RESUMO
The peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) is a critical transcriptional coactivator that maintains metabolic homeostasis and energy expenditure by cooperating with various transcription factors. Recent studies have shown that PGC1α deficiency promotes lung cancer metastasis to the bone through activation of TCF4 and TWIST1-mediated epithelial-mesenchymal transition (EMT), which is suppressed by the inhibitor of DNA binding 1 (ID1); however, it is not clear which transcription factor participates in PGC1α-mediated EMT and lung cancer metastasis. Here, we identified forkhead box A1 (FOXA1) as a potential transcription factor that coordinates with PGC1α and ID1 for EMT gene expression using transcriptome analysis. Cooperation between FOXA1 and PGC1α inhibits promoter occupancy of TCF4 and TWIST1 on CDH1 and CDH2 proximal promoter regions due to increased ID1, consequently regulating the expression of EMT-related genes such as CDH1, CDH2, VIM, and PTHLH. Transforming growth factor beta 1 (TGFß1), a major EMT-promoting factor, was found to decrease ID1 due to the suppression of FOXA1 and PGC1α. In addition, ectopic expression of ID1, FOXA1, and PGC1α reversed TGFß1-induced EMT gene expression. Our findings suggest that FOXA1- and PGC1α-mediated ID1 expression involves EMT by suppressing TCF4 and TWIST1 in response to TGFß1. Taken together, this transcriptional framework is a promising molecular target for the development of therapeutic strategies for lung cancer metastasis.
Assuntos
Transição Epitelial-Mesenquimal , Neoplasias Pulmonares , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição 4/metabolismo , Fatores de Transcrição/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Constitutive activation of the ß-catenin dependent canonical Wnt signaling pathway, which enhances tumor growth and progression in multiple types of cancer, is commonly observed in melanoma. LEF1 activates ß-catenin/TCF4 transcriptional activity, promoting tumor growth and progression. Although several reports have shown that LEF1 is highly expressed in melanoma, the functional role of LEF1 in melanoma growth is not fully understood. While A375, A2058, and G361 melanoma cells exhibit abnormally high LEF1 expression, lung cancer cells express lower LEF1 levels. A luciferase assay-based high throughput screening (HTS) with a natural compound library showed that cinobufagin suppressed ß-catenin/TCF4 transcriptional activity by inhibiting LEF1 expression. Cinobufagin decreases LEF1 expression in a dose-dependent manner and Wnt/ß-catenin target genes such as Axin-2, cyclin D1, and c-Myc in melanoma cell lines. Cinobufagin sensitively attenuates cell viability and induces apoptosis in LEF1 expressing melanoma cells compared to LEF1-low expressing lung cancer cells. In addition, ectopic LEF1 expression is sufficient to attenuate cinobufagin-induced apoptosis and cell growth retardation in melanoma cells. Thus, we suggest that cinobufagin is a potential anti-melanoma drug that suppresses tumor-promoting Wnt/ß-catenin signaling via LEF1 inhibition.
Assuntos
Bufanolídeos/farmacologia , Fator 1 de Ligação ao Facilitador Linfoide/antagonistas & inibidores , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Melanoma/tratamento farmacológico , Células A549 , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismo , Fator de Transcrição 4/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
Cachexia, which is characterised by the wasting of fat and skeletal muscles, is the most common risk factor for increased mortality rates among patients with advanced lung cancer. PTHLH (parathyroid hormone-like hormone) is reported to be involved in the pathogenesis of cancer cachexia. However, the molecular mechanisms underlying the regulation of PTHLH expression and the inhibitors of PTHLH have not yet been identified. The PTHLH mRNA levels were measured using quantitative real-time polymerase chain reaction, while the PTHrP (parathyroid hormone-related protein) expression levels were measured using Western blotting and enzyme-linked immunosorbent assay. The interaction between TCF4 (Transcription Factor 4) and TWIST1 and the binding of the TCF4-TWIST1 complex to the PTHLH promoter were analysed using co-immunoprecipitation and chromatin immunoprecipitation. The results of the mammalian two-hybrid luciferase assay revealed that emodin inhibited TCF4-TWIST1 interaction. The effects of Polygonum cuspidatum extract (Pc-Ex), which contains emodin, on cachexia were investigated in vivo using A549 tumour-bearing mice. Ectopic expression of TCF4 upregulated PTHLH expression. Conversely, TCF4 knockdown downregulated PTHLH expression in lung cancer cells. The expression of PTHLH was upregulated in cells ectopically co-expressing TCF4 and TWIST1 when compared with that in cells expressing TCF4 or TWIST1 alone. Emodin inhibited the interaction between TCF4 and TWIST1 and consequently suppressed the TCF4/TWIST1 complex-induced upregulated mRNA and protein levels of PTHLH and PTHrP. Meanwhile, emodin-containing Pc-Ex significantly alleviated skeletal muscle atrophy and downregulated fat browning-related genes in A549 tumour-bearing mice. Emodin-containing Pc-Ex exerted therapeutic effects on lung cancer-associated cachexia by inhibiting TCF4/TWIST1 complex-induced PTHrP expression.
