This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mavingui, P.
Right arrow Articles by Simonet, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mavingui, P.
Right arrow Articles by Simonet, P.
Agricola
Right arrow Articles by Mavingui, P.
Right arrow Articles by Simonet, P.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, November 2005, p. 6910-6917, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.6910-6917.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Efficient Procedure for Purification of Obligate Intracellular Wolbachia pipientis and Representative Amplification of Its Genome by Multiple-Displacement Amplification

Patrick Mavingui,1* Van Tran Van,1 Estelle Labeyrie,1 Edwige Rancès,1 Fabrice Vavre,2 and Pascal Simonet1

Écologie Microbienne UMR CNRS 5557-USC INRA 1193,1 Biométrie et Biologie Évolutive UMR CNRS 5558, Université Claude Bernard Lyon 1, Villeurbanne, France2

Received 24 December 2004/ Accepted 27 June 2005


arrow
ABSTRACT
 
Bacteria belonging to the genus Wolbachia are obligatorymicroendocytobionts that infect a variety of arthropods and a majority of filarial nematode species, where they induce reproductive alterations or establish a mutualistic symbiosis. Although two whole genome sequences of Wolbachia pipientis, for strain wMel from Drosophila melanogaster and strain wBm from Brugia malayi, have been fully completed and six other genome sequencing projects are ongoing (http://www.genomesonline.org/index.cgi?want=Prokaryotic+Ongoin), genetic analyses of these bacteria are still scarce, mainly due to the inability to cultivate them outside of eukaryotic cells. Usually, a large amount of host tissue (a thousand individuals, or about 10 g) is required in order to purify Wolbachia and extract its DNA, which is often recovered in small amounts and contaminated by host cell DNA, thus hindering genomic studies. In this report, we describe an efficient and reliable procedure to representatively amplify the Wolbachia genome by multiple-displacement amplification from limited infected host tissue (0.2 g or 2 x 107 cells). We obtained sufficient amounts (8 to 10 µg) of DNA of suitable quality for genomic studies, and we demonstrated that the amplified DNA contained all of the Wolbachia loci targeted. In addition, our data indicated that the genome of strain wRi, an obligatory endosymbiont of Drosophila simulans, shares a similar overall architecture with its relative strain wMel.


arrow
INTRODUCTION
 
Wolbachia species are maternally transmitted obligate intracellular bacteria belonging to the alpha subclass of Proteobacteria. These endocytobiotic bacteria are able to invade and maintain themselves in numerous arthropods and nematodes through the induction of reproductive parasitism and the establishment of a mutualistic symbiosis (46). Indeed, in arthropods, they are involved in cytoplasmic incompatibility (36), parthenogenesis (41), feminization (3), and male killing (19). Reproductive alterations induced by Wolbachia have important consequences on host population structure and evolution through sex ratio distortion (10) and reproductive isolation (39). In filarial nematodes and a wasp species, Asobara tabida, Wolbachia bacteria are required for host biology (1, 9). The ability of Wolbachia to control host reproduction and development has promoted applied research dedicated to its use to restrain pest invertebrates (16, 44, 48). However, a rational use of Wolbachia for pest control requires a comprehensive analysis of the genetic and molecular basis of Wolbachia-host interactions. Unfortunately, research in this field has been hampered by the unculturable status of Wolbachia. Thus, the biology of these bacteria is poorly understood, and genomic studies are scarce. Therefore, as with most unculturable bacteria, the development of alternative cellular and molecular technologies for obtaining Wolbachia cells and for purifying their genome is required.

In an effort to study Wolbachia genome architecture, Sun and coworkers (42) had originally developed a method for the isolation of Wolbachia, particularly from Drosophila and the filarial nematodes Brugia malayi and Dirofilaria immitis. This method uses differential centrifugation of a homogenate from thousands of entire host organisms to extract Wolbachia cells and monitor their genome by pulsed-field gel electrophoresis (PFGE). This pioneer work showed that the Wolbachia genome consists of one circular chromosome ranging in size from 0.95 to 1.6 Mb (42), which is consistent with the reduced genome size of strict intracellular microsymbionts (33, 45). Since the description of the method, a genetic map of Wolbachia strain wMelPop, which infects Drosophila melanogaster, has been established (43). Moreover, the same research group purified genomic DNA by PFGE and sequenced the complete genome of another Wolbachia strain, wMel, from D. melanogaster (47). Although this sequencing program was successfully managed, it revealed that the constructed library was highly (36%) contaminated by the host fly genome, thus requiring intensive Wolbachia DNA preparation for genome assembly (47). Recently, the whole genome sequence of Wolbachia strain wBm from Brugia malayi was reported (12), and six genome sequencing projects of different Wolbachia strains are ongoing (http://www.genomesonline.org/index.cgi?want=Prokaryotic+Ongoing+Genomes), in which distinct strategies for obtaining genomic DNA are employed, including PFGE-based extraction and subtractive hybridization to detect cloned Wolbachia DNA from whole host genome libraries as well as long PCR with primers designed from the wMel genome sequence. Despite the success of these different strategies, the progress in sequencing is still slow due to the limited amount of genomic DNA recovered and its contamination by host DNA. In addition, since these methodologies require a relatively large quantity of biological host material, it would be even more difficult to perform for those invertebrates which are difficult to breed and maintain under laboratory conditions. Therefore, these strategies for routine Wolbachia genomic studies are impractical, and alternative procedures are clearly needed.

