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1.
PLoS One ; 14(3): e0213933, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30908529

RESUMO

Eis (Enhanced Intracellular Survival) is an important aminoglycoside N-acetyltransferase enzyme contributing to kanamycin resistance in Mtb clinical isolates. Eis proteins from M. tuberculosis (RvEis) and M. smegmatis (MsEis) have 58% identical and 69% similar amino acid sequences and acetylate aminoglycosides at multiple amines. Both the Eis proteins are hexameric and composed of two symmetric trimers. RvEis has remarkable structural stability and heat-stable aminoglycoside acetyltransferase activity. Although the structure and biochemical properties of MsEis have been studied earlier, the detailed characterization of its acetyltransferase activity and structural stability is lacking. In this study, we have performed comparative analysis of structural stability and aminoglycoside acetyltransferase activity of RvEis and MsEis proteins. Unlike RvEis, MsEis undergoes a three-state unfolding induced by heat or chemical denaturants and involves self-association of partially unfolded oligomers to form high molecular weight soluble aggregates. MsEis is highly susceptible to chemical denaturants and unfolds completely at lower concentrations of GdmCl and urea when compared to RvEis. In contrast to RvEis, the oligomeric forms of MsEis are SDS sensitive. However, SDS treatment resulted in increased helix formation in MsEis than RvEis. MsEis shows lesser thermostable activity with a decreased efficiency of kanamycin acetylation in comparison to RvEis. Furthermore, overexpression of MsEis does not provide thermal resistance to M. smegmatis unlike RvEis. Collectively, this study reveals that homologous proteins from pathogenic and nonpathogenic mycobacteria follow different modes of unfolding and demonstrate differential structural stability and activity despite highly similar sequences and oligomeric organization.


Assuntos
Acetiltransferases/química , Acetiltransferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Acetiltransferases/genética , Proteínas de Bactérias/genética , Humanos , Resistência a Canamicina/genética , Resistência a Canamicina/fisiologia , Cinética , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/patogenicidade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Conformação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Especificidade da Espécie , Espectrometria de Fluorescência , Termodinâmica , Resposta a Proteínas não Dobradas
2.
Plasmid ; 102: 62-70, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30825470

RESUMO

Two B/O plasmids were sequenced. The kanamycin resistance plasmid R805a was found in a Salmonella Typhi strain from a 1972 typhoid fever outbreak in Mexico City. pCERC6, which confers resistance to ampicillin, streptomycin and sulphamethoxazole, was found in a commensal E. coli isolated from a healthy adult in Sydney in 2008. Both plasmids contain the same gene for RNAI, the incompatibility determinant, as the reference B/O plasmid pMU707 and the recently reported plasmid p838B-R, which came from a commensal E. coli isolated in Sydney in 2004. However, three different repA alleles are associated with the IncB/O rnaI. Whereas the repA gene of p838B-R is identical to that of pMU707, R805a and pCERC6 each carry a different repA. Comparison of the plasmid backbones revealed that R805a has a different oriT-nikAB region to that in pCERC6 and p838B-R, encoding different NikA relaxosome accessory factors and NikB relaxases. The different nikA genes were associated with oriT sites that differed in the sequences of their long, imperfect inverted repeats. A further 11 publicly available complete plasmid sequences contain the same B/O rnaI, with either the repA of pMU707/p838B-R or of R805a, and the oriT-nikAB region of p838B-R/pCERC6 or of R805a. All four possible combinations were seen. Extensive variation was also observed in the traY-excA entry exclusion region, and in the locations of mobile elements, including ones carrying antibiotic resistance genes. Plasmids that contain the same rnaI gene would be considered the same by a number of typing methods, but this study has unveiled previously unnoticed mosaicism in the backbones of one such group. This highlights the importance of considering entire plasmid backbones for the typing and tracking of specific lineages.


