【正文】 , Go168。C for 1 h. DNA extraction and purification were performed by the method of Gliesche et al. Cells from 10 liters of lake water (Lake Plussee, SchleswigHolstein, Germany。C, a primerannealing step of 40 s, and an extension step of 40 s at 72176。C and primer annealing and extension for 30 s at 55176。 this may be due to the degree of conservation in the region where the primer should hybridize. From every pure culture tested, at least two different primer binations were applied successfully for nirK and at least three were applied successfully for nirS. The specificity of amplifications was confirmed by sequencing the largest products. Generally, sequencing revealed that the products from the pure cultures with the primer pairs nirK1FnirK5R and nirS1FnirS6R were specific fragments of the genes coding for coppercontaining and cytochrome cd1containing nitrite reductase, respectively. Calculation of the homology revealed that with these primer pairs, PCR products could be obtained from genes showing homology as low as % for nirK and % for nirS to the sequences available for the design of the primers. This indicates that these PCR systems could be reasonable means for a more general detection of denitrifying bacteria. The fragments of nirS investigated here were more heterogeneous than were those of nirK. Inframe deletions or insertions of up to 18 bp could be observed within the sequenced region of nirS. Since these results are not in agreement with the findings that the Cu dNirs were more heterogeneous than the cd1 dNirs , the different molecular weights of the Cu dNir subunits may be a result of processing of the enzyme. The degrees of conservation of the nir genes are very variable. The nirK fragments from eight strains of Ochrobactrum anthropi were to % homologous (data not shown). On the other hand, the sequence of the fragment from two of these strains was 100% homologous to that of the same fragment from Alcaligenes faecalis S6 (data not shown). However, both types of nir genes are distributed among closely related Pseudomonas (RNA group I [35]) and Alcaligenes species. Even among strains of Alcaligenes faecalis, both types could be detected. The distribution of nir genes among denitrifying bacteria could be explained in different ways. The occurrence of the same nir type among phylogeically different groups might be caused by a mon denitrifying ancestor. During evolution, the ability to denitrify may have been lost in some branches, resulting in closely related nondenitrifying and denitrifying strains . The occurrence of different nir types among the same species could be an indication of a horizontal gene transfer . The PCR systems for the nirK and the nirS genes for nitrite reductase, using one generally amplifying primer bination for each nir gene, could be applied successfully to detect populations of denitrifying bacteria in aquatic systems. Due to higher cell densities, detection of such populations from soils by using these PCR systems should be possible as well.