Journal of Experimental Microbiology and Immunology (JEMI) Vol. 4:7-14 Copyright © December 2003, M&I UBC Characterization of the Nitrobacter-specific NIT3 and Nb1000 Probes and Their Use in the Detection of Nitrobacter Species in a Lab Scale Completely Stirred Tank Reactor System KAREN CHAM Department of Microbiology and Immunology, UBC Nitrobacter species are responsible for the oxidation of nitrite to nitrate in the environment. Studies indicated that there is an inconsistent correlation between the amount of nitrifying activity in the bioreactor and the amount of nitrifying bacteria detected by the NIT3 and Nb1000 16S rRNA probes. This study investigates the explanation for the inconsistency, focusing on whether there are any sequence mismatches between the probes and the Nitrobacter 16S ribosomal RNA present in the environmental sample that preclude the detection of Nitrobacter. The samples used in this study were collected from a lab scale completely stirred tank reactor system seeded from the Kent wastewater treatment plant. The techniques employed to analyze samples included RNA isolation, radioactive labelling of 16S rRNA oligonucleotide probes, slot blot hybridization, and the determination of the dissociation temperature for both probes. Nucleic acid hybridization results suggested that Nitrobacter was present at a very low concentration in the stirred tank reactor, which was below the limit of detection using the radioactively labelled NIT3 and Nb1000 probes. Using RNA isolated from pure Nitrobacter strain, the experimentally measured dissociation temperature for the Nb1000 probe was 43ºC and the minimum dissociation temperature for the NIT3 probe was 54ºC. It was concluded that the amount of RNA immobilized onto the membrane should be increased by approximately 400-fold in order to obtain a detectable signal for Nitrobacter. + - Nitrification is a two-step aerobic process involving the oxidation of ammonium ion (NH4 ) to nitrite (NO2 ) and - subsequent oxidation of nitrite to nitrate (NO3 ). This process has great environmental implication as it is the major component of the nitrogen cycle found in ocean water, freshwater lakes, soils, aquaria, and sewage treatment systems (8). Nitrification is beneficial in the sewage treatment system because it helps to protect aquatic life and minimize oxygen depletion by reducing the amount of toxic ammonia in the environment. However, the process also causes pollution with the production of nitrogenous by-products such as nitric oxide, nitrate and nitrite ions. When these compounds are discharged into the receiving water, it has significant concerns related to the loss of fertilizers from soil and the creation of greenhouse effects (2, 11). Bacteria belonging to the genera Nitrosomonas, Nitrosococcus, and Nitrosospira are examples of lithoautotrophs that are capable of converting ammonia to nitrite (6, 7). The second stage of nitrification, nitrite oxidation, is catalyzed by the nitrite-oxidizing bacteria. Nitrobacter and other related chemolithoautotrophic bacteria play important roles in nitrite oxidation. Nitrobacter is one of the well-investigated nitrite-oxidizing alpha Proteobacteria. They are Gram-negative, rod-shaped bacteria present in soil, freshwater, and the marine environments (2). N. winogradskyi, N. hamburgensis and N. spp. belong to the Nitrobacter genus (11). Several techniques have been used in the past to enumerate and detect the nitrifying bacteria. A better understanding of the nitrifying composition of the sewage system would enable engineers to control the nitrification process in the wastewater treatment plants. The most probable number (MPN) technique involves the recording of the presence or absence of bacterial growth in several replicate dilutions of samples, while plating of pure cultures can also be used for quantifying nitrifiers (4). However, these cultivation-dependent techniques have disadvantages which include the time- and labour- intensive enumeration process and the difficulty in the identification of nitrifying organisms due to the slow growth of the bacteria and the small size of the colonies. Improved qualitative and quantitative techniques to study the nitrifying population, including the use of fluorescent in situ hybridization, PCR and amoA gene sequence analysis, and the ribosomal ribonucleic acid analysis, have been developed in the past 10 years. The 16S rRNA-targeted oligonucleotide probes have been specifically designed to detect the presence of a particular bacterial species in the environment (1, 4, 10, 14). The specificities of the probes targeted against 7 Journal of Experimental Microbiology and Immunology (JEMI) Vol. 4:7-14 Copyright © December 2003, M&I UBC particular 16S rRNA sequences can be manipulated to understand the phylogeny and diversity of species within a microbial population. However, recent findings indicated that the amount of activity of the nitrifying bacteria in bioreactors did not consistently correlate to the degree of binding by the 16S rRNA