Evolution of Bacterial Phosphoglycerate Mutases:
Non-Homologous Isofunctional Enzymes Undergoing
Gene Losses, Gains and Lateral Transfers
Jeremy M. Foster*, Paul J. Davis, Sylvine Raverdy, Marion H. Sibley, Elisabeth A. Raleigh, Sanjay Kumar,
Clotilde K. S. Carlow
Division of Parasitology, New England Biolabs, Inc., Ipswich, Massachusetts, United States of America
Background: The glycolytic phosphoglycerate mutases exist as non-homologous isofunctional enzymes (NISE) having
independent evolutionary origins and no similarity in primary sequence, 3D structure, or catalytic mechanism. Cofactor-
dependent PGM (dPGM) requires 2,3-bisphosphoglycerate for activity; cofactor-independent PGM (iPGM) does not. The
PGM profile of any given bacterium is unpredictable and some organisms such as Escherichia coli encode both forms.
Methods/Principal Findings: To examine the distribution of PGM NISE throughout the Bacteria, and gain insight into the
evolutionary processes that shape their phyletic profiles, we searched bacterial genome sequences for the presence of
dPGM and iPGM. Both forms exhibited patchy distributions throughout the bacterial domain. Species within the same
genus, or even strains of the same species, frequently differ in their PGM repertoire. The distribution is further complicated
by the common occurrence of dPGM paralogs, while iPGM paralogs are rare. Larger genomes are more likely to
accommodate PGM paralogs or both NISE forms. Lateral gene transfers have shaped the PGM profiles with intradomain and
interdomain transfers apparent. Archaeal-type iPGM was identified in many bacteria, often as the sole PGM. To address the
function of PGM NISE in an organism encoding both forms, we analyzed recombinant enzymes from E. coli. Both NISE were
active mutases, but the specific activity of dPGM greatly exceeded that of iPGM, which showed highest activity in the
presence of manganese. We created PGM null mutants in E. coli and discovered the DdPGM mutant grew slowly due to a
delay in exiting stationary phase. Overexpression of dPGM or iPGM overcame this defect.
Conclusions/Significance: Our biochemical and genetic analyses in E. coli firmly establish dPGM and iPGM as NISE.
Metabolic redundancy is indicated since only larger genomes encode both forms. Non-orthologous gene displacement can
fully account for the non-uniform PGM distribution we report across the bacterial domain.
Citation: Foster JM, Davis PJ, Raverdy S, Sibley MH, Raleigh EA, et al. (2010) Evolution of Bacterial Phosphoglycerate Mutases: Non-Homologous Isofunctional
Enzymes Undergoing Gene Losses, Gains and Lateral Transfers. PLoS ONE 5(10): e13576. doi:10.1371/journal.pone.0013576
Editor: Niyaz Ahmed, University of Hyderabad, India
Received May 28, 2010; Accepted September 27, 2010; Published October 26, 2010
Copyright: ? 2010 Foster et al. 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 author and source are credited.
Funding: This work was supported by New England Biolabs and by US National Institutes of Health/National Institute for Allergy and Infectious Diseases (SBIR
Grant Number 2R44 A1061865-02). The authors are employees of New England Biolabs; this funder is therefore considered by PLoS ONE to have played a role in
study design, data collection and analysis; however, the authors confirm that the funder did not play a direct role in the study.
Competing Interests: The authors are employees of New England Biolabs; this funder is therefore considered by PLoS ONE to have played a role in study
design, data collection and analysis; however, the authors confirm that the funder did not play a direct role in the study. The authors’ affiliation with the funders
does not alter their adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: firstname.lastname@example.org
Non-homologous ISofunctional Enzymes (NISE) is the pre-
ferred term to accurately describe enzymes that lack detectable
sequence similarity but catalyze the same biochemical reactions
and carry the same Enzyme Classification (EC) number . NISE
have previously been referred to as analogous enzymes [2,3]. In
many cases, NISE also lack structural similarity, this being a more
robust indicator of independent evolutionary routes towards
fulfilling a common metabolic conversion . NISE most likely
evolve by recruitment of existing enzymes that take on a new
cellular function following changes to the substrate binding site or
catalytic mechanism. This scenario is most plausible when one or
both members of a pair of NISE belong to a larger enzyme family
that catalyzes related reactions. For example, gluconate kinase
from Bacillus subtilis has orthologs within the genus Bacillus but is
otherwise unrelated to gluconate kinases from other bacteria or
eukaryotes. However, the Bacillus enzyme belongs to a larger
kinase family that includes xylulose kinase and glycerol kinase in
other taxa. A duplication in the gene encoding either xylulose
kinase or glycerol kinase is presumed to have occurred in the
lineage leading to the Bacilli and been followed by a shift in
substrate specificity to generate the novel gluconate kinase [3,4].
Lateral gene transfer (LGT) events can further shape the
distribution of NISE in different taxonomic groups and introduce
enzyme activities analogous to ones already encoded by the
recipient genome. The protozoan parasite, Trichomonas vaginalis, for
example, encodes distinct forms of malic enzymes, one of which
appears to be the result of LGT from a eubacterium . The
combination of enzyme recruitments and LGTs coupled with
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independent gene losses and gene gains in different lineages can
therefore lead to patchy distributions of NISE forms when viewed
across broad phylogenetic distances.
Phosphoglycerate mutase (PGM; E.C. 220.127.116.11.) catalyzes the
interconversion of 2- and 3-phosphoglycerate (2-PG and 3-PG) in
the glycolytic and gluconeogenic pathways. Two distinct forms of
PGM that have no similarity in protein size, primary sequence,
three-dimensional structure or catalytic mechanism are known to
exist and are considered analogous enzymes (NISE) [1,3,6]. One
form, cofactor-dependent PGM (dPGM), requires the cofactor
2,3-bisphosphoglycerate (2,3-BPG) for activity. The dPGM
enzymes, having a molecular mass of about 27 kD, are usually
active as dimers or tetramers and catalyze the intermolecular
transfer of a phosphoryl group between the monophosphoglyce-
rates and the cofactor via a phosphohistidine intermediate.