Assuntos
Emodina , Fallopia japonica , Neoplasias Pulmonares , Animais , Caquexia/tratamento farmacológico , Caquexia/etiologia , Caquexia/prevenção & controle , Emodina/farmacologia , Emodina/uso terapêutico , Humanos , Neoplasias Pulmonares/complicações , Neoplasias Pulmonares/tratamento farmacológico , Mamíferos/genética , Mamíferos/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteína Relacionada ao Hormônio Paratireóideo/genética , Extratos Vegetais , RNA Mensageiro/metabolismo , Fator de Transcrição 4/genética , Proteína 1 Relacionada a Twist/genéticaRESUMO
Retrospective observational studies have reported that statins improve clinical outcomes in patients previously treated with programmed cell death protein 1 (PD-1)-targeting monoclonal antibodies for malignant pleural mesothelioma (MPM) and advanced non-small cell lung cancer (NSCLC). In multiple mouse cancer models, de novo synthesis of mevalonate and cholesterol inhibitors was found to synergize with anti-PD-1 antibody therapy. In the present study, we investigated whether statins affect programmed death-ligand 1 (PD-L1) expression in cancer cells. Four statins, namely simvastatin, atorvastatin, lovastatin, and fluvastatin, decreased PD-L1 expression in melanoma and lung cancer cells. In addition, we found that AKT and ß-catenin signaling involved PD-L1 suppression by statins. Our cellular and molecular studies provide inspiring evidence for extending the clinical evaluation of statins for use in combination with immune checkpoint inhibitor-based cancer therapy.
Assuntos
Antígeno B7-H1/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , Apoptose , Proliferação de Células , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Células Tumorais CultivadasRESUMO
We developed a multiplex PCR assay for the detection of lactic acid bacteria (LAB) species, and used it to examine the LAB species involved in kimchi fermentation. The LAB profile during kimchi fermentation varied with pH and acidity. Leuconostoc mesenteroides was observed during early fermentation (pH 5.64-4.27 and acidity 0.48-0.89%), and Lactobacillus sakei become dominant later in fermentation (pH
Assuntos
Fermentação/fisiologia , Microbiologia de Alimentos , Lactobacillus/genética , Lactobacillus/isolamento & purificação , Verduras/microbiologia , Concentração de Íons de Hidrogênio , Coreia (Geográfico) , Reação em Cadeia da PolimeraseRESUMO
We developed a transgenic Chinese cabbage (Brassica rapa L. ssp. pekinensis) inbred line, Kenshin, with high tolerance to soft rot disease. Tolerance was conferred by expression of N-acyl-homoserine lactonase (AHL-lactonase) in Chinese cabbage through an efficient Agrobacterium-mediated transformation method. To synthesize and express the AHL-lactonase in Chinese cabbage, the plant was transformed with the aii gene (AHL-lactonase gene from Bacillus sp. GH02) fused to the PinII signal peptide (protease inhibitor II from potato). Five transgenic lines were selected by growth on hygromycin-containing medium (3.7% transformation efficiency). Southern blot analysis showed that the transgene was stably integrated into the genome. Among these five transgenic lines, single copy number integrations were observed in four lines and a double copy number integration was observed in one transgenic line. Northern blot analysis confirmed that pinIISP-aii fusion gene was expressed in all the transgenic lines. Soft rot disease tolerance was evaluated at tissue and seedling stage. Transgenic plants showed a significantly enhanced tolerance (2-3-fold) to soft rot disease compared to wild-type plants. Thus, expression of the fusion gene pinIISP-aii reduces susceptibility to soft rot disease in Chinese cabbage. We conclude that the recombinant AHL-lactonase, encoded by aii, can effectively quench bacterial quorum-sensing and prevent bacterial population density-dependent infections. To the best of our knowledge, the present study is the first to demonstrate the transformation of Chinese cabbage inbred line Kenshin, and the first to describe the effect of the fusion gene pinIISP-aii on enhancement of soft rot disease tolerance.