probes targeted for the nitrifying bacteria. This was found to be true for the Nitrobacter Nb1000 probe, which barely detected the presence of Nitrobacter species even when there was a high level of nitrate formation in the bioreactor (Rob Simm, personal communication, 2003). This inconsistency may be due to the sequence mismatch between the probe and the corresponding nitrifying organism in the reactor. The objective of this experiment was to determine if the discrepancy was due to the presence of novel strains of Nitrobacter present in the bioreactor, or a slightly different sequence between the probe and the 16S rRNA that resulted in a different dissociation temperature of the DNA/RNA hybrid. In addition, the construction of the dissociation temperature profiles for the probes would allow the optimization of the hybridization conditions. MATERIALS AND METHODS Sample Collection. Environmental samples were provided by Robert Simm and were collected from the R5 completely stirred tank lab scale reactor. The bioreactor was seeded from the Kent wastewater treatment plant in Agassiz, British Columbia, Canada. Five millilitres of samples, collected from the bioreactor on May 11, May 20, May 27, June 8, June 11, and June 14 of the year 2003, were stored in RNase-free COD vial. Each sample was centrifuged and the sludge pellet was stored at -80ºC until processing. RNA isolated from Nitrobacter winogradskyi (provided by Rob Simm) was used as a positive control as previous studies (11) reported that this species could be detected by the Nb1000 probe. RNA Extraction. Total RNA was extracted from bacterial cells using the guanidinium thiocyanate-acidic phenol-chloroform (TRIzol® reagent) method as described in the Invitrogen™ Life Technologies manufacturer’s protocol. For samples collected on May 11, 20, 27, and June 8, the pellet resulting from approximately 5 ml of the bioreactor’s mixture was resuspended in 2 ml of TRIzol® reagent. The TRIzol® reagent is a monophasic solution of phenol and guanidine isothiocyanate and it disrupts cells while inactivating RNase activity. Only 1 ml of the mixture was used for RNA extraction. The remainder of the sample was stored at -80ºC for future experiment. For samples collected on June 11 and June 14, the whole pellet resulting from approximately 5 ml of bioreactor’s bacterial mixture was resuspended in 1 ml of RNase-free diethylpyrocarbonate (DEPC)-treated water. The cells were centrifuged at 14,000 rpm at 4ºC for 4 min and the pellet was resuspended in 1 ml of TRIzol® reagent. After the pellet was resuspended in TRIzol® reagent, the suspension was then transferred to a 2-ml screw cap microcentrifuge tube (Fisherband®). This was followed by the addition of 200 µl of chloroform to the suspension. In addition, glass beads of 0.1 mm diameter (BioSpec Products) were added until the microcentrifuge tube was filled to the top. Bacterial cells were broken mechanically by bead beating the suspension at room temperature, for three periods of 1 min each, with the Mini-BeadBeater-1™ (BioSpec Products) set to 4,800 rpm. RNA was precipitated from the upper aqueous layer with 0.5 ml of isopropanol and then washed with 1 ml of 75% ethanol. The pellet was air-dried briefly and re-dissolved in 100 µl of RNase-free water. RNA was quantified by measuring the sample absorbance at 260 nm and 280 nm with a spectrophotometer (Ultrospec®3000; Pharmacia Biotech). For RNA with a A260/A280 ratio of less than 1.8, the concentration of RNA was calculated using the relationship [nucleic acid] (mg/ml) = 0.063 x A260 – 0.036 x A280 (12). An absorbance wave scan from A240 to A300 was obtained as a preliminary assessment of the quality of the isolated RNA. Determination of the Quality of RNA using Gel Electrophoresis. The integrity of the extracted RNA was characterized by gel electrophoresis in 1X TAE buffer (40 mM Tris [pH 8.0], 1.14 ml of glacial acetic acid, 1 mM EDTA) with 1.2% agarose (13). The gels were stained with ethidium bromide and run at 70 V for about 1 hour. The gels were visualized and photographed under a UV transilluminator. Only samples with distinct 23S and 16S rRNA bands in an approximate ratio of 2-to-1 were used for hybridization (11). Slot Blotting. Triplicates of each of the 6 samples (May 11, May 20, May 27, June 8, June 11, June 14), together with RNA isolated from pure Nitrobacter culture as a positive control, RNase-free water as a negative control, were analyzed by slot blotting for each individual probe. The RNA samples were denatured with 3 volumes of 2% glutaraldehyde in 50 mM sodium phosphate (pH 7.0) and incubated for 10 minutes at room temperature. They were then diluted with 1 µg/ml of poly(A) water so that 1 µg of RNA in a final volume of 200 µl was applied to each slot. The diluted RNA was immobilized onto 9 x 12 cm Zeta-Probe® nylon membrane (Bio-Rad Laboratories) with a 48-well slot blotter according to instructions given in the Bio-Dot SF blotting apparatus manual (Bio-Dot® SF Microfiltration Apparatus; Bio-Rad Laboratories).