Group Title: Biology Direct 2010, 5:3
Title: Predicting the pathway involved in post-translational modification of Elongation factor P in a subset of bacterial species
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Title: Predicting the pathway involved in post-translational modification of Elongation factor P in a subset of bacterial species
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Bailly and de Crecy-Lagard Biology Direct 2010, 5:3
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BIOLOGY DIRECT
BIOLOGY DIRECT


Predicting the pathway involved in post-

translational modification of Elongation factor P

in a subset of bacterial species

Marc Bailly, Valerie de Crecy-Lagard*


Abstract
Background: The bacterial elongation factor P (EF-P) is strictly conserved in bacteria and essential for protein
synthesis. It is homologous to the eukaryotic translation initiation factor 5A (elF5A). A highly conserved elF5A lysine
is modified into an unusual amino acid derived from spermidine, hypusine. Hypusine is absolutely required for
elF5A's role in translation in Saccharomyces cerevisiae. The homologous lysine of EF-P is also modified to a
spermidine derivative in Escherichia coil. However, the biosynthesis pathway of this modification in the bacterial EF
P is yet to be elucidated.
Presentation of the Hypothesis: Here we propose a potential mechanism for the post-translational modification
of EF-P. By using comparative genomic methods based on physical clustering and phylogenetic pattern analysis,
we identified two protein families of unknown function, encoded by yjeA and yjeK genes in E. coli, as candidates
for this missing pathway. Based on the analysis of the structural and biochemical properties of both protein
families, we propose two potential mechanisms for the modification of EF-P.
Testing the hypothesis: This hypothesis could be tested genetically by constructing a bacterial strain with a
tagged efp gene. The tag would allow the purification of EF-P by affinity chromatography and the analysis of the
purified protein by mass spectrometry. yjeA or yjeK could then be deleted in the efp tagged strain and the EF-P
protein purified from each mutant analyzed by mass spectrometry for the presence or the absence of the
modification. This hypothesis can also be tested by purifying the different components (YjeK, YjeA and EF-P) and
reconstituting the pathway in vitro.
Implication of the hypothesis: The requirement for a fully modified EF-P for protein synthesis in certain bacteria
implies the presence of specific post-translational modification mechanism in these organisms. All of the 725
bacterial genomes analyzed, possess an efp gene but only 200 (28%) possess both yjeA and yjeK genes. In the
other organisms, EF-P may be modified by another pathway or the translation machinery must have adapted to
the lack of EF-P modification. Our hypotheses, if confirmed, will lead to the discovery of a new post-translational
modification pathway.
Reviewers: This article was reviewed by Celine Brochier-Armanet, Igor B. Zhulin and Mikhail Gelfand. For the full
reviews, please go to the Reviewers' reports section.


Background
Protein translation is dependent on a complete set of
translation factors. Elongation factor P (EF-P) is one of
these factors. It is strictly conserved in bacteria and has
recently been shown to have a role in translation

* Correspondence vcrecy@ufl edu
Department of Microbiology and Cell Science, University of Florida,
Gainesville, FL, USA


initiation by promoting the formation of the first pep-
tide bond [1,2]. Similarly to other initiation factors, EF-P
is present at one copy per ten copies of ribosome [3,4].
The corresponding gene efp is essential in E. coli [5],
but is not a required component of reconstituted in
vitro protein translation systems [6]. EF-P is the bacter-
ial homologue of the conserved eukaryotic/archaeal
translation initiation factor 5A (eIF5A) [2] that has


S 2010 Badly and de Crecy-Lagard; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Bi led Central Creative Commons Attribution License (http//creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.






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recently been shown to be required for ribosome trans-
location in a concerted action with eukaryotic transla-
tion elongation factor 2 [7]. A strictly conserved lysine
of eIF5A (position 51 of the S. cerevisiae eIF5A protein)
is post-translationally modified to hypusine [N"-(4-
amino-2-hydroxybutyl)-lysine] [8]. Hypusine is required
for eIF5A function and the hypusine biosynthesis genes
are essential in S. cerevisiae [9-14]. This unusual amino
acid is not synthesized as a free intermediate but is
exclusively formed by a post-translational modification
of eIF5A involving two enzymatic steps. First, a deoxy-
hypusine synthase transfers the 4-amino butyl moiety of
spermidine to the e-amino group of the conserved Lys
51 of the S. cerevisiae eIF5A precursor, to form a deoxy-
hypusine intermediate [15,16]. This residue is then
hydroxylated to hypusine by deoxyhypusine hydrolase
[17]. No hypusine has ever been identified in bacteria
but it has recently been shown that the E. coli EF-P pro-
tein contains a spermidine modification at the homolo-
gous lysine 34 [18]. The pathway that introduces this
modification in the bacterial EF-P is yet to be
elucidated.
Based on a combination of comparative genomics, lit-
erature mining and phylogenetic analyses, we predict
that the YjeA and YjeK families of proteins are involved
in EF-P modification in a subset of bacterial species. We
also propose two potential mechanisms for EF-P modifi-
cation, based on structural and functional characteristics
of the YjeA and YjeK families of enzymes.

