PCR Fingerprinting of rRNA Intergenic Spacer ...

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16 PCR Fingerprinting of rRNA Intergenic Spacer Regions for Molecular Characterization of Environmental Bacteria Isolates M. Alejandra Martínez and Faustino Siñeriz 1. Introduction The analysis of DNA or RNA molecules has been used in a large number of studies on bacterial taxonomy and typing and for diversity studies. Undoubtedly, these methods presently dominate modern taxonomic studies as a consequence of technological progress, but primarily because the present view on classification is that it should reflect the natural relationships as encoded in the DNA. Some of these methods are not fully sequence-based, although in a number of cases they have provided considerably more information than traditional phenotypic methods (1). Among genotyping methods, analyses of multigene families such as rrn operons and tRNA genes have been demonstrated to be highly useful (2). The rrn operon is a multigene family that frequently presents more than one copy in most bacteria. With some exceptions (3), the rDNA genetic loci in eubacteria include, in 5' to 3' order: 16S, 23S, and 5S rRNA genes, which are separated by intergenic transcribed spacer (ITS) regions. Sequence evaluations of the 16S rRNA have been used frequently as a powerful and accurate method for determining inter- and intraspecific relationships (1). However, owing to their highly conserved nature, closely related organisms are often found to have nearly identical 16S rDNA sequences, limiting its power in resolving close relationships. Indeed, as evolutionary distances decrease, the diversity found in the 16S rRNA gene is often insufficient to define genetic relationships of closely related species (4).

From: Methods in Biotechnology: Environmental Microbiology: Methods and Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer

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The 16S–23S rDNA intergenic regions were denoted as internal transcribed spacers (ITSs; 5,6), as intergenic spacer regions (ISRs; 7), and even long intergenic spacer regions (LISRs; 8). To avoid confusions and also to follow Gürtler and Mayall (2), we will call the 16S–23S rDNA spacers ISRs. ISRs are less conserved than adjacent regions (16S and 23 S rDNA genes) as a result of a lower selective pressure during evolution, thus exhibiting a great deal of sequence and length variation at the genus and species level. The latter is partly because of the variations in the number and type of sequences with functional roles (tRNA genes) that are also found within the ISR (9). As a consequence, analysis of 16S–23S ISR length polymorphism and sequence variability has been shown to be an important supplement to 16S rDNA sequencing for differentiating bacterial species and even strains of prokaryotes, now being more frequently used for microbial typing and evolutionary and diversity studies of many groups of bacteria. Sequences of the 16S–23S regions of several species have become available for comparison. 16S–23S ISRs could be easily targeted by PCR (ISR-PCR) to show spacer length polymorphism in the different rDNA gene operons of the genome, based on their relative electrophoretic mobility on agarose gels. Length varies considerably between species (200–1500 bp), usually showing significant variations at the genus or species level and in some cases intra-species variations (8,10–16). In screening programs to isolate strains of biotechnological interest or in diversity studies of cultivable bacteria, it is necessary to use a fast and easy procedure to characterize and differentiate them, especially when a large number of isolates is to be considered. Among PCR fingerprinting methods, ISRPCR has been successfully used in our laboratory as a fast and reproducible procedure to cluster isolated bacteria according to different band patterns. After clustering different isolates, they could be analyzed and identified according to morphological characteristics, biochemical properties, and several genotyping methods. It is important to note that the use of intergenic spacers is included in genomic approaches to typing, taxonomy, and evolution of bacterial isolates, and also in studies of prokaryotic diversity (most of the references cited). Nevertheless, it is important to note that further work needs to be done to determine the precise role of rrn recombination in phylogenetics, evolution, and typing, since concerted evolution of the rrn multigene family by recombination and mutation in 16S–23S rDNA spacer rearrangements has been described (2,17). This chapter focuses on PCR fingerprinting of rRNA spacers in environmental isolates belonging to the genera Bacillus. We also obtained good results with actinomycetes strains. In our laboratory, different groups work with each bacterial genus, and particular characteristics were used to isolate them.

