Aerobic kinetoplastid flagellate Phytomonas does not require heme for viability.
ABSTRACT Heme is an iron-coordinated porphyrin that is universally essential as a protein cofactor for fundamental cellular processes, such as electron transport in the respiratory chain, oxidative stress response, or redox reactions in various metabolic pathways. Parasitic kinetoplastid flagellates represent a rare example of organisms that depend on oxidative metabolism but are heme auxotrophs. Here, we show that heme is fully dispensable for the survival of Phytomonas serpens, a plant parasite. Seeking to understand the metabolism of this heme-free eukaryote, we searched for heme-containing proteins in its de novo sequenced genome and examined several cellular processes for which heme has so far been considered indispensable. We found that P. serpens lacks most of the known hemoproteins and does not require heme for electron transport in the respiratory chain, protection against oxidative stress, or desaturation of fatty acids. Although heme is still required for the synthesis of ergosterol, its precursor, lanosterol, is instead incorporated into the membranes of P. serpens grown in the absence of heme. In conclusion, P. serpens is a flagellate with unique metabolic adaptations that allow it to bypass all requirements for heme.
- Microbiology 09/2002; 148(Pt 8):2273-82. · 2.85 Impact Factor
Article: The many roles of cytochrome b5.[show abstract] [hide abstract]
ABSTRACT: Four distinct suggestions have been made to explain the mechanism of the cytochrome b(5)-imposed positive modifier action of the cytochrome P450 monooxygenase reaction. The first mechanism involves a direct input of an electron into the monooxygenase cycle. This is the second of the two electrons necessary for activation of molecular oxygen, and appears to be a rate-limiting step in the monooxygenase reaction. P450 monooxygenases all appear to be uncoupled to varying extents, releasing superoxide and hydrogen peroxide instead of oxidized substrate. A second mechanism suggests that cytochrome b(5) acts as a positive modifier of the monooxygenase by decreasing the extent of uncoupling of the monooxygenase reaction. The implication is that a slow input of the second electron allows uncoupling of a superoxide anion instead of formation of two-electron reduced oxygen. Faster input of the second electron via cytochrome b(5) would result in formation of more of the activated oxygen that reacts with substrate to form product. A third suggestion involves formation of a two-hemoprotein complex between cytochrome b(5) and cytochrome P450 that allows acceptance of two electrons from NADPH-cytochrome P450 reductase. Uncomplexed cytochrome P450 accepts an electron from the reductase, dissociates from it, binds oxygen, and re-associates with the reductase to accept another electron. Complexation with cytochrome b(5) enhances the rate of formation of the active oxygen by obviating the need for two interactions with reductase. The fourth mechanism has cytochrome b(5) serving as an effector without a reduction-oxidation role in the monooxygenation reaction. This effector function may be to enhance the breakdown of the oxygenated hemoprotein to products or to facilitate flow of electrons through the system.Pharmacology [?] Therapeutics 03/2003; 97(2):139-52. · 7.79 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Globin-like oxygen-binding proteins occur in bacteria, yeasts and other fungi, and protozoa. The simplest contain protohaem as sole prosthetic group, but show considerable variation in their similarity to the classical animal globins and plant globins. Flavohaemoglobins comprise a haem domain homologous to classical globins and a ferredoxin-NADP+ reductase (FNR)-like domain that converts the globin into an NAD(P)H-oxidizing protein with diverse reductase activities. In Escherichia coli, the prototype flavohaemoglobin (Hmp) is clearly involved in responses to nitric oxide (NO) and nitrosative stress: (i) the structural gene hmp is upregulated by NO and nitrosating agents; (ii) purified Hmp binds NO avidly, but also converts it to nitrate (aerobically) or nitrous oxide (anaerobically); (iii) hmp mutants are hypersensitive to NO and nitrosative stresses. Here, we review recent advances in E. coli and the growing number of microbes in which globins are known, draw particular attention to the essential chemistry of NO and related reactive species and their interactions with globins, and suggest that microbial globins have additional functions unrelated to 'NO' stresses.Molecular Microbiology 06/2000; 36(4):775-83. · 4.96 Impact Factor
Aerobic kinetoplastid flagellate Phytomonas does not
require heme for viability
Ludˇ ek Koˇ renýa,b, Roman Sobotkab,c, Julie Kováˇ rováa,b, Anna Gnipováa,d, Pavel Flegontova,b, Anton Horváthd,
Miroslav Oborníka,b,c, Francisco J. Ayalae,1, and Julius Lukeˇ sa,b,1
aBiology Centre, Institute of Parasitology, Czech Academy of Sciences andbFaculty of Science, University of South Bohemia, 370 05?Ceské Bud? ejovice, Czech
Republic;cInstitute of Microbiology, Czech Academy of Sciences, 379 81 T? rebo? n, Czech Republic;dFaculty of Natural Sciences, Comenius University, 842 15
Bratislava, Slovakia; andeDepartment 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
as a protein cofactor for fundamental cellular processes, such as
electron transport in the respiratory chain, oxidative stress re-
sponse, or redox reactions in various metabolic pathways. Parasitic
kinetoplastid flagellates represent a rare example of organisms
that depend on oxidative metabolism but are heme auxotrophs.
