79 results on '"Spira, Beny"'
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52. Ugp and PitA Participate in the Selection of PHO-Constitutive Mutants
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Iglesias Neves, Henrique, primary, Pereira, Tuanny Fernanda, additional, Yagil, Ezra, additional, and Spira, Beny, additional
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- 2015
- Full Text
- View/download PDF
53. relA Enhances the Adherence of Enteropathogenic Escherichia coli
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Spira, Beny, primary, Ferreira, Gerson Moura, additional, and de Almeida, Luiz Gustavo, additional
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- 2014
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- View/download PDF
54. Antimicrobial Capacity of Plasma from Anurans of the Atlantic Forest
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Assis, Vania Regina de, primary, Titon, Stefanny Christie Monteiro, additional, Barsotti, Adriana Maria Giorgi, additional, Spira, Beny, additional, and Gomes, Fernando Ribeiro, additional
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- 2013
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55. Chromobacterium violaceum adaptation to low-phosphate conditions.
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Costa Vasconcelos, Fernanda, Padilla, Gabriel, and Spira, Beny
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CHROMOBACTERIUM violaceum ,EFFECT of phosphorus on bacteria ,GENE expression in bacteria ,CULTURE media (Biology) ,GENETIC transcription in bacteria ,POLYMERASE chain reaction ,PHYSIOLOGICAL effects of phosphates ,REGULONS - Abstract
Chromobacterium violaceum is a free-living bacterium that inhabits low-nutrient environments such as the Amazon basin. Bacteria respond to phosphate (Pi) shortage by expressing a range of genes involved in Pi uptake and assimilation, known as the PHO regulon. Several PHO regulon genes have been annotated in the genome of C. violaceum. Here we show that C. violaceum is extremely well adapted to low-Pi conditions. Remarkably, this bacterium is able to grow in media containing only traces of Pi. The PHO regulon genes are induced upon Pi depletion, but the bacteria continued to grow under these conditions. Unlike other Proteobacteria hitherto analyzed, neither PstS nor PhoU play a role in the repression of the PHO regulon under Pi excess. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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- View/download PDF
56. The constancy of global regulation across a species: the concentrations of ppGpp and RpoS are strain-specific in Escherichia coli
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Ferenci, Thomas, primary, Galbiati, Heloisa Filus, additional, Betteridge, Thu, additional, Phan, Katherine, additional, and Spira, Beny, additional
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- 2011
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57. Divergence Involving Global Regulatory Gene Mutations in an Escherichia coli Population Evolving under Phosphate Limitation
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Wang, Lei, primary, Spira, Beny, additional, Zhou, Zhemin, additional, Feng, Lu, additional, Maharjan, Ram P., additional, Li, Xiaomin, additional, Li, Fangfang, additional, McKenzie, Christopher, additional, Reeves, Peter R., additional, and Ferenci, Thomas, additional
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- 2010
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58. The effect of sixteen medicinal plants used in the Brazilian pharmacopoeia on the expression and activity of glutathioneS-transferase in hepatocytes and leukemia cells
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Príncipe, Cássia Rosalina, primary and Spira, Beny, additional
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- 2009
- Full Text
- View/download PDF
59. Transcriptional Processing of the pst Operon of Escherichia coli
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Aguena, Meire, primary and Spira, Beny, additional
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- 2008
- Full Text
- View/download PDF
60. Stability of the pstS transcript of Escherichia coli
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Aguena, Meire, primary, Ferreira, Gerson Moura, additional, and Spira, Beny, additional
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- 2008
- Full Text
- View/download PDF
61. Alkaline phosphatase as a reporter of σS levels and rpoS polymorphisms in different E. coli strains
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Spira, Beny, primary and Ferenci, Thomas, additional
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- 2007
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62. Current Methods of Gene Expression Analysis and Quantification
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Spira, Beny, primary
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- 2005
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63. A differential effect of σ S on the expression of the PHO regulon genes of Escherichia coli
- Author
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Taschner, Natalia Pasternak, primary, Yagil, Ezra, additional, and Spira, Beny, additional
- Published
- 2004
- Full Text
- View/download PDF
64. Thioacetamide differentially affects the expression and activity of glutathione-S-transferase in the liver of Wistar rats
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Bueno, Murilo Tadeu Domingues, primary and Spira, Beny, additional
- Published
- 2004
- Full Text
- View/download PDF
65. RT-PCR of long prokaryotic operon transcripts without DNase treatment
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Aguena, Meire, primary and Spira, Beny, additional
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- 2003
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66. Differentially expressed genes in the liver of thioacetamide treated rats
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Spira, Beny, primary and Raw, Isaias, additional
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- 2003
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67. First Characterization of CTX-M-15-Producing Escherichia coliStrains Belonging to Sequence Type (ST) 410, ST224, and ST1284 from Commercial Swine in South America
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Silva, Ketrin C., Moreno, Marina, Cabrera, Carlos, Spira, Beny, Cerdeira, Louise, Lincopan, Nilton, and Moreno, Andrea M.
