The Division of Infectious and Immunological Diseases, British Columbias Childrens Hospital1, and Departments of Paediatrics2 and Pathology & Laboratory Medicine3, University of British Columbia, Vancouver, BC, Canada
British Columbia Cancer Research Center, 601 West 10th Avenue, Vancouver, BC, V5Z 1L3, Canada4
Author for correspondence: Edith M. Dullaghan. Tel: +1 604 875 2491. Fax: +1 604 875 2226. e-mail: dullagha{at}interchange.ubc.ca
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ABSTRACT |
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Keywords: Mycobacterium avium complex, tuberculosis, strain differentiation, fingerprint, two-dimensional DNA electrophoresis
Abbreviations: 2DBGD, two-dimensional bacterial genome display; 2DDE, two-dimensional DNA electrophoresis; DGGE, denaturing gradient gel electrophoresis; MAC, Mycobacterium avium complex
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INTRODUCTION |
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It has become feasible to electrophoretically separate and display, in two dimensions, DNA fragments derived from genomic digests. Separation in the first dimension is by fragment size and in the second dimension separation is by mobility in denaturing gradients. Through the appropriate choice of restriction enzymes, changes as small as single base point mutations can be visualized in 2D gels. This technique has already been successfully used to display microsatellite polymorphisms in the human genome for use in genetic mapping and in studying genomic alterations in animal models and human cancers (Lam et al., 1996 ; Hughes et al., 1998
; Marcinek et al., 1997
). In addition, 2DDE analysis has been used to distinguish different strains of Bordetella pertussis (Malloff et al., 2002
).
We describe a method, two-dimensional bacterial genome display (2DBGD), for producing displays of mycobacterial genomes using 2DDE to separate genomic segments cut with various restriction endonucleases. We demonstrate the utility of this method by detecting genomic differences at the species and strain level and between isogenic mutants.
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METHODS |
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Isolation of genomic DNA from mycobacteria was carried out using the method of Belisle & Sonnenburg (1998) . Genomic DNA was digested with a variety of restriction enzymes that had been initially tested using the genome restriction digest tool of the Comprehensive Microbial Resource (Peterson et al., 2001
) to select ones that would produce an even distribution of fragments ranging from 200 to 2000 bp.
2D DNA electrophoresis.
Five hundred nanograms of digested DNA fragments was treated with calf intestinal alkaline phosphatase (New England Biolabs) prior to radiolabelling with 35 kBq [-32P]ATP (6000 Ci mmol-1; Amersham Pharmacia Biotech) using T4 polynucleotide kinase (New England Biolabs). The resulting fragments were size-fractionated in 5% non-denaturing acrylamide gels in electrophoresis buffer (40 mM Tris, 20 mM sodium acetate, 1 mM Na2EDTA, 0·2%, v/v, glacial acetic acid, pH 7·4) for 1600 volt-hours. Following this, each gel lane was cut and placed on top of a large format (25x20 cm) 6% polyacrylamide denaturing gradient gel that contained an ascending gradient of formamide (1040 %, v/v) and urea (1·87 M) in electrophoresis buffer. In the second dimension, parallel denaturing gradient gel electrophoresis (DGGE) was performed using an ISO-DALT apparatus (Amersham Pharmacia Biotech) for 1700 volt-hours and a constant temperature of 68·5 °C. DGGE gels were run in parallel in the same buffer chamber to ensure uniformity of electrophoretic conditions. A maximum of 10 gels can be run simultaneously in the ISO-DALT apparatus. The gels were dried prior to exposure to film. Alternatively, a gel could be left hydrated prior to electroblotting for Southern analysis.
2D gel comparisons were carried out by visual inspection. Spot constellations were easily aligned when comparing local areas of approximately 4 cm2. Commercially available software for comparing 2D protein gels are suitable for such image analysis and comparison and we tested Malanie II (Bio-Rad) and NIH Image. However, in our experience, 2DBGD images were sufficiently reproducible that spot differences could be detected by simple visual inspection.
DNA probes and Southern hybridizations.
Hybridizations were performed using positively charged nylon membranes (Roche). For Southern transfer of the DNA, 2D gels were electroblotted using a DALT blotting kit in the ISO-DALT electrophoresis tank. DNA probes for hybridization were generated using PCR amplification of DNA from M. intracellulare D673, M. intracellulare D673-19KDa and M. intracellulare D673-Katg. PCR amplification was performed under standard conditions with a programme of 30 cycles of 94 °C for 1 min, 63 °C for 1 min and 72 °C for 1 min and a final cycle of 72 °C for 10 min. Digoxigenin-labelled PCR products were generated for use as probes using oligonucleotides 5'-CACCTACCGCATCCACGAC-3' katG350368, 5'-GGTCTCCTCGTCGTTCAT-3' katG806788, 5'-GTTCGGGTGGTAACAAGTCG-3'19Kdaf and 5'-GCCGCTGATCTTGTAGCTGT-3'19Kda rev. Prehybridization and hybridization was carried out according to the manufacturers instructions (Roche).