Here, we show that heme is fully dispensable for the survival of
Phytomonas serpens, a plant parasite. Seeking to understand the
metabolism of this heme-free eukaryote, we searched for heme-
containing proteins in its de novo sequenced genome and exam-
ined several cellular processes for which heme has so far been con-
sidered indispensable. We found that P. serpens lacks most of the
port in the respiratory chain, protection against oxidative stress, or
desaturation of fatty acids. Although heme is still required for the
synthesis of ergosterol, its precursor, lanosterol, is instead incorpo-
rated into the membranes of P. serpens grown in the absence of
adaptations that allow it to bypass all requirements for heme.
various apoproteins giving rise to functional hemoproteins,
which are ubiquitous in biological systems and exhibit a wide
range of activities. The oxidation state of the iron is important
for most biological roles of heme, but its exact function is ulti-
mately determined by the properties of the polypeptide bound to
it (1). Heme can exist in either the oxidized ferric (Fe3+) or
reduced ferrous (Fe2+) state, which enables it to accept or do-
nate electrons and to function in various redox reactions and
The most abundant group of heme proteins are cytochromes
(2). In aerobic organisms that produce energy mainly through
oxidative phosphorylation, most of the synthesized heme is used
for the formation of the cytochromes functioning in the electron
transport respiratory chain. Other cytochromes, such as the
members of the cytochrome b5or cytochrome P450 family, are
involved in various redox reactions of specific metabolic path-
ways, such as desaturation of fatty acids and sterol biosynthesis,
and also in drug detoxification (3, 4). In catalases, heme func-
tions in the degradation of hydrogen peroxide, whereas in per-
oxidases, it oxidizes a wide variety of organic and inorganic
compounds in the presence of hydrogen peroxide. Through the
consumption of hydrogen peroxide, these enzymes greatly con-
tribute to the oxidative stress defense (5, 6). In addition to its
function as an electron carrier, heme iron has the capacity to
bind diatomic gases. Hemoglobin is well known as the oxygen
transporter in animals, but members of the same protein family
are widespread in all groups of organisms, including anaerobes.
The original roles of globins might have been the responses to
nitric oxide and nitrosative stress (7) or sensing of oxygen, which
was highly toxic to cells before they managed to adapt to an
eme is a tetrapyrrole molecule that consists of a porphyrin
ring coordinated with the iron molecule. It interacts with
aerobic environment (8). In soluble guanylyl cyclase, heme serves
as the nitric oxide sensor, and thus plays an important role in
signal transduction. Heme is also an important regulatory mol-
ecule because it reversibly binds to certain proteins, such as
transcription factors and ion channels, and thus modulates their
The central position of heme in a variety of cellular functions
makes it essential for the viability of virtually all living systems.
There are only a few examples of facultatively anaerobic or
pathogenic bacteria that do not require heme (10–12), but no
eukaryote that can survive without heme has been identified.
Most aerobic organisms synthesize heme by a multistep pathway
that is conserved in all three domains of life: bacteria, archaea,
and eukaryotes. A few eukaryotes that lost this pathway are
known to scavenge heme from external sources. For example,
ticks have easy access to heme from blood (13), whereas parasitic
nematodes uptake it either from their host or from endosymbiotic
bacteria (14). The free-living nematode Caenorhabditis elegans
bacteria it feeds on (15). Even the parasitic protists Entamoeba,
Trichomonas, and Giardia, which dwell in an anaerobic environ-
ment and do not need heme for processes connected to oxidative
metabolism, have retained a few hemoproteins, for which heme is
likely obtained from their hosts (16).
