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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.
Ann Agric Environ Med ; 26(3): 405-408, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31559794

RESUMO

INTRODUCTION: Carbapenemase-producing Enterobacteriaceae have spread rapidly through the countries and continents to become a global concern. One of the main reservoirs of NDM-1 positive strains from the Enterobacteriaceae family is the Indian subcontinent (Bangladesh, Pakistan, India). MATERIAL AND METHODS: During June 2017 - June 2018, rectal swab samples were collected routinely in all patients returning to Poland from South and South-East Asia. During molecular examinations gene blaNDM-1 encoding NDM-1 carbapenemase was detected. RESULTS: 31 patients were examined after returning to Poland from a trip to South and South-East Asia. The presence of New Delhi Metallo-ß-lactamase-1 producing Escherichia coli and Klebsiella pneumoniae was confirmed in three patients (9.7%) returning to Poland from travels to India. All the positive patients were hospitalized during the trip in a New Delhi hospital. CONCLUSIONS: Digestive tract carriage of NDM in a group of Polish travelers is a significant health and epidemiological problem. The study confirms the necessity for screening for carbapenemase-producing Enterobacteriaceae (CPE), particularly among travellers. Rectal swabs should be collected in every case of patients returning from international trips, and the possibility of environment-associated infections should be emphasized.


Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Enterobacteriaceae/microbiologia , Escherichia coli/enzimologia , Klebsiella pneumoniae/enzimologia , Viagem , beta-Lactamases/metabolismo , Adulto , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Feminino , Humanos , Índia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Polônia , beta-Lactamases/genética
3.
BMC Vet Res ; 15(1): 268, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31357996

RESUMO

BACKGROUND: This study investigated changes over time in the epidemiology of extended-spectrum ß-lactamase (ESBL) producing Escherichia coli within a single equine referral hospital in the UK. Faecal samples were collected from hospitalised horses in 2008 and 2017, processed using selective media and standard susceptibility laboratory methods. A novel real-time PCR with high resolution melt analysis was used to distinguish blaCTX-M-1 and blaCTX-M-15 within CTX-M-1 group. RESULTS: In 2008, 457 faecal samples from 103 horses were collected, with ESBL-producing E. coli identified in 131 samples (28.7, 95% CI 24.6-33.1). In 2017, 314 faecal samples were collected from 74 horses with ESBL-producing E. coli identified in 157 samples (50.0, 95% CI 44.5-55.5). There were 135 and 187 non-duplicate ESBL-producing isolates from 2008 and 2017, respectively. In 2008, 12.6% of isolates belonged to CTX-M-1 group, all carrying blaCTX-M-1, whilst in 2017, 94.1% of isolates were CTX-M-1 group positive and of these 39.2 and 60.8% of isolates carried blaCTX-M-1 and blaCTX-M-15, respectively. In addition, the prevalence of doxycycline, gentamicin and 3rd generation cephalosporin resistance increased significantly from 2008 to 2017 while a decreased prevalence of phenotypic resistance to potentiated sulphonamides was observed. CONCLUSIONS: The real-time PCR proved a reliable and high throughput method to distinguish between blaCTX-M-1 and blaCTX-M-15. Furthermore, its use in this study demonstrated the emergence of faecal carriage of CTX-M-15 in hospitalised horses, with an increase in prevalence of ESBL-producing E. coli as well as increased antimicrobial resistance to frequently used antimicrobials.


Assuntos
Infecções por Escherichia coli/veterinária , Fezes/microbiologia , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/microbiologia , beta-Lactamases/metabolismo , Animais , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Cavalos , Hospitais Veterinários/estatística & dados numéricos , Prevalência , Reino Unido/epidemiologia , beta-Lactamases/genética
4.
Biochimie ; 163: 137-141, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31181235

