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Proc Natl Acad Sci U S A. Dec 4, 2007; 104(49): 19392–19397.
Published online Nov 28, 2007. doi:  10.1073/pnas.0708855104
PMCID: PMC2148300
Evolution

Parallel genomic evolution and metabolic interdependence in an ancient symbiosis

Abstract

Obligate symbioses with nutrient-provisioning bacteria have originated often during animal evolution and have been key to the ecological diversification of many invertebrate groups. To date, genome sequences of insect nutritional symbionts have been restricted to a related cluster within Gammaproteobacteria and have revealed distinctive features, including extreme reduction, rapid evolution, and biased nucleotide composition. Using recently developed sequencing technologies, we show that Sulcia muelleri, a member of the Bacteroidetes, underwent similar genomic changes during coevolution with its sap-feeding insect host (sharpshooters) and the coresident symbiont Baumannia cicadellinicola (Gammaproteobacteria). At 245 kilobases, Sulcia's genome is approximately one tenth of the smallest known Bacteroidetes genome and among the smallest for any cellular organism. Analysis of the coding capacities of Sulcia and Baumannia reveals striking complementarity in metabolic capabilities.

Keywords: Bacteroidetes, insects, pyrosequencing, Sharpshooters, genome reduction

Members of the Bacteroidetes are widely distributed in nature, and have been reported as prominent members of environments as diverse as coastal marine waters (1), the human gut (2, 3), and dental plaques (4). In insects, a member of the Bacteroidetes called Sulcia muelleri (Fig. 1) has been shown to be an ancient symbiont of a large group of sap-feeding insects, in which the initial infection was acquired >260 million years ago (5). In addition to Sulcia, these insects have at least one other long-term heritable symbiont (5), exemplified by Baumannia in the case of sharpshooters. These symbioses are models of codiversification over long time periods: Baumannia, Sulcia, and their sharpshooter hosts seem to have diversified through strict vertical association during evolution of this insect group (6).

Fig. 1.
Relationships of Sulcia muelleri to other sequenced members of the Bacteroidetes. Bootstrap values for 100 replicates are shown at the bifurcations. The genome sizes (in megabases) are given in parentheses after the organism name. Those sizes marked with ...

Sharpshooters feed exclusively on xylem sap, which is the most dilute and unbalanced food source used by herbivores (7, 8). Xylem composition varies depending on the plant assayed, but the primary components are typically a dilute mix of the amino acids glutamate, glutamine, aspartate, and asparagine; some simple organic acids (primarily malate); and various sugars (primarily glucose) (7, 8). The genome sequence of Baumannia was recently completed, along with fragments of the Sulcia genome, both from the invasive agricultural pest Homalodisca vitripennis (formerly H. coagulata, also known as the Glassy-Winged Sharpshooter) (9). Analysis of the Baumannia genome revealed that it primarily contributes vitamins and cofactors to the host, while encoding at least partial pathways for two of the 10 essential amino acids (9). The partial sequence obtained for Sulcia suggested that it is primarily responsible for essential amino acid biosynthesis (9).

To fully and unambiguously assess the role of Sulcia in the metabolism of this tripartite symbiosis, we sequenced the genome using pyrosequencing (454 Life Sciences/Roche Applied Science) (10). We attempted to enrich for Sulcia DNA in our sample through dissection of the appropriate bacteriome. Nonetheless, as in previous genome sequencing projects on noncultivable, host-associated microorganisms, we started with a complex sample containing a mixture of DNA from the insect host, Sulcia, and Baumannia, with the host DNA constituting the majority fraction of the sample by weight and the Sulcia DNA representing the majority of the bacterial fraction.

Results

By maximizing the representation of Sulcia in our sample, we succeeded in obtaining deep coverage for this genome. Of the 416 contigs generated from the Newbler (10) assembly, 25 were cleanly separated by a greater average depth of coverage [supporting information (SI) Fig. 4]. Twenty-three of these contigs had gene contents suggesting they belonged to the Sulcia genome and were assembled into a complete circular genome based solely on data generated from the 454 run. This genome included almost all of the sequence contigs that had been assigned to Sulcia previously (9), indicating that the binning criteria were generally accurate but not perfect.

