Synthesis and Scavenging Role of Furan Fatty Acids

Synthesis and scavenging role of furan fatty acids

Rachelle A. S. Lemkea, Amelia C. Petersonb, Eva C. Ziegelhoffera, Michael S. Westphallc, Henrik Tjellströmd,e, Joshua J. Coonb,d,f, and Timothy J. Donohuea,d,1

Departments of aBacteriology, bChemistry, and fBiomolecular Chemistry, University of Wisconsin–Madison, Madison, WI 53706; dGreat Lakes Bioenergy Research Center, Madison, WI 53726; cGenome Center of Wisconsin, Madison, WI 53706; and eDepartment of Plant Biology, Michigan State University, East Lansing, MI 48824

Edited by Carol A. Gross, University of California, San Francisco, CA, and approved May 5, 2014 (received for review March 31, 2014) Fatty acids play important functional and protective roles in living Despite the proposed roles of Fu-FAs, little is known about systems. This paper reports on the synthesis of a previously how they are synthesized (13). We report on proteins needed for unidentified 19 carbon furan-containing fatty acid, 10,13-epoxy- the conversion of cis unsaturated fatty acids to 19Fu-FA. We 1 11-methyl-octadecadienoate (9-(3-methyl-5-pentylfuran-2-yl)nonanoic show that a O2-inducible protein (RSP2144) is an S-adenosyl me- acid) (19Fu-FA), in phospholipids from Rhodobacter sphaeroides. thionine (SAM)-dependent methylase that synthesizes a 19-carbon We show that 19Fu-FA accumulation is increased in cells contain- methylated trans unsaturated fatty acid (19M-UFA) from cis ing mutations that increase the transcriptional response of this vaccenic acid both in vivo and in vitro. We also identify gene 1 bacterium to singlet oxygen ( O2), a reactive oxygen species gen- products needed for the O2-dependent conversion of 19M-UFA 1 erated by energy transfer from one or more light-excited donors to 19Fu-FA. Further, we demonstrate that the presence of O2 to molecular oxygen. We identify a previously undescribed class leads to the disappearance of 19Fu-FA in vivo. Based on our of S-adenosylmethionine-dependent methylases that convert findings, we propose a pathway for Fu-FA synthesis and propose a phospholipid 18 carbon cis unsaturated fatty acyl chain to a 1 a protective role for compounds in the presence of a ROS like O2. 19 carbon methylated trans unsaturated fatty acyl chain (19M-

UFA). We also identify genes required for the O2-dependent Results conversion of this 19M-UFA to 19Fu-FA. Finally, we show that Increased σE Activity Alters Cellular Fatty-Acid Composition. Fatty 1 . thepresenceof O2 leads to turnover of 19Fu-Fa in vivo We acids are targets for direct or indirect damage by ROS (1, 5–8, propose that furan-containing fatty acids like 19Fu-FA can act as 16), particularly when ROS are produced by integral membrane 1 a membrane-bound scavenger of O2, which is naturally produced enzymes in the respiratory chain or the photosynthetic apparatus by integral membrane enzymes of the R. sphaeroides photo- (1, 7, 8, 16, 18). The R. sphaeroides σE protein activates a tran- synthetic apparatus. 1 scriptional stress response to O2, a ROS that is generated by integral membrane proteins of the photosynthetic apparatus (16, radical scavenger | oxygenated fatty acid | fatty acyl methylase 17, 19). At least one ORF, which is a known member of the σE regulon, RSP2144, encodes a protein with amino acid simi- atty acids have crucial, yet diverse, roles in biology. In cells larity to an enzyme predicted to modify fatty acids (16, 17, 19– Fand organelles, fatty acids maintain bilayer stability, provide 21). To test for σE-dependent alterations in fatty acid composition, a permeability barrier, act as secondary messengers in signaling we prepared fatty acid methyl esters (FAMEs) to compare the pathways, and aid the function of integral membrane proteins fatty acid content of wild-type cells and mutant cells (ΔChrR; see (1–3). Fatty acids also help maintain viability in response to Table 1 for strain designations), which have high σE activity when temperature and environmental changes and can be targets for grown aerobically in the absence of light because the antisigma modification by reactive oxygen species or membrane-active – agents (2 8). Fatty acids, or the products derived from them, are Significance valuable as food additives, specialty chemicals, and petroleum substitutes (9–12). Thus, there is considerable interest in un- Fatty acids comprise a large class of compounds that serve derstanding the suite of fatty acids that can be made by native or broad roles in cells and society. These hydrophobic compounds engineered pathways. We are studying the synthesis and role of provide integrity for biological membranes, make them im- fatty acids during stress responses. permeable to solutes and toxins, and modulate the cellular Here, we demonstrate a previously unreported ability of the response to signals or stresses. Fatty acids, or the products photosynthetic bacterium Rhodobacter sphaeroides to produce derived from them, are also important as dietary supplements, furan-containing fatty acids (Fu-FAs), an important, yet poorly lubricants, specialty chemicals, and fuels. Their potential utility understood, class of compounds. The presence of Fu-FAs has in biology or industry could be increased by producing novel been reported previously in plants, fish, and some bacteria (13). classes of fatty acids. This paper reports on the occurrence and Based on their chemical properties, it is proposed that Fu-FAs synthesis of a newly discovered class of furan-containing fatty could provide bilayer protection against radicals or organic per- acid. It also provides evidence that furan-containing fatty acids oxides that reduce membrane function (13–15). The oxygen atom scavenge toxic reactive oxygen species, suggesting a pre- within Fu-FAs also provides a functional group for modifications viously unnoticed role for this class of compounds in bacteria that could increase their industrial value (13). and other cells. We discovered the 19-carbon furan-containing fatty acid 10,13-

epoxy-11-methyl-octadecadienoate (9-(3-methyl-5-pentylfuran- Author contributions: R.A.S.L., A.C.P., E.C.Z., H.T., J.J.C., and T.J.D. designed research; 2-yl)nonanoic acid) (19Fu-FA) in phospholipids isolated from R.A.S.L., A.C.P., E.C.Z., M.S.W., and H.T. performed research; A.C.P., E.C.Z., M.S.W., H.T., and an R. sphaeroides mutant lacking an antisigma factor, ChrR, J.J.C. contributed new reagents/analytic tools; R.A.S.L., A.C.P., M.S.W., H.T., J.J.C., and T.J.D. that has increased transcription of genes that are normally acti- analyzed data; and R.A.S.L., A.C.P., E.C.Z., M.S.W., H.T., J.J.C., and T.J.D. wrote the paper. vated in the presence of the reactive oxygen species (ROS) The authors declare no conflict of interest. 1 1 singlet oxygen ( O2). In this and other phototrophs, O2 is This article is a PNAS Direct Submission. a byproduct of light energy capture in integral membrane com- Freely available online through the PNAS open access option. plexes of the photosynthetic apparatus (5, 16, 17). Consequently, 1To whom correspondence should be addressed. Email: [email protected]. fatty acids or other membrane components are likely targets for This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1 damage by O2 (16, 17). 1073/pnas.1405520111/-/DCSupplemental.

