes that collectively comprise the metalloproteome. Metallostasis considers all cellular processes, notably metal sensing, metalloproteome remodeling, trafficking (or allocation) ensure functional integrity adaptability Bacteria employ both protein RNA-based mechanisms sense intracellular transition bioavailability orchestrate systems-level outputs maintain metallostasis. In this review, we contextualize metallostasis by briefly discussing specialized roles play in biology. We then offer comprehensive perspective on metalloregulatory metal-sensing riboswitches, defining general principles within each sensor superfamily capture how specificity is encoded sequence, selectivity can be leveraged downstream synthetic biology biotechnology applications. This followed discussion recent work highlights selected outputs, including remodeling allocation metallochaperones to client compartments. close places where more needed fill gaps our understanding

Language: Английский

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Targeting Riboswitches with Beta-Axial-Substituted Cobalamins

ACS Chemical Biology, Journal Year: 2023, Volume and Issue: 18(5), P. 1136 - 1147

Published: April 24, 2023

RNA-targeting small-molecule therapeutics is an emerging field hindered by incomplete understanding of the basic principles governing RNA–ligand interactions. One way to advance our knowledge in this area study model systems where these interactions are better understood, such as riboswitches. Riboswitches bind a wide array small molecules with high affinity and selectivity, providing wealth information on how RNA recognizes ligands through diverse structures. The cobalamin-sensing riboswitch particularly useful system, similar sequences show highly specialized binding preferences for different biological forms cobalamin. This also widely dispersed across bacteria therefore holds strong potential antibiotic target. Many synthetic cobalamin have been developed various purposes including therapeutics, but their interaction riboswitches yet be explored. In study, we characterize 11 derivatives three representative using vitro experiments (both chemical footprinting fluorescence-based assay) cell-based reporter assay. productive two riboswitches, demonstrating simultaneous plasticity selectivity within RNAs. observed partially achieved novel structural rearrangement ligand pocket, insight into structures can targeted. As vivo functionality, they serve several lead compounds further drug development.

Language: Английский

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Laboratory evolution ofE. coliwith a natural vitamin B₁₂analog reveals roles for cobamide uptake and adenosylation in methionine synthase-dependent growth

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Jan. 4, 2024

Abstract Bacteria encounter chemically similar nutrients in their environment that impact growth distinct ways. Among such are cobamides, the structurally diverse family of cofactors related to vitamin B 12 (cobalamin), which function as for metabolic processes. Given different environments contain varying abundances bacteria likely cobamides enable them grow robustly well those do not efficiently metabolism. Here, we performed a laboratory evolution cobamide-dependent strain Escherichia coli with pseudocobalamin (pCbl), cobamide E. uses less effectively than cobalamin MetH-dependent methionine synthesis, identify genetic adaptations lead improved less-preferred cobamides. After propagating and sequencing nine independent lines validating results by constructing targeted mutations, found mutations increase expression outer membrane transporter BtuB beneficial during under cobamide-limiting conditions. Unexpectedly, also overexpression adenosyltransferase BtuR confers specific advantage pCbl. Characterization latter phenotype revealed adenosylated contribute optimal growth. Together, these findings improve our understanding how expand potential. Importance In nature, commonly experience fluctuations availability required nutrients. Thus, often contains insufficient quantity or poorly Cobamides, cofactors, ideal investigating influence nutrient quality on bacterial We experiment examine whether can genes uptake modification key shared metabolites microbial communities, reveal insights into interactions competition

Language: Английский

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