Determination of Wolbachia Genome Size by Pulsed-Field Gel Electrophoresis

The sevenWolbachia strains used in this study are listed in Table1. Drosophila simulansRiverside previously treated with tetracycline (DSRT) was used as aWolbachia-free control insect strain.

Drosophila organisms were reared on standard corn flour-sugar-yeast medium at 25°C. Young adults were harvested for extraction of Wolbachia, except for Drosophila melanogaster w¹¹¹⁸, which harbors wMelPop. The infection density in this strain rises dramatically with age (25). Newly emerged adults of this strain were transferred to standard egg-laying bottles for aging. Twenty-day-old flies were harvested for wMelPop purification.

All purification methods published to date have been unable to separateWolbachia from Drosophila mitochondria. In this report, the methods used to purify mitochondria fromDrosophila (28, 35) were modified to prepare DNA from Wolbachia in quantities that could be visualized by ethidium bromide staining of agarose gels. Around 5 ml of adult flies (about 1,000) was collected and then homogenized in buffer as previously described (9), except without Lubrol (90 mM KCl, 55 mM CaCl₂, 15 mM MgSO₄, 30 mM NaCl, 250 mM sucrose) using a Dounce tissue grinder (Wheaton, Millville, N.J.). The homogenate was filtered through a 95-μm-pore-size nylon mesh. The filtrate was centrifuged at 200 × g_maxfor 25 min at 4°C to pellet Drosophila nuclei. The supernatant was then centrifuged at 4,100 × g_max for 5 min at 4°C to pelletWolbachia. The pellet was resuspended at 56°C in a mixture consisting of 1 volume of Tris-EDTA (TE) plus 1 volume of 2% molten GPG low-melting-point agarose (American Bioanalytical, Natick, Mass.), and the resuspension was loaded into a plug module (Bio-Rad, Hercules, Calif.). Plugs were treated with 40 μg of DNase I (Roche, Basel, Switzerland)/ml in DNase I reaction buffer (10 mM Tris-HCl [pH 8.0], 1 mM MgCl₂) for 40 min at room temperature (RT) (25°C). After DNase I treatment, the plugs were incubated overnight at 56°C in the lysis buffer (2) (100 mM EDTA [pH 8.0], 10 mM Tris-HCl [pH 8.0], 1% [wt/vol]N-lauroylsarcosine sodium salt [Sigma, St. Louis, Mo.], 200 μg of proteinase K [Roche]/ml). The plugs were stored in this lysis buffer at 4°C before restriction digestion or electrophoresis.

Adult female nematodes were selected for purification of Wolbachianot only because they are larger than certain other life cycle stages (e.g., microfilariae) that also have high densities of endosymbionts but also because they were more readily and uniformly homogenized using the Dounce tissue grinder. Typically, 5 mature adult females ofDirofilaria immitis or approximately 275 mature females of the smaller Brugia malayi were used for extractions. The purification procedure for Wolbachia from nematodes was essentially the same as the one for Wolbachia from insects, but with the modifications discussed below.

Live worms, supplied by TRS Laboratories (Athens, Ga.), were placed in a petri dish on ice and chopped into small pieces using a sterile razor blade. The homogenization buffer used was physiological saline (0.85% NaCl) supplemented with 0.001% Nonidet P-40 detergent (Sigma). Inclusion of this very low concentration of detergent in the homogenization buffer was found to decrease the amount ofWolbachia pelleting with the worm tissue fragments without causing any noticeable increase in degradation of DNA. The filtrate was passed through two layers of cheesecloth (Veratec, Walpole, Mass.). The first centrifugation was carried out at 350 × g_max for 25 min at 4°C to pellet nematode nuclei. The supernatant was then centrifuged at 4,100 × g_max for 5 min at 4°C to pelletWolbachia. This final pellet was resuspended in an equal volume of saline without detergent and 2 volumes of molten 2% SeaPlaque (low-melting-point) agarose (FMC, Rockland, Maine) in 0.5× Tris-borate-EDTA (TBE) to give a final concentration of 1% agarose. The sample was allowed to set in 75-μl plug molds (Bio-Rad). Following DNase I treatment, the plugs were transferred to proteinase K lysis buffer (6) (0.5 M EDTA [pH 8.0], 1% lauroylsarcosine, sodium salt supplemented with 2 mg of proteinase K [Gibco BRL, Gaithersburg, Md.]/ml) and incubated at 55°C for 48 h. The proteinase K was diffused out of the plugs by performing a minimum of six washes, each of 30 min, in TE at RT. The plugs were stored short term in TE at 4°C.

After formation of plugs, DNase I was used to digest any fragmented DNA produced during homogenization. Several concentrations of DNase I (10, 20, 30, 40, 50, and 60 μg/ml) in combination with a time course (0, 5, 10, 15, 22, 28, 35, 40, or 50 min) were used at RT to determine the best conditions for digestion. The limited amounts of nematode sample precluded optimization of DNase I treatment as was carried out for theWolbachia from Drosophila. The conditions determined optimal for Drosophila (40 μg of DNase I/ml for 40 min at RT) were applied.

