MedicineLakex

Pnas201201089 3808.3813

Aerobic kinetoplastid flagellate Phytomonas does not require heme for viabilityLud ˇek Ko ˇrenýa,b, Roman Sobotkab,c, Julie Ková ˇrováa,b, Anna Gnipováa,d, Pavel Flegontova,b, Anton Horváthd,Miroslav Oborníka,b,c, Francisco J. Ayalae,1, and Julius Luke ˇsa,b,1 aBiology Centre, Institute of Parasitology, Czech Academy of Sciences and bFaculty of Science, University of South Bohemia, 370 05 Ceské Budejovice, CzechRepublic; cInstitute of Microbiology, Czech Academy of Sciences, 379 81 Trebo n, Czech Republic; dFaculty of Natural Sciences, Comenius University, 842 15 Bratislava, Slovakia; and eDepartment of Ecology and Evolutionary Biology, University of California, Irvine, CA 92697 Contributed by Francisco J. Ayala, January 19, 2012 (sent for review December 8, 2011) Heme is an iron-coordinated porphyrin that is universally essential aerobic environment (8). In soluble guanylyl cyclase, heme serves as a protein cofactor for fundamental cellular processes, such as as the nitric oxide sensor, and thus plays an important role in electron transport in the respiratory chain, oxidative stress re- signal transduction. Heme is also an important regulatory mol- sponse, or redox reactions in various metabolic pathways. Parasitic ecule because it reversibly binds to certain proteins, such as kinetoplastid flagellates represent a rare example of organisms transcription factors and ion channels, and thus modulates their that depend on oxidative metabolism but are heme auxotrophs.
functions (9).
Here, we show that heme is fully dispensable for the survival of The central position of heme in a variety of cellular functions Phytomonas serpens, a plant parasite. Seeking to understand the makes it essential for the viability of virtually all living systems.
metabolism of this heme-free eukaryote, we searched for heme- There are only a few examples of facultatively anaerobic or containing proteins in its de novo sequenced genome and exam-ined several cellular processes for which heme has so far been con- pathogenic bacteria that do not require heme (10–12), but no sidered indispensable. We found that P. serpens lacks most of the eukaryote that can survive without heme has been identified.
known hemoproteins and does not require heme for electron trans- Most aerobic organisms synthesize heme by a multistep pathway port in the respiratory chain, protection against oxidative stress, or that is conserved in all three domains of life: bacteria, archaea, desaturation of fatty acids. Although heme is still required for the and eukaryotes. A few eukaryotes that lost this pathway are synthesis of ergosterol, its precursor, lanosterol, is instead incorpo- known to scavenge heme from external sources. For example, rated into the membranes of P. serpens grown in the absence of ticks have easy access to heme from blood (13), whereas parasitic heme. In conclusion, P. serpens is a flagellate with unique metabolic nematodes uptake it either from their host or from endosymbiotic adaptations that allow it to bypass all requirements for heme.
bacteria (14). The free-living nematode Caenorhabditis eleganslacks the capacity to synthesize heme but is able to take it from the cytochromes respiration sterols protist bacteria it feeds on (15). Even the parasitic protists Entamoeba,Trichomonas, and Giardia, which dwell in an anaerobic environ- Heme is a tetrapyrrole molecule that consists of a porphyrin mentanddonotneedhemeforprocessesconnectedtooxidative ring coordinated with the iron molecule. It interacts with metabolism, have retained a few hemoproteins, for which heme is various apoproteins giving rise to functional hemoproteins, likely obtained from their hosts (16).
which are ubiquitous in biological systems and exhibit a wide Flagellates of the order Kinetoplastea, which includes major range of activities. The oxidation state of the iron is important human parasites, depend on oxygen but are unable to produce for most biological roles of heme, but its exact function is ulti- heme. Media for their cultivation must therefore be supplemented mately determined by the properties of the polypeptide bound toit (1). Heme can exist in either the oxidized ferric (Fe3+) or with heme to support their growth (17). Members of the genus reduced ferrous (Fe2+) state, which enables it to accept or do- Trypanosoma lost the entire biosynthetic pathway and extract nate electrons and to function in various redox reactions and heme from host blood (18, 19), whereas Leishmania spp. have electron transport.
retained genes for the last three steps of the pathway, allowing The most abundant group of heme proteins are cytochromes them to synthesize heme from their host-derived precursors (20).