Because pyrosequencing has trouble accurately calling the number of bases in homopolymeric regions, especially for single-base runs longer than 5–8 bases (10, 11), and because Sulcia's high A+T content results in numerous A and T homopolymeric tracts, we expected and found inaccuracies in those regions. Thirty homopolymeric regions that disagreed between the 454- and previously obtained Sanger-generated sequences available for portions of the Sulcia genome (9) were retested by PCR and Sanger sequencing; in all cases, the Sanger prediction was confirmed.

To resolve the homopolymeric regions in the Sulcia genome, 13,564,883 reads of 33 bases in length were generated from one partial run of an Illumina/Solexa Genome Analyzer, which affords many short, highly accurate reads (12). Mapping these reads onto the Sulcia genome corrected 125 additional homopolymeric regions and eliminated almost all frameshifts and in-frame stop codons in the predicted coding regions. However, an in-frame stop in a putative cytochrome c assembly protein was well supported by the Solexa data and was left in the genome, and two homopolymeric tracks (one in the tyrosyl tRNA synthetase tyrS and one in the valyl tRNA synthetase valS) had no Solexa coverage but were changed to extend the reading frame of a protein to match homologs in the sequence databases.

The Sulcia genome is 245,530 bp in length with G+C content of 22.4%. It encodes 228 protein genes, 31 tRNAs representing all 20 aa, one rRNA operon (23S, 16S, and 5S), and one tmRNA (SI Table 3). The coding density is 96.1%, with 61 genes overlapping at an average length of 11.8 bases. A COG-based assignment of functional groups reveals that 33.0% and 21.3% of all protein-coding genes are devoted to translation-related and amino acid biosynthesis functions, respectively (SI Fig. 5).

Analysis of Sulcia's predicted metabolic map (Fig. 2) reveals an organism largely devoted to essential amino acid synthesis. We predict that pathways for leucine, valine, threonine, isoleucine, phenylalanine, and tryptophan are all complete. As observed in Escherichia coli, we predict that the DapC activity in lysine biosynthesis is fulfilled by ArgD (13). The lysine and arginine pathways show evolutionary homology (14), and one Sulcia gene cannot be definitely assigned as either argE or dapE. These two homologous enzymes perform the same chemical reaction (the cleavage of an amide bond) on related molecules, and this one gene may perform both roles in Sulcia. In addition, we predict that the N-acetyltransferase activity of ArgA is fused to the amino terminus of ArgG, not unlike other previous examples of the mobility of this activity (15).

Fig. 2.
The predicted metabolism of Sulcia muelleri. Essential amino acids are shown in red letters, and vitamins/cofactors are shown in blue. Gene names are written in plain letters, and names of selected compounds are written in bold and indicated by a small ...

Sulcia seems to be able to generate reducing power in the form of NADH, to aerobically transmit this energy to a cbb-3 type cytochrome c oxidase-terminated electron transport chain to generate a proton gradient, and to make ATP using this proton motive force. Whereas fragmentary compared with the corresponding gene sets in free-living relatives, Sulcia does have a minimal set of genes to transcribe RNA, translate protein, and replicate its genome, although its set of tRNA synthetases is incomplete (SI Table 4). Genes involved in DNA repair are limited to mutL and mutS. As previously observed for other obligate bacterial symbionts, Sulcia possesses surprisingly few transporters, especially considering the diversity of molecules that likely need to cross the cell membrane. Identifiable transporters are limited to a putative multiple antibiotic resistance-related transporter, an ATP-dependent cation transporter, and a heavy metal ion transporter. It is particularly striking that no transporters for amino acids were found. Both the SecY and the twin-arginine protein translocation pathways are present and apparently functional, although in a minimal form compared with most Bacteria (1618).

