Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 18.896
Filtrar
1.
Enzyme Microb Technol ; 150: 109858, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489018

RESUMO

Actarit is widely regarded as a safe and effective drug for the treatment of rheumatoid arthritis. There is no report on the bioproductin of actarit so far. In this study, we demonstrated for the first time the development of an artificial actarit biosynthetic pathway in Escherichia coli. First, 4-aminophenylacetic acid is selected as precursor substrates for the production of actarit. Second, an N-acetyltransferase that can efficiently catalyse the esterification of acetyl-CoA and 4-aminophenylacetic acid to form actarit was discovered. Subsequently, an engineered E. coli that allows production of actarit from simple carbon sources was established. Finally, we further increased the production of actarit to 206 ± 16.9 mg/L by overexpression of shikimate dehydrogenase ydiB and shikimate kinase aroK.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Vias Biossintéticas , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica , Fenilacetatos
2.
Nat Commun ; 12(1): 4976, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404790

RESUMO

To construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in sophisticated metabolic networks. Here, we take advantage of CRISPR interference (CRISPRi) and omics analyses to systematically identify beneficial genes that can be engineered to promote free fatty acids (FFAs) production in Escherichia coli. CRISPRi-mediated genetic perturbation enables the identification of 30 beneficial genes from 108 targets related to FFA metabolism. Then, omics analyses of the FFAs-overproducing strains and a control strain enable the identification of another 26 beneficial genes that are seemingly irrelevant to FFA metabolism. Combinatorial perturbation of four beneficial genes involving cellular stress responses results in a recombinant strain ihfAL--aidB+-ryfAM--gadAH-, producing 30.0 g L-1 FFAs in fed-batch fermentation, the maximum titer in E. coli reported to date. Our findings are of help in rewiring cellular metabolism and interwoven intracellular processes to facilitate high-titer production of biochemicals.


Assuntos
Escherichia coli/genética , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Ácidos Graxos não Esterificados/biossíntese , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Fermentação , Regulação Bacteriana da Expressão Gênica , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Transcriptoma
3.
Phys Chem Chem Phys ; 23(32): 17656-17662, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34373871

RESUMO

In this manuscript the ability of selenium carbohydrates to undergo chalcogen bonding (ChB) interactions with protein residues has been studied at the RI-MP2/def2-TZVP level of theory. An inspection of the Protein Data Bank (PDB) revealed SeA (A = O, C and S) intermolecular contacts involving Se-pyranose ligands and ASP, TYR, SER and MET residues. Theoretical models were built to analyse the strength and directionality of the interaction together with "Atoms in Molecules" (AIM), Natural Bonding Orbital (NBO) and Non Covalent Interactions plot (NCIplot) analyses, which further assisted in the characterization of the ChBs described herein. We expect that the results from this study will be useful to expand the current knowledge regarding biological ChBs as well as to increase the visibility of the interaction among the carbohydrate chemistry community.


Assuntos
Lectinas/metabolismo , Monossacarídeos/metabolismo , Compostos Organosselênicos/metabolismo , Agaricales/química , Aspergillus oryzae/química , Bases de Dados de Proteínas , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Ligação de Hidrogênio , Lectinas/química , Modelos Moleculares , Monossacarídeos/química , Compostos Organosselênicos/química , Ligação Proteica , Selênio/química , Eletricidade Estática , Termodinâmica
4.
Nat Commun ; 12(1): 4723, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354064

RESUMO

Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.


Assuntos
Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Ribossomos/química , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo
5.
Nat Commun ; 12(1): 5098, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429416

RESUMO

KdpFABC, a high-affinity K+ pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K+ limitation. Here, we apply a combination of cryo-EM, biochemical assays, and MD simulations to illuminate the mechanisms underlying transport and the coupling to ATP hydrolysis. We show that ions are transported via an intersubunit tunnel through KdpA and KdpB. At the subunit interface, the tunnel is constricted by a phenylalanine, which, by polarized cation-π stacking, controls K+ entry into the canonical substrate binding site (CBS) of KdpB. Within the CBS, ATPase coupling is mediated by the charge distribution between an aspartate and a lysine. Interestingly, individual elements of the ion translocation mechanism of KdpFABC identified here are conserved among a wide variety of P-type ATPases from different families. This leads us to the hypothesis that KdpB might represent an early descendant of a common ancestor of cation pumps.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Transporte de Íons/fisiologia , Ácido Aspártico/metabolismo , Sítios de Ligação , Proteínas de Transporte de Cátions/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Lisina/metabolismo , Simulação de Dinâmica Molecular , Mutação , Fenilalanina , Potássio/metabolismo , Subunidades Proteicas , ATPase Trocadora de Sódio-Potássio
6.
J Microbiol ; 59(9): 854-860, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34382147