The main goal of this study was to implement an efficient strategy for facilitating the isolation of Wolbachia from small amounts of infected material and for the recovery of its DNA in large quantities of suitable quality for genetic analysis. Drosophila simulans eggs and the insect cell line Aa23 infected by Wolbachia strain wRi were used as starting biological materials. The combination of differential centrifugation, PFGE, and whole-genome amplification by multiple-displacement amplification (MDA) (7) produced a large amount of Wolbachia DNA. The DNA recovered has been found to contain most of the Wolbachia loci targeted, and thus new avenues for genomic studies of this bacteria have been made possible.


arrow
MATERIALS AND METHODS
 
Drosophila egg collection and Aa23 cell culture.
Drosophila simulans naturally infected by the Wolbachia strain wRi and a derived uninfected line obtained upon tetracycline treatment were kept on a standard medium diet (6) at 20°C with a 12-h light-12-h dark cycle and 70% relative humidity. At emergence, 30 individuals each for infected and uninfected flies were placed into boxes containing disks of sterilized corn flour-based medium, where females deposited eggs. After 20 h of oviposition, eggs corresponding to approximately 0.4 ml were collected per 10 boxes. Aa23 cell lines that were uninfected or infected with Wolbachia wRi were kindly provided by Eric Marsland (University of Kentucky, Lexington); the Aa23 line was originally established from Aedes albopictus embryos (34). Stock cultures of Aa23 cells were maintained at 28°C in growth medium that consisted of an equal volume of Mitsuhashi Maramorosh and Schneider's insect medium (Sigma) supplemented with 10% (vol/vol) fetal bovine serum (Gibco, Invitrogen).

Wolbachia purification.
Infected and uninfected tissues were treated equally. Drosophila eggs (0.2 g in an Eppendorf tube) were dechorionated by immersion in 2.6% sodium hypochlorite solution for 1 min and then rinsed six times with 1 ml sterile water. The eggs were then suspended in 0.3 ml of 1x phosphate-buffered saline (PBS; Tebu-Bio, Le Perray-en-Yvelines, France) and crushed with a pestle for 2 min on ice. The resuspended mixture was centrifuged at 604 x g with a tabletop Eppendorf Mini Spin centrifuge for 1 min, and the supernatant was transferred to a clean Eppendorf tube. The pellet was resuspended again in 0.3 ml PBS and centrifuged as described above. The two supernatants were pooled, and three more resuspension and centrifugation cycles were necessary to eliminate the majority of intact host nuclei, as determined by phase-contrast microscopic observation. The resuspended fraction, of approximately 0.5 ml, was then centrifuged at 12,100 x g for 5 min at 10°C. After the supernatant was discarded, the pellet was suspended in 60 µl of TE buffer (10 mM Tris, pH 7.5, and 0.1 mM EDTA, pH 8.0).

To obtain Wolbachia from the infected Aa23 cell line, a confluent cell monolayer grown for 8 days in 75-cm2 tissue culture flasks (Greiner Bio-one, Frickenhausen, Germany) containing 15 ml of medium was harvested by manual scrapping. The culture was centrifuged (604 x g, 5 min), and the pellet was washed once with PBS and resuspended in 1 ml of PBS. Approximately 2 x 107 viable infected Aa23 cells per flask were obtained, as estimated by counting with trypan blue staining (Sigma) on Mallassez lamé. The resuspended cells were lysed by sonication on ice (90% power, two cycles of 1 min of exposure each), and the homogenate was centrifuged for 5 min at 604 x g to remove cell debris. The elimination of nuclei and recovery of the bacterial fraction were done as described above.

PFGE and genomic DNA purification.
Prior to PFGE, the presence or absence of Wolbachia in cell suspensions was determined by diagnostic PCR amplification with primers (Table 1) specific for the Wolbachia genes wsp (4), ftsZ (17), orf7 (28), groEL (30), and methII (K. Bourtzis, personal communication). An aliquot (5 µl) of cell suspension was diluted in 45 µl STE buffer (0.1 M NaCl, 10 mM Tris-Cl, and 1 mM EDTA [pH 8.0]), and 0.2 mg/ml of proteinase K (Euromedex, Mundolsheim, France) was added. Following incubation at 56°C for 1 h, the proteinase K was inactivated for 15 min at 95°C, and 2 µl of the mixture was used as the template for PCR (see below). For PFGE, genomic DNAs in agarose plugs were prepared according to the method of Mavingui et al. (31), with some modifications. An equal volume of 2% low-melting-point preparative-grade agarose (Bio-Rad) in sterile water was added to 50 µl of cell suspension containing Wolbachia prepared as described above. The mixture was distributed into a mold (100 µl/well) and cooled at 4°C for 10 min. Agarose plugs were treated with 1.4 mg/ml of lysozyme (Eurogentec) for 3 h at 37°C and with 0.2 mg/ml of proteinase K at 56°C for 24 h. After incubation with 0.2 mg/ml of phenylmethylsulfonyl fluoride (Sigma) at 50°C for 1 h to inactivate proteinase K, the agarose plugs were washed three times in TE at 4°C. To improve the release of embedded DNA into the agarose gel, the DNAs were tentatively linearized by digestion with the endonuclease I-CeuI according to the manufacturer's recommendations (New England Biolabs). PFGE was performed using a CHEF DRII apparatus (Bio-Rad) with 1% low-melting-point preparative-grade agarose (Bio-Rad) in 0.5x TAE (40 mM Tris acetate, 1 mM EDTA). The conditions were as follows: the switch time ramped from 60 s to 120 s, the field angle was 120°, the temperature was 14°C, and PFGE was performed for 24 h at 6 V/cm. After electrophoresis, the agarose gel was briefly stained with ethidium bromide, rinsed in water, and photographed under a UV lamp.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Primers and PCR-amplified genes from the Wolbachia wRi genome

Filter blot hybridization was performed as described by Mavingui et al. (32). Briefly, probes consisted of PCR-amplified DNA fragments radiolabeled with 32P by a random priming DNA labeling kit (rediprime; Amersham). Hybridizations were carried out under the following high-stringency conditions: a temperature of 65°C, with washes once in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate at room temperature and twice in 0.1x SSC-0.1% sodium dodecyl sulfate at 60°C.