- Abstract
ABSTRACTWe report for the first time the isolation of CTX-M-15-producing Escherichia colistrains belonging to sequence type (ST) 410, ST224, and ST1284 in commercial swine in Brazil. The blaCTX-M-15gene was located on F-::A9::B1 and C1::A9::B1 IncF-type plasmids, surrounded by a new genetic context comprising the IS26insertion sequence truncated with the ISEcp1element upstream of blaCTX-M-15. These results reveal that commercial swine have become a new reservoir of CTX-M-15-producing bacteria in South America.
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- 2016
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68. relA Enhances the Adherence of Enteropathogenic Escherichia coli.
- Author
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Spira, Beny, Ferreira, Gerson Moura, and de Almeida, Luiz Gustavo
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ESCHERICHIA coli , *BACTERIAL adhesion , *MEMBRANE proteins , *PLASMIDS , *GENETIC transcription in bacteria , *EPITHELIAL cells , *GENE expression in bacteria - Abstract
Enteropathogenic Escherichia coli (EPEC) is a known causative agent of diarrhea in children. In the process of colonization of the small intestine, EPEC synthesizes two types of adhesins, the bundle-forming pilus (BFP) and intimin. The BFP pilus is an adhesin associated with the initial stages of adherence of EPEC to epithelial cells, while the outer membrane protein intimin carries out the intimate adherence that takes place at the third stage of infection. BFP is encoded by the bfp operon located in plasmid EAF, present only in typical EPEC isolates, while eae, the gene that encodes intimin is situated in the LEE, a chromosomal pathogenicity island. Transcription of bfp and eae is regulated by the products of the perABC operon, also present in plasmid EAF. Here we show that deletion of relA, that encodes a guanosine penta and tetraphosphate synthetase impairs EPEC adherence to epithelial cells in vitro. In the absence of relA, the transcription of the regulatory operon perABC is reduced, resulting in lower levels of BFP and intimin. Bacterial adherence, BFP and intimin synthesis and perABC expression are restored upon complementation with the wild-type relA allele. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
69. relA Enhances the Adherence of Enteropathogenic Escherichia coli.
- Author
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Spira, Beny, Ferreira, Gerson Moura, and de Almeida, Luiz Gustavo
- Subjects
ESCHERICHIA coli ,BACTERIAL adhesion ,MEMBRANE proteins ,PLASMIDS ,GENETIC transcription in bacteria ,EPITHELIAL cells ,GENE expression in bacteria - Abstract
Enteropathogenic Escherichia coli (EPEC) is a known causative agent of diarrhea in children. In the process of colonization of the small intestine, EPEC synthesizes two types of adhesins, the bundle-forming pilus (BFP) and intimin. The BFP pilus is an adhesin associated with the initial stages of adherence of EPEC to epithelial cells, while the outer membrane protein intimin carries out the intimate adherence that takes place at the third stage of infection. BFP is encoded by the bfp operon located in plasmid EAF, present only in typical EPEC isolates, while eae, the gene that encodes intimin is situated in the LEE, a chromosomal pathogenicity island. Transcription of bfp and eae is regulated by the products of the perABC operon, also present in plasmid EAF. Here we show that deletion of relA, that encodes a guanosine penta and tetraphosphate synthetase impairs EPEC adherence to epithelial cells in vitro. In the absence of relA, the transcription of the regulatory operon perABC is reduced, resulting in lower levels of BFP and intimin. Bacterial adherence, BFP and intimin synthesis and perABC expression are restored upon complementation with the wild-type relA allele. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
70. The effect of sixteen medicinal plants used in the Brazilian pharmacopoeia on the expression and activity of glutathione S-transferase in hepatocytes and leukemia cells.