Aedes albopictus cell line Aa23 containing Wolbachia strainwAlbB was used (27). Cells were maintained in a 25-mm² flask at 25°C in 5 ml of medium (45% Mitsuhashi and Maramorosch insect medium [Sigma], 45% Schneider medium [Sigma], and 10% heat-inactivated fetal bovine serum). The cells were harvested and washed in phosphate-buffered saline twice. The end pellet was then used to make agarose plugs as described above and directly placed into the lysis buffer and treated at 56°C overnight.

The Drosophila Wolbachia plugs were washed twice with TE buffer, treated twice with TE buffer supplemented with 40 μg of phenylmethylsulfonyl fluoride (Life Technologies, Rockville, Md.)/ml to inactivate the proteinase K, and then washed twice with TE buffer again. All washes were done for 30 min each at RT. For the nematodeWolbachia plugs, the proteinase K was diffused out of the plugs before they were stored in TE buffer.

The Drosophila Wolbachia plugs were equilibrated with restriction endonuclease buffer for 1 h at RT and transferred to fresh buffer for endonuclease digestion overnight. Four restriction enzymes were used: AscI (GG∧CGCGCC),ApaI (GGGCC∧C),FseI (GGCCGG∧CC), and SmaI (CCC∧GGG) (New England Biolabs, Beverly, Mass.). The nematodeWolbachia plugs were equilibrated on ice for 2 h in restriction enzyme buffer containing 50% of the final number of enzyme units. The remaining 50% of the enzyme was then added, and the endonuclease digestion continued for 3 h at the appropriate reaction temperature.

Contour-clamped homogeneous electric field (CHEF) (10) gels were run to separate DNA fragments that included at least one fragment with a size greater than 50 kb, using either a CHEF Mapper XA (Bio-Rad) or a CHEF-DR II (Bio-Rad). For resolution of DNA fragments of less than 50 kb, field inversion gel electrophoresis (7) gels were used with only the CHEF Mapper XA. All of the electrophoresis was carried out at 14°C using 0.5× TBE as the running buffer. Plugs prepared from filarial nematode samples were equilibrated in 0.5× TBE prior to electrophoresis, whileDrosophila Wolbachia plugs underwent no treatment before electrophoresis. The migration profiles were determined using CHEF Mapper XA interactive software, version 1.2 (Bio-Rad). Fragment lengths and the presence of multiple fragments were determined using Gel-doc and Quantity One one-dimensional analysis software (Bio-Rad).

The Wolbachia surface protein (wsp) gene fragment from wRi was amplified by PCR using total DNA from DSRT flies as the template and wsp-specific primers 81F (5′-TGGTCCAATAAGTGATGAAGAAAC-3′) and 691R (5′-AAAAATTAAACGCTACTCCA-3′) as a probe (9). The nematode ftsZ gene fragment was amplified with total DNA from B. malayi as the template and withftsZ-specific primers ftsZ1F (5′-GTTGTCGCAAATACCGATGC-3′) and ftsZ1R (5′-CTTAAGTAAGCTGGTATATC-3′) as a probe (39). The mitochondrial 12S rRNA gene fragment was amplified with primer pair 12SAI (5′-AAACTAGGATTAGATACCCTATTAT-3′) and 12SBI (5′-AAGAGCGACGGGCGATGTGT-3′) (32). The amplification conditions were the same as those previously described (9). Total DNA of Drosophila was extracted using the Holmes-Bonner method (18). PCR products were gel purified with either β-agarase (New England Biolabs) or Qiagen (Valencia, Calif.) gel extraction kits. These probes were radioactively labeled using either the Random Primed DNA labeling kit (Roche) or the NEBlot kit (New England Biolabs) according to the manufacturer's instructions.

A cocktail of seven probes derived from wBma and known from preliminary mapping to be well dispersed around theWolbachia genome was also prepared. These were a 16S rRNA fragment, a 23S rRNA fragment, HSP-60 (GroEL homologue), a DNA mismatch repair protein homologue, the DNA polymerase III γ subunit, the RNA polymerase β subunit, and serine hydroxymethyltransferase. TheseWolbachia sequences had been identified among the expressed sequence tags reported from B. malayi as part of the Filarial Genome Project (see http://neb.com/fgn/filgen1.html ). The sequences were amplified from the appropriate phage stocks representing these cDNA clones using T3 and T7 primers (New England Biolabs). The PCR products were precipitated to remove excess primers, and nucleotides and then labeled by hot PCR (15) with the same primers but using a nucleotide mixture that contained dATP at 1/10 the concentration of the other deoxynucleoside triphosphates but that included [³²P]dATP. The labeled PCR products were purified using QIAquick PCR purification columns (Qiagen) according to the manufacturer's instructions and pooled to make aWolbachia probe cocktail.

After gel electrophoresis, Southern transfer was done with a VacuGene XL vacuum blotting system (Amersham Pharmacia Biotech, Uppsala, Sweden), and the filters were hybridized at either 60 or 65°C and washed under high-stringency conditions.