To extract DNA from the agarose gel, the DNA band corresponding to the Wolbachia genome was excised from the gel slice, washed in TE four times for 45 min each time with gentle agitation at 4°C, and then digested with gelase according to the manufacturer's recommendations (Tebu-Bio). The gel slice was washed with 1x gelase buffer for 1 h, the buffer was removed, and the agarose gel slice was melted at 70°C for 10 min. The melted agarose was cooled to 45°C and then incubated with gelase (1 unit/200 µl agarose solution) for 1 h. The agarose solution was cooled at 4°C for 1 h and then centrifuged at 4,100 x g for 30 min to remove undigested agarose. The supernatant was concentrated in a Centricon YM-100 column (Millipore, Bedford, MA). After being rinsed once with 70% ethanol and once with TE buffer, the retentate containing DNA was eluted by centrifugation at 1,000 x g for 30 min at 25°C. The concentration and purity of the DNA were estimated using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc.). Aliquots of DNA (10 ng/µl) were kept at –80°C until used.

MDA.
Wolbachia genomic DNA extracted by PFGE was used as a template for multiple-displacement amplification employing a GenomiPhi kit (Amersham). One microliter of DNA (1 or 10 ng) or a water control was mixed with 9 µl of sample buffer and heated to 95°C for 3 min. After the mixture cooled on ice for 10 min, 9 µl of the reaction buffer and 1 µl of the Phi29 DNA polymerase enzyme were added. The mixture was incubated at 30°C for 18 h, and then the enzyme was inactivated by incubation for 10 min at 65°C. Samples for MDA were centrifuged briefly, and aliquots were stored at –80°C until used. For restriction analysis, genomic DNA was digested with the endonuclease EcoRI according to the manufacturer's recommendations (Euromedex). After electrophoresis in 0.8% agarose and ethidium bromide staining, DNA was photographed under UV illumination on a BioCaptMw apparatus (Fisher Bioblock Scientific).

PCR amplification, cloning, and sequencing.
PCR primers were designed according to the complete nucleotide sequence of the Wolbachia strain wMel genome (http://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=dmg) and partial sequences of Wolbachia strain wRi (GenBank accession numbers AF348330, AJ012073, and AY040670), using the software Oligo 5.1. The oligonucleotides were synthesized by ProliGo (France). Classical PCR amplifications were done in 25-µl reaction mixtures containing the genomic DNA template (30 ng) in 1x polymerase reaction buffer (Invitrogen), 1.5 mM MgCl2, a 200 µM concentration of each deoxynucleoside triphosphate, a 200 nM concentration of each primer, and 0.5 unit of Taq polymerase (Invitrogen). PCRs were performed in a 9700 thermocycler (Perkin-Elmer) under the following conditions: initial denaturation at 95°C for 1 min; 35 cycles of denaturation (94°C, 1 min), annealing (50 to 57°C, depending on the primers, 1 min), and extension (72°C, 2 min); and a final extension at 72°C for 10 min.

For "long PCR" amplifications, a MasterAmp extra-long PCR kit (Tebu-Bio) was used. The 50-µl reaction volume consisted of the following two solutions: a 25-µl mix containing the template DNA (50 ng), a 300 nM concentration of each primer, and 2.5 units of MasterAmp extra-long DNA polymerase and a 25-µl premix containing 2x MasterAmp extra-long PCR buffer. The thermal cycling parameters were an initial denaturation at 94°C for 1 min followed by 30 amplification cycles of denaturation at 94°C for 1 min, annealing at 49°C (glnAF1-virD4R1) or 57°C (methIIF2-WOR and recFF1-rpoDF1) for 1 min, and extension at 68°C for 10 min. PCR products were monitored by electrophoresis in a 0.8% agarose gel stained with ethidium bromide.

Following purification with a GFX PCR DNA and gel band purification kit (Amersham), PCR products of the expected sizes were either cloned directly (0.4 to 2.2 kb) into the pCR2.1-TOPO vector by using a TOPO-TA cloning kit (Invitrogen) or digested before being cloned into pUC19. Long PCR fragments and cloned inserts were subjected to sequencing at either Genome Express (Meylan, France) or Biofidal (Vaux-en-Velin, France). All sequences were analyzed by the BLASTn program (http://www.ncbi.nlm.gov/BLAST).

Nucleotide sequence accession numbers.
The sequences obtained for this study are available in GenBank under accession numbers AY833061 to AY833078.


arrow
RESULTS AND DISCUSSION
 
Recovery of Wolbachia from host cells.
Infected host cells consisted of D. simulans eggs and the Aa23 cell line harboring Wolbachia pipientis strain wRi. Uninfected cells were used as negative controls. Manual crushing or sonication was employed to dislocate host membranes, and then Wolbachia cells were recovered by differential centrifugation. Diagnostic PCR using Wolbachia-specific primers for the genes wsp, ftsZ, groEL, orf7, and methII resulted in successful amplification, indicating the presence of Wolbachia in the cell preparations (see below). Cell preparations were embedded in agarose, and the resulting plugs were enzymatically treated prior to PFGE. Typically, one plug was made with a cell preparation from 0.2 g (about 5,000) of eggs or 2 x 107 Aa23 cells harboring Wolbachia.