(2). In aerobic organisms that produce energy mainly through Some kinetoplastids that parasitize insects obtain heme from their oxidative phosphorylation, most of the synthesized heme is used bacterial endosymbionts, which can be eliminated by antibiotic for the formation of the cytochromes functioning in the electron treatment, turning these protists into heme auxotrophs (17).
transport respiratory chain. Other cytochromes, such as the Kinetoplastid flagellates of the genus Phytomonas are impor- members of the cytochrome b5 or cytochrome P450 family, are tant yet understudied parasites of plants with a major economic involved in various redox reactions of specific metabolic path- impact in Latin America and the Caribbean (21). They reside in ways, such as desaturation of fatty acids and sterol biosynthesis, carbohydrate-rich tissues, such as phloem, latex, fruits, and seeds; and also in drug detoxification (3, 4). In catalases, heme func- their ATP production is based on glycolysis (22). In the present tions in the degradation of hydrogen peroxide, whereas in per- study, we show that Phytomonas serpens does not require heme for oxidases, it oxidizes a wide variety of organic and inorganic viability and possesses unique metabolic properties that allow it to compounds in the presence of hydrogen peroxide. Through the bypass all functions of this otherwise omnipresent molecule.
consumption of hydrogen peroxide, these enzymes greatly con-tribute to the oxidative stress defense (5, 6). In addition to itsfunction as an electron carrier, heme iron has the capacity to Author contributions: L.K., A.H., M.O., F.J.A., and J.L. designed research; L.K., R.S., J.K., bind diatomic gases. Hemoglobin is well known as the oxygen and A.G. performed research; L.K., P.F., and M.O. analyzed data; and L.K., F.J.A., and J.L.
transporter in animals, but members of the same protein family wrote the paper.
are widespread in all groups of organisms, including anaerobes.
The authors declare no conflict of interest.
The original roles of globins might have been the responses to 1To whom correspondence may be addressed. E-mail: or nitric oxide and nitrosative stress (7) or sensing of oxygen, which This article contains supporting information online at was highly toxic to cells before they managed to adapt to an 3808–3813 PNAS March 6, 2012 vol. 109 no. 10

Growth dependence on availability of heme and quantification of heme b in P. serpens and related flagellates. (A) Growth rate of P. serpens is the same without heme or when heme is supplied up to a concentration of 5 μM; 25 μM heme inhibits growth, likely attributable to toxic effects of free heme. Quite oppositedependence of growth on heme concentration is observed in the closely related C. fasciculata, which grows best when supplied with 25 μM heme and stops growingwhen heme concentration in the media is lowered to 1 μM. (B) Heme b extracted from equal numbers of cells from various kinetoplastids was separated by HPLC anddetected by diode array detector. C. fasciculata was used as a related organism that possesses a complete set of respiratory complexes. It was grown in the samemedium and supplemented with the same amount of heme (5 μM) as P. serpens. The bloodstream stage of T. brucei, which does not express its respiratory complexesIII and IV in this life cycle stage, and thus functionally resembles P. serpens, was used as another control. The absence of respiratory complexes that normally consumemost of heme is reflected in the much lower amount of extracted heme compared with C. fasciculata. The heme content in P. serpens is even lower than in T. brucei,which is in accordance with the lowest number of heme proteins found in the Phytomonas spp. genomes among all kinetoplastids (Table 1). Not even a trace amountof heme is detected in P. serpens grown without heme (dotted line). +H, with heme; −H, without heme.
Results and Discussion into one lacking it did not alter their growth. This is in contrast to We cultivated P. serpens strain 9T in a chemically defined medium related flagellates, such as Crithidia fasciculata, which requires without heme continuously for over a year without heme for growth and was used as a control (Fig. 1A).
noticeable decrease of the growth rate (generation time of ∼8 h), We sought to test the heme biosynthetic capacity of P. serpens compared with parallel cultures supplemented with heme (Fig.