The methods used led to the successful enrichment of Drosophila Wolbachia. When run on a PFGE gel, Wolbachia genomic DNA could be resolved with ethidium bromide staining (Fig. 1a, lane 2, b, lanes 2 and 3, and c, lane 2). This band was not visible in preparations from uninfected Drosophila. Moreover, Southern hybridization using a wsp gene fragment as the probe confirmed that the visible band represented Wolbachiagenomic DNA (Fig. 1a, lane 3, b, lanes 4 and 5, and c, lane 3). This procedure also showed that much of the uncut Wolbachia DNA still remained in the loading wells (Fig. 1). Southern hybridization using labeled total DNA from Wolbachia-free strain DSRT as a probe produced a faint background smear, which indicated the presence of trace amounts of degraded Drosophila DNA but which did not hybridize to any distinct fragments. No extrachromosomal DNA was detected on the gel, indicating an absence of plasmids.

Optimal DNase I reaction conditions were found to be 40 μg of DNase I/ml for 40 min of treatment at RT. A time course study with DNase I (40 μg/ml) showed that the background DNA smear on gels gradually decreased and finally disappeared with the increasing reaction time (data not shown). Southern hybridization with a mitochondrial DNA 12S rRNA gene fragment as the probe showed that the mitochondrial genome migrated at approximately 500 kb (owing to its circular conformation) and was cleared with increasing DNase I treatment (Fig.2). Excessive DNase I treatment was found to digest Wolbachia DNA completely as well. The final conditions used were found to remove mitochondrial DNA while still maintaining a good yield of Wolbachia DNA.

The methods described above also led to the successful enrichment ofWolbachia from the filarial nematodes. Staining pulsed-field gels with ethidium bromide showed bands of around 1,100 and 950 kb for extracts made from B. malayi and D. immitis, respectively (Fig. 3). There was a sufficient amount of Wolbachia DNA in the extracts made from D. immitis worms for hybridization with the ftsZ gene fragment probe, confirming that the 950-kb band representedWolbachia genomic DNA. For the extracts made from the smaller B. malayi worms it was usually necessary to use theWolbachia probe cocktail to demonstrate that the 1,100-kb band was the Wolbachia genome (Fig. 3). The majority of uncut Wolbachia was found to remain in the well of the gel, paralleling the situation observed for extracts fromDrosophila.

The sequences of several genes previously cloned fromWolbachia strains suggest that the genomes of these bacteria are A+T rich (8, 9, 17, 23). Therefore, to find rare-cutting restriction enzymes, we utilized those with six- or eight-base GC recognition sites initially using wMelPop and wRi genomic DNA.

Out of a total of 15 restriction enzymes screened,ApaI, SmaI, AscI, andFseI produced small numbers of fragments that facilitated the calculation of the genome size forwMelPop. Among them, ApaI andSmaI cut wMelPop into multiple fragments,AscI cut this chromosome into two fragments, andFseI cut only once (Fig.4a). The size of the genome ofwMelPop was calculated by digestion with multiple enzymes to be 1.36 Mb (Table 2). Digestions of the closely related wMel andwMelCS strains with AscI indicated genome sizes equal to that for wMelPop (Fig. 4b; Table 2).

The size of the genome of wRi was obtained via restriction enzyme digestion with ApaI, SmaI, andAscI (Fig. 4c and d). The size estimated fromApaI digestion and ApaI and AscI double digestion was 1.66 Mb (Table 2), while that from SmaI digestion and SmaI and AscI double digestion was 1.65 Mb (Table 2). Thus the genome size of wRi was designated 1.66 Mb, the mean of 1.65 and 1.66 Mb.

A panel of restriction enzymes having GC-rich recognition sequences were also tested for their ability to cut the nematodeWolbachia DNA into only one or a few linear fragments. Several enzymes were found not to cut the genomes of wBma orwDim, while others cut the genome into multiple fragments. The optimal enzyme for wBma was found to be ApaI, whose cut produced only four large fragments (Fig. 3), the sum of which was approximately 1,100 kb (Table 2), confirming the genome size determined from analysis of uncut DNA. Similarly, theWolbachia genome from D. immitis extracts was cut into four bands totaling 950 kb by enzyme ApaI, again confirming the genome size by an independent method (Fig. 3; Table 2). For the Wolbachia genomes enriched from nematode tissues it was necessary to hybridize Southern blots of digested DNA with the probe cocktail in order to identify the individual fragments.

To determine if the Wolbachia chromosome was linear or circular, plugs containing intact mosquito cells which harboredWolbachia wAlbB were prepared (27). The plugs of intact Aa23 (wAlbB) were digested withAscI and FseI, respectively. Then, PFGE was performed for both digested and undigested plugs. After PFGE, the gel was Southern blotted. Hybridization with thewRi wsp gene fragment clearly showed that uncut and FseI-digested Wolbachia DNAs were retained in the loading well, while AscI-digested WolbachiaDNA migrated into the gel (Fig. 5). These results suggest that the Wolbachia chromosome is circular.