Sequence and structural analyses of dPGM enzymes place them
in the acid phosphatase superfamily along with enzymes such as
fructose-2,6-bisphosphatase and acid phosphatase [7,8]. On the
other hand, cofactor-independent PGM (iPGM) is typically about
57 kD, active as a monomer, and catalyzes the intramolecular
transfer of the phosphoryl group between monophosphoglycerates
through a phosphoserine intermediate. The iPGM enzymes
belong to the alkaline phosphatase superfamily along with
enzymes such as phosphopentomutases and certain sulfatases to
name a few [7,8,9]. The two forms of PGM can be distinguished
further by the metal ion requirement of iPGM and the sensitivity
of dPGM to vanadate [8,10].
PGM sequences, in particular those of iPGM, appear to be
evolving very slowly  and are generally very well conserved even
across different kingdoms , allowing their identification in
genome sequences from diverse organisms. However, since both
dPGM and iPGM are members of larger phosphatase superfam-
ilies containing diverse enzymes with related sequences, the
identification of PGMs solely by sequence similarity should be
treated with caution. Indeed, a predicted dPGM of Bacillus spp.
was subsequently shown by molecular modeling and enzymatic
analyses of recombinant protein to encode a broad specificity
phosphatase . Small-scale bioinformatic surveys and biochem-
ical studies have indicated that only iPGM is present in plants and
nematodes while only dPGM is found in mammals [6,10,12,13].
However, within other phylogenetic groups the distribution of the
two PGM forms is complex and has been described as appearing
haphazard . Most bacteria, archaea, protozoa and fungi
contain either iPGM or dPGM, while some bacteria such as
Escherichia coli and certain archaea and protozoa contain both
forms. The respective roles of dPGM and iPGM in organisms that
contain both forms of enzyme are uncertain.
In E. coli, at least, distinct PGM activities were reported for both
dPGM and iPGM in crude cell extracts and when expressed in
recombinant form . The dPGM form accounted for the great
majority of activity leaving unanswered questions about the role of
iPGM in E. coli. To gain insight into the respective functions of
dPGM and iPGM in E.coli, we generated null mutants for
phenotypic studies to examine the role of each enzyme. We report
that loss of dPGM leads to delayed growth both in liquid cultures
and on solid medium, apparently due to a delay or defect in
exiting stationary phase. We further show that the wild type
phenotype can be restored by overexpression of either dPGM or
iPGM in dPGM null mutants. We also produced recombinant
dPGM and iPGM for detailed biochemical analyses to address the
specific PGM and phosphatase activities of each enzyme. We
demonstrate that the distinct PGM forms present in E.coli have
overlapping and complementary roles in the cell.
The evolutionary origins of dPGM and iPGM that underlie the
unpredictable distribution of these NISE proteins in bacteria are
not clear [7,8]. However, the abundance of sequenced microbial
genomes provides an unprecedented opportunity to address the
distribution of NISE across hundreds of bacterial species. In the
present study we performed a comprehensive survey of the
distribution of the PGM forms throughout the bacterial domain to
gain insight into the processes and events that appear to have
contributed to their apparently haphazard phyletic profiles.
Materials and Methods
Bioinformatic identification of iPGM and dPGM in
The 702 completed microbial genomes listed in Table S1 were
downloaded from NCBI Refseq (ftp://ftp.ncbi.nih.gov/genomes/
Bacteria/) on October 18th, 2008.
A set of proteins was compiled which encompassed examples of
divergent bacterial dPGM and iPGM proteins, archaeal iPGM
and dPGM, as well as closely related, but functionally divergent,
acid and alkaline phosphatase superfamily members that could
complicate bioinformatic identification of bacterial PGM by
generating false positives. Using TBLASTN , these query
proteins (E. coli GpmA (dPGM), NCBI GI number 50402115;
Chlamydia trachomatis dPGM, 15605455; E. coli GpmM (iPGM),
586733; Ureaplasma parvum iPGM, 13357740; Thermoplasma acid-
ophilum (archaeon) dPGM , 10640690; Pyrococcus furiosus
(archaeon) iPGM , 18894161; E. coli gpmB (dPGM family
member), 67465002; Bacillus subtilis phosphatase, PhoE, 2633370;
Mycobacterium tuberculosis phosphatase , 38490339; E. coli
phosphopentomutase, DeoB, 170083769 and E. coli alkaline
phosphatase, PhoA, 48994877) were aligned against the six-frame
translations of the set of completed microbial genomes. References
to publications that establish the function of the above query
proteins are provided in those instances where original NCBI
functional definition of the query proteins was either lacking or
incorrect. TBLASTN was provided a value of 10,000 for both the
one-line descriptions and alignments parameters and a value of 10
for E-Value cutoff, with all other parameters left at default values.
All TBLASTN alignments with a bit score less than 100.0 were
discarded. The bit score cutoff of 100 was established empirically
by examination of the output produced using a range of bit score
cutoffs (data not shown).
Using the alignments passing the bit score threshold, a list of
automatic PGM assignments was generated for each genome using
the pattern of TBLASTN hits for the query proteins as follows. If
the genome had hits with overlapping genomic coordinates for the
dPGM queries from E. coli and C. trachomatis, it was automatically
called as ‘‘dPGM’’. Similarly, if the genome had overlapping hits
for the iPGM queries from E. coli and U. parvum it was called as
‘‘iPGM’’. If the genome had hits for the two dPGM and two
iPGM queries it was called as ‘‘iPGM plus dPGM’’ (both forms).
The genome coordinates of additional hits arising from any of
the 7 other queries were examined to identify instances where the
protein aligned to the same genomic locus as PGM or one of the
other query proteins. This step served to highlight any cases where
sequence similarity searches failed to differentiate between either
of the PGM forms and functionally diverged proteins from the
same phosphatase superfamily. Genomes for which no assignment
was automatically made usually contained more than one copy of
a given PGM type, or PGM similar to an archaeal PGM query, or
lacked any form of PGM. Such cases were curated following
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To verify the orthology assignments determined by TBLASTN,
we recovered each identified gene and used BLASTP (default
arguments) against the E. coli MG1655 genome (GI: 49175990) to
check that the corresponding PGM form in E. coli MG1655 (iPGM
GI:16131483, dPGM GI:16128723) was the top ranking hit. In all
cases except one, this check was successful. The single exception
was a dPGM gene from Akkermansia muciniphila (GI: 187735276)
that apparently has a full-length dehydrogenase gene (encodes
,330 amino acids) fused to the 39 end of a predicted PGM gene.
In this case, the E. coli dPGM was the second best hit while the top
hit was to the orthologous dehydrogenase.
