Both forward and reverse TCA cycles operate in green sulfur bacteria

Abstract

The anoxygenic green sulfur bacteria (GSBs) assimilate CO(2) autotrophically through the reductive (reverse) tricarboxylic acid (RTCA) cycle. Some organic carbon sources, such as acetate and pyruvate, can be assimilated during the phototrophic growth of the GSBs, in the presence of CO(2) or HCO(3)(-). It has not been established why the inorganic carbonis required for incorporating organic carbon for growth and how the organic carbons are assimilated. In this report, we probed carbon flux during autotrophic and mixotrophic growth of the GSB Chlorobaculum tepidum. Our data indicate the following: (a) the RTCA cycle is active during autotrophic and mixotrophic growth; (b) the flux from pyruvate to acetyl-CoA is very low and acetyl-CoA is synthesized through the RTCA cycle and acetate assimilation; (c) pyruvate is largely assimilated through the RTCA cycle; and (d) acetate can be assimilated via both of the RTCA as well as the oxidative (forward) TCA (OTCA) cycle. The OTCA cycle revealed herein may explain better cell growth during mixotrophic growth with acetate, as energy is generated through the OTCA cycle. Furthermore, the genes specific for the OTCA cycle are either absent or down-regulated during phototrophic growth, implying that the OTCA cycle is not complete, and CO(2) is required for the RTCA cycle to produce metabolites in the TCA cycle. Moreover, CO(2) is essential for assimilating acetate and pyruvate through the CO(2)-anaplerotic pathway and pyruvate synthesis from acetyl-CoA.

FIGURE 1.

Schematic representation of the RTCA cycle (A) and how FAc inhibition occurs in carbon flow via the OTCA cycle (B).

FIGURE 2.

Effect of FAc on the growth of C. tepidum. 0.34% (40 mm) HCO₃⁻ is included in all of the growth media. The growth curve (A) and image of cell cultures (B) of C. tepidum during autotrophic and mixotrophic growth (with either 10 mm acetate or pyruvate) conditions with or without 2 mm FAc is shown. The cell growth (recorded as A₆₂₅) during growth conditions with various concentration of acetate, pyruvate, and FAc is shown (C). F, fluoro.

FIGURE 3.

Shown are gene expression profiles for ACL (subunits A and B are encoded by aclA and aclB, respectively), CS (encoded by gltA), and putative succinate dehydrogenase/fumarate reductase subunit A (sdhA) and subunit B (sdhB) of C. tepidum, and activity assays for ACL and CS. Relative gene expression profiles of aclA/gltA, aclB/gltA, CT2042/CT2267 (putative sdhA), and CT2042/CT2266 (putative sdhB) after 36 h of autotrophic growth (A), the enzymatic activity of ACL and CS in autotrophic and mixotrophic cultures (B), and relative gene expression levels of aclA/gltA, aclB/gltA, aclA/acn (acn encoding aconitase) and aclA/korA (korA encoding α-keto-glutarate:ferredoxin oxidoreductase, α subunit) during various growth stages of mixotrophic cultures with 10 mm acetate are shown (C). The data in this figure were acquired from experiments performed two times in triplicate, and the error bar is the standard deviation of the mean value.

FIGURE 4.

The proposed carbon flux in C. tepidum. The proposed carbon flux during mixotrophic growth with pyruvate (A) and acetate (B) and the proposed FAc effect for the carbon flux (C). F, fluoro.