Flagellates of the order Kinetoplastea, which includes major
human parasites, depend on oxygen but are unable to produce
with heme to support their growth (17). Members of the genus
Trypanosoma lost the entire biosynthetic pathway and extract
heme from host blood (18, 19), whereas Leishmania spp. have
retained genes for the last three steps of the pathway, allowing
them to synthesize heme from their host-derived precursors (20).
Somekinetoplastids that parasitize insects obtain hemefrom their
bacterial endosymbionts, which can be eliminated by antibiotic
treatment, turning these protists into heme auxotrophs (17).
Kinetoplastid flagellates of the genus Phytomonas are impor-
tant yet understudied parasites of plants with a major economic
impact in Latin America and the Caribbean (21). They reside in
carbohydrate-rich tissues, such as phloem, latex, fruits, and seeds;
their ATP production is based on glycolysis (22). In the present
viability andpossesses uniquemetabolic propertiesthatallowitto
bypass all functions of this otherwise omnipresent molecule.
Author contributions: L.K., A.H., M.O., F.J.A., and J.L. designed research; L.K., R.S., J.K.,
and A.G. performed research; L.K., P.F., and M.O. analyzed data; and L.K., F.J.A., and J.L.
wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence may be addressed. E-mail: email@example.com or firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| March 6, 2012
| vol. 109
| no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1201089109
Results and Discussion
We cultivated P. serpens strain 9T in a chemically defined medium
without heme (Table S1) continuously for over a year without
noticeable decrease of the growth rate (generation time of ∼8 h),
compared with parallel cultures supplemented with heme (Fig.
1A). Abrupt transfer of cells grown in heme-containing medium
into one lacking it did not alter their growth. This is in contrast to
related flagellates, such as Crithidia fasciculata, which requires
heme for growth and was used as a control (Fig. 1A).
We sought to test the heme biosynthetic capacity of P. serpens
by measuring the amount of extractable heme. Even using a very
sensitive HPLC assay, we failed to detect any traces of heme in
detected 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 same
of heme is detected in P. serpens grown without heme (dotted line). +H, with heme; −H, without heme.
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
Table 1. Heme proteins of kinetoplastid flagellates
Heme proteinGenBankTBTC LeiCF PSPEPH
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 a3binding subunit of complex IV
Heme-dependent plant peroxidase homolog 1
Heme-dependent plant peroxidase homolog 2
Δ9 Fatty acid desaturase (cytochrome b5domain)
Δ4 Fatty acid desaturase (cytochrome b5domain)
Δ5 Fatty acid desaturase (cytochrome b5domain)
Δ6 Fatty acid desaturase (cytochrome b5domain)
Nitrate reductase (cytochrome b5domain)
Fumarate reductase-like (cytochrome b5domain)
Ferric reductase (cytochrome b561)
Ferric reductase (flavocytochrome b558)
Globin domain of adenylate cyclase-like protein
Cytochromes P450 with unknown function*
Cytochromes b5with 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 proteins
of 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 b5are
not listed individually. The number of these proteins is shown for each taxon.
Ko? rený et al.PNAS
| March 6, 2012
| vol. 109
| no. 10
cells grown in its absence (Fig. 1B). This indicates that P. serpens
is able to survive without heme, which is further supported by the
fact that, with the exception of ferrochelatase, no other genes for
heme synthesis were found in the draft genome of P. serpens
strain 9T obtained for this study. On the other hand, we found
a small amount of heme in cells growing in the medium sup-
plemented with heme (Fig. 1B), which implies that P. serpens is
able 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 using
heme, we decided to test cellular processes experimentally in
which heme is known to be involved. A screen for homologs of
heme-containing proteins in the genome produced only a few
hits, compared with the list of hemoproteins from related flag-
ellates (Table 1 and Table S2). The same results were obtained
for two other recently sequenced Phytomonas genomes (Table
1). 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 of
polyunsaturated fatty acids (23), a nitrate reductase, and two
different 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 the
medium does not increase the resistance of P. serpens against
oxidative stress induced by the superoxide generator paraquat
(Fig. S1). This is the opposite of what was found for the evolu-
tionarily related Trypanosoma brucei, which needs heme for ox-
idative stress defense (18).