The taxonomic designations of all organisms described in this
study are consistent with the NCBI Taxonomy Browser. In the
Results section (Tables and Figures), PGM distribution data is
generally presented at the Class taxon so as to adequately reveal
the non-uniform nature of PGM while limiting the number of
bacterial genomes displayed.
Mapping the likely origin of archaeal PGM genes in
The output from the TBLASTN analysis was also used to find
genes in bacterial genomes that contained hits to the archaeal
iPGM or dPGM queries with a bit score exceeding 100. The
archaeal-like genes were then used as queries against all
completely sequenced archaeal genomes downloaded from NCBI
(ftp://ftp.ncbi.nih.gov/genomes/Bacteria/) on October 18th,
2008 using the same TBLASTN parameters as described above.
The score for each archaeal species was then calculated as the
average bit score of the best blast hits for all PGM query
sequences. Where multiple genome sequences for one species are
available, only the single top bit score from across all sequences
was used in the calculation.
Bacterial strains, media and growth
Recombinant GpmA (dPGM) and GpmM (iPGM) were
expressed in E. coli T7 Express (New England Biolabs). Deletions
of gpmA (dPGM) or gpmM (iPGM) were made in the E. coli K-12
derivative, MG1655 (E. coli Genetic Stock Center). Bacteria were
grown in Luria Bertani (LB) medium (10 g tryptone, 5 g yeast
extract, 5 g NaCl per liter H20, pH 7.2) and in 3-(N-morpholi-
no)propanesulfonic acid (MOPS) minimal medium  (Te-
kNova), supplemented with 0.1% glucose. For production of
recombinant proteins or complementation by plasmid constructs,
ampicillin (100 mg/ml) was included in the growth medium. All
bacterial growth was at 37uC and liquid cultures were shaken at
PGM cloning, expression and enzyme assays
Full-length E. coli iPGM and dPGM were cloned into the pET-
21a vector (Novagen) for expression of recombinant proteins with
C-terminal His6tags in E. coli. The sequences were amplified (see
Table S2 for primers) from genomic DNA from E. coli strain T7
Express using the Expand High Fidelity PCR System (Roche).
Constructs were verified by DNA sequencing before expression of
the recombinant proteins in T7 Express E. coli. Optimal expression
of both iPGM and dPGM was achieved following induction with
0.3 mM isopropyl-1-thio-b-D-galactopyranoside (Sigma) for 3 h at
37uC. The His-tagged proteins were extracted and purified on
nickel columns (Qiagen) under native conditions according to the
Purified recombinant proteins were assayed for PGM activity in
the glycolytic direction (3-PG to 2-PG) using a standard enzyme-
coupled assay as described previously . Briefly, PGM was
added to 1 ml assay buffer (30 mM Tris-HCl, pH 7.0, 5 mM
MgSO4, 20 mM KCl) supplemented with 0.15 mM NADH,
1 mM ADP, 1.5 mM 3-PG substrate (Sigma P8877) and 2.5 units
of each coupling enzyme, namely enolase (Sigma E6126), pyruvate
kinase (Sigma P7768) and L-lactic dehydrogenase (Sigma L2518).
Reactions were at 30uC for 5 min with data collected every 10 s
using a DU 640 spectrophotometer (Beckman). Consumption of
NADH at 340 nm provided an indirect measurement of PGM
activity as the amount of NADH converted to NAD corresponds
to the amount of reaction product, 2-PG. One unit of PGM
activity is defined as the amount of activity necessary to convert
1.0 mmole NADH to NAD per minute under the standard assay
conditions. The effect of manganese ions was studied by adding
manganese chloride to the standard assay buffer to a final
concentration of 1 mM. Sensitivity to vanadate was addressed by
incubating the recombinant enzymes with different concentrations
of sodium metavanadate (Acros) for 15 min. prior to the assay.
The activity of dPGM was determined in the absence of the
cofactor, 2,3-BPG, since the commercially available 3-PG
substrate for PGM assays contains 2,3-BPG as a contaminant in
sufficient amounts to stimulate dPGM activity causing an apparent
lack of dependency on cofactor .
Phosphatase activity was assessed in 200 ml reactions using
10 mg enzyme and 50 mM p-nitrophenyl phosphate (New England
Biolabs) as substrate. Various buffer systems were used: NEBuffer
3, pH 7.9, NEBuffer EcoRI, pH 7.5 (both from New England
Biolabs), PGM assay buffer, pH 7.0 (see above), and 1 M
diethanolamine, 1 mM MgCl2, pH 9.75. The effect of different
metal ions was determined by addition of either ZnCl2or CoCl2to
these four magnesium-containing buffers. Calf intestinal phospha-
tase (New England Biolabs) served as an alkaline phosphatase
positive control in each buffer. Reactions were incubated at 37uC
for 30 min before being stopped by addition of 1 ml 1N NaOH.
The production of p-nitrophenylate was determined spectropho-
tometrically at 405 nm and compared to controls lacking either
substrate or enzyme.
Construction and characterization of E. coli PGM mutant
Separate strains bearing either a deletion of the entire iPGM or
dPGM open reading frame of E. coli MG1655 were prepared by l
Red-mediated recombination . PCR primer pairs were
designed (Table S2) such that their 59 ends corresponded to the
sequence immediately upstream and downstream of each PGM
translational start and stop codon, respectively, while the 39 ends
of each primer pair corresponded to the P1 and P2 priming sites of
the pKD4 plasmid . The gene deletions in the resultant strains,
MG1655DgpmM::FRT1 and MG1655DgpmA::FRT1 (DiPGM and
DdPGM, respectively), were confirmed by PCR with diagnostic
primers and by DNA sequencing. FRT1 indicates a FLP
recombinase recognition site left at each locus after removal of a
kanamycin cassette used during strain construction .
The growth of the DdPGM and DiPGM strains relative to the
MG1655 parental strain was assessed by diluting overnight
cultures grown in MOPS minimal medium supplemented with
0.1% glucose into 10 ml fresh LB medium in Nephelo sidearm
flasks (Bellco Biotechnology) to give initial OD600values of 0.03.
Each strain was grown in triplicate and turbidity monitored using
a photoelectric colorimeter (Klett Summerson). For evaluating
growth on solid media, overnight cultures grown in MOPS
minimal medium containing 0.1% glucose were standardized to
similar optical density, when necessary, then serially diluted and
100 ml of each dilution plated in triplicate to LB agar. The number
of colonies on each plate was recorded after overnight growth.