Presentation of the hypothesis
Genome organization and phyletic distribution of efp,
yjeA and yjeK
The SEED database [19] was used to investigate the EF-
P modification pathway. The results are given in the
"Elongation factor P modification" subsystem in the
public SEED http://theseed.uchicago.edu/FIG/index.cgi.
Among the 725 bacterial genomes analyzed all contain
an efp gene. By using the neighbourhood analysis tool of
SEED database [19], we were able to establish a physical
clustering association between the yjeA, yjeK and efp
genes in 183 genomes. As shown on Fig. 1A and Addi-
tional file 1, efp, yjeA and yjeK genes are physically clus-
tered in phylogenetically distant organisms. 200
genomes out of 725 analyzed (28%) possess both yjeA
and yjeK genes. In 31% of these 200 genomes, the three
genes are organized in an operon as in Vibrio cholerae.
Only two of the three genes are clustered in 60% of the
200 genomes such as efplyjeA in Coxiella burnetii or
efplyjeK in E. coli. Finally, 9% of the 200 genomes do
not show any clustering between any two of these three
genes. Only two organisms Planctomyces limnophilus
and Buchnera aphidicola str. APS (Additional file 1)
only contained a yjeA and not a yjeK homolog. These


are two symbiotic organisms known to shed genes and
pathways [20].
Not all EF-P proteins are predicted to be modified
because some lack the conserved lysine 34 residue Fig.
1C[21]. However, 97% of the EF-P from organisms that
contain homologs of YjeA and YjeK have a lysine resi-
due at position 34 as part of the conserved "PGKG"
motif (Fig. 1B). The only exceptions are organisms that
contain paralogs copies of efp genes such as Geobacter
uraniireducens Rf4, Pelobacter carbinolicus DSM 2380
and Alcanivorax borkumensis SK2 (Additional file 1). In
these cases, one EF-P protein has the conserved Lys,
while in the other it has been replaced by an Ala or His
residue. In the organisms that lack the yjeAlyjeK pair
(Fig. 1C) only 70% have kept the Lys 34 in EF-P. 24.1%
have an Arg, 3.4% a Met, 2.2% a Asn and 0.3% a Gin at
that position. The strong physical clustering between efp
and yjeAlyjeK and the strict correlation between the
presence of yjeA and yjeK and the presence of Lys34 in
EF-P led us to propose that the corresponding gene pro-
ducts might be involved in post-translational modifica-
tion of EF-P. This hypothesis was further explored by
detailed sequence and structural analyses of the YjeK
and YjeA families.
yjeK encodes a truncated 2,3 Lysine aminomutase (LAM)
yjeK encodes a homologue of lysine 2,3 aminomutase
(LAM) involved in lysine catabolism [22]. However, it
was shown in vitro that E. coli YjeK catalyzes the con-
version of (S)-a-lysine to (R)-3-lysine and not of (S)-a-
lysine to (S)-3-lysine like classical LAM enzymes [22].
The E. coli YjeK catalytic efficiency is quite low com-
pared to the LAM catalyzed reaction (0.1% of the activ-
ity of the Clostridium subterminale SB4 LAM [22]). (S)-
a-lysine might not therefore be the real in vivo substrate
of the YjeK enzyme. Primary sequence alignment analy-
sis on 95 LAM/YjeK homologs were performed using
Clustal W2 [23] and revealed that the LAM (YjeK) that
clusters with the efp gene can be separated from the
canonical LAM involved in Lys degradation pathway.
The major difference between the two families is that
the YjeK proteins lack the C-terminal multimerization
domain present in the LAM family of proteins [24] (Fig.
2B). Further phylogenetic analysis on 24 LAM/YjeK
homologs was performed on 118 amino acid sequences
present in the N-terminus active site and conserved
between the YjeK and LAM subfamilies (Fig. 2A). The
amino acid sequences were aligned using the ClustalW2
algorithm with default parameters [23]. Phylogenetic
analyses were carried out by employing the Phylip 3.68
program package [25]. Distance-based matrices were
generated between all pairs of sequences using the
Jones-Taylor-Thornton matrix as employed in Protdist
(Phylip). Phylogenetic trees were generated from these
matrices using the neighbour-joining method as


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7%


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.;- I .4.,,0., ~j n.r .,I
31 % Al :c '00'!


i/rn -L CI


.. 53 % .. H ,nll,.,, n-i







SC I C


Figure 1 Genomic organisation of efp, yjeA and yjeK genes. A-Physical clustering of efp (in brown), yjeA (in pink) and yjeK (in orange) genes
in several organisms. The black lines indicate that the genes are not contiguous in the genome. Examples of organisms and percentages for
each genomic organisation among the 200 genomes that possess both yjeA and yjeK genes are indicated. The full list is given in Additional fle
1. B and C- The sequence logos were created using an alignments of 9 amino acids from the EF-P protein sequences surrounding the position
34 generated in Clustal W2. The Logos were then generated by pasting the alignment in the WebLogo interface version 2.8.2 [39,40]. B- Logo
for EF-P proteins from organisms that possess YjeA and YjeK (the list of sequences used is given in Additional fle 3). C- Logo for EF-P proteins
from organisms deprived of YjeA and YjeK.