E3450–E3457 | PNAS | Published online August 4, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1405520111 Downloaded by guest on September 23, 2021 Table 1. Strains and plasmids PNAS PLUS Strains/plasmids Relevant genotype Source

Strains E. coli DH5α supE44 lacu169(Ф80 lacZ M15) hsdR178 recA1 endA1 gyrA96 thi-1 relA-1 (52) S17-1 C600::RP-4 2-(Tc::Mu) (Kn::Tn7) thi pro hsdR Hsd M+recA (53) BL21(DE3) F− ompT hsdSB (rB- mB-) gal dcm (DE3) Novagen JW1653 cfa::kan of BW25113 Keio Collection (48) RLcfaK49-6 cfa markerless deletion mutant of JW1653 This study R. sphaeroides 2.4.1 Wild type (36) TF18 rpoE::drf (23) ΔChrR chrR::drf (54) ΔRSP2144 RSP2144::Ω SmrSpr (20) RSL1 ΔchrR RSP2144::Ω SmrSpr This study 1091:spR/ΔChrR ΩSpR insertion in RSP1091 coding sequence in ΔChrR This study Delta ΔRSP1091/ ΔChrR In-frame deletion of both RSP1091 and ChrR This study Plasmids pBlueScriptII KS- Apr Agilent Technologies pRS2144 RSP2144 in pBSII (20) r pET-28a+ His6 expression vector, Kn Novagen pRLhisRSP2144 1.2-kb RSP2144 fragment from pRS44 cloned into NdeI/EcoRI-cut pET-28a This study pIND5 pIND4 NcoI site replaced with NdeI site, Knr (20) pRL101 1.3-kb fragment amplified from pRLhisRSP2144 cloned into NdeI/HindIII pIND5 This study pAYW19 E. coli cfa gene on pGEM5, Apr (49) MICROBIOLOGY factor ChrR that normally inhibits σE function has been inacti- mulates in cells with increased σE activity, shows that it has an vated (19, 20, 22, 23). intact molecular ion mass of 322.2502 Da, corresponding to a In wild-type cells, we found the expected major FAME prod- molecular formula of C20H34O3 (Fig. 2A). The fragmentation pat- ucts (C18:1, C18:0, C16:1, C16:0) (Table 2), based on published tern has good correlation with a methyl ester of a 19-carbon furan- fatty-acid analysis of R. sphaeroides (24–27). In ΔChrR cells, we containing fatty acid, 10,13-epoxy-11-methyl-octadecadienoate observed the accumulation of two additional FAME products (9-(3-methyl-5-pentylfuran-2-yl)nonanoic acid), as seen by the (retention times of ∼16.4 and 17.5 min in Fig. 1) and lower levels comparison with the reference spectrum in Fig. 2A [spectrum of the vaccenic acid (C18:1) FAME compared with wild-type M11703; American Oil Chemists’ Society (AOCS) Lipid Library]. cells (Table 2). Thus, we conclude that increased σE activity alters This compound is hereafter referred to as 19Fu-FA. the cellular fatty acid composition. However, neither of the two The other unidentified FAME (retention time ∼16.4 min in E additional FAME products in cells containing increased σE ac- Fig. 1), which also accumulates in cells with increased σ activity, tivity elutes with compounds in bacterial fatty acid standard mix- has an intact molecular ion mass of 310.2866 Da, corresponding B tures so we sought to determine their identity. to an elemental composition of C20H38O2 (Fig. 2 and Fig. S1). The EI mass spectrum of this FAME did not allow a definitive Identification of Additional FAMEs in Cells with Increased σE Activity. assignment of its identity so additional experiments were nec- The electron ionization (EI) (70 eV) mass spectrum of one un- essary. First, hydrogenation of the FAME led to a shift in re- known FAME (retention time ∼17.5 min in Fig. 1), which accu- tention time and an increase in the intact molecular ion mass by

Table 2. Relative cellular fatty acid content † Strain/condition C16:1 C16:0 C18:1 C18:0 19M-UFA* 19Fu-FA N

WT Aero 5.3 (0.6) 21.1 (3.1) 45.9 (6.7) 25.7 (2.9) 1.3 (0.2) 0.6 (0.2) 3 WT Photo 5.1 (0.1) 18.6 (0.1) 48.3 (1.3) 26.3 (0.7) 1.7 (0.1) ND 2 ΔChrR Aero 5.7 (0.3) 23.3 (1.2) 40.0 (2.3) 26.4 (1.1) 2.5 (0.1) 2.3 (0.2) 3 ΔChrR Photo 5.4 (0.3) 21.4 (2.0) 42.8 (4.6) 25.9 (3.4) 4.6 (0.7) ND 3 ΔUfaM Aero 5.0 (0.2) 21.4 (1.3) 47.4 (2.4) 26.0 (1.2) ND ND 3 ΔUfaM Photo 4.2 (0.4) 18.1 (5.1) 51.8 (11.9) 25.5 (6.4) ND ND 2 ΔChrR/ΔUfaM Aero 5.4 (0.8) 24.5 (3.0) 45.3 (6.3) 24.5 (2.5) ND ND 3 ΔChrR/ΔUfaM Photo 4.5 (0.1) 19.1 (0.5) 49.7 (0.6) 26.4 (1.4) ND ND 3 ‡ 1091:spR/ΔChrR2 Aero 5.0 (0.3) 22.6 (0.3) 40.1 (0.7) 23.1 (0.5) 9.2 (0.6) ND 2 1091:spR/ΔChrR2 Photo 5.2 (0.4) 19.9 (0.6) 43.5 (2.6) 24.6 (1.6) 6.8 (0.1) ND 3 Δ1091/ΔChrR2 Aero§ 3.9 (1.2) 21.5 (1.4) 43.4 (3.2) 22.5 (0.4) 8.7 (1.7) ND 3 Δ1091/ΔChrR2 Photo 5.1 (0.2) 21.6 (1.4) 39.5 (2.4) 26.8 (0.4) 7.1 (0.5) ND 3

Percentage of the total fatty acid, with standard deviation in parentheses. Not determined (ND), <0.5% of the total FAME; N,numberofbiologicalreplicates. *19M-UFA is 11-methyl-octadecenoate (n-6). † 19Fu-FA is 10,13-epoxy-11-methyl-octadecadienoate (9-(3-methyl-5-pentylfuran-2-yl)nonanoic acid). ‡ 1091:spR cells contain a polar insertion of a spectinomycin-resistance gene in RSP1091. §Δ1091 cells contain an in-frame deletion in RSP1091.

Lemke et al. PNAS | Published online August 4, 2014 | E3451 Downloaded by guest on September 23, 2021 Int. 2.0E5 acyl methylase (UfaM) activity alters the isomeric state of the fatty A C18:1 acyl molecule as is reported for SAM-dependent methylases involved in mycolic acid biosynthesis (29, 30). To validate the assigned identity of these two FAMEs, we compared the behavior of synthetic standards of 19M-UFA and 19Fu-FA to those present in ΔChrR cells. We found that the C18:0 fragmentation patterns of the synthetic 19M-UFA and 19Fu-FA were indistinguishable from the native 19M-UFA and 19Fu-FA B FAMEs present in ΔChrR cells (Fig. S3). In addition, by using the synthetic FAMEs as quantitative standards, we estimated the relative cellular abundance of the 19M-UFA and 19Fu-FA. In aerobically grown wild-type cells, we found little of either the 19M-UFA 19M-UFA or the 19Fu-FA (Fig. 1 and Table 2), presumably 19Fu-FA because these cells have low σE activity (19). In contrast, in aerobically grown ΔChrR cells (which contain high σE activity), ∼2.5% and ∼2.3% of the total FAME products are 19M-UFA and C 19Fu-FA, respectively. In these cells, there is decreased abundance of vaccenic acid (C18:1) (Fig. 1 and Table 2), suggesting that both of these previously undescribed fatty acids were derived from vaccenic acid.