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RESULTS |
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Species differentiation
Various species of mycobacteria were resolved using 2DBGD. The conditions for resolution of each species were determined empirically. On each occasion, no two species of mycobacteria produced the same display, regardless of the enzymes used. The choice of enzymes for use in this work were selected using the M. tuberculosis H37Rv genome sequence (Cole et al., 1998 ) and the genome restriction digest tool of the Comprehensive Microbial Resource (Peterson et al., 2001
) and included HinfI, AluI, Sau3AI, Sau96AI, AflIII and NcoI. Fig. 1
shows AluI displays of the genomes of M. intracellulare D673 and M. avium 104. The result obtained clearly demonstrates that the resolving power of this method can distinguish between different species of mycobacteria.
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The first mutant, D673-19KDa, was produced by insertional mutagenesis using a cassette containing the 19 kDa antigen (19Ag) gene of M. intracellulare D673 disrupted by the gentamicin-resistance (Gmr) gene of pUC-GM (Mahenthiralingam et al., 1998 ). The mycobacterial 19Ag is a highly expressed glycolipoprotein known to be immunodominant in infected patients and considered a candidate virulence factor (Young & Garbe, 1991
). Southern analysis of AflIII-digested genomic DNA from M. intracellulare D673 and the 19Ag mutant was performed using a digoxigenin-labelled Gmr cassette as probe. As expected, this produced two hybridization signals in the mutant but none in the parental D673 (data not shown). Therefore, this enzyme was a logical choice for resolution of this mutant, although digests using other restriction enzymes were also used to identify differences. Interestingly, regardless of which restriction enzyme was used with this mutant, between five and ten visible differences could always be identified, of which one is illustrated in Fig. 4
.
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DISCUSSION |
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A number of factors determine the resolution limits of 2DBGD; however, 5001000 spots can be typically resolved. Electrophoresis conditions (time, voltage and temperature), steepness of the denaturation gradient and acrylamide concentration can all be adjusted to best resolve any specific size range and G+C content. There are, however, areas of poor resolution present in the displays, for example, at the leading edge of the gel where the density of fragments is too great for resolution. Theoretically, those fragments that are not well resolved under one set of conditions would be located in a different position of the display when an alternative enzyme or enzyme combination is used.
2DBGD is capable of comparing entire bacterial genomes and can identify large and small variations. The utility of 2DBGD, however, in comparing distantly related strains is limited because of the numerous small deletions or point mutations that are unrelated to the phenotype but result in significant differences in genomic displays (Figs 1 and 2
). As an alternative, a new method called bacterial comparative genomic hybridization (BCGH) may facilitate the differentiation of such numerous inconsequential genetic alterations by resolving the reference and the test samples together on the same 2D gel. Following 2DBGD, the gel is then transferred to a positively charged nylon membrane, which is then hybridized sequentially with probes generated from the two samples. This technique produces two superimposable images generated from the same blot, which, when colour coded differentially, reveal the signals that are unique to either one or the other sample (Malloff et al., 2001
).
The utility of 2DBGD lies, mainly, in the identification of variations between the genomes of closely related strains, such as reported in this study for H37Rv versus H37Ra (Fig. 3) and for D673 and its mutants, D673-19KDa and D673-Katg (Figs 4
and 5
). We deliberately selected two mutants constructed in our laboratory to enable us to check the power of 2DBGD to detect minor genetic differences. In addition, the hybridization with probes to the known mutation validated the visual identification in that they confirmed that the spots that differed between wild-type and mutant were fragments of the mutated gene.
Other genome comparison techniques such as microarrays, PFGE and genomic subtractive hybridization are powerful molecular tools. However, these methods are not designed to detect small deletions and insertions, point mutations, or genetic rearrangements. 2DBGD is sensitive enough for detecting such alterations and, unlike array technology, can identify the gain or loss of DNA from both test and reference strains. Furthermore, 2DBGD can identify minor changes in DNA sequence that would not be resolvable with techniques such as DNA microarrays or subtractive hybridization.
We have shown that the high GC genomes of mycobacteria can be resolved by 2DDE and demonstrated the utility of the 2DBGD technology in detecting genetic differences between species, strains and isogenic mutants of mycobacteria. Whilst we recognize that multiple techniques will be required to fully unravel the genetic differences between virulent and avirulent forms of mycobacteria, 2DBGD represents a novel approach to this problem.
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ACKNOWLEDGEMENTS |
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Received 4 April 2002;
revised 22 June 2002;
accepted 25 June 2002.