Group Title: BMC Genomics
Title: Comparative genomics of bacterial and plant folate synthesis and salvage : predictions and validations
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Title: Comparative genomics of bacterial and plant folate synthesis and salvage : predictions and validations
Physical Description: Book
Language: English
Creator: de Crécy-Lagard,Valérie
El Yacoubi, Basma
de la Garza, Rocìo Dìaz
Noiriel, Alexandre
Hanson, Andrew
Publisher: BMC Genomics
Publication Date: 2007
Abstract: BACKGROUND:Folate synthesis and salvage pathways are relatively well known from classical biochemistry and genetics but they have not been subjected to comparative genomic analysis. The availability of genome sequences from hundreds of diverse bacteria, and from Arabidopsis thaliana, enabled such an analysis using the SEED database and its tools. This study reports the results of the analysis and integrates them with new and existing experimental data.RESULTS:Based on sequence similarity and the clustering, fusion, and phylogenetic distribution of genes, several functional predictions emerged from this analysis. For bacteria, these included the existence of novel GTP cyclohydrolase I and folylpolyglutamate synthase gene families, and of a trifunctional p-aminobenzoate synthesis gene. For plants and bacteria, the predictions comprised the identities of a 'missing' folate synthesis gene (folQ) and of a folate transporter, and the absence from plants of a folate salvage enzyme. Genetic and biochemical tests bore out these predictions.CONCLUSION:For bacteria, these results demonstrate that much can be learnt from comparative genomics, even for well-explored primary metabolic pathways. For plants, the findings particularly illustrate the potential for rapid functional assignment of unknown genes that have prokaryotic homologs, by analyzing which genes are associated with the latter. More generally, our data indicate how combined genomic analysis of both plants and prokaryotes can be more powerful than isolated examination of either group alone.
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BMC Genomics

Research article

Comparative genomics of bacterial and plant folate synthesis and
salvage: predictions and validations
Valerie de Crecy-Lagard*1, Basma El Yacoubi1, Rocio Diaz de la Garza2,
Alexandre Noiriel2 and Andrew D Hanson2

Address: 'Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA and 2Department of Horticultural
Sciences, University of Florida, Gainesville, FL 32611, USA
Email: Valerie de Crecy-Lagard*; Basma El Yacoubi; Rocio Diaz de la Garza;
Alexandre Noiriel; Andrew D Hanson
* Corresponding author

d Central

Published: 23 July 2007
8MC Genomics 2007, 8:245 doi:10.1186/1471-2164-8-245

Received: 5 December 2006
Accepted: 23 July 2007

This article is available from:
2007 de Crecy-Lagard et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Folate synthesis and salvage pathways are relatively well known from classical
biochemistry and genetics but they have not been subjected to comparative genomic analysis. The
availability of genome sequences from hundreds of diverse bacteria, and from Arabidopsis thaliana,
enabled such an analysis using the SEED database and its tools. This study reports the results of the
analysis and integrates them with new and existing experimental data.
Results: Based on sequence similarity and the clustering, fusion, and phylogenetic distribution of
genes, several functional predictions emerged from this analysis. For bacteria, these included the
existence of novel GTP cyclohydrolase I and folylpolyglutamate synthase gene families, and of a
trifunctional p-aminobenzoate synthesis gene. For plants and bacteria, the predictions comprised
the identities of a 'missing' folate synthesis gene (folQ) and of a folate transporter, and the absence
from plants of a folate salvage enzyme. Genetic and biochemical tests bore out these predictions.
Conclusion: For bacteria, these results demonstrate that much can be learnt from comparative
genomics, even for well-explored primary metabolic pathways. For plants, the findings particularly
illustrate the potential for rapid functional assignment of unknown genes that have prokaryotic
homologs, by analyzing which genes are associated with the latter. More generally, our data indicate
how combined genomic analysis of both plants and prokaryotes can be more powerful than isolated
examination of either group alone.

Folates are tripartite molecules comprising pterin, p-ami-
nobenzoate (pABA), and glutamate moieties to which
one-carbon units at various oxidation levels can be
attached at the N5 and N10 positions (Figure 1). In natu-
ral folates the pterin ring is in the dihydro or tetrahydro
state, and a short, y-linked polyglutamyl tail of up to

about eight residues is usually attached to the first gluta-

Tetrahydrofolates serve as cofactors in one-carbon transfer
reactions during the synthesis of purines, formylmethio-
nyl-tRNA, thymidylate, pantothenate, glycine, serine, and
methionine [1] (Figure 2). Most folate-dependent

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y-Glu tail

Pterin pABA Glu
0 0 0 COOH
N H-N -N-H
HN .H H \ / H H) COOH
C-N- -CH
11 H I
LO In (CH2)2

Figure I
The structure of tetrahydrofolate. In natural folates, the
pterin ring exists in tetrahydro form (as shown) or in 7,8-
dihydro form (as in DHF). The ring is fully oxidized in folic
acid, which is not a natural folate. Folates usually have a y-
linked polyglutamyl tail of up to about eight residues attached
to the first glutamate. One-carbon units (formyl, methyl, etc.)
can be coupled to the N5 and/or N 10 positions.

enzymes strongly prefer polyglutamates to monogluta-
mates, but the opposite is usually true of folate transport-
ers so that polyglutamylation is generally considered to
favor folate retention within cells and subcellular com-
partments [2,3].

Plants, fungi, certain protests, and most bacteria make
folates de novo, starting from GTP and chorismate, but
higher animals lack key enzymes of the synthetic pathway
and so require dietary folate [4-7]. Folates are crucial to
human nutrition and health [3], and antifolate drugs are
widely used in cancer chemotherapy and as antimicrobi-
als [3,7,8]. For these reasons, folate synthesis and salvage
pathways have been extensively characterized in model
organisms, and the folate synthesis pathway in both bac-
teria and plants has been engineered in order to boost the
folate content of foods [9-11].

