Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543, USA1
Author for correspondence: J. J. Wernegreen. Tel: +1 508 548 3705 x6650. Fax: +1 508 457 4727. e-mail: jwernegreen{at}mbl.edu
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ABSTRACT |
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Keywords: genome reduction, symbiosis, bacteriocytes, asexuality, genetic drift
a The GenBank accession number for the sequence reported in this paper is AF495758.
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INTRODUCTION |
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Like most intracellular pathogens, bacterial endosymbionts of insects have undergone severe genome reduction in the context of their obligate associations with hosts. The small genomes of Buchnera (630650 kb; Charles & Ishikawa, 1999 ; Wernegreen et al., 2000
) and Wigglesworthia (705730 kb; Akman & Aksoy, 2001
) approach the smallest known bacterial genome size (Mycoplasma genitalium, 580 kb; Fraser et al., 1995
) and are very reduced compared to the 4·55·5 Mb genome size range for E. coli (Bergthorsson & Ochman, 1995
, 1998
). Since most bacterial genomes contain primarily coding DNA, genome reduction in endosymbionts must involve the loss of metabolic functions and physiological capacities, with important phenotypic implications (Andersson & Kurland, 1998
; Moran & Wernegreen, 2000
; Ochman & Moran, 2001
). Indeed, full genome sequence data for Buchnera (Shigenobu et al., 2000
) and microarray analysis of gene content in Wigglesworthia (Akman & Aksoy, 2001
) demonstrate the deletion of several loci for metabolic functions that are not essential within the host cellular environment. The loss of these biochemical capabilities may account for the inability to culture insect endosymbionts without the host.
Blochmannia, first discovered in 1887 (Blochmann, 1887 ) and recently assigned to the new genus Candidatus Blochmannia gen. nov. (Sauer et al., 2000
) is the primary endosymbiont of the ant genus Camponotus, the largest genus in the family Formicidae (ants) that includes 931 species in every biogeographical region (Bolton, 1995
). Like most intracellular bacteria, Blochmannia shows accelerated evolutionary rates (unpublished data) and an extremely low G+C content (23 mol%; Dasch, 1975
; Dasch et al., 1984
). Blochmannia occurs exclusively within ant ovaries and within bacteriocytes, specialized host cells that are intercalated among enterocytes of the ant midgut. Blochmannia is widely distributed across most species of Camponotus, and congruence of host and symbiont phylogenies (Schroder et al., 1996
; Sameshima et al., 1999
; Sauer et al., 2000
) suggests the association is evolutionarily stable and as old as the host genus (20 million years; Wilson, 1985
), if not older. In this study, we used PFGE to estimate the genome size of Blochmannia. In the course of this analysis, we further developed methods for isolating genomic DNA of unculturable endosymbionts in sufficient quantity and at sufficient purity for PFGE.
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METHODS |
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Endosymbiont preparation.
Blochmannia was isolated from ant hosts, using a Percoll density-gradient protocol previously described by Charles & Ishikawa (1999) for the isolation of Buchnera cells from aphid hosts. For Blochmannia isolations, we modified the protocol as follows: buffer A was supplemented with 100 mM and 250 mM EDTA, pH 8, and MgCl2 was omitted, to reduce the activity of nucleases. The endosymbiont preparation was performed in buffer A with 250 mM EDTA, except for steps involving Percoll, for which we used buffer A with 100 mM EDTA. Filtration through 5 and 3 µM filters was omitted.
Endosymbiont cells were resuspended in buffer A with 100 mM EDTA and embedded in plugs of InCert agarose (BioWhittaker Molecular Applications) to preserve the structural integrity of chromosomal molecules. DNA preparations were performed in plugs as previously described (Charles & Ishikawa, 1999 ). DNA sequence data support a low genomic G+C content Blochmannia (e.g. Schroder et al., 1996
, and our unpublished results). Therefore, we digested the genomic DNA within plugs using G+C-rich (rare-cutting) restriction enzymes, including ApaI, NotI, SmaI and AscI. Before digestion, plugs were washed twice in the appropriate restriction buffer (30 min per wash). Digestions were performed with an excess of enzyme (40 U per 150 µl reaction) and incubated for 8 h at the appropriate temperature.
PFGE.
PFGE was performed in a contour-clamped homogeneous electric field system (CHEF-XA Mapper; Bio-Rad) at 14 °C using 0·5x TBE, pH 8, and gels of 1% pulsed field gel-grade agarose (Bio-Rad). All gels were run at 6 V cm1, a 120° angle, and a constant ramp time. Switch times and total run times were programmed using the CHEF XA Mapper automated algorithm to span a wide range of fragment sizes (5950 kb; see legend of Fig. 1; Ausubel et al., 1987
). Standard size ladders included high-molecular-mass DNA markers (8·348·5 kb; Life Technologies), the Midrange II PFG ladder (24267 kb; New England BioLabs), the Lambda Ladder PFG marker (48·51018 kb; NEB) and the Yeast Chromosome PFG marker (2251900 kb; NEB). Digested fragments were sized by comparison with standard ladders, using the software package GelPro 2.0 (Image Processing Solutions). Sizes were determined based on the standard ladder closest to the lane of interest. In cases where experimental lanes were equidistant between two standard ladders, fragment sizes based on each ladder were averaged. Sizes of fragments generated by each enzyme were summed to estimate the genome size of Blochmannia.
