Abstract
The haloarchaeon Haloferax volcanii grows on acetate as sole carbon and energy source. The genes and proteins involved in uptake and activation of acetate and in gluconeogenesis were identified and analyzed by characterization of enzymes and by growth experiments with the respective deletion mutants. (i) An acetate transporter of the sodium: solute-symporter family (SSF) was characterized by kinetic analyses of acetate uptake into H. volcanii cells. The functional involvement of the transporter was proven with a Δssf mutant. (ii) Four paralogous AMP-forming acetyl-CoA synthetases that belong to different phylogenetic clades were shown to be functionally involved in acetate activation. (iii) The essential involvement of the glyoxylate cycle as an anaplerotic sequence was concluded from growth experiments with an isocitrate lyase knock-out mutant excluding the operation of the methylaspartate cycle reported for Haloarcula species. (iv) Enzymes involved in phosphoenolpyruvate synthesis from acetate, namely two malic enzymes and a phosphoenolpyruvate synthetase, were identified and characterized. Phylogenetic analyses of haloarchaeal malic enzymes indicate a separate evolutionary line distinct from other archaeal homologs. The exclusive function of phosphoenolpyruvate synthetase in gluconeogenesis was proven by the respective knock-out mutant. Together, this is a comprehensive study of acetate metabolism in archaea.
Highlights
Acetate serves as substrate for catabolism and anabolism of several aerobic and anaerobic bacteria, eukarya and archaea
We identified an acetate transporter, four paralogous AMP-forming acetyl-CoA synthetases that are essential for acetate activation, and describe enzymes that catalyze the formation of PEP from acetyl-CoA
We report the identification and characterization of an acetate transporter and of enzymes involved in acetate activation and in gluconeogenesis
Summary
Acetate serves as substrate for catabolism and anabolism of several aerobic and anaerobic bacteria, eukarya and archaea. The metabolism of acetate in bacteria and eukarya is well studied; it involves uptake of acetate into the cells followed by activation of acetate to acetyl-CoA, the common intermediate for both catabolism and anabolism. In the domain of archaea acetate activation has been reported for the aerobic halophilic archaea Haloarcula marismortui and Haloferax volcanii and for anaerobic methanogens, e.g., Methanosarcina and Methanosaeta species (Thauer et al, 1989; Bräsen and Schönheit, 2001, 2005). Methanosaeta species, M. consilii and M. thermophila, activate acetate via ACS (Ingram-Smith and Smith, 2007; Berger et al, 2012) whereas acetate activation in Methanosarcina species involves the AK-PTA mechanism (Thauer et al, 1989). An ACS from H. marismortui has been characterized (Bräsen and Schönheit, 2005)
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