The de novo folate synthesis pathway has the same steps in
bacteria and plants, and consists of a pterin branch and a
pABA branch (Figure 3, rose and blue color, respectively).
The first enzyme of the pterin branch is GTP cyclohydro-
lase I (GCHY-I, EC, which catalyzes a complex
reaction in which the five-membered imidazole ring of
GTP is opened, C8 is expelled as format, and a six-mem-
bered dihydropyrazine ring is formed using Cl and C2 of
the ribose moiety of GTP 1[5]. The resulting 7,8-dihydrone-
opterin triphosphate is then converted to the correspond-
ing monophosphate by a specific pyrophosphatase [5,12].
Removal of the last phosphate is believed to be mediated
by a non-specific phosphatase [5]. Dihydroneopterin
aldolase (DHNA, EC then releases glycolalde-
hyde to produce 6-hydroxymethyl-7,8-dihydropterin,

which is then pyrophosphorylated by hydroxymethyldi-
hydropterin pyrophosphokinase (HPPK, EC
DHNA also interconverts 7,8-dihydroneopterin and 7,8-
dihydromonapterin, and cleaves the latter to 6-
hydroxymethyl-7,8-dihydropterin. A paralog of DHNA,
FoIX, interconverts the triphosphates of 7,8-dihydroneop-
terin and 7,8-dihydromonapterin, and also catalyzes the
same reactions as DHNA at very slow rates [13].

In the pABA branch of the pathway, chorismate is ami-
nated to aminodeoxychorismate (ADC) by ADC synthase
(EC using the amide group of glutamine as amino
donor [51. ADC is then converted to pABA by ADC lyase
(EC [51.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphate and
pABA moieties are condensed by dihydropteroate syn-
thase (DHPS, EC The resulting dihydropteroate
is glutamylated by dihydrofolate synthase (DHFS, EC giving dihydrofolate (DHF), which is reduced
by dihydrofolate reductase (DHFR, EC to tetrahy-
drofolate (THF). Folylpolyglutamate synthase (FPGS, EC then adds a y-glutamyl tail. In Escherichia coli, it
has been reported that there can also be a linkages in the
distal part of the polyglutamyl tail [14].

Although the biosynthetic steps are the same in plants and
bacteria, the plant pathway is split between three subcel-
lular compartments, with pterin synthesis in the cytosol,
pABA synthesis in chloroplasts, and the other steps in
mitochondria (Figure 4) [6]. FPGS isoforms are present in
all three of these compartments, as are folates themselves
[15,16]. Folates both poly- and monoglutamates are
also found in plant vacuoles [16]. The highly compart-
mented nature of folate synthesis in plants implies the
existence of pterin and folate transporters that are integral
components of the pathway.

Folate-related salvage pathways are of three kinds. The
first ('intact folate salvage') (Figure 3, green color) enables
utilization of supplied folic acid and DHF, and relies on a
DHFR activity to reduce these oxidized folates to THF, and
on an FPGS activity [7]. DHFR activity is also required to
recycle the DHF produced in the reaction catalyzed by
thymidylate synthase (TS, EC The second kind
of salvage ('pterin salvage') (Figure 3, yellow color),
known in Leishmania and other trypanosomatid parasites,
involves the reduction of fully oxidized pterins to the
dihydro and tetrahydro levels by pteridine reductase 1
(PTR1, EC [171. This enables oxidized pterins to
be used (after reduction to dihydro forms) for folate syn-
thesis, and (after reduction to tetrahydro forms) as cofac-
tors for aromatic hydroxylases and other pterin-
dependent enzymes. Finally, some bacteria, plants, and
protests probably carry out a more radical kind of salvage,

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BMC Genomics 2007, 8:245





THF 5-Methyl-THF

thyA tFt SDH Sarcosine
Thymidylate metF SlyA
tA-I glyA
5,10-Methylene-THF o Ser
Ketopantoate BpanB t GCV Gly

5,10-Methenyl-THF lyA 5-Formyl-THF

fo/lD gyA

Purines 4- 10-Formyl-THF Formate
purN purU


fm t


Figure 2
Major folate-dependent reactions of one-carbon metabolism. The gene names are for E. coli (except for sarcosine
dehydrogenase). Note that the formation of 5-formyl-THF from 5,10-methenyl-THF occurs via a second catalytic activity of
serine hydroxymethyltransferase (glyA), and that 5-formyl-THF is reconverted to 5,10-methenyl-THF by 5-formyl-THF cycloli-
gase (ygfA). For simplicity, THF is not shown as a participant in most reactions in which it is consumed or released. GCV, gly-
cine cleavage complex, comprising the products of the gcvT, gcvH, gcvP, and lpd genes; SDH, sarcosine dehydrogenase (not
present in E. coli).

in which the pterin and pABA-glutamate fragments pro-
duced by folate breakdown are recycled for folate synthe-
sis [18]. This type of salvage has been little studied and
will not be considered further in this article.