RSP2144 Is a SAM-Dependent Fatty Acyl Methylase. The accumulation of 19M-UFA and 19Fu-FA and the reduction in vaccenic acid in ΔChrR cells could reflect the use of a monounsaturated D fatty acyl chain as a substrate for synthesis of one or both of these products. RSP2144 is annotated as a SAM-dependent fatty acyl- modifying enzyme with significant amino acid similarity to bacterial cyclopropane fatty acid synthase (16, 19, 21). However, RSP2144 does not appear to catalyze this reaction because ΔChrR cells, which have increased RSP2144 expression (19, 21), 16.2 16.5 16.8 17.1 17.4 do not contain detectable levels of a C19 cyclopropane FAME (Fig. 1 and Table 2). time [min] Thus, we considered the possibility that RSP2144 is a pre- Fig. 1. Alterations in the fatty-acid profile of cells with increased σE activity viously uncharacterized SAM-dependent unsaturated fatty acyl (ΔChrR cells). Gas chromatograms of FAMEs are shown, and those FAMEs methylase (UfaM). To test this hypothesis, we asked whether known to be present in wild-type R. sphaeroides (C18:0 and C18:1) are in- purified recombinant His6-tagged RSP2144 was able to methyl- dicated, as well as two additional FAMEs (19M-UFA and 19Fu-FA) that are ac- ate fatty acids. We found that purified His6-RSP2144 catalyzed cumulated in ΔChrR cells. (A–D) The gas chromatograms of FAMEs from wild- transfer of a 3H-methyl group from methyl-labeled SAM into type (A), ΔChrR (B), ΔRSP2144 (C), and ΔRSP2144 cells in which the RSP2144 β tricholoracetic acid (TCA)-precipitated material when incubated gene is ectopically expressed from an isopropyl -D-1-thiogalactopyranoside R. (IPTG)-inducible plasmid (D), respectively. The y and x axes show the relative with a phospholipid substrate mixture isolated from an abundance and retention time for each species, respectively. sphaeroides ΔRSP2144 mutant. The activity of the recombinant −1 −1 RSP2144 enzyme (Vmax ∼ 331 pmol·min ·mg ) and its apparent affinity for phospholipid substrate (Km ∼ 308 μM) (Fig. 3A) were 2 Da (312.3023 Da, C20H40O2). The increase in the mass of this comparable with other SAM-dependent fatty acyl-modifying FAME after hydrogenation indicates that the untreated mole- enzymes (31, 32). When the FAME products of this in vitro cule is unsaturated (Fig. S1A). The EI mass spectrum of the reaction were analyzed by GC-MS, we observed the accumu- 312-Da hydrogenated unknown contained diagnostic a and b lation of a product with a retention time and fragmentation fragment ions that localized a methyl branch at position 11 pattern identical to the 19M-UFA [methyl 11-methyl-C18:1 on the hydrogenated molecule, and by extension on the non- (n-6)], which accumulates in ΔChrR cells (Fig. 3B). hydrogenated unknown. This spectrum correlates well with the We also found that ectopic expression of His6-RSP2144 in reference spectrum of methyl 11-methyl-octadecanoate [spec- either R. sphaeroides or an Escherichia coli cfa mutant leads to trum 112141; National Institute of Standards and Technology accumulation of 19M-UFA (Fig. 4C). Unlike R. sphaeroides, (NIST) Library] (Fig. S1B). To then localize the position of the E. coli contains significant amounts of C16:1 (n-7) fatty acyl chains double bond in the acyl chain of the 310-Da unsaturated, faster- in its phospholipids (2, 3) so preferential accumulation of 19M- migrating unknown, we used a soft ionization technique (28), UFA and the absence of a detectable methyl C17 FAME in acetonitrile (ACN) positive chemical ionization (PCI), with this host could indicate that RSP2144 has some selectivity for subsequent isolation and MS/MS of a chemical ionization- methylation of vaccenic acid. However, in E. coli, there is a bias derived molecular ion adduct {[M + 1-methyleneimino-1-etheny- for having a C16:1 chain at position 2 of phospholipids (33) so + lium (MIE)] } of the (nonhydrogenated) unknown FAME. The the lack of accumulation of a 17-carbon M-UFA in this bacte- ACN PCI MS/MS fragmentation pattern of this compound con- rium could also reflect a preference for UfaM to methylate acyl tains diagnostic fragment ions, α and ω, that localize the double chains at the 1 position. As a control, we found that ectopic bond in the acyl chain to position 12 and thus identify this un- expression of E. coli cfa in its native host led to accumulation of known FAME as methyl 11-methyl-C18:1 (n-6) (Fig. 2B), here- C17 and C19 cyclopropane FAMEs (Fig. 4B), as expected given after referred to as 19M-UFA. Finally, we showed that 19M-UFA the reported function of this enzyme (31, 32). Thus, we con- has a trans configuration around the double bond (Fig. S2). clude that His6-RSP2144 is a previously uncharacterized SAM- 19M-UFA is derived from cis-vaccenic acid so unsaturated fatty dependent UFA methylase, which we hereafter call UfaM.

E3452 | www.pnas.org/cgi/doi/10.1073/pnas.1405520111 Lemke et al. Downloaded by guest on September 23, 2021 RSP1091 Is Needed for Accumulation of 19Fu-FA. Ectopic expression PNAS PLUS 291 179 279 A of His6-RSP2144 in ΔRSP2144 cells resulted in accumulation of 19M-UFA (Fig. 1D). However, both 19M-UFA and 19Fu-FA were accumulated in ΔChrR cells (Fig. 1B and Table 2), which have increased expression of RSP2144 and other proteins in the 165 109 265 σE regulon (21). One interpretation of these data is that another σE σE C H O Int. 9.55E8 -dependent gene is needed to synthesize 19Fu-FA. Other 11 17 target genes in the putative RSP1091-1087 operon have amino acid sequence similarity to fatty acid-modifying enzymes (16, 19, 21). Thus, we asked whether any of these proteins had a previously unrecognized role in fatty-acid modification. C H O To test this hypothesis, we analyzed the FAME content of 19 31 2 322 aerobic cells that lacked ChrR and RSP1091 (20). For this +• C7H9O C16H25O3 [M] analysis, we used cells containing either an in-frame deletion C H O C H O in the RSP1091 coding sequence or ones that contained an in- C6H7O 8 9 12 19 C H O C H O 17 27 3 20 34 3 sertion in RSP1091 that might have a polar (i.e., negative) effect on expression of the downstream genes RSP1090–1087 (20, 21). 95 179 291 Fatty-acid analysis of either of the ChrR/RSP1091 double mutants 121 265 showed that they lacked detectable levels of 19Fu-FA present in 109 279 322 the ΔChrR mutant (Table 2). However, both the ChrR/RSP1091 double mutants tested contained 19M-UFA that is present in a ΔChrR mutant. Thus, we conclude that RSP1091 is needed for synthesis of 19Fu-FA although we cannot determine from this experiment whether other genes in the putative RSP1091-1087 operon are also involved in the conversion of 19M-UFA to AOCS Lipid Library 19Fu-FA (Discussion). 165 Spectrum M11703 Based on only these data, it was deemed possible that the

