【正文】 ratory strains. The nirK gene could be amplified from Blastobacter denitrificans, Alcaligenes xylosoxidans, and Alcaligenes sp. (DSM 30128)。C under anaerobic conditions, 500 ml of the enrichment was again inoculated and kept under the same growth conditions. Six months later, cells were harvested by centrifugation (6,000 3 g for 60 min at 4176。 Millipore, Bedford, Mass.), and cells were collected on a Durapore filter (pore size, mm。 DNA was extracted with Chelex 100 and further purified with CTAB . DNA from sediment (Lake Kleiner Plo168。C was performed. During the first 10 cycles, the annealing temperature was decreased by 176。C. The sequencing products were blotted with a direct blotting apparatus (GATC) onto a nylon membrane. The separated products were visualized by an enzymelinked streptavidinbiotin coupling assay with a streptavidinalkaline phosphatase conjugate(GATC)and NBT/Xphosphate (Boehringer, Mannheim, Germany) as specified by the manufacturers. The sequences obtained were pared with published nirK and nirS sequences in the EMBL Nucleotide Sequence Database by FASTA analysis of the HUSAR program package based on the Geics Computer Group sequence analysis package . Hybridization analysis of nir products from total DNA of environmental samples. Approximately 100 ng (pure cultures) or 250 ng (environmental samples) of PCR product was analyzed on an agarose gel (2%, wt/vol). After electrophoresis, the DNA was transferred onto a positively charged nylon membrane samples. Approximately 100 ng (pure cultures) or 250 ng (environmental samples) of PCR product was analyzed on an agarose gel (2%, wt/vol). After electrophoresis, the DNA was transferred onto a positively charged nylon membrane (QIAbrane Nylon Plus。已知的近紅外型硝化實(shí)驗(yàn)室培養(yǎng)可以得到證實(shí)。這是每個(gè) nir 基因在本研究開發(fā)的水生生物棲息地的總 DNA 的一個(gè)普遍擴(kuò)增引物組合。反硝化細(xì)菌的系統(tǒng)發(fā)育多樣。 nirK 基因中只有 30％的反硝化細(xì)菌的研究迄今發(fā)現(xiàn)的。與一對引物，針對 NIRS 亞硝酸鹽還原酶基因的 PCR 方法具有較高的特異性比雜交實(shí)驗(yàn)。假單胞菌，產(chǎn)堿菌，副球菌，固氮螺菌菌株和反硝化隔離 IFAM 3698基因組 DNA的提取，增加營養(yǎng)肉湯（ NB?；蚪M DNA提取。C 下厭氧條件下， 500毫升的濃縮再次接種和相同的生長條件下保存。 Sigma Aldrich公司， Steinheim，德國）沉淀步驟去除腐殖酸和碳水 化合物。C）。 集中由切向流過濾（ 31）從 10公升湖水湖 Plussee，石勒 蘇益格 荷爾斯泰因州，德國于 1996年 8月在 9米的深度收集細(xì)胞。 變性步驟 5分鐘后，在 95176。在第 10個(gè)循環(huán)，退火溫度降低 176。測序擴(kuò)增近紅外產(chǎn)品。C。電泳后， DNA被轉(zhuǎn)移到一個(gè)帶正電的尼龍膜（ QIAbrane尼龍加 。C 孵育過夜雜交經(jīng)過雜交，洗膜兩次，在室溫下在 5分鐘 100毫升含有 23 SSC（ 13 SSC是 M氯化鈉加檸檬酸鈉 ） ％（重量 /體積） SDS 和 45兩次的解決方案 176。這里，我們描述了反硝化細(xì)菌檢測水生生物棲息地，由兩個(gè)截然不同的 PCR 系統(tǒng)使用的亞硝酸鹽還原酶基因， nirK基因和 NIRS的第一步。