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1.
Antonie Van Leeuwenhoek ; 113(1): 13-20, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31587117

RESUMO

A Gram-stain negative, rod-shaped bacterial, catalase and oxidase positive strain (83-4T) that formed yellow colonies was isolated from human Meibomian gland secretions. Strain 83-4T belongs to the genus Lysobacter according to phylogenetic analysis based on 16S rRNA gene sequences. The DNA G+C content was 67.1 mol%. The circular genome was 2.6 Mb, which contained 2431 protein-coding sequences, 75 pseudogenes, 46 tRNAs, 3 rRNAs and 4 ncRNAs. A bacteriocin cluster and aryl polyene cluster were also found in the genome. The average nucleotide identity value was 79.6% between isolate 83-4T and the closely related type strain Lysobacter tolerans UM1T. The estimated DNA-DNA hybridization value between strain 83-4T and L. tolerans UM1T was 41.6%. Diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol were the major polar lipids. Iso-C15:0, iso-C11:0 3-OH, iso-C11:0 and summed feature 9 (iso-C17:1ω9c) were the major fatty acids. Ubiquinone (Q-8) was the only respiratory quinone. Therefore, based on the data of phylogenetic analysis, chemotaxonomical and biochemical analyses, it is concluded that strain 83-4T represents a novel species of the genus Lysobacter with the name of Lysobacter oculi sp. nov. The type strain is 83-4T (= CGMCC 1.13464T = NRBC 113451T).

2.
Biotechnol Biofuels ; 12: 180, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31338122

RESUMO

Background: DNA assembly is an essential technique enabling metabolic engineering and synthetic biology. Combining novel DNA assembly technologies with rational metabolic engineering can facilitate the construction of microbial cell factories. Amino acids and derived biochemicals are important products in industrial biotechnology with wide application and huge markets. DNA assembly scenarios encountered in metabolic engineering for the construction of amino acid and related compound producers, such as design-build-test-learn cycles, construction of precise genetic circuits and repetitive DNA molecules, usually require for iterative, scarless and repetitive sequence assembly methods, respectively. Results: Restriction endonuclease (RE)-assisted strategies constitute one of the major categories of DNA assembly. Here, we developed a Type IIP and IIS RE-assisted method named PS-Brick that comprehensively takes advantage of the properties of PCR fragments and REs for iterative, seamless and repetitive sequence assembly. One round of PS-Brick reaction using purified plasmids and PCR fragments was accomplished within several hours, and transformation of the resultant reaction product from this PS-Brick assembly reaction exhibited high efficiency (104-105 CFUs/µg DNA) and high accuracy (~ 90%). An application of metabolic engineering to threonine production, including the release of feedback regulation, elimination of metabolic bottlenecks, intensification of threonine export and inactivation of threonine catabolism, was stepwise resolved in E. coli by rounds of "design-build-test-learn" cycles through the iterative PS-Brick paradigm, and 45.71 g/L threonine was obtained through fed-batch fermentation. In addition to the value of the iterative character of PS-Brick for sequential strain engineering, seamless cloning enabled precise in-frame fusion for codon saturation mutagenesis and bicistronic design, and the repetitive sequence cloning ability of PS-Brick enabled construction of tandem CRISPR sgRNA arrays for genome editing. Moreover, the heterologous pathway deriving 1-propanol pathway from threonine, composed of Lactococcus lactis kivD and Saccharomyces cerevisiae ADH2, was assembled by one cycle of PS-Brick, resulting in 1.35 g/L 1-propanol in fed-batch fermentation. Conclusions: To the best of our knowledge, the PS-Brick framework is the first RE-assisted DNA assembly method using the strengths of both Type IIP and IIS REs. In this study, PS-Brick was demonstrated to be an efficient DNA assembly method for pathway construction and genome editing and was successfully applied in design-build-test-learn (DBTL) cycles of metabolic engineering for the production of threonine and threonine-derived 1-propanol. The PS-Brick presents a valuable addition to the current toolbox of synthetic biology and metabolic engineering.