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Complementation of DdPGM
To examine whether E. coli iPGM or dPGM could complement
the DdPGM growth phenotype, these genes were cloned into the
pKK223-3 expression vector (Amersham Pharmacia Biotech) and
transformed into the DdPGM mutant strain. The sequences were
amplified (see Table S2 for primers) from the pET-21a constructs
described above using Phusion High Fidelity DNA polymerase
(New England Biolabs), then cloned into pKK223-3 and verified
by DNA sequencing. The constructs were designated pKKiPGM
and pKKdPGM. For complementation assays, strains MG1655,
DdPGM, and DdPGM harboring, separately, pKKiPGM and
pKKdPGM were initially grown overnight in MOPS minimal
medium containing 0.1% glucose. These cultures were then
serially diluted and plated in triplicate to LB agar as described
above. Strain DdPGM harboring empty plasmid, designated pKK,
served as a control.
Results and Discussion
Validation of the selected PGM superfamily members as
queries for ortholog detection
The distribution of dPGM and iPGM was previously reported
from small-scale bioinformatic
[6,10,12,13]. Here we took advantage of the abundance of
microbial genome sequences to comprehensively examine the
distribution of the PGM NISE across 702 complete bacterial
genomes (Table S1). We reasoned that use of divergent dPGM and
divergent iPGM queries for our TBLASTN analyses would
maximize identification of their bacterial orthologs and reduce/
eliminate false negatives. We also used a variety of functionally
divergent protein queries from the acid and alkaline phosphatase
superfamilies to which dPGM and iPGM respectively belong.
Since in most cases these superfamily members show sequence
similarity to PGM, careful analysis of their BLAST hits was
necessary to reduce/eliminate false positive identification of PGM.
PGM query proteins.
For identification of dPGM orthologs
in bacterial genomes by TBLASTN analysis, we selected the
experimentally validated E. coli dPGM (GpmA)  and dPGM
from C. trachomatis as queries. The latter dPGM shows considerable
divergence from the E. coli ortholog, but passed our 100 bit score
threshold for assignment as a PGM. The two proteins give
reciprocal best BLAST hits between their genomes establishing
biochemically characterized dPGM from Schizosaccharomyces pombe
than it does to E. coli dPGM. The C. trachomatis protein lacks a
stretch of ,25 amino acids when compared to dPGM from E. coli
and most other organisms. Although this missing loop region is the
least conserved region of dPGMs [11,21], it contains amino acids
important for dimerization or tetramerization . Interestingly,
S. pombe dPGM, which has been characterized in detail, also lacks
this region and is active as a monomer [23,24], suggesting that
certain bacterial dPGM forms, such as that from C. trachomatis, are
also monomeric. We noted that this type of dPGM, lacking the
dimerization/tetramerization domain, is common within the order
Chlamydiales and phylum Cyanobacteria (orders Chroococcales
and Gloeobacteria), as well as the order Rhizobiales (a-
proteobacteria). It was also observed in Pseudoalteromonas atlantica
Sulfurihydrogenibium sp. (Aquificales). However, we found that
members of the order Chlamydia and the cyanobacteria that
lack this region of dPGM generally have an insertion of ,25
amino acids nearer to the N-terminus. The significance, if any, of
this region is unknown. Despite the use of divergent dPGM
the higher similarity to
proteins for our TBLASTN analysis, we determined that the two
queries always generated the same hits (overlapping genome
coordinates) on the bacterial genomes. To identify iPGM
orthologsin the bacterial
experimentally characterized iPGM from E. coli (GpmM) 
and the divergent U. parvum iPGM as queries for our TBLASTN
analyses. These query proteins were also established as orthologs
via reciprocal best BLAST hits. These two iPGM queries also
always showed overlapping hits on the bacterial genomes. These
observations are in agreement with the known well-conserved
nature of the two PGM forms across different taxa and provides
confidence that we identified all (or most) of the bacterial enzymes.
The dPGM and/or iPGM genes identified in each bacterial
genome were verified as orthologs of the characterized E. coli
PGM genes by returning as best BLAST hits the appropriate
PGM gene of E. coli.
We also used the sequences of biochemically characterized
dPGM and iPGM proteins from the archaea T. acidophilum and P.
furiosus, respectively, to query the complete bacterial genome
sequences. We did not detect any archaeal dPGM orthologs in the
bacterial genomes but found several examples of archaeal iPGM.
The loci of the archaeal-like iPGM sequences we identified in
bacterial genomes were in all cases distinct from those revealed by
the bacterial iPGM and dPGM queries, again indicating that our
parameters were sufficiently sensitive to differentiate closely related
Alkaline and acid phosphatase superfamily members as
Although our divergent iPGM and dPGM
query proteins gave clear and consistent results, it is known that
identification of PGM orthologs based on sequence similarity
alone can be unreliable because of their similarity to functionally
more divergent members of the alkaline and acid phosphatase
superfamilies to which they belong . We addressed this
possibility by including as queries for our TBLASTN analysis of
the bacterial genomes, various superfamily member proteins,
which could reveal false positive PGM identification or cases
where functional assignment by sequence similarity is ambiguous.
For this purpose we used well characterized proteins such as phoE,
a broad-specificity phosphatase from B. subtilis, and a Mycobacterium
tuberculosis phosphatase, both of which were originally annotated as
dPGM [11,17]. We included other representative superfamily
members, namely deoB, an E. coli phosphopentomutase, and
phoA, an E. coli alkaline phosphatase, which could also confound
interpretation of the BLAST outputs [9,25]. While these four
additional queries returned hits from various genomes (Table S1),
there was not a single instance where a hit with a BLAST bit score
greater than 100 had overlapping genome coordinates with hits
returned by the dPGM or iPGM queries. This indicates that a bit
score threshold of 100 appears to reliably differentiate the various
superfamily members. We did not use more distant superfamily
members such as SixA phosphoprotein phosphatase and Ais as
queries since these are known not to have significant match to
dPGM in standard BLAST searches . However, a second
dPGM-like gene, phosphoglycerate mutase B (GpmB) has been
noted previously in various Enterobacteriaceae . We identified
candidate orthologs of this protein not only in the c-proteobacteria
but in other diverse bacterial taxa (Table S1). Once again, the
dPGM and GpmB hits on all such genomes were non-overlapping.