Although P. serpens lacks the heme-containing respiratory
complexes III and IV (26–28), the mitochondrial respiratory
chain remains functional, serving to reoxidize NADH produced
during glycolysis (22, 29). Complex I is present in P. serpens (27,
30), which, instead of cytochrome c reductase (complex III) and
cytochrome c oxidase (complex IV), uses alternative oxidase to
reduce oxygen to water (31). We found that succinate de-
hydrogenase (complex II) is also present (Fig. 2), with a con-
served histidine residue in its SDH4 subunit, which supposedly
binds heme in the related Trypanosoma cruzi and other kineto-
plastids (32). Visualization of the P. serpens complex II by in-gel
staining in clear-native gel revealed that its abundance is not
influenced by the availability of heme in the medium (Fig. 2A).
Moreover, its size of ∼600 kDa is unaltered in the heme-de-
prived cells, being almost the same as in T. cruzi (32) and the
related C. fasciculata, used as a control (Fig. 2A), suggesting
a proper assembly of complex II in the absence of heme. To
assess the abundance of its subunits, we generated specific an-
tiserum against one subunit of the kinetoplastid complex II,
SDH1. The amount of the target protein was the same in cells
grown with or without heme (Fig. 2B). Furthermore, the absence
of heme did not affect the capacity of complex II to reduce
ubiquinone (Fig. 2 C and D).
These findings are in line with previous reports showing that
heme is not universally indispensable for the function of complex
II (33, 34). In mammalian cells, the absence of heme disrupts
proper assembly and inhibits the activity of complex II (35).
However, in yeast and Escherichia coli, the homologous com-
plexes retain physiological activity even without heme (33, 34). It
has been suggested that although heme does not participate in
the electron transfer in complex II and is not necessarily required
for the assembly of the complex, it may provide an electron sink
to protect against free radical damage during periods of high
electron flux (34). However, the presence of heme in the proton-
pumping complexes III and IV is indispensable, because it di-
rectly mediates electron transport. However, when enough en-
ergy is produced by glycolysis, these heme-containing complexes,
as well as the soluble cytochrome c, may be bypassed by using the
alternative terminal oxidase, which utilizes nonheme iron to
transfer electrons from ubiquinone directly to oxygen. This is
also known for the bloodstream (mammalian) stage of T. brucei,
which, similar to Phytomonas, dwells in a sugar-rich environment,
whereas the T. brucei procyclic (insect) stage has a fully de-
veloped mitochondrion equipped with the heme-containing
complexes (36). This metabolic switch is impossible in Phyto-
monas, which has lost the genes encoding the subunits of these
complexes from its genome (26, 28) (Table 1).
Because of its capacity to transfer electrons, heme participates
in various redox reactions, some of which are virtually universal
for eukaryotes. One of them is the desaturation of fatty acids. In
eukaryotes, this reaction needs electron equivalents that are
transferred from reduced cytochrome b5, and thus depends on
heme (37, 38). Many desaturases contain cytochrome b5 as
a domain conveniently fused to their N- or C-termini, including
the most widespread one, which creates the double bond in the
Δ9 position (23, 39). Our phylogenetic analyses revealed that this
fusion took place only once in the evolution of eukaryotic Δ9
fatty acid desaturases, specifically at the base of a superclade
comprising fungi, amoebozoans, rhodophytes, choanozoans, and
excavates, including kinetoplastids (Fig. 3A and Fig. S2). Re-
markably, Δ9 desaturase in P. serpens is the only member of this
superclade that conspicuously lacks the cytochrome b5domain,
apparently as a consequence of its secondary loss, a singular
active in P. serpens grown with (+H) or without (−H) heme. (A) Clear native
gel (3–12%) after in-gel staining for succinate dehydrogenase activity; C.