3.
J Struct Biol ; 206(3): 322-334, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30946901

RESUMO

3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) is responsible for the biosynthesis of essential aromatic compounds in microorganisms and plants. It plays a crucial role in the regulation of the carbon flow into the shikimate pathway. Until now, the crystal structures and regulatory mechanisms of dimeric DAHPS enzymes from type Iα subclass have not been reported. Here, we reported dimeric structures of the tyrosine-regulated DAHPS from Escherichia coli, both in its apo form and complex with the inhibitor tyrosine at 2.5 and 2.0 Šresolutions, respectively. DAHPS(Tyr) has a typical (ß/α)8 TIM barrel, which is decorated with an N-terminal extension and an antiparallel ß sheet, ß6a/ß6b. Inhibitor tyrosine binds at a cavity formed by residues of helices α3, α4, strands ß6a, ß6b and the adjacent loops, and directly interacts with residues P148, Q152, S181, I213 and N8*. Although the small angle X-ray scattering profiles from DAHPS(Tyr) with and without tyrosine shows that tyrosine binding leaves most of DAHPS(Tyr) structures unaffected. The comparison of the liganded and unliganded crystal structures reveals that conformational changes of residues P148, Q152 and I213 initiate a transmission pathway to propagate the allosteric signal from the tyrosine-binding site to the active site, which is different from DAHPS(Phe), a phenylalanine-regulated isozyme from E. coli. In addition, mutations of five tyrosine-binding residues P148, Q152, S181, I213 and N8* leads to tyrosine-resistant DAHPS(Tyr) enzymes. These findings provide a new insight into the regulatory mechanism of DAHPS enzymes and a basis for further engineering studies.

4.
Biotechnol Biofuels ; 11: 277, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30337956

RESUMO

Background: The thermotolerant methylotrophic yeast Ogataea polymorpha has been regarded as an important organism for basic research and biotechnological applications. It is generally recognized as an efficient and safe cell factory in fermentative productions of chemicals, biofuels and other bio-products. However, it is difficult to genetically engineer for the deficiency of an efficient and versatile genome editing technology. Results: In this study, we developed a CRISPR-Cas9-assisted multiplex genome editing (CMGE) approach including multiplex genes knock-outs, multi-locus (ML) and multi-copy (MC) integration methods in yeasts. Based on CMGE, various genome modifications, including gene deletion, integration, and precise point mutation, were performed in O. polymorpha. Using the CMGE-ML integration method, three genes TAL from Herpetosiphon aurantiacus, 4CL from Arabidopsis thaliana and STS from Vitis vinifera of resveratrol biosynthetic pathway were simultaneously integrated at three different loci, firstly achieving the biosynthesis of resveratrol in O. polymorpha. Using the CMGE-MC method, ∼ 10 copies of the fusion expression cassette P ScTEF1 -TAL-P ScTPI1 -4CL-P ScTEF2 -STS were integrated into the genome. Resveratrol production was increased ~ 20 fold compared to the one copy integrant and reached 97.23 ± 4.84 mg/L. Moreover, the biosynthesis of human serum albumin and cadaverine were achieved in O. polymorpha using CMGE-MC to integrate genes HSA and cadA, respectively. In addition, the CMGE-MC method was successfully developed in Saccharomyces cerevisiae. Conclusions: An efficient and versatile multiplex genome editing method was developed in yeasts. The method would provide an efficient toolkit for genetic engineering and synthetic biology researches of O. polymorpha and other yeast species.

5.
Infect Drug Resist ; 11: 1729-1740, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30349330

RESUMO

Objective: To explore the composition of the ocular microbiome in normal subjects and patients with Meibomian gland dysfunction (MGD). Subjects and methods: Seventy subjects (140 eyes) were enrolled in our study. Signs of dry eye were evaluated and bacterial species in the conjunctival sac (CS) and Meibomian gland (MG) secretions were then identified by 16S rRNA gene sequencing. Additionally, 17 subjects (34 eyes) were further evaluated to determine differences in the microbiomes in the surface and deep layers of MG using a segmental secretion analysis. Results: The positive bacterial isolation rate was markedly higher in MG secretions than in the CS. The bacterial composition of the control and mild group was simple, whereas the composition of bacteria was more complex as the severity of MGD increased. The positive bacterial isolation rate and number of bacterial types were significantly higher in the severe MGD group than those in the control, mild and moderate MGD groups. Corynebacterium macginleyi was only detected in the severe MGD group, with an isolation rate of up to 26.3%. Furthermore, a new grading system for bacterial severity of MGD was proposed and the severity of MGD appeared to be positively correlated with a higher grade of bacterial severity. The segmental secretion analysis showed severe MGD had a significantly higher incidence of bacterial discordance rate. Conclusion: The severity of MGD was positively correlated with a higher isolation rate, a greater number of bacterial species, and a higher grade of bacterial severity, which implied that MGD might be correlated with bacterial changes. This study provided some basis for the indications of antibiotic in clinical practice.

