Physiological Studies of Chlorobiaceae Suggest that Bacillithiol Derivatives Are the Most Widespread Thiols in Bacteria

Abstract

Low-molecular-weight (LMW) thiols mediate redox homeostasis and the detoxification of chemical stressors. Despite their essential functions, the distribution of LMW thiols across cellular life has not yet been defined. LMW thiols are also thought to play a central role in sulfur oxidation pathways in phototrophic bacteria, including the Chlorobiaceae Here we show that Chlorobaculum tepidum synthesizes a novel LMW thiol with a mass of 412 ± 1 Da corresponding to a molecular formula of C₁₄H₂₄N₂O₁₀S, which suggests that the new LMW thiol is closely related to bacillithiol (BSH), the major LMW thiol of low-G+C Gram-positive bacteria. The Cba. tepidum LMW thiol structure was N-methyl-bacillithiol (N-Me-BSH), methylated on the cysteine nitrogen, the fourth instance of this modification in metabolism. Orthologs of bacillithiol biosynthetic genes in the Cba. tepidum genome and the CT1040 gene product, N-Me-BSH synthase, were required for N-Me-BSH synthesis. N-Me-BSH was found in all Chlorobiaceae examined as well as Polaribacter sp. strain MED152, a member of the Bacteroidetes A comparative genomic analysis indicated that BSH/N-Me-BSH is synthesized not only by members of the Chlorobiaceae, Bacteroidetes, Deinococcus-Thermus, and Firmicutes but also by Acidobacteria, Chlamydiae, Gemmatimonadetes, and Proteobacteria. Thus, BSH and derivatives appear to be the most broadly distributed LMW thiols in biology.IMPORTANCE Low-molecular-weight thiols are key metabolites that participate in many basic cellular processes: central metabolism, detoxification, and oxidative stress resistance. Here we describe a new thiol, N-methyl-bacillithiol, found in an anaerobic phototrophic bacterium and identify a gene that is responsible for its synthesis from bacillithiol, the main thiol metabolite in many Gram-positive bacteria. We show that the presence or absence of this gene in a sequenced genome accurately predicts thiol content in distantly related bacteria. On the basis of these results, we analyzed genome data and predict that bacillithiol and its derivatives are the most widely distributed thiol metabolites in biology.

Keywords: Chlorobaculum tepidum; cellular redox status; chlorobiaceae; low molecular weight thiol; sulfur.

FIG 1

Structures of LMW thiols referred to in the text with the sulfhydryl-bearing motif derived from cysteine or homocysteine colored blue. BSH derivatives are boxed together with the R group on the common backbone indicated. The distributions of previously studied LMW thiols across different organisms are indicated.

FIG 2

Chlorobaculum tepidum contains a novel LMW thiol. (A) Full HPLC chromatogram of bimane extract from a stationary-phase Cba. tepidum culture grown under standard conditions. The arrow indicates the novel thiol U7. (B) U7 and cysteine (Cys) are not detected if extract is treated with N-ethyl-maleimide (dashed line) before mBBr. (C) Glutathione (purified standard, dashed-dotted line) is readily detected in E. coli bimane extract (gray line), but not Cba. tepidum (solid line).

FIG 3

The novel Cba. tepidum U7-bimane adduct (U7mB) is N-Me-BSmB. (A to E) HPLC chromatograms of CysmB and synthetic BSmB (A), synthetic hCys-BSmB (B), synthetic N-Me-BSmB (C), purified Cba. tepidum U7mB (D), mixture of N-Me-BSmB and purified Cba. tepidum U7mB (E). The retention times are different from those shown in Fig. 1, because a different column and elution gradient were used to better separate these compounds.

FIG 4

The proposed biosynthetic pathway for N-Me-BSH (A) and N-Me-BSH pool size (B) in Cba. tepidum deletion mutant strains ΔCT1419 (bshB), ΔCT1040 (putative SAM-dependent methyltransferase), and ΔCT1213 (putative SAM-dependent methyltransferase). The values shown for strains ΔCT1419 and ΔCT1040 indicate the limit of detection for N-Me-BSH. There is currently no evidence to support N-Me-BSH synthesis via N-Me-Cys addition to GlcNMal.

FIG 5

Dynamics of N-Me-BSH pool sizes in Cba. tepidum. (A) Pool size in Cba. tepidum grown for the indicated times (mid log phase, late log phase, early stationary phase) with different electron donor combinations. (B) Pool size in the wild-type cells grown with the indicated light fluxes grown to stationary phase. (C) Pool size in stationary-phase wild-type cells or cells treated with 1 mM trimethyl-TCEP (tmTCEP) or 2 mM diamide. Significant differences are indicated by P values calculated by the Tukey-Kramer HSD test after ANOVA. n.s., not significant (P > 0.05).

FIG 6

Distribution of LMW thiols in bacteria as determined by mBBr derivatization-HPLC (A) and the potential distribution based on an analysis of complete genome sequences (B) for the presence of orthologs encoding complete pathways for GSH (gshA-B), MSH (mshA-D), BSH (bshA-C), or N-Me-BSH (bshA-C plus nmbA).