Hamiltonella defensa, genome evolution of protective bacterial endosymbiont from pathogenic ancestors

Patrick H. Degnan; Yeisoo Yu; Nicholas Sisneros; Rod A. Wing; Nancy A. Moran

doi:10.1073/pnas.0900194106

New Research In

Edited by Edward F. DeLong, Massachusetts Institute of Technology, Cambridge, MA, and approved April 14, 2009 (received for review January 7, 2009)

Abstract

Eukaryotes engage in a multitude of beneficial and deleterious interactions with bacteria. Hamiltonella defensa, an endosymbiont of aphids and other sap-feeding insects, protects its aphid host from attack by parasitoid wasps. Thus H. defensa is only conditionally beneficial to hosts, unlike ancient nutritional symbionts, such as Buchnera, that are obligate. Similar to pathogenic bacteria, H. defensa is able to invade naive hosts and circumvent host immune responses. We have sequenced the genome of H. defensa to identify possible mechanisms that underlie its persistence in healthy aphids and protection from parasitoids. The 2.1-Mb genome has undergone significant reduction in size relative to its closest free-living relatives, which include Yersinia and Serratia species (4.6–5.4 Mb). Auxotrophic for 8 of the 10 essential amino acids, H. defensa is reliant upon the essential amino acids produced by Buchnera. Despite these losses, the H. defensa genome retains more genes and pathways for a variety of cell structures and processes than do obligate symbionts, such as Buchnera. Furthermore, putative pathogenicity loci, encoding type-3 secretion systems, and toxin homologs, which are absent in obligate symbionts, are abundant in the H. defensa genome, as are regulatory genes that likely control the timing of their expression. The genome is also littered with mobile DNA, including phage-derived genes, plasmids, and insertion-sequence elements, highlighting its dynamic nature and the continued role horizontal gene transfer plays in shaping it.

Insects host a wide diversity of noncultivable bacteria, which have important ecological phenotypes ranging from parasitism to mutualism (1, 2). Genome sequencing of noncultivatable parasitic bacteria has revealed possible mechanisms responsible for reproductive manipulations (3–5), whereas genomes of obligate mutualists of ants, aphids, psyllids, tsetse flies, and sharpshooters have documented biosynthetic abilities important to host nutrition (6–10). Heritable endosymbionts that protect their hosts from parasites and pathogens are increasingly being recognized as common. Because they are occasionally transferred horizontally, sometimes between distantly related species, these symbionts provide a conduit for the transfer of highly adaptive and stably inherited traits (resistance and defense) between host species. So far, no such defensive symbiont has been studied using genome sequencing.

Hamiltonella defensa, a gamma-proteobacterium, is a maternally transmitted defensive endosymbiont found sporadically in sap-feeding insects, including aphids, psyllids, and whiteflies (11–13). In pea aphids (Acyrthosiphon pisum), H. defensa can block larval development of the solitary endoparasitoid wasps Aphidius ervi and Aphidius eadyi, rescuing the aphid host (14–16). The reduction in aphid mortality is variable among H. defensa strains and is correlated to the presence of a temperate, lambda-like bacteriophage APSE, which infects H. defensa (17–20). H. defensa occurs sporadically in A. pisum and is beneficial only when parasitoids are present (21). Consequently, infection frequencies increase under strong parasitoid pressure but decrease when parasitoids are absent. H. defensa and APSE can also be transmitted horizontally either intraspecifically [e.g., sexually (22)] or interspecifically (12, 17). Moreover, protection by H. defensa has been shown to be transferable between distantly related aphid species (19).

Although H. defensa confers protection, it also exhibits many attributes of enteric pathogens. Its lifestyle requires that it invade novel hosts, and a preliminary survey of its genome content showed that it contains many pathogenicity factors related to host invasion (18). APSE strains encode toxins, including cytolethal distending toxin and Shiga-like toxin, intimating a role of horizontal gene transfer (HGT) in modulating the protection conferred by H. defensa (18, 23).

To shed light on the interactions of H. defensa, its insect hosts, bacteriophage, and invading parasitoids, we have sequenced the H. defensa genome from a strain previously shown to confer protection to A. pisum (16). The H. defensa genome combines mechanisms known from both symbiotic and pathogenic bacterial species.

Results and Discussion

Both general and specific features of the H. defensa genome reflect its lifestyle as a host-restricted, mutualist symbiont that invades host cells. The moderately reduced genome consists of a 2,110,331-bp circular chromosome and a 59,034-bp conjugative plasmid with average G + C contents of 40.1% and 45.3%, respectively (Table 1, Fig. 1). The chromosome contains a canonical origin of replication (oriC) situated between mnmG (gidA) and mioC. Of the 2,100 predicted coding sequences (CDS), 1,665 (79%) have homologs present in GenBank. Most remaining unique hypothetical proteins (75%) are <100 aa (AA), making their identity as true genes equivocal. In addition, 188 readily identifiable pseudogenes were present; this number is similar to that in Escherichia coli genomes (24).

View this table:

Table 1.

Comparison of H. defensa genome features to those of relevant Enterobacteriaceae

Fig. 1.

Genomic characteristics of H. defensa str. 5AT. (A) H. defensa genome schematic; rings starting from outer to innermost: (i) coordinates in kb; (ii) G + C skew of hexamers using a 1,000-bp window; (iii) Predicted CDS with E. coli hit (red), NR hit (blue), hypothetical (yellow), pseudogene (gray); (iv) ribosomal RNAs (black), and putative virulence loci (pink); (v) mobile genetic elements IS elements (light green), group II introns (dark green), phage (blue), or plasmid (purple) islands. Lines connect repeated phage (blue) or plasmid (purple) blocks that are on the same strand (light) or inverted (dark). Asterisk indicates the location of the APSE prophage and the dashed line in (ii) is the location of the incomplete genome juncture. (B) Schematic of plasmid pHD5AT: outer ring (i) coordinates in kb; (ii) predicted coding sequences (CDS) of plasmid origin (purple), hypothetical (yellow), pseudogene (gray), IS elements (light green), and group II introns (dark green); inner ring (iii) G + C skew of hexamers. (C) Graph of primary functional roles for chromosomal CDS and pseudogenes (stippled).