Genes for all the enzymes of folate synthesis have been
identified in model organisms such as Escherichia coli, Sac-
charomyces cerevisiae, and Arabidopsis thaliana [4-6]. Like-
wise, the intact folate salvage pathway has been well
characterized in mammals, the malaria parasite Plasmo-
dium, and Lactobacillus casei [7,19,20], and pterin salvage
in Leishmania [17]. However, analysis of the distribution
of known folate synthesis and salvage genes in hundreds
of bacterial genomes using the SEED platform [21] reveals
that much remains to be learnt about both synthesis and

The SEED is a freely available, open-source database that
provides efficient ways to discover new genes or pathways,
to generate predictions about gene function, and to
improve annotations, based on a 'functional subsystem
approach' [21]. This approach has much in common with
metabolic reconstruction [22,23]. A functional subsystem
may be defined as a set of functional roles (usually ten to
twenty) jointly involved in a biological process. A typical
subsystem is a group of enzymes, transporters, and regu-
latory components that participate in a metabolic path-
way such as folate synthesis or salvage. Subsystem analysis
examines which components are actually present in a
genome and which should be present but cannot be iden-
tified, and so provides a picture of what is actually missing.
This sets the stage to pursue the 'missing genes', also
termed 'pathway holes' [24-26]. Homology-based

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BMC Genomics 2007, 8:245


I hd I .1-, ... -Ii

IIIh. .1, ,I


Ptin branch and de novo IIL' jII Z11.ii 3Ivagel

pABA salvageIIHF

Figure 3
Folate synthesis and salvage pathways. Gene names are white-on-gray; all except folQ and PTRI are from E coli. The folQ
gene has been identified only in Lactococcus lactis and plants, and PTRI only in Leishmania and other trypanosomatids. Note that
DHN aldolase also mediates epimerization of DHN to 7,8-dihydromonapterin and aldol cleavage of 7,8-dihydromonapterin.
ADC, aminodeoxychorismate; DHFR, dihydrofolate reductase; DHFS, dihydrofolate synthase; DHNA, dihydroneopterin aldo-
lase; DHNTPase, dihydroneopterin triphosphate pyrophosphatase; DHPS, dihydropteroate synthase; FPGS, folylpolyglutamyl
synthase; HPPK, hydroxymethyldihydropterin pyrophosphokinase; NP, nonspecific phosphatase; PTRI, pteridine reductase I;
the subscript x denotes the fully oxidized forms of pterins.

searches alone are usually unable to locate missing genes
that have not been previously identified in any genome
('globally missing genes') or those that are missing due to
non-orthologous gene replacement ('locally missing
genes') [271.

In this study, we first predicted the pathways (de novo
folate synthesis, intact folate salvage, and pterin salvage)
present in around four hundred sequenced bacteria and
identified cases of missing genes for almost every step of
the synthesis pathway. Candidates for such missing genes
in bacteria and plants were then predicted using compar-
ative genomic tools and representative candidates were
tested experimentally.

Results and Discussion
Are folates essential in all bacteria?
As folate-dependent formylation of the initiator tRNA is a
hallmark of bacterial translation and bacteria cannot
import formylmethionyl-tRNA [28], we investigated the
distribution of the fmt gene encoding methionyl-tRNA
formyltransferase (EC as a signature gene for a
folate requirement. Homologs of fmt are found in all
sequenced genomes except Mycoplasma hyopneumoniae
and Onion yellows phytoplasma OY-M (Table 1). We
confirmed the observation [29] that M. hyopneumoniae
lacks all the enzymes of folate-mediated one-carbon
metabolism except for glycine hydroxymethyltransferase
(GlyA), which has aldolase activities that do not require

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Glu THF Glu

THF-Glun THF-Glun


-y *.^




Figure 4
Compartmentation of the folate synthesis pathway in plants. The steps in the pterin branch of the pathway are in
blue, those in the pABA branch are in green, and the others (condensation, glutamylation, and reduction) are in red. Note that
the compartmentation of the pathway and of its folate end products implies the existence of pterin or folate carriers in the
mitochondria, chloroplast, and vacuolar membranes. Pathway intermediates are designated by the symbols used in Figure 3.
THF-Glun, THF polyglutamates.

folate [30]. Another widespread folate-dependent meta-
bolic step is the conversion of dTMP to dUMP, catalyzed
by thymidylate synthase (ThyA, EC This step
can also be performed by a folate- and flavin-dependent
thymidylate synthase (ThyX) [31]. As first observed by
Myllykallio et al. [32], most bacteria have a thyA or a thyX
homolog, some have both, and the few that have neither
- such as M. hyopneumoniae or Ureaplasma parvum con-
tain the tdk gene encoding the thymidine (dT) salvage
enzyme thymidine kinase. Our genomic analysis suggests
that M. hyopneumoniae strains are the only sequenced bac-
teria that do not require folate for initiator tRNA formyla-
tion or thymidylate synthesis. The situation in the
phytoplasma that lacks the fm t gene (Table 1) is different;

it contains a thyA homolog like most Mycoplasma species
and therefore presumably requires intact folates.

Intact folate transport and salvage
As just discussed, folate is most probably essential for all
sequenced bacteria except M. hyopneumoniae. However,
not all bacteria synthesize folate de novo but instead rely
on an external supply [see Additional File 1, variant 001;
see "Methods" for an explanation of the variant code]. To
predict the absence of the de novo synthesis pathway, the
HPPK (FolK) and DHPS (FolP) proteins were used as sig-
nature proteins (for reasons described below). Many bac-
teria lack homologs of both these genes (Table 1) and so
almost certainly rely on reducing and glutamylating intact

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Glu PPi


C tosf^C


BMC Genomics 2007, 8:245



Table I: Examples of bacteria dependent on folate salvage

De novo signature enzymes Folate salvage pathway

Folate requiring enzymes Folate independent dTMP synthesis

FolP Dhfra FolCb ThyA GlyA Fmt

Lactobacillus acidophilus
Pediococcus pentosaceus
Buchnera aphidicola (3 strains)
Rickettsia rickettsit
Bartonella henseloe str. Houston-1I
Mycoplasma hyopneumoniae (3 strains)
Mycoplasma genitalium G-37
Mycoplasma synovice 53
Ureaplasma parvum ATCC 700970
Mesoplasma forum LI
Onion yellows phytoplasma OY-M
Spiroplasma kunkelii CR2-3x
Borrelic burgdorferi B3 I
Treponema pallidum subsp. pallidum

+ +
+ +
+ +
? +

- +
- +
- +
- + +
- + +
- +
- ?
-? +

aCombination of the presence DHFRO, DHFRI or DHFR2; b Combination of the presence of FolC and FolC2; (+): gene identified; blankcell with (-
): no gene identified; (?): predicted missing gene

folates taken up from the environment. These are mainly
host-associated bacteria such as Mycoplasma or Treponema
or organisms that live in folate-rich environments such as
Lactobacilli. Chloroplasts and vacuoles must likewise take
up folates from the cytoplasm (Figure 4), and there is also
evidence for folate uptake by intact plant cells [6].