Assuntos
Bacillus/patogenicidade , Brassica rapa/genética , Doenças das Plantas/genética , Percepção de Quorum , Brassica rapa/imunologia , Brassica rapa/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Imunidade Inata , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/imunologia , Plantas Geneticamente Modificadas/metabolismo , Rhizobium/genéticaRESUMO
An asc operon of Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34) was isolated from a genomic library in a screen for beta-glucosidase activities. Sequence analysis of the 5618-bp cloned DNA fragment (accession number AY622309) showed three open reading frames (ascG, ascF, and ascB) that are predicted to encode 375, 486, and 476 amino acid proteins, respectively. The AscG ORF shared a high similarity with the Escherichia coli AscG repressor. The AscF ORF shared 81% identity with the E. coli AscF PTS enzyme II(asc), while the AscB ORF was highly similar to 6-phospho-beta-glucosidases and is a member of the glycosyl hydrolase family 1. The purified AscB enzyme hydrolyzed salicin, arbutin, pNPG, and MUG. It exhibited maximal activity at pH 7.0 and 40 degrees C, and its activity was enhanced in the presence of Mg(2+) and Ca(2+). The molecular weight of the enzyme was estimated to be 53 000 Da by SDS-PAGE. Two conserved glutamate residues (Glu(182) and Glu(374)) were shown to be important for AscB activity.
Assuntos
Glucosidases , Óperon , Pectobacterium carotovorum/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clonagem Molecular , Glucosidases/química , Glucosidases/genética , Glucosidases/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Pectobacterium carotovorum/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Análise de Sequência de DNA , Relação Estrutura-AtividadeRESUMO
The gene encoding an intracellular alpha-amylase, AmyB (TM1650), from Thermotoga maritima MSB8, a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli. The AmyB enzyme hydrolyzed alpha-1,4 starch linkage. The amyB gene is 1269 bp in length, encoding a protein of 422 amino acids (calculated molecular mass of 50187 Da). The molecular weight of the enzyme was estimated to be 50000 Da by SDS-PAGE after starch-nondenaturing-PAGE. The amino acid sequence of AmyB showed less than 12% identity to other amylases, but contained four regions that are highly conserved among alpha-amylases. The AmyB alpha-amylase exhibited maximal enzymatic activity at pH 7.0 and its optimum temperature for activity was 70 degrees C. Like the alpha-amylases of many other organisms, the thermostability of T. maritima MSB8 alpha-amylase, AmyB expressed in E. coli was enhanced in the presence of Ca(2+) (10 mM).
Assuntos
Clonagem Molecular , Temperatura Alta , Thermotoga maritima/enzimologia , alfa-Amilases/genética , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Dados de Sequência Molecular , Mapeamento por Restrição , Análise de Sequência de DNA , Thermotoga maritima/genética , alfa-Amilases/química , alfa-Amilases/isolamento & purificação , alfa-Amilases/metabolismoRESUMO
The phytopathogenic Pectobacterium chrysanthemi (Pch) PY35 secretes multiple isozymes of plant cell wall degrading enzyme cellulases. We cloned a second cel gene that encodes cellulase in Pch PY35. The inserted 2 kb fragment was subcloned in order to geneate pPY710 (cel8Y). The structural organization of the cel8Y gene consists of an open reading frame (ORF) of 999 bp that encodes 332 amino acid residues with a signal peptide of 23 amino acids. The predicted amino acid sequence of Cel8Y was very similar to that of Cellulomonas uda, but completely different from that of the Cel5Z of Pch PY35. It belonged to the glycoside hydrolase family 8, based on amino acid sequence similarities in contrast to Cel5Z of Pch PY35, which was confirmed as family 5. Cel8Y was not closely related to the known cellulases of Pectobacterium. It had the conserved region of the glycoside hydrolase family 8, ASDGDVLIAWALLKAGNKW. The apparent molecular mass of the Cel8Y protein was calculated to be approximately 34 kDa by a carboxymethylcellulosesodium dodecyl sulfate-polyacrylamide gel electrophoresis (CMC-SDS-PAGE). The Cel8Y had a calculated pl of 6.49. It was optimally active at pH 7 with an approximate optimal temperature around 40 degrees C. The cellulase activity of Cel8Y was lower than that of Cel5Z.