Phylogenies based on single loci place H. defensa in the Enterobacteriaceae, but are otherwise poorly resolved (12, 25). In analyses of multigene alignments of conserved, single-copy, core proteins, H. defensa and another aphid endosymbiont, Regiella insecticola, consistently fell within a clade containing Yersinia spp. and Serratia spp. (Fig. 2). Low bootstrap values near these nodes are elevated by removing Hamiltonella and Regiella from analyses, suggesting that the long branches reduce confidence. Regardless, the phylogeny suggests that Hamiltonella and Regiella form a lineage distinct from the entomopathogenic nematode symbionts Photorhabdus and Xenorhabdus, and from the sequenced tsetse symbiont Sodalis glossinidius.

Fig. 2.

Phylogenetic reconstruction of H. defensa and related Enterobacteriaceae using 88 single-copy orthologous proteins. Bacteria engaged in associations with insects are indicated (I). Support values are reported from 100 bootstrap replicates from RaxML, and PhyML analyses values greater than 80 are indicated by asterisks.

Complementarity of Host and Symbiont Metabolisms.

The metabolism of H. defensa inferred from the genome confirms that it is host-dependent. It is an aerobic heterotroph that shares its central metabolic machinery with that of most free-living enteric bacteria (Fig. 3). Unlike most endosymbionts, Hamiltonella is also capable of the fermentation of pyruvate to lactate (pykF, ldhA) and acetyl-CoA to acetate (pta, ackA). Thus, H. defensa appears able to produce energy even under oxygen-limiting conditions.

Fig. 3.

Metabolic reconstruction of H. defensa indicates that it can complete glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway, in addition to producing both prymidines and purines. Essential (red) and nonessential (green) amino acids are either synthesized de novo or imported by a substrate-specific transporter. Most vitamins and cofactors (blue) are synthesized, although pantothenate and thiamin must be imported. Circles indicate genes in a particular pathway that are present (filled) or absent (open). *Putative “polar” amino acid transporter may transport histidine or threonine.

Biosynthesis of essential amino acids and vitamins is a hallmark of nutritional endosymbionts, exemplified by Buchnera. Based on its gene set, H. defensa synthesizes only 2 essential and 7 nonessential amino acids, but can make most essential vitamins except thiamine (B₁) and pantothenate (B₅) (see Fig. 3). Unlike Buchnera, which lacks most active transport mechanisms, H. defensa likely acquires missing building blocks via substrate-specific transporters.

The essential amino acids that H. defensa requires are largely lacking from the insect diet of phloem sap (26). Our data suggest that both H. defensa and the host insect rely on Buchnera, the required endosymbionts that synthesize essential amino acids from this limited carbon and nitrogen source (9, 10). Except for the glutamate/aspartate transporter (gltP), the H. defensa genome contains no trace of the missing biosynthetic or transporter genes. This suggests that, unlike S. glossinidius, which very recently became host-restricted (27, 28), H. defensa has had a long-term association with insects (Table S1) consistent with previous evidence (12).

Putative Virulence Mechanisms Involved in Symbiosis.

H. defensa's abilities to invade novel insect hosts, to persist in them, and to kill their endoparasites are likely dependent on the presence of numerous loci commonly involved in pathogenicity (18). Our results give a complete inventory of these pathogenicity or symbiosis factors and indicate that some of these loci have been rearranged or disrupted. For example, H. defensa carries two type-3 secretion systems (T3SS), which are similar in gene content and order to T3SS in Salmonella typhimurium LT2 (SPI-1, SPI-2) (18). These protein translocation systems are normally used by pathogens to invade host cells and evade host immune responses (29) and are required for the maintenance of the Sodalis-tsetse fly symbiosis (28, 30). Although both H. defensa T3SS are complete, neither forms a single genomic island. Putative secreted effector proteins are scattered throughout the genome and were probably acquired by multiple HGT events (Table S2).

The most abundant putative virulence factors are RTX (repeats in toxin) toxins: a protein family that includes a variety of exported proteins including α-hemolysin and leukotoxin (31). These proteins have highly variable lengths (800–6,000 AA) and contain a tandemly repeated nonapeptide sequence that is involved in binding calcium. The toxin genes (rtxA) tend to occur in operons containing an activating acyltransferase (rtxC) and an ABC transporter (rtxBD). H. defensa contains 32 CDS with similarity to rtxA, 2 copies of rtxB, and only a single copy of rtxD. These sequences are significantly diverged from known RTX toxins (20–40% AA identity), and several are possibly paralogs (60–92% AA identity). The rtxA copies include both intact (n = 10) and fragmented (n = 22) CDS. Together, these data suggest past duplication and diversification of these toxin genes, followed by mutation and inactivation of some copies.

Response of H. defensa to Changing Environments.

Despite the constrained biosynthetic capabilities of H. defensa, it has considerably more cell structural, DNA replication, recombination, and repair genes than do obligate endosymbionts (2). H. defensa also retains more regulatory genes, including global regulators (e.g., 4 sigma factors), specific regulators of biosynthetic pathways (e.g., for production of biotin, cysteine, fatty acids), 4 pairs of putative 2-component regulators, and 3 genes involved in quorum sensing.

Pathogenic bacteria typically express virulence factors under strict regulatory controls. In H. defensa, putative regulatory genes flank both T3SS, one of which is homologous to hilA, the key regulator for SPI-1 (32). We have also identified homologs of Hha and SlyA, which activate the expression of hemolysins (33, 34), and 2-component regulators and quorum-sensing genes are also known to influence expression of virulence factors. The diversity of regulatory genes suggests a mechanism by which H. defensa copes with changing environments, such as invasion of a new host species or attack of hosts by parasitoids.

Repetitious Genomics.