(i) Transport
Systems that mediate folate uptake in auxotrophs such as
Lactobacillus casei and L. salivarius have been partially bio-
chemically characterized [33,34], but the corresponding
genes remain unknown. Whatever they are, they are most
likely unrelated to mammalian folate carriers (i.e., the
reduced folate carrier, the folate receptor, the intestinal
folate carrier, and the mitochondrial folate carrier) since
these lack close homologs among bacteria and plants.
However, cyanobacteria, which are folate prototrophs,
have a protein with significant similarity to a folate carrier
from Leishmania species (FT1), and the cyanobacterial
protein has a close homolog in plants (52% amino acid
identity), as well as several more distant relatives in plants
and in alpha-, beta-, and gamma-proteobacteria. We
showed first that the cyanobacterial protein (Synechocystis
slr0642) conferred the ability to transport folates and
folate analogs when expressed in E. coli, and then that its
plant homolog (Arabidopsis At2g32040) did the same
[35]. We further showed that the Arabidopsis At2g32040
protein is located in the chloroplast envelope [35]. The
weak slr0642 homolog in some alpha-proteobacteria (Sil-
icibacter, Roseobacter) clusters with the folate-dependent
enzyme sarcosine dehydrogenase, suggesting that this
protein may also be a folate transporter.

Thus, despite progress in identifying folate transporters in
cyanobacteria and in the chloroplast envelope, there are

as yet no candidates for the folate carriers in many folate-
requiring bacterial taxa, or in plant mitochondrial, vacu-
olar, and plasma membranes. These still-missing genes
are future prospects for discovery by comparative genom-
ics methods [36].

(ii) Reduction
As noted above, DHFR is essential in both de novo and sal-
vage pathways. Most bacteria have a folA gene (DHFRO),
but two other bacterial enzymes able to reduce DHF are
now known: FolM (DHFR1) belonging to the short-chain
dehydrogenase/reductase (SDR) family [37], and a flavin-
dependent dihydropteroate reductase that is fused to
dihydropteroate synthase (DHFR2). [381. The trypano-
somatid enzyme PTR1 can also reduce DHF and folic acid
[171. AsfolM occurs in E. coli and other bacteria that also
have afolA gene, its normal function is most probably not
folate reduction, as discussed in a later section. The anno-
tation of DHFRO family members is complicated by their
similarity to pyrimidine dehydrogenase family members
(Pfam01872), which are numerous in Actinomycetes like
Streptomyces coelicolor. At this stage we named them all
DHFRO but further genetic or biochemical analysis is
needed to check these assignments.

Analysis of the distribution of DHFR genes in bacterial
genomes reinforced the conclusions [321 that many bacte-
ria such as Prochlorococcus marinus lack any recognizable
DHFR proteins, and that most of these organisms use
ThyX and not ThyA. Even if a high capacity for DHF reduc-
tion is not needed in ThyX-dependent organisms [39],
these do require some DHFR activity to complete the de
novo or salvage pathways so the corresponding gene(s)
have yet to be identified in these organisms [32] (see
Additional File 1, variants 106, 116, 006).

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(iii) Glutamylation
FolC-like proteins can have FPGS activity alone [20] or
both DHFS and FPGS activities [40], which complicates
annotation. Although the bifunctional type has a unique
dihydropteroate binding site [41], it overlaps the rest of
the substrate binding site and we could not derive a motif
to distinguish mono- and bifunctional enzymes. We
therefore annotated them all as bifunctional. By analogy
with the Lactococcus. lactis situation, we predict that organ-
isms reliant on the salvage pathway (see Additional File 1,
variants 001 and 011) will have a monofunctional FPGS.
The folC gene is missing in the Mycoplasma species that
contain an fmt, a thyA and a folA gene and must therefore
rely on a salvage pathway (Table 1). This absence points
to three possibilities for these species: (a) they import
folate polyglutamates; (b) they have a novel type of FPGS
gene; or (c) they import monoglutamyl folates and poly-
glutamylation is not needed. We favor the last hypothesis
as there is evidence for monoglutamyl folate uptake in
Mycoplasma mycoides [42]. A similar situation must exist in
bacteria such as Borrelia burgdorferi that lack all folate syn-
thesis genes but contain THF-dependent enzymes such as
Fmt (Table 1).

De novo folate biosynthesis
The majority of sequenced bacteria (250 out of 400) con-
tain all genes of the pathway and are therefore predicted
to be prototrophic for folate (see examples in Table 2 and
Additional File 1, variant 111). However, a substantial
minority lack just one or a few genes of the pterin or pABA
branches, and detailed analysis of these cases reveals sev-
eral biologically significant points.

