Pessoa T B A, de Souza S S, Cerqueira A F, Rezende R P, Pirovani C P, Dias J C T
Laboratório de Monitoramento Ambiental, Departamento de Ciências Biológicas, Universidade Estadual de Santa Cruz, Salobrinho, Ilhéus, BA, Brasil.
Genet Mol Res. 2013 Jun 28;12(2):2148-55. doi: 10.4238/2013.June.28.2.
Landfarming biodegradation is a strategy used by the petrochemical industry to reduce pollutants in petroleum-contaminated soil. We constructed 2 metagenomic libraries from landfarming soil in order to determine the pathway used for mineralization of benzene and to examine protein expression of the bacteria in these soils. The DNA of landfarm soil, collected from Ilhéus, BA, Brazil, was extracted and a metagenomic library was constructed with the Copy Control(TM) Fosmid Library Production Kit, which clones 25-45-kb DNA fragments. The clones were selected for their ability to express enzymes capable of cleaving aromatic compounds. These clones were grown in Luria-Bertani broth plus L-arabinose, benzene, and chloramphenicol as induction substances; they were tested for activity in the catechol cleavage pathway, an intermediate step in benzene degradation. Nine clones were positive for ortho-cleavage and one was positive for meta-cleavage. Protein band patterns determined by SDS-polyacrylamide gel electrophoresis differed in bacteria grown on induced versus non-induced media (Luria-Bertani broth). We concluded that the DNA of landfarm soil is an important source of genes involved in mineralization of xenobiotic compounds, which are common in gasoline and oil spills. Metagenomic library allows identification of non-culturable microorganisms that have potential in the bioremediation of contaminated sites.
土地耕作生物降解是石化行业用于减少石油污染土壤中污染物的一种策略。我们从土地耕作土壤中构建了2个宏基因组文库，以确定苯矿化所使用的途径，并检测这些土壤中细菌的蛋白质表达。从巴西巴伊亚州伊列乌斯采集的土地耕作土壤的DNA被提取出来，并用复制控制（TM）粘粒文库构建试剂盒构建了一个宏基因组文库，该试剂盒可克隆25 - 45 kb的DNA片段。根据克隆表达能够裂解芳香族化合物的酶的能力来选择克隆。这些克隆在添加L - 阿拉伯糖、苯和氯霉素作为诱导物质的Luria - Bertani肉汤中培养；对它们在儿茶酚裂解途径（苯降解的中间步骤）中的活性进行了测试。9个克隆对邻位裂解呈阳性，1个克隆对间位裂解呈阳性。通过SDS - 聚丙烯酰胺凝胶电泳测定的蛋白质条带模式在诱导培养基（Luria - Bertani肉汤）和非诱导培养基上生长的细菌中有所不同。我们得出结论，土地耕作土壤的DNA是参与外源化合物矿化的基因的重要来源，这些外源化合物在汽油和石油泄漏中很常见。宏基因组文库能够鉴定出在污染场地生物修复中具有潜力的不可培养微生物。
