Metabolic stasis in an ancient symbiosis: genome-scale metabolic networks from two Blattabacterium cuenoti strains, primary endosymbionts of cockroaches

Abstract

Background: Cockroaches are terrestrial insects that strikingly eliminate waste nitrogen as ammonia instead of uric acid. Blattabacterium cuenoti (Mercier 1906) strains Bge and Pam are the obligate primary endosymbionts of the cockroaches Blattella germanica and Periplaneta americana, respectively. The genomes of both bacterial endosymbionts have recently been sequenced, making possible a genome-scale constraint-based reconstruction of their metabolic networks. The mathematical expression of a metabolic network and the subsequent quantitative studies of phenotypic features by Flux Balance Analysis (FBA) represent an efficient functional approach to these uncultivable bacteria.

Results: We report the metabolic models of Blattabacterium strains Bge (iCG238) and Pam (iCG230), comprising 296 and 289 biochemical reactions, associated with 238 and 230 genes, and 364 and 358 metabolites, respectively. Both models reflect both the striking similarities and the singularities of these microorganisms. FBA was used to analyze the properties, potential and limits of the models, assuming some environmental constraints such as aerobic conditions and the net production of ammonia from these bacterial systems, as has been experimentally observed. In addition, in silico simulations with the iCG238 model have enabled a set of carbon and nitrogen sources to be defined, which would also support a viable phenotype in terms of biomass production in the strain Pam, which lacks the first three steps of the tricarboxylic acid cycle. FBA reveals a metabolic condition that renders these enzymatic steps dispensable, thus offering a possible evolutionary explanation for their elimination. We also confirm, by computational simulations, the fragility of the metabolic networks and their host dependence.

Conclusions: The minimized Blattabacterium metabolic networks are surprisingly similar in strains Bge and Pam, after 140 million years of evolution of these endosymbionts in separate cockroach lineages. FBA performed on the reconstructed networks from the two bacteria helps to refine the functional analysis of the genomes enabling us to postulate how slightly different host metabolic contexts drove their parallel evolution.

Figure 1

The TCA cycle and the enzymatic connections of its intermediates. The only difference between the Bge and the Pam metabolic networks is the absence of citrate synthase, aconitase and isocitrate dehydrogenase in the latter (asterisk labelled steps). Note that, with the exception of their participation in the TCA cycle, citrate and isocitrate are isolated nodes in the network. Each enzymatic step is indicated by its EC number. Double arrows indicate reversible reactions, single arrows indicate irreversible reactions.

Figure 2

Metabolite flow in the metabolic models of the endosymbionts. Metabolites with unconstrained import and export across system boundaries are represented by green arrows (8 metabolites related to usual exchange with extracellular medium) and yellow arrows (9 metabolites supposed to be directly provided by the host). Ammonia is only allowed to leave the system (blue arrow). Other external metabolites (purple arrows) can also alternatively enhance or support (depending on the strain considered) bacterial growth. Abbreviations: nac, nicotinic acid; ppbng, porphobilinogen; thm, thiamin; pan4p, pantotheine-4-phosphate; dhor-S, S-dihydroorotate.

Figure 3

Effect of different metabolites on the performance of the metabolic models. Biomass production rates (mmol g DW^-1 h^-1) in the two networks (strain Bge, green bars; strain Pam, red bars) were measured under minimal conditions (see Fig. 2 and main text) or considering the uptake of different metabolites.

Figure 4

Flux distribution through the TCA cycle and adjacent reactions. FBA simulations of both models (strain Bge, left; strain Pam, right) were performed under minimal medium (green values) or with L-Glu uptake (red values). EC numbers are indicated (for enzyme names, see Fig. 1).

Figure 5

Effect of oxygen and L-Gln uptake on metabolic network performance. Biomass production rates (mmol g DW^-1 h^-1) by the Bge strain model were measured at different uptake rates of oxygen (left) and L-Gln (right).

Figure 6

Sensitivity analysis for the first three reactions of the TCA cycle. Biomass production rates (mmol g DW^-1 h^-1) by the Bge strain model were measured under different metabolic environments (minimal conditions or the uptake of the indicated metabolites, see inset) and diverse reaction flux through the first enzymatic steps of the TCA cycle: citrate synthase, aconitase and isocitrate dehydrogenase.