PFGE of undigested DNA revealed the presence of a clear genomic DNA band of approximately 1.6 Mb that hybridized with the Wolbachia wsp gene probe under high-stringency conditions (Fig. 1A and B, lanes 1 and 2). The smear detected on the bottom was assigned to broken host DNA by Southern hybridization with mitochondrion- and nucleus-specific probes (data not shown). The 1.6-Mb DNA band was not seen in homogenates issued from uninfected tissues (Fig. 1A, lanes 3 and 4). Since no hybridization was observed with uninfected host DNA (Fig. 1B, lanes 3 and 4), signals seen in wells of infected cells might correspond to Wolbachia DNA trapped in plugs. Indeed, the Wolbachia chromosome is circular (12, 42, 47), and as known higher-molecular-weight circular DNAs did not enter the agarose gel, only a fraction of randomly linearized replicons migrated during PFGE. An attempt to linearize the Wolbachia chromosome by digestion with the endonuclease I-CeuI, which is known to cut only once in prokaryotic 23S rRNA genes(25), did not increase the signal of the DNA band (data not shown), suggesting that restriction was not achieved. Although the 23S rRNA gene sequence of wRi has not been reported yet, we detected the presence of three mismatches in the 26-bp site of I-CeuI in the unique wMel 23S rRNA gene, and thus the potential absence of a restriction site might explain the apparent inactivity of the endonuclease.



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 1. PFGE gel of undigested Wolbachia wRi genome (arrows) stained with ethidium bromide (A) and hybridized against a wsp gene probe (B). Lanes: M, yeast chromosomal size marker; 1, Wolbachia genomic DNA plug from infected Aa23 cell line; 2, Wolbachia genomic DNA from infected Drosophila simulans eggs; 3, DNA from uninfected Aa23 cells; 4, DNA from uninfected Drosophila simulans eggs.

As reported previously (42), our result is consistent with the presence of a unique chromosomal 1.6-Mb replicon in Wolbachia strain wRi. In previous studies, 1,000 Drosophila adults or grams of worms (42), as well as large numbers from cell lines (11), have been used to purify Wolbachia in sufficient quantities to be detected by PFGE. For this study, the PFGE method was extended to infected Drosophila eggs and, in addition, was shown to be successful with relatively small amounts (about 0.2 g) of infected host tissues. However, direct extraction of Wolbachia DNA from these cell preparations resulted in low yields and contamination by host DNA. To improve the DNA yield and circumvent host DNA contamination, the development of alternative strategies for extracting Wolbachia genomic DNA in large quantities and at a high quality for genetic analysis is required.

Obtention of large amounts of Wolbachia DNA suitable for genomic studies.
To obtain Wolbachia DNA of adequate quality in large quantities for routine genetic analysis, the 1.6-Mb band of DNA cited above was extracted from pulsed-field gel slices by gelase digestion followed by concentration in a Centricon YM-100 column. Wolbachia DNA has already been extracted after PFGE (47), but no indication was given on the efficiency of recovery. In our work, 50 ng of DNA was routinely recovered from one or two gel slices. Diagnostic PCR with specific primers (wsp, ftsZ, groEL, orf7, and methII) confirmed the presence of Wolbachia DNA without any detectable contaminating host DNA (see below). However, the quantity of DNA recovered was still low for routine genomic analysis such as clone library construction or genome sequencing. To increase the amount of Wolbachia DNA, we used a whole-genome amplification strategy based on MDA, as described by Dean et al. (7, 8). This isothermal amplification method employs the MDA property of the bacteriophage Phi29 DNA polymerase to amplify a long (10 to 70 kb) stretch of circular or linear DNA (2, 26), yielding a huge amount of DNA from a relatively limited quantity of template with minimum sequence bias (7). In addition, Phi29-based MDA was shown to cover most of the loci present in the original DNA template, including those of an entire phage or plasmid as well as a complete genome from one single cell (7, 8, 14, 15, 37). In spite of the success of the MDA methodology with eukaryotes, the applications for prokaryotic organisms have been limited. Recently, MDA has been used to improve the detection of bacteria in mites (20) or to amplify genomic DNA from Salmonella enterica serovar Enteritidis cells (22), but the genetic content was evaluated for only a few loci. In this study, MDA was applied in order to produce considerable quantities of Wolbachia DNA by using GenomiPhi technology and to evaluate the representative content of the genome. To achieve that purpose, the template DNA (1 or 10 ng) extracted after PFGE as mentioned above was heated for 3 min at 95°C and added to the reaction mix. Since the quality of the starting material is crucial for representational gene content, particular caution was taken in handling the DNA solution in order to avoid excessive shearing. MDA yielded 8 to 10 µg of DNA consistently from both 1- and 10-ng templates, whereas no DNA was detected in sterilized and UV-treated water control samples (not shown). To reduce the potential risk of amplifying contaminating DNA from the highly diluted template (23), only the 10-ng template was used further. Consistent with previous data (5, 22, 49), an ethidium bromide stained-gel showed that MDA generated DNA (up to 23 kb) that could be digested by the endonuclease EcoRI (Fig. 2), thus providing the possibility of cloning into a recombinant vector for either structural genetic or gene expression analyses. Diagnostic PCRs with Wolbachia-specific primers for the loci wsp, ftsZ, groEL, orf7, and methII as well as the eubacterial 16S rRNA gene (24, 35) were all positive (Fig. 3). No signal was detected with primers targeting mitochondrial or nuclear host DNA (data not shown).



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 2. Wolbachia genomic DNA obtained by MDA. An ethidium bromide-stained agarose gel of undigested (lanes 1 and 2) and EcoRI-restricted (lanes 3 and 4) MDA products is shown. Starting with 10 ng of PFGE-extracted DNA template from the infected Aa23 cell line (lanes 1 and 3) or Drosophila eggs (lanes 2 and 4), MDA yielded up to 10 µg of DNA per sample, with a fragment size of up to 23 kb, as indicated by the lambda marker (M). EcoRI restriction generated fragments of down to 2 kb. Each lane contains 200 ng of DNA template.