Assuntos
Celulase/genética , Dickeya chrysanthemi/enzimologia , Dickeya chrysanthemi/genética , Genes Bacterianos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Celulase/química , Celulase/metabolismo , Clonagem Molecular , Sequência Conservada , DNA Bacteriano/genética , Evolução Molecular , Glicosídeo Hidrolases/genética , Ponto Isoelétrico , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Filogenia , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
Lactobacillus brevis WCP902 that is capable of biodegrading chlorpyrifos was isolated from kimchi. The opdB gene cloned from this strain revealed 825 bp, encoding 274 aa, and an enzyme molecular weight of about 27 kDa. OpdB contains the same Gly-X-Ser-X-Gly motif found in most bacterial and eukaryotic esterase, lipase, and serine hydrolases, yet it is a novel member of the GDSVG family of esterolytic enzymes. Its conserved serine residue, Ser82, is significantly involved with enzyme activity that may have application for removing some pesticides. Optimum organophosphorus hydrolase (OpdB) activity appeared at pH 6.0 and 35 degrees C and during degradation of chlorpyrifos, coumaphos, diazinon, methylparathion, and parathion.
Assuntos
Arildialquilfosfatase/metabolismo , Levilactobacillus brevis/enzimologia , Compostos Organofosforados/metabolismo , Praguicidas/metabolismo , Sequência de Bases , Primers do DNARESUMO
We examined the role of microorganisms in the degradation of the organophosphorus (OP) insecticide chlorpyrifos (CP) during kimchi fermentation. During the fermentation of kimchi, 30 mg L(-1) of CP was added and its stability assayed during fermentation. CP was degraded rapidly until day 3 (83.3%) and degraded completely by day 9. Four CP-degrading lactic acid bacteria (LAB) were isolated from kimchi fermentation in the presence of 200 mg L(-1) CP and were identified as Leuconostoc mesenteroides WCP907, Lactobacillus brevis WCP902, Lactobacillus plantarum WCP931, and Lactobacillus sakei WCP904. CP could be utilized by these four strains as the sole source of carbon and phosphorus. Coumaphos (CM), diazinon (DZ), parathion (PT), and methylparathion (MPT) were also degraded by WCP907, WCP902, WCP931, and WCP904 when provided as sole sources of carbon and phosphorus.
Assuntos
Brassica/microbiologia , Clorpirifos/metabolismo , Fermentação , Contaminação de Alimentos/análise , Inseticidas/metabolismo , Lactobacillaceae/metabolismo , Biodegradação Ambiental , Qualidade de Produtos para o Consumidor , Ácido Láctico/metabolismo , Lactobacillaceae/genética , Lactobacillaceae/isolamento & purificação , Dados de Sequência MolecularRESUMO
A chromosomal region of Pectobacterium chrysanthemi PY35 that contains of genes for glycogen synthesis was isolated from a cosmid library. The operon consists of glycogen branching enzyme (glgB), glycogen debranching enzyme (glgX), ADP-glucose pyrophosphorylase (glgC), glycogen synthase (glgA), and glycogen phosphorylase (glgP) genes. Gene organization is similar to that of Escherichia coli. The purified ADP-glucose pyrophosphorylase (GlgC) was activated by fructose 1,6-bisphosphate and inhibited by AMP. The constructed glgX::Omega mutant failed to integrate into the chromosome of P. chrysanthemi by marker exchange. Phylogenetic analysis based on the 16S rDNA and the amino acid sequence of Glg enzymes showed correlation with other bacteria. gamma-Proteobacteria have the glgX gene instead of the bacilli glgD gene in the glg operon. The possible evolutionary implications of the results among the prokaryotes are discussed.
