Denaturing gradient gel electrophoresis is a powerful and convenient tool for analyzing the sequence diversity of complex natural microbial populations. To amplify 16S rDNA and PCR product specific primers were used and analyzed by Denaturing gradient gel electrophoresis. Genus- and cluster-specific probes were designed to bind to sequences within the region amplified by these primers. All b-subgroup ammonia oxidizer’s specific sequence could not be identified, but probes specific for Nitrosospira and Nitrosomonas were designed. The presence of Nitrosomonas cluster and Nitrosospira clusters were confirmed by denaturing gradient gel electrophoresis banding patterns, but results were ambiguous because of overlapping banding patterns. Compared to internal control probe, the signal from the Nitrosospira cluster 3 probe decreased significantly, with decreasing pH in the range of 6.5 to 3.8, while Nitrosospira cluster 2 hybridization signals increased with increasing soil acidity. At pH 5.4, signals from Nitrosospira cluster 4 were greatest, decreasing at higher and lower values, while Nitrosomonas cluster signals did not vary significantly with pH. Different sets of PCR primers for amplification of 16S rDNA sequences from soil were used in two studies and the similar findings suggest that PCR bias was unlikely to be a significant factor. The current findings reveals the worth of DGGE and probing for quick analysis of communities of b-subgroup proteobacterial ammonia oxidizers, denotes momentous pH-associated differences in Nitrosospira populations, and indicates that Nitrosospira cluster 2 may be of significance for ammonia-oxidizing activity in acid soils.
Key words: Ammonia oxidizing bacteria, DGGE, PCR, Nitrosospira, Nitrosomonas
Effluents discharged from wastewater treatment plants (WWTPs) into rivers may have detrimental environmental effects, because they are a source of high levels of nutrients, organic matter, and bacteria (Kowalchuk, 2001). The Amla Khadi is greatly affected by the discharge of the effluents from the waste water treatment plant, which treats the wastewater from 6.5 million inhabitant equivalents. These effluents contain large amounts of ammonium (NH4+ ), which is completely oxidized into nitrite (NO2′ ) and subsequently converted to nitrate (NO3′ ) by the nitrification process. Autotrophic nitrification is accomplished in two stages by two distinct groups of bacteria: ammonia oxidizers and nitrite oxidizers. Ammonia oxidation due to chemolithotrophic ammonia-oxidizing bacteria is the first and often the rate limiting step of nitrification; it is essential for the removal of nitrogen from the environment (Eichner, 1999). Aerobic autotrophic ammoniaoxidizing bacteria are found within two phylogenetic groups based on comparative analyses of 16S rRNA sequences (Torsvik, 2002). One group comprises strains of Nitrosomonas and Nitrosospira spp. within the class of ??- Proteobacteria, and the other contains Nitrosococcus oceani and Nitrosococcus halophilus within the class of gamma-Proteobacteria. All ??-proteobacterial ammonia oxidizers belong to a phylogenetically coherent group, within which all the organisms have the same basic physiology as far as we know. A continually expanding database of ammonia oxidizing bacteria 16S ribosomal DNA gene sequences has produced descriptions of distinct lineages and clusters within the genera Nitrosomonas and Nitrosospira of the ??-Proteobacteria (Torsvik, 2002). Pure culture representatives have been isolated for all groups, except for Nitrosospira cluster 1 and Nitrosomonas lineage 5, where only clone sequences are available. A number of studies suggest that there are physiological and ecological differences between the different ammonia oxidizing bacteria genera and lineages and that environmental factors such as salinity, pH, and concentrations of ammonia and suspended particulate matter select for certain species of ammonia oxidizing bacteria (Kim, 2000). These external factors, which include the impact of waste water treatment plant effluents, may therefore influence the range of ammonia oxidizing bacteria diversity and consequently the structure and function of the ammonia-oxidizing community. Most research has focused on ammonia oxidizing bacteria community composition in waste water treatment plant activated sludge (Rowan, 2003). Only a few studies on ammonia oxidizing bacteria diversity in freshwater environments or estuaries are available (Rowan, 2003). The application of molecular techniques, in particular analysis of 16S rRNA genes, provides new opportunities for the assessment of ammonia-oxidizing populations in aquatic sediments. Phylogenetic analysis of 16S rRNA genes of pure and mixed cultures places ammonia oxidizing bacteria in three groups. Molecular analysis of ammonia oxidizer 16S rDNA fragments; amplified from environmental DNA, by denaturing gradient gel electrophoresis characterizes community structure, enables rapid analysis of clone libraries, and, through excision, reamplification, and sequencing of bands from gels, provides information on species composition. This approach has demonstrated differences in populations of Nitrosomonas-like -proteobacterial ammonia oxidizing bacteria associated with polluted marine fish farm sediments (McCaig, 1999) and marine aggregates (Phillips, 1999) and domination by Nitrosospira-like organisms in Arctic Ocean waters (Bano, 2000). Analysis of 16S rDNA does not discriminate between metabolically active and quiescent cells. Analysis of environmental 16S rRNA, using reverse transcription-PCR, provides greater sensitivity, because of the higher target copy number, and may indicate which members of the community are more metabolically active, if active cells contain larger numbers of ribosomes (Nold, 2000). The main purpose of this study was to design and test oligonucleotide probes capable of distinguishing the subgroups within the different clusters of b-subgroup ammonia oxidizers from each other and then to use these probes to identify bands separated by DGGE analysis of PCR products generated from soil with ammonia oxidizer-specific primers.