As suggested previously based on incomplete genomic sequence for Sulcia (9), the metabolic capabilities of Baumannia and Sulcia are broadly complementary in that Sulcia is primarily devoted to amino acid biosynthesis whereas Baumannia is primarily devoted to cofactor and vitamin synthesis. Our findings extend these earlier observations by demonstrating that the amino acid pathways found in Sulcia are complete (with the exception of one gene in the lysine or arginine pathways; see above), in striking contrast to the situation reported for Carsonella ruddii, a symbiont with a tiny genome but with only fragmentary amino acid biosynthetic pathways. In addition, by completing the Sulcia genome, we have confirmed that the predicted capabilities of Baumannia for vitamin biosynthesis are not present in Sulcia. Furthermore, amino acid and vitamin biosynthetic functions are not perfectly partitioned between the two genomes (Fig. 3), and our current data indicate that the complementarity in biosynthetic abilities extends to the exceptions to this general pattern. Thus, Baumannia has a complete pathway for histidine synthesis, and is able to make cysteine from homoserine, but is unable to make homoserine. In this regard, Sulcia shows remarkable complementarity in that it has no genes for the synthesis of either histidine or cysteine, but is able to make homoserine from aspartate. Baumannia has the capability of making CoA from 2-ketovaline, but cannot make 2-ketovaline itself, which can be produced instead by Sulcia, in the valine biosynthetic pathway.

Fig. 3.
The predicted metabolic capabilities of Sulcia and Baumannia are complementary. The major components of xylem sap are shown in green at the top of the figure. Compounds produced by the bacterial symbionts that are needed by the host are shown with large ...

The metabolic complementarity may extend to compounds that are not needed by the insect host. For example, Baumannia has all of the genes normally seen in a complete fatty acid biosynthetic pathway except the β-ketoacyl-ACP synthase II gene fabF, the sole enzymatic gene for fatty acid biosynthesis encoded in the Sulcia genome. (Also present is acpP, the inactive form of the acyl carrier protein of fatty acid biosynthesis.) Baumannia does encode fabB, the β-ketoacyl-ACP synthase I gene, the function of which partially overlaps fabF. Both fabF and fabB can perform all of the elongation steps in saturated fatty acid synthesis, but perform distinct reactions in unsaturated fatty acid biosynthesis (19). Escherichia coli carrying mutations in fabF show deficiencies in the temperature control of fatty acid composition (20), and Haemophilus influenzae Rd, which lacks fabF, is unable to alter the fatty acid content of its membranes over a wide range of growth temperatures (21). It is unclear how, if at all, Sulcia and Baumannia coordinate these distinct roles in fatty acid biosynthesis.

Additionally, whereas Baumannia is primarily responsible for vitamin and cofactor synthesis, it has no genes for the synthesis of ubiquinone or menaquinone. Sulcia has only two genes devoted to vitamin or cofactor synthesis (menA and ubiE); we hypothesize that both are for the production of menaquinone from polyprenyl diphosphate and 1,4-dihydroxy-2-napthoate (DHNA). Baumannia (and possibly the insect host) can make polyprenyl diphosphate, whereas the source of DHNA could be the plant (from the phylloquinone biosynthesis pathway) or the host.

One outstanding issue concerns the source of nitrogen for the entire three-member system (insect-Baumannia-Sulcia). Three possibilities were previously suggested (9): (i) the ammonium present in xylem or generated as waste from the metabolism of the host could be assimilated by Sulcia; (ii) the nonessential amino acids present in xylem sap could supply the needed nitrogen; or (iii) the insect genome could encode enzymes (e.g., glutamine synthase) that allowed incorporation of nitrogen in the form of ammonium. The complete Sulcia genome rules out ammonium assimilation from either of the bacterial symbionts and leaves both the nonessential amino acids and ammonium assimilation by the insect as the potential sources of nitrogen for the system.