RESUMO

Extraintestinal pathogenic Escherichia coli (ExPEC) is an important zoonotic pathogen that places severe burdens on public health and animal husbandry. There are many pathogenic factors in E. coli. The type VI secretion system (T6SS) is a nano-microbial weapon that can assemble quickly and inject toxic effectors into recipient cells when danger is encountered. T6SSs are encoded in the genomes of approximately 25% of sequenced Gram-negative bacteria. When these bacteria come into contact with eukaryotic cells or prokaryotic microbes, the T6SS assembles and secretes associated effectors. In the porcine ExPEC strain PCN033, we identified four classic rearrangement hotspot (Rhs) genes. We determined the functions of the four Rhs proteins through mutant construction and protein expression. Animal infection experiments showed that the Δrhs-1CT, Δrhs-2CT, Δrhs-3CT, and Δrhs-4CT caused a significant decrease in the multiplication ability of PCN033 in vivo. Cell infection experiments showed that the Rhs protein is involved in anti-phagocytosis activities and bacterial adhesion and invasion abilities. The results of this study demonstrated that rhs1, rhs3, and rh4 plays an important role in the interaction between PCN033 and host cell. Rhs2 has contribution to cell and mice infection. This study helps to elucidate the pathogenic mechanism governing PCN033 and may help to establish a foundation for further research seeking to identify potential T6SS effectors.


Assuntos
Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/metabolismo , Doenças dos Suínos/microbiologia , Animais , Aderência Bacteriana , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Escherichia coli Extraintestinal Patogênica/genética , Escherichia coli Extraintestinal Patogênica/metabolismo , Feminino , Intestinos/microbiologia , Camundongos , Família Multigênica , Suínos
7.
Nat Commun ; 12(1): 4909, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389707

RESUMO

In bacteria, trans-translation is the main rescue system, freeing ribosomes stalled on defective messenger RNAs. This mechanism is driven by small protein B (SmpB) and transfer-messenger RNA (tmRNA), a hybrid RNA known to have both a tRNA-like and an mRNA-like domain. Here we present four cryo-EM structures of the ribosome during trans-translation at resolutions from 3.0 to 3.4 Å. These include the high-resolution structure of the whole pre-accommodated state, as well as structures of the accommodated state, the translocated state, and a translocation intermediate. Together, they shed light on the movements of the tmRNA-SmpB complex in the ribosome, from its delivery by the elongation factor EF-Tu to its passage through the ribosomal A and P sites after the opening of the B1 bridges. Additionally, we describe the interactions between the tmRNA-SmpB complex and the ribosome. These explain why the process does not interfere with canonical translation.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Biossíntese de Proteínas/genética , RNA Bacteriano/genética , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura
8.
NPJ Biofilms Microbiomes ; 7(1): 56, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215744

RESUMO

Aryl polyenes (APEs) are specialized polyunsaturated carboxylic acids that were identified in silico as the product of the most widespread family of bacterial biosynthetic gene clusters (BGCs). They are present in several Gram-negative host-associated bacteria, including multidrug-resistant human pathogens. Here, we characterize a biological function of APEs, focusing on the BGC from a uropathogenic Escherichia coli (UPEC) strain. We first perform a genetic deletion analysis to identify the essential genes required for APE biosynthesis. Next, we show that APEs function as fitness factors that increase protection from oxidative stress and contribute to biofilm formation. Together, our study highlights key steps in the APE biosynthesis pathway that can be explored as potential drug targets for complementary strategies to reduce fitness and prevent biofilm formation of multi-drug resistant pathogens.


Assuntos
Biofilmes , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Essenciais , Polienos/metabolismo , Biofilmes/crescimento & desenvolvimento , Transporte Biológico , Vias Biossintéticas , Regulação Bacteriana da Expressão Gênica , Estrutura Molecular , Mutação , Oxirredução , Fenótipo , Polienos/química
9.
Methods Mol Biol ; 2312: 237-251, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228294

RESUMO

Chemical control of protein localization is a powerful approach for manipulating mammalian cellular processes. Self-localizing ligand-induced protein translocation (SLIPT) is an emerging platform that enables control of protein localization in living mammalian cells using synthetic self-localizing ligands (SLs). We recently established a chemogenetic SLIPT system, in which any protein of interest fused to an engineered variant of Escherichia coli dihydrofolate reductase, DHFRiK6, can be rapidly and specifically translocated from the cytoplasm to the inner leaflet of the plasma membrane (PM) using a trimethoprim (TMP)-based PM-targeting SL, mDcTMP. The mDcTMP-mediated PM recruitment of DHFRiK6-fusion proteins can be efficiently returned to the cytoplasm by subsequent addition of free TMP, enabling temporal and reversible control over the protein localization. Here we describe the use of this mDcTMP/DHFRiK6-based SLIPT system for inducing (1) reversible protein translocation and (2) synthetic activation of the Raf/ERK pathway. This system provides a simple and versatile tool in mammalian synthetic biology for temporally manipulating various signaling molecules and pathways at the PM.


