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1.
Microb Cell Fact ; 18(1): 163, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31581944

RESUMO

BACKGROUND: Sustainable production of microbial fatty acids derivatives has the potential to replace petroleum based equivalents in the chemical, cosmetic and pharmaceutical industry. Most fatty acid sources for production oleochemicals are currently plant derived. However, utilization of these crops are associated with land use change and food competition. Microbial oils could be an alternative source of fatty acids, which circumvents the issue with agricultural competition. RESULTS: In this study, we generated a chimeric microbial production system that features aspects of both prokaryotic and eukaryotic fatty acid biosynthetic pathways targeted towards the generation of long chain fatty acids. We redirected the type-II fatty acid biosynthetic pathway of Escherichia coli BL21 (DE3) strain by incorporating two homologues of the beta-ketoacyl-[acyl carrier protein] synthase I and II from the chloroplastic fatty acid biosynthetic pathway of Arabidopsis thaliana. The microbial clones harboring the heterologous pathway yielded 292 mg/g and 220 mg/g DCW for KAS I and KAS II harboring plasmids respectively. Surprisingly, beta-ketoacyl synthases KASI/II isolated from A. thaliana showed compatibility with the FAB pathway in E. coli. CONCLUSION: The efficiency of the heterologous plant enzymes supersedes the overexpression of the native enzyme in the E. coli production system, which leads to cell death in fabF overexpression and fabB deletion mutants. The utilization of our plasmid based system would allow generation of plant like fatty acids in E. coli and their subsequent chemical or enzymatic conversion to high end oleochemical products.


Assuntos
Arabidopsis/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/biossíntese , Engenharia Metabólica , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/síntese química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/síntese química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Ácido Graxo Sintases/genética , Ácidos Graxos/química , Isoenzimas/síntese química , Isoenzimas/genética , Isoenzimas/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo
2.
Sheng Wu Gong Cheng Xue Bao ; 35(9): 1761-1770, 2019 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-31559757

RESUMO

Seamless modification is a popular genomic manipulation technique in genetic engineering. Selection stringency of the counter-selection system determines the efficiency of the seamless modification. Recently, a novel counter-selection system, kil, was constructed. It is reported that the selection selectivity of kil is higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, indicating that recombinants could be selected out more efficiently by combining kil counter-selection system and plasmid pSim6. In order to confirm this speculation, four different loci (lacI, dbpa, ack, glk) in Escherichia coli strains W3110, MG1655 and DH10B were selected for testing: dsDNA fragments of different sizes (500 bp, 1 000 bp, and 2 000 bp) were used to substitute tet/kil. As expected, recombination efficiency was higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, and the results were more obvious with the length of dsDNA increasing. Specifically, recombination efficiency was 1.2 to 2 fold higher in pSim6 harboring bacteria than in pKD46 harboring bacteria when dsDNA fragments were 1 000 bp in length. With the length of dsDNA increasing up to 2 000 bp, the gap increased to 2.2-5 fold. In conclusion, it is easier to perform seamless modification by combining kil counter-selection system and plasmid pSim6 than combining kil and pKD46. An alternative tool in genomic engineering is provided in this study.


Assuntos
Engenharia Genética , Escherichia coli , Proteínas de Escherichia coli , Plasmídeos , Recombinação Genética
3.
J Microbiol ; 57(9): 781-794, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31452043

RESUMO

The phytopathogenic Burkholderia species B. glumae and B. plantarii are the causal agents of bacterial wilt, grain rot, and seedling blight, which threaten the rice industry globally. Toxoflavin and tropolone are produced by these phytopathogens and are considered the most hostile biohazards with a broad spectrum of target organisms. However, despite their nonspecific toxicity, the effects of toxoflavin and tropolone on bacteria remain unknown. RNA-seq based transcriptome analysis was employed to determine the genome-wide expression patterns under phytotoxin treatment. Expression of 2327 and 830 genes was differentially changed by toxoflavin and tropolone, respectively. Enriched biological pathways reflected the down-regulation of oxidative phosphorylation and ribosome function, beginning with the inhibition of membrane biosynthesis and nitrogen metabolism under oxidative stress or iron starvation. Conversely, several systems such as bacterial chemotaxis, flagellar assembly, biofilm formation, and sulfur/taurine transporters were highly expressed as countermeasures against the phytotoxins. In addition, our findings revealed that three hub genes commonly induced by both phytotoxins function as the siderophore enterobactin, an iron-chelator. Our study provides new insights into the effects of phytotoxins on bacteria for better understanding of the interactions between phytopathogens and other microorganisms. These data will also be applied as a valuable source in subsequent applications against phytotoxins, the major virulence factor.


