Uptake and incorporation of ³⁵S-lipoic acid in the presence of Lpa.

<p>(A) KER176 cells that are auxotrophic for lipoic acid were transformed with plasmids encoding wild type Lpa (Lpa) or the active site nucleophile mutant Lpa S259A and were grown in minimal medium supplemented with ³⁵S-lipoic acid. After a 10 hour induction at 20°C, cell samples were normalized by OD₆₀₀ and protein extracts were separated by SDS-PAGE and analyzed by autoradiography. The assignment of the labeled species to the three lipoylated proteins in <i>E. coli</i>, the PDH, KDH, and H-protein, is indicated. (B) Scintillation counting was used to quantify ³⁵S-lipoic acid taken up by the KER176 cultures shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007392#pone-0007392-g002" target="_blank">Figure 2A</a>. Counts per minute (CPM) correspond to the uptake of cells from 5 µl of culture with an OD₆₀₀ of 1.1.</p

Growth of lipoylation deficient <i>E. coli</i> expressing Lpa and Lpa active site mutants.

<p>Growth assay of TM136 strain cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007392#pone.0007392-Morris1" target="_blank">[16]</a> expressing Lpa (squares), Lpa S235A (upward triangles), and Lpa S259A (circles). Growth curves at 37°C (solid symbols) and 20°C (open symbols are shown with cultures induced with 0.4 mM IPTG highlighted in red. In each condition, the growth curve corresponding to cells expressing wild type Lpa is shown with a thickened line.</p

Lipoylation and biotinylation sites.

<p>(A) Amino acid sequence alignments of lipoylation and biotinylation sites in <i>E. coli</i>. The lysine that is involved in lipoic acid or biotin attachment is marked in bold. Residues corresponding to conserved glycine and glutamine residues are shaded <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007392#pone.0007392-Fujiwara2" target="_blank">[33]</a>. Residues forming the biotinylation consensus site are underlined. (B) ClustalW comparison of amino acid sequences surrounding the site of lipoate attachment for lipoylated proteins found in <i>E. faecalis</i>. The substitution of the Glu three residues amino-terminal to the lipoyl lysine with Gln (underlined residues) is a common motif in the BCDH E2.</p

Lpa expression at 20 and 37°C.

<p>(A) Growth assay of <i>E. coli</i> cells expressing lipoamidase at 20°C (open triangles) and 37°C (closed triangles) relative to cells containing vector alone at 20°C (open circles) and 37°C (closed circles). The OD₆₀₀ of cultures was measured at the time of induction with IPTG and then 2, 4, 6, and 10 hours post-induction. Error bars represent the standard deviation of three replicates. (B) Anti-His western blot of cells expressing Lpa at 20°C and 37°C. Samples were taken from cultures at the time of induction and at 2, 4, 6, and 10 hours post-induction.</p

Effect of protein expression on biotinylation.

<p>(A) Affinity blot analysis of biotinylation in <i>E. coli</i> BL21 cells expressing Lpa and Lpa S259A. Samples were normalized by OD₆₀₀ and the protein extracts from whole cell lysate were separated by SDS-PAGE and blotted onto nitrocellulose. The biotinylation status of the BCCP, the sole biotinylated protein in <i>E. coli</i>, was analyzed by streptavidin-HRP western blot. (B) Densitometry analysis of the biotinylation level in cells expressing Lpa and Lpa S259A. Streptavidin-HRP signal was normalized to the HSP70 loading control signal for each sample. Within each blot, the fraction of biotinylation relative to cells expressing vector alone was determined. Error bars represent the SEM for fraction of biotinylation for three independent western blots. (C) Affinity blot analysis of biotinylation in <i>E. coli</i> BL21 cells expressing Lpa and the unrelated proteins MBP-MCAT and MBP-KASII. Samples were analyzed by the methods described in part A of this figure.</p

Growth, lipoylation, and biotinylation of cells expressing Lpa and Lpa active site mutants.

<p>(A) Growth assay of <i>E. coli</i> BL21 cells expressing Lpa (open triangles) compared to vector alone (open circles) and the three active site mutants: Lpa K159A (open squares), Lpa S235A (closed circles), and Lpa S259A (closed triangles). The OD₆₀₀ of cultures was measured at the time of induction with IPTG and at 2, 4, 6, and 10 hours post-induction. Error bars represent the standard deviation of three replicates from a representative growth assay. (B) Anti-lipoic acid and streptavidin affinity blots to determine levels of PDH and KDH lipoylation and BCCP biotinylation in <i>E. coli</i> expressing empty vector, Lpa, and Lpa active site mutants. (C) Densitometry analysis of PDH and KDH lipoylation in cells expressing Lpa and Lpa active site mutants. To determine the level of KDH and PDH lipoylation, the anti-lipoic acid signal was normalized to the anti-HSP70 loading control signal. The fraction of lipoylation in cells expressing Lpa or the Lpa active site mutants relative to cells expressing vector alone was then determined. Error bars represent the SEM for the fraction of lipoylation for three independent western blots. (D) Autoradiograph of KER176 cells expressing vector alone, the Lpa active site mutants, and MBP-MCAT grown in minimal medium supplemented with ³⁵S-lipoic acid. After a 6 hour induction at 20°C, cell samples were normalized by OD₆₀₀ and protein extracts were separated by SDS-PAGE and analyzed by autoradiography. Equal sample loading was assessed by anti-HSP70 western blot.</p

Primer sequences.