- Author
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Príncipe, Cássia Rosalina and Spira, Beny
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PHARMACOPOEIAS , *MEDICINAL plants , *GLUTATHIONE , *LIVER cells , *LEUKEMIA - Abstract
Glutathione S-transferase (GST) is a family of enzymes involved in the detoxification of electrophilic compounds. Different classes of GST are expressed in various organs, such as liver, lungs, stomach and others. Expression of GST can be modulated by diet components and plant-derived compounds. The importance of controlling GST expression is twofold: increasing levels of GST are beneficial to prevent deleterious effects of toxic and carcinogenic compounds, while inhibition of GST in tumor cells may help overcoming tumor resistance to chemotherapy. A screening of 16 plants used in the Brazilian pharmacopoeia tested their effects on GST expression in hepatocytes and Jurkat (leukemia) T-cells. The methanol extracts of five plants inhibited GST expression in hepatocytes. Three plants significantly inhibited and four others induced GST expression in Jurkat cells. Among these, the extracts of Bauhinia forficata Link. (Leguminosae) and Cecropia pachystachya Tréc. (Urticaceae) inhibited GST expression at relatively low concentrations. With the exception of B. forficata, all plants were cytotoxic when administered to Jurkat cells at high doses (1 mg/mL) and some extracts were considerably cytotoxic even at lower concentrations. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
71. Alkaline phosphatase as a reporter of σS levels and rpoS polymorphisms in different E. coli strains.
- Author
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Spira, Beny and Ferenci, Thomas
- Subjects
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ALKALINE phosphatase , *ESCHERICHIA coli , *RNA polymerases , *GENETIC polymorphisms , *PHOSPHATASES , *PROMOTERS (Genetics) , *TRANSFERASES , *MICROBIAL mutation , *GENETIC mutation , *MICROBIOLOGY - Abstract
σS is responsible for the transcriptional regulation of genes related to protection against stresses and bacterial survival and it accumulates in the cell under conditions of stress, such as nutrient limitation. An increase in the levels of σS causes a reduction in the expression of genes that are transcribed by RNA polymerase associated with the principal sigma factor, σ70. phoA, that encodes alkaline phosphatase (AP) is expressed under phosphate shortage conditions, and is also repressed by σS. Here we show that in a Pi-limited chemostat, accumulation of rpoS mutations is proportional to the intrinsic level of σS in the cells. Acquisition of mutations in rpoS relieves repression of the PHO genes. We also devised a non-destructive method based on the rpoS effect on AP that differentiates between rpoS + and rpoS mutants, as well as between high and low-σS producers. Using this method, we provide evidence that σS contributes to the repression of AP under conditions of Pi excess and that AP variation among different strains is at least partly due to intrinsic variation in σS levels. Consequently, a simple and non-destructive AP assay can be employed to differentiate between strains expressing different levels of σS on agar plates. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
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72. The effect of IHF on σ S selectivity of the phoA and pst promoters of Escherichia coli.
- Author
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Taschner, Natalia Pasternak, Yagil, Ezra, and Spira, Beny
- Subjects
ESCHERICHIA coli ,ESCHERICHIA ,BACTERIA ,OPERONS ,GENETIC transcription ,PROMOTERS (Genetics) - Abstract
The pst operon, a member of the PHO regulon of Escherichia coli, encodes a high-affinity phosphate transport system whose expression is induced when the cells enter a phase of phosphate starvation. The expression of pst is stimulated by the integration host factor (IHF). Transcription of the PHO regulon genes is initiated by the RNA polymerase complexed with σ
D (E σD ). Owing to a cytosine residue at position −13 of the pst promoter its transcription can also be initiated by E σS . Here, we show that inactivation of IHF in vivo abolishes the σS -dependent transcription initiation of the pst operon, indicating that both −13C residue and IHF are required to confer on pst the ability to be transcribed by E σS . Introduction of a −13C residue in the promoter region of phoA, another PHO regulon gene that is not directly affected by IHF, did not affect its exclusive transcription initiation by E σD . [ABSTRACT FROM AUTHOR]- Published
- 2006
- Full Text
- View/download PDF
73. Identificación de una mutación en una cepa no adherente de escherichia coli enteropatogenica epec
- Author
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Spira, Beny, Corredor Fuentes, Ingrid Johana, Spira, Beny, and Corredor Fuentes, Ingrid Johana
