【正文】 ches, 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)的開發(fā)。C for nirK and 46176。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. 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。C and primer annealing and extension for 30 s at 55176。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. After a denaturing step of 4 min at 94176。C until it reached a touchdown at 40176。C was performed. During the first 10 cycles, the annealing temperature was decreased by 176。C, a primerannealing step of 40 s, and an extension step of 40 s at 72176。C, a “touchdown” PCR was performed (Thermocycler 2400。 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。 DNA was extracted with Chelex 100 and further purified with CTAB . DNA from sediment (Lake Kleiner Plo168。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) and thawed (5 min at 30176。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。 Millipore, Bedford, Mass.), and cells were collected on a Durapore filter (pore size, mm。 collected in April 1996) was filtered through a cellulose filter (Sartorius, Go168。 Sigma Aldrich, Steinheim, Germany) precipitation step to remove humic acids and carbohydrates. Surface water (30 liters) from Lake Kleiner Plo168。n (SchleswigHolstein, Germany) was pelleted (13600 3 g for 10 min at 4176。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。 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。C. For