In fact we noticed overlap of the hit coordinates for the GpmB and
Enterobacteriales but also in the Clostridiales, suggesting that
GpmB may be an acid phosphatase. These analyses increase our
confidence that our identification of PGM orthologs was robust
since they showed that the distribution and genomic loci of
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orthologs of known PGM superfamily members, or other
sequences closely related to PGM, had no overlap with those of
dPGM or iPGM.
Overview of distribution of dPGM and iPGM across the
After removal of duplicate genomic sequences for some
bacterial strains, we calculated that the dPGM queries had 447
hits on 410 genomes (,1.1 hits/genome) with a range of 0 to 3 hits
per genome (Table S1). Thirty-four genomes had more than one
dPGM. Of the 410 genomes containing dPGM, 115 also had at
least one iPGM hit (Fig. 1). No eubacterial genomes had hits above
the bit score threshold of 100 when the biochemically character-
ized dPGM from the archaeon Thermoplasma acidophilum  was
used a query. There were 430 iPGM hits on 391 genomes (,1.1
hits per genome) with a range of 0 to 4 hits per genome. However,
only in 4 diverse bacteria (discussed below) was more than one
iPGM identified by the two bacterial iPGM queries used.
Considering only these ‘‘bacterial type’’ iPGMs, we report 380
hits on 373 genomes (,1.0 hit/genome). The experimentally
validated archaeal iPGM from Pyrococcus furiosus  identified 50
archaeal-like iPGM sequences in 43 bacterial genomes (Fig. 1),
presumably as a result of LGT, thereby increasing the apparent
frequency and number of iPGM hits per genome. The genome
coordinates of the archaeal iPGM hits were distinct from those for
the two bacterial iPGM queries in all cases. Of interest, eighteen
bacterial genomes contained archaeal type iPGM as their only
PGM form (Table 1; Fig. 1; Table S1).
Sixteen genomes did not contain any form of PGM (Table 1;
Fig.1). These organisms included the a-proteobacterial Rickettsia
spp and closely related Orientia spp., together with Candidatus
Sulcia muelleri (Flavobacterium), Candidatus Carsonella ruddii (c-
proteobacterium) and Candidatus Phytoplasma mali (Mollicute)
(Table 1; Table S1). These are all intracellular bacteria with
reduced genomes ranging from 2.1 Mb (O. tsutsugamushi) to the
smallest known bacterial genome of 160 kb (Candidatus Carsonella
ruddii) that lack all or part of the glycolytic pathway.
Examination of the presence of the PGM NISE across different
bacterial taxa revealed a strikingly non-uniform distribution
(Table 1) as noted previously . This was generally most evident
for taxa such as the a-, d- and c-proteobacteria, the Clostridia and
the Bacilli which contain greater numbers of fully sequenced
genomes. Other groups often contained very few sequenced
genomes or a limited diversity of sequenced species thereby
potentially masking PGM heterogeneity within those groups. For
example, the 12 completed genomes within the order Prochlorales
are from different strains of the same species. However, even
different strains of Prochlorococcus  and other species [28,29,30]
may have considerable variation in their gene content. In the case
of Frankia spp., as many as 3,500 genes (,50% of the predicted
ORFs) may differ between strains [29,31]. The non-uniform
distribution of PGM NISE did not appear to correlate with any
obvious trait such as aerobic/anaerobic metabolism, pathogenic-
ity, or Gram staining.
PGM Diversity within bacterial taxa
We found that much of the PGM heterogeneity observed in
certain classes of bacteria (Table 1) stratified when individual
families and genera were considered. For example, the diversity
observed in the class Bacilli (Table 1) was resolved by examination
of different families and genera (Fig. 2). Although a comparison
between different families or genera revealed divergent PGM
profiles, of 9 represented families, only the Bacillaceae exhibited
diversity within its PGM profile, and of 13 genera, only the genus
Bacillus (6 iPGM; 10 iPGM plus dPGM) had a non-uniform
distribution (Fig. 2). Similarly, the 66 genomes from the family
Enterobacteriaceae (c-proteobacteria) (12 dPGM; 54 dPGM +
iPGM) come from 17 genera, each of which is internally
homogeneous: either a genus had exclusively dPGM or it had
dPGM plus iPGM (Fig. S1). Nonetheless, the different lineages
within the classes Bacilli and c-proteobacteria still showed
considerable variation in their PGM profiles, as depicted by the
shading in Fig. 2 and Fig. S1. For example, of the 3 species within
the family Alteromonadaceae (c-proteobacteria), one contains
dPGM, another contains iPGM and the third contains both.
Variation also existed even at the species level: of two species of
Pseudoalteromonas (c-proteobacteria), one contains iPGM while the
other has both dPGM and iPGM (Fig. S1, Table S1). Other classes
of bacteria such as the Clostridia and a-proteobacteria showed yet
more variation in their PGM profiles (Figs 3, 4). All 19 Clostridium
spp. genomes contain iPGM but 3 of these additionally contain
dPGM. Similarly, amongst the 7 genomes within the order
Thermoanaerobacterales (Clostridia) examples exist of those
containing just dPGM or iPGM or both. All 3 species of
Thermoanaerobacter contain dPGM but 2 of them also have iPGM
(Fig. 3, Table S1). The order Rhizobiales (a-proteobacteria) has a
particularly haphazard PGM distribution with individual species
in 2 genera (Bradyrhizobium and Methylobacterium) showing variable
PGM profiles. However, the iPGM identified in Bradyrhizobium sp.
BTAi1 consists of only the N-terminal 225 amino acids and is
followed by a transposase so we considered it a pseudogene. Of the
6 sequenced strains of Rhodopseudomonas palustris, 4 contain only
iPGM while the remaining 2 have only dPGM (Fig. 4, Table S1).