Assuntos
Engenharia Celular , Proteínas de Escherichia coli/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas Genéticas , Biologia Sintética , Tetra-Hidrofolato Desidrogenase/genética , Trimetoprima/farmacologia , Técnicas de Cultura de Células , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Tetra-Hidrofolato Desidrogenase/metabolismo , Quinases raf/metabolismo
10.
Nat Commun ; 12(1): 4476, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294706

RESUMO

CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.


Assuntos
Sistemas CRISPR-Cas , Endodesoxirribonucleases/química , Edição de Genes , Bacteriófagos/enzimologia , Bacteriófagos/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Clivagem do DNA , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , RNA Guia/genética , RNA Guia/metabolismo , RNA Viral/genética , RNA Viral/metabolismo
11.
Commun Biol ; 4(1): 841, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230602

RESUMO

Characterizing protein-protein interactions (PPIs) is an effective method to help explore protein function. Here, through integrating a newly identified split human Rhinovirus 3 C (HRV 3 C) protease, super-folder GFP (sfGFP), and ClpXP-SsrA protein degradation machinery, we developed a fluorescence-assisted single-cell methodology (split protease-E. coli ClpXP (SPEC)) to explore protein-protein interactions for both eukaryotic and prokaryotic species in E. coli cells. We firstly identified a highly efficient split HRV 3 C protease with high re-assembly ability and then incorporated it into the SPEC method. The SPEC method could convert the cellular protein-protein interaction to quantitative fluorescence signals through a split HRV 3 C protease-mediated proteolytic reaction with high efficiency and broad temperature adaptability. Using SPEC method, we explored the interactions among effectors of representative type I-E and I-F CRISPR/Cas complexes, which combining with subsequent studies of Cas3 mutations conferred further understanding of the functions and structures of CRISPR/Cas complexes.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Mapas de Interação de Proteínas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Endopeptidase Clp/genética , Enterovirus/enzimologia , Enterovirus/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteólise , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
12.
Nat Commun ; 12(1): 4349, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272394

RESUMO

Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein "Wzx-Wzy" system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.


Assuntos
Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Membrana/química , Periplasma/metabolismo , Polissacarídeos Bacterianos/metabolismo , Proteínas Tirosina Quinases/química , Motivos de Aminoácidos , Domínio Catalítico , Microscopia Crioeletrônica , Citoplasma/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Periplasma/química , Fosforilação , Conformação Proteica em alfa-Hélice , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Tirosina/química , Tirosina/metabolismo
13.
Nat Commun ; 12(1): 4363, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272395

RESUMO

Conformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the ß-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Eletrofisiologia/métodos , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Canais Iônicos/metabolismo , Porinas/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Bicamadas Lipídicas/química , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Porinas/genética , Porinas/metabolismo , Conformação Proteica , Conformação Proteica em Folha beta , Proteínas Recombinantes , Análise Espectral , Relação Estrutura-Atividade
14.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200244

RESUMO

Ribosome biogenesis is a highly coordinated and complex process that requires numerous assembly factors that ensure prompt and flawless maturation of ribosomal subunits. Despite the increasing amount of data collected, the exact role of most assembly factors and mechanistic details of their operation remain unclear, mainly due to the shortage of high-resolution structural information. Here, using cryo-electron microscopy, we characterized 30S ribosomal particles isolated from an Escherichia coli strain with a deleted gene for the RbfA factor. The cryo-EM maps for pre-30S subunits were divided into six classes corresponding to consecutive assembly intermediates: from the particles with a completely unresolved head domain and unfolded central pseudoknot to almost mature 30S subunits with well-resolved body, platform, and head domains and partially distorted helix 44. The structures of two predominant 30S intermediates belonging to most populated classes obtained at 2.7 Å resolutions indicate that RbfA acts at two distinctive 30S assembly stages: early formation of the central pseudoknot including folding of the head, and positioning of helix 44 in the decoding center at a later stage. Additionally, it was shown that the formation of the central pseudoknot may promote stabilization of the head domain, likely through the RbfA-dependent maturation of the neck helix 28. An update to the model of factor-dependent 30S maturation is proposed, suggesting that RfbA is involved in most of the subunit assembly process.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Bactérias/fisiologia , Ribossomos/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica/métodos , Proteínas de Escherichia coli/genética , Modelos Moleculares , Ligação Proteica , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores de Bactérias/ultraestrutura , Ribossomos/ultraestrutura
15.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200430