(i) The pterin branch
The first enzyme of this branch, GCHY-I, is encoded in E.
coli by the folE gene. A recent analysis of the distribution
of folE genes among bacterial genomes showed the folE
gene to be locally missing in one-third of them [43].
Another protein family, COG1469, was found to respon-
sible for 7,8-dihydroneopterin triphosphate formation in
these organisms. This protein was named GCHY-IB and
the corresponding gene folE2 [43] (Table 2). Further anal-
ysis revealed that a few bacteria such as Wolbachia,
Chlamydia, and Chlamydophila species lack both folE and
folE2 homologs whereas they contain the signature genes
of the pathway folKP (see Table 2 and additional File 1,
variants 701, 702), suggesting that another family of
GCHY-I enzymes has yet to be identified. For instance, at
least certain Chlamydia species are known to synthesize
folates de novo [44], but lackfolE and folE2. A candidate for
the missing GCYH-I enzyme was the Chlamydia trachoma-
tis protein CT610 and its homologs, which cluster with
the folABKP folate genes in Chlamydia and Wolbachia spe-
cies (Figure 5A). The protein is homologous to the pyr-
roloquinoline quinone (PQQ) biosynthesis protein PqqC

that catalyzes an overall eight-electron oxidation, leading
to a pyrrole and pyridine ring, but their active sites are not
conserved, consistent with a different enzymatic activity
[45]. The CT610 gene was cloned in pBAD24 but failed to
complement the dT auxotrophy of the E. colifolE mutant.
The strong linkage of CT610 homologs with folate genes
certainly points to a function in folate metabolism as
other de novo folate genes thanfolE are missing in chlamy-
diae such as folQ or pabAabc (Table 2), but further studies
are needed to determine its functional role.

The second step of folate synthesis is the removal of pyro-
phosphate. Although an enzyme mediating this step had
been demonstrated in E. coli [46], no gene was known
from any organism. We identified a DHNTP pyrophos-
phatase (FolQ) candidate in L. lactis as part of the
folKEPQC gene cluster (Table 2) [12]. FolQ belongs to the
Nudix (Nucleoside diphosphate X) hydrolase family [47].
Biochemical and genetic tests confirmed DHNTP pyro-
phosphatase activity [12]. Furthermore, the closest Arabi-
dopsis homolog of L. lactis FolQ was also shown to have
this activity [12].

Since the Nudix family is large and functionally heteroge-
neous it is not very amenable to projection of annotations
just by homology. FolQ homologs with a high homology
score occur in rather few bacteria, so that the DHNTP
pyrophosphatase gene is still missing in most genomes,
including E. coli. Other putative phosphohydrolases unre-
lated to FolQ, FolQ2 members of the HDIG superfamily,
are found in some folate-related gene clusters (Figure 5B),
such as CPE1020 in Clostridium perfringens; these genes are
good candidates for alternatives to FolQ but again have a
limited phylogenetic distribution leaving the problem
open in most bacterial species (Table 2).

The third specific enzyme of the pathway, DHNA, is
encoded in E. coli by the folB gene. This gene and its para-
log folX [13] appear to be missing in many phylogeneti-
cally diverse bacteria such as Geobacter metallireducens.
Genome and functional context analysis allows the pre-
diction that the DHNA role is played by members of the
transaldolase (EC family (e.g. DVU1658 inDesul-
fovibrio vulgaris). Specifically, about half the bacteria that
lack DHNA have a transaldolase encoding gene that clus-
ters with folK genes in several organisms (Figure 5C). This
prediction awaits experimental validation as this transal-
dolase family is broad and only some of its members
might encode a DHNA aldolase. Some genomes such as
Rickettsia felis lack both FolB and transaldolase homologs
while containing all the other de novo enzymes (see Table
2 and additional File 1, variant 401), again suggesting that
another family of FolB enzymes has yet to be identified
unless the pathway is on its way to elimination in these
organisms specifically.

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Table 2: Examples of bacteria capable of de novo folate synthesis and of genes that are still missing

De novo early steps

De novo


FolE FolE2 FolQ FolQ2* FolB FolB2*

De novo late steps



FolP FolK FolC FolC2 FolA FolM Dhfr2

Escherichia coli K12
Staphylococcus aureus
Chlamydia trachomatis
Lactococcus lactis
Parachiamydia sp. UWE25
Helicobacter pylori
Prochlorococcus marinus
Clostridium perfringens
Rickettsia felis
Geobacter metallireducens
Mycobacterium leprae

Shewanella denitrificans

Xyella fastidioso

* Prediction not experimentally verified; # Fused proteins; Underlined and
predicted missing gene.

HPPK (FolK) and DHPS (FolP) are distinctive proteins
found in all organisms that make folate de novo and so, as
noted above, these were used as pathway signature genes.
A few sporadic organisms apparently lack one of the two
genes, but further analysis shows that this is usually
because of a gene-calling problem (a homolog can be
found using the tblastn algorithm) or because the corre-
sponding genome is still incomplete. Some organisms,
however, have two folP genes or two folK genes (Table 2).
Are these functionally redundant or catalyzing different
reactions? In most cases one paralog is clustered with
folate genes and the other clusters with genes involved in
different pathways (see Table 2 and additional File 1). For
instance, in the high-GC gram-positive group the second
folP (folP2) clusters with cell wall synthesis genes. In Myco-
bacterium leprae the folP2 gene does not complement an E.
coli folP mutant whereas the copy that clusters with the
folate genes (folP1) does, suggesting thatfolP2 is involved
in another pathway [49].

FolK is duplicated in many organisms. In most cases such
as Shewanella denitrificans (Table 2), one copy is in a folate
operon and the other in a pantothenate operon but there
are several cases where both genes are close to other folate
biosynthesis genes (see also Additional File 1). Only
experimental testing will show whether both copies are
active. Itis of note that an internal duplication of FolK and
fusion with FolB is found in Bifidobacterium longum.