RESUMO

RNA amplification has extensive applications on biochemistry and its related fields. Here, we present an isothermal strategy named rolling circle reverse transcription-mediated RNA amplification (RCRT-MRA) to amplify small RNAs with accurate length and sequence. The target RNA and complementary DNA were circularized to serve as amplicons replicated via the rolling circle mechanism. The transcription product consisting of tandemly repeated RNA units, was monomerized by site-specific cleavage to generate amplified RNA with authentic length and sequence. T4 DNA ligase was chosen to circularize RNA template for its high efficiency and low cost. SuperScript IV reverse transcriptase was found to be able to catalyze the RCRT reaction on the circular RNA template, and the reaction efficiency was enhanced by adding the nicking enzyme, Nb.BbvCI to the RCRT system. E. Coli RNA polymerase, instead of the commonly used T7 RNA polymerase, was applied to synthesize long-strand RNA product for its high universality and processivity. Under the optimized conditions, small RNAs can be precisely amplified by 105∼6 folds. The fidelity of the established method was demonstrated by the accordance of the sequencing result and the initial RNA sequences. Free from expensive thermal cycler (necessary for RT-PCR-based amplification), precise replication of the initial RNA and high fidelity will enable the established strategy to be applied in RNA-seq, mRNA profiling, microarray analysis and RNA-based SELEX as well.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/metabolismo , Transcrição Reversa , DNA Circular/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo
5.
Chemotherapy ; 64(1): 22-27, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31167192

RESUMO

BACKGROUND: Pantothenate, the fundamental precursor to coenzyme A, is required for optimal growth and virulence of microbial pathogens. It is synthesized by the enzyme-catalyzed condensation of ß-alanine and pantoate, which has shown susceptibility to inhibition by analogs of its molecular constituents. Accordingly, analogs of ß-alanine are gaining inquiry as potential antimicrobial chemotherapeutics. METHODS: We synthesized and evaluated 35 derivatives of ß-alanine, substituted at the α, ß, amine, and carboxyl sites, derived from in silico, dynamic molecular modeling to be potential competitive inhibitors of pantothenate synthetase. Employing the Clinical Laboratory Standards M7-A6 broth microdilution method, we tested these for inhibition of growth in Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. RESULTS: All compounds proved entirely ineffective in all species tested, with no inhibition of growth being observed up to 200 µM/mL. CONCLUSIONS: Upon revision of the literature, we conclude that high enzyme selectivity or external salvage mechanisms may render this strategy futile against most bacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Peptídeo Sintases/metabolismo , beta-Alanina/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Simulação de Acoplamento Molecular , Peptídeo Sintases/antagonistas & inibidores , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , beta-Alanina/análogos & derivados , beta-Alanina/farmacologia
6.
Chem Commun (Camb) ; 55(52): 7498-7501, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31187106

RESUMO

We explored a collection of 2-deoxyribose-5-phosphate aldolases (DERAs) from biodiversity for their nucleophile substrate promiscuity. The DERAs were screened using as nucleophiles propanone, propanal, cyclobutanone, cyclopentanone, dihydroxyacetone, and glycolaldehyde with l-glyceraldehyde-3-phosphate as an electrophile in aldol addition. A DERA from Arthrobacter chlorophenolicus (DERAArthro) efficiently allowed the synthesis of the corresponding aldol adducts in good yields, displaying complementarity in terms of configuration and substrate specificity with fructose-6-phosphate aldolase, the only previously known aldolase with a large nucleophile tolerance.


Assuntos
Aldeído Liases/metabolismo , Proteínas de Bactérias/metabolismo , Aldeído Liases/genética , Aldeídos/química , Aldeídos/metabolismo , Arthrobacter/enzimologia , Proteínas de Bactérias/genética , Biocatálise , Biodiversidade , Escherichia coli/enzimologia , Gliceraldeído 3-Fosfato/metabolismo , Especificidade por Substrato
7.
Vet Microbiol ; 233: 52-60, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176413