6.
Clin Ther ; 40(3): 440-449, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29519716

RESUMO

PURPOSE: We designed a prospective, multicenter, randomized, controlled study to assess a 5-month regimen compared with the standard regimen on previously treated patients with pulmonary tuberculosis (TB). METHODS: We enrolled 917 sputum smear-positive patients undergoing additional treatment in 27 major tuberculosis hospitals in China. Patients were randomly assigned to a test group (n = 626)treated with a 5-month regimen of moxifloxacin, pasiniazid, rifabutin, ethambutol, and pyrazinamide or a reference group (n = 291) treated with an 8-month regimen of isoniazid, rifampicin, and streptomycin. All patients with a favorable response were followed up for 5 years after the end of treatment. FINDINGS: Of the study patients, 61 in the test group and 19 in the reference group had multidrug-resistant (MDR) TB. The treatment success rate in the study group was 74.12%, which was significantly higher than the 67.70% in the reference group (P = 0.04), whereas the treatment success rate of patients with MDR-TB was not significantly different between the test and reference groups (70.5% vs 63.1%, P =0.79). The adverse effects rates in the test and reference groups were 7.4% and 3.1%, respectively (P = .01). The difference in the TB recurrence rates between the group arm (9.6%) and the reference group (21.8%) was statistically significant (P < 0.001). IMPLICATIONS: The moxifloxacin, pasiniazid, rifabutin, ethambutol, and pyrazinamide test regimen yielded higher success and lower recurrence rates than the currently recommended isoniazid, rifampicin, and streptomycin regimen, but the rate of adverse effects was higher. ClinicalTrials.gov identifier: NCT02331823.


Assuntos
Antituberculosos/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Pulmonar/tratamento farmacológico , Adulto , Antituberculosos/administração & dosagem , China , Quimioterapia Combinada , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Recidiva , Resultado do Tratamento , Adulto Jovem
7.
ACS Synth Biol ; 7(1): 98-106, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-28968490

RESUMO

Scarless genetic manipulation of genomes is an essential tool for biological research. The restriction-modification (R-M) system is a defense system in bacteria that protects against invading genomes on the basis of its ability to distinguish foreign DNA from self DNA. Here, we designed an R-M system-mediated genome editing (RMGE) technique for scarless genetic manipulation in different microorganisms. For bacteria with Type IV REase, an RMGE technique using the inducible DNA methyltransferase gene, bceSIIM (RMGE-bceSIIM), as the counter-selection cassette was developed to edit the genome of Escherichia coli. For bacteria without Type IV REase, an RMGE technique based on a restriction endonuclease (RMGE-mcrA) was established in Bacillus subtilis. These techniques were successfully used for gene deletion and replacement with nearly 100% counter-selection efficiencies, which were higher and more stable compared to conventional methods. Furthermore, precise point mutation without limiting sites was achieved in E. coli using RMGE-bceSIIM to introduce a single base mutation of A128C into the rpsL gene. In addition, the RMGE-mcrA technique was applied to delete the CAN1 gene in Saccharomyces cerevisiae DAY414 with 100% counter-selection efficiency. The effectiveness of the RMGE technique in E. coli, B. subtilis, and S. cerevisiae suggests the potential universal usefulness of this technique for microbial genome manipulation.


Assuntos
Bacillus subtilis/genética , Enzimas de Restrição-Modificação do DNA/genética , Escherichia coli/genética , Edição de Genes/métodos , Genoma Bacteriano , Sistemas de Transporte de Aminoácidos Básicos/deficiência , Sistemas de Transporte de Aminoácidos Básicos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enzimas de Restrição do DNA/genética , Enzimas de Restrição-Modificação do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Mutação Puntual , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
8.
Am J Physiol Regul Integr Comp Physiol ; 314(2): R147-R152, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29046312

RESUMO

Relaxin (RLX) is a pleiotropic peptide hormone with marked renal vasodilatory actions that are physiologically important during pregnancy. RLX also has potent antifibrotic actions and is being tested therapeutically in various fibrotic diseases, including chronic kidney disease (CKD). Since renal vasodilation may expose the glomerulus to increased blood pressure [glomerular capillary pressure (PGC)], which exacerbates progression of CKD, we assessed the glomerular hemodynamic actions of acute (0.89 µg·100 g body wt-1·h-1 iv over 75 min) and chronic (1.5 µg·100 g body wt-1·h-1 sc) administration of RLX. Both acute and chronic RLX produced marked renal vasodilation and increased renal plasma flow (RPF) in euvolemic, anesthetized male rats. Glomerular filtration rate also increased with RLX, but the magnitude of the rise was much less than the increase in RPF due to concomitant decreases in filtration fraction. The fall in filtration fraction was the result of significant decreases in PGC, despite a slight increase in mean arterial blood pressure (MAP) with acute RLX and no net change in MAP with chronic RLX. This fall in PGC occurred because of the "in-series" arrangement of the afferent and efferent arteriolar resistance vessels, which can regulate PGC independently of MAP. With both acute and chronic RLX, efferent arteriolar resistance vessels relaxed to a greater extent than afferent arteriolar resistance vessels, thus producing falls in PGC. Based on this finding, RLX has a beneficial hemodynamic impact on the kidney, which, together with the antifibrotic actions of RLX, suggests a strong therapeutic potential for use in CKD.