RESUMO

The virus-host interaction requires a complex interplay between the phage strategy of reprogramming the host machinery to produce and release progeny virions, and the host defense against infection. Using RNA sequencing, we investigated the phage-host interaction to resolve the phenomenon of improved lytic development of P1vir phage in a DksA-deficient E. coli host. Expression of the ant1 and kilA P1vir genes in the wild-type host was the highest among all and most probably leads to phage virulence. Interestingly, in a DksA-deficient host, P1vir genes encoding lysozyme and holin are downregulated, while antiholins are upregulated. Gene expression of RepA, a protein necessary for replication initiating at the phage oriR region, is increased in the dksA mutant; this is also true for phage genes responsible for viral morphogenesis and architecture. Still, it seems that P1vir is taking control of the bacterial protein, sugar, and lipid metabolism in both, the wild type and dksA- hosts. Generally, bacterial hosts are reacting by activating their SOS response or upregulating the heat shock proteins. However, only DksA-deficient cells upregulate their sulfur metabolism and downregulate proteolysis upon P1vir infection. We conclude that P1vir development is enhanced in the dksA mutant due to several improvements, including replication and virion assembly, as well as a less efficient lysis.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/patogenicidade , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Interações entre Hospedeiro e Microrganismos/genética , Transcriptoma , Proteínas de Bactérias/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/virologia , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Virulência
16.
Nat Commun ; 12(1): 4644, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330903

RESUMO

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Elongação Traducional da Cadeia Peptídica/genética , Fator G para Elongação de Peptídeos/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Ribossomos/genética , Biocatálise , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/metabolismo , Conformação Proteica , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
17.
Nat Commun ; 12(1): 4415, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285228

RESUMO

Complex dynamical fluctuations, from intracellular noise, brain dynamics or computer traffic display bursting dynamics consistent with a critical state between order and disorder. Living close to the critical point has adaptive advantages and it has been conjectured that evolution could select these critical states. Is this the case of living cells? A system can poise itself close to the critical point by means of the so-called self-organized criticality (SOC). In this paper we present an engineered gene network displaying SOC behaviour. This is achieved by exploiting the saturation of the proteolytic degradation machinery in E. coli cells by means of a negative feedback loop that reduces congestion. Our critical motif is built from a two-gene circuit, where SOC can be successfully implemented. The potential implications for both cellular dynamics and behaviour are discussed.


Assuntos
Engenharia Celular/métodos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Engenharia Genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Retroalimentação Fisiológica , Modelos Genéticos , Proteólise , Análise de Célula Única
18.
Nat Commun ; 12(1): 4174, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234105

RESUMO

The folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the ß-barrel assembly machinery (BAM). How lateral opening in the ß-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrolases/metabolismo , Lipossomos/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Microscopia Crioeletrônica , Difusão Dinâmica da Luz , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/ultraestrutura , Hidrolases/genética , Hidrolases/isolamento & purificação , Hidrolases/ultraestrutura , Metabolismo dos Lipídeos , Lipossomos/ultraestrutura , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
19.
FASEB J ; 35(7): e21647, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34165206

RESUMO

The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron-electron double resonance, and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. Especially the C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to a hypothetical protein receptor may support the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface.


Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Linfócitos/metabolismo , Proteínas Recombinantes/metabolismo , Yersinia pseudotuberculosis/química , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Células HeLa , Humanos , Conformação Proteica , Proteínas Recombinantes/genética
20.
Commun Biol ; 4(1): 678, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083706

RESUMO

Toxicity mechanisms of metal oxide nanoparticles towards bacteria and underlying roles of membrane composition are still debated. Herein, the response of lipopolysaccharide-truncated Escherichia coli K12 mutants to TiO2 nanoparticles (TiO2NPs, exposure in dark) is addressed at the molecular, single cell, and population levels by transcriptomics, fluorescence assays, cell nanomechanics and electrohydrodynamics. We show that outer core-free lipopolysaccharides featuring intact inner core increase cell sensitivity to TiO2NPs. TiO2NPs operate as membrane strippers, which induce osmotic stress, inactivate cell osmoregulation and initiate lipid peroxidation, which ultimately leads to genesis of membrane vesicles. In itself, truncation of lipopolysaccharide inner core triggers membrane permeabilization/depolarization, lipid peroxidation and hypervesiculation. In turn, it favors the regulation of TiO2NP-mediated changes in cell Turgor stress and leads to efficient vesicle-facilitated release of damaged membrane components. Remarkably, vesicles further act as electrostatic baits for TiO2NPs, thereby mitigating TiO2NPs toxicity. Altogether, we highlight antagonistic lipopolysaccharide-dependent bacterial responses to nanoparticles and we show that the destabilized membrane can generate unexpected resistance phenotype.


Assuntos
Vesículas Citoplasmáticas/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Pressão Osmótica/efeitos dos fármacos , Titânio/toxicidade , Vesículas Citoplasmáticas/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Microscopia de Força Atômica/métodos , Mutação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...