Assuntos
Mosaicismo , Plasmídeos/genética , Sequência de Bases , Mapeamento Cromossômico , Replicação do DNA/genética , Elementos de DNA Transponíveis/genética , Resistência a Canamicina/genética , Mapeamento por Restrição
3.
Biochem Biophys Res Commun ; 505(1): 333-337, 2018 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-30245132

RESUMO

Escherichia coli ß-lactamase TEM-1 is potentially useful in the antibody-directed enzyme/prodrug therapy (ADEPT), converting nontoxic prodrugs to toxic agents. The produced toxin would kill cancer cells, when the enzyme is attached to a tumor-antigen-specific antibody. However, the off-site reaction possibly occurring in the blood or normal tissues raises safety concern. In the present study, we engineered TEM-1 variants preferentially active at pH 5.8-6.2, near the pH of the acidic microenvironment of tumor. A library of randomly mutagenized variants was screened for the ability to confer an antibiotic resistance on E. coli cells in acidic growth media and not in neutral media, to isolate a variant with a Thr-to-Ile substitution at position 160. An extensive mutagenesis study was then conducted in the proximity of this position, to show that a Leu162Glu mutation also causes the acid preference. Kinetic analyses indicated that the overall activity of the wild-type TEM-1 hardly changes over a pH range from 5.8 to 7.0, whereas TEM-1(T160I) is 1.5-times as active at pH 6.2 than pH 7.0, and TEM-1(T160I) is 3.1-fold as active at pH 5.8 than pH 7.0. A further mutagenesis study suggested that a change in the overall structure of the enzyme underlies the pH dependency of the variants.


Assuntos
Substituição de Aminoácidos , Proteínas de Escherichia coli/genética , Resistência a Canamicina/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Biocatálise , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Canamicina/farmacologia , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios Proteicos , Engenharia de Proteínas/métodos , beta-Lactamases/química , beta-Lactamases/metabolismo
4.
PLoS One ; 13(3): e0193435, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29513730

RESUMO

While antimicrobial resistance in Salmonella enterica is mainly attributed to large plasmids, small plasmids may also harbor antimicrobial resistance genes. Previously, three major groups of ColE1-like plasmids conferring kanamycin-resistance (KanR) in various S. enterica serotypes from diagnostic samples of human or animals were reported. In this study, over 200 KanR S. enterica isolates from slaughter samples, collected in 2010 and 2011 as a part of the animal arm of the National Antimicrobial Resistance Monitoring System, were screened for the presence of ColE1-like plasmids. Twenty-three KanR ColE1-like plasmids were successfully isolated. Restriction fragment mapping revealed five major plasmid groups with subgroups, including two new groups, X (n = 3) and Y/Y2/Y3 (n = 4), in addition to the previously identified groups A (n = 7), B (n = 6), and C/C3 (n = 3). Nearly 75% of the plasmid-carrying isolates were from turkey and included all the isolates carrying X and Y plasmids. All group X plasmids were from serotype Hadar. Serotype Senftenberg carried all the group Y plasmids and one group B plasmid. All Typhimurium isolates (n = 4) carried group A plasmids, while Newport isolates (n = 3) each carried a different plasmid group (A, B, or C). The presence of the selection bias in the NARMS strain collection prevents interpretation of findings at the population level. However, this study demonstrated that KanR ColE1-like plasmids are widely distributed among different S. enterica serotypes in the NARMS isolates and may play a role in dissemination of antimicrobial resistance genes.


Assuntos
Resistência a Canamicina , Carne/microbiologia , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/isolamento & purificação , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Bovinos/microbiologia , Galinhas/microbiologia , Monitoramento Epidemiológico , Escherichia coli , Canamicina/farmacologia , Resistência a Canamicina/genética , Plasmídeos , RNA Bacteriano/metabolismo , Salmonella enterica/genética , Alinhamento de Sequência , Sus scrofa/microbiologia , Perus/microbiologia
5.
Biosci Biotechnol Biochem ; 82(7): 1169-1171, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29521166

RESUMO

Plasmid vector and allelic exchange mutagenesis systems were established for the genetic analysis of a phenanthrene-degrading mycobacterial strain, Mycobacterium sp. EPa45. Successful application of these systems revealed the necessity of the EPa45 phdI gene for the degradation of 1-hydroxy-2-naphthoate, which has been proposed to be an intermediate product in the degradation pathway of phenanthrene.