74. A differential effect of σS on the expression of the PHO regulon genes of Escherichia coli.
- Author
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Taschner, Natalia Pasternak, Yagii, Ezra, and Spira, Beny
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ESCHERICHIA coli , *RNA polymerases , *ALKALINE phosphatase , *PHOSPHATASES , *MICROBIOLOGY , *CARRIER proteins - Abstract
The RNA polymerase core associated with σS transcribes many genes related to stress or to the stationary phase. When cells enter a phase of phosphate starvation, the transcription of several genes and operons, collectively known as the PHO regulon, is strongly induced. The promoters of the PHO genes hitherto analysed are recognized by σD-associated RNA polymerase. A mutation in the gene that encodes σS, rpoS, significantly increases the level of alkaline phosphatase activity and the overproduction of σS inhibits it. Other PHO genes such as phoE and ugpB are likewise affected by σS. In contrast, pstS, which encodes a periplasmic phosphate-binding protein and is a negative regulator of PHO, is stimulated by σS. The effect of σS on the PHO genes is at the transcriptional level. It is shown that a cytosine residue at position -13 is important for the positive effect of σS on pst. The interpretation of these observations is based on the competition between σS and σD for the binding to the core RNA polymerase. [ABSTRACT FROM AUTHOR]
- Published
- 2004
- Full Text
- View/download PDF
75. Caracterização do mecanismo de Quorum Sensing (QS) em Zymomonas Mobilis em resposta ao auto-indutor do tipo 2 (AI-2)
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Silva, Juliana de Fatima dos Santos, Nunes, Luiz Roberto, Spira, Beny, Ribeiro, César Augusto João, Arruda, Denise Costa, and Rodrigues, Tiago
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ZYMOMONAS MOBILIS ,QUORUM SENSING ,TRANSCRIPTÔMICA ,PROGRAMA DE PÓS-GRADUAÇÃO EM BIOSSISTEMAS - UFABC ,TRANSCRIPTOME - Abstract
Orientador: Prof. Dr. Luiz Roberto Nunes Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, Sâo Bernardo do Campo, 2022. A bactéria Gram-negativa Zymomonas mobilis apresenta diversas características que a tornam atrativa para a produção industrial de diferentes bioprodutos, incluindo o bioetanol. Técnicas de manipulação genética foram desenvolvidas para esta bactéria, que vem sendo modificada geneticamente e/ou adaptada para trabalhar em sistemas de bioprocessamento integrado, produção de diversos produtos de alto valor agregado além de incorporar fontes alternativas de carbono às suas vias metabólicas. Dessa forma, Z. mobilis vem sendo vista como um microrganismo de grande potencial para a indústria de bioconversão e um melhor entendimento acerca dos mecanismos por ela utilizados para o controle de sua expressão gênica pode auxiliar no desenvolvimento de novas linhagens geneticamente manipuladas, com elevada capacidade de produção industrial. O autoindutor 2 (ou AI-2) é uma das moléculas utilizadas pelas bactérias para desencadear a resposta Quorum Sensing (QS), que ativa a expressão de genes envolvidos em uma série de mecanismos alternativos, quando as células atingem altas densidades populacionais (incluindo bioluminescência, motilidade, formação de biofilme, resistência ao estresse, fatores de patogenicidade, entre outros). Ao contrário da maioria dos autoindutores, o AI-2 pode induzir respostas QS tanto em bactérias Gram-negativas quanto Gram-positivas. Desta forma, esta molécula se apresenta como um sistema trans-específico de comunicação, capaz de afetar até mesmo bactérias que não podem produzir esse autoindutor. Neste trabalho, demonstramos que a bactéria Gram-negativa etanologênica Z. mobilis (não produtora de AI-2) responde ao AI-2 exógeno, modulando a expressão de genes envolvidos em mecanismos tipicamente associados a QS em outras bactérias como motilidade, Reparação de DNA e fixação de nitrogênio. Além disso, o metabolismo de células de Z. mobilis induzidas por AI-2 parece favorecer a produção de etanol sobre o acúmulo de biomassa, provavelmente como uma adaptação à alta demanda energética de fixação de N2. Do