Discussion

All of the previously sequenced genomes from insect symbionts have been from members of the Gammaproteobacteria division of Bacteria (9, 2229). These genomes share many features in common: small genome sizes, low G+C contents, and increased substitution rates compared with their free-living relatives. Our results show parallel evolution of these features in a symbiotic lineage outside the Gammaproteobacteria (Table 1). At ≈245 kb, the Sulcia genome is the second-smallest bacterial genome sequenced; correspondingly, its 22.4% G+C content is one of the most biased base compositions among bacterial genomes (9, 2229). An increased rate of sequence evolution is also evident: phylogenetic analysis shows that Sulcia occurs on a long branch, indicating a higher rate of sequence evolution compared with its free-living relatives within the Bacteroidetes (Fig. 1).

Table 1.
General genomic properties of representative free-living and symbiotic Gammaproteobacteria and Bacteroidetes

Recent work has suggested that there are only 11 replication-related and 6 transcription-related genes universally conserved between genomes of symbionts and free-living Bacteria (30). Extension of these results to include three recent small genomes [Buchnera aphidicola Cc (25), Carsonella ruddii (28), and Sulcia] reduces this number even further (Table 2): only gidA, the glucose-inhibited division protein, and dnaE, the α-subunit of DNA polymerase III (the subunit that contains the polymerase activity), are universally conserved in replication. In RNA transcription, only the RNA polymerase core enzyme (rpoA, the α subunit; rpoB, the β subunit; and rpoC, the β′ subunit) and rpoD, the sigma 70 factor are universally conserved. Thus, the only replicative gene functions that seem to be universal in disparate Bacteria are the polymerization of dNTPs (DnaE) and the ability to turn this polymerization on and off (GidA), although the role of GidA replication control is controversial (31). In transcription, only the core polymerase (RpoA, RpoB, and RpoC) and its strongest binding partner, the sigma 70 factor (RpoD), seem to be universally distributed.

Table 2.
Conserved genes for replication and transcription

In contrast to all other insect symbiont genomes, Sulcia and Carsonella have incomplete sets of tRNA synthetases (SI Table 4). However, they both have complete sets of tRNA genes encoding all 20 aa (28 tRNAs in Carsonella and 31 in Sulcia). This situation is not unprecedented, as the Archaeon Methanocaldococcus jannaschii has no identifiable cysteinyl-tRNA synthetase and has alternative biochemical pathways for the synthesis of glutamine- and asparagine-charged tRNAs (32). There are a number of different mechanisms that Sulcia and Carsonella might use to overcome this deficiency: shuttling tRNAs out to be aminoacylated by Baumannia or the host; importing the needed tRNA synthetases into the cell; broadening the specificity of some tRNA synthetases to perform more than one aminoacylation reaction [again, this mechanism is not unprecedented; the prolyl-tRNA synthetase from Deinococcus radiodurans can charge both prolyl-tRNA with proline and cysteinyl-tRNA with cysteine (33)]; and using alternative biochemical pathways as in the case of M. jannaschii described above. Additionally, some of the hypothetical proteins in the Sulcia and Carsonella genomes could encode novel tRNA synthetases, as was demonstrated for lysyl-tRNA synthetase in the Archaeon Methanococcus maripaludis (32).

The highly reduced genomes of Sulcia and Carsonella (28) raise a number of interesting questions regarding the minimal gene content required for cellular life (3436). Sulcia seems to be able to make NADH from NAD, and to use this reducing power to generate ATP. It has genes to replicate its genome, transcribe RNA, and translate this mRNA into protein, although it is missing many genes thought to be essential in these processes. Of course, Sulcia is not “free-living”: it very likely cannot be cultured outside of the host, and it lacks most genes for membrane synthesis and cell division control [and has a strange elongated cell shape, sometimes reaching 80 μm (5)]. Nevertheless, it retains a stable and independently evolving genome that contains some genes for most required cell functions, and it displays corresponding compartmentalization of its cellular components. Perhaps the most interesting question regarding highly reduced symbiont genomes such as that of Sulcia is the extent to which they are achieved through transferring genes to the host genome (coupled with importation of gene products back into the symbiont cell) versus through gene loss and modification of the retained genes to enable life with a very small but sufficient gene set.