The genome of H. defensa is riddled with mobile DNA. Insertion sequences (IS), group II introns, integrated prophage, and plasmids comprise 21% of the genome (444,936 bp) (see Fig. 1). Estimates of genetic diversity for the most prevalent, intact IS elements are very low (π = 0.000–0.040), suggesting recent transpositional activity or gene conversion (see Table S3). The single active group II intron also appears to have undergone recent retrotransposition (see Fig. 1, and Table S3). The lack of site specificity has resulted in retrotransposition within and between genes, as well as into previously retrotransposed group II introns. PCR screens of H. defensa strains from different hosts showed that ISHde1, ISHde2, and ISHde3 were widespread, whereas ISHde4 and the group II intron were in fewer than half of tested strains (see Table S3). Proliferation of repeats is expected in intracellular bacteria, as they tend to have small effective population sizes (N_e) because of recurrent transmission bottlenecks, increasing the level of genetic drift (35).

Genome evolution and virulence in H. defensa, as in many free-living bacteria, has been influenced by interactions with bacteriophage (23). Apart from the APSE prophage, H. defensa contains 22 phage-like gene blocks (153,384 bp), several of which have undergone partial duplication (see Fig. 1 and Table S4). The prophage islands were readily identified because of both gene content (e.g., phage integrases) and elevated G + C% (mean 46.5%). Except for APSE, the prophage appear to be inactive, as all of the islands are fragmentary and most contain inactivated or truncated genes. Mobile elements were probably involved in the inactivation, rearrangement, and duplication of the gene blocks, most of which (16 of 22) are flanked on one or more sides by either an IS element or group II intron.

H. defensa bears a conjugative IncFII plasmid pHD5AT. The type IV secretion system (T4SS) and pilus it encodes are similar to the tra and pil loci from the Serratia entomophila plasmid pADAP. These loci underlie the mobilization and dissemination of pADAP, which carries genes responsible for the cessation of insect feeding (36). In contrast to pADAP, the pHD5AT plasmid has no genes implicated in virulence or resistance.

Integrated plasmid genes represent 9% of the H. defensa genome (197,022 bp) (see Fig. 1 and Table S4). They share features with the prophage blocks, including elevated G + C content (44.2%), a large fraction of pseudogenes or truncated proteins, and flanking IS elements or group II introns. Two of the islands are the result of chromosomal integration and decay of pHD5AT, as indicated by missing or inactivated genes (Fig. S1 and SI Methods). Two other plasmid islands are inactivated T4SS, yet are phylogenetically distinct from the tra locus on pHD5AT (see Table S4). The remaining islands contain a variety of plasmid-associated genes, but precise assignation of fragments to plasmids or integration events are difficult because of recombination.

H. defensa Proteome.

To explore the expression of H. defensa genes and proteins, we performed a proteomics experiment on a sample of purified H. defensa cells, using the genome sequence for peptide and protein identification. Implementing conservative identity cutoffs, we identified 89 expressed proteins (Fig. 4 and Table S5). Several phage APSE proteins and one T3SS protein (SseC) were recovered. Among the most highly expressed proteins were those involved bacterial responses to stress and membrane components. Indeed, the most abundant protein, GroEL (Hsp60, MopA) or chaperonin, is also the most abundant protein in other obligate and facultative endosymbionts (37). Other recovered H. defensa proteins include ones involved in core processes (e.g., transcription, translation) and conserved or hypothetical proteins encoded in the genome but having unknown functions.

Fig. 4.

Functional distribution of H. defensa proteins recovered from MudPit analysis. (A) The 89 identified proteins are divided by principle functional roles. Numbers in parentheses indicate the number of expressed (open, <1.0) and highly expressed (stippled, >1.0) proteins in each category based on exponentially modified protein abundance index (emPAI) values. (B) Table of the 12 highly expressed proteins, the number of peptides recovered for each protein, and emPAI values. Colors correspond to the assigned functional roles in (A).

Conclusions

The reduced size and compositional bias in the genome of H. defensa reflects a long-term, stable association with its insect hosts. In this respect, the H. defensa genome is similar to Wolbachia genomes, which are small, have highly reduced biosynthetic capabilities, and encode an abundance of mobile genetic elements (3, 5). Whereas Wolbachia is known mostly as a reproductive parasite and antagonist of its hosts, H. defensa protects hosts from parasites. Genes for toxins, effector proteins, and 2 T3SS are likely to be critical elements underlying this mutualistic role. The presence of numerous homologs of known virulence factors, which have homologs in other insect symbionts and in mammalian and plant pathogens, reiterates how conserved genetic mechanisms involved in bacterial-eukaryotic cellular interactions can result in vastly different outcomes. Some of the virulence-gene homologs (e.g., rtxA) are not intact, suggesting a changing role for the toxins in this symbiosis. These shifting gene sets likely reflect the inherent dynamism of antagonistic interactions, which impose ongoing selection for counter-adaptations in parasites, hosts, and symbionts. Gene losses and inactivations in H. defensa are tempered by gene gains via HGT, evidenced by the abundance of plasmid and phage islands. Although the variable toxins encoded by the phage APSE appear to contribute to parasitoid protection, the H. defensa genome reveals a history of association with other phage and plasmids that likely played an earlier role in resorting ecologically important genes among H. defensa strains and possibly other bacteria.

Methods

DNA Isolation and Construction of Libraries.

We used 2 complementary sequencing strategies to complete the H. defensa genome: (i) subcloning and Sanger sequencing a large insert BAC library and (ii) pyrosequencing (Fig. S2). Intact H. defensa cells were purified from whole insects to minimize contamination with aphid and Buchnera DNA, as described previously (18). A BAC library was constructed, fingerprinted, and minimal tiling paths were chosen (as in ref. 38). Individual BACs were then subcloned, sequenced bi-directionally with ABI3730xl sequencers, and assembled using Phred, Phrap, and Consed (39–41). Overlapping and validated BACs were then merged.

Bacterial genomes contain nonclonable fragments, so we performed pyrosequencing as an unbiased sequencing method. High molecular weight DNA was isolated directly from the purified H. defensa cells using the Puregene Tissue Core Kit B (Qiagen). We generated a standard and paired-end single-stranded template DNA (sstDNA) library using the GS DNA Library Preparation Kits (Roche Applied Sciences) that were then amplified by emPCR and sequenced on a GS-FLX (454 Life Sciences). The 454 reads were assembled with Newbler (v1.1.03.24) using default parameters.