Assuntos
Pressão Arterial/efeitos dos fármacos , Arteríolas/efeitos dos fármacos , Glomérulos Renais/irrigação sanguínea , Relaxina/administração & dosagem , Insuficiência Renal Crônica/tratamento farmacológico , Fluxo Plasmático Renal/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/administração & dosagem , Animais , Arteríolas/fisiopatologia , Taxa de Filtração Glomerular/efeitos dos fármacos , Infusões Intravenosas , Masculino , Ratos Sprague-Dawley , Insuficiência Renal Crônica/fisiopatologia , Fatores de Tempo , Resistência Vascular/efeitos dos fármacos
9.
ACS Synth Biol ; 7(3): 822-831, 2018 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-28094982

RESUMO

Manipulating the bacterial genomes in an efficient manner is essential to biological and biotechnological research. Here, we reprogrammed the bacterial TA systems as the toxin counter-selectable cassette regulated by an antitoxin switch (TCCRAS) for genetic modifications in the extensively studied and utilized Gram-positive bacteria, B. subtilis and Corynebacterium glutamicum. In the five characterized type II TA systems, the RelBE complex can specifically and efficiently regulate cell growth and death by the conditionally controlled antitoxin RelB switch, thereby serving as a novel counter-selectable cassette to establish the TCCRAS system. Using a single vector, such a system has been employed to perform in-frame deletion, functional knock-in, gene replacement, precise point mutation, large-scale insertion, and especially, deletion of the fragments up to 194.9 kb in B. subtilis. In addition, the biosynthesis of lycopene was first achieved in B. subtilis using TCCRAS to integrate a 5.4-kb fusion cluster ( P spac- crtI- crtE- crtB). The system was further adapted for gene knockdown and replacement, and large-scale deletion of the fragments up to 179.8 kb in C. glutamicum, with the mutation efficiencies increased by 0.8-1.0-fold compared to the conventional SacB method. TCCRAS thus holds promise as an effective and versatile genome-scale engineering technology for metabolic engineering and synthetic genomics research in a broad range of the Gram-positive bacteria.


Assuntos
Antitoxinas/genética , Bacillus subtilis/genética , Toxinas Bacterianas/genética , Corynebacterium glutamicum/genética , Edição de Genes/métodos , Genoma Bacteriano , Sequência de Bases , Cromossomos Bacterianos/genética , Deleção de Genes , Genes Bacterianos , Engenharia Genética , Óperon/genética , Mutação Puntual/genética
10.
Nutrients ; 9(9)2017 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-28891983

RESUMO

Oxidative stress and inflammation are well-documented pathological factors in alcoholic liver disease (ALD). Artichoke (Cynara scolymus L.) is a healthy food and folk medicine with anti-oxidative and anti-inflammatory properties. This study aimed to evaluate the preventive effects of ethanolic extract from artichoke against acute alcohol-induced liver injury in mice. Male Institute of Cancer Research mice were treated with an ethanolic extract of artichoke (0.4, 0.8, and 1.6 g/kg body weight) by gavage once daily. Up to 40% alcohol (12 mL/kg body weight) was administered orally 1 h after artichoke treatment. All mice were fed for 10 consecutive days. Results showed that artichoke extract significantly prevented elevated levels of aspartate aminotransferase, alanine aminotransferase, triglyceride, total cholesterol, and malondialdehyde. Meanwhile, the decreased levels of superoxide dismutase and glutathione were elevated by artichoke administration. Histopathological examination showed that artichoke attenuated degeneration, inflammatory infiltration and necrosis of hepatocytes. Immunohistochemical analysis revealed that expression levels of toll-like receptor (TLR) 4 and nuclear factor-kappa B (NF-κB) in liver tissues were significantly suppressed by artichoke treatment. Results obtained demonstrated that artichoke extract exhibited significant preventive protective effect against acute alcohol-induced liver injury. This finding is mainly attributed to its ability to attenuate oxidative stress and suppress the TLR4/NF-κB inflammatory pathway. To the best of our knowledge, the underlying mechanisms of artichoke on acute ALD have been rarely reported.