The sequenced chlamydiae all lack homologs of folC
(DHFS/FPGS) but have folPK homologs (see Table 2 and

bold: physical clustering; (+): gene identified; (-): no identified gene; (?):

additional File 1), making folC a locally missing gene in
this group. Inspection revealed that a member of gene
family COG1478 is clustered in chlamydiae with folate
biosynthesis genes (Figure 5A, folC2). This COG1478
family contains the F420:y-glutamyl ligase CofE of Archaea
and Mycobacteria [50]. CofE catalyzes the GTP-dependent
successive addition of two y-linked L-glutamates to the L-
lactyl phosphodiester of 7,8-didemethyl-8-hydroxy-5-
deazariboflavin (F420), a reaction analogous to that medi-
ated by FolC. Chlamydiae almost certainly do not make
F420 since they lack all the other known cof genes [50]. We
accordingly predicted that the CofE homolog in chlamy-
diae has FolC activity. A cofE homolog (CT611) was
shown to complement the methionine and glycine
requirements of the E. colifolC mutant SF4 [40] indicating
that CT611 can indeed functionally replace FolC (Figure
6). The E. colifolC gene from the ASKA collection [51] was
used as a positive control.

(ii) The pABA branch
We adopted the nomenclature of Xie et al. [52] for the
pABA branch genes. These genes are hard to annotate for
several reasons. In the first place, they can be fused in var-
ious combinations. A fusion between the subunits ofADC
synthase (PabAa and PabAb) is a common arrangement,
as is fusion between PabAa and ADC lyase (PabAc). In
one genome, Corynebacterium diphtheriae, our analysis
indicated a triple fusion. The functions of this PabAa-
PabAb-PabAc fusion gene (DIP1790) were tested experi-
mentally. The gene was cloned into an expression vector
and introduced into an E. coli pabAa pabAb mutant (strain

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+ FolKI
+ FolKI

+ +

+ +

BMC Genomics 2007, 8:245

BMC Genomics 2007, 8:245



folB folKP folA folC2 pqqC-like


Chlamydophl Clostd um

Rickettsia Carboxydothernus
flels '- /hydrogenoformans
Wolbachia pipientiss Desulfitobacterium
quinquefasciatus ohafniense

Wolbachia sp in.
(Fly endosymblont)

folK folB2

folM folK folB
Methyl.oc u
folX folE

Desulfovibrio ulrs P eudomonas

G. sulfurreducens

jo_~ UW2

Xylel. f-tidioss 1

Figure 5
Clustering of predicted folate-related genes with known folate synthesis genes. Gene names are as described in the
text or given below. [For full gene and genome names, see Additional File I.] Matching colors correspond to orthologous
genes. Pale grey arrows are non-folate related genes. A. Clustering of folC2 and pqqC-like genes. 5-fcl, 5-formyl-THF cycloligase.
B. Clustering of fo/Q2 genes. fhs, formate-tetrahydrofolate ligase; dhfr, dihydrofolate reductase. C. Clustering of foIB2 (fructose-
6-phosphate aldolase-like) genes. D. Clustering of folM genes.

BN1 163), which cannot grow on minimal medium unless
it expresses a recombinant enzyme with ADC synthase
activity. A bifunctional PabAa-PabAb ADC synthase pro-
tein from Arabidopsis served as a positive control. Like the
positive control, expression of the DIP1790 protein
restored pABA prototrophy (Figure 7). This result shows
that the DIP1790 protein has ADC synthase activity but
does not demonstrate ADC lyase activity because the
BN1163 strain has endogenous ADC lyase (PabAc).
Enzyme assays were therefore used to test DIP1790 for
ADC lyase activity. BN1163 cultures harboring plasmids
encoding DIP1790, Arabidopsis ADC synthase, and E. coli
PabAc were grown and induced, and proteins were
extracted. Extracts of cells expressing DIP1790 were incu-
bated with chorismate and glutamine, without or with E.

coli PabAc; pABA was formed in the absence of PabAc
whereas, as expected, Arabidopsis PabAa-PabAb formed
pABA only if PabAc was added. Reaction rates (nmol
pABA h-1 mg-1 protein) were: DIP1790 PabAc, 7.0;
DIP170 + PabAc, 6.0; Arabidopsis ADCS PabAc, <0.01;
Arabidopsis ADCS + PabAc, 4.0. These data establish that
DIP1790 has ADC lyase as well as ADC synthase activity.

Another difficulty in annotating the pabAabc genes is that
most organisms contain paralogs of pabAa and pabAb
(trpAa and trpAb, respectively) that participate in tryp-
tophan biosynthesis [52], and in some cases the PabAb
(amidotransferase) subunit is shared between the pABA
and tryptophan pathways [53]. Finally, PabAc belongs to
the large branched-chain amino acid aminotransferase

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pabAab pabAc folE folQ2 folP FoIBK


+ Gly/Met, IPTG/Cm + Gly/Met, Ara/Amp Gly/Met, Ara/IPTG

Figure 6
Complementation of folC function by Chlamydia trachomatis CT61 I. Complementation of E. coli folC mutant SF4 by a
pBAD24 plasmid harboring CT61 I from Chlamydia trachomatis on MS minimal medium with or without glycine plus methionine.
E coli folC was included as a positive control. I, pCA24N::EcfolC; 2, pBAD24::CT6 /I; 3, pBAD24 alone. SF4 shows slower
growth even in the presence of the added amino acids. The appropriate antibiotics and inducers were included in the media as
indicated. Amp, ampicillin; Ara, arabinose; Cm, chloramphenicol.

family (EC and is hard to distinguish from these
enzymes. These problems mean that the current SEED
annotation of the pABA branch of folate synthesis should
be taken as tentative. That said, analysis of the distribution
of these genes reveals that most bacteria make pABA from
chorismate. As expected, many intracellular bacteria lack
all pabA genes. In cases where the organisms have the
pterin branch but lack all enzymes of the pABA branch,
annotation problems cannot be ruled out but an alterna-
tive pathway for the biosynthesis of pABA, starting for
example with dehydroquinate instead of chorismate,
could also be the answer [54].