Assuntos
Antibacterianos/toxicidade , Burkholderia/química , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Doenças das Plantas/microbiologia , Pirimidinonas/toxicidade , Triazinas/toxicidade , Tropolona/toxicidade , Antibacterianos/metabolismo , Burkholderia/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Oryza/microbiologia , Pirimidinonas/metabolismo , Transcriptoma/efeitos dos fármacos , Triazinas/metabolismo , Tropolona/metabolismo
4.
Mol Biol (Mosk) ; 53(4): 674-684, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397441

RESUMO

Acriflavine resistance protein B (AcrB) serves as prototype for multidrug resistance (MDR) efflux transporters of resistance nodulation division (RND) superfamily. AcrB has been proven as potential drug target with many synthetic and natural inhibitors have been identified such as those belonging to pyranopyridine, naphthamide and pimozide classes. The plant derived alkaloid inhibitors represented by reserpine has been found to inhibit both ATP binding cassette and major facilitator efflux transporters. In this study we report the reserpine induced inhibition of RND transporter AcrB. The preliminary docking analysis hints that reserpine shares its binding site with ciprofloxacin, a known substrate of AcrB and could possibly act as competitive inhibitor. For in vitro validation, AcrB from Salmonella typhi was cloned under the control of tac promoter and resulting vector was introduced into E. coli C41(DE3). Under autoinduced conditions, cells overexpressing AcrB transporter were subjected to combined dose of ciprofloxacin and reserpine. The combined exposure resulted in enhanced ciprofloxacin-induced growth inhibition of cells expressing AcrB transporter as compared to control cells transformed with vector of backbone sequence. Time kill analysis further confirmed these findings. To the best of our knowledge, this is first study to show that exposure to reserpine induces inhibition of AcrB. The assay developed in this study allows simple and reproducible detection of substrate/inhibitor effects upon AcrB and related efflux transporters.


Assuntos
Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Reserpina/farmacologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Testes de Sensibilidade Microbiana , Reprodutibilidade dos Testes
5.
Chemistry ; 25(50): 11635-11640, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31368214

RESUMO

Disulfide-containing detergents (DCDs) are introduced, which contain a disulfide bond in the hydrophobic tail. DCDs form smaller micelles than corresponding detergents with linear hydrocarbon chains, while providing good solubilization and reconstitution of membrane proteins. The use of this new class of detergents in structural biology is illustrated with solution NMR spectra of the human G protein-coupled receptor A2A AR, which is an α-helical protein, and the ß-barrel protein OmpX from E. coli.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Detergentes/química , Proteínas de Escherichia coli/química , Hidrolases/química , Receptor A2A de Adenosina/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Dissulfetos/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Humanos , Hidrolases/metabolismo , Micelas , Ressonância Magnética Nuclear Biomolecular , Estabilidade Proteica , Receptor A2A de Adenosina/metabolismo
6.
Sheng Wu Gong Cheng Xue Bao ; 35(7): 1247-1255, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31328481

RESUMO

L-tyrosine is one of three aromatic amino acids that are widely used in food, pharmaceutical and chemical industries. The transport system engineering provides an important research strategy for the metabolic engineering of Escherichia coli to breed L-tyrosine producing strain. The intracellular transport of L-tyrosine in E. coli is mainly regulated by two distinct permeases encoded by aroP and tyrP genes. The aroP and tyrP gene knockout mutants were constructed by CRISPR-Cas technique on the basis of L-tyrosine producing strain HGXP, and the effects of regulating transport system on L-tyrosine production were investigated by fermentation experiments. The fermentation results showed that the aroP and tyrP knockout mutants produced 3.74 and 3.45 g/L L-tyrosine, respectively, which were 19% and 10% higher than that of the original strain. The optimum induction temperature was determined to be 38 °C. Fed-batch fermentation was carried out on a 3-L fermentor. The L-tyrosine yields of aroP and tyrP knockout mutants were further increased to 44.5 and 35.1 g/L, respectively, which were 57% and 24% higher than that of the original strain. The research results are of great reference value for metabolic engineering of E. coli to produce L-tyrosine.


