GntR Family Regulator DasR Controls Acetate Assimilation by Directly Repressing the acsA Gene in Saccharopolyspora erythraea

ABSTRACT The GntR family regulator DasR controls the transcription of genes involved in chitin and N -acetylglucosamine (GlcNAc) metabolism in actinobacteria. GlcNAc is catabolized to ammonia, fructose-6-phosphate (Fru-6P), and acetate, which are nitrogen and carbon sources. In this work, a DasR-responsive element ( dre ) was observed in the upstream region of acsA1 in Saccharopolyspora erythraea . This gene encodes acetyl coenzyme A (acetyl-CoA) synthetase (Acs), an enzyme that catalyzes the conversion of acetate into acetyl-CoA. We found that DasR repressed the transcription of acsA1 in response to carbon availability, especially with GlcNAc. Growth inhibition was observed in a dasR -deleted mutant (Δ dasR ) in the presence of GlcNAc in minimal medium containing 10 mM acetate, a condition under which Acs activity is critical to growth. These results demonstrate that DasR controls acetate assimilation by directly repressing the transcription of the acsA1 gene and performs regulatory roles in the production of intracellular acetyl-CoA in response to GlcNAc. IMPORTANCE Our work has identified the DasR GlcNAc-sensing regulator that represses the generation of acetyl-CoA by controlling the expression of acetyl-CoA synthetase, an enzyme responsible for acetate assimilation in S. erythraea . The finding provides the first insights into the importance of DasR in the regulation of acetate metabolism, which encompasses the regulatory network between nitrogen and carbon metabolism in actinobacteria, in response to environmental changes.

Genetic Plasticity and Ethylmalonyl Coenzyme A Pathway during Acetate Assimilation inRhodospirillum rubrumS1H under Photoheterotrophic Conditions

ABSTRACTPurple nonsulfur bacteria represent a promising resource for biotechnology because of their great metabolic versatility.Rhodospirillum rubrumhas been widely studied regarding its metabolism of volatile fatty acid, mainly acetate. As the glyoxylate shunt is unavailable inRs. rubrum, 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 ofRs. rubrumin 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.IMPORTANCEPurple 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.Rhodospirillum rubrumS1H 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. InRs. rubrum, 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.

Malate Synthase and β-Methylmalyl Coenzyme A Lyase Reactions in the Methylaspartate Cycle in Haloarcula hispanica

ABSTRACT Haloarchaea are extremely halophilic heterotrophic microorganisms belonging to the class Halobacteria (Euryarchaeota). Almost half of the haloarchaea possesses the genes coding for enzymes of the methylaspartate cycle, a recently discovered anaplerotic acetate assimilation pathway. In this cycle, the enzymes of the tricarboxylic acid cycle together with the dedicated enzymes of the methylaspartate cycle convert two acetyl coenzyme A (acetyl-CoA) molecules to malate. The methylaspartate cycle involves two reactions catalyzed by homologous enzymes belonging to the CitE-like enzyme superfamily, malyl-CoA lyase/thioesterase (haloarchaeal malate synthase [hMS]; Hah_2476 in Haloarcula hispanica) and β-methylmalyl-CoA lyase (haloarchaeal β-methylmalyl-CoA lyase [hMCL]; Hah_1341). Although both enzymes catalyze the same reactions, hMS was previously proposed to preferentially catalyze the formation of malate from acetyl-CoA and glyoxylate (malate synthase activity) and hMCL was proposed to primarily cleave β-methylmalyl-CoA to propionyl-CoA and glyoxylate. Here we studied the physiological functions of these enzymes during acetate assimilation in H. hispanica by using biochemical assays of the wild type and deletion mutants. Our results reveal that the main physiological function of hMS is malyl-CoA (not malate) formation and that hMCL catalyzes a β-methylmalyl-CoA lyase reaction in vivo. The malyl-CoA thioesterase activities of both enzymes appear to be not essential for growth on acetate. Interestingly, despite the different physiological functions of hMS and hMCL, structural comparisons predict that these two proteins have virtually identical active sites, thus highlighting the need for experimental validation of their catalytic functions. Our results provide further proof of the operation of the methylaspartate cycle and indicate the existence of a distinct, yet-to-be-discovered malyl-CoA thioesterase in haloarchaea. IMPORTANCE Acetate is one of the most important substances in natural environments. The activated form of acetate, acetyl coenzyme A (acetyl-CoA), is the high-energy intermediate at the crossroads of central metabolism: its oxidation generates energy for the cell, and about a third of all biosynthetic fluxes start directly from acetyl-CoA. Many organic compounds enter the central carbon metabolism via this key molecule. To sustain growth on acetyl-CoA-generating compounds, a dedicated assimilation (anaplerotic) pathway is required. The presence of an anaplerotic pathway is a prerequisite for growth in many environments, being important for environmentally, industrially, and clinically important microorganisms. Here we studied specific reactions of a recently discovered acetate assimilation pathway, the methylaspartate cycle, functioning in extremely halophilic archaea.