RESUMO

The spread of extended-spectrum ß-lactamases (ESBLs) in Escherichia coli is a major public health issue and ESBL-producing bacteria are frequently reported in livestock. For the assessment of the role of the foodborne transmission pathway in Germany, detailed data on the prevalence and characteristics of isolates of food origin are necessary. The objective of this study was to describe the prevalence of cefotaxime resistant E. coli as well as ESBL/pAmpC-producing E. coli and their characteristics in foods in Germany. Out of 2256 food samples, the highest prevalence of cefotaxime resistant E. coli was observed in chicken meat (74.9%), followed by turkey meat (40.1%). Prevalence in beef, pork and minced meat was considerably lower (4.2-15.3%). Whereas 18.0% of the raw milk samples, collected at farm level were positive, this was true only for few cheese samples (1.3%). In one out of 399 vegetable samples a cefotaxime-resistant E. coli was isolated. ESBL resistance genes of the CTX-M-group (10.1% of all samples) were most frequently detected, followed by genes of the pAmpC (2.6%), SHV (2.0%) and TEM (0.8%) families. Distribution of ESBL/AmpC-encoding E. coli resistance genes and E. coli phylogroups was significantly different between the chicken related food samples and all other food items. Our study results reflect that consumers might get exposed to ESBL/pAmpC-producing E. coli through several food chains. These results together with those collected at primary production and in the human population in other studies will allow more detailed analysis of the foodborne pathways, considering transmission from livestock populations to food at retail and to consumers in Germany.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Microbiologia de Alimentos , Carne/microbiologia , beta-Lactamases/genética , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Cefotaxima/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/enzimologia , Infecções por Escherichia coli/transmissão , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Alemanha , Gado/microbiologia , Aves Domésticas/microbiologia , Prevalência , Carne Vermelha/microbiologia , Verduras/microbiologia , beta-Lactamases/biossíntese
8.
Vet Microbiol ; 233: 61-67, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176414

RESUMO

The incidence of infections with extended spectrum ß-lactamase producing Escherichia coli (ESBL-E) is increasing both in humans and animals. There is a paucity of data about the rate of faecal carriage of ESBL-E in pets. In this study, faecal swabs collected from 586 pets (225 cats; 361 dogs) in Auckland, New Zealand, were analysed for the presence of ESBL-E by culture, and a questionnaire was delivered to the owners. The ESBL-E were characterised and data elicited by the questionnaires were used for a multivariable analysis, to investigate the factors associated with faecal ESBL-E carriage. The prevalence of ESBL-E in faecal swabs was 6.4%. The ß-lactamase genes detected in the ESBL-E were the blaCTX-M-14 (n = 2) and blaCMY-2 (n = 34). Several isolates displayed multilocus sequence types (ST) associated with human and animal infections. Multiple isolates sharing the same ST displayed different antibiograms and ß-lactamase genes, reflecting horizontal gene transfer between and within ST. Variables independently associated with increased odds of ESBL-E carriage were: animal received systemic antimicrobial treatment in the six months before the sampling; presence of household members working in veterinary clinics; presence of household members travelling overseas in the six months before the sampling. We conclude that pets are colonised by ESBL-E which are genotypically similar to the bacteria found to infect humans and animals. The statistical analysis suggested a number of eco-epidemiological factors associated with ESBL-E carriage. In particular, they suggest veterinary clinics may represent hot-spots of antimicrobial resistance.


Assuntos
Portador Sadio/veterinária , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/veterinária , Escherichia coli/genética , Animais de Estimação/microbiologia , beta-Lactamases/genética , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Portador Sadio/epidemiologia , Gatos/microbiologia , Cães/microbiologia , Escherichia coli/enzimologia , Infecções por Escherichia coli/epidemiologia , Fezes/microbiologia , Feminino , Transferência Genética Horizontal , Genética Populacional , Genótipo , Hospitais Veterinários , Humanos , Masculino , Tipagem de Sequências Multilocus , Nova Zelândia/epidemiologia , Prevalência , beta-Lactamases/biossíntese
9.
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
10.
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
11.
Future Microbiol ; 14: 691-704, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31148474

