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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
Environ Sci Pollut Res Int ; 26(22): 22305-22311, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31154643

RESUMO

The dissemination of extended spectrum beta-lactamases (ESBL) genes through gene transfer attracts wide attention. Bentonite is widely used as a feed additive in an animal-breeding environment. In order to obtain a better understanding of the effect of bentonite on Escherichia coli carrying ESBL gene, proteomic analysis was carried out to screen the key proteins. The results showed that a total of 31 proteins were differentially expressed, including 21 up-regulated proteins and 10 down-regulated proteins. These proteins were involved in biosynthetic process, metabolic process, stress response, transport, anaerobic respiration, proteolysis, hydrolase, protein folding, transcription, salvage, and other. The transcriptional level of four genes (mipA, gntY, tldD, and arcA) was in consensus with proteomic results. This study revealed the differentially expressed proteins involved when E. coli was incubated under bentonite and PBS condition, which implied the possibility that bentonite may promote the transfer of ESBL gene between E. coli.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , beta-Lactamases/genética , Animais , Bentonita/química , Bentonita/farmacologia , Escherichia coli/química , Infecções por Escherichia coli , Proteínas de Escherichia coli/química , Humanos , Proteômica , beta-Lactamases/análise
9.
Microb Cell Fact ; 18(1): 98, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31151455

RESUMO

BACKGROUND: Farnesol is an acyclic sesquiterpene alcohol present in the essential oils of various plants in nature. It has been reported to be valuable in medical applications, such as alleviation of allergic asthma, gliosis, and edema as well as anti-cancerous and anti-inflammatory effects. Coenzyme Q10 (CoQ10), an essential cofactor in the aerobic respiratory electron transport chain, has attracted growing interest owing to its clinical benefits and important applications in the pharmaceutical, food, and health industries. In this work, co-production of (E,E)-farnesol (FOH) and CoQ10 was achieved by combining 3 different exogenous terpenes or sesquiterpene synthase with the RNA interference of psy (responsible for phytoene synthesis in Rhodobacter sphaeroides GY-2). RESULTS: FOH production was significantly increased by overexpressing exogenous terpene synthase (TPS), phosphatidylglycerophosphatase B (PgpB), and sesquiterpene synthase (ATPS), as well as RNAi-mediated silencing of psy coding phytoene synthase (PSY) in R. sphaeroides strains. Rs-TPS, Rs-ATPS, and Rs-PgpB respectively produced 68.2%, 43.4%, and 21.9% higher FOH titers than that of the control strain. Interestingly, the CoQ10 production of these 3 recombinant R. sphaeroides strains was exactly opposite to that of FOH. However, CoQ10 production was almost unaffected in R. sphaeroides strains modified by psy RNA interference. The highest FOH production of 40.45 mg/L, which was twice as high as that of the control, was obtained from the TPS-PSYi strain, where the exogenous TPS was combined with the weakening of the phytoene synthesis pathway via psy RNA interference. CoQ10 production in TPS-PSYi, ATPS-PSYi, and PgpB-PSYi was decreased and lower than that of the control strain. CONCLUSIONS: The original flux that contributed to phytoene synthesis was effectively redirected to provide precursors toward FOH or CoQ10 synthesis via psy RNA interference, which led to weakened carotenoid synthesis. The improved flux that was originally involved in CoQ10 production and phytoene synthesis was redirected toward FOH synthesis via metabolic modification. This is the first reported instance of FOH and CoQ10 co-production in R. sphaeroides using a metabolic engineering strategy.


Assuntos
Carotenoides/metabolismo , Farneseno Álcool/metabolismo , Engenharia Metabólica/métodos , Rhodobacter sphaeroides/metabolismo , Ubiquinona/análogos & derivados , Alquil e Aril Transferases/genética , Vias Biossintéticas , Escherichia coli , Proteínas de Escherichia coli/genética , Geranil-Geranildifosfato Geranil-Geraniltransferase/genética , Fosfatidato Fosfatase/genética , Interferência de RNA , Rhodobacter sphaeroides/enzimologia , Rhodobacter sphaeroides/genética , Ubiquinona/biossíntese , Ubiquinona/metabolismo
10.
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
11.
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
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.
Braz J Infect Dis ; 23(3): 203-206, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31228460