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 3. Gene content of Wolbachia wRi DNA generated by the MDA strategy. Primers were selected to amplify genes that are representatively present on the Wolbachia wMel chromosome. Amplification reactions were performed by classical PCR, as indicated (see Materials and Methods), and the products were analyzed by electrophoresis on an agarose gel stained with ethidium bromide. The identity of the genes was ascertained by sequencing and BLASTn analysis (see the text and Table 1). Lanes: M, molecular DNA size marker; 1, Wolbachia MDA sample from infected Aa23 cell line; 2, Wolbachia MDA sample from infected D. simulans eggs; 3, water control MDA sample.

Analysis of genomic content of Wolbachia wRi DNA generated by MDA.
Whole-genome amplification by MDA has been shown to representatively amplify nearly complete and unbiased eukaryotic genomes (7, 14, 18, 37). To investigate the representative content of the Wolbachia wRi genome amplification by MDA, the partial wRi sequences (GenBank accession numbers AF348330, AJ012073, and AY040670) and the complete genome of Wolbachia wMel (http://www.tigr.org /tigr-scripts/CMR2/GenomePage3.spl?database=dmg) were used as a basis for analysis. Since comparative analyses between published wRi and wMel sequences suggested good syntheny (data not shown), different sets of primers (Table 1) targeting genes positioned at distinct locations around the wMel chromosomal map (Fig. 4) were used for PCR amplification employing MDA genomic DNA as the template. All 25 primer sets tested gave PCR products of the expected sizes (Fig. 3). Attempts to amplify ISW1, the first insertion sequence detected in several copies in Wolbachia strain wTai (29), from wRi DNA failed. This failure might be linked to the absence of ISW1 in wRi DNA. Indeed, an analysis of the wMel genome sequence did not reveal an ISW1 homologue, suggesting a closer relationship between wRi and wMel than between wRi and wTai. To improve the analysis of the quality of wRi genomic MDA, a long PCR was performed with the combination of three pairs of primers (glnAF1-virD4R1, methIIF2-WOR, and recFF1-rpoDF1) which had been successful in classical PCR amplification (see above). These three regions were chosen to represent the start (glnAF1-virD4R1), middle (methIIF2-WOR), and end (recFF1-rpoDF1) of the wMel chromosome (Fig. 4). As predicted, amplification yielded PCR products of the expected molecular sizes, i.e., about 12.4, 10.3, and 8.3 kb for recFF1-rpoDF1, glnAF1-virD4R1, and methIIF2-WOR, respectively (Fig. 5).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 4. Schematic representation of Wolbachia wMel chromosome with locations of gene loci amplified by MDA of wRi DNA. All of the genes reported on the chromosomal map (black circle) were efficiently amplified by classical and long PCR with specific primers (see Fig. 3 and 5) and then sequenced. Their identity to published Wolbachia sequences was validated by BLASTn analysis (Table 1).



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 5. Amplification of long DNA regions from Wolbachia wRi DNA. Ethidium bromide-stained agarose gels of long PCR fragments obtained from Wolbachia wRi DNA generated by MDA (see Materials and Methods) are shown. recFF1-rpoDF1, glnAF1-virD1, and methIIF2-WOR indicate the pairs of primers used. M, molecular DNA size marker.

To ascertain the identity of the PCR products, all of the amplicons were cloned into the pCR2.1-TOPO vector and sequenced. For long PCR products, the borders of the fragments were sequenced using the corresponding specific primers, except for the methIIF2-WOR fragment, from which internal sequences were also obtained. BLASTn analyses showed that all sequences matched the corresponding published Wolbachia genes or DNA regions, with homologies of >95% (Table 1) and similarities of almost 100% with wRi sequences present in databases. Interestingly, the 8.3-kb long PCR fragment methIIF2-WOR contains similar clustered loci (not shown) for phage WO which were already reported for different Wolbachia strains (13, 27, 47). As depicted in Fig. 4, the distribution of amplified genes on the wMel chromosomal map strongly suggests that the genome of wMel might be representatively present in wRi MDA samples and that both strains might share a similar overall genomic architecture. Although the overall genomic architecture seems to be conserved between the two strains, the results confirmed that the genome of wRi is larger (~1,600 kb) than that of wMel (1,267 kb [47]). Thus, some DNA segments (approximately 350 kb) present in wRi might be lacking in wMel, suggesting that genomic changes have also occurred in the two Wolbachia strains. Genome rearrangements such as deletion, duplication, or insertion are known to occur in bacteria (38, 40). The presence of highly repeated DNA and many transposable elements, which represent up to 15% of the Wolbachia genome (12, 47), as well as most genes involved in recombination, supports the notion that the Wolbachia genome is prone to genomic DNA rearrangements. Indeed, an inversion in one chromosomal region has already been reported between two closely related Wolbachia strains, wMel and wMelPop (43).

In summary, genomic studies require large amounts of high-quality DNA, which is often difficult to obtain from unculturable bacteria such as obligate intracellular microsymbionts. We have shown in this work that by using a combination of bacterial purification from relatively few host cells, excision of DNA after PFGE, and whole-genome amplification by MDA, large quantities of high-quality DNA can be generated for genomic analysis. Indeed, the DNA generated by MDA was shown to be useful for restriction analysis, classical and long PCR, and sequencing. Although the Wolbachia wMel genome sequence used to design PCR primers was not fully covered, the positions of the genes targeted and the successful amplification of a long DNA tract suggested strongly that MDA-based DNA samples of Wolbachia strain wRi contain a nearly complete subset of the wMel chromosome. To our knowledge, this is the first time that MDA was successfully applied to representatively amplify a bacterial genome. When the genome sequence of wRi is publicly available, information from that sequence will allow a global analysis of the wRi genome in the MDA-based samples obtained in this study. In particular, experiments will be done to amplify supplementary DNA regions that are present in wRi but absent in wMel. Nevertheless, the results obtained support the proposed strategy for the production of whole genomic DNAs of strict intracellular bacteria, such as Wolbachia, from small quantities of host material. As reported previously (15, 22, 49), we expect that if bacterial cells can be fully separated from host material, for example, by performing effective gradients or immunotrapping, only a few cells will be required in order to amplify the whole genome directly by MDA, thus avoiding the PFGE step, which can be difficult with some bacterial genomes.


arrow
ACKNOWLEDGMENTS
 
We are grateful to Marie-France Grasset for help in cell culture implementation and to Timothy M. Vogel and two anonymous reviewers for critical readings of the manuscript.

