Onderstepoort Veterinary Institute, Onderstepoort, 0110, Republic of South Africa1
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164, USA2
Author for correspondence: E. P. de Villiers. Tel: +31 30 253 6923. Fax: +31 30 254 0784. e-mail: e.devilliers{at}vet.uu.nl
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ABSTRACT |
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Keywords: Cowdria ruminantium, Rickettsiales, genome size, genetic map
Abbreviations: MACS, magnetic cell separation
a Present address: Division of Parasitology and Tropical Veterinary Medicine, Faculty of Veterinary Medicine, Utrecht University, PO Box 80.165, 3508 TD Utrecht, The Netherlands.
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INTRODUCTION |
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All Ehrlichia species are parasites of eukaryotic cells and many species cause important diseases of domestic animals and of humans (Rikihisa, 1991 ). Molecular genetic analysis of the Ehrlichieae, with the exception of Anaplasma spp., has been severely impeded because of their obligate intracellular habitat. The organisms are therefore difficult to separate from host-cell components, especially the nuclei, which makes genomic library construction and large-fragment DNA separation difficult.
Although a few genes from C. ruminantium have been identified and cloned (Mahan et al., 1994 ; Van Vliet et al., 1994
; Lally et al., 1995
; Allsopp et al., 1997
; Brayton et al., 1997
), nothing was previously known about the structure and organization of the genome. Recent advances in the purification of C. ruminantium (De Villiers et al., 1998
), using a combination of Percoll density-gradient centrifugation (Tamura et al., 1982
) and high-gradient magnetic cell separation (MACS) (Miltenyi et al., 1990
), have allowed us to study some of the basic molecular genetics of this organism. The objectives of this study were to determine the genome size of the Welgevonden isolate of C. ruminantium by PFGE analysis and restriction endonuclease digestion, and to construct a combined physical and genetic map by integrating PFGE and Southern blot analytical data.
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METHODS |
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Restriction endonuclease digestion.
Individual plugs containing intact C. ruminantium DNA were digested with restriction endonucleases (Roche Molecular Biochemicals). Agarose plugs were equilibrated with 100 µl suitable restriction endonuclease buffer at 4 °C for 20 min. The buffer was exchanged with 100 µl fresh buffer containing 10 units of restriction endonuclease and the mixture incubated for 4 h at the appropriate temperature. Reactions were stopped by the addition of 50 µl 0·5 M EDTA (pH 8·0).
PFGE.
DNA plugs were loaded into wells of a 1% (w/v) FastLane agarose gel (FMC Bioproducts) in 0·025 M TBE (2·25 mM Tris base, 2·25 mM boric acid, 0·05 mM EDTA, pH 8·5). Electrophoresis was carried out at 13 °C in a Rotaphor Type 5 PFGE apparatus (Biometra) using the following separation conditions: voltage 120125 V (logarithmic ramp); pulse rate 590 s (linear ramp); rotation angle 110125° (logarithmic ramp) with a duration of 37 h. Saccharomyces cerevisiae chromosomal DNA markers (New England Biolabs) and Low Range PFG markers (New England Biolabs) were used as molecular size markers. Gels were stained with ethidium bromide and restriction fragments photographed and analysed with a Lumi-Imager F1 Workstation (Roche Molecular Biochemicals). Band sizes were calculated from at least three independent separations of restriction fragments.
Two-dimensional electrophoresis was carried out according to the method of Römling & Tümmler (1991) . A lane containing restriction endonuclease digested DNA was cut from a PFGE gel, incubated with 200 U of a second restriction endonuclease in 3 ml of appropriate reaction buffer and loaded across the width of a second PFGE gel. This gel was then electrophoresed at right angles to the sample strip. DNA spots were visualized with ethidium bromide staining and subsequently photographed and analysed with the Lumi-Imager F1 Workstation.
Southern hybridization.
Gels were depurinated for 15 min in 0·25 M HCl, washed twice for 30 min in denaturing solution (0·5 M NaOH, 1·5 M NaCl) and for 1 h in neutralizing solution (1·5 M NaCl, 0·5 M Tris/HCl, 1 mM EDTA, pH 7·5) and then transferred to MagnaCharge nylon transfer membrane (MSI) with 20x SSC [1x SSC is 150 mM NaCl plus 75 mM sodium citrate (pH 7·0)] (Sambrook et al., 1989 ). After transfer, DNA was fixed to the membrane by baking at 80 °C for 2 h. DNA probes were labelled using a Megaprime labelling kit (Amersham) and [
-32P]dATP (3·7 x 108 Bq ml-1, Amersham) according to the instructions of the manufacturer. Partially purified organisms were further purified by high-gradient MACS (Miltenyi et al., 1990
) as previously described (De Villiers et al., 1998
) to obtain genomic DNA that contained <10% bovine DNA for use as probes. Cloned genes (see Table 4
) and gel-purified restriction endonuclease fragments were also used as probes. Filters were prehybridized in 0·5 M sodium phosphate (pH 7·4), 7% SDS, for at least 1 h and hybridized overnight at 64 °C in the same buffer following addition of probe. Excess probe was removed by washing at room temperature (1x SSC, 0·5% SDS; 2 x10 min) followed by washing in the same buffer (1x10 min at 64 °C). Blots were stripped with boiling 0·5% SDS and stored at 4 °C until re-use. Hybridization results were visualized by autoradiography and analysed with the Lumi-Imager F1 Workstation.
