Evidence for Autotrophic CO ₂ Fixation via the Reductive Tricarboxylic Acid Cycle by Members of the ε Subdivision of Proteobacteria

Abstract

Based on 16S rRNA gene surveys, bacteria of the subdivision of proteobacteria have been identified to be important members of microbial communities in a variety of environments, and quite a few have been demonstrated to grow autotrophically. However, no information exists on what pathway of autotrophic carbon fixation these bacteria might use. In this study, Thiomicrospira denitrificans and Candidatus Arcobacter sulfidicus, two chemolithoautotrophic sulfur oxidizers of the subdivision of proteobacteria, were examined for activities of the key enzymes of the known autotrophic CO 2 fixation pathways. Both organisms contained activities of the key enzymes of the reductive tricarboxylic acid cycle, ATP citrate lyase, 2-oxoglutarate: ferredoxin oxidoreductase, and pyruvate:ferredoxin oxidoreductase. Furthermore, no activities of key enzymes of other CO 2 fixation pathways, such as the Calvin cycle, the reductive acetyl coenzyme A pathway, and the 3-hydroxypropionate cycle, could be detected. In addition to the key enzymes, the activities of the other enzymes involved in the reductive tricarboxylic acid cycle could be measured. Sections of the genes encoding the ␣-and ␤-subunits of ATP citrate lyase could be amplified from both organisms. These findings represent the first direct evidence for the operation of the reductive tricarboxylic acid cycle for autotrophic CO 2 fixation in -proteobacteria. Since -proteobacteria closely related to these two organisms are important in many habitats, such as hydrothermal vents, oxic-sulfidic interfaces, or oilfields, these results suggest that autotrophic CO 2 fixation via the reductive tricarboxylic acid cycle might be more important than previously considered.Almost all major groups of prokaryotes include representatives that are able to grow autotrophically (33). These organisms play an essential role in ecosystems by providing a continuous supply of organic carbon for heterotrophs. The Calvin-Benson-Bassham cycle (Calvin cycle) represents the most important extant autotrophic carbon fixation pathway (43, 50). Despite its global significance, it is restricted to organisms with high-energy yield from a chemotrophic or phototrophic lifestyle. Microorganisms present in extreme environments, e.g., high temperature or anaerobic or acidic conditions, generally utilize different CO 2 fixation pathways (17, 33). At present, there are three alternative pathways known: the reductive tricarboxylic acid (TCA) cycle, the reductive acetyl coenzyme A (CoA) pathway, and the 3-hydroxypropionate cycle (4, 33).It has been proposed that the first autotrophic pathway was akin to either the reductive TCA cycle or the reductive acetylCoA pathway (11,17,35,45,58). The reductive TCA cycle has the characteristics of an autocatalytic cycle and leads to a complex cyclic reaction network from which other anabolic pathways could have evolved (11, 58): e.g., the oxidative TCA cycle (8,45). Based upon biochemical and isotopic analyses, the reductive TCA cycle appears to operate in phylogenetically diverse aut...

Microbial diversity in the deep sea and the underexplored “rare biosphere”

“…only recently cultured from hydrothermal vents, and those isolates characterized are either mesophilic or moderately thermophilic sulfur reducers that grow autotrophically with hydrogen under anaerobic conditions and many use the reverse tricarboxylic acid cycle to fix carbon (36,(39)(40)(41). These organisms likely serve as an important member of the microbial community in both the shallow and deep subseafloor biosphere.…”

Culture dependent and independent analyses of 16S rRNA and ATP citrate lyase genes: a comparison of microbial communities from different black smoker chimneys on the Mid-Atlantic Ridge

“…Cell extracts of C. mediatlanticus were prepared using a mixer mill 151 (type MM 301, Retsch, Haare, Germany) according to (Hügler et al 2005). Protein 152 concentrations in cell extracts were determined by the method of (Bradford 1976) using 153 bovine serum albumine as standard.…”

Energy flow and the organization of life

“…Apart from some uncertainty about phosphates, all of these species are known to be produced in the hydrothermal vent environments of the modern earth. Vent energy sources support communities of organisms today [11] in the same way we propose they supported the earliest proto-metabolic chemical systems, except that modern organisms do not require energetic phosphates from the environment because they can recycle phosphate internally using redox energy and the machinery of oxidative phosphorylation.…”

“…If the first polymers were rare in a world where a nascent metabolic order already existed, their sequences would originally have been selected on the basis of their interactions with metabolites. 11 Only at a later stage, when many such sequences were present, would we expect them to have taken on relations to each other that aided in their replication. Under this order of selective forces the commitment to metabolism would have been made before replication became a coordinated macromolecular process, and we would expect the resulting metabolism to preserve the prior geochemical pathways.…”

“…We have good reason to believe that the 11 We thus dissociate the source of selective advantage from the process of molecular replication, to the point where the two could have been completely independent. Note that this premise is directly opposite the usual premise adopted for an RNA world [40]: that differential ability of macromolecules to self-replicate was the first source of selective advantage.…”