E.L. is a postdoctoral fellow supported by funds provided by the Swiss National Science Foundation.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Écologie Microbienne UMR CNRS 5557-USC INRA 1193, Université Claude Bernard Lyon 1, Bat. G. Mendel, 43 bd du 11 Novembre 1918, 69622 Villeurbanne, France. Phone: (33) 472431378. Fax: (33) 472431223. E-mail: mavingui{at}biomserv.univ-lyon1.fr. Back


arrow
REFERENCES
 
    1
  1. Bandi, C., A. J. Trees, and N. W. Brattig.2001 . Wolbachia in filarial nematodes: evolutionary aspects and implications for the pathogenesis and treatment of filarial diseases. Vet. Parasitol. 98:215-238.[CrossRef][Medline]
    2. 2
  2. Blanco, L., A. Bernard, J. M. Lazaro, G. Martin, C. Garmendia, and M. Salas. 1989. Highly efficient DNA synthesis by the phage phi29 DNA polymerase. Symmetrical mode of DNA replication.J. Biol. Chem. 264:8935-8940.[Abstract/Free Full Text]
    3. 3
  3. Bouchon, D., T. Rigaud, and P. Juchault. 1998. Evidence for widespread Wolbachia infection in isopod crustaceans: molecular identification and host feminization. Proc. R. Soc. Lond. B Biol. Sci. 1401:1081-1090.
    4. 4
  4. Braig, H. R., W. Zhou, S. L. Dobson, and S. L. O'Neill. 1998. Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis. J. Bacteriol. 180:2373-2378.[Abstract/Free Full Text]
    5. 5
  5. Cheung, V. G., and S. F. Nelson. 1996. Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA. Proc. Natl. Acad. Sci. USA 93:14676-14679.[Abstract/Free Full Text]
    6. 6
  6. David, J., and M. F. Clavel. 1965. Interaction entre le génotype et le milieu d'élevage: conséquences sur les caractéristiques du développement de la drosophile. Bull. Biol. Fr. Belg. 93:369-378.
    7. 7
  7. Dean, F. B., S. Hosono, L. Fang, X. Wu, A. F. Faruqi, P. Bray-Ward, Z. Sun, Q. Zong, Y. Du, J. Du, M. Driscoll, W. Song, S. F. Kingsmore, M. Egholm, and R. S. Lasken.2002 . Comprehensive human genome amplification using multiple displacement amplification. Proc. Natl. Acad. Sci. USA 99:5261-5266.[Abstract/Free Full Text]
    8. 8
  8. Dean, F. B., J. R. Nelson, T. L. Giesler, and R. S. Lasken. 2001. Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11:1095-1099.[Abstract/Free Full Text]
    9. 9
  9. Dedeine, F., F. Vavre, F. Fleury, B. Loppin, M. E. Hochberg, and M. Boulétreau. 2001. Removing symbiotic Wolbachia bacteria specifically inhibits oogenesis in a parasitic wasp. Proc. Natl. Acad. Sci. USA 98:6247-6252.[Abstract/Free Full Text]
    10. 10
  10. Dyson, E. A., and G. D. Hurst. 2004. Persistence of an extreme sex-ratio bias in a natural population.Proc. Natl. Acad. Sci. USA 101:6520-6523.[Abstract/Free Full Text]
    11. 11
  11. Fenollar, F., B. La Scola, H. Inokuma, J. S. Dumler, M. J. Taylor, and D. Raoult. 2003. Culture and phenotypic characterization of a Wolbachia pipientis isolate.J. Clin. Microbiol. 41:5434-5441.[Abstract/Free Full Text]
    12. 12
  12. Foster, J., M. Ganatra, I. Kamal, J. Ware, K. Makarova, N. Ivanova, A. Bhattacharyya, V. Kapatral, S. Kumar, J. Posfai, T. Vincze, J. Ingram, L. Moran, A. Lapidus, M. Omelchenko, N. Kyrpides, E. Ghedin, S. Wang, E. Goltsman, V. Joukov, O. Ostrovskaya, K. Tsukerman, M. Mazur, D. Comb, E. Koonin, and B. Slatko.2005 . The Wolbachia genome of Brugia malayi: endosymbiont evolution within a human pathogenic nematode.PloS Biol. 3:e121.[CrossRef][Medline]
    13. 13
  13. Fujii, Y., T. Kubo, H. Ishikawa, and T. Sasaki. 2004. Isolation and characterization of the bacteriophage WO from Wolbachia, an arthropod endosymbiont. Biochem. Biophys. Res. Commun. 317:1183-1188.[CrossRef][Medline]
    14. 14
  14. Gorrochotegui-Escalante, N., and W. C. Black IV. 2003. Amplifying whole insect genomes with multiple displacement amplification.Insect. Mol. Biol. 12:195-200.[CrossRef][Medline]
    15. 15
  15. Hellani, A., S. Coskun, M. Benkhalifa, A. Tbakhi, N. Sakati, A. Al-Odaib, and P. Ozand. 2004. Multiple displacement amplification on a single cell and possible PGD applications. Mol. Hum. Reprod. 10:847-852.[Abstract/Free Full Text]
    16. 16
  16. Hoerauf, A., S. Mand, L. Volkmann, M. Büttner, Y. Marfo-Debrekyei, M. Taylor, O. Adjei, and D. W. Büttner.2003 . Doxycycline in the treatment of human onchocerciasis: kinetics of Wolbachia endobacteria reduction and of inhibition of embryogenesis in female Onchocerca worms.Microbes Infect. 5:261-273.[CrossRef][Medline]
    17. 17
  17. Holden, P. R., J. F. Brookfield, and P. Jones.1993 . Cloning and characterization of an ftsZ homologue from a bacterial symbiont of Drosophila melanogaster.Mol. Gen. Genet. 240:213-220.[CrossRef][Medline]
    18. 18
  18. Hosono, S., A. F. Faruqi, F. B. Dean, Y. Du, Z. Sun, X. Wu, J. Du, S. F. Kingsmore, M. Egholm, and R. S. Larken. 2003. Unbiased whole-genome amplification directly from clinical samples. Genome Res. 13:954-964.[Abstract/Free Full Text]
    19. 19
  19. Hurst, G. D., J. H. Graf von der Schulenburg, T. M. Majerus, D. Bertrand, I. A. Zakharov, J. Baungaard, W. Volkl, R. Stouthamer, and M. E. Majerus.1999 . Invasion of one insect species, Adalia bipunctata, by two different male-killing bacteria. Insect Mol. Biol. 8:133-139.[CrossRef][Medline]
    20. 20
  20. Jeyaprakash, A., and M. A. Hoy. 2004. Multiple displacement amplification in combination with high-fidelity PCR improves detection of bacteria from single females or eggs of Metaseiulus occidentalis (Nesbitt) (Acari:Phytoseiidae).J. Invertebr. Pathol. 86:111-116.[CrossRef][Medline]
    21. 21
  21. Kang, L., H. Zhu, Q. Cheng, W. Zhou, L. Sun, L. Cai, X. Ma, S. Zhao, and C. Li. 2002. Cloning and characterization of a gene encoding glutathione-regulated potassium-efflux system protein kefKL.DNA Seq. 13:375-381.[Medline]
    22. 22
  22. Kwon, Y. M., and M. M. Cox. 2004. Improved efficacy of whole genome amplification from bacterial cells.BioTechniques 37:1-3.
    23. 23