fasciculata (Cf) served as a control. Ferritin (monomeric and dimeric forms)
was used as a molecular weight marker (M). (B) Lysates from the same cells
as in A were analyzed by SDS/PAGE and immunoblotted with specific anti-
serum against the T. brucei subunit of complex II, SDH1. (C) Activity of suc-
cinate dehydrogenase in P. serpens grown without heme. The decrease in
absorbance (A600) with time (curve 2) was caused by the addition of ubi-
quinone to the reaction, which mediated the electron transfer from succi-
nate to 2,6-dichlorophenolindophenol. The activity was specifically inhibited
using malonate (curve 3). Curve 1 represents the background without ubi-
quinone. (D) Activity did not significantly differ between P. serpens grown
with (+H) or without (−H) heme. Medium values were calculated from three
Respiratory complex II (succinate dehydrogenase) is assembled and
| www.pnas.org/cgi/doi/10.1073/pnas.1201089109Ko? rený et al.
event among all known eukaryotes. To assess the ability of P.
serpens grown in the absence of heme to desaturate fatty acids,
we analyzed their composition by gas chromatography. We found
that P. serpens contains unsaturated fatty acids and that their
composition is virtually the same regardless of the presence or
absence of heme (Fig. 3B). These findings indicate that for the
desaturation of fatty acids, P. serpens is able to use an electron
donor other than cytochrome b5. It may likely be ferredoxin,
which serves this role for the desaturases of some bacteria and
plant plastids. For example, the plastid Δ12 fatty acid desaturase
of plants and diatoms depends on ferredoxin as an electron
donor, whereas a homologous desaturase with the same function
in the endoplasmic reticulum of the same organisms, as well as in
other eukaryotes, uses cytochrome b5(40). The possibility that
these redox molecules could substitute for each other has been
experimentally demonstrated in E. coli and in yeast expressing
cyanobacterial Δ6 fatty acid desaturase (41). Although ferre-
doxin is the natural electron donor for this desaturase, cyto-
chrome b5 fully complemented its function when fused or
coexpressed with the desaturase enzyme. Three different ferre-
doxin homologs were identified in the genomic sequences of P.
serpens (Table S2).
The oxidative 14α-demethylation of lanosterol, another key
reaction in the eukaryotic cell, fully depends on heme. Its sub-
stitution by means of analogous nonheme enzyme has never been
documented. This reaction is a crucial step in the synthesis of
sterols, such as cholesterol in animals or ergosterol in fungi, as
well as in protists, including kinetoplastid flagellates (42). It is
catalyzed by lanosterol 14α-demethylase (CYP51), which belongs
to the cytochrome P450 family, found in most eukaryotes, in-
cluding Phytomonas spp. (Table 1). No eukaryotic cell can
function without sterols or their analogs in its membranes; in-
hibition of this enzymatic step is thus frequently lethal (43).
Consequently, CYP51 is a popular target of fungicides and other
drugs, which are also effective against kinetoplastids (44). Until
now, the only kinetoplastid known to be naturally resistant to
inhibitors of CYP51 is Leishmania braziliensis, a flagellate closely
related to Phytomonas, which seems to be able to incorporate
14-methyl sterols into its membranes (45). We have found that
P. serpens possesses this unique capability as well. Based on the
TLC analysis, the cells synthesized a sterol that corresponded to
the ergosterol standard only when heme was added to the growth
medium. In contrast, they accumulated lanosterol in the absence
of heme with no impact on cell viability (Fig. 3C). The fact that
certain eukaryotes are able to use lanosterol but others are not is
very interesting and implies the existence of some regulatory
mechanism. Cholesterol-deficient human T cells can adapt to
growth with lanosterol; the initial growth of these cells dropped
10-fold when cholesterol was depleted, yet their prolonged cul-
tivation resulted in a growth rate ∼65% that of the cholesterol-
supplemented cells (46). A study on yeast revealed that what
regulates the incorporation of lanosterol in the membranes is the
level of synthesized heme (47). The growth of P. serpens in the
absence of heme precludes the activity of CYP51; thus, this
flagellate meets the two conditions that are required in yeast for
lanosterol utilization (low heme levels and CYP51 inhibition).
Overall, there are several cellular processes for which heme is
crucial in a typical eukaryote, yet it is dispensable in P. serpens.
