ABSTRACT
Metagenomic library PP1 was obtained from Antarctic soil samples. Both functional and genotypic metagenomic screening were used for the isolation of novel cold-adapted enzymes with potential applications, and for the detection of genetic elements associated with gene mobilization, respectively. Fourteen lipase/esterase-, 14 amylase-, 3 protease-, and 11 cellulase-producing clones were detected by activity-driven screening, with apparent maximum activities around 35 °C for both amylolytic and lipolytic enzymes, and 35-55 °C for cellulases, as observed for other cold-adapted enzymes. However, the behavior of at least one of the studied cellulases is more compatible to that observed for mesophilic enzymes. These enzymes are usually still active at temperatures above 60 °C, probably resulting in a psychrotolerant behavior in Antarctic soils. Metagenomics allows to access novel genes encoding for enzymatic and biophysic properties from almost every environment with potential benefits for biotechnological and industrial applications. Only intI- and tnp-like genes were detected by PCR, encoding for proteins with 58-86 %, and 58-73 % amino acid identity with known entries, respectively. Two clones, BAC 27A-9 and BAC 14A-5, seem to present unique syntenic organizations, suggesting the occurrence of gene rearrangements that were probably due to evolutionary divergences within the genus or facilitated by the association with transposable elements. The evidence for genetic elements related to recruitment and mobilization of genes (transposons/integrons) in an extreme environment like Antarctica reinforces the hypothesis of the origin of some of the genes disseminated by mobile elements among "human-associated" microorganisms.
A partir de muestras de suelo antártico se obtuvo la metagenoteca PP1. Esta fue sometida a análisis funcionales y genotípicos para el aislamiento de nuevas enzimas adaptadas al frío con potenciales aplicaciones, y para la detección de elementos génicos asociados a la movilización de genes, respectivamente. Por tamizaje fenotípico se detectaron 14, 14, 3 y 11 clones productores de lipasas/esterasas, proteasas, amilasas y celulasas, respectivamente, con actividades máximas aparentes de 35 °C para las amilasas y lipasas, y de 35-55 °C para las celulasas, tal como se observó para otras enzimas adaptadas al frío. Sin embargo, una celulasa parece ser compatible con enzimas mesófilas, las que usualmente se mantienen activas hasta por sobre 60 °C. Este hecho probablemente esté asociado a un comportamiento psicrotolerante en los suelos antárticos. La metagenómica permite acceder a una nueva miríada de productos metabólicos con potenciales beneficios para aplicaciones biotecnológicas e industriales. Se detectaron los genes tipo intI y tnp por PCR, y sus productos génicos deducidos tuvieron identidades del 58 al 86 % y del 58 al 73 % con secuencias conocidas, respectivamente. Dos clones, BAC 27A-9 y BAC 14A-5, parecen presentar organizaciones sintéticas únicas, lo cual sugiere la existencia de rearreglos génicos probablemente debidos a divergencias evolutivas dentro del género o facilitados por la asociación de elementos de transposición. La evidencia de elementos génicos relacionados con el reclutamiento y la movilización de genes en ambientes extremos como la Antártida refuerza la hipótesis sobre el origen de algunos genes diseminados por elementos móviles entre los microorganismos asociados al ser humano.
