The alternative electron acceptor tetrathionate supports B12-dependent anaerobic growth of Salmonella enterica serovar typhimurium on ethanolamine or 1,2-propanediol

Abstract

Synthesis of cobalamin de novo by Salmonella enterica serovar Typhimurium strain LT2 and the absence of this ability in Escherichia coli present several problems. This large synthetic pathway is shared by virtually all salmonellae and must be maintained by selection, yet no conditions are known under which growth depends on endogenous B12. The cofactor is required for degradation of 1,2-propanediol and ethanolamine. However, cofactor synthesis occurs only anaerobically, and neither of these carbon sources supports anaerobic growth with any of the alternative electron acceptors tested thus far. This paradox is resolved by the electron acceptor tetrathionate, which allows Salmonella to grow anaerobically on ethanolamine or 1,2-propanediol by using endogenously synthesized B12. Tetrathionate provides the only known conditions under which simple cob mutants (unable to make B12) show a growth defect. Genes involved in this metabolism include the ttr operon, which encodes tetrathionate reductase. This operon is globally regulated by OxrA (Fnr) and induced anaerobically by a two-component system in response to tetrathionate. Salmonella reduces tetrathionate to thiosulfate, which it can further reduce to H2S, by using enzymes encoded by the genes phs and asr. The genes for 1,2-propanediol degradation (pdu) and B12 synthesis (cob), along with the genes for sulfur reduction (ttr, phs, and asr), constitute more than 1% of the Salmonella genome and are all absent from E. coli. In diverging from E. coli, Salmonella acquired some of these genes unilaterally and maintained others that are ancestral but have been lost from the E. coli lineage.

FIG. 1

Reduction of tetrathionate to sulfide. The tetrathionate reductase (Ttr) described here performs the initial reduction to thiosulfate (S₂O₃²⁻). This area of metabolism has been reviewed by Barrett and Clark (4).

FIG. 2

Metabolism of ethanolamine and of propanediol. The upper part of this diagram outlines the known metabolism of ethanolamine and indicates the proposed role of various proteins encoded by the ethanolamine (eut) operon (31). The lower part of the diagram outlines the known metabolism of propanediol as described in references and .

FIG. 3

Stimulation of anaerobic growth by ethanolamine or propanediol. Cells of wild-type serovar Typhimurium, strain LT2, were grown anaerobically on minimal NCE medium supplemented with 0.2% yeast extract (YE) to provide a carbon source with or without 80 mM propanediol or 98 mM ethanolamine as an energy source.

FIG. 4

Anaerobic growth on ethanolamine plus B₁₂ or on propanediol with the electron acceptor tetrathionate (S₄O₆) or nitrate (NO₃). Additions to NCE minimal medium were as follows: sodium tetrathionate (40 mM) or potassium nitrate (10 mM), ethanolamine (10 mM), and B₁₂ (0.2 mM) (A and C) or propanediol (50 mM) (B and D). Growth was monitored on the basis of absorbance at 650 nm (A and B), viable cell counts (filled symbols in panels C and D), and microscopic counts (open symbols in panels C and D) using a Petroff-Hausser bacterial cell counter (C and D). The data for cells grown with S₄O₆ but no carbon source are replotted in graphs A and B (for turbidity) and C and D (for microscopic and viable cell counts).

FIG. 5

Cell chain and granule formation during growth of wild-type serovar Typhimurium on ethanolamine or propanediol plus tetrathionate. Cells were viewed on a Zeiss Axioplan phase-contrast microscope. The scale bar in panel A represents 2 μm, and all photos are at the magnification indicated in panel A. Photographs are from stationary-phase cultures grown as described in the legend to Fig. 4. Panels A to C show cells (A and B) and refractile granules (B and C) observed in an ethanolamine-tetrathionate culture after 44 h of incubation at 37°C. (D and E) Cells and refractile granules observed in propanediol-tetrathionate culture after 67 h of incubation at 37°C. (F) Cells grown on propanediol plus NO₃. Neither cell chains nor refractile granules were seen in either ethanolamine- or propanediol-grown cultures when nitrate was the electron acceptor.

FIG. 6

Effect of cobamides on anaerobic growth on ethanolamine or propanediol. Wild-type serovar Typhimurium (A and C) and a mutant with an insertion in cbiD (Cob⁻) (B and D) were grown anaerobically on ethanolamine plus tetrathionate (A and B) or propanediol plus tetrathionate (C and D). Additions were as follows: cyano-B₁₂ (0.2 μM in panels A, B, and D; 15 nM in panel C), cobinamide dicyanide (15 nM), and AdoB₁₂ (15 nM).

FIG. 7

Map of the Sallmonella ttr region and analogous region of the E. coli chromosome. Regions present in only one genome are represented as raised triangles. The map is not to scale; the sizes of various fragments, in kilobases, are shown in parentheses. The SPI2 region is represented by a dashed line to indicate its foreign evolutionary origin. Genes are represented by arrows that point in the direction of transcription. The hatched box represents the region of the Salmonella chromosome that was sequenced during the course of this work.

FIG. 8

Proximal regulation of the ttrBCA operon. The key to inserted elements is shown on the lower right. In the model presented below the map, tetrathionate is sensed by the TtrS protein, which autophosphorylates and then transfers the phosphate group to activate TtrR. Activated TtrR cooperates with the global regulator OxrA (Fnr) to positively regulate expression of the ttrBCA operon.