Materials & Methods:
Sample collection and pretreatment: Five to 10 liters of water were collected for chemical, biochemical, and molecular analyses and were brought to the laboratory within 2 to 3 h. The water was filtered through glass fiber membrane filters and frozen until it could be analyzed for inorganic nitrogen compounds. Ammonium, nitrite, and nitrate levels were determined spectrophotometrically; ammonium and nitrite concentrations were determined according to the procedure of Slawyck and MacIsaac (1972), and nitrate levels were determined after Cd reduction to nitrite (Avrahami, 2003). Suspended matter was weighed on GF/F filters dried at 450??C. Potential nitrifying activities were determined by two methods directly after the sampling. For molecular analysis, 150 to 250 ml of water was filtered through 0.22- ??m-pore-size nitrocellulose filters in triplicate, and the filters were frozen (’20 ??C) until DNA extraction. The bacterial samples of the influents and effluents were harvested by centrifugation of 1.5 ml samples at 10000??g for 30 min. The cell pellets were washed once with phosphate-buffered saline (93 mmol Na2HPO4, 7 mmol NaH2PO4) before being re-suspended in 50 ??l of sterile distilled water, and then frozen at ’20 ??C. The cell suspensions were subjected to three freeze-thaw cycles and then used for whole-cell PCR amplification (Ovreas, 1997).
Nucleic Acid Extraction: DNA was extracted from the filters by a bead-beating method with the Fast DNA spin kit for soil according to the manufacturer’s instructions. DNA extracts were then stored at ’20 ??C until purification on a Sephadex G-200 column and ethanol precipitation. Nucleic acids were quantified by comparison between 1 ^l of an undiluted sample and a range of known DNA concentrations on an agarose gel stained with ethidium bromide. To obtain suitable PCR amplicons, 10- to 100- fold dilutions of crude DNA were used as templates for subsequent PCRs. DNA was visualized by UV transillumination. Digital images of the gels were obtained with a charge-coupled device camera controlled by Quantity One software.
Oligonucleotide Probe design: Primers and oligonucleotide probes were developed through the manual alignment of full-length 16S rDNA sequences and utilization of the ARB probe design tool and probe match programs (Table 1). The specificity of the oligonucleotide probes was verified with BLAST (Altschul, 1990) and CHECK_PROBE (Maidak, 1999). The probes were labeled at the 5′ end with indocarbocyanine dye (Cy-3) and/or with fluorescein isothiocyanate. The specificity of the designed primers was determined by amplifying template from inserts of known ammonia oxidizing bacteria 16S rDNA clones or pure bacterial cultures. Stepwise increments of annealing temperatures from 46 to 60 ??C were used to determine the optimal primer annealing temperature. A general ammonia oxidizing bacteria PCR was used to screen DNA extracted from nitrifying biomass for the presence of ammonia oxidizing bacteria using two published oligonucleotides, CTO189f (Kowalchuk, 1997) and NITROSO4Er (Hovanec, 1996), at an annealing temperature of 57 ??C for 30 cycles. Samples were also analyzed for the presence of specific strains of ammonia oxidizing bacteria using primers developed in this study. Nitrosomonas marina-like ammonia oxidizing bacteria were detected with NSMR71f and NSMR74r (54 ??C annealing temperature, 35 cycles), Nitrosospira tenuis-like AOB were detected with NSMR32f and NSMR33r (56 ??C, 35 cycles), Nitrosomonas europaea-like AOB were detected with NSMR52f and NSMR53r (56 ??C, 35 cycles), and Nitrosococcus mobilis-like ammonia oxidizing bacteria were detected with NMOB1f and NMOB1r (56??C, 30 cycles) (Table 1)
Probe Hybridization Analysis: PCR products recovered with the CTO primers were subjected to DGGE according to the protocol of Muyzer et al. (1996) as adapted by Kowalchuk et al. (1997) for the study of ammonia-oxidising bacteria. Gels contained a 38-50% gradient of denaturing chemicals with 100% denaturant defined as 7 M urea and 40% formamide. DNA was visualized after ethidium bromide staining by UV transillumination, and gel images were stored using `The Imager’ system. DNA in the polyacrylamide gels was blotted to Hybond-N. Nucleic Acid Transfer Membranes using a Transblot SD according to Muyzer et al. (1996). After completion of the transfer, the DNA was denatured (DNA-side down) on Whatman 3MM filter paper soaked with 0.4 M NaOH; 0.6 M NaCl and similarly neutralised with 1 M NaCl; 0.5 M Tris (pH 8). Membranes were sealed in plastic and stored at 4??C until further use. Hybridization analyses were conducted using the oligonucleotide probes and hybridization conditions described by Stephen et al. (1998). Specifically, the probes CTO189f, NITROSO4Er, NSMR71f, NSMR74r and NSMR32f were used to detect 16S rDNA fragments from all ammonia oxidisers, Nitrosospira, Nitrosomonas, cluster 3 Nitrosospira and cluster 4 Nitrosospira, respectively. No attempts to quantify the intensity of radioactive signals were made during the course of this study. 