Figure 1.
Coenzyme-B12-dependent ethanolamine utilization (eut) genes of Salmonella enterica.
(A) eut operon in S. enterica. eutS, eutM, eutN, eutL and eutK encode BMC shell proteins that are proposed to form the Eut microcompartment (yellow and orange) [20]. Asterisks indicate genes that encode for enzymes with predicted N-terminal signal sequences that target them to the BMC interior [19]. Transcription is induced from the PI promoter in the presence of both ethanolamine and vitamin B12, while the promoter PII regulates weak constitutive expression of the transcription factor EutR [49]. (B) Model for catabolism of ethanolamine by the Eut BMC. Ethanolamine enters the microcompartment and is metabolized to ethanol, acetyl-phosphate and acetyl-CoA, which can enter the tricarboxylic acid cycle [7]. Eut BMC prevents dissipation of acetaldehyde, a volatile and toxic reaction intermediate (red) [21]. Enzymes assumed to reside in the BMC lumen include coenzyme-B12-dependent ethanolamine ammonia lyase (EAL, EutBC), EAL reactivase (EutA), alcohol dehydrogenase (EutG), aldehyde dehydrogenase (EutE), and phosphotransacetylase (EutD).
Figure 2.
Formation of engineered protein shells by expression of S. enterica Eut shell proteins in E. coli.
Transmission electron micrographs of thin sections of S. enterica and recombinant E. coli. (A) S. enterica grown on glycerol. (B) S. enterica grown on ethanolamine. (C) E. coli C2566 expressing recombinant EutSMNLK. (D) E. coli JM109 expressing recombinant EutSMNLK. (E) E. coli C2566 expressing recombinant EutS. (F) E. coli JM109 expressing recombinant EutS. (G) E. coli C2566 expressing recombinant EutMNLK. (H) E. coli JM109 expressing recombinant EutMNLK. Arrows indicate the location of recombinant BMCs. (Scale bar: 200 nm).
Figure 3.
Distribution of EGFP bearing putative N-terminal Eut BMC-targeting signal sequences in S. enterica.
S. enterica cells containing constructs for constitutive expression of EGFP, EutC1–19-EGFP or EutG1–19-EGFP were cultured with either glycerol or ethanolamine. Distribution of green fluorescence within the cells was observed by fluorescence microscopy. DIC images show the cell boundaries.
Figure 4.
Localization of EutC1–19-EGFP in recombinant E. coli expressing S. enterica Eut shell proteins.
Fluorescence microscopy images of E. coli C2566 cells co-expressing EGFP or EutC1–19-EGFP with EutS (wild type or the G39V mutant), EutMNLK or EutSMNLK. Cell boundaries are shown by the DIC images. (see Table S2 for the quantification of EGFP localization in recombinant E. coli, and Fig. S4 for the localization of EutC1–19-EGFP in the E. coli JM109 strain).
Figure 5.
Purification of Eut compartments.
(A) Silver stained SDS-PAGE gel showing purification of (lane 1) Eut BMCs from S. enterica cells harboring EutC1–19-EGFP, (lane 2) recombinant EutSMNLK BMCs, and (lane 3) recombinant EutS BMCs from E. coli C2566 cells co-expressing EutC1–19-EGFP. Calculated protein sizes are as follows: EutS (11.6 kDa), EutM (9.8 kDa), EutN (10.4 kDa), EutL (22.7 kDa), EutK (17.5 kDa), EutC1–19-EGFP (29.1 kDa). (B) Transmission electron micrographs of isolated native and recombinant Eut compartments. From left to right: Eut BMCs from S. enterica, EutSMNLK shells from E. coli C2566, EutS shells from E. coli C2566. (Scale bar: 100 nm).
Figure 6.
EutC1–19-EGFP is sequestered in the recombinant EutSMNLK compartment.
(A) Anti-GFP immunogold TEM of a thin section of E. coli JM109 cells co-expressing EutSMNLK and EutC1–19-EGFP. Gold particles are localized to a protein shell. (Scale bar: 200 nm). (B) Native gel electrophoresis followed by anti-GFP western blot analysis of broken (b) and intact (i) Eut shells, harboring EutC1–19-EGFP.
Figure 7.
Hydrolysis of X-gal by E. coli co-expressing EutC1–19-β-galactosidase and recombinant Eut shell proteins.
E. coli C2566 cells with constructs for constitutive expression of β-galactosidase (β-gal) or EutC1–19-β-gal and different combinations of Eut shell proteins were grown with the β-gal substrate X-gal. Intracellular accumulation of the insoluble X-gal cleavage product was observed by Differential Interference Contrast (DIC) microscopy. Arrows point to intracellular indole deposits.