Distribution of EGFP bearing putative N-terminal Eut BMC-targeting signal sequences in <i>S. enterica</i>.

<p><i>S. enterica</i> cells containing constructs for constitutive expression of EGFP, EutC^1–19-EGFP or EutG^1–19-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.</p

Localization of EutC^1–19-EGFP in recombinant <i>E. coli</i> expressing <i>S. enterica</i> Eut shell proteins.

<p>Fluorescence microscopy images of <i>E. coli</i> C2566 cells co-expressing EGFP or EutC^1–19-EGFP with EutS (wild type or the G39V mutant), EutMNLK or EutSMNLK. Cell boundaries are shown by the DIC images. (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342.s013" target="_blank">Table S2</a></b> for the quantification of EGFP localization in recombinant <i>E. coli</i>, and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342.s004" target="_blank">Fig. S4</a></b> for the localization of EutC^1–19-EGFP in the <i>E. coli</i> JM109 strain).</p

Coenzyme-B₁₂-dependent ethanolamine utilization (<i>eut</i>) genes of <i>Salmonella enterica.</i>

<p>(<b>A</b>) <i>eut</i> operon in <i>S. enterica</i>. <i>eutS</i>, <i>eutM</i>, <i>eutN</i>, <i>eutL</i> and <i>eutK</i> encode BMC shell proteins that are proposed to form the Eut microcompartment (yellow and orange) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342-Kofoid1" target="_blank">[20]</a>. Asterisks indicate genes that encode for enzymes with predicted N-terminal signal sequences that target them to the BMC interior <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342-Fan1" target="_blank">[19]</a>. Transcription is induced from the P_I promoter in the presence of both ethanolamine and vitamin B₁₂, while the promoter P_II regulates weak constitutive expression of the transcription factor EutR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342-Roof1" target="_blank">[49]</a>. (<b>B</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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342-Kerfeld1" target="_blank">[7]</a>. Eut BMC prevents dissipation of acetaldehyde, a volatile and toxic reaction intermediate (red) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342-Penrod1" target="_blank">[21]</a>. 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).</p

Hydrolysis of X-gal by <i>E. coli</i> co-expressing EutC^1–19-β-galactosidase and recombinant Eut shell proteins.

<p><i>E. coli</i> C2566 cells with constructs for constitutive expression of β-galactosidase (β-gal) or EutC^1–19-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.</p

Purification of Eut compartments.

<p>(<b>A</b>) Silver stained SDS-PAGE gel showing purification of (lane 1) Eut BMCs from <i>S. enterica</i> cells harboring EutC^1–19-EGFP, (lane 2) recombinant EutSMNLK BMCs, and (lane 3) recombinant EutS BMCs from <i>E. coli</i> C2566 cells co-expressing EutC^1–19-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), EutC^1–19-EGFP (29.1 kDa). (<b>B</b>) Transmission electron micrographs of isolated native and recombinant Eut compartments. From left to right: Eut BMCs from <i>S. enterica</i>, EutSMNLK shells from <i>E. coli</i> C2566, EutS shells from <i>E. coli</i> C2566. (Scale bar: 100 nm).</p

EutC^1–19-EGFP is sequestered in the recombinant EutSMNLK compartment.

<p>(<b>A</b>) Anti-GFP immunogold TEM of a thin section of <i>E. coli</i> JM109 cells co-expressing EutSMNLK and EutC^1–19-EGFP. Gold particles are localized to a protein shell. (Scale bar: 200 nm). (<b>B</b>) Native gel electrophoresis followed by anti-GFP western blot analysis of broken (b) and intact (i) Eut shells, harboring EutC^1–19-EGFP.</p

Formation of engineered protein shells by expression of <i>S. enterica</i> Eut shell proteins in <i>E. coli</i>.

<p>Transmission electron micrographs of thin sections of <i>S. enterica</i> and recombinant <i>E. coli</i>. (<b>A</b>) <i>S. enterica</i> grown on glycerol. (<b>B</b>) <i>S. enterica</i> grown on ethanolamine. (<b>C</b>) <i>E. coli</i> C2566 expressing recombinant EutSMNLK. (<b>D</b>) <i>E. coli</i> JM109 expressing recombinant EutSMNLK. (<b>E</b>) <i>E. coli</i> C2566 expressing recombinant EutS. (<b>F</b>) <i>E. coli</i> JM109 expressing recombinant EutS. (<b>G</b>) <i>E. coli</i> C2566 expressing recombinant EutMNLK. (<b>H</b>) <i>E. coli</i> JM109 expressing recombinant EutMNLK. Arrows indicate the location of recombinant BMCs. (Scale bar: 200 nm).</p