RSP1091 protein either directly converts vaccenic acid to 19Fu-FA MICROBIOLOGY 10075 150 200 250 300 350 or, alternatively, that 19M-UFA produced by the RSP2144 protein m/z couldbeanintermediateinaRSP1091-dependent pathway for Fu-FA synthesis. To distinguish between these possibilities, we α compared the FAME content of cells lacking both ChrR and B (m/z 308) = C16H29O2 + C3H4N + H RSP2144 with cells lacking only ChrR. Analysis of FAMEs from the ChrR/RSP2144 double mutant showed that it lacked both 266194 the 19M-UFA and 19Fu-FA that are accumulated in ΔChrR ω (m/z 180) = C H + C H N + H cells (Table 2). Thus, we concluded that 19M-UFA, as a product 9 17 3 4 of RSP2144 activity, is needed to produce 19Fu-FA. In addition, ACN PCI MS [M-MeOH]+ Int. 2.34E8 we concluded that the RSP1091 protein, either alone or in E C H O conjunction with another σ target gene, is needed to convert 19 39 19M-UFA into 19Fu-FA. [M+H]+

C20H39O2 O2 Is Needed for Accumulation of 19Fu-FA. O2 is one potential source of the oxygen moiety in Fu-FAs (13), but experimental + [M-MeOH-H2O] evidence in support of this notion is lacking. To test whether + O was needed for accumulation of this bacterial 19Fu-FA, we C19H33 [M+MIE] 2 compared the FAME profile of cells containing increased σE C H O N 23 42 2 activity (ΔChrR cells) that were grown aerobically (30% O2 in the dark) or anaerobically (in the light by photosynthesis). ACN PCI Int. 3.00E6 Analysis of the FAME profile showed that 19Fu-FA is detected MS/MS 25 eV only when cells were grown in the presence of O2. In addition, we found that 19M-UFA is accumulated when this strain is grown + 266 [M+MIE-MeOH] either in the presence or absence of O2, suggesting that RSP2144 ω (m/z 180) C H ON activity does not require O2 (Table 2). We conclude that O2 acts C H O N 22 38 16 28 2 as a source of oxygen in this bacterial 19Fu-FA. C12H22N α (m/z 308) 194 1O Causes Turnover of 19Fu-FA. The above experiments showed C19H34O2N 2 accumulation of 19M-UFA and 19Fu-FA in ΔChrR cells that C13H24N have increased σE activity. We wanted to determine whether changes in fatty acid content were observed when wild-type cells 100 150 200 250 300 350 400 1 E were exposed to O2, a signal that induces σ activity (16, 19, 20). m/z 1 When we exposed wild-type cells to O2 as a way to increase σE activity (16, 19, 20), there was no detectable accumulation of Fig. 2. (A) Identification of one of the unknown FAMEs (retention time ∼17.5 min in Fig. 1). Electron ionization (EI) spectrum and interpretation of major fragment ions (Upper), and comparison with reference library spectrum for methyl 10,13-epoxy-11-methyl-octadecadienoate (Lower). Library MS spectrum of this molecule (Upper) indicating key ACN PCI adducts of the + spectrum adapted from the AOCS Lipid Library, spectrum number M11703. intact species. MS/MS spectrum of the [M+MIE] ion of this molecule at 25 eV (B) Identification of the other unknown FAME (retention time ∼16.4 min in (Lower), showing key fragments that localize the double bond to position Fig. 1) using acetonitrile (ACN) positive chemical ionization (PCI). Full-scan 12, as indicated in the diagram.

Lemke et al. PNAS | Published online August 4, 2014 | E3453 Downloaded by guest on September 23, 2021 A 300 Discussion This report demonstrates the accumulation of methylated and furan-containing fatty acids in R. sphaeroides and shows that a 200 previously unidentified class of a SAM-dependent methylase H-methyl

3 (RSP2144, UfaM) and hypothetical protein(s) (RSP1091), re- 3 spectively, are needed for their production. Our data indicate that Vmax 333.1 ± 18.8 pmol H-methyl incorporated/ 100 min/mg protein both 19M-UFA and 19Fu-FA are synthesized from unsaturated fatty acids in cellular phospholipids using a previously unchar- incorporated/min] K 308 ± 4 µM phospholipid Rate [pmol m acterized set of enzymes (Fig. 6). In addition, we show that 1 0 formation of the ROS O2 leads to loss of 19Fu-FA, and we 0 0.2 0.4 0.6 0.8 1.0 1.2 propose that this fatty acyl chain acts to scavenge reactive and Concentration phospholipid [mM] 1 potentially damaging products present in the bilayer upon O2 B Int. 7.05E5 Int. 9.13E3 formation. Below, we place these observations in context, provide an explanation for our findings, and propose future ex- 10.03 periments to answer questions posed by these results. Negative Identification of Gene Products Needed to Produce 19M-UFA and 10.16 Int. 1.03E5 ω (m/z 180) 19Fu-FA. We identified 19M-UFA and 19Fu-FA as unknown [M+MIE]+ C12H22N FAMEs present in a mutant strain of the photosynthetic bacte- C H O N 23 42 2 rium R. sphaeroides. This mutant strain constitutively expresses 2 hr stress response genes, such as RSP2144 and RSP1091, shown 1 previously to be required for survival in the presence of O2 (16, Int. 1.45E5 [M+MIE-MeOH]+ 19, 20). We show that RSP2144 is a SAM-dependent methylase C H ON 22 38 that synthesizes M-UFA in R. sphaeroides, both in vitro when α (m/z 308) a recombinant protein is incubated with purified native phos- C H O N 19 34 2 pholipids, and in vivo when heterologously expressed in E. coli. Overnight RSP2144 was previously annotated as a cyclopropane fatty acyl 9.9 10.1 10.3 80 130 180 230 280 330 380 synthase; however, it does not produce detectable levels of cy- time [min] m/z clopropane fatty acids (CFAs) in vivo or in vitro under any conditions tested. Instead, our data indicate that RSP2144 is a Fig. 3. Activity of RSP2144 in vitro. (A) Properties of a recombinant RSP2144 protein when assayed for incorporation of 3H-methyl–labeled SAM into previously undescribed enzyme that produces a 19-carbon methy- tricholoroacetic acid insoluble material using micelles containing native R. lated UFA product: thus, the name UfaM. In addition, UfaM sphaeroides phospholipids as a substrate. (B) FAME products obtained using could have a preference for methylating vaccenic acid (C18:1)

R. sphaeroides lipids in the absence (negative) or presence of His6-RSP2144 because we observed only a C19 methyl product when this protein protein (UfaM) and SAM (2 h and overnight time points). The chromato- graphic response of lipids before and after 2 h or overnight incubation with UfaM in vitro shows an increase in 19M-UFA concentration when incubated + with UfaM (shaded in gray). The spectra (Right) show the ACN PCI [M+MIE] Int. 2.0E5 MS/MS (25 eV) spectra collected at the apex of the 19M-UFA peak in all three A C16:0 C18:1 samples, with key fragment ions labeled. No 19M-UFA was detected in the reactions lacking UfaM (negative). C16:1

