Osmoregulation in the Halophilic Bacterium Halomonas elongata: A Case Study for Integrative Systems Biology

Abstract

Halophilic bacteria use a variety of osmoregulatory methods, such as the accumulation of one or more compatible solutes. The wide diversity of compounds that can act as compatible solute complicates the task of understanding the different strategies that halophilic bacteria use to cope with salt. This is specially challenging when attempting to go beyond the pathway that produces a certain compatible solute towards an understanding of how the metabolic network as a whole addresses the problem. Metabolic reconstruction based on genomic data together with Flux Balance Analysis (FBA) is a promising tool to gain insight into this problem. However, as more of these reconstructions become available, it becomes clear that processes predicted by genome annotation may not reflect the processes that are active in vivo. As a case in point, E. coli is unable to grow aerobically on citrate in spite of having all the necessary genes to do it. It has also been shown that the realization of this genetic potential into an actual capability to metabolize citrate is an extremely unlikely event under normal evolutionary conditions. Moreover, many marine bacteria seem to have the same pathways to metabolize glucose but each species uses a different one. In this work, a metabolic network inferred from genomic annotation of the halophilic bacterium Halomonas elongata and proteomic profiling experiments are used as a starting point to motivate targeted experiments in order to find out some of the defining features of the osmoregulatory strategies of this bacterium. This new information is then used to refine the network in order to describe the actual capabilities of H. elongata, rather than its genetic potential.

Anaplerotic pathways inHalomonas elongata: the role of the sodium gradient

“…Thermodynamics-based Metabolic Flux Analysis (TMFA) A simple model of the TCA cycle and the anaplerotic pathways was formulated based on previous stoichiometric models [17] and additional thermodynamic information [2,12]. The stoichiometric and thermodynamic constraints were combined within the well establish TMFA framework [15,16] into a Mixed Integer Linear Program (MILP) that enables the calculation of optimal flux distributions.…”

“…The different stoichiometries of the antiporters provide different degrees of coupling between proton and sodium transport just like the gears in a gearbox. Calculating the optimal flux distributions to maximize the production of Oxaloacetate points at Pck and Mae as the most advantageous anaplerotic reactions from a stoichiometric point of view, as has already been pointed out [17,30]. In spite of their reversibility within the established in vivo ranges for metabolite concentrations no thermodynamically viable flux distributions could be obtained where Pck or the Malic enzymes played an anaplerotic role.…”

“…As can be seen in figure 4, sodium plays multiple roles in this metabolic network beyond driving Oad. The electron transport chain of H. elongata has a sodium traslocating NADH ubiquinone oxidoreductase (Na-Nqr) [17] which couples respiration to the sodium gradient. Moreover, as many other halophilic and halotolerant bacteria, H. elongata has a high number of sodium-proton antiporters that can be used to built a proton gradient or to use one to pump sodium out of the cell.…”

“…Only a strict control of the expression and activity of this enzymes can prevent them from destroying the carefully balanced redox state of the two electron currencies in H. elongata. Enyme assays with cell free extract have shown very low activities for Mae using NADP as cofactor [17]. No activity could be detected using NAD (personal communication, Irina Bagyan).…”

“…Cytoplasmic versions of this enzyme are considered to be irreversible, thermodynamically unable to function in the anaplerotic sense and usually involved in gluconeogenesis [25]. In H. elongata, however, Oad is a membrane bound sodium pump [9] and it has been hypothesized [17] that it can operate as an anaplerotic reaction, thus using the considerable sodium gradient available under high salt concentrations to drive the carboxylation of pyruvate in H. elongata. To the extent of our knowledge, such a function has never been observed in vivo.…”