by measuring the amount of extractable heme. Even using a very 1A). Abrupt transfer of cells grown in heme-containing medium sensitive HPLC assay, we failed to detect any traces of heme in Heme proteins of kinetoplastid flagellates Lanosterol 14α-demethylase (cytochrome P450) Heme-binding subunit of the respiratory complex II Soluble cytochrome c of the respiratory chain Cytochrome b subunit of the respiratory complex III Cytochrome c1 subunit of the respiratory complex III Heme a and heme a3 binding subunit of complex IV Heme-dependent plant peroxidase homolog 1 Heme-dependent plant peroxidase homolog 2 Δ9 Fatty acid desaturase (cytochrome b5 domain) Δ4 Fatty acid desaturase (cytochrome b5 domain) Δ5 Fatty acid desaturase (cytochrome b5 domain) Δ6 Fatty acid desaturase (cytochrome b5 domain) Nitrate reductase (cytochrome b5 domain) Fumarate reductase-like (cytochrome b5 domain) Ferric reductase (cytochrome b561) Ferric reductase (flavocytochrome b558) Globin domain of adenylate cyclase-like protein Cytochromes P450 with unknown function* Cytochromes b5 with unknown function* The presence/absence data for PE and PH were kindly provided by Michel Dollet (CIRAD-BIOS, Montpellier, France) and Patrick Wincker (Genoscope, Evry, France). GenBank accession numbers are quoted for the proteinsof Leishmania major. CF, Crithidia fasciculata; Lei, Leishmania spp.; PE, Phytomonas sp. strain EM1; PH, Phyto-monas sp. strain Hart1; PS, Phytomonas serpens; TB, Trypanosoma brucei; TC, Trypanosoma cruzi.; (−), heme-binding domain is missing, but the rest of the protein is present.
*Proteins that do not have known function but were identified as either cytochrome P450 or cytochrome b5 arenot listed individually. The number of these proteins is shown for each taxon.
Korený et al.
PNAS March 6, 2012 vol. 109 no. 10 3809

cells grown in its absence (Fig. 1B). This indicates that P. serpensis able to survive without heme, which is further supported by thefact that, with the exception of ferrochelatase, no other genes forheme synthesis were found in the draft genome of P. serpensstrain 9T obtained for this study. On the other hand, we founda small amount of heme in cells growing in the medium sup-plemented with heme (Fig. 1B), which implies that P. serpens isable to uptake this compound from the medium.
To find out how P. serpens can survive without the key heme- dependent activities and possibly identify any functions still usingheme, we decided to test cellular processes experimentally inwhich heme is known to be involved. A screen for homologs ofheme-containing proteins in the genome produced only a fewhits, compared with the list of hemoproteins from related flag-ellates (Table 1 and ). The same results were obtainedfor two other recently sequenced Phytomonas genomes (Table1). Unlike other kinetoplastids, Phytomonas spp. have an ap-parent lack of respiratory cytochromes, heme-dependent perox-idases, and several enzymes that possess heme-binding domains,such as front-end fatty acid desaturases for the production ofpolyunsaturated fatty acids (23), a nitrate reductase, and twodifferent ferric reductases, one of which was shown to be in-volved in the iron uptake of related Leishmania (24) (Table 1).
The absence of heme peroxidases in P. serpens, exceptional even among the kinetoplastids, most of which lack catalase (25)(Table 1), corresponds to our finding that heme added to themedium does not increase the resistance of P. serpens against Respiratory complex II (succinate dehydrogenase) is assembled and oxidative stress induced by the superoxide generator paraquat active in P. serpens grown with (+H) or without (−H) heme. (A) Clear native This is the opposite of what was found for the evolu- gel (3–12%) after in-gel staining for succinate dehydrogenase activity; C.
fasciculata (Cf) served as a control. Ferritin (monomeric and dimeric forms) tionarily related Trypanosoma brucei, which needs heme for ox- was used as a molecular weight marker (M). (B) Lysates from the same cells idative stress defense (18).