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RESULTS |
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The restriction endonuclease KspI produced five fragments (Fig. 1a, lane 5), which added up to 1551±114 kb (Table 1
). SmaI produced six fragments (Fig. 1a
, lane 6) with a total length of 1545±143 kb (Table 1
). Based on the data obtained by these three enzyme digestions, we estimated the C. ruminantium genome size at 1576±91 kb.
Construction of a physical map
Both physical methods and hybridization analysis were used in the construction of a physical map for C. ruminantium (Welgevonden). To supplement the data obtained with restriction endonuclease digestion of whole genomic DNA with KspI, RsrII and SmaI, partial digestions with these endonucleases were obtained either by limiting the amount of restriction endonuclease added to a reaction or by limiting the reaction time of the digestions (Fig. 2a).
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DISCUSSION |
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In the exception noted above, Sulsona et al. (1999) found that the map-1 sequence of the Welgevonden isolate (GenBank accession number AF125274) grown in their lab for a number of years was 86·88% identical to the earlier Welgevonden sequence (GenBank accession number U49843). The sequence they obtained was reported to be identical to a new Zimbabwean isolate called Lemco (GenBank accession number AF125277). However, examination of the sequences reported in GenBank show these sequences to share only 99·6% identity. This is the only known report of two different pathogenic isolates of C. ruminantium having identical map-1 sequences. All other laboratories that have sequenced the map-1 gene from the Welgevonden isolate have obtained the same sequence as U49843 (Brayton et al., 1997
; A. Bensaid & D. Martinez, personal communication, 1999).
PFGE separation of intact C. ruminantium chromosomal DNA indicated that the chromosome was circular, in common with most other prokaryotic organisms, with an estimated size of 1576 kb. A physical map was constructed by a combination of complementary techniques. Single, double and partial digests separated with normal PFGE and two-dimensional PFGE in combination with hybridization experiments with genetic markers and isolated restriction fragments. A total of 13 restriction fragment sites were positioned on the map (5 KspI, 2 RsrII and 6 SmaI sites) with a mean resolution of 290 kb. Similar methods were used to construct the Haemophilus influenzae (Lee et al., 1989 ) and Mycoplasma mycoides (Pyle & Finch, 1988
) physical maps.
Relatively few genes of C. ruminantium have been identified and therefore only a limited genetic map could be constructed by Southern hybridization with nine probes (Table 4). Interestingly, the majority of these probes were located on one half of the map with the three stable cosmids from a SuperCos1 library clustered on the 271 kb KspISmaI fragment together with the 16S rRNA and the 28G ORF.
Previously, using PFGE of uncut circular DNA, we estimated the C. ruminantium chromosome size at 1900 kb (De Villiers et al., 1998 ). The discrepancy is probably due to the fact that circular DNA does not migrate predictably during PFGE (Birkelund & Stephens, 1992
) and the large molecular mass markers behaved erratically under the PFGE conditions used (New England Biolabs, Yeast Chromosome PFG Marker technical bulletin). In our earlier study, a 815 kb extrachromosomal element was also identified, which upon sequencing of the PCR-amplified product turned out to be an unknown Mycoplasma sp., most closely related to Mycoplasma indiense. All cultures used in this study were tested and shown to be free of mycoplasma contamination by three different methodologies as described earlier.
Several close relatives of C. ruminantium genomes have been determined with PFGE. The closest relative of C. ruminantium is the group III Ehrlichia sp. Ehrlichia chaffeensis with a genome size of 1225·8 kb (Rydkina et al., 1999 ). Very close in size to the C. ruminantium chromosome is the unnamed agent of human granulocytic ehrlichiosis (HGE), which has a genome size of 1494 kb (Rydkina et al., 1999
). Anaplasma marginale has a slightly smaller genome, between 1200 and 1260 kb (Alleman et al., 1993
). Ehrlichia sennetsu and Ehrlichia risticii, on the other hand, have substantially smaller genomes of 878 kb and 880 kb, respectively (Rydkina et al., 1999
). Unfortunately, no physical or genetic maps are available for these organisms. Rickettsia prowazekii, the causative agent of epidemic typhus and only distantly related to the Ehrlichieae, is the only rickettsial organism to have its genome completely mapped and sequenced (Andersson et al., 1998
). This parasite has a genome size of 1111523 bp. The size of the C. ruminantium genome, therefore, appears to be close to that of its relatives, whose small genomes are consistent with their obligate intracellular lifestyles. Due to the limited amount of genetic data available for C. ruminantium and the fact that only a small number of genes can be compared with this method, it is impossible to make further comparisons between our map and that of R. prowazekii.
However, the C. ruminantium physical map has already been shown to be valuable for comparative genome analyses of C. ruminantium isolates. The restriction endonucleases identified for the construction of the physical map were applied in macrorestriction profile analysis by PFGE to distinguish seven isolates of C. ruminantium from geographically different areas (De Villiers et al., 2000 ). Statistical analysis of the macrorestriction profiles indicated that all isolates were indeed distinct from each other. These data will contribute to a better understanding of the molecular epidemiology of this pathogen and may be further exploited for the identification of genotype-specific DNA probes. In addition, the estimated genome sizes for the seven isolates ranged from 1546 to 1675 kb, in agreement with the genome size calculated in this paper.
The physical and genetic map of the C. ruminantium genome constitutes a significant step forward in the study of the molecular biology of this organism, and will considerably aid the completion of the genome sequencing project which is currently under way.
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ACKNOWLEDGEMENTS |
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Received 22 March 2000;
revised 26 June 2000;
accepted 12 July 2000.