<p>Primer sequences.</p

Growth and lipoylation of Lpa constructs containing only the amidase domain.

<p>(A) Growth assay of <i>E. coli</i> cells expressing Lpa_t471 (closed triangles) and Lpa_t521 (open squares) compared to expression of Lpa (open triangles) and vector alone (open circles). The OD₆₀₀ of cultures was measured at the time of induction with IPTG and at 2, 4, 6, and 10 hours post-induction. Error bars represent the standard deviation of three replicates from a representative growth assay. (B) Anti-lipoic acid and streptavidin affinity blots to determine levels of lipoylation and biotinylation in <i>E. coli</i> expressing empty vector, Lpa, and Lpa truncation mutants. (C) Densitometry analysis of PDH and KDH lipoylation in cells expressing Lpa and Lpa truncation mutants. The fraction of lipoylation in cells expressing Lpa or the Lpa active site mutants relative to cells expressing vector alone was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007392#pone-0007392-g004" target="_blank">Figure 4C</a>. Error bars represent the SEM for the fraction of lipoylation for three independent western blots. (D) Solubility of Lpa and Lpa truncation mutants expressed at 20°C and 37°C. After induction of protein expression, cells were grown at 20°C for 10 hours or 37°C for four hours. Lpa in the insoluble (I) and soluble (S) fractions was analyzed by anti-His western blot.</p

FeS cluster biogenesis pathways in eukaryotic pathogens.

<p>Eukaryotic pathogens contain an ISC (<u>I</u>ron-<u>S</u>ulfur <u>C</u>luster formation) pathway in the mitochondrion or mitochondrion-like organelle (MLO), a CIA (<u>C</u>ytosolic <u>I</u>ron-sulfur protein <u>A</u>ssembly) pathway in the cytosol, and plastid-containing organisms such as <i>Plasmodium</i> have the SUF (<u>SU</u>l<u>F</u>ur mobilization) pathway in this organelle. FeS cluster biogenesis is absolutely essential in mitochondria and MLOs, and is likely the driving force for retention of these organelles. <i>Entamoeba</i> is the exception that proves the rule: it has dispensed with the ISC machinery, but replaced it with a bacterial NIF (<u>NI</u>trogen <u>F</u>ixation) pathway acquired by lateral gene transfer that appears to fulfill the requirement for FeS cluster synthesis in the mitosome.</p

The Suf Iron-Sulfur Cluster Synthesis Pathway Is Required for Apicoplast Maintenance in Malaria Parasites

<div><p>The apicoplast organelle of the malaria parasite <i>Plasmodium falciparum</i> contains metabolic pathways critical for liver-stage and blood-stage development. During the blood stages, parasites lacking an apicoplast can grow in the presence of isopentenyl pyrophosphate (IPP), demonstrating that isoprenoids are the only metabolites produced in the apicoplast which are needed outside of the organelle. Two of the isoprenoid biosynthesis enzymes are predicted to rely on iron-sulfur (FeS) cluster cofactors, however, little is known about FeS cluster synthesis in the parasite or the roles that FeS cluster proteins play in parasite biology. We investigated two putative FeS cluster synthesis pathways (Isc and Suf) focusing on the initial step of sulfur acquisition. In other eukaryotes, these proteins can be located in multiple subcellular compartments, raising the possibility of cross-talk between the pathways or redundant functions. In <i>P. falciparum</i>, SufS and its partner SufE were found exclusively the apicoplast and SufS was shown to have cysteine desulfurase activity in a complementation assay. IscS and its effector Isd11 were solely mitochondrial, suggesting that the Isc pathway cannot contribute to apicoplast FeS cluster synthesis. The Suf pathway was disrupted with a dominant negative mutant resulting in parasites that were only viable when supplemented with IPP. These parasites lacked the apicoplast organelle and its organellar genome – a phenotype not observed when isoprenoid biosynthesis was specifically inhibited with fosmidomycin. Taken together, these results demonstrate that the Suf pathway is essential for parasite survival and has a fundamental role in maintaining the apicoplast organelle in addition to any role in isoprenoid biosynthesis.</p></div