Strains of this species are known to have variable gene contents
and the two strains that contain only dPGM are more similar to
each other than to the other isolates . Other classes of bacteria
showed variable levels of PGM heterogeneity (Tables 1, S1). Of 53
Actinobacteria genomes all but 2 contain solely dPGM. However,
Rubrobacter xylanophilus contains iPGM of archaeal origin as its only
PGM, while Streptomyces coelicolor has both bacterial iPGM and
dPGM. The sister species, S. avermitilis and S. griseus, have only
dPGM. Within the d-proteobacteria, a similar species-level
variability was observed in the genus Geobacter where all 5
sequenced genomes encode both bacterial and archaeal iPGM,
but 3 genomes additionally contain dPGM. A further interesting
example of PGM diversity was seen between the two Candidatus
Phytoplasma spp. (Mollicutes). Candidatus P. australiense has iPGM
and an intact glycolytic pathway, whereas Candidatus P. mali has
Figure 1. Distribution of dPGM, iPGM and orthologs of
archaeal iPGM across 702 completed bacterial genome se-
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an incomplete glycolytic pathway that terminates in glyceralde-
hyde-3-phosphate and consequently lacks any form of PGM.
Bacteria encoding more than one dPGM protein
As mentioned above, 34 genomes contained more than one
dPGM gene, and frequently members of the same genus differed
in this respect. For example, Bacteroides thetaiotaomicron and B.
vulgatus (Bacteroidetes) each contain 2 dPGM genes, while the
different strains of B. fragilis have only one (Table S1). Similar
numerical dPGM variations exist between different species of
and Rhizobium (both a-proteobacteria), and
between different strains of Frankia (Actinobacteria) and Bacillus
cereus (Bacilli) (Table S1). In the case of Rhizobium spp, the two
sequenced strains of R. etli each have 2 dPGM genes, while R.
leguminosarum has one. In each of the R. etli genomes, the additional
dPGM sequence is encoded by one of the extrachromosomal
plasmids. Although R. leguminosarum contains 6 plasmids none
encodes a second dPGM. Most species of Burkholderia (b-
proteobacteria) have 2 or 3 chromosomes with or without
additional plasmids. We determined that of the 21 sequenced
species or strains, only B. xenovorans has 2 dPGM genes and that
one copy is located on a plasmid. Other species also have their
Table 1. Summary of dPGM and iPGM distribution across different bacterial taxa.
D+ +I Total DTotal I
Proteobacteria Alphaproteobacteria 89133 43 3000
Gammaproteobacteria1841 57 468000
Deltaproteobacteria21071 13 110
Epsilonproteobacteria 21011 1900
Actinobacteria Actinobacteria5301 51111
Firmicutes Bacilli960 32531100
Clostridia 37052 3070
Chlamydiae Chlamydiae1300 13000
Spirochaetes Spirochaetes 1600 10600
Cyanobacteria Chroococcales15010 1400
Prochlorales 12000 1200
Tenericutes Mollicutes22100 2100
The number of genomes in each taxon identified as containing only iPGM, only dPGM, both iPGM and dPGM, and no PGM are given. The number of bacterial genomes
containing archaeal type iPGM are given and are a subset of the total iPGM and/or total iPGM and dPGM categories. Genomes containing archaeal iPGM as their only
PGM form are also enumerated. The taxonomic groupings shown in bold type are those used predominantly in this study and are taken from the NCBI Taxonomy
Browser. All are classes except for the orders Chroococcales, Nostocales, Oscillatoriales and Prochlorales (from the phylum Cyanobacteria and lacking any class
designation in the NCBI taxonomy database), and the phylum Bacteroidetes, which encompasses 7 genomes from the class Bacteroidia plus one incompletely classified
Bacteroidete member. Four species with incomplete lineage designations are grouped at bottom of the table as ‘‘Unclassified’’.
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different dPGM genes encoded by different molecules. For
example, Cyanothecae sp. (Chroococcales) has both a circular and
linear chromosome plus 4 plasmids and each of the chromosomes
encodes dPGM. Similarly, the a-proteobacterium, Phenylobacterium
zucineum, has 3 dPGM genes, one located on the chromosome and
two on the single large plasmid. The presence of 2 or more dPGM
genes appeared to correlate with larger genome sizes since no
occurrence of duplicate dPGM genes was found in the smallest
bacterial genomes (about 20% of all genomes). The smallest
genomes with 2 dPGM genes were those found in the order
Lactobacillales (smallest genome ,1.8 Mb). Excluding these, all
remaining examples were over ,3.7 Mb and occurred in the top
45% of genomes ranked by size (Table S1). This observation is
consistent with previous data correlating greater numbers of
paralogous protein families with larger genome sizes .
Lateral Gene Transfers
We reasoned that the patchy phyletic profiles of dPGM and
iPGM we observed across the bacterial domain could be partly
attributable to LGTs. However, inference of LGT events based on
similarity search analysis has several limitations [33,34]. A
combination of methods such as BLAST search, phylogenetic
tree construction, nucleotide composition comparisons and gene
distribution pattern analyses generally provide more robust
predictions of LGTs. However, phenomena including gene loss,
differing evolutionary rates, convergence, selection, mutation and
polymorphisms plague all these methods to various extents .
For large data sets similarity searches still provide a reasonable and
quick indication of LGT events.
Examination of genomes with two or more predicted
Initially we examined genomes with two or more
copies of either PGM form to highlight likely occurrences of LGT.
Therefore we examined in detail the duplicate iPGMs identified
by our bacterial iPGM queries in only 4 of the 702 genomes
(described above). One of the 2 iPGMs of Acidithiobacillus
ferrooxidans matched closely to related c-proteobacteria while the
second copy had only one c-proteobacterial hit (other than to
itself) among the 20 best hits, representing 14 different genera.
These top hits for this second dPGM had comparable BLAST bit
scores and were almost exclusively to certain members of the order
Clostridiales and to d-proteobacteria but included the archaeal
organism, Methanosaeta thermophila. We observed that the PGMs
Figure 2. Distribution of PGM types across 96 completed genome sequences from the Class Bacilli. Taxonomic nodes (left to right) are
Class, Order, Family, Genus. Taxa with genomes containing only iPGM are shaded yellow, those with only dPGM are shaded blue, those with both
iPGM and dPGM are shaded green while taxa with non-uniform PGM profiles are shaded pink. The numbers in boxes accompanying each taxon
identifier correspond to (left to right) number of genomes with only dPGM, only iPGM, both dPGM and iPGM, and no PGM.