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RESULTS AND DISCUSSION |
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In this study, we demonstrate that the Blochmannia genome is severely reduced compared to closely related free-living bacteria such as E. coli. We estimated the size of the Blochmannia genome by summing the sizes of linear DNA fragments resolved by PFGE (Fig. 1, Table 1
). Based on digestions with four different endonucleases, we determined that the genome size of Blochmannia associated with C. pennsylvanicus ranges between approximately 790 and 813 kb, the strongest support being for a genome size of
809 kb. This estimate is based on several independent digestion reactions and pulsed field gels, examples of which are shown in Fig. 1
.
Furthermore, we confirmed that the agarose plugs used in PFGE are pure samples of BlochmanniaC. pennsylvanicus by amplifying and sequencing the 16S rRNA gene of the embedded bacterial DNA. Results of BLASTN (http://www.ncbi.nlm.nih.gov) show that the resulting sequence (assigned GenBank accession no. AF495758) is most similar to published Blochmannia 16S rRNA sequences. Phylogenetic analysis of this and other 16S rRNA genes (data not shown) demonstrated that our samples are most closely related to the published C. pennsylvanicus endosymbiont 16S rRNA gene (AJ245598; Schroder et al., 1996 ). These sequence data confirm that the agarose plugs used for genome size analysis contained pure samples of BlochmanniaC. pennsylvanicus DNA.
PFGE analysis of obligate endosymbionts such as Blochmannia poses distinct experimental challenges. First, the inability to culture Blochmannia outside the host required that we isolate Blochmannia cells from C. pennsylvanicus workers prior to DNA preparation within agarose plugs. This direct isolation from hosts necessarily limited the sample size (e.g. the number of independent restriction enzyme digestions) that we could attain compared to PFGE analyses of culturable bacteria. Furthermore, difficulty in preparing highly concentrated Blochmannia DNA resulted in a low intensity of small chromosomal fragments (less than 15 kb). The SmaI digestion may include faint, small fragments that are difficult to size accurately (Fig. 1e
). If so, these small fragments would account for the slightly smaller genome size estimate based on this enzyme (790 kb). Despite these inherent limitations of PFGE analysis of obligately intracellular bacteria, independent restriction digestions gave remarkably similar estimates of Blochmannia genome size in this study. Our results clearly demonstrate that the Blochmannia genome is severely reduced, and totals less than one-fifth the genome size of E. coli.
Genome reduction in Blochmannia and other obligately intracellular bacteria is consistent with a shift in the selectionmutation balance in these species. The persistence of a gene within a bacterial lineage depends on the selection coefficient for its maintenance, the efficacy of this selection, as determined by effective population size (Ne), and mutation rates (Lawrence, 1999 ). Each of these three factors may shift in obligate endosymbionts. First, selective coefficients for the maintenance of genes for metabolic diversity may be reduced in an intracellular environment, resulting in the loss of genes for the biosynthesis of compounds they can acquire from the host cytoplasm. For example, the full genome sequence of Buchnera sp. APS (Shigenobu et al., 2000
) demonstrates that the exchange of gene products between the symbiont and host is complementary and mutually dependent. Buchnera provides the host with essential amino acids and, as expected, possesses a complete set of genes for the biosynthesis of essential amino acids. However, Buchnera lacks genes for the biosynthesis of non-essential amino acids that it must import from the host cytoplasm. In addition, the pantothenate
pyruvate
CoA pathway illustrates the mutual dependency between host and symbiont genomes. Buchnera performs the pantothenate
pyruvate reaction while the host converts pyruvate to CoA.
Second, endosymbionts may have small Ne due to severe bottlenecks upon transmission to host offspring, and few, if any, opportunities for recombination with genetically distinct strains. According to the nearly neutral theory (Ohta, 1973 ), reduced Ne is expected to increase the effect of genetic drift and to reduce the efficacy of selection. Previous studies show elevated rates of fixation of slightly deleterious mutations at particular genes of endosymbionts, consistent with a strong effect of genetic drift (Moran, 1996
). On a genome-wide level, genetic drift may have a cumulative effect of reducing genome size if it increases the selective coefficient required to maintain a given gene (Lawrence, 1999
; Lawrence & Roth, 1999
).
Third, small genomes of intracellular bacteria may reflect stronger effects of mutational biases due to the loss of several DNA-repair genes (Moran & Wernegreen, 2000 ). Mutational bias toward deletions may account for shorter lengths of endosymbiont genes (Charles et al., 1999
), and recent work suggests that deletion biases in bacteria may have greater effects on endosymbiont genomes, in which more sites are effectively neutral (Mira et al., 2001
).
Further genomic studies of Blochmannia may inform our understanding of the functional significance of this endosymbiont, which is currently unknown. The proximity of Blochmannia to the ant midgut, and the ability to cure Camponotus of Blochmannia if the host is fed a very complex diet (Boursaux-Eude & Gross, 2000 ) both suggest that Blochmannia may provide the host with essential nutrients (Dasch et al., 1984
). However, the insect and plant diet of Camponotus (Hölldobler & Wilson, 1990
) is more complex than the unbalanced diets of other insects with bacteriocyte-associated symbionts (e.g. the phloem diet of aphids and other sap-sucking insects, and the blood diet of tsetse flies). Given the relative complexity of the Camponotus diet, it is quite possible that Blochmannia provides alternative, non-nutritional functions for the host. Notably, we found that the Blochmannia genome is slightly larger than those of the closely related insect endosymbionts Buchnera and Wigglesworthia. Genes that are present in Blochmannia but absent from related symbionts will provide promising candidates for loci with functional significance for this particular host association.
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ACKNOWLEDGEMENTS |
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Received 18 February 2002;
revised 21 April 2002;
accepted 23 April 2002.