implemented in Neighbor (Phylip). Reliability of
branches was determined with the bootstrap method of
1000 replicates using Seqboot (Phylip). The final tree
was generated with Consense (Phylip). This analysis
showed that despite the fact that YjeK from E. coli is
33% identical to LAM from C. subterminale SB4, the
two enzyme subfamilies form two distinct clades on the
YjeK/LAM phylogenetic tree separated by bootstrap
scores ranging from 923 to 906 (Fig. 2A). Genome


neighbourhood analyses were also used to split the two
families. We observed that ablA (LAM) genes physically
cluster mainly with other lysine degradation gene such
as ablB (j3-lysine acetyltransferase) in several organisms
(See Fig. 2A and Additional file 1). On the contrary,
yjeK genes cluster mainly with efp homologs but never
with ablB genes (See Fig. 2A and Additional file 1). In
genomes such as Syntrophus aciditrophicus and Desul-
furomonas acetoxidans, where both yjeK and ablA genes


A li-Lysine
EFP YjeK YjeA LAM acetyltranferase
t Peodiclyonluteolum
SProsthecochoris vbniformis [ 0 *
Syntrophus0cItdirophiau E
Desulfomicrobiumbaculatu D 0
I Desufomonascerridans *
S Desulloholobiumretbaense ,a a
923 Desulfovibriodesulfuricans 0 a
Pelobacter carbinoius
s Peobacter propionicus *

EBcherichicollK12 E E





^06 -------- I- iiijiiii;" ***'""^----------- *D ^c'?
p m illii *i r r.. j. ..',i- ,.|..r.,. .Q Q ( j
UU ED
SBacillusubtilisD 0 E
Figure 2 Phylogenetic and structural analysis of the LAM family of proteins. A- Phylogenic tree generated with a subset YjeK and LAM
proteins. Methods for alignment and tree construction are described in the text. This analysis shows that YjeK (in orange) and LAM (in blue)
proteins forms distinct clades with relevant bootstrap values (923 for the LAM clade and 906 for the YjeK clade). The boxes correspond to the
presence of the genes encoding for the protein indicated on top of the figure in the corresponding organism, white for genes present but not
involved in a clustering, orange for genes that cluster with efp, and blue for genes that cluster with 0-lysine acetyltransferase (Lysine degradation
pathway). Accession numbers for the protein used can be found in Additional fle 1.B- Three dimensional structure of LAM from Clostridium
subterminale SB4 [24] (PDB: 2A5H) in blue with the C-terminal multimerization domain in pink, and 3D-model of YjeK from Acinetobacter baylyi
based on C subterminale SB4. The 3D model was build by using the homology method on the SWISS-MODEL web server [41-43].


yIeK e y e






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are present, one was found to cluster with efp and the
other with ablB respectively (Fig. 2A). The combination
of structural, phylogenetic and physical clustering pat-
tern differences between YjeK and LAM enzymes were
used to split the LAM family of proteins into two subfa-
milies that will be referred to as LAM and YjeK from
hereon and suggest that these families have distinct
functions, the first in lysine catabolism, the second
related to EF-P.
yjeA encodes a truncated Lysyl-tRNA synthetase of
Class II
yjeA in E. coli encodes a homolog of Class II lysyl-tRNA
synthetase (LysRS2) sharing 31% identity with the cano-
nical E. coli LysRS2 [26] (Additional file 2). Pleiotropic
phenotypes have been associated with the absence of
this protein [27,28] but its exact function is not known.
Analysis of the three dimensional structure of E. coli
YjeA (Fig. 3A) revealed that this protein contains only
the catalytic core of LysRS2 deprived of the anticodon
binding domain (ABD), responsible for tRNALys recogni-
tion (Fig. 3B). The structural alignment of YjeA and
LysRS2 from E. coli shows that their catalytic cores are
very similar: the lysine binding residues are conserved
and in the same spatial location [except for two residues
surrounding the lysine substrate that are slightly dis-
placed in the catalytic pocket (red circles in Fig. 3C)].
The absence of the anticodon binding domain YjeA
points to a function different from tRNA aminoacylation
as already observed for other aminoacyl-tRNA synthe-
tase (aaRS) catalytic core homologs. Examples include
the catalytic core homolog of E. coli glutamyl-tRNA
synthetase (Glu-Q-RS) involved queuosine glutamylation


of the tRNAAsp anticodon [29-32], and the catalytic core
homolog of Pyrococcus abyssi AsnRS2 (archaeal Asn
synthetase, AS-AR) involved in asparagine biosynthesis
[33].
Potential mechanisms involved in EF-P modification
The analysis presented above led to the hypothesis that
YjeK and YjeA are involved in the modification of Lys34
of EF-P to a spermidine-like molecule [18]. We propose
two possible mechanisms for the insertion of this
modification.
In the first model (Fig. 4A), YjeA activates (S)-a-lysine
into (S)-a-lysyl.AMP in presence of ATP and magne-
sium. This is coherent with the presence of AMP in the
YjeA three dimensional structure (PDB: 3G1Z) and the
conservation of the lysine binding residues in the YjeA
active site (Fig. 3C). The activated (S)-a-lysine could
then be transferred to the conserved Lys34 of EF-P to
form an iso-peptidic bond between the Lys34 ENH2 and
the a-COOH of the activated Lys. The ability of acti-
vated amino acids to make isopeptidic bonds with the
ENH2 moiety of Lys lateral chains has already been
demonstrated for S. cerevisiae AspRS [34]. Finally, YjeK
could convert the EF-P bound a-lysine into 3-lysine in
presence of S-adenosyl methionine (SAM) and pyridoxal
phosphate (PLP) (Fig 4A). The low activity of the E. coli
YjeK on free (S)-a-lysine noted above could be due to
the fact that the natural substrate is linked to EF-P [22].
The absence of the C-terminal dimerization domain in
YjeK might allow it to access the EF-P-bound substrate.
In the second model (Fig. 4B), YjeK would first con-
vert the (S)-a-lysine into (R)-3-lysine in presence of
SAM and PLP [22] with a low activity to prevent