19Fu-FA. This result was somewhat surprising because the C18:0 1 C17:0 conditions used to produce O2 are known to be sufficient to increase σE activity (19, 20) so we expected to see accumulation of 19Fu-FA. B cycC17:0 1 O2 can directly oxidize furan moieties and produce fatty acyl radicals from unsaturated fatty acids so it has been proposed that Fu-FAs can act as a scavenger for this and other ROS (14, 15, 34, cycC19:0 35). Thus, the failure to observe alterations of the fatty-acid 1 content when wild-type cells were exposed to O2 could reflect the ability of 19Fu-FA to scavenge this ROS or products of its action on bilayer constituents. To test this hypothesis, we mon- C 1 itored the effect of O2 on the fatty-acid content of ΔChrR cells that accumulate the furan fatty acid because they have increased 19M-UFA σE activity (Table 2). We saw a time-dependent decrease in the 1 abundance of 19Fu-FA after exposing ΔChrR cells to O2 generated by adding methylene blue to aerobically grown cultures in the presence of light (Fig. 5). This decrease in abundance of 19Fu-FA was not observed in a control aerobically grown ΔChrR culture that was exposed to methylene blue in the dark (Fig. 5) or 181716 19 20 21 22 23 when aerobically grown cells were transferred to dark anaerobic time [min] conditions at time 0 (Fig. 5). Thus, we conclude that this ob- Fig. 4. RSP2144 produces 19M-UFA in vivo. (A–C) Chromatograms of FAMEs served decrease in 19Fu-FA abundance required conditions that Δ Δ 1 accumulated in an E. coli Cfa mutant (JW1653) (A), an E. coli Cfa mutant result in O2 formation. One explanation for this observation is containing E. coli cfa on a plasmid (B), and an E. coli ΔCfa mutant containing that 19Fu-FA acts as a scavenger of fatty acyl radicals or other RSP2144 on a plasmid (C). The y and x axes show the relative abundance and 1 compounds that are produced in the presence of O2 (Discussion). retention time for each species, respectively.

E3454 | www.pnas.org/cgi/doi/10.1073/pnas.1405520111 Lemke et al. Downloaded by guest on September 23, 2021 4 – PNAS PLUS 3.4 in photosynthetic bacteria and other microbes (38 40). Pre- 1 3.1 3.1 3.3 3.3 vious studies have shown that O2 kills cells lacking either 3.0 UfaM (RSP2144) or RSP1091 proteins (20, 41). We now know 3 3.0 2.9 3.1 3.0 3.0 3.0 2.8 that both of these strains are unable to make 19Fu-FA. Com- 2.6 2.7 2.6 bined, these observations indicate that synthesis of 19Fu-FA 1 2.3 is required for viability in the presence of O2, possibly be- 2 cause they can also scavenge and minimize cellular damage 1.9 by this ROS. 1.7 1.6

log10 (% Total Fatty Acids) Fatty Total log10 (% Potential Role of 19Fu-FA as a Bacterial Second Messenger. It is not MB+Light MB in Dark Switch to Anaerobic growth surprising that previous analysis of the fatty-acid content of wild-type cells did not detect the presence of 19Fu-FA (24– 1 27). Transcription of the genesneededtosynthesize19Fu-FA 00.51 2 3 4 5 requires high activity of the alternative sigma factor σE,but,in Methylene Blue Exposure [hr] 1 E the absence of O2, σ activity is inhibited because it is bound 1 Fig. 5. Impact of O2 exposure on 19Fu-FA abundance. Time-dependent to an antisigma factor, ChrR (16, 19, 22). We have shown that Δ + 1 changes in the cellular abundance of 19Fu-FA in ChrR cells that are (MB O2 formation leads to 19Fu-FA turnover in dChrR cells, ■ ● 1 Light, ) and are not (MB in dark, ) exposed to O2 with a control switched explaining why one does not observe time-dependent changes to anaerobic growth at time 0 (▲). 1 in levels of 19Fu-FA after exposing wild-type cells to O2. In contrast to the situation in wild-type cells, mutants lacking σE was expressed in E. coli (which contains more C16:1 than C18:1 either UfaM or RSP1091 have defects in increasing tran- fatty acyl chains). Furthermore, the production of the trans scriptional activity (20, 41). Based on this observation and the isomer of 19M-UFA from cis-vaccenic acid predicts that SAM- results of the experiments in this study, we propose that a product of either gene is needed to promote dissociation of a σE–ChrR com- dependent fatty acyl methylation by UfaM uses a reaction 1 mechanism similar to methylases involved in mycolic acid bio- plex (20). For example, the ability of 19Fu-FA to scavenge O2 could lead to accumulation of lipid peroxides that act as a second synthesis (29, 30). σE– This report also demonstrates that another σE target gene, messenger to promote dissociation of the ChrR complex. In RSP1091 this model, the subsequent ChrR proteolysis in the presence of MICROBIOLOGY (16, 19, 21), is needed for conversion of 19M-UFA to 1 19Fu-FA (Fig. 6). We show that this conversion requires growth O2 (20, 41, 42) could be promoted by direct modification of this antisigma factor or by the activation of one or more pro- of cells under aerobic conditions, suggesting that O2 is the source of the oxygen moiety in the furan ring. RSP1091 is annotated as teases by a lipid peroxide. ufaM (RSP2144) and the genes in the RSP1091-1087 operon a protein of unknown function (16, 19, 21), but it is predicted to α γ contain an N-terminal Rossman fold (putative pyridine nucleo- are present across a wide group of - and -proteobacteria (16, tide binding domain), a flavin-binding domain, and to be a fatty acyl-modifying enzyme (36, 37). RSP1091 has yet to be char- acterized, but the presence of flavin and pyridine nucleotide cis-octadec-11-enoic acid cofactors could permit conversion of 19M-UFA into cognate 19Fu-FA in an O2-dependent manner. RSP1090 is also unchar- HO 12 acterized (19, 21, 36) so it is possible that RSP1091, along with RSP1090 and other proteins in the putative RSP1091-1087 op- 11 eron, are needed for conversion of 19M-UFA into 19Fu-FA. Our data show that synthesis of 19Fu-FA requires the ability of cells UfaM Methylation & + to make 19M-UFA because the loss of UfaM prevents synthesis allylic migration of 19Fu-FA. In this regard, it appears that methylation of the SAM UFA creates a tertiary carbon in the acyl chain that is needed for subsequent conversion of 19M-UFA to 19Fu-FA.

Protective Role of 19Fu-FA in Scavenging ROS-Mediated Damage. We HO 1 show that the conditions that lead to formation of O2 also result trans-11-methyl-octadec-12-enoic acid (19M-UFA) in turnover of 19Fu-FA in vivo. Under the conditions we used, ∼50% of 19Fu-FA is removed in one cell doubling (∼3hfor R. sphaeroides). This decrease is probably an underestimate of 1 RSP1091 Furan ring the turnover of this fatty acid in the presence of O2 because + these cells are also capable of synthesizing new 19Fu-FA under formation O2 these conditions. In addition, it is unclear precisely how much 1 O2 is formed inside or outside the cells under the conditions used. Thus, it is possible that the reactivity of 19Fu-FA is underestimated because fatty acyl chains in the inner or outer membrane of this Gram-negative bacterium are in the im- HO 1 mediate vicinity of O2. From the chemical properties of Fu-FAs, it is proposed that 1 they can scavenge lipid peroxides, fatty acyl radicals, or even O2 1 10,13-epoxy-11-methyl-octadecadienoic acid (19Fu-FA) (13–15). The loss of 19Fu-FA when cells generate O2 is, to our knowledge, the first report of their potential role as scav- R. sphaeroides Fig. 6. Proposed pathway for 19Fu-FA synthesis. In this model, UfaM (RSP2144) engers of ROS in bacteria. Wild-type retains is a SAM-dependent methlyase that produces 19M-UFA from vaccenic acid. 1 growth after formation of O2 (19), and carotenoids have typi- In an O2-dependent reaction, RSP1091 alone or in combination with other cally been considered the major route for quenching this ROS gene products converts 19M-UFA to 19Fu-FA (see Discussion).

