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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): 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
3.
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
4.
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
5.
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
6.
Gene ; 803: 145890, 2021 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-34375634

RESUMO

Escherichia coli Nissle 1917 (EcN) is an efficient probiotic strain extensively used worldwide because of its several health benefits. Adhesion to the intestinal cells is one of the prerequisites for a probiotic strain. To identify the genes essential for the adhesion of EcN on the intestinal cells, we utilized a quantitative genetic footprinting approach called transposon insertion sequencing (INSeq). A transposon insertion mutant library of EcN comprising of ~17,000 mutants was used to screen the adherence to the intestinal epithelial cells, Caco-2. The transposon insertion sites were identified from the input and output population by employing next-generation sequencing using the Ion torrent platform. Based on the relative abundance of reads in the input and output pools, we identified 113 candidate genes that are essential for the fitness of EcN during the adhesion and colonization on the Caco-2 cells. Functional categorization revealed that these fitness genes are associated with carbohydrate transport and metabolism, cell wall/membrane/envelope biogenesis, post-translational modification, stress response, motility and adhesion, and signal transduction. To further validate the genes identified in our INSeq analysis, we constructed individual knock-out mutants in five genes (cyclic di-GMP phosphodiesterase (gmp), hda, uidC, leuO, and hypothetical protein-coding gene). We investigated their ability to adhere to Caco-2 cells. Evaluation of these mutants showed reduced adhesion on Caco-2 cells, confirming their role in adhesion. Understanding the functions of these genes may provide novel insights into molecular regulation during colonization of probiotic bacteria to the intestinal cells, and useful to develop designer probiotic strains.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/fisiologia , Mutagênese Insercional , Análise de Sequência de DNA/métodos , Aderência Bacteriana , Células CACO-2 , Elementos de DNA Transponíveis , Escherichia coli/genética , Aptidão Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Probióticos
7.
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
8.
Front Cell Infect Microbiol ; 11: 701625, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34395312

RESUMO

Resistance to colistin, especially mobilized colistin resistance (mcr), is a serious threat to public health since it may catalyze a return of the "pre-antibiotic era". Outer membrane vesicles (OMVs) play a role in antibiotic resistance in various ways. Currently, how OMVs participate in mcr-1-mediated colistin resistance has not been established. In this study, we showed that both OMVs from the mcr-1 negative and positive Escherichia coli (E. coli) strains conferred dose-dependent protection from colistin. However, OMVs from the mcr-1 positive strain conferred attenuated protection when compared to the OMVs of a mcr-1 negative strain at the same concentration. The attenuated protective effect of OMVs was related to the reduced ability to absorb colistin from the environment, thus promoting the killing of colistin sensitive E. coli strains. Lipid A modified with phosphoethanolamine was presented in the OMVs of the mcr-1 positive E. coli strain and resulted in decreased affinity to colistin and less protection. Meanwhile, E. coli strain carrying the mcr-1 gene packed more unmodified lipid A in OMVs and kept more phosphoethanolamine modified lipid A in the bacterial cells. Our study provides a first glimpse of the role of OMVs in mcr-1 -mediated colistin resistance.


Assuntos
Colistina , Proteínas de Escherichia coli , Antibacterianos/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Testes de Sensibilidade Microbiana , Plasmídeos
9.
Mol Cell ; 81(16): 3310-3322.e6, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34416138

RESUMO

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product-the alarmone nucleotide (p)ppGpp-through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.


Assuntos
Regulação Alostérica/genética , Proteínas de Escherichia coli/genética , GTP Pirofosfoquinase/genética , Guanosina Pentafosfato/genética , Pirofosfatases/genética , Aminoácidos/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Domínio Catalítico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrolases/genética , Ribossomos/genética , Ribossomos/metabolismo , Inanição/genética , Inanição/metabolismo
10.
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
11.
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
12.
Molecules ; 26(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34200016

RESUMO

The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the rne mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of E. coli rne mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting rne mRNA with antisense oligonucleotides.