RESUMO

Aim: To determine the prevalence of New Delhi metallo-ß-lactamase (NDM)-producing Gram-negative pathogens isolated from children's samples. Materials & methods: Carbapenem-resistant clinical isolates (n = 117) were confirmed by VITEK® 2 compact system, matrix-assisted laser desorption ionization-time of flight and multilocus sequence typing. MIC (µg/ml) of various antibiotics was determined by VITEK 2 compact system. Molecular characterization of the isolates was performed by PCR, DNA sequencing, PFGE and DNA hybridization. Results: Out of 117 carbapenemase producers, 37 (31.6%) and 29 (24.7%) were Klebsiella pneumoniae and Acinetobacter baumannii, respectively. 72 (61.5%) isolates were NDM positive and among these 60, 9 and 3 were NDM-1, -5 and -7, respectively. Majority of the NDM-producing K. pneumoniae belonged to ST11 and ST273 while most of the Escherichia coli belonged to ST405 and ST101. blaNDM were mainly located on 150kb plasmids. MIC displayed high resistance against ß-lactams drugs including carbapenems, and the most sensitive drugs were tigecycline and colistin. Conclusion: Dissemination of blaNDM-producing pathogens, particularly in children clinical settings, is a matter of great public health concern.


Assuntos
Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/genética , beta-Lactamases/biossíntese , beta-Lactamases/genética , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Criança , DNA Bacteriano/análise , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Feminino , Perfilação da Expressão Gênica , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/epidemiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Paquistão/epidemiologia , Plasmídeos , Análise de Sequência de DNA
12.
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
13.
Ultrasonics ; 98: 72-81, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31207474

RESUMO

The ultrasonication-mediated cell disruption of recombinant E. coli was modeled using three machine learning techniques namely Multiple linear regression (MLR), Multi-layer perceptron (MLP) and Sequential minimal optimization (SMO). The four attributes were cellmass concentration (g/L), acoustic power (A), duty cycle (%) and treatment time of sonication (min). For the three responses (nitrilase, total protein release and cell disruption) MLP model was found to be at par with RSM model in terms of generalization as well as prediction capability. Nitrilase release was significantly influenced by the cellmass concentration so was in case of total protein release. Fraction of cells disrupted was heavily influenced by acoustic power and sonication time. Almost 32 U/mL nitrilase could be released for 300 g/L cellmass concentration when sonicated at 225 W for 1 min with 20% duty cycle.


Assuntos
Aminoidrolases/metabolismo , Escherichia coli/enzimologia , Aprendizado de Máquina , Sonicação , Redes Neurais (Computação) , Fatores de Tempo
14.
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
15.
Subcell Biochem ; 92: 337-366, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31214992

RESUMO

The inner membrane of Gram-negative bacteria is a ~6 nm thick phospholipid bilayer. It forms a semi-permeable barrier between the cytoplasm and periplasm allowing only regulated export and import of ions, sugar polymers, DNA and proteins. Inner membrane proteins, embedded via hydrophobic transmembrane α-helices, play an essential role in this regulated trafficking: they mediate insertion into the membrane (insertases) or complete crossing of the membrane (translocases) or both. The Gram-negative inner membrane is equipped with a variety of different insertases and translocases. Many of them are specialized, taking care of the export of only a few protein substrates, while others have more general roles. Here, we focus on the three general export/insertion pathways, the secretory (Sec) pathway, YidC and the twin-arginine translocation (TAT) pathway, focusing closely on the Escherichia coli (E. coli) paradigm. We only briefly mention dedicated export pathways found in different Gram-negative bacteria. The Sec system deals with the majority of exported proteins and functions both as a translocase for secretory proteins and an insertase for membrane proteins. The insertase YidC assists the Sec system or operates independently on membrane protein clients. Sec and YidC, in common with most export pathways, require their protein clients to be in soluble non-folded states to fit through the translocation channels and grooves. The TAT pathway is an exception, as it translocates folded proteins, some loaded with prosthetic groups.