Lemke et al. PNAS | Published online August 4, 2014 | E3455 Downloaded by guest on September 23, 2021 21). In addition, in these other organisms, homologs of these hexane before analysis by GC-MS. Equal cell numbers, of up to 4 mL of cell genes are often predicted to be transcribed by a homolog of culture, were added to 8 mL of 1:1 vol/vol methanol:chloroform (9) con- μ μ R. sphaeroides σE, suggesting that they are members of a core reg- taining 50 g or 100 g of pentadecanoic acid as a recovery standard ulon that is conserved across the bacterial phylogeny (21). Thus, it (pentadecanoic acid is not detectable in R. sphaeroides fatty acids). The 1 suspension was vigorously agitated and centrifuged at low speed to sepa- would seem that 19Fu-FA synthesis in the presence of O2 and rate the phases. The organic phase was removed, dried under N2, and ly- the potential use of the products of UfaM and RSP1091 activity as ophilized for 1 h. FAMEs were prepared by resuspending the dried second messengers are conserved across bacteria. materials in 600 μL of anhydrous methanol (Sigma), adding 100 μLofso- In sum, we identified conditions and enzymes needed for dium methoxide (Sigma), and incubating at room temperature for 3 h (47). bacterial synthesis of 19Fu-FA. Compounds predicted to be The reaction was stopped by adding 600 μL of 2 M HCl, and FAMEs were 19Fu-FA and 19M-UFA have been provisionally identified extracted with 600 μL of hexane. Then, 1 μL of each sample was analyzed on an in bacteria before (43, 44), but information on their cellular Agilent 7890A/5975C GC-MS with differing split ratios with an HP-5ms capillary abundance, the enzymes needed for their synthesis, and their column and He carrier gas (20 cm/s at 150 °C) using one of two oven programs: (i) 150 °C isothermal for 4 min, 4 °C/min ramp to 250 °C, and isothermal at cellular role have not yet been reported. We also identified 250 °C for 5 min; or (ii) 150 °C isothermal for 4 min, 6 °C/min ramp to 245 °C, conditions to increase production of 19Fu-FA in both native isothermal at 245 °C for 2 min, 80 °C/min ramp to 325 °C, and isothermal at and foreign hosts, such as E. coli. These studies pave the way 325 °C for 2 min. Chromatograms and mass spectra were analyzed using to identify and characterize enzymes needed to synthesize large Agilent GC-MS ChemStation (version E.02.00.493) and MassHunter software quantities of 19Fu-FA in bacterial systems. With large amounts (version B.06.00; Agilent Technologies) and compared with the NIST MS 1 Search 2011b library. For quantification, a set of appropriate FAME of 19Fu-FA available, one can probe the interaction of O2 with this Fu-FA, identify potential secondary messengers, and test the standard curves were created from a mix of Supelco C8-C24 standards (for utility of Fu-FAs as food, chemical, or fuel additives. C16:0, C16:1, C18:0), C15:0, C18:1 (Sigma), methyl 11-methyl-octadecenoate (n-6) (19M-UFA), and 10,13-epoxy-11-methyl-octadecadienoate (19Fu-FA) > Materials and Methods (Larodan). The MassHunter integration peak filter was set to 5% of the largest peaks; peak area was integrated for ions diagnostic for each FAME Bacterial Strains and Growth. E. coli and R. sphaeroides strains were grown (m/z 74 for C15:0, C16:0, C18:1, and C18:0; m/z 55 for C16:1; m/z 69 for 19M- Δ Δ R Δ as described (19). Mutant strains 1091/ ChrR and 1091:sp / ChrR were UFA; and m/z 165.1 for 19Fu-FA). The integrated areas were normalized to made using methods described previously (20). the recovery standard (C15:0), and each FAME was converted to a percentage of the total fatty acids, followed by averaging data from technical Purification of His6-RSP2144 Protein. pRLhisRSP2144 was generated by cloning duplicates. Biological duplicates were averaged, and the SD was calculated. the RSP2144 coding region into the NdeI and EcoRI sites of pET-28a(+)to produce an N-terminally hexahistidine-tagged protein (His6-RSP2144). A 500- Ectopic Expression of RSP2144 in R. sphaeroides and E. coli. pRL101 was created mL culture of log phase BL21DE3 E. coli cells, containing pRLhisRSP2144, was by cloning the His -RSP2144 gene from pRLhisRSP2144 into the NdeI and β 6 exposed to 1 mM isopropyl -D-1-thiogalactopyranoside (IPTG) for 4 h at 28 °C HindIII sites of pIND5. This plasmid and pAYW19 (containing E. coli cfa) were to induce expression of His6-RSP2144. The cells were harvested by centrifuga- then transformed into the E. coli strain JW1653, which lacks cfa (48, 49). tion, and the resulting pellet was resuspended in lysis buffer [25 mM Hepes (pH JW1653 was obtained from the Keio collection, and the Knr gene was re- 7.5), 150 mM KCl, 20 mM imidazole, 10% glycerol (vol/vol), 1 mg/mL lysozyme, moved before use (48). Triplicate biological cultures were separately treated × and 1 Halt protease inhibitor (Pierce)], sonicated on ice, pulsing every 20 s, for with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) before preparing FAMEs 7 min, and centrifuged for 1 h at 50,000 × g. The resulting supernatant was (see Fatty Acid Methyl Ester Content section in Materials and Methods). passed over a 4-mL Ni-NTA agarose column (Novagen) and washed with 50 mL of wash buffer [25 mM Hepes (pH 7.5), 150 mM KCl, 50 mM imidazole, Hydrogenation of FAME Samples. FAMEs were dried under N2, dissolved in and 10% glycerol], and protein was removed with a 16-mL elution buffer [25 10 mL of (1:2 vol/vol) chloroform:methanol with 15 mg of 5% platinum on mM Hepes (pH 7.5), 150 mM KCl, 250 mM imidazole, and 10% glycerol]. activated charcoal (50). The reaction tubes were fitted with stoppers and Fractions containing the most protein were combined and concentrated sparged with a 95% N2/5% H2 gas mixture for 1 h. The tubes were centri- using a YM10 centrifugal filter (Millipore) and dialyzed into 50 mM Hepes, fuged twice to remove the charcoal, filtered through glass wool in a Pasteur 10 mM sodium bicarbonate, and 50% glycerol. Small portions were ali- pipet, and analyzed by GC-MS. quotted and stored at −80 °C. Protein concentration was estimated using the Bradford Assay (Bio-Rad). Identification of Unknown FAMEs. Gas chromatography was performed on a Trace GC Ultra (Thermo Electron) equipped with a CTC Analytics GC PAL In Vitro Assay of His -RSP2144 Activity. The phospholipid substrate was pu- 6 autosampler (Zwingen) using a 30 m × 0.25 mm (i.d.) × 0.25 μm(df) Cross- rified from a ΔRSP2144 strain, and a phospholipid micelle solution (in water) bond 5% diphenyl/95% dimethyl polysiloxane column (Restek Rxi-5Sil MS) was created (45) and quantitated by a lipid phosphorous assay (46). Each and He as carrier gas. Mass spectrometry was performed on a breadboard – μ enzyme reaction contained 0.06 1.04 mM phospholipid, 4.4 MHis6-RSP2144 GC/quadrupole-Orbitrap MS. protein, 20 mM potassium phosphate buffer (pH 7.4), 0.5 mg/mL BSA, and A FAME mix of 26 compounds in methyl caproate was used for chro- μ μ μ 750 M SAM (Sigma) with a specific activity of 25 Ci/ mol (Perkin-Elmer). matographic and MS source optimization (Sigma). Samples in hexane (1 μL) The reactions were incubated at 30 °C, and individual time points were were injected via the hot-needle technique at various split ratios depending μ taken by placing 100- L aliquots into 1 mL of 10% trichloroacetic acid (vol/vol). on sample concentration, with an injector temperature of 250 °C, He flow The solutions were filtered over Whatman GF/c glass filter fibers on a 1225 rate of 1 mL/min, and the following oven program: 1 min isothermal at 150 °C, sampling manifold (Millipore), followed by three washes with 1 mL of 10% 15 °C/min to 250 °C, 1 min isothermal at 250 °C, 80 °C/min to 320 °C, and 2 min trichloroacetic acid and three washes with 1 mL of water. The filters were isothermal at 320 °C. The transfer line and source temperatures were 280 °C put into 5 mL of Optiphase scintillation fluid (Perkin-Elmer) and incubated at and 250 °C, respectively. Samples were ionized via EI or positive CI (PCI) room temperature overnight before determining radioactivity on a scintil- using acetonitrile (ACN) as the reagent gas (70 eV). Full-scan analyses lation counter. The results of duplicate assays were averaged, and the used a scan range of 75–400 Th, resolution of 17,500, automated gain reaction rate was calculated by plotting radioactivity versus time for each control (AGC) target of 1E6, and maximum injection time of 100 ms. concentration of phospholipid. The rates were averaged between two Targeted MS/MS analyses used a 3 Th isolation width, normalized collision independent experiments. energy of 25 eV, resolution of 17,500, AGC target of 1E6, and maximum injection time of 250 ms. 1 1 Exposure to O2. R. sphaeroides strains were exposed to O2 as described (19). To enable ACN PCI, a 250-μm (i.d.) fused silica capillary connected an ACN Then, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to cells reservoir (6 mL) directly to the MS source through the heated transfer line. 1 1 h before O2 exposure to induce protein expression in cells containing A two-holed ferrule was used to permit entry of both the GC column and a plasmid-encoded His6-RSP2144 protein. Cells were grown anaerobically by ACN capillary into the transfer line. Although the column extended into sparging cultures with a 95% N2/5% CO2 gas mixture. the source, the ACN capillary was set back ∼5 cm from the source to prevent interference with the GC eluent. A medium-flow metering valve Fatty Acid Methyl Ester Content. All samples from methylene blue-treated (Swagelok) was placed between the reservoir and transfer line to reg- cells were kept in the dark until FAMEs were generated and extracted into ulate the flow of ACN into the source. A source pressure of 7.1E−5