2.5. Sequence analysis of bands excised from denaturing gradient gel electrophoresis gels Bands chosen for sequence analysis was carefully excised from the denaturing gradient gel electrophoresis gel with a scalpel. Only the centremost 50% of each band was excised in order to avoid the lane edges where smearing was observed. DNA extraction, reamplification and DNA sequencing were as described by Kowalchuk et al. (1997). DNA sequence manipulations were performed using the SeqApp program, version 1.9a169 (Kowalchuk, 1997) and phylogenetic analyses were implemented through PHYLIP 5.57 (Stephen, 1998). Distance matrix analyses were according to the method of Shah MP (2014) with a masking function to exclude ambiguous data, and phylogenetic tree construction was by neighbour joining (Shah MP, 2014). Phylogenetic analysis was performed for 287 positions which could be unambiguously aligned for all sequences used in the analysis. Bootstrapping was conducted with 100 replicates using the program SeqBoot (Shah MP, 2014). Recovered sequences were also tested for homology to known sequences in the EMBL databank using the FastA program (Shah MP, 2014). Bands whose nucleotide sequences were determined have been given labels in Figures 2 and 3 which correspond to the sequence names beginning with a `B’ in Figure 1. The addition of an asterisk to a band label (Figures 2 and 3) indicates a difference of one base pair from the given numbered sequence. These differences have been shown to be introduced at the ambiguous position of the reverse primer by PCR  and have not been included in the phylogenetic analysis.DGGE, membrane transfer, and hybridization: The PCR products examined by denaturing gradient gel electrophoresis included the products obtained after specific nested PCR and RT-PCR. The fragment used spanned 465 bp of the 16S rRNA gene and included a 36-bp GC clamp (Sheffield, 1989) introduced during the PCR. DGGE was performed with a D-Gene system by using the protocol of Muyzer et al. (26) as adapted for analysis of b-subgroup proteobacterial ammonia oxidizers (19). DNA fragments with known ammonia oxidizer sequence cluster affinities (Fig. 1) (42) were also electrophoresed on DGGE gels (38 to 50% denaturant) as controls for subsequent hybridization analyses (data not shown). The DNA was stained with ethidium bromide and rinsed twice in deionized water prior to UV transillumination. DNA was transferred from the polyacrylamide gels to Hybond-N1 nucleic acid transfer membranes by using a Bio-Rad model SD semidry transblotter as described by Muyzer et al. (1996). The DNA was subsequently denatured (DNA side down) and simultaneously cross-linked to a membrane by soaking it in 0.4 M NaOH’0.6 M NaCl on 3MM filter paper. The membranes were similarly neutralized with 1 M NaCl’0.5 M Tris-HCl (pH 8). Hybridization analyses were conducted by using the oligonucleotide probes and hybridization conditions described by Stephen et al. (1998) (Fig. 1 shows the sequence clusters). No attempts were made to quantify the intensities of the hybridization signals obtained.
Quantification of relative abundances of sequence types: Gels were first probed with b’AO233 to quantify the total b-proteobacterial ammonia oxidizer signal in PCR products. Bands for clusters 4 and 6a are well separated, and the relative abundances of PCR products represented in these clusters in each environmental sample were calculated as the percentages of the total hybridization signal to b-AO233 within each lane represented by the cluster 4 or cluster 6a bands. The combined relative abundance of clusters 2 and 3 was calculated by subtraction from the total hybridization signal. Relative abundances of clusters 2 and 3 were determined by probing gels with NspCL2_458 and NspCL3_454, specific for clusters 2 and 3, respectively. Gels probed with b’AO233 showed that controls for clusters 2 and 3 were loaded at equal levels (within 0.5% of each other). Differences in binding efficiency and other factors between cluster 2 and 3 probes were determined by comparison of the hybridization signals for the respective controls, consisting of representative clones. Resultant correction factors were applied to hybridization signals for environmental samples to calculate relative proportions of cluster 2 and 3 signals. Finally, these values were expressed as proportions of the combined cluster 2 and 3 signal to give relative abundances of each cluster as a percentage of the total ammonia oxidizer signal. All analyses were carried out on two independent sets of soil samples, and results are expressed as means of duplicates.