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Because several copies of the rDNA operon are present in the bacterial genome, a simple and fast chromosomal DNA isolation procedure for all groups of bacteria has been shown to be adequate to ensure target sequences suitable for PCR amplification. 2. Materials 2.1. Culture Media For Bacillus strains: nutrient broth composed of 5.0 g/L peptone, 3.0 g/L meat extract, 15 g/L agar, if necessary; pH 7.0. To grow alkaliphilic strains, pH of the medium was raised to approx 10.0 after sterilization by the addition of sterile 1% NaHCO3 (1 mL in 10 mL) from a stock solution, or 1 M Na-sesquicarbonate solution prepared with 4.2 g NaHCO3 and 5.3 g Na2CO3 anhydrous and distilled water to 100 mL.

2.2. DNA Isolation 1. Lysis buffer TEC-SDS: 10 mM Tris-HCl, pH 8.0; 10 mM EDTA; 100 mM NaCl, and 2% (w/v) SDS. 2. 20 mg/mL Proteinase K in distilled water. 3. 3 M Sodium acetate, pH 5.2. 4. TE-buffer-saturated phenol (18). 5. Chloroform:isoamyl alcohol, 24:1 (v/v) (18). 6. Isopropanol and 70% ethanol 7. RNAase A solution (10 mg/mL stock) in distilled water (18).

2.3. PCR Amplification 1. Taq polymerase and 10X STR buffer (Promega). 2. Thermal cycler (PE 9700, Applied Biosystems, CA). 3. 100 mM Stock solution of primers ISR-1494 (5'–GTCGTAACAAGG TAGCCGTA–3') and ISR-35 (5'–CAAGGCATCCACCGT–3') (12) (see Note 1).

2.4. Agarose Gel Electrophoresis 1. 2. 3. 4. 5.

TAE buffer (1X): 0.04 M Tris-acetate, 0.001 M EDTA, pH 8.0. 2% Agarose in 1X TAE buffer. Ethidium bromide staining solution (18). Molecular weight markers: 1 kb and 100 bp DNA Ladders (Promega). Loading buffer (Promega) or prepared according to Sambrook et al. (18).

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3. Methods 3.1. DNA Isolation 1. Transfer 1.5 mL of an overnight culture to a microcentrifuge tube and spin down 2 min. Discard the supernatant. 2. Resuspend the pellet in 750 mL of lysis buffer by repeated up and down pipetting. Add 15 mL of 20 mg/mL proteinase K, mix by inverting the tube carefully, and incubate 30 min to 2 h at 55rC (see Notes 2 and 3). 3. Add an equal volume of TE-buffer-saturated phenol and mix by inverting the tube several times. 4. Centrifuge (10,000g, 5 min). Transfer the aqueous phase (upper) to a new tube and repeat phenol extraction once. 5. Transfer the upper aqueous phase to a clean tube and add an equal volume of chloroform:isoamyl alcohol. Again mix well and centrifuge (10,000g, 5 min). Repeat this extraction three times. 6. Transfer the aqueous phase to a new tube. Add 1/10 vol of 3 M sodium acetate. 7. Add 0.6 to 1 vol of 2-propanol and mix gently until the DNA precipitates. If a low amount was recovered, precipitation could be favored by centrifugation (15,000g, 10 min). 8. Discard the supernatant and wash DNA with 500 mL of 70% ethanol to remove residual salts and isopropanol. Centrifuge (15,000g, 5 min), carefully discard the ethanol, and dry until ethanol has been removed (see Notes 4 and 5). 9. Resuspend DNA in 100–200 mL double-distilled sterile water and 1–2 mL RNAase A. Allow to dissolve at 37rC for several hours or overnight at 4rC (see Note 6).

3.2. PCR Amplification 1. Reaction mixture. Prepare 20 mL for each sample in one mixture considering total number of samples plus an additional control tube that will not include template DNA. Each tube should contain 0.5 mL of isolated DNA, 2 mL of 10X STR buffer, 0.2 mL of Taq Blue polymerase (1 U), and 0.1 mL of each primer (0.5 mM final concentration). Double-distilled sterile water to 20 mL. Dispense 19.5 mL for all samples tested into PCR tubes and add samples (see Notes 7 and 8). 2. Amplification conditions: initial denaturation at 94rC for 4 min followed by 30 cycles each consisting of 94rC for 1 min, 55rC for 2 min, and 72rC for 2 min, with a final extension step at 72rC for 7 min (see Note 9). 3. Evaluation of PCR fingerprint obtained: runs should be made in 2% agarose gels using appropriate markers in the range of 100 bp to 2000 bp. Electrophoresis should be performed at 50 V for 2 h (see Note 10). Stain the gel in ethidium bromide and observe under UV light (see Notes 11 and 12).