ponto de vista ambiental, esta estratégia pode estar ligada a mecanismos de sobrevivência em ambientes altamente competitivos onde Z. mobilis ativa a fixação de nitrogênio para sobreviver em meios escassos desta molécula e se torna mais eficaz na relação de produção glicose-etanol. Com estes achados, são abertas novas possibilidades de empregar AI-2 durante a produção industrial de etanol de segunda geração, como forma de aumentar a fixação de N2 por essas bactérias sem comprometer a produção de etanol. The Gram-negative bacterium Zymomonas mobilis has several characteristics that make it attractive for the industrial production of different bioproducts, including bioethanol. Genetic manipulation techniques were developed for this bacterium, which has been genetically modified and/or adapted to work in integrated bioprocessing systems, producing several products with high added value, in addition to incorporating alternative sources of carbon to its metabolic pathways. Thus, Z. mobilis has been seen as a microorganism of great potential for the bioconversion industry and a better understanding of the mechanisms used by it to control its gene expression can help in the development of new genetically manipulated strains, with high capacity of industrial production. Autoinducer 2 (or AI-2) is one of the molecules used by bacteria to trigger the Quorum Sensing (QS) response, which activates the expression of genes involved in a series of alternative mechanisms when cells reach high population densities (including bioluminescence, motility, biofilm formation, stress resistance, pathogenicity factors, among others). Unlike most autoinducers, AI-2 can induce QS responses in both Gramnegative and Gram-positive bacteria. In this way, this molecule presents itself as a trans-specific communication system, capable of affecting even bacteria that cannot produce this autoinducer. In this work, we demonstrate that the ethanologenic Gram-negative bacterium Z. mobilis (nonAI-2 producing) responds to exogenous AI-2, modulating the expression of genes involved in mechanisms typically associated with QS in other bacteria such as motility, DNA repair and nitrogen fixation. Furthermore, AI-2-induced metabolism of Z. mobilis cells seems to favor ethanol production over biomass accumulation, probably as an adaptation to the high energy demand of N2 fixation. From an environmental point of view, this strategy may be linked to survival mechanisms in highly competitive environments where Z. mobilis activates nitrogen fixation to survive in the scarce media of this molecule and becomes more efficient in the glucose-ethanol production relationship. With these findings, new possibilities are opened to use AI-2 during the industrial production of second-generation ethanol, as a way of increasing N2 fixation by these bacteria without compromising ethanol production.
- Published
- 2022
76. Ugp and PitA Participate in the Selection of PHO-Constitutive Mutants.
- Author
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Neves, Henrique Iglesias, Pereira, Tuanny Fernanda, Yagil, Ezra, and Spira, Beny
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ESCHERICHIA coli , *GLYCERIN , *HISTIDINE kinases , *PROTEINS , *CARBON - Abstract
Mutations that cause the constitutive expression of the PHO regulon of Escherichia coli occur either in the pst operon or in the phoR gene, which encode, respectively, a high-affinity Pi transport system and a histidine kinase sensor protein. These mutations are normally selected on glycerol-2-phosphate (G2P) as the carbon source in the presence of excess Pi. The emergence of early PHO-constitutive mutants, which appear after growth for up to 48 h on selective medium, depends on the presence of phoA, which codes for a periplasmic alkaline phosphatase, while late mutants, which appear after 48 h, depend both on phoA and on the ugp operon, which encodes a glycerophosphodiester transport system. The emergence of the late mutants hints at an adaptive mutation process. PHO-constitutive phoR mutants appear only in a host that is mutated in pitA, which encodes an alternative Pi transport system that does not belong to the PHO regulon. The conserved Thr217 residue in the PhoR protein is essential for PHO repression. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