RESUMO

Global dissemination of mcr-like genes represents a serious threat to public health since it jeopardizes the effectiveness of colistin, an antibiotic used as a last-resort treatment against highly antibiotic-resistant bacteria. In 2017, a mcr-1-positive isolate of Escherichia coli was found in Chile for the first time. Herein we report the genetic features of this strain (UCO-457) by whole-genome sequencing (WGS) and conjugation experiments. The UCO-457 strain belonged to ST4204 and carried a 285 kb IncI2-type plasmid containing the mcr-1 gene. Moreover, this plasmid was transferred by conjugation to an E. coli J53 strain at high frequency. The isolate harbored the cma, iroN, and iss virulence genes and did carry resistance genes to trimethoprim/sulfamethoxazole and fluoroquinolones. Other antibiotic resistance determinants such as ß-lactamases-encoding genes were not detected, making the isolate highly susceptible to these antibiotics. Our results revealed that such susceptible isolates could be acting as platforms to disseminate plasmid-mediated colistin resistance. Based on this evidence, we consider that mcr-like prevalence deserves urgent attention and should be examined not only in highly resistant bacteria but also in susceptible isolates.


Assuntos
Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Antibacterianos/farmacologia , Chile , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Escherichia coli/efeitos dos fármacos , Humanos , Pacientes Ambulatoriais
14.
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
15.
Nat Commun ; 10(1): 1995, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040281

RESUMO

Uropathogenic E. coli experience a wide range of osmolarity conditions before and after successful infection. Stress-responsive regulatory proteins in bacteria, particularly proteins of the Hha family and H-NS, a transcription repressor, sense such osmolarity changes and regulate transcription through unknown mechanisms. Here we use an array of experimental probes complemented by molecular simulations to show that Cnu, a member of the Hha protein family, acts as an exquisite molecular sensor of solvent ionic strength. The osmosensory behavior of Cnu involves a fine-tuned modulation of disorder in the fourth helix and the three-dimensional structure in a graded manner. Order-disorder transitions in H-NS act synergistically with molecular swelling of Cnu contributing to a salt-driven switch in binding cooperativity. Thus, sensitivity to ambient conditions can be imprinted at the molecular level by tuning not just the degree of order in the protein conformational ensemble but also through population redistributions of higher-order molecular complexes.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Ligação Proteica , Conformação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
16.
Microb Cell Fact ; 18(1): 80, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064376

RESUMO

BACKGROUND: Bacteria are widely used as hosts for recombinant protein production due to their rapid growth, simple media requirement and ability to produce high yields of correctly folded proteins. Overproduction of recombinant proteins may impose metabolic burden to host cells, triggering various stress responses, and the ability of the cells to cope with such stresses is an important factor affecting both cell growth and product yield. RESULTS: Here, we present a versatile plasmid-based reporter system for efficient analysis of metabolic responses associated with availability of cellular resources utilized for recombinant protein production and host capacity to synthesize correctly folded proteins. The reporter plasmid is based on the broad-host range RK2 minimal replicon and harbors the strong and inducible XylS/Pm regulator/promoter system, the ppGpp-regulated ribosomal protein promoter PrpsJ, and the σ32-dependent synthetic tandem promoter Pibpfxs, each controlling expression of one distinguishable fluorescent protein. We characterized the responsiveness of all three reporters in Escherichia coli by quantitative fluorescence measurements in cell cultures cultivated under different growth and stress conditions. We also validated the broad-host range application potential of the reporter plasmid by using Pseudomonas putida and Azotobacter vinelandii as hosts. CONCLUSIONS: The plasmid-based reporter system can be used for analysis of the total inducible recombinant protein production, the translational capacity measured as transcription level of ribosomal protein genes and the heat shock-like response revealing aberrant protein folding in all studied Gram-negative bacterial strains.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Reporter/genética , Plasmídeos/genética , Proteínas Recombinantes/biossíntese , Clonagem Molecular
17.
mSphere ; 4(3)2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068437

RESUMO

Whole-genome sequencing of trimethoprim-resistant Escherichia coli strains MF2165 and PF9285 from healthy Swiss fattening calves revealed a so far uncharacterized dihydrofolate reductase gene, dfrA35 Functionality and association with trimethoprim resistance were demonstrated by cloning and expressing dfrA35 in E. coli The DfrA35 protein showed the closest amino acid identity (49.4%) to DfrA20 from Pasteurella multocida and to the Dfr determinants DfrG (41.2%), DfrD (40.8%), and DfrK (40.0%) found in Gram-positive bacteria. The dfrA35 gene was integrated within a florfenicol/chloramphenicol-sulfonamide resistance ISCR2 element (floR-ISCR2-dfrA35-sul2) next to a Tn21-like transposon that contained genes with resistance to sulfonamides (sul1), streptomycin (aadA1), gentamicin/tobramycin/kanamycin (aadB), and quaternary ammonium compounds (qacEΔ1). A search of GenBank databases revealed that dfrA35 was present in 26 other E. coli strains from different origins as well as in Acinetobacter IMPORTANCE The presence of dfrA35 associated with ISCR2 in Escherichia coli from animals, as well as its presence in other E. coli strains from different sources and countries and in Acinetobacter, highlights the global spread of this gene and its potential for further dissemination. The genetic link of ISCR2-dfrA35 with other antibiotic and disinfectant resistance genes showed that multidrug-resistant E. coli may be selected and maintained by the use of either one of several antimicrobials.