Assuntos
Alelos , Biodegradação Ambiental , Genes Bacterianos , Vetores Genéticos , Mutagênese , Mycobacterium/metabolismo , Naftóis/metabolismo , Fenantrenos/metabolismo , Plasmídeos , Sequência de Bases , Cloranfenicol/farmacologia , Cromossomos Bacterianos , Troca Genética , Resistência a Canamicina/genética , Mycobacterium/genética , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Recombinação Genética , Rhodococcus/genética , Poluentes do Solo/metabolismo , Resistência a Tetraciclina/genética
6.
Molecules ; 22(12)2017 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-29257114

RESUMO

Aminoglycosides are a group of antibiotics used since the 1940s to primarily treat a broad spectrum of bacterial infections. The primary resistance mechanism against these antibiotics is enzymatic modification by aminoglycoside-modifying enzymes that are divided into acetyl-transferases, phosphotransferases, and nucleotidyltransferases. To overcome this problem, new semisynthetic aminoglycosides were developed in the 70s. The most widely used semisynthetic aminoglycoside is amikacin, which is refractory to most aminoglycoside modifying enzymes. Amikacin was synthesized by acylation with the l-(-)-γ-amino-α-hydroxybutyryl side chain at the C-1 amino group of the deoxystreptamine moiety of kanamycin A. The main amikacin resistance mechanism found in the clinics is acetylation by the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib], an enzyme coded for by a gene found in integrons, transposons, plasmids, and chromosomes of Gram-negative bacteria. Numerous efforts are focused on finding strategies to neutralize the action of AAC(6')-Ib and extend the useful life of amikacin. Small molecules as well as complexes ionophore-Zn+2 or Cu+2 were found to inhibit the acetylation reaction and induced phenotypic conversion to susceptibility in bacteria harboring the aac(6')-Ib gene. A new semisynthetic aminoglycoside, plazomicin, is in advance stage of development and will contribute to renewed interest in this kind of antibiotics.


Assuntos
Amicacina/farmacologia , Antibacterianos/farmacologia , Amicacina/química , Antibacterianos/química , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Genes Bacterianos , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/genética , Humanos , Resistência a Canamicina/genética
7.
Tuberculosis (Edinb) ; 107: 1-4, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29050755

RESUMO

Rapid detection of resistance to the second-line drugs is essential for early initiation of appropriate anti-tubercular treatment regimen among multi-drug tuberculosis (MDR-TB). In this study, we applied a multiplex allele-specific PCR (MAS-PCR) to identify the mutations on codons 90 and 94 of gyrA and nucleotide 1401 of rrs for detecting ofloxacin (OFX) and kanamycin (KAN) resistance in 139 MDR-TB isolates from China. Using the traditional phenotypic method as the reference, MAS-PCR detected resistance to OFX and KAN with sensitivities of 67.3% and 76.5%, respectively, and specificities of 100.0%. Therefore, MAS-PCR assays can be used for rapid detection of second-line drug resistance among MDR-TB in China, enabling early administration of appropriate treatment regimens to the affected MDR-TB patients.


Assuntos
Técnicas Bacteriológicas , Análise Mutacional de DNA/métodos , Farmacorresistência Bacteriana Múltipla/genética , Reação em Cadeia da Polimerase Multiplex , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Antituberculosos/uso terapêutico , China , DNA Girase/genética , Genótipo , Humanos , Canamicina/uso terapêutico , Resistência a Canamicina/genética , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Ofloxacino/uso terapêutico , Valor Preditivo dos Testes , Proteínas Ribossômicas/genética , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico
8.
G3 (Bethesda) ; 7(12): 3955-3966, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29046437