Assuntos
Escherichia coli , Proteínas de Escherichia coli , Técnicas de Inativação de Genes , Engenharia Metabólica , Tirosina
7.
Gene ; 713: 143951, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31269464

RESUMO

Rifampicin (RIF) is still a first line of antibiotic in the treatment of bacterial diseases, in particular the Mycobacterial infections. The antimicrobial activity of RIF is attributed to its ability to inhibit transcription by binding to the ß subunit of bacterial RNA polymerase (encoded by rpoB). Continued use of this drug resulted in the emergence of RIF resistant rpoB mutations in a high frequency that compels the use of RIF almost exclusively in drug combinations. As of date, a broad array of rif mutations have been isolated and characterized by different research groups. Studies on rpoB mutations strengthen the view that the ß subunit of RNA polymerase (RNAP) is very crucial in modulating transcription thereby leading to differential gene expression. Very recently we have reported the transcriptome profile of rpoB12 mutant that provides molecular evidence that presence of rpoB12 mutation modulates the transcription of about 450 genes. Here we present a maiden report that rpoB mutations that substitute Tyr at the Rif binding pocket (RBP) of ß subunit of RNA polymerase are able to suppress the over-production of colanic acid capsular polysaccharide (Ces phenotype) in Δlon mutant of Escherichia coli. Further analyses of the rif mutants involving their growth pattern on LB at higher temperature (42 °C), LB media without NaCl, survival in LB media with acidic pH (pH - 3) and motility revealed that only rpoB12 (His526Tyr) and rpoB137 (Ser522Tyr) affected all the above mentioned physiological parameters in addition to the elicitation of Ces phenotype. These two rif mutations confer fast movement to RNAP and they bear Tyr as the substituted amino acid in the RBP. This is perhaps the first study that brings out the possible role of Tyr in the RBP and its participation in the global gene expression. This study also envisages the point that amino acid residues that share the properties of Tyr in the RBP can be employed as a tool to bring out differential gene expression which would certainly have basic and applied values for the mankind.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Mutação , Rifampina/farmacologia , Tirosina/metabolismo , Antibióticos Antituberculose/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fenótipo , RNA Bacteriano , Tirosina/genética
8.
J Agric Food Chem ; 67(28): 7908-7915, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31268314

RESUMO

In this study, to obtain higher agmatine yields using the previously developed E. coli strain AUX4 (JM109 ΔspeC ΔspeF ΔspeB ΔargR), the genes encoding glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), phosphoenolpyruvate carboxylase (ppc), aspartate aminotransferase (aspC), transhydrogenase (pntAB), and biosynthetic arginine decarboxylase (speA) were sequentially overexpressed by replacing their native promoters with the heterologous strong trp, core-trc, or 5Ptacs promoters to generate the plasmid-free E. coli strain AUX11. The fermentation results obtained using a 3-L bioreactor showed that AUX11 produced 2.93 g L-1 agmatine with the yield of 0.29 g agmatine g-1 glucose in the batch fermentation, and the fed-batch fermentation of AUX11 allowed the production of 40.43 g L-1 agmatine with the productivity of 1.26 g L-1 h-1 agmatine. The results showed that the engineered E. coli strain AUX11 can be used for the industrial fermentative production of agmatine.


Assuntos
Agmatina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas de Cultura Celular por Lotes , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fermentação , Glucose/metabolismo , Engenharia Metabólica
9.
BMC Infect Dis ; 19(1): 571, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266450