  23. Lage, J. M., J. H. Leamon, T. Pejovic, S. Hamann, M. Lacey, D. Dillon, R. Segraves, and B. Vossbrinck.2003 . Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-CGH. Genome Res. 13:294-307.[Abstract/Free Full Text]
    24. 24
  24. Lipski, A., U. Friedrich, and K. Altendorf. 2001. Application of rRNA-targeted oligonucleotide probes in biotechnology. Appl. Microbiol. Biotechnol. 56:40-57.[Medline]
    25. 25
  25. Liu, S.-L., A. Hessel, and K. E. Sanderson. 1993. Genomic mapping with I-Ceu I, an intron-encoded endonuclease specific for genes for ribosomal RNA, in Salmonella spp., Escherichia coli, and other bacteria.Proc. Natl. Acad. Sci. USA 90:6874-6878.[Abstract/Free Full Text]
    26. 26
  26. Lizardi, P. M. November 2000. U.S. patent 6,143,495.
    27. 27
  27. Masui, S., H. Kuroiwa, T. Sasaki, M. Inui, T. Kuroiwa, and H. Ishikawa.2001 . Bacteriophage WO and virus-like particles in Wolbachia, an endosymbiont of arthropods. Biochem. Biophys. Res. Commun. 283:1099-1104.[CrossRef][Medline]
    28. 28
  28. Masui, S., S. Kamoda, T. Sasaki, and H. Ishikawa. 2000. Distribution and evolution of bacteriophage WO in Wolbachia, the endosymbiont causing sexual alterations in arthropods. J. Mol. Evol. 51:491-497.[Medline]
    29. 29
  29. Masui, S., S. Kamoda, T. Sasaki, and H. Ishikawa. 1999. The first detection of the insertion sequence ISW1 in the intracellular reproductive parasite Wolbachia. Plasmid 42:13-19.[CrossRef][Medline]
    30. 30
  30. Masui, S., T. Sasaki, and H. Ishikawa. 1997. groE-homologous operon of Wolbachia, an intracellular symbiont of arthropods: a new approach for their phylogeny. Zoolog. Sci. 14:701-706.[Medline]
    31. 31
  31. Mavingui, P., M. Flores, X. Guo, G. Davila, X. Perret, W. J. Broughton, and R. Palacios. 2002. Dynamics of genome architecture in Rhizobium sp. strain NGR234. J. Bacteriol. 184:171-176.[Abstract/Free Full Text]
    32. 32
  32. Mavingui, P., M. Flores, D. Romero, E. Martinez-Romero, and R. Palacios.1997 . Generation of Rhizobium strains with improved symbiotic properties by random DNA amplification (RDA).Nat. Biotechnol. 15:564-569.[CrossRef][Medline]
    33. 33
  33. Moran, N. A., and G. R. Plague. 2004. Genomic changes following host restriction in bacteria. Curr. Opin. Genet. Dev. 14:627-633.[CrossRef][Medline]
    34. 34
  34. O'Neill, S. L., M. M. Pettigrew, S. P. Sinkins, H. R. Braig, T. G. Andreadis, and R. B. Tesh. 1997. In vitro cultivation of Wolbachia pipientis in an Aedes albopictus cell line. Insect Mol. Biol. 6:33-39.[CrossRef][Medline]
    35. 35
  35. O'Neill, S. L., R. Giordano, A. M. E. Colbert, T. L. Karr, and H. M. Robertson.1992 . 16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects.Proc. Natl. Acad. Sci. USA 89:2699-2702.[Abstract/Free Full Text]
    36. 36
  36. O'Neill, S. L., and T. L. Karr. 1990. Bidirectional incompatibility between conspecific populations of Drosophila simulans. Nature 348:178-180.[CrossRef][Medline]
    37. 37
  37. Paez, J. G., M. Lin, R. Beroukhim, J. C. Lee, X. Zhao, D. J. Richter, S. Gabriel, P. Herman, H. Sasaki, D. Altshuler, C. Li, M. Meyerson, and W. R. Sellers.2004 . Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification. Nucleic Acids Res. 32:e71.[Abstract/Free Full Text]
    38. 38
  38. Petes, T. D., and C. W. Hill. 1988. Recombination between repeated genes in microorganisms. Annu. Rev. Genet. 22:147-168.[CrossRef][Medline]
    39. 39
  39. Rokas, A. 2000. Wolbachia as a speciation agent.Trends Ecol. Evol. 15:44-45.[CrossRef][Medline]
    40. 40
  40. Romero, D., and R. Palacios. 1997. Gene amplification and genomic plasticity in prokaryotes. Annu. Rev. Genet. 31:91-111.[CrossRef][Medline]
    41. 41
  41. Stouthamer, R., R. F. Luck, and W. D. Hamilton.1990 . Antibiotics cause parthenogenetic Trichogramma (Hymenoptera: Trichogrammatidae) to revert to sex. Proc. Natl. Acad. Sci. USA 87:2424-2427.[Abstract/Free Full Text]
    42. 42
  42. Sun, L. V., J. M. Foster, G. Tzertzinis, M. Ono, C. Bandi, B. E. Slatko, and S. L. O'Neill.2001 . Determination of Wolbachia genome size by pulsed-field gel electrophoresis. J. Bacteriol. 183:2219-2225.[Abstract/Free Full Text]
    43. 43
  43. Sun, L. V., M. Riegler, and S. L. O'Neill.2003 . Development of a physical and genetic map of the virulent Wolbachia strain wMelPop. J. Bacteriol. 185:7077-7084.[Abstract/Free Full Text]
    44. 44
  44. Taylor, M. J., C. Bandi, A. M. Hoerauf, and J. Lazdins.2000 . Wolbachia bacteria of filarial nematodes: a target for control? Parasitol. Today 16:179-180.[CrossRef][Medline]
    45. 45
  45. van Ham, R. C., J. Kamerbeek, C. Palacios, C. Rausell, F. Abascal, U. Bastolla, J. M. Fernandez, L. Jimenez, M. Postigo, F. J. Silva, J. Tamames, E. Viguera, A. Latorre, A. Valencia, F. Moran, and A. Moya. 2003. Reductive genome evolution in Buchnera aphidicola. Proc. Natl. Acad. Sci. USA 100:581-586.[Abstract/Free Full Text]
    46. 46
  46. Werren, J. H. 1997. Biology of Wolbachia.Annu. Rev. Entomol. 42:587-609.[CrossRef][Medline]
    47. 47
  47. Wu, M., L. V. Sun, J. Vamathevan, M. Riegler, R. Deboy, J. C. Brownlie, E. A. McGraw, W. Martin, C. Esser, N. Ahmadinejad, C. Wiegand, R. Madupu, M. J. Beanan, L. M. Brinkac, S. C. Daugherty, A. S. Durkin, J. F. Kolonay, W. C. Nelson, Y. Mohamoud, P. Lee, K. Berry, M. B. Young, T. Utterback, J. Weidman, W. C. Nierman, I. T. Paulsen, K. E. Nelson, H. Tettelin, S. L. O'Neill, and J. A. Eisen. 2004. Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements. PLoS Biol. 2:e69.
    48. 48
  48. Zabalou, S., M. Riegler, M. Theodorakopoulou, C. Stauffer, C. Savakis, and K. Bourtzis. 2004. Wolbachia-induced cytoplasmic incompatibility as a means for insect pest population control.Proc. Natl. Acad. Sci. USA 101:15042-15045.[Abstract/Free Full Text]
    49. 49
  49. Zhang, L., X. Cui, K. Schmitt, R. Hubert, W. Navidi, and N. Arnheim.1992 . Whole genome amplification from a single cell: implications for genetic analysis. Proc. Natl. Acad. Sci. USA 89:5847-5851.[Abstract/Free Full Text]