Assuntos
Membrana Celular/enzimologia , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras/metabolismo , Canais de Translocação SEC/metabolismo , Sistema de Translocação de Argininas Geminadas/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Transporte Proteico
16.
J Chem Phys ; 150(19): 195101, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31117770

RESUMO

In chemoradiation therapy, the synergy between the radiation and the chemotherapeutic agent (CA) can result in a super-additive treatment. A priori, this increased effectiveness could be estimated from model calculations, if absolute cross sections (ACSs) involved in cellular damage are substantially higher, when the CA binds to DNA. We measure ACSs for damages induced by 10 eV electrons, when DNA binds to the CA cisplatin as in chemotherapy. At this energy, DNA is damaged essentially by the decay of core-excited transient anions into bond-breaking channels. Films of cisplatin-DNA complexes of ratio 5:1 with thicknesses 10, 15, and 20 nm were irradiated in vacuum during 5-30 s. Conformation changes were quantified by electrophoresis and yields extrapolated from exposure-response curves. Base damages (BDs) were revealed and quantified by enzymatic treatment. The ACSs were generated from these yields by two mathematical models. For 3197 base-pair plasmid DNA, ACS for single strand breaks, double strand breaks (DSBs), crosslinks, non-DSB cluster damages, and total BDs is 71 ± 2, 9.3 ± 0.4, 10.1 ± 0.3, 8.2 ± 0.3, and 115 ± 2 ×10-15 cm2, respectively. These ACSs are higher than those of nonmodified DNA by factors of 1.6 ± 0.1, 2.2 ± 0.1, 1.3 ± 0.1, 1.3 ± 0.3, and 2.1 ± 0.4, respectively. Since LEEs are produced in large quantities by radiolysis and strongly interact with biomolecules, we expect such enhancements to produce substantial additional damages in the DNA of the nucleus of cancer cells during concomitant chemoradiation therapy. The increase damage appears sufficiently large to justify more elaborate simulations, which could provide a quantitative evaluation of molecular sensitization by Pt-CAs.


Assuntos
Cisplatino/efeitos da radiação , Complexos de Coordenação/efeitos da radiação , Dano ao DNA , DNA/efeitos da radiação , Elétrons , DNA/química , DNA-Formamidopirimidina Glicosilase/química , Desoxirribonuclease (Dímero de Pirimidina)/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Plasmídeos
17.
J Enzyme Inhib Med Chem ; 34(1): 1010-1017, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31072165

RESUMO

The Mur ligases form a series of consecutive enzymes that participate in the intracellular steps of bacterial peptidoglycan biosynthesis. They therefore represent interesting targets for antibacterial drug discovery. MurC, D, E and F are all ATP-dependent ligases. Accordingly, with the aim being to find multiple inhibitors of these enzymes, we screened a collection of ATP-competitive kinase inhibitors, on Escherichia coli MurC, D and F, and identified five promising scaffolds that inhibited at least two of these ligases. Compounds 1, 2, 4 and 5 are multiple inhibitors of the whole MurC to MurF cascade that act in the micromolar range (IC50, 32-368 µM). NMR-assisted binding studies and steady-state kinetics studies performed on aza-stilbene derivative 1 showed, surprisingly, that it acts as a competitive inhibitor of MurD activity towards D-glutamic acid, and additionally, that its binding to the D-glutamic acid binding site is independent of the enzyme closure promoted by ATP.


Assuntos
Trifosfato de Adenosina/antagonistas & inibidores , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Ligases/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Cinética , Ligases/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade
18.
Carbohydr Res ; 478: 10-17, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31039450

RESUMO

A series of novel tricyclic quinazolinone-iminosugars 1 (a-c) were synthesized from the benzyl protected sugars through three steps. Firstly, the benzyl protected sugar (aldehyde) 5 reacted with o-aminobenzamide by the iodine-induced oxidative condensation to afford the corresponding aldo-quizanolinone 6. Secondly, through the intramolecular cyclization of the unprotected OH and the amide NH in 6, the tricyclic compounds 7 and 8 were constructed by the key Mitsunobu reaction. Finally, removal of the benzyl group gave the target tricyclic quinazolinone-iminosugars 1. The protocol was effective for the preparation of the tricyclic iminosugars in satisfactory yield. Interestingly, an unusual C-2 epimerization was observed with d-mannose and d-ribose compounds under the conditions of the Mitsunobu reaction that generated the products having the trans configuration at the C-2 and C-3 positions. Unfortunately, such tricyclic quinazolinone-iminosugars showed no inhibitory effects on the tested five glycosidases.