77. Construção de uma linhagem geneticamente modificada de Zymomonas Mobilis com vistas à produção de álcoois de cadeia longa
- Author
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Farnezio, Vinícius Manganaro, Nunes, Luiz Roberto, Spira, Beny, and Puzer, Luciano
- Subjects
MICROBIOLOGY ,ÁLCOOL DE CADEIA LONGA ,ZYMOMONAS MOBILIS ,PROGRAMA DE PÓS-GRADUAÇÃO EM BIOSSISTEMAS - UFABC ,MICROBIOLOGIA ,ENGENHARIA GENÉTICA ,LONG CHAIN ALCOHOL ,BIOFUEL ,GENETIC ENGINEERING ,BIOCOMBUSTÍVEL - Abstract
Orientador: Prof. Dr. Luiz Roberto Nunes Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, Santo André, 2021. A bactéria Gram-negativa Zymomonas mobilis possui diversas características que a tornam atrativa para a produção industrial de diversos bioprodutos, incluindo o bioetanol. Por exemplo, Z. mobilis pode ser cultivada tanto em condições aeróbicas como anaeróbicas, é capaz de converter glicose em etanol com maior eficiência do que a levedura Saccharomyces cerevisiae e tolera concentrações mais elevadas deste álcool, durante o processo de fermentação. No entanto, essa bactéria pode ser geneticamente modificada para produzir outros tipos de biocombustíveis e produtos de alto valor agregado, como o isobutanol, além de outros álcoois de cadeia longa (Long Chain Alcohols, ou LCAs), que possuem maior densidade energética do que o etanol. O presente projeto visa empregar uma abordagem de manipulação genética, originalmente desenvolvida em Escherichia coli, para construir uma linhagem de Z. mobilis modificada, que poderá ser posteriormente utilizada para a produção de álcoois de cadeia longa. Para tanto, dois genes exógenos foram clonados em Z. mobilis: uma 2-cetoácido descarboxilase de amplo espectro (gene kivd, de Lactococcus lactis), capaz de converter 2-cetoácidos em aldeídos e uma álcool desidrogenase de amplo espectro (gene adh2 de S. cerevisiae), que reduz esses aldeídos aos seus respectivos álcoois. Essa nova cepa de Z. mobilis, denominada ZmLCA1, poderá ser utilizada em abordagens de engenharia metabólica, visando a produção de isobutanol e outros LCAs. Além disso, dada a alta toxicidade do isobutanol, esforços foram feitos para a obtenção de uma cepa de Z. mobilis com maior resistência a este LCA, com o auxílio de uma abordagem de evolução dirigida, baseada no uso de cultura contínua, em um biorreator. The Gram-negative bacterium Zymomonas mobilis has several characteristics that make it attractive for the industrial production of different bioproducts, including bioethanol. For example, Z. mobilis can be grown in both aerobic and anaerobic conditions, is capable of converting glucose to ethanol more efficiently than the yeast Saccharomyces cerevisiae and tolerates higher concentrations of this alcohol during the fermentation process. However, this bacterium can be genetically modified to produce other types of biofuels and value-added products, such as isobutanol and other long-chain alcohols (LCAs), which have a higher energy density than ethanol. The current project aims to employ a genetic manipulation approach, originally developed in Escherichia coli, to construct a modified Z. mobilis strain that can be further utilized for the production of long chain alcohols. For that, two exogenous genes were cloned into Z. mobilis: a broad-spectrum 2-keto acid decarboxylase (kivd gene, from Lactococcus lactis), capable of converting 2-keto acids into aldehydes and a broad-spectrum alcohol dehydrogenase (adh2 gene from S. cerevisiae), which reduces these aldehydes to their respective alcohols. This new Z. mobilis strain, called ZmLCA1, can hopefully be used in metabolic engineering approaches, aimed at the production of isobutanol and other LCAs. Moreover, given the high toxicity of isobutanol, efforts were made to obtain a Z. mobilis strain with increased resistance to this LCA, with the aid of a directed evolution approach, based on the use of continuous culturing, in a bioreactor.