Assuntos
Antibacterianos/farmacologia , Endorribonucleases/genética , Escherichia coli/enzimologia , Oligonucleotídeos/farmacologia , Sistema Livre de Células , Endorribonucleases/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Oligonucleotídeos/síntese química , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , RNA Mensageiro/antagonistas & inibidores
13.
C R Biol ; 344(2): 111-126, 2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-34213850

RESUMO

The operon model was proposed six decades ago. And yet, despite all this time, the lactose operon repressor, LacI, remains a subject of major interest. While it is well established that LacI can exist in two functional forms, one that renders the operon inactive via binding of LacI to DNA and another, bound to an inducer that does not allow repression, how it switches from one to the other is still not well understood. The construction of a library of several tens of thousands of LacI mutants has revealed some unexpected features. In particular, the transition implemented in some of them reveals a new type of transcription regulation: band-pass (OFF/ON/OFF) and band-stop (ON/OFF/ON) filters. This makes it natural to think that it is the network of hydrogen bonds associated with the water bound to the molecule that allows the remote interconnection between the binding site to an inducer molecule and the one that binds it to the DNA.


Assuntos
Proteínas de Escherichia coli , Sítios de Ligação , DNA , Proteínas de Escherichia coli/genética , Óperon Lac , Repressores Lac/genética , Repressores Lac/metabolismo
14.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205216

RESUMO

Two independent, complementary methods of structural analysis were used to elucidate the effect of divalent magnesium and iron cations on the structure of the protective Dps-DNA complex. Small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) demonstrate that Mg2+ ions block the N-terminals of the Dps protein preventing its interaction with DNA. Non-interacting macromolecules of Dps and DNA remain in the solution in this case. The subsequent addition of the chelating agent (EDTA) leads to a complete restoration of the structure of the complex. Different effect was observed when Fe cations were added to the Dps-DNA complex; the presence of Fe2+ in solution leads to the total complex destruction and aggregation without possibility of the complex restoration with the chelating agent. Here, we discuss these different responses of the Dps-DNA complex on the presence of additional free metal cations, investigating the structure of the Dps protein with and without cations using SAXS and cryo-EM. Additionally, the single particle analysis of Dps with accumulated iron performed by cryo-EM shows localization of iron nanoparticles inside the Dps cavity next to the acidic (hydrophobic) pore, near three glutamate residues.


Assuntos
Proteínas da Membrana Bacteriana Externa/ultraestrutura , DNA/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Ferro/química , Magnésio/química , Sequência de Aminoácidos/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Cátions/química , Microscopia Crioeletrônica , DNA/química , DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Espalhamento a Baixo Ângulo , Difração de Raios X
15.
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
16.
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
17.
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
18.
Curr Microbiol ; 78(9): 3464-3473, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34264362

RESUMO

Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of diarrhea in children globally, and thus suitable vaccines are desired. Antigen display on lactic acid bacteria is a reliable approach for efficient oral vaccination and preventing bowel diseases. To develop an oral vaccine against ETEC, the gene of the binding domain from heat-labile toxin (LTB), a key ETEC virulence factor, was codon-optimized and cloned into a construct containing a signal peptide and an anchor for display on L. lactis. Bioinformatics analysis showed a codon adaptation index of 0.95 for the codon-optimized gene. Cell surface expression of LTB was confirmed by transmission electron microscopy and blotting. White New Zealand rabbits were immunized per os (PO) with the recombinant L. lactis, and the antibody titers were assayed with ELISA. In vitro neutralization assay was performed using mouse adrenal tumor cells and rabbit ileal loop test was performed as the in vivo assay. ELISA results indicated that oral administration of the engineered L. lactis elicited a significant production of IgA in the intestine. In vitro neutralization assay showed that the effect of the toxin could be neutralized with 500 µg/ml of IgG isolated from the oral vaccine group. Furthermore, the dose of ETEC causing fluid accumulation in the ileal loop test showed a tenfold increase in rabbits immunized with either recombinant L. lactis or LTB protein compared to other groups. Our results imply that recombinant L. lactis could potentially be an effective live oral vaccine against ETEC toxicity.


Assuntos
Escherichia coli Enterotoxigênica , Infecções por Escherichia coli , Proteínas de Escherichia coli , Lactococcus lactis , Administração Oral , Animais , Anticorpos Antibacterianos , Escherichia coli Enterotoxigênica/genética , Enterotoxinas/genética , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/genética , Humanos , Lactococcus lactis/genética , Camundongos , Coelhos , Vacinação
19.
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
20.
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
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