Pterin salvage
The Leishmania pterin reductase PTR1 is a member of the
SDR family, but has a highly characteristic motif
TGX3RXG (in place of the TGX3GXG motif that is typical
of this family) [55]. This motif is shared with E. coli FolM
and similar SDR family proteins in a variety of bacterial
taxa. Several of thefolM-like genes are clustered with genes
of the pterin branch of folate synthesis (Figure 8), suggest-
ing a function in folate or pterin synthesis Since E. coli
[56] and other bacteria [57] are known to contain tetrahy-
dromonapterin or other tetrahydropterins that could
serve as cofactors for pterin-dependent enzymes, we pre-
dict that folM-like genes are not primarily involved in
folate synthesis but rather are pteridine reductases that,
like PTR1, produce and/or reduce 7,8-dihydropterins.

(Note that such reductases are distinct from 6,7-dihydrop-
terin reductases [also termed quinonoid pteridine reduct-
ases], of which E. coli has two [58,59].)

Consistent with this prediction, the recombinant FolM
protein catalyzes reduction of dihydrobiopterin to the tet-
rahydro form; unlike PTR1, however, it does not mediate
reduction of fully oxidized biopterin to the dihydro form
[37]. Supporting the latter observation, we found that an
E. coli GCHY-I mutant (which is unable to make pterins)
can use the dihydro but not the oxidized forms of neop-
terin, monapterin, or 6-hydroxymethylpterin to support
folate synthesis [60]. Futhermore, expression of a typical
folM-like gene (Xylellafastidiosa PD0677, Figure 5D, Table
2) from a plasmid did not enable this mutant to use oxi-
dized pterins, indicating that like FolM the PD0677
gene product does not act on oxidized pterins (Figure 8).
In control experiments in which Leishmania PTR1 was
expressed from a plasmid, the mutant was able to use oxi-
dized pterins, confirming that it is oxidized pterin reduc-
tion (and not uptake) that is lacking in E. coli (Figure 8)

Searching the Arabidopsis genome revealed some 86
members of the SDR family, of which none had the
TGX3RXG motif. This led to the prediction that Arabidop-
sis would be unable to salvage oxidized pterins, which
was verified by showing that 6-hydroxymethylpterin was

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Figure 7
Complementation of pabAa pabAb function by
Corynebacterium diphtheriae DIP 1790. Complementation
of E. coli pabAa pabAb double mutant BN I 163 by a
pLOI707HE plasmid harboring DIPI 790 from Corynebacterium
diphtheriae on M9 minimal medium with or without pABA.
Arabidopsis ADCS was included as a positive control. I,
pLOI707HE::ADCS; 2, pLOI707HE::DIPi790; 3, pLOI707HE
alone. The medium contained IPTG and appropriate antibiot-

not reduced in vivo or in vitro, and was not incorporated
into folates [60].

This analysis and integration study demonstrates that sim-
ple phylogenomic analysis of a biochemical pathway -
even a well-known one can unearth globally missing
(e.g., folQ) or locally missing (e.g., folE2 orfolC2) genes in
bacteria and plants and reveals that many open questions
remain (such as the missing folQ, folB, folE cases listed in
Table 2). It can also identify, or suggest functions for,
additional genes related to the pathway (e.g., folM). Such
analysis can thus lead to discovery of potential new drug
or herbicide targets such as GCHY-IB, which occurs in
many pathogenic bacteria but not in mammals, or the
chloroplast folate carrier that is likewise absent from

It should be noted that content of the current SEED folate
subsystem captures the present status of an ongoing anno-
tation effort, that the content will be refined and
improved as more bacterial and plant genomes are added,
and that further predictions are expected to emerge.
Finally, we emphasize that the predictions herein are
offered with the hope that others will find them useful in
their own research.

Analysis of the folate subsystem was performed in the
SEED database [61]. Results are available in the 'Folate
biosynthesis sub-system' on the public SEED server at
[62]. The snapshot of this analysis on the SEED database
is given in the additional file. Phylogenetic pattern
searches were made on the NMPDR SEED server at [63] to
find candidates for the missing folE and folC genes. We
also used the Blast tools and resources at NCBI [64] and
the comparative genomics platforms STRING [65] for
additional gene clustering analysis tools.

Annotations for paralog families were made using physi-
cal clustering on the chromosome when possible or by
building phylogenetic trees using the ClustalW tool [66]
integrated in SEED or deriving specific protein motifs.
Pseudogenes (i.e., those encoding clearly aberrant pro-
teins) were ignored; these are not uncommon in the folate
pathways of intracellular parasites undergoing genome
reduction [67].

The 'variant code' is used in SEED to schematize the type
of pathways found in a given organism [21]. A three-digit
code was used. Digit one describes the pterin branch of
the pathway: 1 = complete, 0 = HPPK and DHPS missing,
4 = DHNA missing, 7 = GCHY-I missing. Digit two
describes the pABA branch: 1 = two or three of the pabAabc
genes present, 0 = all pabAabc genes missing or just one
present. Digit three describes the salvage pathway: 1 =
complete; 0 = FPGS and DHFR missing; 2 = FPGS missing;
6 = DHFR missing. Variant -1 represents genomes with no
pathway genes but no need for them because no folate-
dependent enzymes are present. Particular care was given
to annotation of fused proteins, which are common in
both branches of the pathway; SEED has annotation tools
to deal with fusion proteins [21].

