Chemoorganoautotrophic lifestyle of the anaerobic enrichment culture N47 growing on naphthalene

Abstract

In almost all respiratory organisms, organic substrates are degraded via catabolic processes to the central metabolite acetyl-CoA which is then oxidized to CO₂ for energy metabolism or used as a building block for anabolism. Most microorganisms have either the closed tricarboxylic acid cycle or the complete Wood-Ljungdahl-pathway for acetyl-CoA oxidation, but the sulfate-reducing, naphthalene-degrading culture N47 possesses both completely. Combining ¹³C- labeled substrates and mass-specific GC-MS analysis of amino acids and fatty acids with enzyme activity assays suggests that N47 has a chemoorganoautotrophic metabolism degrading complex organic substrates such as naphthalene. Surprisingly, however, the biomass is mainly produced from acetyl-CoA generated de novo via CO₂-fixation. This metabolism probably requires both a complete Wood-Ljungdahl pathway for acetyl-CoA oxidation and a reverse tricarboxylic acid cycle for CO₂ fixation. Based on genome analysis, this chemoorganoautotrophic metabolism seems to also occur in other sulfate-reducers and anaerobic ammonium-oxidizers.

Fig. 1. Overview of the metabolic pathways of fatty acids and some amino acids.

Thick red lines indicate adjacent ¹³C-atoms (e.g., ¹³C₂ bodies, two labeled carbon atoms in the same molecule). Blue dots indicate ¹³C-atoms derived from ¹³CO₂ (one labeled carbon atom). ¹³CO₂ can react with ¹²CO₂ in the WLP to form acetyl-CoA with a mass increase of one, marked with an asterisk *(C₂ body, one labeled carbon atom). Due to the symmetry of succinate, it is not possible to observe if the label is on C1 and C2 or C3 and C4, respectively, which is indicated with a thinner red line and (*). 1: naphthalene, 2: acetyl-CoA, 3: exemplary fatty acid, 4: pyruvate, 5: alanine, 6: citrate, 7: 2-oxoglutarate, 8: glutamate, 9: succinate, 10: oxaloacetate, 11: aspartate.

Fig. 2. Expected and observed label distribution in GC-MS measurements of amino- and fatty acids.

The expected A and observed B results of experiments with ¹³C-labeled naphthalene and the expected C and observed D results of experiments with ¹³CO₂. Red lines indicate an expected increase by 2 mass units resulting from degradation of fully labeled naphthalene into acetyl-CoA and the position of the two labeled carbon atoms. A thin red line indicates that the found labeling is not as high as it was expected. Blue dots indicate an increase by 1 mass unit (M+1) and the position of the labeled carbon atom. The columns next to the amino or fatty acids show the measured ratio of M+2 (red) and M+1 (blue). With 1: naphthalene, 2: acetyl-CoA, 3: exemplary fatty acid, 5: alanine.

Fig. 3. Raman spectra and enlargement of the glycogen associated bands of culture N47 grown with ¹³C enrichment or only natural abundance of ¹³C.

Raman spectra of culture N47 grown with either A 30 mM ¹³C-labeled bicarbonate (50 atom% ¹³C) or B non-labeled bicarbonate (1% ¹³C, natural abundance). C, enlargement of the glycogen-associated Raman band of cells grown with ¹³C-labeled bicarbonate (50 atom%) or D, non-labeled bicarbonate. Blue lines mark the characteristic bands representing polyphosphate, while the band marked in red is a prominent characteristic of glycogen. The presented spectra depict means of 20 spectra measured from different cells each.