Ribosomal RNA (rRNA) intergenic spacer analysis (RISA) is a microbial community analysis method. It is used in comparing microbial cultures which differ due to environmental or treatment method. It can work without bias as of in culture-dependent approaches. It is also known as community fingerprinting. The method involves amplification of a part of the rRNA gene operon. It is done between the small (16s) and large subunits (23S) and the subunits are called intergenic space region.
RISA is applied in agriculture field for the study of environmental sustainability of the soil sample. Biological nitrogen fixation (BNF) is mediated by rhizobia. It is one of the important factors in soil fertility as an increase in BNF may reduce the use of chemical nitrogen fertilizer. Rhizobia-host plant interaction is highly specific. Therefore diversity in the rhizobia population can increase the efficiency of natural BNF. In a given environmental condition-specific related group of rhizobia population will be dominant.
In the particular example the treatments are labeled as no-tillage (NT) and conventional tillage (CT) agricultural systems. The applicability and impact of the tillage can be studied using RISA methodology. The extent of natural rhizobial communities as bioindicators for legume cropping is primary in assessing the efficiency of the agricultural practices. Monitoring rhizobial communities can be applied to study the effects of treatment. Traditional microbiological techniques are not adequate for monitoring the rhizobial community as isolating and enumerating the rhizobia involves the use of trap plants. The other drawback in microbial techniques is it does not represent the entire population, as the plants only interact with a selected or specific group of rhizobial community. Direct soil based molecular techniques are used to overcome the limitations of traditional culture-based methods. PCR combined the rRNA cloning and sequencing, Denatured gradient gel electrophoresis (DGGE) of PCR amplified DNA fragments is used in the study of complex bacterial population.
Development of validation of a soil DNA based method for generating 16S-23S rDNA IGS fragments specific for rhizobial groups that commonly nodulate bean is discussed below.
Materials and methods
Bacterial strains and growth conditions
Rhizobium strains were grown in yeast–mannitol (YM) broth (0.5 g KH2PO4, 0.2 g MgSO4.7H2O, 0.1 g NaCl, 0.5 g yeast extract, 10 g mannitol and 0.5% bromothymol blue liter-1 distilled water) for 48 h at 280C under constant shaking. Long-term storage was at 800C in 50% (v/v) glycerol and by lyophilization.`
Field experiment and soil samples
Soil samples were collected and analyzed for parameters like pH, organic matter, Dry Weight, etc. The experimental area comprised four plots established at random: two no-tillage treatment plots (NT I and NT II) and two plots under conventional tillage (CT I and CT II). In the NT plots, the remains from the previous soybean crop were treated with 2.0 L ha-1 of glyphosate and 1.5 L ha-1 of 2,4-dichlorophenoxyacetate-amine 20 days prior to sowing common bean, and it was not ploughed or disc harrowed. In the CT plots, the remains of the previous crop were incorporated into the soil by ploughing and disc narrowing the topsoil. Common bean was sown in both the fields. It is supplemented with 300 kg ha-1 N-P-K fertilizer.
Thirty days after sowing, ten individual samples (approx. 100 g each) from each plot were collected at random, from the topsoil layers (20 cm), between the bean rows. These samples were pooled per plot and stored at 20oC for subsequent DNA extraction. A pooled sample is also taken prior to the experimentation. It is labeled as T0 sample.
Extraction of DNA from pure cultures and soil
Total genomic DNA was isolated from pure cultures of reference strains. Gel electrophoresis is used as the control of the genomic DNA. The high molecular weight genomic DNA was extracted to sufficient purity by PCR method. It is done to all the samples under the study. Direct extraction of the soil DNA is done using mechanical lysis.
Genomic DNA extraction procedure
1) The sample (100 mg, fresh weight) was ground in 500 μL of a guanidium-based buffer (4.2 mol L–1 guanidinium thiocyanate, 100 mmol L–1 Tris-HCl [pH 7.5], 0.05% sarcosyl) and 5 μL of 2-mercaptoethanol with a mortar and pestle.
2) After 3 min at 60°C, the supernatant was collected after centrifugation (19 000 g, 5 min, 4°C).
3) An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added to the sample and shaken gently for 15 min at room temperature.
4) The upper phase was re-extracted two more times after centrifugation (19 000 g, 10 min).
5) The tube was shaken back and forth after adding 0.1 volume of 3 mol L–1 sodium acetate and 2 volumes of –20°C 100% ethanol. Then it was immediately centrifuged at 19 000 g for 3 min.
6) The precipitate was washed by centrifugation (19 000 g, 3 min) with 1 mL of 70% ethanol and dried in a vacuum desiccator for 2 min.
7) The pellet was suspended in 600 μL of TE buffer (10 mmol L–1 Tris-HCl, 1 mmol L–1 EDTA [ethylenediamine tetraacetic acid], pH 8.0) and mixed with a wide bore pipette.
8) To remove RNA, the sample was incubated overnight at 37°C with 2 μL of RNase (10 mg mL–1, DNase-free grade).