Assuntos
Cynara scolymus/química , Hepatopatias Alcoólicas/tratamento farmacológico , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Doença Aguda , Alanina Transaminase/sangue , Animais , Antioxidantes/farmacologia , Aspartato Aminotransferases/sangue , Colesterol/sangue , Glutationa/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Malondialdeído/sangue , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/genética , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fitoterapia , Superóxido Dismutase/sangue , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Triglicerídeos/sangue
11.
Clin Cancer Res ; 23(21): 6673-6685, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28765327

RESUMO

Purpose: Bladder cancer is one of the most common urinary malignancies worldwide characterized by a high rate of recurrence and no targeted therapy method. Bladder cancer stem cells (BCSCs) play a crucial role in tumor initiation, metastasis, and drug resistance. However, the regulatory signaling and self-renewal mechanisms of BCSCs remain largely unknown. Here, we identified a novel signal, the KMT1A-GATA3-STAT3 circuit, which promoted the self-renewal and tumorigenicity of human BCSCs.Experimental Design: In a discovery step, human BCSCs and bladder cancer non-stem cells (BCNSCs) isolated from primary bladder cancer samples #1 and #2, and the bladder cancer cell line EJ were analyzed by transcriptome microarray. In a validation step, 10 paired bladder cancer and normal tissues, different tumor cell lines, the public microarray datasets of human bladder cancer, and The Cancer Genome Atlas database were applied for the verification of gene expression.Results: KMT1A was highly expressed and responsible for the increase of tri-methylating lysine 9 of histone H3 (H3K9me3) modification in BCSCs compared with either BCNSCs or normal bladder tissue. GATA3 bound to the -1710∼-1530 region of STAT3 promoter and repressed its transcription. H3K9me3 modification on the -1351∼-1172bp region of the GATA3 promoter mediated by KMT1A repressed the transcription of GATA3 and upregulated the expression of STAT3. In addition, the activated STAT3 triggered self-renewal of BCSCs. Furthermore, depletion of KMT1A or STAT3 abrogated the formation of BCSC tumorspheres and xenograft tumors.Conclusions: KMT1A positively regulated the self-renewal and tumorigenicity of human BCSCs via KMT1A-GATA3-STAT3 circuit, in which KMT1A could be a promising target for bladder cancer therapy. Clin Cancer Res; 23(21); 6673-85. ©2017 AACR.


Assuntos
Fator de Transcrição GATA3/genética , Metiltransferases/genética , Proteínas Repressoras/genética , Fator de Transcrição STAT3/genética , Neoplasias da Bexiga Urinária/genética , Adulto , Idoso , Linhagem Celular Tumoral , Autorrenovação Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/patologia , Transdução de Sinais/genética , Neoplasias da Bexiga Urinária/patologia
12.
Protein Cell ; 7(4): 267-280, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26960409

RESUMO

Magnetotactic bacteria (MTB), a group of phylogenetically diverse organisms that use their unique intracellular magnetosome organelles to swim along the Earth's magnetic field, play important roles in the biogeochemical cycles of iron and sulfur. Previous studies have revealed that the bacterial actin protein MamK plays essential roles in the linear arrangement of magnetosomes in MTB cells belonging to the Proteobacteria phylum. However, the molecular mechanisms of multiple-magnetosome-chain arrangements in MTB remain largely unknown. Here, we report that the MamK filaments from the uncultivated 'Candidatus Magnetobacterium casensis' (Mcas) within the phylum Nitrospirae polymerized in the presence of ATP alone and were stable without obvious ATP hydrolysis-mediated disassembly. MamK in Mcas can convert NTP to NDP and NDP to NMP, showing the highest preference to ATP. Unlike its Magnetospirillum counterparts, which form a single magnetosome chain, or other bacterial actins such as MreB and ParM, the polymerized MamK from Mcas is independent of metal ions and nucleotides except for ATP, and is assembled into well-ordered filamentous bundles consisted of multiple filaments. Our results suggest a dynamically stable assembly of MamK from the uncultivated Nitrospirae MTB that synthesizes multiple magnetosome chains per cell. These findings further improve the current knowledge of biomineralization and organelle biogenesis in prokaryotic systems.


