Genetic Plasticity and Ethylmalonyl Coenzyme A Pathway during Acetate Assimilation in Rhodospirillum rubrum S1H under Photoheterotrophic Conditions

Abstract

Purple nonsulfur bacteria represent a promising resource for biotechnology because of their great metabolic versatility. has been widely studied regarding its metabolism of volatile fatty acid, mainly acetate. As the glyoxylate shunt is unavailable in, the citramalate cycle pathway and the ethylmalonyl-coenzyme A (CoA) pathway are proposed as alternative anaplerotic pathways for acetate assimilation. However, despite years of debate, neither has been confirmed to be essential. Here, using functional genomics, we demonstrate that the ethylmalonyl-CoA pathway is required for acetate photoassimilation. Moreover, an unexpected reversible long-term adaptation is observed, leading to a drastic decrease in the lag phase characterizing the growth of in the presence of acetate. Using proteomic and genomic analyses, we present evidence that the adaptation phenomenon is associated with reversible amplification and overexpression of a 60-kb genome fragment containing key enzymes of the ethylmalonyl-CoA pathway. Our observations suggest that a genome duplication and amplification phenomenon is not only involved in adaptation to acute stress but can also be important for basic carbon metabolism and the redox balance. Purple nonsulfur bacteria represent a major group of anoxygenic photosynthetic bacteria that emerged as a promising resource for biotechnology because of their great metabolic versatility and ability to grow under various conditions. S1H has notably been selected by the European Space Agency to colonize its life support system, called MELiSSA, due to its capacity to perform photoheterotrophic assimilation of volatile fatty acids (VFAs), mainly acetate. VFAs are valuable carbon sources for many applications, combining bioremediation of contaminated environments with the generation of added-value products. Acetate is one of the major volatile fatty acids generated as a by-product of fermentation processes. In, purple nonsulfur bacteria, the assimilation of acetate is still under debate since two different pathways have been proposed. Here, we clearly demonstrate that the ethylmalonyl-CoA pathway is the major anaplerotic pathway for acetate assimilation in this strain. Interestingly, we further observed that gene duplication and amplification, which represent a well-known phenomenon in antibiotic resistance, also play a regulatory function in carbon metabolism and redox homeostasis.

Global Proteomic Analysis Reveals High Light Intensity Adaptation Strategies and Polyhydroxyalkanoate Production in Rhodospirillum rubrum Cultivated With Acetate as Carbon Source

“…Indeed, in case of sudden light increase, the resulting excess in proton motive force has been shown to increase the reverse flux through NADH dehydrogenase, reducing NAD + to NADH (Herter et al, 1998;Golomysova et al, 2010). Supporting this redox imbalance hypothesis, a reduction in the lag phase could also be obtained using a higher concentration of bicarbonate ions (50 mM) in the medium (De Meur et al, 2018), suggesting that the bicarbonate fixation, which act as an electron sink, helped cells to start growing. Importantly, proteomic data showed lower relative abundance of the Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO), key enzyme of the Calvin-Benson-Bassham-(CBB) cycle in the acetate condition compared to the succinate condition, ruling out involvement of CBB cycle in electron sinking in this case.…”

“…The medium was supplemented with acetate (62.5 mM) or succinate (31.25 mM) as the carbon source and a defined amount of bicarbonate ions (0-3-50 mM). Pre-cultures used for the different experiments were grown in presence of succinate excepted for acetate acclimated strains where pre-cultures were cultivated in presence of acetate in order to avoid the reported deacclimatation of the strain (De Meur et al, 2018). Cultures were subjected to 50 µmol photons/m 2 s (Sencys; 10 W; 100 lumens; 2,650 K) and incubated with rotary shaking at 180 rpm at 30 • C. To perform light stress experiments (see below), this intensity was elevated from 50 µmol photons/m 2 s to 150 µmol photons/m 2 s. Growth was monitored by measuring the optical density at 680nm.…”

“…It is well known that the assimilation of acetate, propionate, butyrate or valerate as a sole source of carbon in anaerobic environments is associated with the consumption of HCO 3 − (Rinne et al, 1965;Porter and Merrett, 1972;Ehrenreich and Widdel, 1994;Laguna et al, 2011). In the case of acetate, carbonate ions were shown to drastically reduce the lag phase encountered at the onset of growth with acetate as sole carbon source (De Meur et al, 2018). Not only bicarbonate concentration can help bacterial growth start up, since we also demonstrated that a reduced lag phase could be obtained by inoculating the culture at a high OD 680 nm (Leroy et al, 2015).…”

“…Proteomic analysis revealed a higher abundance of the corresponding enzymes, but also of most of the proteins of the amplified gene cluster. The resulting phenotype showed a drastically reduced lag phase in the presence of acetate (De Meur et al, 2018). Due to the reduced lag phase, this newly adapted strain named "acetate-competent strain" also constitutes a great interest for biotechnological processes.…”

Phototrophic Lactate Utilization by Rhodopseudomonas palustris Is Stimulated by Coutilization with Additional Substrates

“…However, we believe that several mechanisms can be excluded. First, there are instances where cosubstrates enable anaerobic growth by acting as alternative electron acceptors and thereby contributing to cellular redox balance (28)(29)(30). The contribution of cosubstrates to electron balance during lactate coutilization is unlikely because (i) the same pattern of lactate utilization was observed under two conditions that differentially allow N 2 fixation (Fig.…”