RESUMO
Using a new method based on a combination of bisulfite reaction, the repair enzyme uracil-DNA glycosylase, and synthetic oligodeoxyribonucleotides, the methylation site of DNA-methyltransferase NlaX (M.NlaX) from Neisseria lactamica was established to be the inner cytosine in the double-stranded pentanucleotide recognition sequence 5'-CCNGG-3' (where N = any nucleoside). 5-Methylcytosine (m5C) type modification by M-N1aX was confirmed by the use of oligonucleotide substrates that contain 5-fluoro-2'-deoxycytidine.
Assuntos
Técnicas de Química Analítica/métodos , DNA Glicosilases , Metilação de DNA , Metilases de Modificação do DNA/química , N-Glicosil Hidrolases/química , Reagentes de Ligações Cruzadas/química , Metilases de Modificação do DNA/isolamento & purificação , DNA Bacteriano/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Neisseria/química , Neisseria/enzimologia , Neisseria/genética , Sulfitos/química , Uracila-DNA GlicosidaseRESUMO
A new gene cluster, designated sepABC and a divergently transcribed sepR, was found downstream of the two-component todST phosphorelay system that regulates toluene degradation (the tod pathway) in Pseudomonas putida F1 (PpF1). The deduced amino acid sequences encoded by sepABC show a high homology to bacterial proteins known to be involved in solvent efflux or multidrug pumps. SepA, SepB and SepC are referred to be periplasmic, inner membrane and outer membrane efflux proteins respectively. Effects on growth of various PpF1 mutants compared to that of the wild type in the presence of toluene indicated a possible protective role of the solvent efflux system in a solvent-stressed environment. Growth tests with the complemented mutants confirmed the involvement of the Sep proteins in conferring solvent tolerance. The sepR gene encodes a 260-residue polypeptide that is a member of the E. coli IclR repressor protein family. The repressor role of SepR was established by conducting tests with a sep-lacZ transcriptional fusion in Escherichia coli and PpF1, expression of SepR as a maltose-binding fusion protein in a DNA binding assay, and mRNA analysis. Southern hybridization experiments and analysis of the P. putida KT2440 genome sequence indicated that sepR is a relatively rare commodity compared to homologues of the sepABC genes. We developed a whole-cell bioluminescent biosensor, PpF1G4, which contains a chromosomally based sep-lux transcriptional fusion. The biosensor showed significant induction of the sepABC genes by a wide variety of aromatic molecules, including benzene, toluene, ethylbenzene, and all three isomers of xylene (BTEX), naphthalene, and complex mixtures of aliphatic and aromatic hydrocarbons. PpF1G4 represents a second-generation biosensor that is not based on a catabolic promoter but is nonetheless inducible by aromatic pollutants and moreover functional under nutrient-rich conditions.
Assuntos
Proteínas de Bactérias/genética , Técnicas Biossensoriais , Hidrocarbonetos Aromáticos/farmacologia , Proteínas de Membrana Transportadoras/genética , Pseudomonas putida/genética , Proteínas Repressoras/genética , Solventes/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Poluentes Ambientais/análise , Poluentes Ambientais/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Genes Reporter , Teste de Complementação Genética , Hidrocarbonetos Aromáticos/análise , Hidrocarbonetos Aromáticos/metabolismo , Óperon Lac , Medições Luminescentes , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Pseudomonas putida/metabolismo , Proteínas Repressoras/metabolismo , Análise de Sequência de DNA , Homologia de Sequência , Solventes/análise , Solventes/metabolismo , Tolueno/metabolismo , Tolueno/farmacologia , beta-Galactosidase/metabolismoRESUMO
We report on microbial contamination of embryos and semen cryopreserved in sealed plastic straws and stored for 6-35 years in liquid nitrogen. There were 32 bacterial and 1 fungal species identified from randomly drawn liquid nitrogen, frozen semen, and embryos samples stored in 8 commercial and 8 research facility liquid nitrogen (LN) tanks. The identified bacteria represented commensal or environmental microorganisms and some, such as Escherichia coli, were potential or opportunistic pathogens for humans and animals. Stenotrophomonas maltophilia was the most common contaminant identified from the samples and was further shown to significantly suppress fertilization and embryonic development in vitro. Analysis of the strains by pulsed field gel electrophoresis revealed restriction patterns with no relatedness indicating that there was no apparent cross-contamination of S. maltophilia strains between the germplasm and liquid nitrogen samples. In addition, no transmission of bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1) from infected semen and embryos straws to clean germplasm stored in the same LN tanks or LN was detected.