E3456 | www.pnas.org/cgi/doi/10.1073/pnas.1405520111 Lemke et al. Downloaded by guest on September 23, 2021 Torr, ∼0.2 ms reagent injection time (at a 1E6 AGC target) and m/z 42 following oven program was run: 3 min isothermal at 140 °C, 5 °C /min to 230 °C, PNAS PLUS (protonated ACN)-to-m/z 54 (1-methyleneimino-1-ethenylium, or MIE) ratio and isothermal at 230 °C for 3 min. Injector and detector were maintained at of 5:1 were found to be optimal for generation of molecular ion MIE 250 °C throughout the analysis. Isomers in biological samples were identified adducts of unsaturated FAMEs. by retention time comparison with FAME standards (51).

Identification of Fatty Acyl Isomers. Identification of isomer configuration of ACKNOWLEDGMENTS. We thank Dr. John Ohlrogge (Michigan State Univer- 11-methyl-octadecanoate was determined by gas chromatography equipped sity) for helpful discussions, Dr. John Cronan (University of Illinois) for providing the pAYW19 plasmid, and Becky Ciske for technical assistance early in the with a flame ionization detector (6890N; Agilent technologies). Commercial project. This work was supported in part by National Institute of General Δ cis Δ cis Δ trans trans FAME standards (18:0, 18:1 9 , 18:1 11 , 18:1 9 , and M-UFA ) and Medical Sciences Grants GM075273 (to T.J.D.) and GM107199 (to J.J.C.) and biological samples were separated on a DB-23 capillary column, 30 m × 0.25 mm by Department of Energy, Office of Science Great Lakes Bioenergy Research (i.d.) and 0.25-μm film thickness. The He flow rate was 1.5 mL/min, and the Center Grant DE-FC02-07ER64494 (to T.J.D.).

1. Mueller S, et al. (2008) General detoxification and stress responses are mediated 28. Michaud AL, Diau G-Y, Abril R, Brenna JT (2002) Double bond localization in minor by oxidized lipids through TGA transcription factors in Arabidopsis. Plant Cell 20(3): homoallylic fatty acid methyl esters using acetonitrile chemical ionization tandem 768–785. mass spectrometry. Anal Biochem 307(2):348–360. 2. Cronan JE (2002) Phospholipid modifications in bacteria. Curr Opin Microbiol 5:202–205. 29. Grogan DW, Cronan JE (1997) Cyclopropane ring formation in membrane lipids of 3. Cronan JE (2003) Bacterial membrane lipids: Where do we stand? Annu Rev Microbiol bacteria. Microbiol Mol Biol Rev 61(4):429–441. 57(1):203–224. 30. Yuan Y, Crane DC, Musser JM, Sreevatsan S, Barry CE (1997) MMAS-1, the branch 4. Chang YY, Cronan JE, Jr (1999) Membrane cyclopropane fatty acid content is a major point between cis- and trans-cyclopropane-containing oxygenated mycolates in – factor in acid resistance of Escherichia coli. Mol Microbiol 33(2):249–259. Mycobacterium tuberculosis. J Biol Chem 272(15):10041 10049. ’ 5. Girotti AW, Kriska T (2004) Role of lipid peroxides in photo-oxidative stress signalling. 31. Guianvarc h D, Drujon T, Leang TE, Courtois F, Ploux O (2006) Identification of new Antioxid Redox Signal 6:301–310. inhibitors of E. coli cyclopropane fatty acid synthase using a colorimetric assay. – 6. Imlay JA (2003) Pathways of oxidative damage. Annu Rev Microbiol 57:395–418. Biochim Biophys Acta 1764(8):1381 1388. 7. Sayre LM, De L, Quan Y, Xiaochun Z, Xiaoxia T (2006) Protein adducts generated from 32. Iwig DF, Uchida A, Stromberg JA, Booker SJ (2005) The activity of Escherichia coli products of lipid oxidation: Focus on HNE and ONE*. Drug Metab Rev 38(4):651–675. cyclopropane fatty acid synthase depends on the presence of bicarbonate. J Am Chem – 8. Watabe N, Ishida Y, Ochiai A, Tokuoka Y, Kawashima N (2007) Oxidation de- Soc 127(33):11612 11613. 33. Magnuson K, Jackowski S, Rock CO, Cronan JE (1993) Regulation of fatty acid bio- composition of unsaturated fatty acids by singlet oxygen in phospholipid bilayer synthesis in Escherichia coli. Microbiol Rev 57(3):522–542. membranes. J Oleo Sci 56(2):73–80. 34. White DC, et al. (2005) Phospholipid furan fatty acids and ubiquinone-8: Lipid bio- 9. Lennen RM, Braden DJ, West RA, Dumesic JA, Pfleger BF (2010) A process for mi- markers that may protect dehalococcoides strains from free radicals. Appl Environ crobial hydrocarbon synthesis: Overproduction of fatty acids in Escherichia coli and Microbiol 71(12):8426–8433. catalytic conversion to alkanes. Biotechnol Bioeng 106(2):193–202.