Applied and Environmental Microbiology, November 2005, p. 6910-6917, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.6910-6917.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Zouache, K., Voronin, D., Tran-Van, V., Mavingui, P. (2009). Composition of Bacterial Communities Associated with Natural and Laboratory Populations of Asobara tabida Infected with Wolbachia. Appl. Environ. Microbiol. 75: 3755-3764 [Abstract] [Full Text]  
  • Rances, E., Voronin, D., Tran-Van, V., Mavingui, P. (2008). Genetic and Functional Characterization of the Type IV Secretion System in Wolbachia. J. Bacteriol. 190: 5020-5030 [Abstract] [Full Text]  
  • Rasgon, J. L., Gamston, C. E., Ren, X. (2006). Survival of Wolbachia pipientis in Cell-Free Medium. Appl. Environ. Microbiol. 72: 6934-6937 [Abstract] [Full Text]  
  • Sanguin, H., Remenant, B., Dechesne, A., Thioulouse, J., Vogel, T. M., Nesme, X., Moenne-Loccoz, Y., Grundmann, G. L. (2006). Potential of a 16S rRNA-Based Taxonomic Microarray for Analyzing the Rhizosphere Effects of Maize on Agrobacterium spp. and Bacterial Communities.. Appl. Environ. Microbiol. 72: 4302-4312 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mavingui, P.
Right arrow Articles by Simonet, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mavingui, P.
Right arrow Articles by Simonet, P.
Agricola
Right arrow Articles by Mavingui, P.
Right arrow Articles by Simonet, P.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»