RESUMO

BACKGROUND: Carbapenemase-producing organisms (CPOs) have emerged as antibiotic-resistant bacteria of global concern. Here we assessed the performance of the Carba (beta) assay, a multiplex real-time PCR assay developed by SpeeDx for the detection of key carbapenemase-encoding genes: KPC, NDM, OXA-48-like, IMP-4-like, and VIM. METHODS: DNA extracts of 180 isolates were tested with the Carba (beta) assay, using previously validated in-house TaqMan probe assays for the relevant carbapenemase genes as the reference standard. The Carba (beta) assay was then directly used to screen 460 DNA extracts of faecal specimens, with positive results subjected to the aforementioned in-house assays plus Sanger sequencing. RESULTS: The Carba (beta) assay correctly identified the presence of the respective carbapenemase genes in 154 of 156 isolates and provided negative results for all 24 non-CPO isolates. Two isolates provided positive results for OXA-48-like carbapenemase by the Carba (beta) assay only. The Carba (beta) assay had sensitivities of 100% for all targets, and specificities of 100% for KPC, NDM, IMP-4-like, and VIM targets, and 98.5% for OXA-48-like targets. When applied directly to faecal specimens, eight samples were positive by the Carba (beta) assay, two of which were confirmed by in-house TaqMan probe PCR or DNA sequencing. CONCLUSIONS: The Carba (beta) assay is highly sensitive and specific for detecting key carbapenemase genes in isolates. Further testing is required to assess this assay's suitability for direct screening of clinical specimens.


Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , beta-Lactamases/genética , Antibacterianos , Bactérias/efeitos dos fármacos , Técnicas Bacteriológicas/métodos , Proteínas de Escherichia coli/genética , Fezes/microbiologia , Humanos , Sensibilidade e Especificidade
10.
BMC Vet Res ; 15(1): 234, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286936

RESUMO

BACKGROUND: Enterotoxigenic Escherichia coli K88 (E. coli K88) are considered as a major cause of diarrhea and death in newly weaned piglets. Oral passive immunization with chicken egg yolk immunoglobulins (IgY) have attracted considerable attention for treatment of gastrointestinal infection due to its high specificity. In this study it was estimated the protective effect of anti-K88 fimbriae IgY against E. coli K88 adhesion to piglet intestinal mucus in vitro and to investigate the potential use of IgY for controlling E. coli-induced diarrhea in weaned piglets in vivo. RESULTS: E. coli K88 was incubated with IgY for 24 h, and the bacterial growth profiles showed that specific IgY with a concentration higher than 5 mg/mL was observed to significantly inhibit the growth of E. coli K88 compared to nonspecific yolk powder in a liquid medium. Moreover, pretreatment with 50 mg/mL of IgY was found to significantly decrease the adhesion ability of E. coli K88 to porcine jejunal and ileal mucus, further supported by the observations from our immunofluorescence microscopic analysis. In vivo, administration of IgY successfully protected piglets from diarrhea caused by E. coli K88 challenge. Additionally, IgY treatment efficiently alleviated E. coli-induced intestinal inflammation in piglets as the gene expression levels of inflammatory cytokines TNF-α, IL-22, IL-6 and IL-1ß in IgY-treated piglets remained unchanged after E. coli K88 infection. Furthermore, IgY significantly prevented E. coli K88 adhering to the jejunal and ileal mucosa of piglets with E. coli infection and significantly decreased E. coli and enterotoxin expression in colonic contents. CONCLUSION: Outcome of the study demonstrated that IgY against the fimbrial antigen K88 was able to significantly inhibit the growth of E. coli K88, block the binding of E. coli to small intestinal mucus, and protect piglets from E. coli-induced diarrhea. These results indicate that passive immunization with IgY may be useful to prevent bacterial colonization and to control enteric diseases due to E. coli infection. The study has great clinical implication to provide alternative therapy to antibiotics in E coli induced diarrhea.


Assuntos
Aderência Bacteriana/efeitos dos fármacos , Diarreia/etiologia , Diarreia/prevenção & controle , Escherichia coli Enterotoxigênica/efeitos dos fármacos , Infecções por Escherichia coli/complicações , Imunoglobulinas/farmacologia , Animais , Antígenos de Bactérias/imunologia , Citocinas/genética , Diarreia/microbiologia , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/patologia , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/imunologia , Proteínas de Fímbrias/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Imunização Passiva , Imunoglobulinas/uso terapêutico , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Ligação Proteica/efeitos dos fármacos , Suínos
11.
Biochemistry (Mosc) ; 84(4): 407-415, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228932