Page 4 of 11


YjeA Lysyl-tRNA synthetase (Class II) Overview Active site

Figure 3 Structural analysis of YjeA protein family. A- Three dimensional structure of YjeA from Salmonella typhimurium (PDB: 3G1Z) in pink
and class II lysyl-tRNA synthetase (LysRS) from Escherichia coli [26] (PDB: 1BBU) in purple. The domains that constitute LysRS are indicated:
catalytic core and anticodon binding domain (ABD). B- Merging of the two previous global structures and zoom into the active site and catalytic
residues. The residues responsible for ysie binding are in pink for YjeA and in purple for the LysRS2. The lysine substrate present in LysRS2
structure is indicated in orange and the AMP present in YjeA structure is indicated in yellow. Red circles highlight the residues for which the 3
dimensional positions are not conserved.






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potential use of (R)-3-lysine by other enzymes and a
potential toxicity of this intermediate in cellular metabo-
lism. Then YjeA would recognize (R)-3-lysine as a sub-
strate and activate this non cognate amino acid in
presence of ATP and magnesium prior to its transfer on
the EF-P Lys34. The use of non cognate amino acid by
aaRS or truncated aaRS is well known [35] and the fact
that YjeA active site is slightly different than the LysRS2
site may allow for the accommodation (R)-3-lysine.
Both reactions scheme would generate an EF-P modi-
fied on Lys34 by a P-lysine (146 Da) compatible with
the 144 Da modification that was experimentally
observed [18].

Testing the hypothesis
Our hypotheses could be investigated in vivo by using a
strain of Acinetobacter baylyi sp. ADP1 in which the efp


gene has been replaced by a tagged version by using
genomic replacement as described previously in Metzgar
et al [36]. The tag would allow the purification of EF-P
from the crude extract of A. baylyi and the subsequent
analysis of the factor by mass spectrometry. Then the
yjeK or yjeA genes could be deleted in this strain and
the EF-P purified to analyze the factor for the presence
or the absence of the modification.
This method seems to be an ideal way to test our
model, but several issues make it technically challenging.
First, EF-P is present in the cell in a lower ratio than the
elongation factors (one EF-P per ten ribosomes [4,37]),
thus EF-P might always be in complex with the ribo-
some and inaccessible for our affinity purification
method. Moreover, the method used to release the EF-P
bound to purified ribosomes requires the use of high
salt concentration and low magnesium concentration,


(S)-a-Lys


+ ATP


EF-P
~ ~co


AMP Yje


SAEF-P- a-Lys



SAM, PLPIYjeK


EF-P* J-Lys


(S)-a-Lys



SAM, PLPIYjeK



(R)- -Lys

ATP

EF-P YjeA ,
)P?


EF-P

AMP AYeA Lys34

AMPEF-P -Lys

EF-P* P-Lys


Figure 4 Potential EF-P modification pathways. A- Mechanism in which YjeA acts first on free lysine (Lys) and attaches it to EF-P Lys34 which
is then modified on EF-P into p-lysine by YjeK. B- Mechanism in which YjeK acts first to modify free lysine into p-lysine which is subsequently
activated by YjeA and attached to EF-P lysine 34. EF-P N-terminal loop is indicated in yellow, Lys 34 is indicated in red, the modification appear
on light brown and the AMP generated by YjeA during the activation of the Lys residue appear in blue. Potential and known substrates and
cofactors of each enzyme are indicated.


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but high salt concentration is not compatible with most
affinity purification and leads to the loss of 80% of the
factor during the purification step. Finally, deleting yjeK
and yjeA in A. baylyi gives rise to severe growth pheno-
types [36] that decreases the amount of EF-P available
for the affinity purification step and makes the in vivo
approach difficult to realize.
Our hypothesis could also be tested using an in vitro
approach. EF-P would need to be purified in the apo
form from a AyjeK or AyjeA E. coli strain. Then, the
YjeA and YjeK enzymes could be over-expressed and
purified and the putative activities presented in Fig. 4
could be followed by using radiolabeled (S)-a-lysine as a
substrate.

