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

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.

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).

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.

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.

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).