RESUMO

Proton-translocating FOF1-ATP synthase (F-type ATPase, F-ATPase or FOF1) performs ATP synthesis/hydrolysis coupled to proton transport across the membrane in mitochondria, chloroplasts, and most eubacteria. The ATPase activity of the enzyme is suppressed in the absence of protonmotive force by several regulatory mechanisms. The most conserved of these mechanisms is noncompetitive inhibition of ATP hydrolysis by the MgADP complex (ADP-inhibition) which has been found in all the enzymes studied. When MgADP binds without phosphate in the catalytic site, the enzyme enters an inactive state, and MgADP gets locked in the catalytic site and does not exchange with the medium. The degree of ADP-inhibition varies in FOF1 enzymes from different organisms. In the Escherichia coli enzyme, ADP-inhibition is relatively weak and, in contrast to other organisms, is enhanced rather than suppressed by phosphate. In this study, we used site-directed mutagenesis to investigate the role of amino acid residues ß139, ß158, ß189, and ß319 of E. coli FOF1-ATP synthase in the mechanism of ADP-inhibition and its modulation by the protonmotive force. The amino acid residues in these positions differ in the enzymes from beta- and gammaproteobacteria (including E. coli) and FOF1-ATP synthases from other eubacteria, mitochondria, and chloroplasts. The ßN158L substitution produced no effect on the enzyme activity, while substitutions ßF139Y, ßF189L, and ßV319T only slightly affected ATP (1 mM) hydrolysis. However, in a mixture of ATP and ADP, the activity of the mutants was less suppressed than that of the wild-type enzyme. In addition, mutations ßF189L and ßV319T weakened the ATPase activity inhibition by phosphate in the presence of ADP. We suggest that residues ß139, ß189, and ß319 are involved in the mechanism of ADP-inhibition and its modulation by phosphate.


Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Difosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Cinética , Mutagênese Sítio-Dirigida , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Força Próton-Motriz , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/genética , Alinhamento de Sequência
12.
Biochemistry (Mosc) ; 84(4): 426-434, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228934

RESUMO

The bacterium Escherichia coli has seven σ subunits that bind core RNA polymerase and are necessary for promoter recognition. It was previously shown that the σ70 and σ38 subunits can also interact with the transcription elongation complex (TEC) and stimulate pausing by recognizing DNA sequences similar to the -10 element of promoters. In this study, we analyzed the ability of the σ32, σ28, and σ24 subunits to induce pauses in reconstituted TECs containing corresponding -10 consensus elements. It was found that the σ24 subunit can induce a transcriptional pause depending on the presence of the -10 element. Pause formation is suppressed by the Gre factors, suggesting that the paused complex adopts a backtracked conformation. Some natural promoters contain potential signals of σ24-dependent pauses in the initially transcribed regions, suggesting that such pauses may have regulatory functions in transcription.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Transcrição Genética/fisiologia , Sequência de Bases , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Proteínas de Escherichia coli/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo
13.
Nat Commun ; 10(1): 2393, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160557

RESUMO

Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains (NBDs) power polypeptide translocation through a central channel comprised of a hexameric spiral of protomers that contact substrate via conserved pore-loop interactions. Here we report cryo-EM structures of a hyperactive ClpB variant bound to the model substrate, casein in the presence of slowly hydrolysable ATPγS, which reveal the translocation mechanism. Distinct substrate-gripping interactions are identified for NBD1 and NBD2 pore loops. A trimer of N-terminal domains define a channel entrance that binds the polypeptide substrate adjacent to the topmost NBD1 contact. NBD conformations at the seam interface reveal how ATP hydrolysis-driven substrate disengagement and re-binding are precisely tuned to drive a directional, stepwise translocation cycle.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Caseínas/metabolismo , Endopeptidase Clp/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Escherichia coli/metabolismo , Proteínas de Choque Térmico/ultraestrutura , Transporte Proteico , Domínio AAA , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Hidrólise , Modelos Moleculares , Peptídeos/metabolismo , Agregados Proteicos , Subunidades Proteicas/metabolismo
14.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 545-553, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205017