as in A were analyzed by SDS/PAGE and immunoblotted with specific anti- Although P. serpens lacks the heme-containing respiratory serum against the T. brucei subunit of complex II, SDH1. (C) Activity of suc- complexes III and IV (26–28), the mitochondrial respiratory cinate dehydrogenase in P. serpens grown without heme. The decrease in chain remains functional, serving to reoxidize NADH produced absorbance (A600) with time (curve 2) was caused by the addition of ubi- during glycolysis (22, 29). Complex I is present in P. serpens (27, quinone to the reaction, which mediated the electron transfer from succi- 30), which, instead of cytochrome c reductase (complex III) and nate to 2,6-dichlorophenolindophenol. The activity was specifically inhibited cytochrome c oxidase (complex IV), uses alternative oxidase to using malonate (curve 3). Curve 1 represents the background without ubi-quinone. (D) Activity did not significantly differ between P. serpens grown reduce oxygen to water (31). We found that succinate de- with (+H) or without (−H) heme. Medium values were calculated from three hydrogenase (complex II) is also present (Fig. 2), with a con- served histidine residue in its SDH4 subunit, which supposedlybinds heme in the related Trypanosoma cruzi and other kineto-plastids (32). Visualization of the P. serpens complex II by in-gel as well as the soluble cytochrome c, may be bypassed by using the staining in clear-native gel revealed that its abundance is not alternative terminal oxidase, which utilizes nonheme iron to influenced by the availability of heme in the medium (Fig. 2A).
transfer electrons from ubiquinone directly to oxygen. This is Moreover, its size of ∼600 kDa is unaltered in the heme-de- also known for the bloodstream (mammalian) stage of T. brucei, prived cells, being almost the same as in T. cruzi (32) and the which, similar to Phytomonas, dwells in a sugar-rich environment, related C. fasciculata, used as a control (Fig. 2A), suggesting whereas the T. brucei procyclic (insect) stage has a fully de- a proper assembly of complex II in the absence of heme. To veloped mitochondrion equipped with the heme-containing assess the abundance of its subunits, we generated specific an- complexes (36). This metabolic switch is impossible in Phyto- tiserum against one subunit of the kinetoplastid complex II, monas, which has lost the genes encoding the subunits of these SDH1. The amount of the target protein was the same in cells complexes from its genome (26, 28) (Table 1).
grown with or without heme (Fig. 2B). Furthermore, the absence Because of its capacity to transfer electrons, heme participates of heme did not affect the capacity of complex II to reduce in various redox reactions, some of which are virtually universal ubiquinone (Fig. 2 C and D).
for eukaryotes. One of them is the desaturation of fatty acids. In These findings are in line with previous reports showing that heme is not universally indispensable for the function of complex eukaryotes, this reaction needs electron equivalents that are II (33, 34). In mammalian cells, the absence of heme disrupts transferred from reduced cytochrome b5, and thus depends on proper assembly and inhibits the activity of complex II (35).
heme (37, 38). Many desaturases contain cytochrome b5 as However, in yeast and Escherichia coli, the homologous com- a domain conveniently fused to their N- or C-termini, including plexes retain physiological activity even without heme (33, 34). It the most widespread one, which creates the double bond in the has been suggested that although heme does not participate in Δ9 position (23, 39). Our phylogenetic analyses revealed that this the electron transfer in complex II and is not necessarily required fusion took place only once in the evolution of eukaryotic Δ9 for the assembly of the complex, it may provide an electron sink fatty acid desaturases, specifically at the base of a superclade to protect against free radical damage during periods of high comprising fungi, amoebozoans, rhodophytes, choanozoans, and electron flux (34). However, the presence of heme in the proton- excavates, including kinetoplastids (Fig. 3A and Re- pumping complexes III and IV is indispensable, because it di- markably, Δ9 desaturase in P. serpens is the only member of this rectly mediates electron transport. However, when enough en- superclade that conspicuously lacks the cytochrome b5 domain, ergy is produced by glycolysis, these heme-containing complexes, apparently as a consequence of its secondary loss, a singular Korený et al.

Heme is not needed for desaturation of fatty acids but is required for ergosterol biosynthesis in P. serpens. (A) Schematic phylogenetic tree of Δ9-fatty acid desaturases (FADS). P. serpens is the only organism that secondarily lost the cytochrome b5 domain. The full phylogenetic tree of Δ9-fatty acid desaturaseis shown in . (B) Analyses of fatty acid composition by gas chromatography demonstrate that in P. serpens, the desaturation of fatty acids is not affectedby the absence of heme. (C) Analysis of sterol composition by TLC. Ergosterol, which is the major membrane sterol of Trypanosomatida, and lanosterol, theprecursor of heme-dependent demethylation, were used as standards (S). C. fasciculata (Cf) served as a control. P. serpens synthesized a sterol that corre-sponded to the ergosterol standard only when heme was added to the growth medium (+H). Cells grown without heme (−H) accumulated lanosterol.
event among all known eukaryotes. To assess the ability of P.