RESUMO

Evolve and resequence experiments have provided us a tool to understand bacterial adaptation to antibiotics. In our previous work, we used short-term evolution to isolate mutants resistant to the ribosome targeting antibiotic kanamycin, and reported that Escherichia coli develops low cost resistance to kanamycin via different point mutations in the translation Elongation Factor-G (EF-G). Furthermore, we had shown that the resistance of EF-G mutants could be increased by second site mutations in the genes rpoD/cpxA/topA/cyaA Mutations in three of these genes had been discovered in earlier screens for aminoglycoside resistance. In this work, we expand our understanding of these second site mutations, the goal being to understand how these mutations affect the activities of the mutated gene products to confer resistance. We show that the mutation in cpxA most likely results in an active Cpx stress response. Further evolution of an EF-G mutant in a higher concentration of kanamycin than what was used in our previous experiments identified the cpxA locus as a primary target for a significant increase in resistance. The mutation in cyaA results in a loss of catalytic activity and probably results in resistance via altered CRP function. Despite a reduction in cAMP levels, the CyaAN600Y mutant has a transcriptome indicative of increased CRP activity, pointing to an unknown role for CyaA and / or cAMP in gene expression. From the transcriptomes of double and single mutants, we describe the epistasis between the mutation in EF-G and these second site mutations. We show that the large scale transcriptomic changes in the topoisomerase I (FusAA608E-TopAS180L) mutant likely result from increased negative supercoiling in the cell. Finally, genes with known roles in aminoglycoside resistance were present among the misregulated genes in the mutants.


Assuntos
Toxina Adenilato Ciclase/genética , Toxinas Bacterianas/genética , Proteínas de Escherichia coli/genética , Resistência a Canamicina/genética , Fator G para Elongação de Peptídeos/genética , Transcriptoma/genética , Antibacterianos/efeitos adversos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Canamicina/efeitos adversos , Mutação , Transcrição Genética/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos
9.
Anal Biochem ; 525: 44-45, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28249723

RESUMO

Kanamycin resistance is the most frequently used antibiotic-resistance marker for Arabidopsis transformations, however, this method frequently causes escape of untransformed plants, particularly at the high seedling density during the selection. Here we developed a robust high-density selection method using top agar for Arabidopsis thaliana. Top agar effectively suppressed growth of untransformed wild-type plants on selection media at high density. Survival of the transformed plants during the selection were confirmed by production of green true leaves and expression of a firefly luciferase reporter gene. Top agar method allowed selection using a large amount of seeds in Arabidopsis transformation.


Assuntos
Ágar/química , Antibacterianos/farmacologia , Arabidopsis/metabolismo , Engenharia Genética/métodos , Resistência a Canamicina/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Ágar/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Ensaios de Triagem em Larga Escala , Luciferases/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Sementes/efeitos dos fármacos , Sementes/genética , Sementes/metabolismo , Transformação Genética
10.
Methods Mol Biol ; 1498: 483-490, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27709596

RESUMO

Directed evolution is a powerful strategy for gene mutagenesis, and has been used for protein engineering both in scientific research and in the biotechnology industry. The routine method for directed evolution was developed by Stemmer in 1994 (Stemmer, Proc Natl Acad Sci USA 91, 10747-10751, 1994; Stemmer, Nature 370, 389-391, 1994). Since then, various methods have been introduced, each of which has advantages and limitations depending upon the targeted genes and procedure. In this chapter, a novel alternative directed evolution method which combines mutagenesis PCR with dITP and fragmentation by endonuclease V is described. The kanamycin resistance gene is used as a reporter gene to verify the novel method for directed evolution. This method for directed evolution has been demonstrated to be efficient, reproducible, and easy to manipulate in practice.


Assuntos
Mutagênese/genética , Biotecnologia/métodos , Desoxirribonuclease (Dímero de Pirimidina)/genética , Evolução Molecular Direcionada/métodos , Genes Reporter/genética , Resistência a Canamicina/genética , Mutagênese Sítio-Dirigida/métodos , Reação em Cadeia da Polimerase/métodos , Engenharia de Proteínas/métodos
11.
Antimicrob Agents Chemother ; 60(12): 7522-7523, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27736762

RESUMO

We studied the significance of particular eis mutations on Mycobacterium tuberculosis drug resistance using a specialized transduction strategy. Recombinant strains harboring eis promoter mutations C14T, C12T, and G10A exhibited kanamycin resistance with MICs of 40, 10, and 20 µg/ml, respectively, while recombinant strains harboring C14G and C15G mutations were kanamycin susceptible (MIC, 2.5 to 5 µg/ml). Each of the eis mutants tested remained amikacin susceptible (MIC, 0.5 to 4 µg/ml). The identification of specific eis mutations is needed for accurate genotypic susceptibility testing for kanamycin.