Assuntos
Proteínas de Bactérias/genética , Dickeya chrysanthemi/genética , Glicogênio/biossíntese , Óperon , Sequência de Aminoácidos , Archaea/classificação , Archaea/genética , Bactérias/classificação , Bactérias/genética , Proteínas de Bactérias/classificação , Sequência de Bases , Cosmídeos/genética , Dickeya chrysanthemi/classificação , Dickeya chrysanthemi/enzimologia , Enzimas/genética , Enzimas/metabolismo , Evolução Molecular , Biblioteca Gênica , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/classificação , RNA Ribossômico 16S/genéticaRESUMO
Pectobacterium chrysanthemi PY35 secretes the endoglucanase Cel5Z, an enzyme of the glycoside hydrolase family 5. Cel5Z is a 426 amino acid, signal peptide (SP)-containing protein composed of two domains: a large N-terminal catalytic domain (CD; 291 amino acids) and a small C-terminal cellulose binding domain (CBD; 62 amino acids). These two domains are separated by a 30 amino acid linker region (LR). A truncated cel5Z gene was constructed with the addition of a nonsense mutation that removes the C-terminal region of the protein. A truncated Cel5Z protein, consisting of 280 amino acid residues, functioned as a mature enzyme despite the absence of the SP, 11 amino acid CD, LR, and CBD region. In fact, this truncated Cel5Z protein showed an enzymatic activity 80% higher than that of full-length Cel5Z. However, cellulase activity was undetectable in mature Cel5Z proteins truncated to less than 280 amino acids.
Assuntos
Celulases/química , Celulases/metabolismo , Dickeya chrysanthemi/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Bacterianos , Expressão Gênica , Dados de Sequência Molecular , Conformação Proteica , Engenharia de ProteínasRESUMO
Endophytic Bacillus sp. CY22 was previously isolated from the root interior of the balloon flower (Platycodon grandiflorum) (Cho et al., Biosci. Biotechnol. Biochem., 66, 1270-1275 (2002)). Three-month-old balloon flower seedlings were inoculated with 10(7) cfu/ml of strain CY22R3, a rifampicin-resistant strain of CY22, and external and internal root colonization was assessed 2 and 4 weeks later. After inoculation, large numbers of bacteria were observed on the root surface by scanning electron microscopy. More detailed studies using optical and transmission electron microscopy confirmed that Bacillus sp. CY22 was endophytically established within intercellular spaces, cortical cells, and aerenchymas of root. Also, Bacillus sp. CY22 showed antibiotic activities against several phytopathogens by producing the antibiotic iturin A. In the pot test, root rot of balloon flower seedlings caused by Rhizoctonia solani was suppressed when the Bacillus sp. CY22R3 was inoculated into the soil.
Assuntos
Antifúngicos/biossíntese , Bacillus/crescimento & desenvolvimento , Platycodon/microbiologia , Antifúngicos/farmacologia , Bacillus/metabolismo , Microscopia Eletrônica de Varredura , Peptídeos/farmacologia , Peptídeos Cíclicos , Doenças das Plantas/microbiologia , Raízes de Plantas/citologia , Raízes de Plantas/microbiologia , Rhizoctonia/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Phylogenetic analysis of archaea in the rumen ecosystem was analysed by PCR of 16S rDNA from the bovine rumen using archaea-specific primers. The libraries were constructed from rumen fluid (AF), rumen solid (AS), and rumen epithelium (AE) from a rumen-fistulated Korean cow (Hanwoo). The 45 AF clones could be divided into three groups and the largest group was affiliated with the Methanomicrobiaceae family (96% of clones). The AF clones contained a high proportion of unidentifiable clones (67%). The 39 AE clones could be divided into two groups and the largest group was also affiliated with the Methanomicrobiaceae family (95% of clones). The AE clones contained a low proportion of unidentifiable clones (5%). The 20 AS clones could be divided into two groups that were affiliated with either the Methanobacteriaceae family (55%) or the Methanomicrobiaceae family (45%). The AS clones contained a moderate proportion of unidentifiable clones (40%). The predominant family of whole rumen archaea was found to belong to the Methanomicrobiaceae (85%). Methanomicrobiaceae were predominant in the rumen epithelium and the rumen fluid while Methanobacteriaceae were predominant in the rumen solid. One clone from the rumen fluid and two clones from the rumen epithelium contained rDNA sequences of Non-Thermophilic-Crenarchaeota (NTC) and Thermophilic-Crenarchaeota (TC), respectively, which have not previously been described from the rumen.