Assuntos
Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Imino Açúcares/farmacologia , Quinazolinonas/farmacologia , Aspergillus niger/enzimologia , Canavalia/enzimologia , Configuração de Carboidratos , Café/enzimologia , Ciclização , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Glicosídeo Hidrolases/metabolismo , Imino Açúcares/síntese química , Imino Açúcares/química , Prunus dulcis/enzimologia , Quinazolinonas/síntese química , Quinazolinonas/química
19.
J Agric Food Chem ; 67(22): 6285-6291, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31117501

RESUMO

N-Acetyl-d-neuraminic acid (Neu5Ac) is a potential baby nutrient and the key precursor of antiflu medicine Zanamivir. The Neu5Ac chemoenzymatic synthesis consists of N-acetyl-d-glucosamine epimerase (AGE)-catalyzed epimerization of N-acetyl-d-glucosamine (GlcNAc) to N-acetyl-d-mannosamine (ManNAc) and aldolase-catalyzed condensation between ManNAc and pyruvate. Herein, we cloned and characterized BT0453, a novel AGE, from a human gut symbiont Bacteroides thetaiotaomicron. BT0453 shows the highest soluble fraction among the AGEs tested. With GlcNAc and sodium pyruvate as substrates, Neu5Ac production by coupling whole cells expressing BT0453 and Escherichia coli N-acetyl-d-neuraminic acid aldolase was explored. After 36 h, a 53.6% molar yield, 3.6 g L-1 h-1 productivity and 42.9 mM titer of Neu5Ac were obtained. Furthermore, for the first time, the T7- BT0453-T7- nanA polycistronic unit was integrated into the E. coli genome, generating a chromosome-based biotransformation system. BT0453 protein engineering and metabolic engineering studies hold potential for the industrial production of Neu5Ac.


Assuntos
Aldeído Liases/genética , Proteínas de Bactérias/genética , Bacteroides thetaiotaomicron/enzimologia , Carboidratos Epimerases/genética , Proteínas de Transporte/genética , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Ácido N-Acetilneuramínico/biossíntese , Aldeído Liases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Carboidratos Epimerases/química , Carboidratos Epimerases/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Cinética , Engenharia Metabólica
20.
Vet Microbiol ; 231: 100-106, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30955795

RESUMO

Extended-spectrum beta-lactamase (ESBL) and plasmidic AmpC (pAmpC) producing Escherichia coli are found in the poultry production even without antibiotic use. The spread of these bacteria has been suggested to occur via imported parent birds, enabling transmission to production level broilers vertically via eggs. We studied transmission of ESBL/pAmpC-producing E. coli and E. coli without antibiotic selection by sampling imported parent birds (n = 450), egg surfaces prior to and after the incubation period (n = 300 and n = 428, respectively) and the laying house environment (n = 20). Samples were additionally taken from embryos (n = 422). To study the prevention of transmission, a competitive exclusion (CE) solution was added onto freshly laid eggs prior to incubation period (n = 150). Results showed carriage of ESBL/pAmpC-producing E. coli in parent birds (26.7%), the environment (5%) and egg surfaces before the incubation period (1.3%), but not from egg surfaces or embryos after the incubation period. Whole genome sequencing revealed ESBL/pAmpC-producing E. coli isolates belonging to clonal lineages ST429 and ST2040. However, the finding of E. coli cultured without antibiotic selection in two (2.2%) embryos strengthens the need to study E. coli transmission in poultry production in more depth. Since ESBL/pAmpC-producing E. coli seem not to persist on egg surfaces, there is no need to use CE solution ex ovo as a prevention method. The results indicate that other routes, such as for example transmission through fomites or horizontal gene transfer by other bacterial species, could be more important than vertical transmission in the spread of resistance in broiler production.


Assuntos
Galinhas/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli/isolamento & purificação , Transmissão Vertical de Doença Infecciosa/veterinária , Doenças das Aves Domésticas/transmissão , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Cloaca/microbiologia , Escherichia coli/enzimologia , Infecções por Escherichia coli/transmissão , Genoma Bacteriano , Óvulo/microbiologia , Aves Domésticas/microbiologia , Doenças das Aves Domésticas/microbiologia , Sequenciamento Completo do Genoma , beta-Lactamases/biossíntese
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