Assuntos
Antígenos de Bactérias/genética , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Canamicina/farmacologia , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Acetiltransferases , Amicacina/farmacologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Expressão Gênica , Genótipo , Resistência a Canamicina/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Transdução Genética
12.
Nucleic Acids Res ; 44(21): 10367-10376, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27655632

RESUMO

Casposons are a recently discovered group of large DNA transposons present in diverse bacterial and archaeal genomes. For integration into the host chromosome, casposons employ an endonuclease that is homologous to the Cas1 protein involved in protospacer integration by the CRISPR-Cas adaptive immune system. Here we describe the site-preference of integration by the Cas1 integrase (casposase) encoded by the casposon of the archaeon Aciduliprofundum boonei Oligonucleotide duplexes derived from the terminal inverted repeats (TIR) of the A. boonei casposon as well as mini-casposons flanked by the TIR inserted preferentially at a site reconstituting the original A. boonei target site. As in the A. boonei genome, the insertion was accompanied by a 15-bp direct target site duplication (TSD). The minimal functional target consisted of the 15-bp TSD segment and the adjacent 18-bp sequence which comprises the 3' end of the tRNA-Pro gene corresponding to the TΨC loop. The functional casposase target site bears clear resemblance to the leader sequence-repeat junction which is the target for protospacer integration catalyzed by the Cas1-Cas2 adaptation module of CRISPR-Cas. These findings reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the prokaryotic adaptive immunity systems.


Assuntos
Sítios de Ligação , Sistemas CRISPR-Cas , Elementos de DNA Transponíveis/genética , Endodesoxirribonucleases/metabolismo , Ordem dos Genes , Resistência a Canamicina/genética , Mutagênese Insercional , Conformação de Ácido Nucleico , Plasmídeos/genética , Sequências Repetidas Terminais
13.
BMC Microbiol ; 16(1): 202, 2016 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-27595984

RESUMO

BACKGROUND: The Formosan subterranean termite, Coptotermes formosanus is an invasive urban pest in the Southeastern USA. Paratransgenesis using a microbe expressed lytic peptide that targets the termite gut protozoa is currently being developed for the control of Formosan subterranean termites. In this study, we evaluated Trabulsiella odontotermitis, a termite-specific bacterium, for its potential to serve as a 'Trojan Horse' for expression of gene products in termite colonies. RESULTS: We engineered two strains of T. odontotermitis, one transformed with a constitutively expressed GFP plasmid and the other engineered at the chromosome with a Kanamycin resistant gene using a non- disruptive Tn7 transposon. Both strains were fed to termites from three different colonies. Fluorescent microscopy confirmed that T. odontotermitis expressed GFP in the gut and formed a biofilm in the termite hindgut. However, GFP producing bacteria could not be isolated from the termite gut after 2 weeks. The feeding experiment with the chromosomally engineered strain demonstrated that T. odontotermitis was maintained in the termite gut for at least 21 days, irrespective of the termite colony. The bacteria persisted in two termite colonies for at least 36 days post feeding. The experiment also confirmed the horizontal transfer of T. odontotermitis amongst nest mates. CONCLUSION: Overall, we conclude that T. odontotermitis can serve as a 'Trojan Horse' for spreading gene products in termite colonies. This study provided proof of concept and laid the foundation for the future development of genetically engineered termite gut bacteria for paratransgenesis based termite control.