9) Then 6 μL of 20 mg mL–1 proteinase K was added and incubated at 50°C for 2h.
10) To remove contaminating polysaccharides, the sample was purified with Genomic-tip.Genomic-tips are gravity-flow, anion-exchange tips that allow efficient purification of genomic DNA from a wide range of biological samples. It was important to dilute genomic DNA lysate with an equal volume of TE buffer and to vortex for 10 s at maximum speed to prevent clogging of the Genomic-tip prior to sample loading.
11) The preparation was measured with a spectrophotometer.
12) Electrophoresis was conducted using 0.8 Agarose H for 15 h at 1.7 V/cm
(constant volts) in TAE buffer (40 mmol L–1 Tris-acetate, 1 mmol L–1 EDTA, pH 8.0).
PCR amplification of 16S–23S rDNA IGS fragments
A nested PCR approach was used with soil DNA to amplify R. leguminosarum and/or R. tropici IGS fragments. Two sets of primers were used. Independent nested PCRs were performed using community DNA from each soil. In the first PCR reaction, bacterial 16S–23S rDNA IGS sequences were generically amplified using the conserved primers pHr and p23Suni322anti. PCR was performed in 50 mL reaction volumes. Phage T4 gene 32 protein and formamide were added to improve the efficiency of target amplification when using soil DNA. PCR amplifications were carried out using initial denaturation at 950C for 2 min, followed by 30 cycles of 1 min at 940C, 1 min at 600C and 3 min at 720C; and a final extension at 720C for 5 min, in a thermal cycler. In the second PCR, the primer sets I (rhizo2f/rhizo3r) and II (trop1f/rhizo3r) were employed, in separate reactions, to amplify R. leguminosarum/R. tropici and R. tropici-specific rDNA spacer fragments, respectively. Products for subsequent DGGE analyses were prepared with the GC-clamped reverse primer rhizo3r. Aliquots of 1 mL from the first PCR were used as templates in the subsequent (50 mL) reactions, from which formamide or phage T4 gene 32 protein were omitted. Touchdown PCR was performed in order to optimize both specificity and sensitivity. An initial denaturation step at 950C for 2 min and a final extension step at 720C for 10 min were performed for all samples. For primer set I, after denaturation at 940C for 1 min, the annealing temperature was initially set at 620C for 30 s, and then decreased to 600C by 10C every 3 cycles, followed by 26 additional cycles at 580C; primer extension was performed at 720C for 45 sec. For primer set II, after denaturation at 940C for 1 min, the annealing temperature was set at 55oC for 1 min and then decreased to 51oC by 20C every 3 cycles; then 26 additional cycles were carried out at 500C; primer extension was performed at 720C for 1 min. The same amplification conditions were used to amplify IGS fragments from the reference strains, using 50 ng of genomic DNA.
On the basis of an analysis of database sequences, the expected fragment sizes were 200–320 bp (primer set I) and 240–400 bp (primer set II). The amplicons were checked by electrophoresis on 1.4% agarose gels in 0.5 strength TBE buffer and stored at 20oC for subsequent cloning and DGGE analyses.
Construction and analysis of IGS fragment clone libraries
Clone libraries were constructed on the basis of IGS fragments generated with both primer sets I and II from soil DNA obtained from treatments T0, NT and CT. The PCR products were first purified using the High-pure PCR product purification kit. They were then ligated into the pGEM-T vector cloning kit. Competent Escherichia coli cells were transformed with the ligation mixes. Using blue/white screening, for each treatment 19 clones with putative inserts were selected, and the presence of inserts was verified by specific PCR. The products generated from the clones were (1) assessed for migration on DGGE gel (after re-amplification to introduce the GC clamp), and (2) electrophoresed on an agarose gel to assess their sizes. Clones with inserts of the expected sizes were selected for sequencing. About 190–200 bp of high-quality sequence was obtained per clone, and all analyses were based on this sequence information. These clones are used for phylogenetic analysis and tree construction.
DGGE was carried out using a linear denaturing gradient of urea and formamide ranging from 45% to 65%. Gels were run at 100 Vand 600C for 16 h in 0.5 X TAE buffer. A DGGE marker, composed of a mixture of 16S rDNA fragments from Enterobacter cloacae BE1; Listeria innocua ALM105; R. leguminosarum bv. trifolii R62; Arthrobacter sp. Ar1 and Burkholderia cepacia P2 (listed in order of migration on DGGE gel) generated with primer set U968-GC and L140, was used as the reference in the gels. Gels were stained and documented. DGGE patterns were analyzed by using Gel electrophoresis software.
