Solving a Bloody Mess: B-Vitamin Independent Metabolic Convergence among Gammaproteobacterial Obligate Endosymbionts from Blood-Feeding Arthropods and the Leech Haementeria officinalis

Abstract

Endosymbiosis is a common phenomenon in nature, especially between bacteria and insects, whose typically unbalanced diets are usually complemented by their obligate endosymbionts. While much interest and focus has been directed toward phloem-feeders like aphids and mealybugs, blood-feeders such as the Lone star tick (Amblyomma americanum), Glossina flies, and the human body louse (Pediculus humanus corporis) depend on obligate endosymbionts which complement their B-vitamin-deficient diets, and thus are required for growth and survival. Glossiphoniid leeches have also been found to harbor distinct endosymbionts housed in specialized organs. Here, we present the genome of the bacterial endosymbiont from Haementeria officinalis, first of a glossiphoniid leech. This as-yet-unnamed endosymbiont belongs to the Gammaproteobacteria, has a pleomorphic shape and is restricted to bacteriocytes. For this bacterial endosymbiont, we propose the name Candidatus Providencia siddallii. This symbiont possesses a highly reduced genome with high A+T content and a reduced set of metabolic capabilities, all of which are common characteristics of ancient obligate endosymbionts of arthropods. Its genome has retained many pathways related to the biosynthesis of B-vitamins, pointing toward a role in supplementing the blood-restricted diet of its host. Through comparative genomics against the endosymbionts of A. americanum, Glossina flies, and P. humanus corporis, we were able to detect a high degree of metabolic convergence among these four very distantly related endosymbiotic bacteria.

Keywords: B-vitamin; Haementeria officinalis; Providencia siddallii; blood-feeder; genome reduction; leech endosymbiont.

Fig. 1.—

Haementeria officinalis, bacteriome, and resident Pr. siddallii Off bacteria. (A) View of an H. officinalis leech. (B) Bacteriomes of a dissected H. officinalis leech indicated by black arrowheads. (C) Providencia siddallii Off’s pleomorphic cells present in the bacteriomes of H. officinalis. Putative symbiosomes boundaries are marked with black arrowheads. (D) Magnification showing bacterial cells (B) and mitochondrion (m), evidencing the intracellular localization of Pr. siddallii Off. Scale-bars are presented in different colors to be easily distinguished.

Fig. 2.—

Providencia siddallii strain officinalis-GTOCOR genome. Left: Pr. siddallii Off genome plot. Bottom right: Barplot of COG categories relative abundance. The highest amount of genes involved in metabolism are the ones present in category H (coenzyme metabolism), to which B-vitamin biosynthetic genes belong to.

Fig. 3.—

Providencia siddallii Off phylogenetic positioning. Providencia siddallii Off phylogenetic positioning in a cladogram according to a Bayesian reconstruction done using 55 concatenated single-copy core genes present in diverse Gammaproteobacteria using phylobayes. Providencia siddallii Off is identified as arising from within the Providencia clade. Also, four different origins for obligate endosymbionts from blood-suckers are evident. Sodalis glossinidius is considered as facultative. The full phylogeny can be found as supplementary fig. S2, Supplementary Material online. Red dotted box highlights the position of Pr. siddallii Off. Asterisks denote a posterior probability of 1.

Fig. 4.—

Providencia siddallii Off genetic reduction from a free-living Providencia strain. (A) Venn diagram of the pangenome of free-living Providencia strains and Pr. siddallii Off as calculated by OrthoMCL. It is evident Pr. siddallii Off’s genetic repertoire mainly represents a subset of Providencia’s. (B) Functional profile divergence of Pr. siddallii Off compared with free-living Providencia strains. The most disparate categories are located on the top and bottom rows, going to the most similar in the central rows. Psid: Pr. siddallii Off; Pret: Providencia rettgeri; Pstu: Pr. stuartii.

Fig. 5.—

Metabolic reconstruction of Pr. siddallii Off. Metabolic reconstruction of Pr. siddallii Off as done by PathwayTools. Intact pathways are shown in solid black lines, while almost-complete ones are shown in gray. Compounds whose biosynthesis is not explained by any intact routes present in the organism are shown in faded coloring. Importers are shown using green ovals, whereas exporters and exporters/importers are shown in blue. Essential amino acids and nonessential ones are shown in red and purple coloring, respectively. Cofactors and B-vitamins are shown in blue coloring. The three cellular compartments typical of a gram-negative bacterium are represented in different coloring.

Fig. 6.—

B-vitamin biosynthetic pathways retained by gammaproteobacterial endosymbionts from blood-feeders. Pathways representing the conserved production of diverse B-vitamins and cofactors by blood-feeders endosymbionts. Arrows represent reactions catalyzed by the enzymes represented by the names above and below them. A dash in the names of the enzymes represent that both enzymes are involved in the reaction(s). Dotted lines represent absent reactions in all organisms. Bright red lines indicate pathways present in all organisms but W. glossinidia strain brevipalpis. The rest of the color codes are represented in the Venn-like diagram on the left. Rped: R. pediculicola; Psid: Pr. siddallii Off; Wigg: Wigglesworthia endosymbionts.