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1.
PLoS Genet ; 20(5): e1011064, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38709821

RESUMEN

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered the biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.


Asunto(s)
Oxidación-Reducción , Fenazinas , Microbiología del Suelo , Fenazinas/metabolismo , Transporte de Electrón/genética , Citrobacter/genética , Citrobacter/metabolismo , Anaerobiosis/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética
2.
Nature ; 629(8014): 1165-1173, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38720076

RESUMEN

The nucleus is highly organized, such that factors involved in the transcription and processing of distinct classes of RNA are confined within specific nuclear bodies1,2. One example is the nuclear speckle, which is defined by high concentrations of protein and noncoding RNA regulators of pre-mRNA splicing3. What functional role, if any, speckles might play in the process of mRNA splicing is unclear4,5. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs and higher co-transcriptional splicing levels than genes that are located farther from nuclear speckles. Gene organization around nuclear speckles is dynamic between cell types, and changes in speckle proximity lead to differences in splicing efficiency. Finally, directed recruitment of a pre-mRNA to nuclear speckles is sufficient to increase mRNA splicing levels. Together, our results integrate the long-standing observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a crucial role for dynamic three-dimensional spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.


Asunto(s)
Genoma , Motas Nucleares , Precursores del ARN , Empalme del ARN , ARN Mensajero , Empalmosomas , Animales , Humanos , Masculino , Ratones , Genes , Genoma/genética , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Motas Nucleares/genética , Motas Nucleares/metabolismo , Precursores del ARN/metabolismo , Precursores del ARN/genética , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Empalmosomas/metabolismo , Transcripción Genética
3.
Biochemistry ; 63(2): 219-229, 2024 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-38085650

RESUMEN

Carboxysomes are protein microcompartments that function in the bacterial CO2 concentrating mechanism (CCM) to facilitate CO2 assimilation. To do so, carboxysomes assemble from thousands of constituent proteins into an icosahedral shell, which encapsulates the enzymes Rubisco and carbonic anhydrase to form structures typically > 100 nm and > 300 megadaltons. Although many of the protein interactions driving the assembly process have been determined, it remains unknown how size and composition are precisely controlled. Here, we show that the size of α-carboxysomes is controlled by the disordered scaffolding protein CsoS2. CsoS2 contains two classes of related peptide repeats that bind to the shell in a distinct fashion, and our data indicate that size is controlled by the relative number of these interactions. We propose an energetic and structural model wherein the two repeat classes bind at the junction of shell hexamers but differ in their preferences for the shell contact angles, and thus the local curvature. In total, this model suggests that a set of specific and repeated interactions between CsoS2 and shell proteins collectively achieve the large size and monodispersity of α-carboxysomes.


Asunto(s)
Proteínas Bacterianas , Anhidrasas Carbónicas , Proteínas Bacterianas/química , Dióxido de Carbono/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismo , Péptidos/metabolismo , Anhidrasas Carbónicas/metabolismo , Orgánulos/metabolismo
4.
bioRxiv ; 2023 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-38014283

RESUMEN

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.

5.
Nat Struct Mol Biol ; 27(3): 281-287, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32123388

RESUMEN

Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO2-concentrating mechanism by facilitating high CO2 concentrations near the carboxylase Rubisco. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the α-carboxysome, there is little molecular insight into protein-protein interactions that drive the assembly process. Here, studies on the α-carboxysome from Halothiobacillus neapolitanus demonstrate that Rubisco interacts with the N terminus of CsoS2, a multivalent, intrinsically disordered protein. X-ray structural analysis of the CsoS2 interaction motif bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophysical measurements indicate that this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate that CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome formation.


Asunto(s)
Proteínas Bacterianas/química , Halothiobacillus/química , Proteínas Intrínsecamente Desordenadas/química , Orgánulos/química , Ribulosa-Bifosfato Carboxilasa/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Ciclo del Carbono/fisiología , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Halothiobacillus/genética , Halothiobacillus/metabolismo , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Modelos Moleculares , Orgánulos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribulosa-Bifosfato Carboxilasa/genética , Ribulosa-Bifosfato Carboxilasa/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Electricidad Estática
6.
Nat Microbiol ; 4(12): 2204-2215, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31406332

RESUMEN

Bacterial autotrophs often rely on CO2 concentrating mechanisms (CCMs) to assimilate carbon. Although many CCM proteins have been identified, a systematic screen of the components of CCMs is lacking. Here, we performed a genome-wide barcoded transposon screen to identify essential and CCM-related genes in the γ-proteobacterium Halothiobacillus neapolitanus. Screening revealed that the CCM comprises at least 17 and probably no more than 25 genes, most of which are encoded in 3 operons. Two of these operons (DAB1 and DAB2) contain a two-gene locus that encodes a domain of unknown function (Pfam: PF10070) and a putative cation transporter (Pfam: PF00361). Physiological and biochemical assays demonstrated that these proteins-which we name DabA and DabB, for DABs accumulate bicarbonate-assemble into a heterodimeric complex, which contains a putative ß-carbonic anhydrase-like active site and functions as an energy-coupled inorganic carbon (Ci) pump. Interestingly, DAB operons are found in a diverse range of bacteria and archaea. We demonstrate that functional DABs are present in the human pathogens Bacillus anthracis and Vibrio cholerae. On the basis of these results, we propose that DABs constitute a class of energized Ci pumps and play a critical role in the metabolism of Ci throughout prokaryotic phyla.


Asunto(s)
Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Anhidrasas Carbónicas/metabolismo , Proteínas Portadoras/metabolismo , Células Procariotas/metabolismo , Archaea/enzimología , Archaea/genética , Archaea/metabolismo , Bacillus anthracis/metabolismo , Bacterias/enzimología , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/genética , Dióxido de Carbono/metabolismo , Anhidrasas Carbónicas/genética , Elementos Transponibles de ADN/genética , Compuestos de Diazonio , Genes Bacterianos/genética , Genes Esenciales , Halothiobacillus/genética , Halothiobacillus/metabolismo , Mutagénesis , Operón , Ácidos Sulfanílicos , Vibrio cholerae/metabolismo
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