P. serpens possesses this unique capability as well. Based on the serpens grown in the absence of heme to desaturate fatty acids, TLC analysis, the cells synthesized a sterol that corresponded to we analyzed their composition by gas chromatography. We found the ergosterol standard only when heme was added to the growth that P. serpens contains unsaturated fatty acids and that their medium. In contrast, they accumulated lanosterol in the absence composition is virtually the same regardless of the presence or of heme with no impact on cell viability (Fig. 3C). The fact that absence of heme (Fig. 3B). These findings indicate that for the certain eukaryotes are able to use lanosterol but others are not is desaturation of fatty acids, P. serpens is able to use an electron very interesting and implies the existence of some regulatory donor other than cytochrome b5. It may likely be ferredoxin, mechanism. Cholesterol-deficient human T cells can adapt to which serves this role for the desaturases of some bacteria and growth with lanosterol; the initial growth of these cells dropped plant plastids. For example, the plastid Δ12 fatty acid desaturase 10-fold when cholesterol was depleted, yet their prolonged cul- of plants and diatoms depends on ferredoxin as an electron tivation resulted in a growth rate ∼65% that of the cholesterol- donor, whereas a homologous desaturase with the same function supplemented cells (46). A study on yeast revealed that what in the endoplasmic reticulum of the same organisms, as well as in regulates the incorporation of lanosterol in the membranes is the other eukaryotes, uses cytochrome b5 (40). The possibility that level of synthesized heme (47). The growth of P. serpens in the these redox molecules could substitute for each other has been absence of heme precludes the activity of CYP51; thus, this experimentally demonstrated in E. coli and in yeast expressing flagellate meets the two conditions that are required in yeast for cyanobacterial Δ6 fatty acid desaturase (41). Although ferre- lanosterol utilization (low heme levels and CYP51 inhibition).
doxin is the natural electron donor for this desaturase, cyto- Overall, there are several cellular processes for which heme is chrome b5 fully complemented its function when fused or crucial in a typical eukaryote, yet it is dispensable in P. serpens.
coexpressed with the desaturase enzyme. Three different ferre- Somewhat lower dependence of a typical kinetoplastid on heme doxin homologs were identified in the genomic sequences of P.
has been noted when cystathionine-β-synthase, a hemoprotein of animals and amoebae that is essential for cysteine formation, was The oxidative 14α-demethylation of lanosterol, another key reaction in the eukaryotic cell, fully depends on heme. Its sub- shown to lack heme in kinetoplastids (48). However, P. serpens is stitution by means of analogous nonheme enzyme has never been unique, because it lacks most hemoproteins that are present even documented. This reaction is a crucial step in the synthesis of in closely related protists. Moreover, the few retained in the P.
sterols, such as cholesterol in animals or ergosterol in fungi, as serpens genome are not crucial for its survival, at least under well as in protists, including kinetoplastid flagellates (42). It is culture conditions. In addition to CYP51 and the SDH4 subunit catalyzed by lanosterol 14α-demethylase (CYP51), which belongs of respiratory complex II, we identified 13 proteins that sup- to the cytochrome P450 family, found in most eukaryotes, in- posedly bind heme, because they are homologous to cytochrome cluding Phytomonas spp. (Table 1). No eukaryotic cell can b5 (Table 1). Their functions are unknown, however, and 5 of function without sterols or their analogs in its membranes; in- them lack the HPGG heme-binding motif typical for cytochrome hibition of this enzymatic step is thus frequently lethal (43).
b5 (41). Thus, it is by no means certain that these proteins ac- Consequently, CYP51 is a popular target of fungicides and other tually bind heme in vivo. One of them is a protein recently drugs, which are also effective against kinetoplastids (44). Until identified in the flagellar proteome of T. brucei, shown to be now, the only kinetoplastid known to be naturally resistant to indispensable for the bloodstream stage but nonessential for the inhibitors of CYP51 is Leishmania braziliensis, a flagellate closely procyclic stage (49). Therefore, the only process for which heme, related to Phytomonas, which seems to be able to incorporate if present, was found to be actively used by P. serpens, is the 14-methyl sterols into its membranes (45). We have found that 14α-demethylation of lanosterol in the ergosterol biosynthetic Korený et al.