Assuntos
Antibacterianos/farmacologia , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/genética , Resistência a Trimetoprima/genética , Trimetoprima/farmacologia , Fatores Etários , Animais , Bovinos , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia
18.
Nat Commun ; 10(1): 2142, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086174

RESUMO

Metabolic engineers endeavor to create a bio-based manufacturing industry using microbes to produce fuels, chemicals, and medicines. Plant natural products (PNPs) are historically challenging to produce and are ubiquitous in medicines, flavors, and fragrances. Engineering PNP pathways into new hosts requires finding or modifying a suitable host to accommodate the pathway, planning and implementing a biosynthetic route to the compound, and discovering or engineering enzymes for missing steps. In this review, we describe recent developments in metabolic engineering at the level of host, pathway, and enzyme, and discuss how the field is approaching ever more complex biosynthetic opportunities.


Assuntos
Produtos Biológicos/metabolismo , Engenharia Metabólica/métodos , Microrganismos Geneticamente Modificados/metabolismo , Plantas/metabolismo , Vias Biossintéticas/genética , Enzimas/genética , Enzimas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica/tendências , Microrganismos Geneticamente Modificados/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos , Biologia Sintética/tendências
19.
Sci Total Environ ; 659: 540-547, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31096383

RESUMO

Mercury is a potentially toxic trace metal that poses threats to aquatic life and to humans. In this study, a mercury-binding peptide was displayed on the surface of Escherichia coli cells using an N-terminal region ice nucleation protein anchor. The surface-engineered E. coli facilitated selective adsorption of mercury ions (Hg2+) from a solution containing various metal ions. The Hg2+ adsorption capacity of the surface-engineered cell was four-fold higher than that of the original E. coli cells. Approximately 95% of Hg2+ was removed from solution by these whole-cell sorbents. The transformed strains were fed to Carassius auratus, so that the bacteria could colonize fish intestine. Engineered bacteria-fed C. auratus showed significantly less (51.1%) accumulation of total mercury when compared with the group that had not been fed engineered bacteria. The surface-engineered E. coli effectively protected fish against the toxicity of Hg2+ in aquatic environments by adsorbing more Hg2+. Furthermore, the surface-engineered E. coli mitigated microbial diversity changes in the intestine caused by Hg2+ exposure, thereby protecting the intestinal microbial community. This strategy is a novel approach for controlling Hg2+ contamination in fish.


Assuntos
Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Carpa Dourada/metabolismo , Mercúrio/metabolismo , Peptídeos/metabolismo , Poluentes Químicos da Água/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Engenharia Genética , Mucosa Intestinal/metabolismo , Microrganismos Geneticamente Modificados/genética , Peptídeos/genética
20.
Sci Total Environ ; 683: 308-316, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31132710

RESUMO

Multidrug resistance associated with pigs not only affects pig production but also threatens human health by influencing the farm surrounding and contaminating the food chain. This paper focused on the occurrence and prevalence of high-risk resistance genes (using blaNDM and mcr-1 as marker genes) in two Chinese swine farms, and investigated their fate and seasonal changes in piggery wastewater treatment systems (PWWTSs). Results revealed that blaNDM and mcr-1 were prevalent in both confined swine farms, and even prevailed through various processing stages of PWWTSs. Moreover, the abundance of blaNDM and mcr-1 in winter was higher than that in summer, with 0.01-1.01 logs variation in piggery wastewater. Of concern is that considerable amounts of blaNDM and mcr-1 were present in final effluent that is applied to farmland (up to 102-104copies/mL), raising the risk of propagation to indigenous bacteria. Worse still, those pig-derived isolates harboring the blaNDM/mcr-1 gene were confirmed to spread multidrug resistance to other bacteria, which further increased their dissemination potential in agricultural environment. This study highlights the prevalence of blaNDM and mcr-1 in swine farms, meanwhile, also emphasizes the necessary to mitigate the release and propagation of these high-risk genes from swine farms following land fertilization and wastewater usage.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Genes Bacterianos , Águas Residuárias/microbiologia , Animais , Escherichia coli , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/genética , Humanos , Testes de Sensibilidade Microbiana , Suínos , Eliminação de Resíduos Líquidos , Microbiologia da Água , beta-Lactamases/análise , beta-Lactamases/genética
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