RESUMO

Spermidine N-acetyltransferase (SpeG) transfers an acetyl group from acetyl-coenzyme A to an N-terminal amino group of intracellular spermidine. This acetylation inactivates spermidine, reducing the polyamine toxicity that tends to occur under certain chemical and physical stresses. The structure of the SpeG protein from Vibrio cholerae has been characterized: while the monomer possesses a structural fold similar to those of other Gcn5-related N-acetyltransferase superfamily members, its dodecameric structure remains exceptional. In this paper, structural analyses of SpeG isolated from Escherichia coli are described. Like V. cholerae SpeG, E. coli SpeG forms dodecamers, as revealed by two crystal structures of the ligand-free E. coli SpeG dodecamer determined at 1.75 and 2.9 Šresolution. Although both V. cholerae SpeG and E. coli SpeG can adopt an asymmetric open dodecameric state, solution analysis showed that the oligomeric composition of ligand-free E. coli SpeG differs from that of ligand-free V. cholerae SpeG. Based on these data, it is proposed that the equilibrium balance of SpeG oligomers in the absence of ligands differs from one species to another and thus might be important for SpeG function.


Assuntos
Acetiltransferases/química , Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/química , Modelos Moleculares , Estrutura Quaternária de Proteína , Cristalização , Cristalografia por Raios X/métodos
15.
Nat Commun ; 10(1): 2551, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186428

RESUMO

Respiratory complex I plays a central role in cellular energy metabolism coupling NADH oxidation to proton translocation. In humans its dysfunction is associated with degenerative diseases. Here we report the structure of the electron input part of Aquifex aeolicus complex I at up to 1.8 Å resolution with bound substrates in the reduced and oxidized states. The redox states differ by the flip of a peptide bond close to the NADH binding site. The orientation of this peptide bond is determined by the reduction state of the nearby [Fe-S] cluster N1a. Fixation of the peptide bond by site-directed mutagenesis led to an inactivation of electron transfer and a decreased reactive oxygen species (ROS) production. We suggest the redox-gated peptide flip to represent a previously unrecognized molecular switch synchronizing NADH oxidation in response to the redox state of the complex as part of an intramolecular feed-back mechanism to prevent ROS production.


Assuntos
Complexo I de Transporte de Elétrons/química , Escherichia coli/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Bactérias/química , Bactérias/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas com Ferro-Enxofre/química , Mutagênese Sítio-Dirigida , NAD/química , Oxirredução
16.
Nat Chem Biol ; 15(7): 669-671, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209348

RESUMO

Fatty acid synthases are dynamic ensembles of enzymes that can biosynthesize long hydrocarbon chains efficiently. Here we visualize the interaction between the Escherichia coli acyl carrier protein (AcpP) and ß-ketoacyl-ACP-synthase I (FabB) using X-ray crystallography, NMR, and molecular dynamics simulations. We leveraged this structural information to alter lipid profiles in vivo and provide a molecular basis for how protein-protein interactions can regulate the fatty acid profile in E. coli.


Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Proteína de Transporte de Acila/metabolismo , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintase Tipo II/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , Proteína de Transporte de Acila/química , Cristalografia por Raios X , Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Ácido Graxo Sintase Tipo II/química , Modelos Moleculares , Ligação Proteica
17.
Nat Commun ; 10(1): 2868, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253770

RESUMO

Prokaryotes and eukaryotes alike endogenously generate the gaseous molecule hydrogen sulfide (H2S). Bacterial H2S acts as a cytoprotectant against antibiotics-induced stress and promotes redox homeostasis. In E. coli, endogenous H2S production is primarily dependent on 3-mercaptopyruvate sulfurtransferase (3MST), encoded by mstA. Here, we show that cells lacking 3MST acquire a phenotypic suppressor mutation resulting in compensatory H2S production and tolerance to antibiotics and oxidative stress. Using whole genome sequencing, we identified a non-synonymous mutation within an uncharacterized LacI-type transcription factor, ycjW. We then mapped regulatory targets of YcjW and discovered it controls the expression of carbohydrate metabolic genes and thiosulfate sulfurtransferase PspE. Induction of pspE expression in the suppressor strain provides an alternative mechanism for H2S biosynthesis. Our results reveal a complex interaction between carbohydrate metabolism and H2S production in bacteria and the role, a hitherto uncharacterized transcription factor, YcjW, plays in linking the two.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Sulfeto de Hidrogênio/metabolismo , Substituição de Aminoácidos , Antibacterianos/farmacologia , Mapeamento Cromossômico , DNA Bacteriano , Dissacarídeos/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/fisiologia , Ligação Proteica , RNA Mensageiro , Regulon , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Nat Commun ; 10(1): 2858, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253794