Materials and Methods

DNA Preparation and Sequencing.

The “yellow” portion of the adult H. vitripennis bacteriome was dissected in 100% ethanol from frozen (−80°C) animals caught in June 2001 and June 2004 in a lemon orchard in Riverside, CA. Samples from the same collection were used previously to sequence the Baumannia genome (9). The bacteriomes were spun down briefly in a tabletop centrifuge, the ethanol was removed, and DNA was prepared by using Qiagen DNeasy Blood and Tissue Kits. For 454 sequencing, 5 μg of DNA was prepared as described in ref. 10 using a kit supplied by Roche Applied Science and sequenced on a Roche GS-FLX 100 using 454 technology. For Solexa sequencing, 5 μg of DNA was prepared in a kit supplied by Solexa/Illumina and sequenced as directed by the manufacturer. All Solexa/Illumina work was done at the Genome Sequencing Center at the Washington University School of Medicine.

Genome Assembly.

The 454 sequencing was carried out at the Arizona Genomics Institute at the University of Arizona. The run generated 26,711,618 nt of sequence in 118,380 reads with an average read length of 225.6 nt. The Newbler (software version 1.1.01.20, standard running parameters with ace file output selected) (10) assembly of these data resulted in 416 contigs greater than 500 bp in length totaling 984,315 nt with an average G+C content of 31.0%. A summary of these data is presented in SI Fig 4.

Twenty-five contigs representing 245,748 nt of sequence with an average G+C content of 22.5% were clearly separated based on average depth. Twenty-three of these contigs generated high-scoring tblastx alignments to proteins from Bacteroidetes genomes. These 23 contigs could be linked up into a putative circular chromosome using the “_to” and “_from” information appended to the read name in the ace file generated from the Newbler assembly, which were visualized by using the HAWKEYE assembly viewer (37). [The two remaining high-depth contigs had no significant tblastx hits to any protein in the GenBank nonredundant protein database (nr).] These 23 contigs represented 243,948 nt of sequence with an G+C content of 22.1% and had an average depth of coverage of 26.6X.

The Solexa/Illumina Genome Analyzer System generated 14,035,386 reads of 33 nt for 463,167,738 nt of sequence from one partial run (three of eight channels) using the Solexa Analysis Pipeline version 0.2.2.5. After removing any read containing an N (3.4% of the reads), 13,564,883 reads were left for analysis. These reads were mapped onto the Sulcia genome using NCBI blastn (with the parameters −G 2 −E 1 −F F −e 1e-8 −W 5 −b 1 −v 1 −a 2). The average coverage of the genome was 132-fold in Solexa reads, although these reads were not distributed evenly with some small regions having no coverage and some regions showing very high coverage.

Genome Annotation.

The Sulcia genome has no detectable GC skew nor a dnaA gene, two common ways of positioning the origin of replication. The putative origin of replication was therefore based on a weak transition in oligonucleotide skew using the originx (38) program.

Protein-coding genes were predicted by using the g3-iterated.csh script contained within version 3 of the GLIMMER (39) software package. [The new GLIMMER module that was developed to discriminate between host and symbiont DNA (39) was not needed because of the strong signal from the differences in the depth of coverage in the assembled contigs described above.] These predicted protein-coding genes were annotated by combining results from a NCBI blastp (parameters: −F “m S” −e 1 −b 5 −v 5 −a 2) search against the GenBank nonredundant database (downloaded May 15, 2007), an hmmpfam (HMMER version 2.3.2, default parameters, http://hmmer.janelia.org/) search against the Pfam 21.0 database (40), an hmmpfam search against the TIGRFAM 6.0 database (41), and a blastp search (parameters: −F “m S” −e 1 −b 5 −v 5) against the COG database (42) (downloaded on March 2, 2007).

tRNAs were identified with tRNAscan-SE (43), using the bacterial tRNA model. The 16S and 23S rRNAs were identified by using blastn against the GenBank nonredundant nucleotide database. The 5S rRNA was identified by using the web version of the profile stochastic context-free grammar search on the Rfam 8.0 database (44). The lone tmRNA (also known as 10Sa RNA) was identified by using BRUCE version 1.0 (45).