Assuntos
Actinas/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Actinas/química , Trifosfato de Adenosina/metabolismo , Bactérias/classificação , Proteínas de Bactérias/química , Magnetospirillum/classificação , Magnetospirillum/metabolismo , Nucleotídeos/metabolismo , Filogenia , Especificidade por Substrato
13.
Sheng Wu Gong Cheng Xue Bao ; 31(5): 611-20, 2015 May.
Artigo em Chinês | MEDLINE | ID: mdl-26571683

RESUMO

Raman spectroscopy has generated many branches during the development for more than 90 years. Surface enhanced Raman spectroscopy (SERS) improves SNR by using the interaction between tested materials and the surface of rough metal, as to quickly get higher sensitivity and precision spectroscopy without sample pretreatment. This article describes the characteristic and classification of SERS, and updates the theory and clinical application of SERS. It also summarizes the present status and progress of SERS in various disciplines and illustrates the necessity and urgency of its research, which provides rationale for the application for SERS in microbiology.


Assuntos
Microbiologia , Análise Espectral Raman
14.
Appl Microbiol Biotechnol ; 99(12): 5151-62, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25750031

RESUMO

Bacillus subtilis and its closely related species are important strains for industry, agriculture, and medicine. However, it is difficult to perform genetic manipulations using the endogenous recombination machinery. In many bacteria, phage recombineering systems have been employed to improve recombineering frequencies. To date, an efficient phage recombineering system for B. subtilis has not been reported. Here, we, for the first time, identified that GP35 from the native phage SPP1 exhibited a high recombination activity in B. subtilis. On this basis, we developed a high-efficiency GP35-meditated recombineering system. Taking single-stranded DNA (ssDNA) as a recombineering substrate, ten recombinases from diverse sources were investigated in B. subtilis W168. GP35 showed the highest recombineering frequency (1.71 ± 0.15 × 10(-1)). Besides targeting the purine nucleoside phosphorylase gene (deoD), we also demonstrated the utility of GP35 and Beta from Escherichia coli lambda phage by deleting the alpha-amylase gene (amyE) and uracil phosphoribosyltransferase gene (upp). In all three genetic loci, GP35 exhibited a higher frequency than Beta. Moreover, a phylogenetic tree comparing the kinship of different recombinase hosts with B. subtilis was constructed, and the relationship between the recombineering frequency and the kinship of the host was further analyzed. The results suggested that closer kinship to B. subtilis resulted in higher frequency in B. subtilis. In conclusion, the recombinase from native phage or prophage can significantly promote the genetic recombineering frequency in its host, providing an effective genetic tool for constructing genetically engineered strains and investigating bacterial physiology.


Assuntos
Bacillus subtilis/genética , Bacteriófagos/enzimologia , DNA Bacteriano/genética , DNA de Cadeia Simples/genética , Engenharia Genética/métodos , Recombinases/metabolismo , Recombinação Genética , Proteínas Virais/metabolismo , Bacillus subtilis/metabolismo , Bacteriófagos/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Reação em Cadeia da Polimerase , Recombinases/genética , Proteínas Virais/genética
15.
Am J Physiol Regul Integr Comp Physiol ; 308(11): R945-56, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25810384

RESUMO

Oxidative stress and inflammation are risk factors for hypertension in pregnancy. Here, we examined the 24-h mean arterial pressure (MAP) via telemetry and the nitric oxide (NO) and redox systems in the kidney cortex, medulla, and aorta of virgin and pregnant rats treated with a high-fat/prooxidant Western diet (HFD), ANG II, and TNF-α. Female Sprague-Dawley rats were given a normal diet (ND) or a HFD for 8 wk before mating. Day 6 of pregnancy and age-matched virgins were implanted with minipumps infusing saline or ANG II (150 ng·kg(-1)·min(-1)) + TNF-α (75 ng/day) for 14 days. Groups consisted of Virgin + ND + Saline (V+ND) (n = 7), Virgin + HFD +ANG II and TNF-α (V+HFD) (n = 7), Pregnant + ND + Saline (P+ND) (n = 6), and Pregnant + HFD + ANG II and TNF-α (P+HFD) (n = 8). After day 6 of minipump implantation, V+HFD rats displayed an increase in MAP on days 7, 8, and 10-15 vs. V+ND rats. P+HFD rats, after day 6 of minipump implantation, showed an increase in MAP only on day 7 vs. P+ND rats. P+HFD rats had a normal fall in 24-h MAP, hematocrit, plasma protein concentration, and osmolality at late pregnancy. No change in kidney cortex, medulla, or aortic oxidative stress in P+HFD rats. P+HFD rats displayed a decrease in nNOSß abundance, but no change in kidney cortex NOx content vs. P+ND rats. Pregnant rats subjected to a chronic HFD and prooxidant and proinflammatory insults have a blunted increase in 24-h MAP and renal oxidative stress. Our data suggest renal NO bioavailability is not altered in pregnant rats treated with a HFD, ANG II, and TNF-α.


