Alternative fates of the acyl aldehyde intermediate produced by Aar.

<p>Acyl-ACP reductase (Aar) converts the end-product of fatty acid biosynthesis, acyl-ACP, into an acyl aldehyde. This intermediate can be converted into alkanes or free fatty acids by the endogenously encoded aldehdye decarbonylase (AD) and AldE enzymes, respectively; or into wax esters by co-expression of alcohol dehydrogenase and wax ester synthase (WS) transgenes. Previously described pathways in <i>S. elongatus</i> are in black; pathways described in this paper are in gray.</p

orf0489 is required for Aar-induced FFA production and to mitigate Aar toxicity.

<p><b>A.</b> Growth curve of the indicated strains (log OD₇₅₀ vs time). For clarity, only cultures induced with IPTG are shown. There was no significant difference in growth rate in any of the uninduced strains compared to WT. Values are the average +/− SD of three independent experiments. <b>B.</b> Colony forming units per OD₇₅₀ (CFU/OD₇₅₀) of the indicated strains were determined 48 h post-induction. WT is the average of five independent experiments; the remaining strains are the average of three independent experiments. Error bars are the standard deviation. <b>C.</b> 0.5 OD₇₅₀-equivalents from samples collected 24 and 48 hours post-induction were resolved by TLC using polar solvents (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058307#s4" target="_blank">Materials and Methods</a>). PA: palmitic acid. <b>D.</b> Total FAMES 24 and 48 h post-induction of the indicated strains. <b>E.</b> In a separate experiment from A-D, samples from biological triplicates were collected 24 h post-induction, and 1 OD₇₅₀-equivalents were resolved on TLC using non-polar solvents (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058307#s4" target="_blank">Materials and Methods</a>). A C16∶0 fatty aldehyde standard (i.e. hexadecanal) was included between lanes and on the flanking lanes.</p

Overexpression of Aar (encoded by orf1594) leads to increased free fatty acid production in <i>S. elongatus</i> PCC 7942.

<p><b>A.</b> qPCR analysis of <i>aar</i> expression levels. WT and IPTG-inducible Aar strains were grown as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058307#s4" target="_blank">materials and methods</a>, and induced with 1 mM IPTG at t = 0. “I” indicates induction; “U” indicates uninduced. Total cellular RNA was obtained from all samples at 24 hour post-induction. RNA was converted to cDNA and relative <i>aar</i> transcript levels were assessed by quantitative PCR. RNA input for each sample was normalized to <i>rnpB</i>, and <i>aar</i> expression is presented as the fold change in expression compared to endogenous <i>aar</i> from a WT strain. The data is the average of 6 independent experiments; error bars are the standard deviation. <b>B.</b> Growth curves of the indicated strains (log OD₇₅₀ vs. time). The WT strain is the average of 6 independent experiments; Aar_U and Aar_I are the average of 10 independent experiments. Error bars are the standard deviations. <b>C.</b> 0.5 OD₇₅₀-equivalents from samples collected at 24, 48, 72 and 120 hours post-induction were separated by thin layer chromatography (TLC) using polar solvent for the mobile phase. 10 µg of hexadecanol (HD) and 5 µg of palmitic acid (PA; C16∶0 free fatty acid) were included as standards. MGDG: monogalactosyldiacylglycerol; DGDG: digalactosyldiacylglycerol; PG: phosphatidylglycerol. <b>D.</b> Total fatty acid methyl esters (FAMES) assessed by GC. Each timepoint is the average of three measurements; error bars are standard deviations. The numbers above the Aar_I bars indicate the fold-increase of total FAMES compared to WT samples. <b>E.</b> Individual constituent FAMES were quantified. The observed increase in total FAMES is due almost entirely to accumulation of C16∶0 free fatty acids (solid black).</p

Kinetic parameters of h₆-AldE from <i>S. elongatus</i> PCC7942¹.

1<p>Results are from three independent experiments and are expressed as average ± standard deviation.</p

Aar-dependent production of wax esters in PCC7942.

<p>Strains containing the WS/DGAT enzyme from <i>A. baylyii</i> (“DGAT”); DGAT and Aar; DGAT, Aar and slr1192 (“triple 1); or DGAT, Aar and ACIAD3612 (“triple 2″) were analyzed 24 hours post-induction by TLC using nonpolar solvents to resolve WE and TAG (<b>A</b>) or polar solvents to resolve fatty acids (<b>B</b>). slr1192 and ACIAD3612 are long-chain aldehyde dehydrogenases from <i>Synechocystis</i> and <i>A. baylyii</i>, respectively. 0.5- OD₇₅₀ equivalents were loaded per well. WE: wax ester; TAG: triacylglycerol; PA: palmitic acid. <b>C.</b> Electron micrographs of the indicated strains 24 hours post-induction. Lipid bodies are present in DGAT-containing samples and appear as white globules.</p

Overexpression of two copies of Aar leads to increased FFA production, but at a growth cost that is alleviated by AldE overexpression.

