Evidence for autotrophic CO2 fixation via the reductive tricarboxylic acid cycle by members of the epsilon subdivision of proteobacteria

Abstract

Based on 16S rRNA gene surveys, bacteria of the epsilon 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 epsilon 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 alpha- and beta-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 epsilon-proteobacteria. Since epsilon-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.

FIG. 1.

Outline of the reductive citric acid cycle for autotrophic CO₂ fixation. The reactions catalyzed by key enzymes are indicated by bold arrows. Enzyme activities: 1, malate dehydrogenase (EC 1.1.1.37); 2, fumarate hydratase (fumarase) (EC 4.2.1.2); 3, fumarate reductase; 4, succinyl-CoA synthetase (EC 6.2.1.5); 5, 2-oxoglutarate:ferredoxin oxidoreductase (EC 1.2.7.3); 6, isocitrate dehydrogenase (EC 1.1.1.42); 7, aconitate hydratase (aconitase) (EC 4.2.1.3); 8, ATP citrate lyase (EC 2.3.3.8); and 9, pyruvate:ferredoxin oxidoreductase (EC 1.2.7.1). Fd_red, reduced ferredoxin.

FIG. 2.

Phylogenetic tree based on a 333-bp-long fragment (306 positions considered for the analyses) of the gene coding for the β-subunit of ATP citrate lyase (aclB). The tree depicts the phylogenetic relationship of aclB of Candidatus Arcobacter sulfidicus and Thiomicrospira denitrificans to other bacterial sequences derived directly either from the environment or other cultures. The tree was constructed by using distance analysis in PAUP*, version 4.0b10, with minimum evolution as the optimal criterion and the distance measure set to maximum likelihood. Trees constructed with other reconstruction algorithms (parsimony and maximum likelihood) resulted in the same overall topology. The numbers at the nodes are bootstrap confidence values expressed as percentages of 1,000 bootstrap replications. Bootstrap values from both distance analysis (first number) and parsimony analysis (second number) are depicted. Bootstrap values less than 50% are not shown. The scale bar represents 10 estimated nucleotide changes.

FIG. 3.

Phylogenetic tree based on a 1,016-bp-long fragment (952 positions considered for the analyses) of the gene coding for the α-subunit of ATP citrate lyase (aclA). The tree depicts the phylogenetic relationship of aclA of Candidatus Arcobacter sulfidicus and Thiomicrospira denitrificans to other bacterial sequences (two Chlorobium species and two sequences [7G3 and 6C6] derived from the epibiont community of Alvinella pompejana), as well as eukaryotic sequences. The tree was constructed by using maximum-likelihood analysis in PAUP*, version 4.0b10. Trees constructed with other reconstruction algorithms (distance analyses and parsimony) resulted in the same overall topology. The numbers at the nodes are bootstrap confidence values expressed as percentages of 1,000 bootstrap replications. Bootstrap values from both distance analysis (first number) and parsimony analysis (second number) are depicted. Bootstrap values less than 50% are not shown. The positions of the sequences derived from the two Chlorobium species could not be confidently resolved with any treeing method due to their divergence from the other sequences. The scale bar represents 0.1 estimated change per nucleotide.