- Published
- 2021
78. Characterization of Xanthomonas citri subsp. citri extracytoplasmic function sigma factor SigF
- Author
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Lidia dos Passos Lima, Alvarez-Martinez, Cristina Elisa, 1976, Silva Neto, José Freire da, Spira, Beny, Universidade Estadual de Campinas. Instituto de Biologia, Programa de Pós-Graduação em Genética e Biologia Molecular, and UNIVERSIDADE ESTADUAL DE CAMPINAS
- Subjects
Regulação da expressão gênica ,Gene expression regulation ,Protein kinases ,Sigma factor ,Fator sigma ,Xanthomonas citri subsp. citri ,Proteínas quinases - Abstract
Orientador: Cristina Elisa Alvarez Martinez Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Xanthomonas citri subsp. citri (Xac) é uma bactéria Gram-negativa responsável pela doença do cancro cítrico em uma grande variedade de plantas cítricas, causando grandes perdas econômicas. Xac sobrevive como epífita nas superfícies foliares e causa a doença após a penetração e crescimento dentro do parênquima mesofílico de plantas suscetíveis. Como uma bactéria associada a plantas, Xac precisa se adaptar às condições ambientais em constante mudança. Fatores sigma alternativos da família extracitoplásmica (ECF) estão envolvidos na regulação da expressão gênica em resposta ao estresse ambiental. O genoma de Xac306 codifica 9 fatores sigmas ECF, o que provavelmente reflete seu estilo de vida e sugere um papel importante desta família de reguladores transcricionais na adaptação de Xac. O objetivo deste estudo é caracterizar a função de um fator sigma ECF encontrado no genoma de Xac, denominado sigF. Para isso, foram obtidas duas linhagens com níveis alterados de sigF: uma linhagem mutante (?sigF) por deleção em fase do gene e uma linhagem com cópia extra do gene em vetor multicópia, o que promove a super-expressão deste sigma. Ensaios de virulência em hospedeiro suscetível demonstraram que sigF não é necessário para a virulência em Xac. A sensibilidade da linhagem ?sigF a diferentes tipos de estresse ambiental também foi analisada, incluindo estresse oxidativo, por radiação UV, deficiência de ferro e tratamento com o agente quelante de metais ácido fítico, não sendo observadas diferenças na viabilidade quando comparada com a linhagem selvagem. Além disso, as linhagens com níveis alterados de sigF não apresentaram alterações na capacidade de formar biofilmes e aderir às folhas da planta. Curiosamente, observamos que SigF é essencial para a sobrevivência de Xac a predação pela ameba de vida livre Dictyostelium discoideum. Resultados de ensaios de RT-PCR quantitativo e RNA-Seq demonstram que a super-expressão de uma versão que mimetiza um estado fosforilado de SigF causa indução da expressão de genes que codificam componentes de um sistema de secreção do tipo VI (SSVI) de Xac,que também tem papel essencial na sobrevivência a D.discoideum. Ensaios preliminares de fosforilação in vitro pela incubação de SigF purificada com a proteína quinase PknB, codificada por um gene localizado em provável operon com sigF, reforçam o modelo proposto, no qual SigF é regulado em nível pós-traducional por fosforilação. Além disto, nossos resultados demonstram que SigF regula positivamente a expressão do SSVI de Xac Abstract: Xanthomonas citri subsp. citri (Xac) is a Gram-negative bacterium responsible for citrus canker disease in a wide variety of citrus plants, causing major economic losses. Xac survives as epiphyte on leaf surfaces and causes the disease after penetration and growth inside mesophyll parenchyma of susceptible plants. As a plant-associated bacterium, Xac needs to adapt to constantly changing environmental conditions. Alternative sigma factors of the extracytoplasmic family (ECF) are involved in the regulation of gene expression in response to environmental stress. The genome of Xac strain 306 encodes 9 sigma factors of the ECF family, which probably reflects its lifestyle and suggests an important role of this family of transcriptional regulators in Xac adaptation. The aim of this study is to characterize the function of one ECF sigma factor found in Xac genome, named sigF. For that, we have constructed a sigF mutant strain (?sigF) by in frame deletion of the gene and an overexpression strain, which contains an extra copy of sigF in a multicopy plasmid. Results from virulence assays in a susceptible host have shown that sigF is not required for Xac virulence. We've also tested sensitivity of the ?sigF strain to distinct types of environmental stress, including oxidative stress, UV