RESUMO
The phytopathogenic bacterium Pectobacteium chrysanthemi PY35 secretes Cel5Z endoglucanase belonging to the glycoside hydrolase family 5 of EC 3.2.1.4. The mutation of cel5Z::Omega gene was constructed by cloning the 2.0-kb SmaI fragment containing the streptomycin/spectinomycin-resistance gene of pHP45(Omega) into the BalI site of pPY100. The insertion of Omega fragment generated a new stop codon, removing the Ser/Thr-rich linker region and the cellulose binding domain (CBD) in the C-terminal region of cel5Z gene. By subsequent subcloning from this 4.9-kb fragment (pPY1001), a 1.0-kb (pPY1002) fragment was obtained and designated as cel5Z::Omega. The cel5Z::Omega gene had an open reading frame (ORF) of 1011 bp, encoding 336 amino acids, starting with an ATG codon and ending with a new TGA stop codon. The molecular mass of the Cel5Z::Omega protein in E. coli transformant appeared to be 32 kDa by SDS-PAGE analysis in the presence of carboxymethyl-cellulose (CMC). The Cel5Z::Omega protein hydrolyzed CMC with 1.7-fold higher activity than the intact Cel5Z cellulase.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Celulase/genética , Celulase/metabolismo , Dickeya chrysanthemi/enzimologia , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Celulase/química , Ativação Enzimática , Escherichia coli/genética , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Peso Molecular , Mutagênese Insercional , Estrutura Terciária de Proteína , Mapeamento por Restrição , Deleção de Sequência , TemperaturaRESUMO
The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli. The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da). The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX. The enzyme was 43-69% sequence identical with other glycogen branching enzymes. The enzyme is the most similar to GlgB of E. coli and contained the four regions conserved among the alpha-amylase family. The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE. The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C.
Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/genética , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Genes Bacterianos , Sistema da Enzima Desramificadora do Glicogênio/genética , Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Sequência Conservada , DNA Bacteriano/genética , Escherichia coli/genética , Dados de Sequência Molecular , Peso Molecular , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C15:0 (37%), C17:0 (5.1%), and iso-C15:0 (27.7%). DNA G+C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.
Assuntos
Bacillus/classificação , Bacillus/isolamento & purificação , Raízes de Plantas/microbiologia , Platycodon/microbiologia , Bacillus/genética , Bacillus/fisiologia , DNA Bacteriano/genética , DNA Ribossômico/genética , Fenótipo , Filogenia , RNA Ribossômico 16S/genéticaRESUMO
A putative bgl operon of Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34) was isolated. Sequence analysis of the 5,557 bp cloned DNA fragment (accession no. AY542524) showed three open reading frames (bglT, bglP, and bglB) predicted to encode 287, 633, and 468 amino acid proteins respectively. BglT and BglP ORFs show high similarity to that of the Pectobacterium chrysanthemi ArbG antiterminator and ArbF permease respectively. Also, the latter contains most residues important for phosphotransferase activity. The amino acid sequence of BglB showed high similarity to various beta-glucosidases and is a member of glycosyl hydrolase family 1. The purified BglB enzyme hydrolyzed salicin, arbutin, pNPG, and MUG. The molecular weight of the enzyme was estimated to be 53,000 Da by SDS-PAGE. The purified beta-glucosidase exhibited maximal activity at pH 7.0 and 40 degrees C, and its activity was enhanced in the presence of Mg(2+). Two glutamate residues (Glu(173) and Glu(362)) were found to be essential for enzyme activity.