PLoS ONE | www.plosone.org7 October 2010 | Volume 5 | Issue 10 | e13576
from these Clostridial, d-protoebacterial and Methanosarcinale
organisms, many of which are thermophilic, frequently grouped
together in our TBLASTN outputs indicating their sequence
similarity, as noted previously [15,35]. Many archaea belonging to
the order Methanosarcinales are found in fresh water and marine
sediments so it is perhaps not surprising to find genes shared with
anaerobic soil bacteria such as Clostridium spp. Indeed, one-third of
the ORFs from Methanosarcina mazei, including a predicted iPGM,
have their closest homolog in the bacterial domain, indicative of
widespread LGTevents . Thus itappears that one iPGM copy in
A. ferrooxidans may be the result of an ancient LGT. Of the two iPGM
copies in the d-proteobacterium Sorangium cellulosum, one shared
proteobacterial groups. However, the second copy had greatest
similarity with a very restricted set of bacteria (3 other d-
proteobacterial species, 1 c-proteobacterium and 3 species of the
spirochaete Leptospira), but was otherwise most similar to kinetoplastid
protozoans and plants. The phylogenetic relatedness of plants and
kinetoplastids is known and many kinetoplastid proteins, including
iPGM, are believed to have a plant or cyanobacterial origin [36,37].
However, the S. cellulosum gene had little similarity to any extant
sequenced cyanobacterium.Interestingly,the trypanosomatid
phosphate dehydrogenase, appear to have spirochaete origins
leading to the suggestion that various trypanosomatid housekeeping
genes may have been acquired by an ancestral LGT from
spirochaetes . It is likely that the second iPGM copy we
detected inS. cellulosum is also the resultof an LGTfrom a spirochaete
although the possibility of an interdomain LGT from eukaryotes is
not ruled out. We determined that one iPGM copy in Pseudomonas
putida F1 contained an in-frame stop codon and should therefore be
considered a pseudogene. This finding makes the P. putida F1 strain
similar to other sequenced strains in having just one full-length iPGM
open reading frame. The two iPGM copies in the Clostridial
bacterium Desulfotomaculatum reducens appeared to be the result of a
gene duplication, with the predicted proteins sharing 90% similarity
and generating almost identical TBLASTN results. Therefore, of the
fourinstancesoftwo ‘‘bacterial-like’’iPGMsin one bacterialgenome,
one is explained by a pseudogene, one represents probable gene
duplication while two appear to be the result of LGT.
Examination of genomes with two or more predicted
dPGM genes or phylogenetically aberrant PGM profiles.
We also examined genomes with unusual PGM composition in
comparison to closely related species, and genomes with two or
Figure 3. Distribution of PGM types across 37 completed genome sequences from the Class Clostridia. Taxonomic nodes (left to right)
are Class, Order, Family, Genus. Taxa with genomes containing only iPGM are shaded yellow, those with only dPGM are shaded blue, those with both
iPGM and dPGM are shaded green while taxa with non-uniform PGM profiles are shaded pink. The numbers in boxes accompanying each taxon
identifier correspond to (left to right) number of genomes with only dPGM, only iPGM, both dPGM and iPGM, and no PGM.
PLoS ONE | www.plosone.org8 October 2010 | Volume 5 | Issue 10 | e13576
PLoS ONE | www.plosone.org9 October 2010 | Volume 5 | Issue 10 | e13576
more dPGM genes, for candidate LGT events. As mentioned
above, of 53 Actinobacteria genomes, Streptomyces coelicolor was the
only species that contained bacterial-like iPGM. This protein had
similarity to a variety of other bacterial groups but predominantly
to proteins from cyanobacteria, fimicutes and d-proteobacteria,
indicating a likely LGT event. Similarly, the dPGM of
similarity to proteins from the Chroococcales, Chlamydiae and
plants as well as to a single member of the Aquificae. The ancient
ancestral relationship of cyanobacteria (eg. Chroococcales),
Chlamydiaceae and plant chloroplasts is known , but the
unusual finding of a gene with high similarity to members of these
groups within the c-proteobacteria is suggestive of a LGT. We
found that the TBLASTN results for one dPGM protein from
those species having more than one dPGM gene, or that have
dPGM when closely related species do not, were often broadly
similar.For example,one dPGM
proteobacterium Nitrosomonas europaea had similarity to dPGM
proteins from Janthinobacterium sp., Herminiimonas arsenicoxydans
(both b-proteobacteria with two dPGM genes) and to only the 3
species of Geobacter (d-proteobacterium) that contain dPGM in
addition to iPGM. We also observed that many of the highest-
ranking hits from these various dPGM queries were to members
of the Chlorobia, suggestive of either a shared ancestry or LGT
events. Many of these bacterial dPGM queries also showed
similarity to dPGMs from lower eukaryotes, notably the slime
mold Dictyostelium discoideum, the hydrozoan Hydra magnipapillata,
and the protozoan Trichomonas vaginalis. In many cases (eg.
Burkholderia xenovorans, Nitrosomonas europaea, Geobacter spp.), the hits
to these eukaryotic dPGMs were amongst the top 6 BLAST hits.
We analyzed these eukaryotic proteins in more detail and
determined that in all cases their own top BLAST hits were to
bacteria (Chlorobia members in the cases of T. vaginalis and D.
discoideum; b-proteobacteria in the case of H. magnipapillata).
Interestingly, T. vaginalis also contains iPGM and clustering of this
protein with bacterial iPGM has been noted while other
protozoans with iPGM formed a monophyletic group .
Other inter-domain LGTs have been described or implicated
previously for PGM [15,35,37,40].
Archaeal type PGMs in bacterial genomes.
evidence of archaeal type dPGM genes in bacteria. The 43 bacterial
genomes that contained the 50 archaeal type iPGM genes were not
randomly distributed throughout the bacterial domain. Classes such
as the Deinococci, Aquificae and Thermotogae that contain
predominantly or exclusively thermophilic species accounted for
many of the archaealtypeiPGMs (Tables 1,S1). With theexception
of Deinococcus radiodurans and 3 Dehalococcoides spp., all 18 bacteria
with archaeal iPGM as their only PGM form are thermophilic. Of
the bacterial orders with larger numbers of sequenced genomes,
only the Bacteroidetes, Clostridia and d-proteobacteria had
representatives with archaeal type iPGM, and even within these
groups, somespeciessuch as
Pelotomaculum thermopropionicum are thermophiles. Genome analyses
have previously indicated massive gene exchange between
thermophilic bacteria and archaea [41,42] with as much as 25%
of the bacterial proteome being most similar to archaeal proteins.