Somewhat lower dependence of a typical kinetoplastid on heme
has been noted when cystathionine-β-synthase, a hemoprotein of
animals and amoebae that is essential for cysteine formation, was
shown to lack heme in kinetoplastids (48). However, P. serpens is
unique, because it lacks most hemoproteins that are present even
in closely related protists. Moreover, the few retained in the P.
serpens genome are not crucial for its survival, at least under
culture conditions. In addition to CYP51 and the SDH4 subunit
of respiratory complex II, we identified 13 proteins that sup-
posedly bind heme, because they are homologous to cytochrome
b5(Table 1). Their functions are unknown, however, and 5 of
them lack the HPGG heme-binding motif typical for cytochrome
b5(41). Thus, it is by no means certain that these proteins ac-
tually bind heme in vivo. One of them is a protein recently
identified in the flagellar proteome of T. brucei, shown to be
indispensable for the bloodstream stage but nonessential for the
procyclic stage (49). Therefore, the only process for which heme,
if present, was found to be actively used by P. serpens, is the
14α-demethylation of lanosterol in the ergosterol biosynthetic
acid desaturases (FADS). P. serpens is the only organism that secondarily lost the cytochrome b5domain. The full phylogenetic tree of Δ9-fatty acid desaturase
is shown in Fig. S1. (B) Analyses of fatty acid composition by gas chromatography demonstrate that in P. serpens, the desaturation of fatty acids is not affected
by the absence of heme. (C) Analysis of sterol composition by TLC. Ergosterol, which is the major membrane sterol of Trypanosomatida, and lanosterol, the
precursor 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.
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
Ko? rený et al.PNAS
| March 6, 2012
| vol. 109
| no. 10
pathway (Fig. 3C). Surprisingly, however, in vitro growth remains
unaffected by the lack of this activity.
It is conceivable that some anaerobic eukaryotes possessing only
a few of the known hemoproteins may survive without heme as
well; however, this will be hard to test, because, so far, none of
these anaerobic protists can be grown in a chemically defined
medium. Furthermore, anaerobic protists need to obtain some
products of heme-dependent enzymes, such as cholesterol and
fatty acids from their environment; thus, their existence cannot be
considered to be independent of heme (50). To the best of our
knowledge, P. serpens is the only eukaryote that can survive
without heme and yet depends on oxidative metabolism. This
unique metabolic property, a feature likely developed as an ad-
aptation to the carbohydrate-rich environment of plant sap, makes
it an ideal model to study different cellular functions in a heme-
free background, which may shed further light on the exact roles
and essentiality for life of the otherwise omnipresent heme.
Materials and Methods
Cultivation Conditions and Growth Curves. Both P. serpens and C. fasciculata
were grown in a chemically defined medium (Table S1) supplemented with
different concentrations of hemin at 27 °C and shaking at 80 rpm, daily
diluted with fresh media to the density of 6 × 106cells per milliliter. Cell
concentration was measured daily using a Beckman Coulter Z2 counter.
Quantification of Heme b. In total, 2 × 109cells of P. serpens, C. fasciculata, and
the bloodstream form of T. brucei were filtrated through a DEAE-cellulose
column and washed five times with PBS buffer to remove all traces of heme
from the media. The cell pellets were extracted with methanol/0.2% NH4OH,
and heme was extracted from the delipidated cells with acetone/2% HCl (vol/
vol) and separated by HPLC ona Nova-Pak C18 column (4-μm particle size, 3.9 ×
150 mm; Waters) using linear gradient 25–100% (vol/vol) acetonitrile/0.1% tri-
fluoroacetic acid at a flow rate of 1.1 mL/min at 40 °C. Heme b was detected by
diode array detector (Agilent 1200; Agilent Technologies) and quantified using
authentic hemin standard (Sigma–Aldrich) and extinction coefficient as de-
scribed previously (51).
Analysis of Fatty Acids. Lipids were extracted from P. serpens cell pellets by
amodified method of Bligh andDyer (52) with dichloromethane used instead
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-
TR-FAME capillary column for the separation of Fatty Acid Methyl Esters
(FAMEs) (60-m, 0.25-mm inner diameter and 0.25-μm film thickness; Thermo
Scientific). Hydrogen was used as the carrier gas with a pressure of 200 kPa.