由于誘導(dǎo)性質(zhì)的酶，抗血清可檢測主動脫硝的條件下非常有用。這可能是因?yàn)?PCR 擴(kuò)增引物雜交區(qū)域的同源性是決定性的，而基因探針雜交，可以檢測，如果探頭任何地區(qū)，顯示出足夠的同源性。發(fā)現(xiàn)這兩個(gè)基因是在一個(gè)給定的應(yīng)變相互排斥，這是一致的。在幀刪除或插入至 18基點(diǎn)，可觀察到測序區(qū)域內(nèi)的近紅外光譜。相同的近紅外系統(tǒng)發(fā)育不同群體之間的類型的發(fā)生可能是由于硝化通過一個(gè)共同的祖先。 NIR之間同一物種不同類型的發(fā)生可能是水平基因轉(zhuǎn)移的指示。nir 基因的保護(hù)程度是非常多變。從每一個(gè)純文化測試，至少有兩個(gè)不同的引物組合成功應(yīng)用于 nirK 基因至少有三個(gè)成功應(yīng)用于近紅外光譜。這兩個(gè)基因的保守區(qū)域不能被發(fā)現(xiàn)，因?yàn)槊傅慕Y(jié)構(gòu)不同。 nirS基因的不同擁有純培養(yǎng)更廣泛的響應(yīng)與抗血清相比，通過使用特定的基因探針。 rRNA基因定位探頭的使用已成功地應(yīng)用于迄今只有探索脫氮活性污泥法社會的菌株和特定群體。C下 15分鐘 100毫升含 3 SSC和 ％（重量 /體積） SDS溶液的近紅外光譜。 DNA的交聯(lián)膜的紫外燈在 302納米（ 45秒）。分離出的產(chǎn)品是由鏈霉親和素酶聯(lián) 1鏈霉親和堿性磷酸共軛（ GATC）和 NBT / X磷酸鹽（勃林格，曼海姆，德國），由廠家指定的耦合分析的可視化。直接測序的 PCR 產(chǎn)物純化循環(huán)測序試劑盒（的 GATC，康斯坦茨，德國）和 Thermosequenase （ Amersham公司， Braunschweig，德國），由廠家指定的 DNA 序列測定。每一個(gè)周期開始，在 45176。在PerkinElmer， Branchburg，新 澤西州）。 Chelex 100（ 28）提取 DNA，并與 CTAB進(jìn)一步純化。由的 Smalla建議的修改由 Gliesche 等方法，在細(xì)胞裂解。哥廷根，德國）通過過濾去除顆粒大于 100毫米然后通過玻璃纖維過濾器（孔徑 3毫米 。C）和懸浮在 400毫升雙蒸水。分光光度計(jì)的 DNA 制劑的純度和濃度測定。培養(yǎng)基上生長的根瘤菌株（ YEM [27]）。我們用幾個(gè)不同的引物對，以確定反硝化菌的近紅外型。幾種不同的方法被用來確定在實(shí)驗(yàn)室純培養(yǎng)的亞硝酸鹽還原酶的類型。作為一個(gè)生理群的定義，這些兼性厭氧菌可以切換由氧氣，氮氧化物作為終端電子受體在缺氧條件下保存時(shí)。隨之而來的氣體終端產(chǎn)品一氧化氮，氧化亞氮，和氮?dú)獗会尫?。通過 nirK 基因擴(kuò)增產(chǎn)堿桿菌。C. Hybridization was performed with 10 ml of DIGEasy Hyb solution containing the specific probe (25 ng ml21) and by incubation overnight at 42176。C until it reached a touchdown at 40176。 collected in April 1996) was isolated by the method of van Elsas and Smalla with an additional proteinase K treatment ( ml of a 20mg ml21 solution) after the incubation with SDS. PCR amplification of the nir genes. PCR amplifications from pure cultures and environmental samples were performed in a total volume of 50 ml containing 5 ml of 103 PCR buffer (500 mM KCl, 25 mM MgCl2, 200 mM TrisHCl [pH ], % Triton X100), 200 mM each deoxyribonucleoside triphosphate, U of Taq polymerase (5 U ml21。C) in 100 ml of filtered lake water (pore size, mm) containing mM EDTA. The cells were then harvested by centrifugation (8,000 3 g for 45 min at 4176。n (SchleswigHolstein, Germany) was pelleted (13600 3 g for 10 min at 4176。 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 。 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。ner See (SchleswigHolstein, Germany。C) and kept on ice with 1 volume of chilled acetone for 30 min. The pellet (after centrifugation at 4000 for 10 min) was dried, resuspended in 5 ml of SET buffer containing 5 mg of lysozyme, and inc