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Fig. 1. ISR-PCR from Bacillus spp. strains. Lanes: M1, 1 kb DNA Ladder; 1, B. subtilis 1 A1 (obtained from BGSCa); 2, B. halodurans DSM497T (purchased as DSMZb). From lanes 3 to 10, Bacillus sp. alkaliphilic strainsc: 3, MIR32; 4, MRL1; 5, MRL2; 6, MRL22; 7, MRL33; 8, MRL4; 9, MRL5; 10, MRL5; 11, control reaction without DNA template; M2, 100 bp DNA Ladder. Band between 200 and 600 bp are obtained for members of subtilis and cereus groups (Martinez, unpublished data), and patterns including long ISR spacers, generally between 600 and 1200 bp are typical for most alkaliphilic Bacillus spp. tested (see Note 13). a = Bacillus Genentic Stock Center (BGSC); b = German Collection of Microorganisms (DSMZ); c = Isolated in soda lakes in Kenya, Africa (Breccia, personal communication).

4. Notes 1. Concerning primer sequences, the ones given were useful in Bacillus and even in actinomycetes strains, but they could be used to check other possibilities such as L1 5'–CAAGGCATCCACCGT–3' and G1 5'–GAAGTCGTAACAACG–3' (6,19), which mainly differ in the position of the primer complementary to 23S sequence (3,7,9,20–22, and others). 2. When setting up DNA isolation and PCR reactions, wear gloves to minimize the risks of DNAase contamination. In the case of PCR preparation, gloves should be powder-free because powder inhibits DNA polymerases. Precautions must be taken when handling dangerous solutions such as phenol, chloroform, and ethidium bromide. 3. Information on this topic can be found at Material Safety Data Sheets (www.sigma-aldrich.com).

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4. DNA isolation. It could be necessary to optimize lysis conditions when handling wild-type isolates. Spore-forming bacterial strains might need to be processed before the end of the exponential growth phase. If poor clarification is achieved after lytic treatment, proteinase K treatment could be extended. Nevertheless, further phenol treatment ensures enough DNA suitable for PCR. 5. After two ethanol wash steps, liquid can be discarded by inverting tubes, followed by a spin down of 1 min to allow residual liquid in the walls to be collected by carefully pipeting from the bottom of the tubes. Do not overdry DNA pellets; this can make resuspension difficult. 6. DNA samples can be stored for several weeks at 4rC. Aliquots of samples can be kept at –20rC, taking into account that repeated freezing and thawing of the samples can damage DNA. 7. DNA samples obtained by this procedure are usually suitable for PCR amplification. Concentration and quality can be tested by gel electrophoresis and absorbance measurements (18). Nevertheless, OD measurements tend to overestimate DNA concentration. After electrophoretic evaluation, concentration of the samples can be equalized by repeating steps 5–8. 8. Total PCR reaction volume should be loaded to allow detection of all bands, even the less intense ones. Inclusion of controls without template is important because possible PCR artifacts should be considered when analyzing bands of low size (100–300 bp). 9. The use of longer annealing times and temperature ramps has been described (11), although we obtained good results as indicated previously. 10. Electrophoresis runs of 2–3% agarose gels should be relatively slow (40–50 V). Migrations of approx 10–11 cm of the front marker should be done to allow a discrete band pattern to develop. 100 bp Ladder and 1 kb Ladder (Promega) could be loaded in a separate well or in the same well. It is important to consider that bromophenol blue contained in gel-loading buffer migrates at approximately the same rate as DNA 300 bp in length. 11. The use of capture-image systems and software are useful when several bands are obtained to precisely determine the number and size of bands. 12. Absence of amplification could be caused by residual phenol or ethanol in the final preparations. To test reagents, a positive (amplifiable) control should be included in all assays. Samples can be recovered by repeating steps 5–9. 13. Frequently, Bacillus species yield several bands, and intra-species variations are sometimes observed. For example, alkaliphilic strains present larger spacers (Fig. 1). Evaluation of intergenic length polymorphism of hypervariable parts of conserved genomic regions revealed us typically large 16S–23S intergenic regions in alkaliphilic Bacillus strains in comparison with those described for other Bacillus species and lane 3 (11,2,3).

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