亞硝酸鹽擴(kuò)增 PCR 引物系統(tǒng)的發(fā)展 一個(gè)特定的亞硝酸鹽還原酶基因片段的 PCR 擴(kuò)增反硝化細(xì)菌檢測(cè)系統(tǒng)的開(kāi)發(fā)。隨后測(cè)序證實(shí)擴(kuò)增產(chǎn)物的基因。此外， 反硝化細(xì)菌引起的，其中硝化耦合硝化過(guò)程從廢水中含氮化合物的去除。雖然結(jié)構(gòu)不同，這兩種酶的類型，功能和生理等效。軟弱無(wú)力的反應(yīng)也發(fā)生 nirK 基因基因探針與 DNA 的一些其他 NIR型反硝化 。株生長(zhǎng)需氧 27176。 Difco公司實(shí)驗(yàn)室，底特律，密歇根州）種植，脫氮貧營(yíng)養(yǎng)培養(yǎng)基上生長(zhǎng)（ PYGV [25]）輔以 25‰ 的人工海水，蛋白胨酵母生長(zhǎng)脫氮沒(méi)有維生素中提取的葡萄糖培養(yǎng)基，即 PYGV。N （石勒蘇益格 荷爾斯泰因州，德國(guó)） 100毫升。C 10分鐘，沉淀空氣干燥和懸浮在 ％ NaCl溶液。C），細(xì)菌細(xì)胞從過(guò)濾器中刪除。C 孵育 1小時(shí)的 SET 緩沖。從單純的文化和環(huán)境樣品進(jìn)行PCR擴(kuò)增量在 50毫升含 5 PCR緩沖液 103毫升（ 500毫米氯化鉀，氯化鎂 ， 25毫米200毫米的 TrisHCl [pH值 ， ％曲拉通 X100）的總 200毫米， Taq聚合酶鈾（ 5252。經(jīng)過(guò) 30個(gè)循環(huán)，最后 7分鐘的潛伏期為 72176。C。C 和 30秒引物退火和延伸，在 55176。電泳后，DNA被轉(zhuǎn)移到一個(gè)帶正電的尼龍膜樣品。膜預(yù)雜交 2小時(shí) 20毫升辛易 HYB 解決方案（勃林格）在 42176。 AJ224902通過(guò) AJ224913。那么具體的反應(yīng)，可以得到組合血清對(duì)斯氏假單胞菌 JM300和綠膿桿菌（ hemetype dNirs。從三個(gè) NIRS側(cè)翼一個(gè)保守的中部地區(qū) nirS基因序列的引物對(duì)純培養(yǎng)中的應(yīng)用，發(fā)現(xiàn)比與抗血清的使用，但還不如利用基因探針令人滿意的反應(yīng)更廣泛。使用這兩個(gè)基因的不同組合的引物和 PCR 方法在低溫的嚴(yán)格條件，近紅外片段的擴(kuò)增，可以測(cè)試所有反硝化菌株。這表明，這些 PCR 系統(tǒng)可能是一個(gè)更普遍的反硝化細(xì)菌檢測(cè)的合理手段。即使在糞產(chǎn)堿桿菌菌株，這兩種類型可以被檢測(cè)出來(lái)。由于細(xì)胞密度較高，從土壤等人群使用這些 PCR系統(tǒng)檢測(cè)應(yīng)盡可能 。另一方面，從這些菌株的兩個(gè)片段序列從糞產(chǎn)堿菌的 S6（數(shù)據(jù)未顯示）的相同片段的同源性為 100％。一般來(lái)說(shuō)，測(cè)序顯示，從底漆的雙 nirK1F nirK5R和nirS1FnirS6R 純文化的產(chǎn)品含銅和細(xì)胞色素 CD1含有亞硝酸鹽還原酶編碼基因的特異片段。設(shè)計(jì)簡(jiǎn)并引物，兩側(cè)的兩個(gè)基因的高度保守的 C 端區(qū)域。富集的文化，不同 人群的 denitri fiers 可檢測(cè)限制性內(nèi)切酶消化籌備工作的總 DNA（酶切）與近紅外光譜探頭。為此，亞硝酸鹽還原酶，其基因已被用于幾位作者，因?yàn)檫@是在反硝化過(guò)程中的關(guān)鍵酶。核苷酸序列號(hào)。近紅外產(chǎn)品進(jìn)行純化，由生產(chǎn)廠家指定使用 QIAquick 凝膠提取試劑盒（ Qiagen）洗脫瓊脂糖凝膠帶。 從環(huán)境樣品總 DNA雜交分析，近紅外產(chǎn)品。經(jīng)過(guò) 4分鐘的變性步驟 94176。C。C間， 40秒一步的引物退火，延伸 40秒，在 72176。收集在 1996年 4月），是由與一個(gè)額外的蛋白酶 K 處理方法（ 50毫升 20毫克 ML21解決方案），面包車 Elsas 和 Smalla 分離后與 SDS 的潛伏期。C） 1冰鮮丙酮體積為 30分鐘，并保持在冰上。 Millipore公司）。 168。 準(zhǔn)備從 5個(gè)樣品的 DNA。紅假單胞菌 FSP。 材料與方法 細(xì)菌的生長(zhǎng)條件。非常具體的檢測(cè)，主要是在應(yīng)變水平，可實(shí)現(xiàn)對(duì)異化硝酸鹽還原酶的血清（ dNirS ， 24，29和 dNirK ）。催化亞硝酸鹽還原為 NO可以由兩個(gè)不同的亞硝酸鹽還原酶基因的產(chǎn)品：一個(gè)產(chǎn)品含有銅（ nirK基因產(chǎn)品），和其他含有色素 CD1（ NIRS產(chǎn)品）。它會(huì)導(dǎo)致在肥料應(yīng)用的主要農(nóng)業(yè)土壤的氮素?fù)p失。對(duì)于每個(gè)的兩個(gè)基因，成功擴(kuò)增引物組合至 少有一個(gè)為所有測(cè)試菌株。C for nirK and 46176。C. The amplification products were analyzed by electrophoresis on 2% (wt/vol) agarose gels (Boehringer, Ingelheim, Germany) followed by a 15min staining with ethidium bromide ( mg liter21). Sequencing of amplified nir products. For DNA sequencing, amplified PCR products from pure cultures were purified with the QIAquick PCR purification kit (Qiagen, Hilden, Germany) as specified by the manufacturer. DNA sequences were determined by direct sequencing of purified PCR products with the cyclesequencing kit (GATC, Konstanz, Germany) and Thermosequenase (Amersham, Braunschweig, Germany) as specified by the manufacturers. Labeling was performed by terminating the polymerization with biotinlabeled dideoxynucleoside triphosphates.