Implication of the hypothesis
Using a comparative genomic approach, we predict that
the yjeK and yjeA genes of unknown function that phy-
sically cluster with efp gene are potential candidates of
EF-P modification genes. We propose two potential
mechanisms based on further structural and functional
data mining and analysis of YjeK and YjeA protein
families. However, among the 725 bacterial genomes
used in our analysis, only 200 (28%) possess both yjeA
and yjeK genes. The taxonomic distribution of these
organisms spans several phyla but are mainly repre-
sented by members of the protebacteria (gamma, alpha
and delta) and by scattered representatives of the Aqui-
ficales, Chlamydiae, Chlorofexi, Planctomycetes and
Sphirochetes phyla (Additional file 1). It is totally absent
in Actinomycetes, Firmicutes and Cyanobacteria (Addi-
tional file 1). A more extensive phylogenetic analysis
beyond the scope of this article could establish if the
common ancestor of bacteria had a EF-P modification
pathway that was lost in specific phyla or if it appeared
later on, as for example in the proteobacteria ancestor,
and then was horizontally transferred in other clades.
In organisms that lack the yjeK/yjeA genes only 70%
of the EF-P proteins have retained a Lys residue at the
34 position (Fig. 1C). This implies that the translation
machinery can adapt to the lack of EF-P modification.
Indeed, this adaptation happens as shown for Thermus
ril ...i...''iil" EF-P [38]. In this case, an arginine (Arg32)
replaces the corresponding E. coli Lys34. This Arg32 lies
close to the peptidyl transferase center (PTC) and inter-
acts with the C75 position of the CCA end of the accep-
tor stem of the initiator tRNA and with the phosphates
of positions C2064 and C2065 of the 23S ribosomal
RNA (rRNA) [38]. The authors propose that the hypu-
sine moiety in eukaryotes/archaea and the spermidine
(or p-lysine) moiety in bacteria could allow closing of
the distance between EF-P and the PTC and enable
proper positioning and stabilization of the initiator
tRNA in the peptidyl site of the ribosome [38]. How T.


thermophilus and other organisms that lack the modifi-
cation achieve this is not known but favors an adapta-
tion of the translation machinery, particularly of the 23S
rRNA and initiator tRNA. Finally, our hypotheses if con-
firmed will lead to the discovery of a new post-transla-
tional modification mechanism, 3-lysinylation.

Reviewers'reports
Reviewer 1
Celine Brochier Armanet, ILaboratoire de Chimie
Bacterienne (CNRS UPR9043) Marseille, IFrance
In the paper entitled "Predicting the pathway involved
in post-translational modification of Elongation factor P
in a subset of bacterial species" Bailly et de Cr(cy-Lar-
gard identify two genes (yjeA and yjeK) as candidates for
the posttranslational modification of lysine 34 of EfP to
a spermidine derivative. This prediction is based on var-
ious in silico analyses genomicc clustering of Ef-P coding
genes, the presence or the absence of a lysine at the tar-
geted position in Ef-P protein, functional annotations,
structural comparisons etc).
I think that the work presented is very convincing and
is a good illustration of the predictive power of in silico
approaches for functional prediction. Accordingly I
strongly recommend its publication in Biology Direct.
However, I have few suggestions that may increase the
value of the work (especially from an evolutionary point
of view).
A) The authors say that EfP is strictly conserved in
Bacteria and accordingly they quote two references.
However, these papers are "old" from a genomic point
of view (e.g. in 1992, less than 70 bacterial genomes
were available and many bacterial phyla were not repre-
sented in genomic databases). It would be interesting to
verify that this important assumption is still verified by
available data.
Authors' response: In the revised version of the manu-
script we included a sentence stating that efp was found
in all 725 genomes analyzed. And two additional files 3
and 4 provide all the EF-P sequences used in the
analysis.
Reviewer 1
B) Also important is the presence of the lysine at posi-
tion 34. The authors say that some EfP sequences lack
this residue quoting a paper published in 1992. As pre-
viously, I think it would be interesting to precise how
many and which EfP sequences harbour this conserved
lysine and their taxonomic distribution across bacterial
phyla.
Authors' response: In the revised version we provided
an additional figure (Fig. 1B) that shows that the EF-P
Lys-34 residue is nearly strictly conserved in all the
organisms that have the yjeA/yjeK pair. The only excep-
tions are found in organisms that have two EF-P


