A polycyclic terpenoid that alleviates oxidative stress

Abstract

Polycyclic terpenoid lipids such as hopanes and steranes have been widely used to understand ancient biology, Earth history, and the oxygenation of the ocean-atmosphere system. Some of these lipids are believed to be produced only by aerobic organisms, whereas others actually require molecular oxygen for their biosynthesis. A persistent question remains: Did some polycyclic lipids initially evolve in response to certain environmental or metabolic stresses, including the presence of oxygen? Here, we identify tetracyclic isoprenoids in spores of the bacterium Bacillus subtilis. We call them sporulenes. They are produced by cyclization of regular polyprenes, a reaction that is more favorable chemically than the formation of terpenoids such as hopanoids and steroids from squalene. The simplicity of the reaction suggests that the B. subtilis cyclase may be analogous to evolutionarily ancient cyclases. We show that these molecules increase the resistance of spores to a reactive oxygen species, demonstrating a specific physiological role for a nonpigment bacterial lipid biomarker. Geostable derivatives of these compounds in sediments could thus be used as direct indicators of oxidative stress and aerobic environments.

Fig. 1.

Expression of sqhC and the intracellular localization of SqhC. (A) Organization of the sqhC operon. Hairpin symbols represent transcriptional terminators. (B) Accumulation of colorimetric marker-enzyme β-galactosidase expressed from PsqhC-lacZ in a wild-type strain (amyE::P_sqhC-lacZ, TB12; open diamond) and in a strain lacking the sporulation-dependent RNA polymerase subunit σ^E but harboring PsqhC-lacZ (spoIIGA::tet, amyE::P_sqhC-lacZ, TB19; filled square). Samples were collected at the indicated times after the beginning of sporulation (hour 0). Error bars correspond to 1σ error on the mean value from triplicate samples. (C) Fluorescence micrograph of representative cells producing green fluorescent protein (GFP) fused in-frame to the C terminus of SqhC (amyE::P_Hyspank-shqC-gfp, TB29) 4 h after the beginning of the sporulation. The expression of the fusion was driven by 1 mM IPTG 2.5 h after the induction of sporulation. (D) The same field of view as in C in transmitted light.

Fig. 2.

SqhC produces polycyclic terpenoids. (A) Total ion chromatogram of the lipid extract of wild-type spores (PY79). Three conspicuous peaks (I, II, and III) were never present in the extracts of the strain lacking SqhC (ΔsqhC ΔsodF::tet, TB10). (B) Acyclic tetraprenyl curcumenes (IV and V) were abundant in the spores of both strains. (C) The radioactivity of eighty lipid fractions extracted from the cell lysates of the strain overexpressing SqhC (ΔsqhC ΔsodF::tet amyE::PHyperspank-sqhC, TB28; filled diamond) and the strain mutant for sqhC (TB10; open triangle) after incubation with ³H-FPP. Fractions derived from TB28 that contained I–III were three times as radioactive as the corresponding fractions derived from TB10 (*). These fractions from TB10 still contained a nonnegligible amount of radioactivity that is probably due to the presence of unidentified polyprenoid lipids other than I–III. (D) Structure of acyclic C-35 polyprenoid tetraprenyl-ar-curcumene (IV) detected in the spores of B. subtilis both in the presence and the absence of the putative cyclase. (E) Structure of acyclic C-35 polyprenoid tetraprenyl-β-curcumene (IV) detected in the spores of B. subtilis in the presence and the absence of the putative cyclase. (F) Proposed structure of isomer (II) of tetracyclic C-35 polyprenoids. Isomers I–III are detected only in B. subtilis spores that contained the putative squalene hopene cyclase.

Fig. 3.

Spores containing sqhC are more resistant to hydrogen peroxide. Wild-type colorimetrically marked cells (amyE::PSpac-lacZ) (TB38) and cells lacking sqhC (ΔsqhC ΔsodF::tet) (TB10) were mixed in 10:90 initial ratio in liquid sporulation medium. Purified spores were treated with heat and hydrogen peroxide. A small aliquot of spores was used to determine the ratio of wild-type and mutant cells in the population and the rest was used to inoculate the next cycle of the experiment for eight consecutive cycles. The treatment included the incubation of spores in the presence of 1% H₂O₂ (open squares) and the absence of H₂O₂ (filled triangles). The same experiment was repeated with a mixture of TB38 cells and cells mutant for sqhC and sodF that contained a functional copy of sqhC (ΔsqhC ΔsodF::tet, thrC::PsqhC-sqhC) (TB71, filled squares). The observed trend was not influenced by the presence of the gene encoding for the colorimetric marker lacZ (Fig. S5).