Strains, growth conditions, and cloning
Bacteria were routinely grown at 37 C in LB medium (BD
Diagnostic Systems), in minimal medium [68] supple-
mented with 0.2% (v/v) glycerol, or in M9 medium [35].
Agar (BD Diagnostic Systems) concentration in plates was
15 g 1-1. Transformations were by standard procedures
[69,70]. Thymidine (dT, 300 riM), ampicillin (100 |ig ml-
1), kanamycin (50 or 100 ig ml-1), tetracycline (10 ig ml-
1), isopropyl-P-D-thiogalactopyranoside (IPTG, 0.5 or 1
mM), methionine (100 |ig ml-1), glycine (100 |ig ml-1),
pABA (0.5 ig ml-1) and L-arabinose (0.02-0.2%, w/v)
were added as required. Strains TopolO (Invitrogen),
BL21-CodonPlus (DE3)-RIL (Stratagene), DH10B, or
DH5a were used for cloning and expression. SF4 (F-strA
recA folC srlC::TnlO) [40], BN1163 (pabAl, pab-B::Kan,
rpsL704, ilvG-, rfb-50, rph-1) (B. Nichols, University of

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+ dT dT


Figure 8
Pterin utilization by an E. coli folE deletant harboring Xylella fastidiosa PD0677 or Leishmania major PTRI.
Deletant cells transformed with pBluescript (pBS) alone, or pBS harboring Xylella fastidiosa Temeculal PD0677 or Leishmania
major PTRI, were streaked on LB medium containing IPTG and appropriate antibiotics, without or with 300 iM thymidine (dT)
or 19 iM 6-hydroxymethylpterin (HMPt). For PD0677, neopterin, monapterin, and pterin-6-aldehyde were also tested and
found not to support growth (not shown). I, pBS::PD0677; 2, pBS; 3, pBS::PTRI.

Chicago), and MG1655 ( Ifo'l klan'l) [35] were used for
complementation tests.

The Chlamydia trachomatis CT610 and CT611 genes were
cloned in pBAD24 [71] using the following primers:
3'. Genomic DNA from C. trachomatis, LGV-II, strain 434
was obtained from ABI (Maryland). PCR products were
obtained and purified as described [43], then digested
with NcoI/HindIII before ligation into plasmid pBAD24
digested with the same enzymes and transformation into
Topo 10 cells (Invitrogen). The respective plasmids named
pBY149.9 (expressing CT610) and pBY143.1 (expressing
CT611) were checked by sequencing.

The Corynebacterium diphtheriae DIP 1790 gene was cloned
into pGEM-T Easy (Promega), after amplification from
genomic DNA (obtained from the American Type Culture
Collection) using the primers 5'-GCGGCCCCCACAG-
the gene was ablated by PCR using the internal primers 5'-
TCAaGtTCGGTGATG-3' (changed nucleotides in lower
case). The modified gene was ligated into pGEM-T Easy

and verified by sequencing. It was then excised with NotI
and SstI and ligated into pLOI707HE [72]. This construct
was used to transform E. coli BN 1163. Complementation
tests were made using minimal medium, appropriately
supplemented as above.

The Xylella fastidiosa Temeculal PD0677 amplicon pre-
ceded by a Shine-Dalgarno sequence and a stop codon in
frame with LacZa was cloned between the EcoRI and KpnI
sites of pBluescript SK-. The PCR template was genomic
DNA from the American Type Culture Collection; primers
CAGCGTGCGGCC-3'; amplification was with KOD HiFi
polymerase. The deduced amino acid sequence differed
from that published in having serine not isoleucine at
position 57. The PTRI construct was as described [60].
The constructs were introduced into E. coli folE deletant
cells [35]. Transformants were grown on LB plates supple-
mented appropriately as above.

ADCSIADCL enzyme assays
Protein extracts were prepared from IPTG-induced cul-
tures as described [73] from strain BN1163 harboring
three pLOI707HE constructs DIP1790, Arabidopsis
ADCS [73], or vector alone and from BL21-CodonPlus
(DE3)-RIL harboring E. coli PabC cloned in pJMG30 [74].
Glutamine-dependent pABA synthesis activity was

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assayed as described [73]. PabC extract (3.8 lig protein)
was added when indicated. Assays were incubated for 1 h
at 37 oC, stopped with 20 [l of 75 % (v/v) acetic acid, held
on ice for 1 h, then stored at -80 C until analysis. pABA
was estimated by HPLC with fluorescence detection [73].

Authors' contributions
BElY carried out the complementation studies on the
Chlamydia cofE homolog. RDdelaG made the complemen-
tation and biochemical assays on the Corynebacterium
pABA synthesis protein. AN carried out the studies on the
Xylella folM homolog. VdeC-L and ADH conceived the
study, carried out bioinformatic work, and drafted the
manuscript. All authors read and approved the final man-

Additional material

Additional File 1
Spreadsheet summarizing the distribution, clustering, and fusions of bac-
terial and plant folate synthesis genes. This table represents a snapshot for
the record of the "BMCgenomics 2007" table that can be found in the
Folate Biosynthesis sub-system on the public SEED website ii ',.
seed. uchicago. edulFIG/index. cgi. Clustering is shown by similar color
backgrounds. Genome and protein IDs are from the SEED database.
Abbreviations for the functional roles are given in the first page of the
spreadsheet, gene distribution in all analyzed genomes in the second page.
Note that the SEED table is the primary source to which the reader is
directed; it is not static but develops with time as new genomes become
available and predictions are tested and validated.
Click here for file

We thank B. Shane for the SF4 strain, B. Nichols for the BN 1163 strain, H.
Mori for the pfolCEc plasmid, and G. Basset for help with enzyme assays. We
thank A. Osterman for insightful suggestions and help with figure design.
This work was supported in part by National Institutes of Health grants RO I
GM70641-01 (to V. de C.-L.) and ROI GM071382 (to A.D.H.), by the
National Research Initiative of the USDA Cooperative State Research, Edu-
cation, and Extension Service, grant number 2005-35318-15228 (to
A.D.H.), and by an endowment from the C.V. Griffin, Sr. Foundation.

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