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encoding genes (one with the conserved lysine, one with-
out). The corresponding list of sequences is given in S2.
Conversely, Fig. 1C shows that in organisms that lack
yjeA/yjeK the Lys can be replaced by other residues (Arg,
Met, Asn or Gln). Here again the corresponding protein
sequences are given in S3. A section of the taxonomic dis-
tribution of organisms that have or not the yjeA/yjeK
pair has been added to the implication section and this
information is also included in Additionalfile 1.
Reviewer 1
C) More important, based on the SEED database the
authors say that 125 on 722 bacterial genomes analysed
harbour both yjeA and yjeK genes. I think it is very
important to provide and to comment their taxonomic
distribution: are these genomes closely related? Or are
they well distributed among bacterial phyla? Even if I
think that this will not change the main conclusions of
the paper, I think it would be interesting to develop this
point. Indeed, these two situations have very different
evolutionary implications. The presence of these two
genes only in closely related genomes may suggest a
recent appearance of this modification during bacterial
evolution. On the contrary, if these genes are present in
distantly related genomes (e.g. genomes from different
bacterial phyla), this may point to either an ancient ori-
gin of this modification in Bacteria followed by numer-
ous independent secondary losses, or to a recent origin
followed by horizontal gene transfer across bacterial
phyla. To my point of view, the addition of this informa-
tion is important and may interest many readers.
Authors' response: The information on the taxonomy
of the organisms that contain the yjeK/yjeA pair is given
in Additional file land the analysis of this taxonomic
distribution has also been added to the "Implications"
section in the revised version of the manuscript.
Reviewer 1
D) Finally, it is important to show the genomes that
harbour only yjeA or yjeK.
Authors' response: Only two organisms Planctomyces
limnophilus and Buchnera aphidicola str. APS (Addi-
tionalfile 1) contained a yjeA and not a yjeK homolog.
These are two symbiotic organisms known to shed genes
and pathways. This information was added to the text.
Reviewer 1
To sum up, my main request concerns the inclusion of
raw results in the paper (and not only their analysis).
This may be easily done by the inclusion of a single sup-
plementary table, showing for each bacterial genome
analysed: (1) their taxonomic affiliation, (2) the presence
of efP coding genes (with their accession numbers), (3)
the conservation of lysine-34 in the corresponding pro-
teins, (4) the presence of yjeA and yjeK genes (with their
accession numbers) and (5) their physical clustering on
the chromosome.


Authors' response: The requested information covering
points 1, 2, 4 and 5 are now included in the Additional
file 1. For point 3 we included a new Fig 1B and Fig 1C
giving the distribution of Lys residues in organisms that
have yjeK/yjeA and in organisms that do not and added
the list of sequences used for this analysis as Additional
files 3 and 4.
Reviewer 1
Other remarks:
1) Precise if the two lysines that are modified in the
bacterial efP and in the eukaryotic/archaeal eIF5A are
homologous or analogous. Please use one of these two
terms instead of "equivalent" (p.3 line 1). If they are
homologous, it would be nice to show an alignment of a
subsets efP and eIF5A as supplementary material.
Authors' response: The two proteins are homologous
as stated in the background section EF-P "is the bacterial
homologue of the conserved eukaryotic/archaeal transla-
tion initiation factor 5A (eIF5A) [2]". The referenced
paper by Kyrpedes and Woese had discovered this
homology in 1998. We replaced the p. 3 equivalent by
homologous in the revised version.
Reviewer 1
2) P.4 the authors say that clustalW2 were used to con-
struct the phylogenetic tree showed as Fig. 2A. ClustalW
is an alignment software that provides guide trees (but
these are not phylogenetic trees). Thus, I suggest to the
authors to re-compute their phylogenetic tree using
typical tree reconstruction software (e.g. PhyML,
MrBayes, Phylip, etc). Moreover, the authors should
provide the methods and the parameters used for the
phylogenetic reconstruction.
Authors' response: We redid the phylogenetic tree
using the Phylip software and the methods and para-
meters were added to the text.
Reviewer 1
3) P.4, it would be interesting to provide an alignment
of primary sequences of LysRS2 and YjeA as supple-
mentary material.
Authors' response: This alignment was included as
Additional file 2.
Reviewer 1
4) P.6 In the sentence "First Ef-P is present in the cell in
a lower ratio than the elongation factors... do the
authors mean that Ef-P is present in the cell in a lower
ratio than ribosomes?
Authors' response: Yes all initiation factors are used
only once per protein synthesis cycle and can be recycled.
Reviewer 2
Igor B. Zhulin, University of Tennessee Oak Ridge
National Laboratory, Oak Ridge, TN, USA
Synopsis:
This paper identifies two proteins that may be
involved in post-translational modification of the


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bacterial EF-P translation elongation factor. The two
proposed candidate proteins, YjeA and YjeK, were iden-
tified by genome neighborhood analysis that showed the
genes coding for these proteins were adjacent to the efp
gene (encoding EF-P) on 125 of 722 bacterial genomes.
All genomes with the conserved motif for lysine modifi-
cation of EF-P encoded homologs of these proteins. The
YjeK homologs share common enzymatic domains with
a protein involved in lysine catabolism, lysine aminomu-
tase (LAM). The YjeK homologs are shown by phyloge-
netic analysis to form a distinct clade from the LAM
proteins. YjeA is a homolog of lysine t-RNA synthase
and the authors demonstrate a high degree of overlap
between the 3D structures of YjeA from S. typhimurium
and class II lysyl-tRNA synthetase (LysRS) from E. coli.
Based on the chemistries of LAM and LysRS2, the
authors propose how YjeA and YjeK could carry out the
observed lysine modification of EF-P and discuss the
implications of this hypothesis.
In general, this is an interesting paper and it offers a
testable hypothesis, which can lead to better under-
standing the mechanisms of post-translational modifica-
tion in bacteria.
I am not sure about the terms "bacteria" and "bacter-
ial" that are used throughout this paper. I recall they
were used to contrast "archaebacteria" that we now call
"archaea". Thus, simply "bacteria" and "bacterial" will
do.
Authors' response: Eubacterial and eubacteria was
replaced by bacterial and bacteria in the revised version.
Reviewer 2
Analysis:
Chromosomal clustering of efp and yjeA/K
The presentation of yjeA and yjeK can be improved.
In the current version of the paper these genes first
come from nowhere (Genome organization and phy-
letic distribution ofefp, yjeA and yjeK: the second
sentence). It would be helpful to mention that in order
to find potential members of the pathway, investigators
decided to look into the genomic neighborhoods of the
efp gene. There, they have found yjeA and yjeK on many
occasions, indicating a likely link. The initial chromoso-
mal clustering study seems solid, although "clustering"
as used is not a perfect term. "Clustering" can be a part
of a very different type of sequence analysis (grouping
sequences by similarity). "Chromosomal clustering",
"chromosomal proximity", "genome context", genomicc
neighborhood" seem to be better terms. Well, after all,
there is a reference to "physical clustering" in the Fig. 1
legend. Still, there is a need for consistency in
terminology.
Authors' response: As suggested clustering was
replaced by physical clustering when adequate through-
out the revised manuscript.