<p>Strains were constructed containing a single copy of Aar in neutral site 1 (NS1) or neutral site 2 (NS2); two copies of Aar (2X); or two copies of Aar and an additional copy of AldE (“Aar(2X)/AldE”). <b>A.</b> Growth curve of the indicated strains. For clarity only the Aar (2X) and Aar(2X)/AldE strains are presented; Aar(NS1) and Aar(NS2) strains were processed in parallel for FAMES analysis. <b>B.</b> Transcript levels of <i>aar</i> and <i>aldE</i> were assessed by qPCR from samples collected 24 hours post-induction and are presented as the fold increase relative to endogenous expression of each gene from a WT strain. The data are the average of three experiments +/− SD. <b>C.</b> Total FAMES of the indicated strains.</p

Siderocalin/Lcn2/NGAL/24p3 Does Not Drive Apoptosis Through Gentisic Acid Mediated Iron Withdrawal in Hematopoietic Cell Lines

<div><p>Siderocalin (also lipocalin 2, NGAL or 24p3) binds iron as complexes with specific siderophores, which are low molecular weight, ferric ion-specific chelators. In innate immunity, siderocalin slows the growth of infecting bacteria by sequestering bacterial ferric siderophores. Siderocalin also binds simple catechols, which can serve as siderophores in the damaged urinary tract. Siderocalin has also been proposed to alter cellular iron trafficking, for instance, driving apoptosis through iron efflux via BOCT. An endogenous siderophore composed of gentisic acid (2,5-dihydroxybenzoic acid) substituents was proposed to mediate cellular efflux. However, binding studies reported herein contradict the proposal that gentisic acid forms high-affinity ternary complexes with siderocalin and iron, or that gentisic acid can serve as an endogenous siderophore at neutral pH. We also demonstrate that siderocalin does not induce cellular iron efflux or stimulate apoptosis, questioning the role siderocalin plays in modulating iron metabolism.</p> </div

Stably-induced expression of Scn does not drive apoptosis in FL5.12 cells.

<p>(A) FL5.12 cells were transduced with the pCVL-SFFV-muScn-IRES-GFP lentivirus and GFP mean fluorescence intensity was determined one-week post-transduction by cytometry, confirming transgene functionality. (B) A Western blot of supernatants, concentrated from 32 µL, from FL5.12 cells shows that the transduced cells constitutively express Scn, while parental cells in the presence or absence of IL-3 do not secrete detectable amounts of Scn after 72 h in culture. (C) Transduced FL5.12 were incubated with a variety of siderophores in order to assess the role of exogenous siderophores on cell viability (NT: no treatment). The hexadentate chelators DFO and Ent at 100 µM produce robust apoptosis, while the bidentate chelators at 300 µM do not affect viability.</p

Scn has no effect on iron release or iron uptake from HeLa cells.

<p>Control HeLa/X7 (transfected with empty vector) or HeLa/24p3R-L cells were labeled with either (A) 2.5 µM ⁵⁹FeCl₃ or (B) 0.75 µM ⁵⁹FeTf and re-incubated with 2 µM murine Scn or control medium for 5 h (dotted columns) or 24 h (checked columns); 100 µM DFO was used as a positive control. Expression of <i>BOCT</i> in transfected HeLa/24p3R-L cells was confirmed by RT-PCR (C). In (C), a typical result from three experiments is shown. In (D), control HeLa/X7 (white columns) and HeLa/24p3R-L cells (black columns) were incubated for 4 h in the presence of 2 µM ⁵⁹FeCl₃, 2 µM ⁵⁹FeEnt, 2 µM murine Scn with bound ⁵⁹FeEnt (⁵⁹FeEnt+Scn) or in the presence of 2 µM ⁵⁹FeEnt plus 2 µM human albumin (⁵⁹FeEnt+Alb). Internalized ⁵⁹Fe was determined by γ-counting. Albumin was added in (D) as an additional control for non-specific binding. Error was calculated as the standard deviation among three experiments.</p

Added Scn does not affect the expression of iron responsive genes.

<p>Expression of <i>H-ferritin</i> (<i>FTH-1</i>) and <i>NDRG1</i> in HeLa/X7 and HeLa/24p3R-L cells was assayed by RT-PCR (A) and Western blot (B). Cells were untreated or treated with 2 µM murine Scn or DFO (100 µM or 250 µM) for 24 h. Densitometry results (right) were calculated relative to β-actin; error was calculated from the standard deviation among three experiments; a typical result from three experiments is shown in (A) and (B).</p