PNAS March 6, 2012 vol. 109 no. 10 3811

pathway (Fig. 3C). Surprisingly, however, in vitro growth remains (80 μg of proteins per line) by incubating in a staining solution [50 mM NaPi unaffected by the lack of this activity.
(pH 7.4), 84 mM sodium succinate, 0.2 mM N-methylphenazonium methyl It is conceivable that some anaerobic eukaryotes possessing only sulfate, 4.5 mM EDTA (pH 8.5), 10 mM potassium cyanide, 2 mg/mL Nitro- a few of the known hemoproteins may survive without heme as tetrazolium blue chloride) for 3 h at room temperature in dark. Nitro-tetrazolium blue chloride changes color on accepting electrons from succinate well; however, this will be hard to test, because, so far, none of via N-methylphenazonium methyl sulfate, a process catalyzed by complex II.
these anaerobic protists can be grown in a chemically defined SDH1 subunit of complex II was detected by Western blot analysis using 10% medium. Furthermore, anaerobic protists need to obtain some (wt/vol) SDS/PAGE and a specific polyclonal antiserum generated against the products of heme-dependent enzymes, such as cholesterol and oligopeptide SHLSKAYPVIDHTFDC [SDH1 subunit of T. brucei (Tb927.8.6580) fatty acids from their environment; thus, their existence cannot be in a rabbit].
considered to be independent of heme (50). To the best of our Specific succinate dehydrogenase activity was measured using the fol- knowledge, P. serpens is the only eukaryote that can survive lowing protocol: 5 μL of mitochondrial protein lysate was incubated with without heme and yet depends on oxidative metabolism. This 1 mL of succinate dehydrogenase solution [25 mM KPi (pH 7.2), 5 mM MgCl2, unique metabolic property, a feature likely developed as an ad- 20 mM sodium succinate] for 10 min at 30 °C. This mixture was transferred in aptation to the carbohydrate-rich environment of plant sap, makes the cuvette, and antimycin A (2 μg/mL), rotenone (2 μg/mL), potassium cy- it an ideal model to study different cellular functions in a heme- anide (2 mM), and 2,6-dichlorphenolindophenol (50 μM) were added.
free background, which may shed further light on the exact roles Background absorbance at 600 nm was then measured for 5 min. The re-action was triggered by adding 65 μM coenzyme Q and essentiality for life of the otherwise omnipresent heme.
2, and the absorbance at 600 nm was measured every 20 s for 5 min. Change in absorbance was Materials and Methods caused by the electron transfer from succinate via coenzyme Q2 to 2,6-dichlorophenolindophenol. The activity was specifically inhibited by the Cultivation Conditions and Growth Curves. Both P. serpens and C. fasciculata addition of 1 mM sodium malonate.
were grown in a chemically defined medium (supplemented withdifferent concentrations of hemin at 27 °C and shaking at 80 rpm, daily Genome Sequencing, Assembly, and Protein Search. P. serpens nuclear DNA diluted with fresh media to the density of 6 × 106 cells per milliliter. Cell fraction was sequenced using Illumina technology at BGI-Hong Kong (HiSeq concentration was measured daily using a Beckman Coulter Z2 counter.
2000 sequencing system, average insert size of 500 bp, read length of 90 bp). Adataset of 1.62 Gbp was obtained after basic filtering of low-quality reads.
Quantification of Heme b. In total, 2 × 109 cells of P. serpens, C. fasciculata, and Genome assembly with MIRA 3.4rc2 (54) produced 5,399 contigs longer than the bloodstream form of T. brucei were filtrated through a DEAE-cellulose 500 bp (N50 contig size of 6,781 bp) with average coverage 60 (genome as- column and washed five times with PBS buffer to remove all traces of heme sembly deposited in National Center for Biotechnology Information BioProject from the media. The cell pellets were extracted with methanol/0.2% NH4OH, database under accession no. PRJNA80957). Translated reads and contigs were and heme was extracted from the delipidated cells with acetone/2% HCl (vol/ screened using tblastn 2.2.24+ with e-value cutoffs at 10−3 and 10−10, re- vol) and separated by HPLC on a Nova-Pak C18 column (4-μm particle size, 3.9 × spectively, against Leishmania major heme-binding proteins (Table 1) and 150 mm; Waters) using linear gradient 25–100% (vol/vol) acetonitrile/0.1% tri- heme-synthesis enzymes. Conserved protein domains were identified using fluoroacetic acid at a flow rate of 1.1 mL/min at 40 °C. Heme b was detected by InterPro database. Draft genome sequences of C. fasciculata were kindly diode array detector (Agilent 1200; Agilent Technologies) and quantified using provided by Stephen M. Beverley (Washington University School of Medicine, authentic hemin standard (Sigma–Aldrich) and extinction coefficient as de- St. Louis, MO), produced by The Genome Center at Washington University scribed previously (51).