Final Assembly and Genome Closure.

Putative H. defensa contigs generated from the 454 reads and distinct from the finished BACs were sorted and oriented using linking information from the paired ends. PCR primers were designed at the contig ends, and products were amplified and sequenced using standard protocols described elsewhere (23). The 454 reads for each scaffold were then reassembled with Newbler, and Sanger reads were incorporated in Consed using Phrap.

Genome Annotation.

Genes were predicted for the finished H. defensa genome using Glimmer v3.02 (protein-coding genes), tRNAscan-SE (tRNAs) and BlastN (structural and ribosomal RNAs). Putative CDS greater than 30 AA were annotated using consensus of BlastP similarity searches to NR, all microbial genomes, and E. coli str. K12 and protein domain searches using Hmmr and the Pfam_ls database (42). CDS without hits having expectation values less than 10⁻¹⁰ (BlastP) and 10⁻⁴ (Pfam) were annotated as hypothetical, and CDS with conflicting results were assigned as putative. Predicted start codons were adjusted manually using alignments to the top 5 NR hits and the E. coli best hit if present. Intergenic regions were rescreened with BlastX for possible CDS missed by Glimmer. CDS with truncations >40% length or fragmented CDS were designated pseudogenes in the final annotation. Boundaries of multicopy repeats (e.g., insertion sequences, group II introns) were identified by consensus alignments. Gene functions were inferred from those of identified homologs, and the integration of genes into metabolic pathways was determined using EcoCyc (43).

Whole Genome Phylogeny.

Multigene phylogenetic reconstruction was used to determine the relationship of H. defensa with other gamma-proteobacteria. Briefly, we identified 88 of 203 single copy orthologs (SICO) in H. defensa and 29 other genomes (Table S6) (44). Protein sequences of each ortholog were aligned in Muscle v3.6 (45), and all invariant and gap-containing columns were removed. Individual protein alignments were then concatenated into 4 alignments (Table S7). Alignments without H. defensa and R. insecticola sequences were also generated to assess impact of of long-branch attraction or other artifacts. Each dataset was analyzed with RaXML and PhyML (46, 47), and unique topologies were compared using the SH-test in TREE-PUZZLE 5.2 (48). The topology with the lowest log likelihood and that disagreed with the fewest datasets is presented. Support values were estimated from 100 nonparametric bootstrap replicates.

Protein Expression.

Briefly, H. defensa cells were isolated as above and immediately frozen at –80 °C. The cell pellet was thawed, homogenized, and centrifuged, and proteins were precipitated. The resulting pellet was dissolved and run on a 10% SDS/PAGE gel, and the lane was divided into sections and subjected to alkylation and in-gel tryptic digestion. The tryptic peptides were extracted from each gel section, concentrated, and injected into an LC-MS/MS system. Resultant tandem mass spectra were processed and analyzed with Mascot 2.2 (Matrix Science), using a database of H. defensa, B. aphidicola, and A. pisum protein sequences. The results were filtered using a Mascot significance threshold of 0.05 and Mowse ion score cutoff of >31, and the false-discovery rate for H. defensa peptides was 0.2%.

Acknowledgments

The authors thank K. Hammond, B. Nankivell, K. Sunitsch and J. Currie, T.R. Mueller, K. Collura, R. He, and J.L. Goicoechea of the Arizona Genomics Institute. We also thank J. Ewbank and H. Goodrich-Blair for access to unpublished genome data and Q. Lin at the University of Albany Proteomics Facility for running the protein sample. This research was supported by National Science Foundation Grant 0313737 (to N.A.M.). P.H.D. received funding from National Science Foundation Integrative Graduate Education and Research Traineeship Fellowship in Evolutionary and Functional Genomics, the Center for Insect Science at the University of Arizona, and National Science Foundation Doctoral Dissertation Improvement Grant Award 0709992.

Footnotes

¹To whom correspondence should be addressed. E-mail: pdegnan{at}email.arizona.edu
Author contributions: P.H.D., Y.Y., R.A.W., and N.A.M. designed research; P.H.D. and N.S. performed research; P.H.D. analyzed data; and P.H.D. and N.A.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. CP001277, CP001278).
This article contains supporting information online at www.pnas.org/cgi/content/full/0900194106/DCSupplemental.