Assuntos
Enterobacteriaceae/genética , Técnicas de Transferência de Genes , Engenharia Genética/métodos , Isópteros/microbiologia , Animais , Biofilmes/crescimento & desenvolvimento , Elementos de DNA Transponíveis , Sistema Digestório/microbiologia , Sistema Digestório/patologia , Enterobacteriaceae/metabolismo , Enterobacteriaceae/fisiologia , Microbioma Gastrointestinal , Genes Bacterianos , Canamicina/farmacologia , Resistência a Canamicina/genética , Controle Biológico de Vetores/métodos , Recombinação Genética , Transformação Bacteriana
14.
BMC Infect Dis ; 16: 458, 2016 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-27576542

RESUMO

BACKGROUND: Rapid molecular diagnostics, with their ability to quickly identify genetic mutations associated with drug resistance in Mycobacterium tuberculosis clinical specimens, have great potential as tools to control multi- and extensively drug-resistant tuberculosis (M/XDR-TB). The Qiagen PyroMark Q96 ID system is a commercially available pyrosequencing (PSQ) platform that has been validated for rapid M/XDR-TB diagnosis. However, the details of the assay's diagnostic and technical performance have yet to be thoroughly investigated in diverse clinical environments. METHODS: This study evaluates the diagnostic performance of the PSQ assay for 1128 clinical specimens from patients from three areas of high TB burden. We report on the diagnostic performance of the PSQ assay between the three sites and identify variables associated with poor PSQ technical performance. RESULTS: In India, the sensitivity of the PSQ assay ranged from 89 to 98 % for the detection of phenotypic resistance to isoniazid, rifampicin, fluoroquinolones, and the injectables. In Moldova, assay sensitivity ranged from 7 to 94 %, and in South Africa, assay sensitivity ranged from 71 to 92 %. Specificity was high (94-100 %) across all sites. The addition of eis promoter sequencing information greatly improved the sensitivity of kanamycin resistance detection in Moldova (7 % to 79 %). Nearly all (89.4 %) sequencing reactions conducted on smear-positive, culture-positive specimens and most (70.8 %) reactions conducted on smear-negative, culture-positive specimens yielded valid PSQ reads. An investigation into the variables influencing sequencing failures indicated smear negativity, culture negativity, site (Moldova), and sequencing of the rpoB, gyrA, and rrs genes were highly associated with poor PSQ technical performance (adj. OR > 2.0). CONCLUSIONS: This study has important implications for the global implementation of PSQ as a molecular TB diagnostic, as it demonstrates how regional factors may impact PSQ diagnostic performance, while underscoring potential gene targets for optimization to improve overall PSQ assay technical performance. TRIAL REGISTRATION: ClinicalTrials.gov ( #NCT02170441 ). Registered 12 June 2014.


Assuntos
Antituberculosos/farmacologia , Tuberculose Extensivamente Resistente a Medicamentos/diagnóstico , Mycobacterium tuberculosis/genética , Antituberculosos/uso terapêutico , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Fluoroquinolonas , Genes Bacterianos , Humanos , Isoniazida/farmacologia , Canamicina/farmacologia , Resistência a Canamicina/genética , Testes de Sensibilidade Microbiana , Técnicas de Diagnóstico Molecular , Tipagem Molecular , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Regiões Promotoras Genéticas , Rifampina/farmacologia , Sensibilidade e Especificidade , Análise de Sequência de DNA
16.
Plant Biotechnol J ; 14(4): 1151-60, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26426390

RESUMO

Genome modification by homology-directed repair (HDR) is an attractive tool for the controlled genetic manipulation of plants. Here, we report the HDR-mediated gene exchange of expression cassettes in tobacco BY-2 cells using a designed zinc finger nuclease (ZFN). The target contained a 7-kb fragment flanked by two ZFN cutting sites. That fragment was replaced with a 4-kb donor cassette, which integrates gene markers for selection (kanamycin resistance) and for scoring targeting (red fluorescent protein, RFP). Candidates resulting from cassette exchange were identified by molecular analysis of calli generated by transformation via direct DNA delivery. The precision of HDR-mediated donor integration was evaluated by Southern blot analysis, sequencing of the integration locus and analysis of RFP fluorescence by flow cytometry. Screening of 1326 kanamycin-resistant calli yielded 18 HDR events, 16 of which had a perfect cassette exchange at the insert junction and 13 of which produced functional RFP. Our results demonstrate that ZFN-based HDR can be used for high frequency, precise, targeted exchange of fragments of sizes that are commercially relevant in plants.