School of Medicine in St. Louis, and can be obtained from tritryp database.
Two of the heme-proteins of C. fasciculata (lanosterol 14α-demethylase and Δ6 Analysis of Fatty Acids. Lipids were extracted from P. serpens cell pellets by fatty acid desaturase) were not identified in the draft genome sequences, but a modified method of Bligh and Dyer (52) with dichloromethane used instead their partial sequences were amplified by PCR assay from C. fasciculata and of chloroform. The methyl esters were prepared by trans-esterifying the lipid extract with BF3-CH3OH at 85 °C for 1 h and analyzed using a gas chromato-graph (HRGC 5300; Carlo Erba) equipped with a flame ionization detector and Phylogenetic Analysis. Amino acid sequences of Δ9 fatty acid desaturases TR-FAME capillary column for the separation of Fatty Acid Methyl Esters from different eukaryotic lineages and bacteria were aligned using MAFFT (FAMEs) (60-m, 0.25-mm inner diameter and 0.25-μm film thickness; ThermoScientific). Hydrogen was used as the carrier gas with a pressure of 200 kPa.
6.717b (55) and manually edited using BioEdit (56). A maximum likelihood The following temperature ramp was used: 140 °C to 240 °C with a rate of tree was constructed with RAxML 7.0.3 using the PROTGAMMALG model 4 °C per min−1 and holding at 240 °C for 10 min. The flame ionization detector (57) (1,000 replications). The bootstrap supports of individual branches were was isothermal at 260 °C, and the injector was set to 250 °C. Separated fatty calculated using the same model after 1,000 iterations.
acids were identified by comparison of their retention times with knownstandards (37-component fatty acid methyl ester mix 47885-U, Supelco; Oxidative Stress Assay. The sensitivity of cells to oxidative stress was measured polyunsaturated fatty acid no. 3, menhaden oil).
by exposing them to paraquat added to the cultivation medium in a widerange of concentrations, ranging from 10−8 to 100 mM. After 44 h of in- Analysis of Sterols. Sterols were extracted and separated on TLC silica gel cubation, resazurin was added to each culture, and after 4 h, the viability of plates as described previously (43) and visualized by spraying the plates with cells was established by measurement of fluorescence. Obtained data were a water solution of 0.05% ferric chloride/5% (vol/vol) acetic acid/5% (vol/vol) analyzed by GraphPad Prism software using nonlinear regression (curve fit) sulfuric acid and heating to 100 °C for 15 min.
with a sigmoidal dose–response analysis (58, 59).
Detection and Activity Measurements of Respiratory Complex II. Mitochondria ACKNOWLEDGMENTS. We thank Martin Lukes for his help with gas chro- were isolated by hypotonic lysis as described previously (53). Protein lysates matography analysis of fatty acids. Michel Dollet and Patrick Wincker kindlyprovided the absence/presence data for selected genes in the Phytomonas were prepared by digitonin lysis (4 mg of digitonin per 1 mg of proteins, 1 h EM1 and Hart1 genomes. This work was supported by the Grant Agency of on ice) for native gel electrophoresis and histochemical staining and by the Czech Republic (Grants 204/09/1667, 206/08/1423, and P305/11/2179), dodecylmaltoside lysis (40 μL of 0.5 M aminocaproic acid and 10 μL of 10% (wt/ a Praemium Academiae award (to J.L.), the Algatech Project (CZ.1.05/ vol) dodecylmaltoside, 1 h on ice) for spectroscopic activity measurements and 2.1.00/03.0110), and the Scientific Grant Agency of the Slovak Ministry of SDS/PAGE. Whole-complex II was detected in 3–12% (wt/vol) clear native gel Education and the Academy of Sciences (Grant 1/0393/09).
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