Reviewer 2
Of the 125 genomes with yjeK and yjeA genes, 94% are
chromosomally clustered with efp. Additional evidence
comes from the fact that all EF-P proteins with the con-
served lysine motif also contained homologs of YjeA
and YjeK. Unfortunately, this critical piece of supporting
data is not shown in any way. No mention is made of
how many genomes contain only one of these two pro-
teins (in which case one or the other enzymes could
have been replaced by another function). I strongly
recommend producing a supplementary table that
would show the results. Column subheadings for such a
table should include: 1) Species name; 2) Genome acces-
sion number; 3) efp ID (locus, GI or accession number.
Locus tags would be the best because they will show
proximity of neighboring genes); 4) YjeA (the same
information or blank if absent); 5) yjeK (the same infor-
mation or blank if absent).
Authors' response: This information was included in
the revised version as Additionalfile 1.
Reviewer 2
YjeK/LAM homology:
YjeK and LAM were previously reported as homologs.
The multiple sequence alignment (MSA) and phyloge-
netic analysis of these homologs seem a bit simplistic,
carried out only using Clustal W. I am not familiar with
the improvements made in Clustal W2, but the original
Clustal W is certainly not the best program for MSA
construction. No examples of the alignment are shown,
so it is impossible to judge how well the Clustal W2
output looks like.
There is a similar problem with a tree shown in Fig. 2.
Which method was used for its construction? I presume
it is NJ, but this should be explicitly stated.
The results indicate that LAM and YjeK form two dis-
tinct clades (Fig. 2). However, LAM contains an addi-
tional domain not found in YjeK. From the figure
legend, it appears that the full length proteins were used
for the alignment (rather than just the conserved
domain). If so, this will obviously strongly favor creation
of a separate clade. The genome context analysis does
support the idea of separate functional groups for LAM
and YjeK proteins. This whole issue must be better clar-
ified/supported.
Authors' response: we thank the reviewer for pointing
this to us as we had done the mistake of doing the align-
ment with the whole protein and not only the common
domain. We redid the alignment using 118 amino acid
sequence present in the N terminus active site and con-
served between the YjeK and LAM subfamilies. Then we
ran a phylogenetic analysis using the Phylip package
[25]. using neighbour-joining method, with 1000 boot-
strap and this analysis shown clear separations with
high bootsrap values between the two clades. This


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analysis also helped us better separate YjeK/LAM para-
logs in several genomes that we had not been able to
really separate in our previous work that is why we have
now 200 genomes that have both yjeA/yjeK s instead of
125 in the previous analysis.
Reviewer 2
YjeA/LysRS2 similarity:
The structural overlay of YjeA and LysRS2 clearly
shows the structural similarity. The very close overlap is
suggestive of similar function.
Potential mechanisms:
This section is plausible and can provide a starting
point for experiments, but is clearly highly speculative.
Testing the hypothesis:
This section is weak. The authors point out a scheme
to make an affinity tagged version of the protein and sub-
stitute the normal chromosomal copy in an Acinetobacter
species. This is a very standard technique that could be
summarized in a phrase instead of a paragraph. No rea-
son is listed for carrying out these experiments in Acine-
tobacter instead of E. coli or another model organism
(although the referenced paper claims that Acinetobacter
is now a good model). The section does clarify the pro-
blems of dealing with a low copy essential gene, but no
ideas are presented to overcome these difficulties.
While the paper spends almost one page of the manu-
script on the detailed mechanistic hypothesis, no men-
tion is made of experiments that could be used to
differentiate these mechanisms from other possible
mechanisms. Likewise, no experiments are proposed to
more carefully examine the proposed phylogenetic rela-
tionships of YjeK and LAM.
To the authors' defence, I do not think this part of the
paper should even be there. In this, I disagree with the
requirements of Biology Direct. Experimenters, not
computational scientists should think about testing a
hypothesis, because the latter are not quite familiar with
modern experimental approaches and techniques (espe-
cially the newer ones).
Authors' response: a section was added on the in vitro
testing of the hypothesis in the revised version.
Reviewer 2
Implications:
This section seems to offer a reasonable interpretation
of the possible implications possibly identifying a new
post-translational modification mechanism while
acknowledging that this is not a ubiquitous mechanism
used by all prokaryotes.
Reviewer 3
Mikhail Gelfand, Research and Training Center on
Bioinformatics, Moscow, Russia
Overall this is a nice paper describing an interesting
hypothesis. I feel, however, that the authors have nit
exhausted the possibilities of comparative genomic


