【正文】 畢業(yè)設(shè)計 (論文 )外文資料翻譯 系 別： 電子信息系 專 業(yè)： 自動化 班 級 姓 名： 學 號： 外文出處： 電子電器類外文文獻 附 件： 1. 原文； 2. 譯文 年月 Development of PCR Primer Systems for Amplification of Nitrite Reductase Genes (nirK and nirS) To Detect Denitrifying Bacteria in Environmental Samples A system was developed for the detection of denitrifying bacteria by the amplification of specific nitrite reducetase gene fragments with PCR. Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirK and nirS) after parative sequence analysis. Whenever amplification was triedwiththese primers, the known nir type of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of the nir type of five laboratory strains. The nirK gene could be amplified from Blastobacter denitrificans, Alcaligenes xylosoxidans, and Alcaligenes sp. (DSM 30128)。 the nirS gene was amplified from Alcaligenes eutrophus DSM 530 and from the denitrifying isolate IFAM 3698. For each of the two genes,at least one primer bination amplified successfully for all of the test strains. Specific amplification products were not obtained with nondenitrifying bacteria or with strains of the other nir type. The specificityof the amplified products was confirmed by subsequent sequencing. These results suggest the suitability of themethod for the qualitative detection of denitrifying bacteria in environmental samples. This was shown byapplying one generally amplifying primer bination for each nir gene developed in this study to total DNApreparations from aquatic habitats. Denitrification is a dissimilatory process of bacteria in which oxidized nitrogen pounds are used as alternative electron acceptors for energy production. The gaseous end products NO, N2O, and N2 are released conitantly. In the environment,denitrification is responsible for the release of fixed nitrogen into the atmosphere in form of N2 . It causes major nitrogen losses in agricultural soils to which fertilizers are applied. Accumulation of the greenhouse gases NO and N2O leads to the destruction of the ozone layer . Also, denitrifying bacteria cause the removal of nitrogen pounds from waste water, where denitrification is coupled to the nitrification process . Bioremediation of environmental pollutants can be achieved under denitrifying conditions . Denitrifying bacteria are phylogeically diverse. They belong to all major physiological groups except for the Enterobacteriaceae, obligate anaerobes, and grampositive bacteria other than Bacillus spp. Defined as a physiological group, these facultative anaerobes can switch from oxygen to nitrogen oxides as terminal electron acceptors when kept under anoxic conditions. Nitrite reductase is the key enzyme in the dissimilatory denitrification process. The reduction of nitrite to NO can be catalyzed by the products of two different nitrite reductase genes: one product contains copper (the nirK product), and the other contains cytochrome cd1 (the nirS product). The two genes seem to occur mutually exclusively in a given strain, but both types have been found in different strains of the same species . Although structurally different, both enzyme types are functionally and physiologically equivalent . nirS is more widely distributed。 nirK is found in only 30% of the denitrifiers studied so far. However, nirK is found in a wider range of physiological groups . Several different approaches were used to determine the type of nitrite reductase in laboratory pure cultures. Diethyldithiocarbamate has been used to identify nirKcontaining denitrifiers . Very specific detection, mostly at the strain level, could be achieved with antisera against dissimilatory nitrite reductase . Another approach was the use of gene probes for nirK or nirS , which were generally specific for the strains investigated. Weak reactivity also occurred for the nirK gene probe with DNA from some of the other nirtype denitrifiers 。 the nirS probe, on the other hand, hybridized with a more limited variety of strains . A PCR method with one primer pair to target the nirS nitrite reductase gene showed higher specificity than hybridization experiments . In the present study, we report on the application of new primer systems for both types of nitrite reductase genes. We used several different primer pairs to determine the nir type of denitrifying strains. Using samples from aquatic habitats, we amplified nir fragments and used the most reliable primer pairs for nirK or nirS, respectively, to successfully detect, in these aquatic samples, different populations of denitrifying bacteria. MATERIALS AND METHODS Bacteria and growth conditions. A variety of denitrifying and nondenitrifying bacterial strains were used to evaluate the specificity of designed PCR primers. All strains were grown aerobically at 27176。C. For genomic DNA isolation, Pseudomonas, Alcaligenes, Ochrobactrum, Paracoccus, and Azospirillum strains and the denitrifying isolate IFAM 3698 were grown on nutrient broth (NB。 Merck, Darmstadt, Germany). Rhizobium strains were grown on yeast extract medium . Hyphomicrobium zavarzinii IFAM ZV622T was grown on 337B1 medium with % (vol/vol) methanol. Rhodobacter sphaeroides f. sp. denitrificans was grown on trypticase soy broth (TSB。 Difco Laboratories, Detroit, Mich.), Roseobacter denitrificans wa