radiation, iron deprivation and treatment with the metal chelating agent phytic acid and no difference in viability was observed when compared to the wild type strain, indicating the SigF is not required under these conditions. In addition, we have not observed differences in biofilm formation and adhesion to plant leaves caused by deletion or overexpression of sigF. Interestingly, our results show that SigF is required for Xac survival to predation by the amoeba Dictiostelium discoideum. In addition, results from qRT-PCR analysis and RNA-seq experiments have shown that overexpression of a SigF phospho-mimicking mutant version promotes induction of genes from a Xac type VI secretion system required for survival to D. discoideum. Prelimary in vitro phosphorylation assays using purified SigF and PknB Ser/thr kinase encoded in the same operon as sigF corroborates with a model in which SigF is post-translational regulated by phosphorylation. Our results also suggest that SigF positively regulates Xac SSVI Mestrado Microbiologia Mestra em Genética e Biologia Molecular FAPESP 2014/19720-6
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- 2017
79. Ciprofloxacin-Mediated Mutagenesis Is Suppressed by Subinhibitory Concentrations of Amikacin in Pseudomonas aeruginosa
- Author
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Beny Spira, Rodrigo S. Galhardo, Fernanda Esposito, Jesús Blázquez, Estela Y. Valencia, Fundação de Amparo à Pesquisa do Estado de São Paulo, Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brasil), Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (España), Red Española de Investigación en Patología Infecciosa, European Commission, Blázquez Gómez, Jesús [0000-0003-0495-3848], Galhardo, Rodrigo [0000-0002-5686-9704], [Valencia, Estela Ynes] Univ Sao Paulo, Inst Biomed Sci, Dept Microbiol, Sao Paulo, Brazil, [Spira, Beny] Univ Sao Paulo, Inst Biomed Sci, Dept Microbiol, Sao Paulo, Brazil, [Galhardo, Rodrigo S.] Univ Sao Paulo, Inst Biomed Sci, Dept Microbiol, Sao Paulo, Brazil, [Esposito, Fernanda] Univ Sao Paulo, Sch Pharm, Dept Clin Anal, Sao Paulo, Brazil, [Blazquez, Jesus] Inst Biomed Sevilla IBIS CSIC, Seville, Spain, [Blazquez, Jesus] CSIC, Ctr Nacl Biotecnol, Madrid, Spain, Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil (FAPESP), Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil CNPq, postdoctoral fellowship from CNPq, Plan Nacional de, Instituto de Salud Carlos III, Subdireccion General de Redes y Centros de Investigacion Cooperativa, Ministerio de Economia y Competitividad, Spanish Network for Research in Infectious Diseases, European Regional Development Fund (ERDF), Blázquez Gómez, Jesús, and Galhardo, Rodrigo
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0301 basic medicine ,Target ,SOS response ,Antibiotics ,genetic processes ,Gene Expression ,Levofloxacin ,medicine.disease_cause ,Plasmid ,Disk Diffusion Antimicrobial Tests ,Genes, Reporter ,Ciprofloxacin ,Pharmacology (medical) ,Luciferases ,Reca protein ,Inhibition ,Chemistry ,Vectors ,Anti-Bacterial Agents ,Infectious Diseases ,Damage ,Amikacin ,Combination ,Pseudomonas aeruginosa ,recA ,medicine.drug ,Plasmids ,medicine.drug_class ,030106 microbiology ,Chromosome ,Microbiology ,03 medical and health sciences ,SOS Response (Genetics) ,Mechanisms of Resistance ,medicine ,SOS Response, Genetics ,Pharmacology ,Mutagenesis ,DNA ,biochemical phenomena, metabolism, and nutrition ,enzymes and coenzymes (carbohydrates) ,Rec A Recombinases ,Mutation ,bacteria ,Antibiotic-resistance - Abstract
Resistance to antibiotics is a global health problem. Activation of the SOS response, and the subsequent elevation in mutagenesis, contributes to the appearance of resistance mutations. Among currently used drugs, quinolones are the most potent inducers of the SOS response. In the present study, we show that amikacin inhibits ciprofloxacin-mediated SOS induction and mutagenesis in Pseudomonas aeruginosa., This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (FAPESP; grant 2014/15982-6), and Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (CNPq; grant 407259/2013-9). E.Y.V. was funded by a postdoctoral fellowship from CNPq (grant 151264/2014-7). J.B. was supported by Plan Nacional de I+D+i and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD12/0015), cofinanced by the European Regional Development Fund (ERDF) “A way to achieve Europe” and grant FIS PI13/00063.
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- 2017
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