35. Wakimoto T, et al. (2011) Furan fatty acid as an anti-inflammatory component from MICROBIOLOGY 10. Peralta-Yahya PP, Zhang F, del Cardayre SB, Keasling JD (2012) Microbial engineering the green-lipped mussel Perna canaliculus. Proc Natl Acad Sci USA 108(42):17533–17537. for the production of advanced biofuels. Nature 488(7411):320–328. 36. Kontur WS, et al. (2012) Revised sequence and annotation of the Rhodobacter 11. Connor MR, Liao JC (2009) Microbial production of advanced transportation fuels in sphaeroides 2.4.1 genome. J Bacteriol 194:7016–7017. – non-natural hosts. Curr Opin Biotechnol 20(3):307 315. 37. Mackenzie C, et al. (2001) The home stretch, a first analysis of the nearly completed – 12. Lands B (2012) Consequences of essential fatty acids. Nutrients 4(9):1338 1357. genome of Rhodobacter sphaeroides 2.4.1. Photosynth Res 70(1):19–41. 13. Spiteller G (2005) Furan fatty acids: Occurrence, synthesis, and reactions. Are furan 38. Armstrong G, Hearst J (1996) Carotenoids 2: Genetics and molecular biology of fatty acids responsible for the cardioprotective effects of a fish diet? Lipids 40(8): carotenoid pigment biosynthesis. FASEB J 10(2):228–237. – 755 771. 39. Cogdell RJ (2000) How carotenoids protect bacterial photosynthesis. Philos Trans R 14. Okada Y, Kaneko M, Okajima H (1996) Hydroxyl radical scavenging activity of natu- Soc Lond B Biol Sci 355:1345–1349. rally occurring furan fatty acids. Biol Pharm Bull 19(12):1607–1610. 40. Krinsky NI (1989) Antioxidant functions of carotenoids. Free Radic Biol Med 7(6): 15. Okada Y, Okajima H, Konishi H (1990) Antioxidant effect of naturally occurring furan 617–635. fatty acids on oxidation of linoleic acid in aqueous dispersion. J Am Oil Chem Soc 67: 41. Nuss AM, et al. (2013) DegS and RseP homologous proteases are involved in singlet 858–862. oxygen dependent activation of RpoE in Rhodobacter sphaeroides. PLoS ONE 8(11): 16. Ziegelhoffer EC, Donohue TJ (2009) Bacterial responses to photo-oxidative stress. Nat e79520. Rev Microbiol 7(12):856–863. 42. Greenwell RS, Nam TW, Donohue TJ (2011) Aspects of the zinc metalloprotein ChrR 17. Glaeser J, Nuss AM, Berghoff BA, Klug G (2011) Singlet oxygen stress in micro- required for dissociation of σE/ChrR complexes. J Mol Biol 407:477–491. organisms. Advances in Microbial Physiology,edPooleRK(Academic,NewYork), 43. Shirasaka N, Nishi K, Shimizu M (1995) Occurrence of a furan fatty acid in marine Vol 58, pp 141–173. bacteria. Biochim Biophys Acta 1258(3):225–227. 18. Koopman WJ, et al. (2010) Mammalian mitochondrial complex I: Biogenesis, regula- 44. Shirasaka N, Nishi K, Shimizu S (1997) Biosynthesis of furan fatty acids (F-acids) by tion, and reactive oxygen species generation. Antioxid Redox Signal 12(12):1431–1470. a marine bacterium, Shewanella putrefaciens. Biochim Biophys Acta 1346(3):253–260. 19. Anthony JR, Warczak KL, Donohue TJ (2005) A transcriptional response to singlet 45. Courtois F, Guerard C, Thomas X, Ploux O (2004) Escherichia coli cyclopropane fatty – oxygen, a toxic byproduct of photosynthesis. Proc Natl Acad Sci USA 102(18):6502–6507. acid synthase. Eur J Biochem 271(23-24):4769 4778. 20. Nam TW, Ziegelhoffer EC, Lemke RAS, Donohue TJ (2013) Proteins needed to activate 46. Rouser G, Fkeischer S, Yamamoto A (1970) Two dimensional then layer chromato- a transcriptional response to the reactive oxygen species singlet oxygen. MBio 4(1): graphic separation of polar lipids and determination of phospholipids by phosphorus – e00541–12. analysis of spots. Lipids 5(5):494 496. th 21. Dufour YS, Landick R, Donohue TJ (2008) Organization and evolution of the bi- 47. Christie WW, Han X (2010) Lipid Analysis (Oily, Bridgwater, UK), 4 Ed, p 148. 48. Baba T, et al. (2006) Construction of Escherichia coli K-12 in-frame, single-gene ological response to singlet oxygen stress. J Mol Biol 383(3):713–730. knockout mutants: The Keio collection. Mol Syst Biol 2:2006 0008. 22. Anthony JR, Newman JD, Donohue TJ (2004) Interactions between the Rhodobacter 49. Wang AY, Grogan DW, Cronan JE (1992) Cyclopropane fatty acid synthase of sphaeroides ECF sigma factor, σE, and its anti-sigma factor, ChrR. J Mol Biol 341: Escherichia coli: Deduced amino acid sequence, purification, and studies of the 345–360. enzymeactivesite.Biochemistry 31(45):11020–11028. 23. Newman JD, Falkowski MJ, Schilke BA, Anthony LC, Donohue TJ (1999) The Rhodo- 50. Montanari C, Sado Kamdem SL, Serrazanetti DI, Etoa FX, Guerzoni ME (2010) bacter sphaeroides ECF sigma factor, σE, and the target promoters cycA P3 and rpoE Synthesis of cyclopropane fatty acids in Lactobacillus helveticus and Lactobacillus P1. J Mol Biol 294(2):307–320. sanfranciscensis and their cellular fatty acids changes following short term acid and 24. Donohue TJ, Cain BD, Kaplan S (1982) Purification and characterization of an cold stresses. Food Microbiol 27(4):493–502. N-acylphosphatidylserine from Rhodopseudomonas sphaeroides. Biochemistry 51. Tjellström H, Strawsine M, Silva J, Cahoon EB, Ohlrogge JB (2013) Disruption of plastid – 21(11):2765 2773. acyl:acyl carrier protein synthetases increases medium chain fatty acid accumulation 25. Hands AR, Bartley W (1962) The fatty acids of Rhodopseudomonas particles. Biochem J in seeds of transgenic Arabidopsis. FEBS Lett 587(7):936–942. – 84:238 239. 52. Bethesda Research Laboratories (1986) BRL pUC host: Escherichia coli DH5α compe- 26. Qureshi N, Honovich JP, Hara H, Cotter RJ, Takayama K (1988) Location of fatty acids tent cells. Bethesda Res Lab Focus 8:9–10. in lipid A obtained from lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 53. Simon R, Priefer U, Puhler A (1983) A broad host range mobilization system for in 17023. J Biol Chem 263(12):5502–5504. vitro genetic engineering: Transposon mutagenesis in Gram negative bacteria. 27. Russell NJ, Harwood JL (1979) Changes in the acyl lipid composition of photosynthetic Nature Biotech 1:748–791. bacteria grown under photosynthetic and non-photosynthetic conditions. Biochem J 54. Schilke BA, Donohue TJ (1995) ChrR positively regulates transcription of the Rhodo-

181(2):339–345. bacter sphaeroides cytochrome c2 gene. J Bacteriol 177(8):1929–1937.

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