Subject(s)
Cold Climate , Enzymes/genetics , Interspersed Repetitive Sequences/genetics , Metagenome , Soil Microbiology , Adaptation, Physiological , Amino Acid Sequence , Antarctic Regions , Cloning, Molecular , Chromosomes, Artificial, Bacterial/genetics , Enzymes/isolation & purification , Fertilizers , Gasoline , Gene Library , Molecular Sequence Data , Petroleum , Sequence Alignment , Sequence Homology, Amino Acid , Soil PollutantsABSTRACT
phiC31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of phiC31 integrase system for alveolar type II cells. Luciferase and beta-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase using a CMV promoter resulted in highest integrase activity, whereas in MLE12 cells, both CAG and CMV promoter were equally effective. Effect of polyA site was observed only in A549 cells, where replacement of SV40 polyA by bovine growth hormone (BGH) polyA site resulted in an enhancement of integrase activity. Addition of a C-terminal SV40 nuclear localization signal (NLS) did not result in any significant increase in integrase activity. Long-term expression studies with AZA and TSA, provided evidence for post-integrative gene silencing. In MLE12 cells, both DNA methylases and HDACs played a significant role in silencing, whereas in A549 cells, it could be attributed majorly to HDAC activity. Donor plasmids comprising cellular promoters ubiquitin B (UBB), ubiquitin C (UCC) and elongation factor 1alpha (EF1alpha) in an improved backbone prevented post-integrative gene silencing. In contrast to A549 and MLE12 cells, no silencing could be observed in human bronchial epithelial cells, BEAS-2B. Donor plasmid coding for murine erythropoietin under the EF1alpha promoter when combined with phiC31 integrase resulted in higher long-term erythropoietin expression and subsequently higher hematocrit levels in mice after intravenous delivery to the lungs. These results provide evidence for cell specific post integrative gene silencing with phiC31 integrase and demonstrate the pivotal role of donor plasmid in long-term expression attained with this system.
Subject(s)
Animals , Chick Embryo , Female , Humans , Mice , Bacteriophages/genetics , Cell Line , Gene Expression , Gene Silencing , Genetic Therapy , Genes, Reporter , Genetic Vectors/genetics , Integrases/genetics , Mice, Inbred BALB C , Plasmids/genetics , Alveolar Epithelial Cells/metabolism , Promoter Regions, Genetic , Streptomyces/virology , TransfectionABSTRACT
Objective Since integrons play an important role in the spread of antibiotic resistance genes in bacteria,the characterization of a new resistance gene cassette in class 1 integron positive strains of E.coli was analyzed. Methods The presence and genetic content of class 1 integron were examined by PCR and sequencing.The sequence was analyzed by using some bioinformatics softwares.Results 5 class 1 integron positive strains were amplified by primers of in-F and in-B which were set for amplifying the region of antibiotics resistance genes.Among the 5 strains,an amplicon of 1009 bp was yielded.Sequencing analysis revealed that amplicon of 1009 bp harbored a 780 bp ORF.Further analysis with bioinformatics software showed that it was 99.6% and 99.5% identical to the known aadA23 and aadA21 cassette,and was just 66.4% identical to the known aadA5 cassette.It was conferring resistant to spectinomycin and streptomycin,and was given a new name aadA23b.Conclusions Multi-drug resistance genes has been proved changeable in E.coli clinical strains.The result not only stressed the need for continuing surveillance of antibiotic resistance in the molecular level,but also the need caring for genetic variation of drug resistance gene cassettes.
ABSTRACT
OBJECTIVE To investigate the genes of carbapenemases and integrases in multi-drug resistant Acinetobacter baumannii(MDRAba).METHODS PCR was used for detection of genes of carbapenemases: OXA-23-like,OXA-24-like,OXA-51-like,and OXA-58-like,and integrases Ⅰand Ⅱ from 70 clinical strains of carbapenem-resistant A.baumannii(CRAba).PCR products of OXA-23-like and OXA-58-like were analyzed by sequencing.Agar dilution method was carried out for antimicrobial susceptibility test.RESULTS Genes for OXA-23-like and OXA-51-like were positive from 56 and 69 isolates,accounted for 80.0% and 98.6%, respectively;gene of OXA-58-like was detected from only one strain.Sixty one strains showed positive for integrase Ⅰ gene.OXA-23 or OXA-58 was the exact gene type by sequencing.All 70 strains were highly resistant to ciprofloxacin,ceftazidime,and gentamicin,but susceptible to polymyxin B.CONCLUSIONS CRAba strains distributed in General Hospital of PLA mainly possess OXA-23 type carbapenemase and integraseⅠ.