Assuntos
Desoxirribonucleases/metabolismo , Marcação de Genes/métodos , Tabaco/genética , Southern Blotting , Desoxirribonucleases/genética , Citometria de Fluxo/métodos , Resistência a Canamicina/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células Vegetais , Plantas Geneticamente Modificadas , Reparo de DNA por Recombinação/genética , Tabaco/citologia , Dedos de Zinco
17.
PLoS One ; 10(10): e0139414, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26436944

RESUMO

Aminoglycosides, amikacin (AK) and kanamycin (KM) are second line anti-tuberculosis drugs used to treat tuberculosis (TB) and resistance to them affects the treatment. Membrane and membrane associated proteins have an anticipated role in biological processes and pathogenesis and are potential targets for the development of new diagnostics/vaccine/therapeutics. In this study we compared membrane and membrane associated proteins of AK and KM resistant and susceptible Mycobacterium tuberculosis isolates by 2DE coupled with MALDI-TOF/TOF-MS and bioinformatic tools. Twelve proteins were found to have increased intensities (PDQuest Advanced Software) in resistant isolates and were identified as ATP synthase subunit alpha (Rv1308), Trigger factor (Rv2462c), Dihydrolipoyl dehydrogenase (Rv0462), Elongation factor Tu (Rv0685), Transcriptional regulator MoxR1(Rv1479), Universal stress protein (Rv2005c), 35kDa hypothetical protein (Rv2744c), Proteasome subunit alpha (Rv2109c), Putative short-chain type dehydrogenase/reductase (Rv0148), Bacterioferritin (Rv1876), Ferritin (Rv3841) and Alpha-crystallin/HspX (Rv2031c). Among these Rv2005c, Rv2744c and Rv0148 are proteins with unknown functions. Docking showed that both drugs bind to the conserved domain (Usp, PspA and SDR domain) of these hypothetical proteins and GPS-PUP predicted potential pupylation sites within them. Increased intensities of these proteins and proteasome subunit alpha might not only be neutralized/modulated the drug molecules but also involved in protein turnover to overcome the AK and KM resistance. Besides that Rv1876, Rv3841 and Rv0685 were found to be associated with iron regulation signifying the role of iron in resistance. Further research is needed to explore how these potential protein targets contribute to resistance of AK and KM.


Assuntos
Amicacina/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/fisiologia , Resistência Microbiana a Medicamentos/fisiologia , Canamicina/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Proteômica , Tuberculose/microbiologia , Motivos de Aminoácidos , Antituberculosos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Sequência Conservada , Sistemas de Liberação de Medicamentos , Resistência Microbiana a Medicamentos/genética , Eletroforese em Gel Bidimensional , Humanos , Ferro/fisiologia , Resistência a Canamicina/genética , Resistência a Canamicina/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Modelos Moleculares , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Conformação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Ubiquitinas/metabolismo
18.
Plant Sci ; 238: 53-63, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26259174