The following temperature ramp was used: 140 °C to 240 °C with a rate of
4°Cpermin−1andholding at 240°C for10 min.The flameionizationdetector
was isothermal at 260 °C, and the injector was set to 250 °C. Separated fatty
acids were identified by comparison of their retention times with known
standards (37-component fatty acid methyl ester mix 47885-U, Supelco;
polyunsaturated fatty acid no. 3, menhaden oil).
Analysis of Sterols. Sterols were extracted and separated on TLC silica gel
plates as described previously (43) and visualized by spraying the plates with
a water solution of 0.05% ferric chloride/5% (vol/vol) acetic acid/5% (vol/vol)
sulfuric acid and heating to 100 °C for 15 min.
Detection and Activity Measurements of Respiratory Complex II. Mitochondria
were isolated by hypotonic lysis as described previously (53). Protein lysates
were prepared by digitonin lysis (4 mg of digitonin per 1 mg of proteins, 1 h
on ice) for native gel electrophoresis and histochemical staining and by
SDS/PAGE. Whole-complex II was detected in 3–12% (wt/vol) clear native gel
(80 μg of proteins per line) by incubating in a staining solution [50 mM NaPi
(pH 7.4), 84 mM sodium succinate, 0.2 mM N-methylphenazonium methyl
sulfate, 4.5 mM EDTA (pH 8.5), 10 mM potassium cyanide, 2 mg/mL Nitro-
tetrazolium blue chloride) for 3 h at room temperature in dark. Nitro-
tetrazolium blue chloride changes color on accepting electrons from succinate
via N-methylphenazonium methyl sulfate, a process catalyzed by complex II.
SDH1 subunit of complex II was detected by Western blot analysis using 10%
(wt/vol) SDS/PAGE and a specific polyclonal antiserum generated against the
oligopeptide SHLSKAYPVIDHTFDC [SDH1 subunit of T. brucei (Tb927.8.6580)
in a rabbit].
Specific succinate dehydrogenase activity was measured using the fol-
lowing protocol: 5 μL of mitochondrial protein lysate was incubated with
1 mL of succinate dehydrogenase solution [25 mM KPi (pH 7.2), 5 mM MgCl2,
20 mM sodium succinate] for 10 min at 30 °C. This mixture was transferred in
the cuvette, and antimycin A (2 μg/mL), rotenone (2 μg/mL), potassium cy-
anide (2 mM), and 2,6-dichlorphenolindophenol (50 μM) were added.
Background absorbance at 600 nm was then measured for 5 min. The re-
action was triggered by adding 65 μM coenzyme Q2, and the absorbance at
600 nm was measured every 20 s for 5 min. Change in absorbance was
caused by the electron transfer from succinate via coenzyme Q2 to 2,6-
dichlorophenolindophenol. The activity was specifically inhibited by the
addition of 1 mM sodium malonate.
Genome Sequencing, Assembly, and Protein Search. P. serpens nuclear DNA
fraction was sequenced using Illumina technology at BGI-Hong Kong (HiSeq
2000 sequencing system, average insert size of 500 bp, read length of 90 bp). A
dataset of 1.62 Gbp was obtained after basic filtering of low-quality reads.
Genome assembly with MIRA 3.4rc2 (54) produced 5,399 contigs longer than
500 bp (N50 contig size of 6,781 bp) with average coverage 60 (genome as-
sembly deposited in National Center for Biotechnology Information BioProject
database under accession no. PRJNA80957). Translated reads and contigs were
screened using tblastn 2.2.24+ with e-value cutoffs at 10−3and 10−10, re-
spectively, against Leishmania major heme-binding proteins (Table 1) and
heme-synthesis enzymes. Conserved protein domains were identified using
InterPro database. Draft genome sequences of C. fasciculata were kindly
provided by Stephen M. Beverley (Washington University School of Medicine,
St. Louis, MO), produced by The Genome Center at Washington University
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
fatty acid desaturase) were not identified in the draft genome sequences, but
their partial sequences were amplified by PCR assay from C. fasciculata and
sequenced (Table S2).
Phylogenetic Analysis. Amino acid sequences of Δ9 fatty acid desaturases
from different eukaryotic lineages and bacteria were aligned using MAFFT
6.717b (55) and manually edited using BioEdit (56). A maximum likelihood
tree was constructed with RAxML 7.0.3 using the PROTGAMMALG model
(57) (1,000 replications). The bootstrap supports of individual branches were
calculated using the same model after 1,000 iterations.