References

↵
1. Buchner P
(1965) Endosymbiosis of Animals with Plant Microorganisms (John Wiley and Sons, New York).
↵
1. Moran NA,
2. McCutcheon JP,
3. Nakabachi A
(2008) Genomics and evolution of heritable bacterial symbionts. Annu Rev Genet 42:165–190.
OpenUrl CrossRef PubMed
↵
1. Klasson L,
2. et al.
(2008) Genome evolution of Wolbachia strain wPip from the Culex pipiens group. Mol Biol Evol 25:1877–1887.
OpenUrl Abstract/FREE Full Text
↵
1. Sinkins SP,
2. et al.
(2005) Wolbachia variability and host effects on crossing type in Culex mosquitoes. Nature 436:257–260.
OpenUrl CrossRef PubMed
↵
1. Wu M,
2. et al.
(2004) Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements. PLoS Biol 2:e69.
OpenUrl CrossRef PubMed
↵
1. Akman L,
2. et al.
(2002) Genome sequence of the endocellular obligate symbiont of tsetse flies, Wigglesworthia glossinidia. Nat Genet 32:402–407.
OpenUrl CrossRef PubMed
↵
1. Degnan PH,
2. Lazarus AB,
3. Wernegreen JJ
(2005) Genome sequence of Blochmannia pennsylvanicus indicates parallel evolutionary trends among bacterial mutualists of insects. Genome Res 15:1023–1033.
OpenUrl Abstract/FREE Full Text
↵
1. McCutcheon JP,
2. Moran NA
(2007) Parallel genomic evolution and metabolic interdependence in an ancient symbiosis. Proc Natl Acad Sci USA 104:19392–19397.
OpenUrl Abstract/FREE Full Text
↵
1. Nakabachi A,
2. et al.
(2006) The 160-kilobase genome of the bacterial endosymbiont Carsonella. Science 314:267.
OpenUrl Abstract/FREE Full Text
↵
1. van Ham RC,
2. et al.
(2003) Reductive genome evolution in Buchnera aphidicola. Proc Natl Acad Sci USA 100:581–586.
OpenUrl Abstract/FREE Full Text
↵
1. Clark MA,
2. et al.
(1992) The eubacterial endosymbionts of whiteflies (Homoptera: Aleyrodoidea) constitute a lineage distinct from the endosymbionts of aphids and mealybugs. Curr Microbiol 25:119–123.
OpenUrl CrossRef
↵
1. Russell JA,
2. Latorre A,
3. Sabater-Muñoz B,
4. Moya A,
5. Moran NA
(2003) Side-stepping secondary symbionts: widespread horizontal transfer across and beyond the Aphidoidea. Mol Ecol 12:1061–1075.
OpenUrl CrossRef PubMed
↵
1. Sandström JP,
2. Russell JA,
3. White JP,
4. Moran NA
(2001) Independent origins and horizontal transfer of bacterial symbionts of aphids. Mol Ecol 10:217–228.
OpenUrl CrossRef PubMed
↵
1. Bensadia F,
2. Boudreault S,
3. Guay J-F,
4. Michaud D,
5. Cloutier C
(2005) Aphid clonal resistance to a parasitoid fails under heat stress. J Insect Phys 52:146–157.
OpenUrl CrossRef PubMed
↵
1. Ferrari J,
2. Darby AC,
3. Daniell TJ,
4. Godfray HCJ,
5. Douglas AE
(2004) Linking the bacterial community in pea aphids with host-plant use and natural enemy resistance. Ecol Entomol 29:60–65.
OpenUrl CrossRef
↵
1. Oliver KM,
2. Russell JA,
3. Moran NA,
4. Hunter MS
(2003) Facultative bacterial symbionts in aphids confer resistance to parasitic wasps. Proc Natl Acad Sci USA 100:1803–1807.
OpenUrl Abstract/FREE Full Text
↵
1. Degnan PH,
2. Moran NA
(2008) Evolutionary genetics of a defensive facultative symbiont of insects: exchange of toxin-encoding bacteriophage. Mol Ecol 17:916–929.
OpenUrl PubMed
↵
1. Moran NA,
2. Degnan PH,
3. Santos SR,
4. Dunbar HE,
5. Ochman H
(2005) The players in a mutualistic symbiosis: insects, bacteria, viruses, and virulence genes. Proc Natl Acad Sci USA 102:16919–16926.
OpenUrl Abstract/FREE Full Text
↵
1. Oliver KM,
2. Moran NA,
3. Hunter MS
(2005) Variation in resistance to parasitism in aphids is due to symbionts not host genotype. Proc Natl Acad Sci USA 102:12795–12800.
OpenUrl Abstract/FREE Full Text
↵
1. van der Wilk F,
2. Dullemans AM,
3. Verbeek M,
4. van den Heuvel JF
(1999) Isolation and characterization of APSE-1, a bacteriophage infecting the secondary endosymbiont of Acyrthosiphon pisum. Virology 262:104–113.
OpenUrl CrossRef PubMed
↵
1. Oliver KM,
2. Campos J,
3. Moran NA,
4. Hunter MS
(2008) Population dynamics of defensive symbionts in aphids. Proc Roy Soc 275:293–299.
OpenUrl CrossRef
↵
1. Moran NA,
2. Dunbar HE
(2006) Sexual acquisition of beneficial symbionts in aphids. Proc Natl Acad Sci USA 103:12803–12806.
OpenUrl Abstract/FREE Full Text
↵
1. Degnan PH,
2. Moran NA
(2008) Diverse-phage encoded toxins in a protective insect endosymbiont. Appl Environ Microbiol 74:6782–6791.
OpenUrl Abstract/FREE Full Text
↵
1. Lerat E,
2. Ochman H
(2004) ψ-φ: Exploring the outer limits of bacterial pseudogenes. Genome Res 14:2273–2278.
OpenUrl Abstract/FREE Full Text
↵
1. Moran NA,
2. Russell JA,
3. Koga R,
4. Fukatsu T
(2005) Evolutionary relationships of three new species of Enterobacteriaceae living as symbionts of aphids and other insects. Appl Environ Microbiol 71:3302–3310.
OpenUrl Abstract/FREE Full Text
↵
1. Sandström JP,
2. Pettersson J
(1994) Amino acid composition of phloem sap and the relation to intraspecific variation in pea aphid (Acyrthosiphon pisum) performance. J Insect Physiol 40:947–955.
OpenUrl CrossRef
↵
1. Darby AC,
2. et al.
(2005) Extrachromosomal DNA of the symbiont Sodalis glossinidius. J Bacteriol 187:5003–5007.
OpenUrl Abstract/FREE Full Text
↵
1. Toh H,
2. et al.
(2006) Massive genome erosion and functional adaptations provide insights into the symbiotic lifestyle of Sodalis glossinidius in the tsetse host. Genome Res 16:149–156.
OpenUrl Abstract/FREE Full Text
↵
1. Hueck CJ
(1998) Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62:379–433.
OpenUrl Abstract/FREE Full Text
↵
1. Dale C,
2. Young SA,
3. Haydon DT,
4. Welburn SC
(2001) The insect endosymbiont Sodalis glossinidius utilizes a type III secretion system for cell invasion. Proc Natl Acad Sci USA 98:1883–1888.
OpenUrl Abstract/FREE Full Text
↵
1. Lally ET,
2. Hill B,
3. Kieba IR,
4. Korostoff J
(1999) The interaction between RTX toxins and target cells. Trends Microbiol 7:356–361.
OpenUrl CrossRef PubMed
↵
1. Baja V,
2. Hwang C,
3. Lee CA
(1995) hilA is a novel ompR/toxR family member that activates the expression of Salmonella typhimurium invasion genes. Mol Microbiol 18:715–727.
OpenUrl CrossRef PubMed
↵
1. Nieto JM,
2. et al.
(1997) Construction of a double hha hns mutant of Escherichia coli: effect on DNA supercoiling and α-haemolysin production. FEMS Microbiol Lett 155:39–44.
OpenUrl PubMed
↵
1. Wyborn NR,
2. et al.
(2004) Regulation of Escherichia coli hemolysin E expression by H-NS and Salmonella SlyA. J Bacteriol 186:1620–1628.
OpenUrl Abstract/FREE Full Text
↵
1. Moran NA,
2. Plague GR
(2004) Genomic changes following host restriction in bacteria. Curr Opin Genet Dev 14:627–633.
OpenUrl CrossRef PubMed
↵
1. Hurst MRH,
2. Glare TR,
3. Jackson TA
(2004) Cloning Serratia entomophila antifeeding genes–a putative defective prophage active against the grass grub Costelytra zealandica. J Bacteriol 186:5116–5128.
OpenUrl Abstract/FREE Full Text
↵
1. Fares MA,
2. Moya A,
3. Barrio E
(2004) GroEL and the maintenance of bacterial endosymbiosis. Trends Genet 20:413–416.
OpenUrl CrossRef PubMed
↵
1. Peterson DG,
2. Tomkins JP,
3. Frisch DA,
4. Wing RA,
5. Paterson AH
(2000) Construction of plant bacterial artificial chromosome (BAC) libraries: An illustrated guide. J Agr Genomics, 5.
↵
1. Ewing B,
2. Green P
(1998) Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res 8:186–194.
OpenUrl Abstract/FREE Full Text
↵
1. Ewing B,
2. Hillier L,
3. Wendel MC,
4. Green P
(1998) Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 8:175–185.
OpenUrl Abstract/FREE Full Text
↵
1. Gordon D,
2. Abajian C,
3. Green P
(1998) Consed: a graphical tool for sequence finishing. Genome Res 8:195–202.
OpenUrl Abstract/FREE Full Text
↵
1. Bateman A,
2. et al.
(2004) The Pfam protein families database. Nucleic Acids Res 32:D138–D141.
OpenUrl Abstract/FREE Full Text
↵
1. Karp PD,
2. et al.
(2007) Multidimensional annotation of the Escherichia coli K-12 genome. Nucleic Acids Res 22:7577–7590.
OpenUrl
↵
1. Lerat E,
2. Daubin V,
3. Moran NA
(2003) From gene trees to organismal phylogeny in prokaryotes: the case of the γ-Proteobacteria. PLoS Biol 1:e19.
OpenUrl PubMed
↵
1. Edgar RC
(2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797.
OpenUrl Abstract/FREE Full Text
↵
1. Guindon S,
2. Gascuel O
(2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704.
OpenUrl Abstract/FREE Full Text
↵
1. Stamatakis A
(2006) RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690.
OpenUrl Abstract/FREE Full Text
↵
1. Schmidt HA,
2. Strimmer K,
3. Vingron M,
4. von Haeseler A
(2002) TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18:502–504.
OpenUrl Abstract/FREE Full Text