Sequencing of clone inserts and DGGE bands
Sequences from the IGS fragment clones are obtained in this step. They are specific PCR products were generated by primer sets I and II. To obtain sequences from DGGE bands, Small blocks of acrylamide gel containing fragments of interest were excised from the gel. DNA was extracted by using the crush and soak method. Pellets were resuspended in 15 μL TE buffer for subsequent PCR and cloning. The DNA from selected bands was then diluted and subjected to PCR using the DGGE primer set. The dilution that yielded one single band with migration distance equivalent to the band of interest in DGGE was then used as the template in subsequent PCR reactions, using primers without GC clamp. PCR products (2 μL) were cloned into the pCRR vector. Sequencing of the inserts of IGS clones was performed on PCR products generated from the respective clones. Sequencing of DGGE bands was performed on both strands. The Thermo sequenase fluorescently-labelled primer cycle sequencing kit was used with 7-deaza-dGTP in an automatic sequence analyzer.
Phylogenetic analysis of 16S–23S rDNA IGS fragments
Sequences of the 16S–23S rDNA IGS fragments generated in this study were compared with reference sequences that showed highest similarity values in BLAST-N searches as well as a selected outgroup sequence, all recovered from GenBank. The sequences were aligned using the CLUSTAL-X program and analyzed using PAUP. Evolutionary distances were calculated using the Kimura 2p DNA substitution model with settings for gap spacing of 10 and gap extension of 5. The phylogenetic reconstruction was done using the neighbor-joining algorithm, with bootstrap values calculated from 1000 replicate runs, using the routines included in the PAUP software.
Soil rhizobial population are low in numbers. Therefore direct PCR methods based on soil DNA are useful in their detection. Temple concentrations are the limiting factor in these methods. Nested PCR is recommended in this case. This enhances the sensitivity of PCR-based fingerprinting of spectral bacterial groups. First, the amplification is targeting the bacterial r DNA 16S-23S IGS region is done. In the second round of amplification two rhizobial specific primer sets are done. The two-step process helps in amplifying targeted IGS sequences of species of interest. Nested PCR is necessary since direct PCR did not yield any detectable signal in any soil test.
The analysis of the sequences in the clone library obtained from the soil confirms the specificity of the PCR based method for species related to the target. After the first amplification the sequences showed a broad range of species around the targeted one and after the second amplification, the sequences are centered around the targetted species. The close relatedness of the spectrum is tested by IGS sequences clone library. Specificity of detection based on the IGS sequences was supported by BLAST-N and phylogenetic analysis.
RISA application in monitoring the snail population the methodology is discussed below.
The majority of snails used were reared and maintained at room temperature under identical conditions in aquaria with running water, sterilized earth, and calcium carbonate, except some field populations. The snails were identified by means of comparative morphology based on the reproductive organs and shells accordingly.
Total DNA was extracted from both the foot and eggs of the snails essential. Briefly, the snails foot or eggs were mechanically disrupted in 50 mM Tris– HCl, pH 8.0, 100 mM NaCl, 50 mM EDTA, 0.5% SDS and incubated overnight at 37oC with 50 mg/ml proteinase K. Following phenol/chloroform extraction and ethanol precipitation, DNA was resuspended in 10 mM, Tris–HCl, pH 8.0, 1 mM EDTA, and DNA concentrations were estimated by comparison with known standards on 2% ethidium bromide-stained agarose gels.
rRNA-Internal transcribes spacer (ITS) amplification
The entire ITS was amplified using the primers ITS1 (58-TAACAAGGTTTCCGTAGGTGAA-38) and ITS2 (58-TGCTTAAGTTCAGCGGGT-38) anchored, respectively, in the conserved extremities of the 18S and 28S ribosomal genes. PCR amplification was undertaken in a volume of 10 ml consisting of 1–10 ng template DNA, 10 mM Tris–HCl, pH 8.5, 200 mm each dNTP, 1.5 mM MgCl2, 0.8 U of Taq DNA polymerase, 50 mM KCl, together with 5.0 pmol of each primer. The reactions were covered with a drop of mineral oil and subjected to the following cycle program: initial denaturation step for 3 min at 95oC and then 32 cycles with annealing at 540C for 1 min, extension at 720C for 2 min, denaturation at 950C for 45 s, and a final extension step at 720C for 5 min. A negative control (no DNA) was included in all experiments. Three microliters of the amplification products were visualized on 0.8% ethidium bromide-stained agarose gels to check the quality of amplification. The remaining 7 ml was mixed with 93 ml of water and divided into 10-ml samples for enzyme digestion.
Production and evaluation of the rRNA-ITS RFLP profiles
To evaluate possible enzymes that might yield informative RFLPs of the regions of Biomphalaria, eight restriction enzymes with 4-bp recognition site were used—AluI, DdeI, HaeIII, HinfI, MnlI, MspI, RsaI, and Sau3aI. One microliter (10–12 units) was used to for each digestion reaction, together with 1.2 ml of the respective enzyme buffer in a final volume of 12.2 ml. The digestion was performed for 2 to 3.5 h at 370C, and the digestion products were evaluated on 6 to 8% silver-stained polyacrylamide gels after phenol/chloroform extraction. The results were recorded with Polaroid film.A control for the activity of each enzyme was performed by the ITS digesting 150 ng of pUC18 simultaneously with the samples being evaluated.