Assuntos
Angiotensina II , Pressão Arterial , Dieta Hiperlipídica , Dieta Ocidental , Hipertensão/prevenção & controle , Córtex Renal/metabolismo , Estresse Oxidativo , Fator de Necrose Tumoral alfa , Animais , Antioxidantes/metabolismo , Aorta/metabolismo , Aorta/fisiopatologia , Peso ao Nascer , Modelos Animais de Doenças , Feminino , Hipertensão/etiologia , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Tamanho da Ninhada de Vivíparos , Óxido Nítrico/metabolismo , Gravidez , Ratos Sprague-Dawley , Telemetria , Fatores de Tempo
16.
ISME J ; 8(12): 2463-77, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24914800

RESUMO

Magnetotactic bacteria (MTB) of the genus 'Candidatus Magnetobacterium' in phylum Nitrospirae are of great interest because of the formation of hundreds of bullet-shaped magnetite magnetosomes in multiple bundles of chains per cell. These bacteria are worldwide distributed in aquatic environments and have important roles in the biogeochemical cycles of iron and sulfur. However, except for a few short genomic fragments, no genome data are available for this ecologically important genus, and little is known about their metabolic capacity owing to the lack of pure cultures. Here we report the first draft genome sequence of 3.42 Mb from an uncultivated strain tentatively named 'Ca. Magnetobacterium casensis' isolated from Lake Miyun, China. The genome sequence indicates an autotrophic lifestyle using the Wood-Ljungdahl pathway for CO2 fixation, which has not been described in any previously known MTB or Nitrospirae organisms. Pathways involved in the denitrification, sulfur oxidation and sulfate reduction have been predicted, indicating its considerable capacity for adaptation to variable geochemical conditions and roles in local biogeochemical cycles. Moreover, we have identified a complete magnetosome gene island containing mam, mad and a set of novel genes (named as man genes) putatively responsible for the formation of bullet-shaped magnetite magnetosomes and the arrangement of multiple magnetosome chains. This first comprehensive genomic analysis sheds light on the physiology, ecology and biomineralization of the poorly understood 'Ca. Magnetobacterium' genus.


Assuntos
Bactérias/genética , Genoma Bacteriano , Bactérias/classificação , Bactérias/metabolismo , Bactérias/ultraestrutura , Ciclo do Carbono/genética , Óxido Ferroso-Férrico/metabolismo , Ilhas Genômicas , Genômica , Ferro/metabolismo , Magnetossomos/metabolismo , Magnetossomos/ultraestrutura , Dados de Sequência Molecular , Nitrogênio/metabolismo , Filogenia , Enxofre/metabolismo
17.
J Microbiol Biotechnol ; 24(2): 197-208, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24150493

RESUMO

A novel protease gene from Bacillus gibsonii, aprBG, was cloned, expressed in B. subtilis, and characterized. High-level expression of aprBG was achieved in the recombinant strain when a junction was present between the promoter and the target gene. The purified recombinant enzyme exhibited similar N-terminal sequences and catalytic properties to the native enzyme, including high affinity and hydrolytic efficiency toward various substrates and a superior performance when exposed to various metal ions, surfactants, oxidants, and commercial detergents. AprBG was remarkably stable in 50% organic solvents and retained 100% activity and stability in 0-4 M NaCl, which is better than the characteristics of previously reported proteases. AprBG was most closely related to the high-alkaline proteases of the subtilisin family with a 57-68% identity. The secretion and maturation mechanism of AprBG was dependent on the enzyme activity, as analyzed by site-directed mutagenesis. Thus, when taken together, the results revealed that the halo-solvent-tolerant protease AprBG displays significant activity and stability under various extreme conditions, indicating its potential for use in many biotechnology applications.


Assuntos
Bacillus/metabolismo , Engenharia Metabólica , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Bacillus/genética , Clonagem Molecular , Análise por Conglomerados , Análise Mutacional de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Ativadores de Enzimas/metabolismo , Inibidores Enzimáticos/metabolismo , Estabilidade Enzimática , Expressão Gênica , Hidrólise , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/metabolismo
18.
Mol Plant Microbe Interact ; 27(2): 101-12, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24200074