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Proceedings of the National Academy of Sciences: 106 (22)

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Article Classifications

Biological Sciences
Microbiology

[1] ↵
Buchner P
(1965) Endosymbiosis of Animals with Plant Microorganisms (John Wiley and Sons, New York).

[2] Buchner P

[3] ↵
Moran NA,
McCutcheon JP,
Nakabachi A
(2008) Genomics and evolution of heritable bacterial symbionts. Annu Rev Genet 42:165–190.
OpenUrl CrossRef PubMed

[4] Moran NA,

[5] McCutcheon JP,

[6] Nakabachi A

[7] ↵
Klasson L,
et al.
(2008) Genome evolution of Wolbachia strain wPip from the Culex pipiens group. Mol Biol Evol 25:1877–1887.
OpenUrl Abstract/FREE Full Text

[8] Klasson L,

[9] et al.

[10] ↵
Sinkins SP,
et al.
(2005) Wolbachia variability and host effects on crossing type in Culex mosquitoes. Nature 436:257–260.
OpenUrl CrossRef PubMed

[11] Sinkins SP,

[12] et al.

[13] ↵
Wu M,
et al.
(2004) Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements. PLoS Biol 2:e69.
OpenUrl CrossRef PubMed

[14] Wu M,

[15] et al.

[16] ↵
Akman L,
et al.
(2002) Genome sequence of the endocellular obligate symbiont of tsetse flies, Wigglesworthia glossinidia. Nat Genet 32:402–407.
OpenUrl CrossRef PubMed

[17] Akman L,

[18] et al.

[19] ↵
Degnan PH,
Lazarus AB,
Wernegreen JJ
(2005) Genome sequence of Blochmannia pennsylvanicus indicates parallel evolutionary trends among bacterial mutualists of insects. Genome Res 15:1023–1033.
OpenUrl Abstract/FREE Full Text

[20] Degnan PH,

[21] Lazarus AB,

[22] Wernegreen JJ

[23] ↵
McCutcheon JP,
Moran NA
(2007) Parallel genomic evolution and metabolic interdependence in an ancient symbiosis. Proc Natl Acad Sci USA 104:19392–19397.
OpenUrl Abstract/FREE Full Text

[24] McCutcheon JP,

[25] Moran NA

[26] ↵
Nakabachi A,
et al.
(2006) The 160-kilobase genome of the bacterial endosymbiont Carsonella. Science 314:267.
OpenUrl Abstract/FREE Full Text

[27] Nakabachi A,

[28] et al.

[29] ↵
van Ham RC,
et al.
(2003) Reductive genome evolution in Buchnera aphidicola. Proc Natl Acad Sci USA 100:581–586.
OpenUrl Abstract/FREE Full Text

[30] van Ham RC,

[31] et al.

[32] ↵
Clark MA,
et al.
(1992) The eubacterial endosymbionts of whiteflies (Homoptera: Aleyrodoidea) constitute a lineage distinct from the endosymbionts of aphids and mealybugs. Curr Microbiol 25:119–123.
OpenUrl CrossRef

[33] Clark MA,

[34] et al.