Repeat sequences were identified by using the web version of Tandem Repeats Finder (http://tandem.bu.edu/trf/trf.html) (46). Of the 126 repeats found using the program with default parameters, only 4 are annotated in the genome. No repeats with <5 copies were considered, and many were rejected because they were completely contained within genes, were very degenerate, or were highly AT-biased.

The preceding results were overlaid and organized by using the Artemis annotation tool (47) for the final annotation of the Sulcia genome.

Metabolic Pathway Construction.

Sulcia's metabolic pathways (Fig. 2) were built by hand, using Ecocyc (48) and Metacyc (49) as guides.

Phylogenetic Tree Construction.

The tree in Fig. 1 was calculated from a concatenated set of 10 protein sequences (a subset of proteins suggested in ref. 50) that were first aligned using CLUSTALW (51) and then concatenated. All columns with a gap character were removed, leaving 5,520 usable characters. A maximum-likelihood tree was generated with proml from the PHYLIP package (52) using the JTT model of amino acid change. Bootstrap values for 100 replicates were calculated. The proteins used in the analysis were as follows: DNA polymerase III, α subunit; initiation factor IF-2; leucyl-tRNA synthetase; phenylalanyl-tRNA synthetase, β subunit; valyl-tRNA synthetase; elongation factor Tu; RNA polymerase, β subunit; and ribosomal proteins L2, S5, and S11. The GenBank accession numbers for the genomes are: Croceibacter atlanticus HTCC2559, AAMP00000000; Gramella forsetii KT0803, CU207366; Flavobacteria bacterium BBFL7, AAPD00000000; Flavobacterium sp. MED217, AANC00000000; Cellulophaga sp. MED134, AAMZ00000000; Flavobacteriales bacterium HTCC2170, AAOC00000000; Robiginitalea biformata HTCC2501, AAOI00000000; Flavobacterium johnsoniae UW101, CP000685; Flavobacteria bacterium BAL38, AAXX00000000; Polaribacter irgensii 23-P, AAOG00000000; Bacteroides thetaiotaomicron VPI-5482, AE015928; Bacteroides fragilis NCTC 9343, CR626927; Bacteroides fragilis YCH46, AP006841; Porphyromonas gingivalis W83, AE015924; Algoriphagus sp. PR1, AAXU00000000; Microscilla marina ATCC 23134, AAWS00000000; Cytophaga hutchinsonii ATCC 33406, CP000383; Salinibacter ruber DSM 13855, CP000159.

COG Analysis.

COG categories were assigned to the Sulcia genome as part of the annotation process. The COG assignments for the C. ruddii genome were obtained from A. Nakabachi (personal communication), the values for B. aphidicola Cc were extracted from GenBank record CP000263, and the COG values for Baumannia cicadellinicola, Escherichia coli K12, Bacteroides thetaiotaomicron, and Porphyromonas gingivalis W83 were obtained from *.cog files at the National Center for Biotechnology Information Bacterial genomes ftp site (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/).

Supplementary Material

Supporting Information:

Acknowledgments

We thank Vince Magrini, Matt Hickenbotham, and Elaine Mardis at the Washington University Genome Sequencing Center for the Solexa run; Yeisoo Yu and Rod Wing at the Arizona Genome Institute at the University of Arizona for the 454 sequencing run; Patrick Degnan for making the 454 library; and Becky Nankivell for help in making the figures. This work was funded by National Science Foundation Microbial Genome Sequencing award 0626716 (to N.A.M.). J.P.M. is funded by the University of Arizona's Center for Insect Science through National Institutes of Health Training Grant 1K 12 GM00708.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequence of Sulcia muelleri has been deposited in the GenBank/EMBL/DDBJ database (accession no. CP000770).

This article contains supporting information online at www.pnas.org/cgi/content/full/0708855104/DC1.

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