Minimal functions and physiological conditions required for growth of salmonella enterica on ethanolamine in the absence of the metabolosome

Abstract

During growth on ethanolamine, Salmonella enterica synthesizes a multimolecular structure that mimics the carboxysome used by some photosynthetic bacteria to fix CO(2). In S. enterica, this carboxysome-like structure (hereafter referred to as the ethanolamine metabolosome) is thought to contain the enzymatic machinery needed to metabolize ethanolamine into acetyl coenzyme A (acetyl-CoA). Analysis of the growth behavior of mutant strains of S. enterica lacking specific functions encoded by the 17-gene ethanolamine utilization (eut) operon established the minimal biochemical functions needed by this bacterium to use ethanolamine as a source of carbon and energy. The data obtained support the conclusion that the ethanolamine ammnonia-lyase (EAL) enzyme (encoded by the eutBC genes) and coenzyme B(12) are necessary and sufficient to grow on ethanolamine. We propose that the EutD phosphotransacetylase and EutG alcohol dehydrogenase are important to maintain metabolic balance. Glutathione (GSH) had a strong positive effect that compensated for the lack of the EAL reactivase EutA protein under aerobic growth on ethanolamine. Neither GSH nor EutA was needed during growth on ethanolamine under reduced-oxygen conditions. GSH also stimulated growth of a strain lacking the acetaldehyde dehydrogenase (EutE) enzyme. The role of GSH in ethanolamine catabolism is complex and requires further investigation. Our data show that the ethanolamine metabolosome is not involved in the biochemistry of ethanolamine catabolism. We propose the metabolosome is needed to concentrate low levels of ethanolamine catabolic enzymes, to keep the level of toxic acetaldehyde low, to generate enough acetyl-CoA to support cell growth, and to maintain a pool of free CoA.

FIG. 1.

The ethanolamine utilization (eut) operon of S. enterica. The eutR gene encodes an AraC-type protein that activates eut operon transcription in response to AdoCbl and ethanolamine. eutR expression is controlled independently from the operon.

FIG. 2.

Transmission electron micrographs of S. enterica strains grown on ethanolamine as carbon and energy source. A. JE8392 (eut⁺) grown in 30 mM ethanolamine viewed at 40,000-fold magnification. B. JE8392 grown in 22 mM glycerol viewed at 40,000-fold magnfication. C. Strain JE8217 (Δeut peutABC⁺ peutE⁺) grown in 30 mM ethanolamine, viewed at 53,000-fold magnification.

FIG. 3.

Complementation studies. In all panels VOC means vector-only control. A. A strain lacking the eutABC genes encoding the EAL reactivase EutA and the large and small subunits of EAL, respectively, was tested for the ability to use ethanolamine as sole carbon and energy source. Expression of genes encoded by plasmid pEUT34 (P_araBAD-eutABC⁺) was induced with l-(+)-arabinose (JE8138) (100 μM) (solid diamonds); empty cloning vector pBAD33 was introduced into JE8095 (ΔeutABC1163) and the growth behavior of the resulting strain (JE8139) was also analyzed in the presence of l-(+)-arabinose (100 μM) (solid squares). Strain JE8392 (eut⁺/pBAD33) (solid circles) was used as a positive control. B. Strain JE8094 (ΔeutBC) was tested for its ability to grow on ethanolamine. Strain JE8411 (JE8094/pEUT42 (P_araBAD-eutBC⁺) was grown on ethanolamine in the absence of l-(+)-arabinose (solid diamonds). Strain JE8412 (JE8094/pBAD33) (solid squares) was used negative control. Strain JE8392 (eut⁺/pBAD33) (solid circles) was used as positive control. C. Strain JE7973 (ΔeutE) was tested for the ability to grow on ethanolamine. Strain JE8191 (JE7973/pEUT40 P_tac-eutE⁺) was grown in ethanolamine medium containing 100 μM IPTG (solid diamonds). Strain JE8425 (JE7973/pTAC-85) (solid squares) was used as the negative control, and strain JE8424 (TR6583/pTAC-85) (solid circles) was used as the positive control.

FIG. 4.

Minimal functions for the ethanolamine catabolism. For all panels, VOC means vector-only control, strain JE8142 (Δeut/pBAD33 pTAC-85) was used as the negative control (open squares), and strain JE8298 (eut⁺/pBAD33 pTAC-85) (solid circles) was used as the positive control. When added, arabinose or IPTG was at 100 μM. A. Strain JE2261 (Δeut) was transformed with plasmids pEUT34 (P_araBAD-eutABC⁺) and pEUT40 (P_tac-eutE⁺) and phenotypes assessed. For growth on ethanolamine as the sole source of carbon and energy, 2.5 mM oxidized GSSG (open triangles) or no GSSG (inverted solid triangles) was added to the medium. B. Strain JE2261 (Δeut) was transformed with plasmids pEUT42 (P_araBAD-eutBC⁺) and plasmid pEUT40 (P_tac-eutE⁺), and phenotypes were assessed. For growth on ethanolamine as the sole source of carbon and energy: plus GSSG (solid diamonds) or without GSSG (solid triangles) addition to the medium. C. Strain JE2261 (Δeut) was transformed with plasmids pEUT34 (P_araBAD-eutABC⁺) and pTAC-85 and phenotypes were assessed: plus GSSG (solid diamonds) or without GSSG (solid triangles) addition to the medium.

FIG. 5.

Conditional requirement of eutA function. Strain JE8093 (ΔeutA) was transformed with either plasmid pEUT33 [gene expression induced with 100 μM l-(+)-arabinose] (open diamonds) or pBAD33 (solid inverted triangles). Strain JE8392 (solid circles) was used as a positive control. In both panels, VOC means vector-only control. A. The experiment was performed in 5-ml cultures in 16- by 150-mm borosilicate tubes. B. The experiment was performed in a microtiter plate with medium supplemented with ethanolamine (30 mM) and glycerol (0.5 mM). Glycerol was needed to prime cell growth; in its absence the onset of exponential growth was drastically delayed. Strain JE8142 (Δeut/pBAD33 pTAC-85) was used as the negative control.

FIG. 6.

Model for ethanolamine catabolism in S. enterica. The metabolosome representation is not to scale.