[35] ↵
Russell JA,
Latorre A,
Sabater-Muñoz B,
Moya A,
Moran NA
(2003) Side-stepping secondary symbionts: widespread horizontal transfer across and beyond the Aphidoidea. Mol Ecol 12:1061–1075.
OpenUrl CrossRef PubMed

[36] Russell JA,

[37] Latorre A,

[38] Sabater-Muñoz B,

[39] Moya A,

[40] Moran NA

[41] ↵
Sandström JP,
Russell JA,
White JP,
Moran NA
(2001) Independent origins and horizontal transfer of bacterial symbionts of aphids. Mol Ecol 10:217–228.
OpenUrl CrossRef PubMed

[42] Sandström JP,

[43] Russell JA,

[44] White JP,

[45] Moran NA

[46] ↵
Bensadia F,
Boudreault S,
Guay J-F,
Michaud D,
Cloutier C
(2005) Aphid clonal resistance to a parasitoid fails under heat stress. J Insect Phys 52:146–157.
OpenUrl CrossRef PubMed

[47] Bensadia F,

[48] Boudreault S,

[49] Guay J-F,

[50] Michaud D,

[51] Cloutier C

[52] ↵
Ferrari J,
Darby AC,
Daniell TJ,
Godfray HCJ,
Douglas AE
(2004) Linking the bacterial community in pea aphids with host-plant use and natural enemy resistance. Ecol Entomol 29:60–65.
OpenUrl CrossRef

[53] Ferrari J,

[54] Darby AC,

[55] Daniell TJ,

[56] Godfray HCJ,

[57] Douglas AE

[58] ↵
Oliver KM,
Russell JA,
Moran NA,
Hunter MS
(2003) Facultative bacterial symbionts in aphids confer resistance to parasitic wasps. Proc Natl Acad Sci USA 100:1803–1807.
OpenUrl Abstract/FREE Full Text

[59] Oliver KM,

[60] Russell JA,

[61] Moran NA,

[62] Hunter MS

[63] ↵
Degnan PH,
Moran NA
(2008) Evolutionary genetics of a defensive facultative symbiont of insects: exchange of toxin-encoding bacteriophage. Mol Ecol 17:916–929.
OpenUrl PubMed

[64] Degnan PH,

[65] Moran NA

[66] ↵
Moran NA,
Degnan PH,
Santos SR,
Dunbar HE,
Ochman H
(2005) The players in a mutualistic symbiosis: insects, bacteria, viruses, and virulence genes. Proc Natl Acad Sci USA 102:16919–16926.
OpenUrl Abstract/FREE Full Text

[67] Moran NA,

[68] Degnan PH,

[69] Santos SR,

[70] Dunbar HE,

[71] Ochman H

[72] ↵
Oliver KM,
Moran NA,
Hunter MS
(2005) Variation in resistance to parasitism in aphids is due to symbionts not host genotype. Proc Natl Acad Sci USA 102:12795–12800.
OpenUrl Abstract/FREE Full Text

[73] Oliver KM,

[74] Moran NA,

[75] Hunter MS

[76] ↵
van der Wilk F,
Dullemans AM,
Verbeek M,
van den Heuvel JF
(1999) Isolation and characterization of APSE-1, a bacteriophage infecting the secondary endosymbiont of Acyrthosiphon pisum. Virology 262:104–113.
OpenUrl CrossRef PubMed

[77] van der Wilk F,

[78] Dullemans AM,

[79] Verbeek M,

[80] van den Heuvel JF

[81] ↵
Oliver KM,
Campos J,
Moran NA,
Hunter MS
(2008) Population dynamics of defensive symbionts in aphids. Proc Roy Soc 275:293–299.
OpenUrl CrossRef

[82] Oliver KM,

[83] Campos J,

[84] Moran NA,

[85] Hunter MS

[86] ↵
Moran NA,
Dunbar HE
(2006) Sexual acquisition of beneficial symbionts in aphids. Proc Natl Acad Sci USA 103:12803–12806.
OpenUrl Abstract/FREE Full Text

[87] Moran NA,

[88] Dunbar HE

[89] ↵
Degnan PH,
Moran NA
(2008) Diverse-phage encoded toxins in a protective insect endosymbiont. Appl Environ Microbiol 74:6782–6791.
OpenUrl Abstract/FREE Full Text

[90] Degnan PH,

[91] Moran NA

[92] ↵
Lerat E,
Ochman H
(2004) ψ-φ: Exploring the outer limits of bacterial pseudogenes. Genome Res 14:2273–2278.
OpenUrl Abstract/FREE Full Text

[93] Lerat E,

[94] Ochman H

[95] ↵
Moran NA,
Russell JA,
Koga R,
Fukatsu T
(2005) Evolutionary relationships of three new species of Enterobacteriaceae living as symbionts of aphids and other insects. Appl Environ Microbiol 71:3302–3310.
OpenUrl Abstract/FREE Full Text

[96] Moran NA,

[97] Russell JA,

[98] Koga R,

[99] Fukatsu T

[100] ↵
Sandström JP,
Pettersson J
(1994) Amino acid composition of phloem sap and the relation to intraspecific variation in pea aphid (Acyrthosiphon pisum) performance. J Insect Physiol 40:947–955.
OpenUrl CrossRef

[101] Sandström JP,

[102] Pettersson J

[103] ↵
Darby AC,
et al.
(2005) Extrachromosomal DNA of the symbiont Sodalis glossinidius. J Bacteriol 187:5003–5007.
OpenUrl Abstract/FREE Full Text

[104] Darby AC,

[105] et al.

[106] ↵
Toh H,
et al.
(2006) Massive genome erosion and functional adaptations provide insights into the symbiotic lifestyle of Sodalis glossinidius in the tsetse host. Genome Res 16:149–156.
OpenUrl Abstract/FREE Full Text

[107] Toh H,

[108] et al.