analysis, nor presented their results in the best possible
form.
The main problem with the hypothesis is that the sug-
gested candidates are not universal. The abstract says:
"In the other organisms, EF-P may be modified by
another pathway or the translation machinery must
have adapted to the lack of EF-P modification", but this
means nothing. At that, how strictly conserved is the
modification pathways in eukaryotes? How strictly con-
served is the lysine: are there any examples when the
corresponding position is occupied by another residue?
Authors' response: The elFSA modification pathway is
strictly conserved in eukaryotes. The predicted modifica-
tion pathway is clearly not conserved in prokaryotes. We
changed the Implication section to reflect this and also
included the data on the distribution of residues other
than Lys in bacterial EF-P in Fig. 1C.
Reviewer 3
Since only 125 of 722 studied bacterial species have
both candidate genes, an obvious question is, whether
these genes always co-occur (if they are often found
solo, this would be suspicious), whether they occur in
species without the conserved lysine (if they do, what
are they doing there?), and how many of 597 = 722-125
genomes have this lysine. Such analysis would allow the
authors to verify the conclusions obtained by positional
clustering using the phylogenetic profiles. The answer is
best represented by a Wenn diagram (Fig. 5).
In fact, the authors write: "all organisms that contain
EF-P proteins with a lysine residue at position 34 as
part of the conserved "PGKG" motif contain homologs


Figure 5 Wenn diagram. Red representing bacteria with efp;
orange, a subset of those with the conserved, lysine; blue, bacteria
with yjeA; and green, bacteria with yjeK (the colors, of course, are
arbitrary, whereas the topology is not).


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of YjeA and YjeK", but this crucial bit of their case is
largely ignored: "(data not shown)".
Authors' response:as discussed in response to reviewer
1 and 2 this information was added not as Wenn dia-
gram but in the text, as Additional file 1 and as Fig
1Band 1C.
Reviewer 3
Not being a specialist in mass-spec, I cannot judge the
validity of the the sentence: "Both reactions scheme
would generate an EF-P modified on Lys34 by a P-lysine
(146 Da) compatible with the 144 Da modification", is
144 = 146 within the accepted error margins?
Authors' response: The data describing the presence of
the modification in E. coli EF-P in Aoki H et al publica-
tion (Febs J 1998, 275:671-681)is quite crude based only
on predicted mass of tryptic peptide, so we feel we are in
the accepted error margins.
Reviewer 3
Fig. 4 is rather difficult to comprehend. The notation
and the distribution of proteins and substrates as sum-
mands at the top, or additional summands in intermedi-
ate stages, or catalyzing enzymes is unclear. I feel a
more traditional form of presentation would be more
useful here. Finally, for some reason the right panel is a
mirror reflection of the left one: it may create a nice-
looking symmetry, but for a less informed reader may
create a minor, but annoying problem.
Authors' response: we simplified the figure but kept
the mirror image organization as we felt that was not
really a problem.
Reviewer 3
A technical comment: I cannot deduce the meaning of
parentheses "(red circle Fig. 3B zoom merge)" and "(Fig.
3B active site zoom merge)" on page 5.
Authors' response: We clarified references to Fig. 3 in
the text in the revised manuscript.


Additional file 1: Table S1 Distribution and clustering of efp, yjeK, yjeA,
ablB and ablA in the 725 organisms analyzed
Click here for file
[ http//wwwbiomedcentra com/content/supplementary/l 7456150-5-3-
51 XLS]
Additional file 2: Fig. 54 Multiple Alignment of YjeA and LysRS2
sequences
Click here for file
[ http//wwwbiomedcentral com/content/supplementary/l 7456150-5-3-
2 PDF]
Additional file 3: Text S2 List of EF-P sequences from organisms that
have
Click here for file
[ http//wwwbiomedcentral com/content/supplementary/l 7456150-5-3-
S3 DOC]
Additional file 4: Text 53 List of EF-P sequences from organisms that
do not have have
Click here for file
[ http//wwwbiomedcentra com/content/supplementary/l 7456150-5-3-
54 DOC]


Acknowledgements
We thank Paul Schimmel, John Reader and David Metzgar for stimulating
discussion at the beginning of the project We also thank Daniel Kern and
Bruno Senger for helpful discussions This work was supported by the
National Institutes of Health (grant no NIAID-R01AI66244-01, subcontract to
V de C-L from A Osterman), and Marc Bailly is a recipient from a
postdoctoral fellowship from Human Frontier Scientific Program (HFSP)

Authors' contributions
MB did the detailed alignment analysis and all the figures and proposed the
mechanisms VdCL performed the comparative genomic analysis and made
the functional predictions Both authors wrote the paper and read and
approved the final manuscript

Competing interests
The author declare that they have no competing interests

Received: 23 December 2009
Accepted: 13 January 2010 Published: 13 January 2010

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doi:10.1186/1745-6150-5-3
Cite this article as: Bailly and de Crecy-Lagard Predicting the pathway
involved in post-translational modification of Elongation factor P in a
subset of bacterial species. Biology Direct 2010 53


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