RESUMO

T-DNA insertion mutants play a crucial role in elucidating Arabidopsis gene function. In some cases, two or more T-DNA mutants are combined to study genetic interactions between homologous genes or genes hypothesized to act in the same pathway. We studied the significance of protein-protein interactions between CSN5A and ROP11 by crossing three independent rop11 T-DNA insertion mutants with csn5a-2, a partial loss-of-function intronic T-DNA insertion mutant. The csn5a-2 single mutant is severely stunted, but double rop11 csn5a-2mutants were rescued and exhibited increased CSN5A transcript and protein levels. The rescued phenotype was maintained in non-Mendelian fashion when the csn5a-2 single mutant was re-isolated from the rop11-1 csn5a-2 double mutant, and was sensitive to two inhibitors of DNA methylation. Loss of kanamycin resistance was also observed in re-isolated csn5a-2. These findings indicate that the rescue of csn5a-2 resulted from a trans T-DNA-mediated epigenetic effect on the csn5a-2 intronic T-DNA, similar to recent reports involving the intronic T-DNA mutants ag-TD, ben1-1, and cob-6. Thus the work reported here provides further support for the recommendation that mutants created through novel combinations of T-DNA alleles should be carefully evaluated for evidence of epigenetic modification of T-DNA before final conclusions are drawn.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , DNA Bacteriano/genética , Epigênese Genética , Íntrons/genética , Mutação/genética , Proteínas de Arabidopsis/metabolismo , Western Blotting , Complexo do Signalossomo COP9 , Citidina/análogos & derivados , Citidina/farmacologia , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Resistência a Canamicina/efeitos dos fármacos , Resistência a Canamicina/genética , Mutagênese Insercional/efeitos dos fármacos , Mutagênese Insercional/genética , Fenótipo , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas rho de Ligação ao GTP/metabolismo
19.
Environ Pollut ; 206: 342-51, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26232739

RESUMO

Antibiotic resistance genes may be considered as environmental pollutants if anthropogenic emission and manipulations increase their prevalence above usually occurring background levels. The prevalence of aph(3')-IIa/nptII and aph(3')-IIIa/nptIII - frequent marker genes in plant biotechnology conferring resistance to certain aminoglycosides - was determined in Austrian soils from 100 maize and potato fields not yet exposed to but eligible for GMO crop cultivation. Total soil DNA extracts were analysed by nptII/nptIII-specific TaqMan real time PCR. Of all fields 6% were positive for nptII (median: 150 copies/g soil; range: 31-856) and 85% for nptIII (1190 copies/g soil; 13-61600). The copy-number deduced prevalence of nptIII carriers was 14-fold higher compared to nptII. Of the cultivable kanamycin-resistant soil bacteria 1.8% (95% confidence interval: 0-3.3%) were positive for nptIII, none for nptII (0-0.8%). The nptII-load of the studied soils was low rendering nptII a typical candidate as environmental pollutant upon anthropogenic release into these ecosystems.


Assuntos
Antibacterianos/análise , Produtos Agrícolas/crescimento & desenvolvimento , Farmacorresistência Bacteriana/genética , Genes Bacterianos , Microbiologia do Solo , Poluentes do Solo/análise , Solo/química , Áustria , Produtos Agrícolas/genética , DNA Bacteriano/genética , Resistência a Canamicina/genética , Solo/normas , Solanum tuberosum/genética , Solanum tuberosum/crescimento & desenvolvimento , Zea mays/genética , Zea mays/crescimento & desenvolvimento
20.
Methods Mol Biol ; 1223: 299-310, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25300850

RESUMO

We established improved methods for Agrobacterium-mediated transformation of cucumber (Cucumis sativus L.) and kabocha squash (Cucurbita moschata Duch). Vacuum infiltration of cotyledonary explants with Agrobacterium suspension enhanced the Agrobacterium infection efficiency in the proximal regions of explants. Wounding treatment was also essential for kabocha squash. Cocultivation on filter paper wicks suppressed necrosis of explants, keeping regeneration efficacy. Putative transgenic plants were screened by kanamycin resistance and green fluorescent protein (GFP) fluorescence. These putative transgenic plants grew normally and T1 seeds were obtained, and stable integration and transmission of the transgene in T1 generations were confirmed by Southern hybridization and PCR. The average transgenic efficiency for cucumber and kabocha squash was 11.9 ± 3.5 and 9.2 ± 2.9 %, respectively.


Assuntos
Cucumis sativus/genética , Cucurbita/genética , Técnicas Genéticas , Plantas Geneticamente Modificadas , Agrobacterium tumefaciens/genética , Southern Blotting , Técnicas de Cocultura , Cotilédone/genética , Cucumis sativus/efeitos dos fármacos , Cucurbita/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Canamicina/farmacologia , Resistência a Canamicina/genética , Reação em Cadeia da Polimerase/métodos , Sementes/genética , Transformação Bacteriana
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