[109] ↵
Hueck CJ
(1998) Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62:379–433.
OpenUrl Abstract/FREE Full Text

[110] Hueck CJ

[111] ↵
Dale C,
Young SA,
Haydon DT,
Welburn SC
(2001) The insect endosymbiont Sodalis glossinidius utilizes a type III secretion system for cell invasion. Proc Natl Acad Sci USA 98:1883–1888.
OpenUrl Abstract/FREE Full Text

[112] Dale C,

[113] Young SA,

[114] Haydon DT,

[115] Welburn SC

[116] ↵
Lally ET,
Hill B,
Kieba IR,
Korostoff J
(1999) The interaction between RTX toxins and target cells. Trends Microbiol 7:356–361.
OpenUrl CrossRef PubMed

[117] Lally ET,

[118] Hill B,

[119] Kieba IR,

[120] Korostoff J

[121] ↵
Baja V,
Hwang C,
Lee CA
(1995) hilA is a novel ompR/toxR family member that activates the expression of Salmonella typhimurium invasion genes. Mol Microbiol 18:715–727.
OpenUrl CrossRef PubMed

[122] Baja V,

[123] Hwang C,

[124] Lee CA

[125] ↵
Nieto JM,
et al.
(1997) Construction of a double hha hns mutant of Escherichia coli: effect on DNA supercoiling and α-haemolysin production. FEMS Microbiol Lett 155:39–44.
OpenUrl PubMed

[126] Nieto JM,

[127] et al.

[128] ↵
Wyborn NR,
et al.
(2004) Regulation of Escherichia coli hemolysin E expression by H-NS and Salmonella SlyA. J Bacteriol 186:1620–1628.
OpenUrl Abstract/FREE Full Text

[129] Wyborn NR,

[130] et al.

[131] ↵
Moran NA,
Plague GR
(2004) Genomic changes following host restriction in bacteria. Curr Opin Genet Dev 14:627–633.
OpenUrl CrossRef PubMed

[132] Moran NA,

[133] Plague GR

[134] ↵
Hurst MRH,
Glare TR,
Jackson TA
(2004) Cloning Serratia entomophila antifeeding genes–a putative defective prophage active against the grass grub Costelytra zealandica. J Bacteriol 186:5116–5128.
OpenUrl Abstract/FREE Full Text

[135] Hurst MRH,

[136] Glare TR,

[137] Jackson TA

[138] ↵
Fares MA,
Moya A,
Barrio E
(2004) GroEL and the maintenance of bacterial endosymbiosis. Trends Genet 20:413–416.
OpenUrl CrossRef PubMed

[139] Fares MA,

[140] Moya A,

[141] Barrio E

[142] ↵
Peterson DG,
Tomkins JP,
Frisch DA,
Wing RA,
Paterson AH
(2000) Construction of plant bacterial artificial chromosome (BAC) libraries: An illustrated guide. J Agr Genomics, 5.

[143] Peterson DG,

[144] Tomkins JP,

[145] Frisch DA,

[146] Wing RA,

[147] Paterson AH

[148] ↵
Ewing B,
Green P
(1998) Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res 8:186–194.
OpenUrl Abstract/FREE Full Text

[149] Ewing B,

[150] Green P

[151] ↵
Ewing B,
Hillier L,
Wendel MC,
Green P
(1998) Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 8:175–185.
OpenUrl Abstract/FREE Full Text

[152] Ewing B,

[153] Hillier L,

[154] Wendel MC,

[155] Green P

[156] ↵
Gordon D,
Abajian C,
Green P
(1998) Consed: a graphical tool for sequence finishing. Genome Res 8:195–202.
OpenUrl Abstract/FREE Full Text

[157] Gordon D,

[158] Abajian C,

[159] Green P

[160] ↵
Bateman A,
et al.
(2004) The Pfam protein families database. Nucleic Acids Res 32:D138–D141.
OpenUrl Abstract/FREE Full Text

[161] Bateman A,

[162] et al.

[163] ↵
Karp PD,
et al.
(2007) Multidimensional annotation of the Escherichia coli K-12 genome. Nucleic Acids Res 22:7577–7590.
OpenUrl

[164] Karp PD,

[165] et al.

[166] ↵
Lerat E,
Daubin V,
Moran NA
(2003) From gene trees to organismal phylogeny in prokaryotes: the case of the γ-Proteobacteria. PLoS Biol 1:e19.
OpenUrl PubMed

[167] Lerat E,

[168] Daubin V,

[169] Moran NA

[170] ↵
Edgar RC
(2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797.
OpenUrl Abstract/FREE Full Text

[171] Edgar RC

[172] ↵
Guindon S,
Gascuel O
(2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704.
OpenUrl Abstract/FREE Full Text

[173] Guindon S,

[174] Gascuel O

[175] ↵
Stamatakis A
(2006) RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690.
OpenUrl Abstract/FREE Full Text

[176] Stamatakis A

[177] ↵
Schmidt HA,
Strimmer K,
Vingron M,
von Haeseler A
(2002) TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18:502–504.
OpenUrl Abstract/FREE Full Text

[178] Schmidt HA,

[179] Strimmer K,

[180] Vingron M,

[181] von Haeseler A

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Hamiltonella defensa, genome evolution of protective bacterial endosymbiont from pathogenic ancestors

Abstract

Results and Discussion

Complementarity of Host and Symbiont Metabolisms.

Putative Virulence Mechanisms Involved in Symbiosis.

Response of H. defensa to Changing Environments.

Repetitious Genomics.

H. defensa Proteome.

Conclusions

Methods

DNA Isolation and Construction of Libraries.

Final Assembly and Genome Closure.

Genome Annotation.

Whole Genome Phylogeny.

Protein Expression.

Acknowledgments

Footnotes

References

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Hamiltonella defensa, genome evolution of protective bacterial endosymbiont from pathogenic ancestors

Abstract

Results and Discussion

Complementarity of Host and Symbiont Metabolisms.

Putative Virulence Mechanisms Involved in Symbiosis.

Response of H. defensa to Changing Environments.

Repetitious Genomics.

H. defensa Proteome.

Conclusions

Methods

DNA Isolation and Construction of Libraries.

Final Assembly and Genome Closure.

Genome Annotation.

Whole Genome Phylogeny.

Protein Expression.

Acknowledgments

Footnotes

References

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