RESUMO

In bacterial tRNAs, 5-carboxymethoxyuridine (cmo5U) and its derivatives at the first position of the anticodon facilitate non-Watson-Crick base pairing with guanosine and pyrimidines at the third positions of codons, thereby expanding decoding capabilities. However, their biogenesis and physiological roles remained to be investigated. Using reverse genetics and comparative genomics, we identify two factors responsible for 5-hydroxyuridine (ho5U) formation, which is the first step of the cmo5U synthesis: TrhP (formerly known as YegQ), a peptidase U32 family protein, is involved in prephenate-dependent ho5U formation; and TrhO (formerly known as YceA), a rhodanese family protein, catalyzes oxygen-dependent ho5U formation and bypasses cmo5U biogenesis in a subset of tRNAs under aerobic conditions. E. coli strains lacking both trhP and trhO exhibit a temperature-sensitive phenotype, and decode codons ending in G (GCG and UCG) less efficiently than the wild-type strain. These findings confirm that tRNA hydroxylation ensures efficient decoding during protein synthesis.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Biossíntese de Proteínas/fisiologia , RNA de Transferência/metabolismo , Proteínas de Escherichia coli/genética , Evolução Molecular , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/fisiologia , Filogenia , RNA Bacteriano
19.
J Microbiol Biotechnol ; 29(6): 923-932, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154747

RESUMO

Current strategies of strain improvement processes are mainly focused on enhancing the synthetic pathways of the products. However, excessive metabolic flux often creates metabolic imbalances, which lead to growth retardation and ultimately limit the yield of the product. To solve this problem, we applied a dynamic regulation strategy to produce L-phenylalanine (LPhe) in Escherichia coli. First, we constructed a series of Phe-induced promoters that exhibited different strengths through modification of the promoter region of tyrP. Then, two engineered promoters were separately introduced into a Phe-producing strain xllp1 to dynamically control the expression level of one pathway enzyme AroK. Batch fermentation results of the strain xllp3 showed that the titer of Phe reached 61.3 g/l at 48 h, representing a titer of 1.36- fold of the strain xllp1 (45.0 g/l). Moreover, the L-Phe yields on glucose of xllp3 (0.22 g/g) were also greatly improved, with an increase of 1.22-fold in comparison with the xllp1 (0.18 g/ g). In summary, we successfully improved the titer of Phe by using dynamic regulation of one key enzyme and this strategy can be applied for improving the performance of strains producing other aromatic amino acids and derived compounds.


Assuntos
Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Microbiologia Industrial/métodos , Engenharia Metabólica/métodos , Fenilalanina/biossíntese , Sistemas de Transporte de Aminoácidos Neutros/genética , Vias Biossintéticas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fermentação , Glucose/metabolismo , Análise do Fluxo Metabólico , Mutação , Fenilalanina/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Regiões Promotoras Genéticas
20.
J Microbiol Biotechnol ; 29(6): 839-844, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154751

RESUMO

Anthranilate derivatives have been used as flavoring and fragrant agents for a long time. Recently, these compounds are gaining attention due to new biological functions including antinociceptive and analgesic activities. Three anthranilate derivatives, N-methylanthranilate, methyl anthranilate, and methyl N-methylanthranilate were synthesized using metabolically engineered stains of Escherichia coli. NMT encoding N-methyltransferase from Ruta graveolens, AMAT encoding anthraniloyl-coenzyme A (CoA):methanol acyltransferase from Vitis labrusca, and pqsA encoding anthranilate coenzyme A ligase from Pseudomonas aeruginosa were cloned and E. coli strains harboring these genes were used to synthesize the three desired compounds. E. coli mutants (metJ, trpD, tyrR mutants), which provide more anthranilate and/or S-adenosyl methionine, were used to increase the production of the synthesized compounds. MS/MS analysis was used to determine the structure of the products. Approximately, 185.3 µM N-methylanthranilate and 95.2 µM methyl N-methylanthranilate were synthesized. This is the first report about the synthesis of anthranilate derivatives in E. coli.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , ortoaminobenzoatos/metabolismo , Vias Biossintéticas , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/metabolismo , Ruta/enzimologia , Ruta/genética , Vitis/enzimologia , Vitis/genética , ortoaminobenzoatos/química
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