RESUMO

PDZ domain-containing proteases, also known as HtrA family proteases, play important roles in bacterial cells by modulating disease pathogenesis and cell-envelope stress responses. These proteases have diverse functions through proteolysis- and nonproteolysis-dependent modes. Here, we report that the genome of the causative agent of rice bacterial blight, Xanthomonas oryzae pv. oryzae, encodes seven PDZ domain-containing proteins. Systematic inactivation of their encoding genes revealed that PXO_01122 and PXO_04290 (prc) are involved in virulence. prc encodes a putative HtrA family protease that localizes in the bacterial periplasm. Mutation of prc also resulted in susceptibility to multiple environmental stresses, including H2O2, sodium dodecylsulfate, and osmolarity stresses. Comparative subproteomic analyses showed that the amounts of 34 periplasmic proteins were lower in the prc mutant than in wild-type. These proteins were associated with proteolysis, biosynthesis of macromolecules, carbohydrate or energy metabolism, signal transduction, and protein translocation or folding. We provide in vivo and in vitro evidence demonstrating that Prc stabilizes and directly binds to one of these proteins, DppP, a dipeptidyl peptidase contributing to full virulence. Taken together, our results suggest that Prc contributes to bacterial virulence by acting as a periplasmic modulator of cell-envelope stress responses.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Oryza/microbiologia , Doenças das Plantas/microbiologia , Xanthomonas/fisiologia , Proteínas de Bactérias/genética , Peróxido de Hidrogênio/farmacologia , Mutação , Pressão Osmótica , Domínios PDZ , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Periplasma/metabolismo , Fenótipo , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteômica , Dodecilsulfato de Sódio/farmacologia , Virulência , Xanthomonas/efeitos dos fármacos , Xanthomonas/genética , Xanthomonas/patogenicidade
19.
J Bacteriol ; 195(23): 5334-42, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24056108

RESUMO

Corynebacterium glutamicum is equipped with abundant membrane transporters to adapt to a changing environment. Many amino acid transporters have been identified in C. glutamicum, but histidine uptake has not been investigated in detail. Here, we identified the aromatic amino acid transporter encoded by aroP as a histidine transporter in C. glutamicum by a combination of the growth and histidine uptake features. Characterization of histidine uptake showed that AroP has a moderate affinity for histidine, with a Km value of 11.40 ± 2.03 µM, and histidine uptake by AroP is competitively inhibited by the aromatic amino acids. Among the four substrates, AroP exhibits a stronger preference for tryptophan than for tyrosine, phenylalanine, and histidine. Homology structure modeling and molecular docking were performed to predict the substrate binding modes and conformational changes during substrate transport. These results suggested that tryptophan is best accommodated in the binding pocket due to shape compatibility, strong hydrophobic interactions, and the lowest binding energy, which is consistent with the observed substrate preference of AroP. Furthermore, the missense mutations of the putative substrate binding sites verified that Ser24, Ala28, and Gly29 play crucial roles in substrate binding and are highly conserved in the Gram-positive bacteria. Finally, the expression of aroP is not significantly affected by extracellular histidine or aromatic amino acids, indicating that the physiological role of AroP may be correlated with the increased fitness of C. glutamicum to assimilate extracellular amino acid for avoiding the high energy cost of amino acid biosynthesis.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Sistemas de Transporte de Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica
20.
Am J Physiol Renal Physiol ; 305(5): F727-33, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23825068

RESUMO

The rat kidney ablation and infarction (A/I) model of subtotal or 5/6th nephrectomy is the most commonly studied model of nondiabetic chronic kidney disease (CKD). The A/I kidney at 1 wk exhibits reductions in kidney function, as determined by glomerular filtration rate, and diminished metabolic efficiency as determined by oxygen consumption per sodium transport (QO2/TNa). As renoprotective AMPK activity is affected by metabolic changes and cellular stress, we evaluated AMPK activity in this model system. We show that these early pathophysiological changes are accompanied by a paradoxical decrease in AMPK activity. Over time, these kidney parameters progressively worsen with extensive kidney structural, functional, metabolic, and fibrotic changes observed at 4 wk after A/I. We show that induction of AMPK activity with either metformin or 5-aminoimidazole-4-carboxamide ribonucleotide increases AMPK activity in this model and also corrects kidney metabolic inefficiency, improves kidney function, and ameliorates kidney fibrosis and structural alterations. We conclude that AMPK activity is reduced in the subtotal nephrectomy model of nondiabetic CKD, that altered regulation of AMPK is coincident with the progression of disease parameters, and that restoration of AMPK activity can suppress the progressive loss of function characteristic of this model. We propose that induction of AMPK activity may prove an effective therapeutic target for the treatment of nondiabetic CKD.


Assuntos
Adenilato Quinase/biossíntese , Insuficiência Renal Crônica/fisiopatologia , Animais , Modelos Animais de Doenças , Indução Enzimática , Masculino , Metformina/farmacologia , Nefrectomia , Ratos , Ratos Wistar
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