【正文】 在進(jìn)化過程中，反硝化的能力可能已經(jīng)失去了一些分支機(jī)構(gòu)，致使密 切相關(guān) nondenitrifying和反硝化菌。可能是由于這些結(jié)果是在不與協(xié)議的結(jié)果，的的銅 dNirs 是超過 CD1 dNirs，不同的的銅 dNir 亞基分子量的異構(gòu)酶處理的結(jié)果。組合包含 nirK4R或含 nirS5R導(dǎo)致在特定產(chǎn)品中最小的數(shù)量，這可能是由于在該地區(qū)的底漆應(yīng)雜交程度的保護(hù)。在本研究 中， PCR nirK基因或的 NIRS從數(shù)據(jù)庫每六個序列為基礎(chǔ)的系統(tǒng)的承諾更一般的方法。相比之下，接近目標(biāo) nirK 基因或 nirS 基因檢測的反硝化反硝化條件不論。由于反硝化不 密切的親緣關(guān)系的定義，涉及 16S rRNA分子的做法是不適合這種生理組一般在環(huán)境檢測。為 nirK基因和 46176。 Qiagen公司），毛細(xì)轉(zhuǎn)移。測序產(chǎn)品涂抹直接印跡儀器到尼龍膜（ GATC）。用于 DNA 測序，從純培養(yǎng)擴(kuò)增 PCR產(chǎn)物純化 QIAquick PCR純化試劑盒（ Qiagen，希爾登，德國），由生產(chǎn)廠家指定的。231。C 時， “著陸 ”聚合酶鏈反應(yīng)（熱循環(huán) 2400。 100毫升細(xì)胞懸液加入 MilliQ水200毫升 。風(fēng)干顆粒懸浮在 10毫升（ 5％蔗糖， 50 mM的 EDTA， 50毫米的 TrisHCl， pH值 ]）設(shè)置緩沖。 地表水從克萊納普羅湖（ 30公升） “NER教廷（德國石勒蘇益格 荷爾斯泰因州，于 1996年 4月收集）纖維素過濾器（賽多利斯，轉(zhuǎn)到 168。半年后，細(xì)胞，離心收集（ 6,000 3克 60分鐘，在 4176。溶菌酶，蛋白酶 K，十二烷基硫酸鈉（ SDS）的治療之后氯仿抽提和隨后的乙醇沉淀基因組 DNA，獲得純培養(yǎng)。默克公司，德國達(dá)姆施塔特）。在本研究中，我們報告的亞硝酸鹽還原酶基因的兩種類型的新的引物系統(tǒng)中的應(yīng)用。然而， nirK 基因被發(fā)現(xiàn)在更廣泛的生理組。他們屬于腸桿菌，厭氧菌和革蘭氏陽性菌，芽孢桿菌屬以外的其他所有主要的生理團(tuán)體除外。 脫氮是一氧化氮化合物作為替代能源生產(chǎn)的電子受體的細(xì)菌的異化過程。同樣，該方法允許 NIR 五個實驗室培養(yǎng)菌株類型的決心。 Qiagen) by capillary transfer (24). The DNA was crosslinked to the membrane with UV light (45 s at 302 nm). Products generated with the primer bination nirK1FnirK5R from genomic DNA from Alcaligenes xylosoxidans NCIMB 11015 and with the bination nirS1FnirS6R from genomic DNA from Pseudomonas stutzeri ATCC 14405 were used as probes for nirK and nirS, respectively, to determine the specificity of nir products amplified from total environmental DNA. The nir products were purified by eluting the bands from an agarose gel by using the QIAquick gel extraction kit (Qiagen) as specified by the manufacturer. The probes were labeled randomly with digoxigenin by using the digoxigenin DNA labeling and detection kit (Boehringer) as specified by the manufacturer. The membrane was prehybridized in 20 ml of DIGEasy Hyb solution (Boehringer) for 2 h at 42176。C every cycle, starting at 45176。ner See。 Millipore). Bacterial cells were removed from the filter by shaking it (100 rpm for 5 h at 4176。C) and resuspended in 400 ml of doubledistilled water. The DNA was extracted with Chelex 100 . A volume of activated sludge from a sewage treatment plant in Plo168。 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。 Difco Laboratories, Detroit, Mich.), Roseobacter denitrificans was grown on oligotrophic medium supplemented with 25‰ artificial seawater , and Blastobacter denitrificans was grown on peptone yeast extract glucose medium, ie., PYGV without vitamins. Nondenitrifying strains of the Enterobacteriaceae were grown on Luria broth . Extraction of genomic DNA. Genomic DNA was obtained from pure cultures by lysozymeproteinase Ksodium dodecyl sulfate (SDS) treatment followed byphenolchloroform extraction and subsequent ethanol precipitation . The purity and concentration of the DNA preparations were determined spectrophotometrically. Preparation of total DNA from an enrichment culture and four environmental samples. DNA was prepared from five samples. A 500ml volume of medium 337B1 with % (wt/vol) KNO3 for the enrichment of denitrifying methylotrophic bacteria was inoculated with 100 ml of activated sludge from a sewage treatment plant near Plo168。 collected in April 1996) was filtered through a cellulose filter (Sartorius