Oxidative Stress Assay. The sensitivity of cells to oxidative stress was measured
by exposing them to paraquat added to the cultivation medium in a wide
range of concentrations, ranging from 10−8to 100 mM. After 44 h of in-
cubation, resazurin was added to each culture, and after 4 h, the viability of
cells was established by measurement of fluorescence. Obtained data were
analyzed by GraphPad Prism software using nonlinear regression (curve fit)
with a sigmoidal dose–response analysis (58, 59).
ACKNOWLEDGMENTS. We thank Martin Luke? s for his help with gas chro-
matography analysis of fatty acids. Michel Dollet and Patrick Wincker kindly
provided the absence/presence data for selected genes in the Phytomonas
EM1 and Hart1 genomes. This work was supported by the Grant Agency of
the Czech Republic (Grants 204/09/1667, 206/08/1423, and P305/11/2179),
a Praemium Academiae award (to J.L.), the Algatech Project (CZ.1.05/
2.1.00/03.0110), and the Scientific Grant Agency of the Slovak Ministry of
Education and the Academy of Sciences (Grant 1/0393/09).
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Ko? rený et al. 10.1073/pnas.1201089109
Hemin added to the medium did not improve the resistance against oxidative stress, but it worsened it. This is likely attributable to the toxic effects of free
heme, which itself causes the generation of reactive oxygen species.
Dependence of the viability of P. serpens grown in different concentrations of hemin on the concentration of the superoxide generator paraquat.
Ko? rený et al. www.pnas.org/cgi/content/short/12010891091 of 8
Fig. S2.Phylogenetic tree of Δ9-fatty acid desaturase.
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Table S1.Defined medium for cultivation of P. serpens
β-Glycerophosphate × 5H2O, Na2
Citric acid × H2O
K3citrate × H2O
L-Histidine × HCl × H2O
L-Cystine × 2HCl
D-Pantothenic acid × ½Ca
Trace elements solution (1,000×)
Na4EDTA × 4H2O
Na2EDTA × 2H2O
0.06 (0.002) g/L†
2.2 g/100 mL
1.1 g/100 mL
0.5 g/100 mL
0.5 g/100 mL
0.16 g/100 mL
0.16 g/100 mL
0.11 g/100 mL
6.5 g/100 mL
0.77 g/100 mL
*Added in the form of amino acid mixture: MEM Amino acid solution
(M5550; Sigma–Aldrich), 3× concentration.
†Added in the form of vitamin mixture: MEM Vitamin solution (M6895;
Sigma–Aldrich), 1× concentration.
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Table S2. Newly generated sequences that are referred to in the main text
Organism/name of protein Sequence
P. serpens/ferrochelatase atgtcaaaccctctttgtctgccggcggagtacaacacacggcgacgcta
P. serpens/lanosterol 14α-demethylase
P. serpens/heme-binding subunit of the
respiratory complex II
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Organism/name of proteinSequence
P. serpens/Δ9 fatty acid desaturaseatgtctctaaaacaaaatccaaatgggcttgaaacccatgaaaaacctcctcaaca
P. serpens/cytochrome b5homolog 1
P. serpens/cytochrome b5homolog 2
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Organism/name of proteinSequence
P. serpens/cytochrome b5homolog 3 atgaaggaagtgaaagttacaaccagaggcgataacgttgcaagcatg
P. serpens/cytochrome b5homolog 4
P. serpens/cytochrome b5homolog 5
P. serpens/cytochrome b5homolog 6
P. serpens/cytochrome b5homolog 7
P. serpens/cytochrome b5homolog 8
P. serpens/cytochrome b5homolog 9
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Organism/name of proteinSequence
P. serpens/cytochrome b5homolog 10atgaacacttatgataaagcatccgaagagacctccaaggtctctgatgat
P. serpens/cytochrome b5homolog 11
P. serpens/cytochrome b5homolog 12
P. serpens/cytochrome b5homolog 13
P. serpens/ferredoxin homolog 1
P. serpens/ferredoxin homolog 2
P. serpens/ferredoxin homolog 3
C. fasciculata/lanosterol 14α-demethylase
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Organism/name of proteinSequence
C. fasciculata/Δ6 fatty acid desaturase
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