We found no
Of 19 Clostridia spp., only 3 had archaeal iPGM (Table S1). The
gene in C. phytofermentans, although similar to that from C.
thermocellum, contains an in-frame stop codon and is considered a
pseudogene. The predicted proteins of C. themocellum and C. novyi
have relatively low similarity to each other and gave quite different
TBLASTN results, showing highest similarity to different groups
of archaea, indicative of different ancestral origins. The 3
Dehalococcoides spp. all have two archaeal type iPGM genes.
Although comparisons between species showed that the gene pairs
are very similar, comparison of the two predicted proteins in any
species again points to different phylogenies. Similarly, the single
archaeal iPGM in Pelobacter propionicus (d-proteobacteria) is similar
to one of two such genes in P. carbinolicus. However, the second
archaeal iPGM in P. carbonolicus is quite divergent. The two iPGMs
of Thermodesulfovibrio yellowstonii also appeared to have different
archaeal origins. The d-proteobacterium Syntrophus aciditrophicus
encodes 3 archaeal type iPGMs, which share only about 45%
amino acid similarity and also appear to derive from different
groups of archaea.
We developed a bioinformatic approach to investigate the
archaeal groups that have greatest similarity to the archaeal-like
iPGMs identified in bacterial genomes. We used the 50 archaeal-
like iPGM proteins as queries of all complete archaeal genome
sequences that represent 48 distinct archaeal species (Table S3).
We determined that overall, the archaeal iPGMs from bacterial
genomes had greatest similarity with members of the phylum
Euryarchaeota, most notably, in decreasing order, to the classes
Methanobacteria, Methanomicrobia and Methanococci (Fig. S2).
However, the highest scoring individual hits were to the
Methanomicrobial species Methanococcoides burtonii, Methanosarcina
spp., and Methanosaeta thermophila. This is consistent with the
reported high similarity of iPGM from these archaea and iPGM
from bacteria, and the observation that Methanosarcina mazei and its
close relatives appear to have exchanged genetic information by
LGT with the bacteria that share their environment on multiple
Bacterial genomes encoding both dPGM and iPGM
Both PGM forms were detected in 115 genomes (16% of total)
(Fig. 1; Table 1). While an archaeal iPGM never accompanied
dPGM in the absence of bacterial type iPGM, 10 genomes contain
all 3 types. (Fig. 1; Table S1) With the exception of the Clostridium
phytofermentans pseudogene (discussed above), the remaining 9
genomes were restricted to the Bacteroidetes and d-proteobacteria.
The majority of species with both bacterial type PGM NISE, but
not an archaeal-type example, were found within the Bacilli and c-
(Table 1), but this observation is mostly accounted for by the
large numbers of sequenced genomes for genera such as Bacillus,
Staphylococcus, Escherichia, Salmonella, Klebsiella and Yersinia.
In looking at the dPGM and iPGM proteins predicted by each
genome that encodes both forms, we noted that frequently the
dPGM had unusual BLAST matches, similar to several of the
dPGM proteins encoded by genomes with two or more dPGM
genes (see above). For example, within the phylum Firmicutes
(Clostridia/Bacilli), all Listeria spp and several species of Clostridium,
Figure 4. Distribution of PGM types across 89 completed genome sequences from the Class a-proteobactria. Taxonomic nodes (left to
right) are Class, Order, Family, Genus. Taxa with genomes containing only iPGM are shaded yellow, those with only dPGM are shaded blue, those with
both iPGM and dPGM are shaded green while taxa with non-uniform PGM profiles are shaded pink. Taxa with no PGM are unshaded. The numbers in
boxes accompanying each taxon identifier correspond to (left to right) number of genomes with only dPGM, only iPGM, both dPGM and iPGM, and
PLoS ONE | www.plosone.org 10October 2010 | Volume 5 | Issue 10 | e13576
Bacillus and Thermoanaerobacter have both PGM NISE forms; their
dPGM proteins showed high similarity to various members of the
Chlorobia as well as to lower eukaryotes such as D. discoideum and
H. magnipapillata. Notably, the dPGM protein from Desulfovibrio
desulfuricans had best BLAST match to the dPGM from the
eukaryote H. magnipapillata followed by various Chlorobia
members rather than to other d-proteobacteria and might
represent another candidate LGT event. We observed that
another subset of the dPGM proteins predicted by genomes with
both NISE forms had similarity to the same restricted set of
bacteria and to certain yeasts (eg S. pombe), and some lower
eukaryotes. Closer inspection of the BLAST results for Parvibaculum
lavamentivorans, Methylobacterium spp (both a-proteobacteria) and
Myxococcus xanthus (d-proteobacteria) for example, revealed that
these similarities were at least in part accounted for by the proteins
resembling the characterized S. pombe dPGM [23,24] in lacking a
,25 aa region involved in dimerization/tetramerization. This
finding further supports our notion that several bacterial dPGM
proteins are active as monomers.
There appeared to be a strong correlation between the presence
of both PGM NISE forms and genome size (Table S1). We found
that of 115 genomes encoding both dPGM and iPGM, 85 were
larger than 4 Mb. In fact, only 3 such genomes were smaller than
2.5 Mb (2 Thermoanaerobacter spp, ,2.4 Mb and the unclassified
bacterium Elusimicrobium minutum, ,1.6 Mb). These genomes were
the only examples found in bottom third of the list of 702
sequenced genomes ranked by size. This correlation is similar to
the one we observed linking duplicate dPGM genes with larger
genomes (see above) and supports the published observation that
smaller genomes encode disproportionally fewer analogous
enzymes (NISE) [1,3]. Our data indicate that the presence of
PGM paralogs or both NISE forms is a feature predominantly
enjoyed by bacteria with larger genomes.
Characterization of the PGM NISE forms of E. coli
The co-occurrence of dPGM and iPGM in the same organism is
found in diverse bacterial groups (Table 1), yet only in E. coli has
the PGM activity of both forms been investigated . Biochemical
and genetic studies are ultimately necessary to verify NISE
predictions made by bioinformatic means. We therefore produced
recombinant E. coli PGM enzymes for a more detailed
characterization and exploited the genetic tractability of E. coli
to create strains deficient for each PGM protein so as to gain
further insight into their cellular roles and their status as functional
Expression and activity of E. coli dPGM and iPGM
Recombinant dPGM and iPGM were abundantly overex-
pressed in E.coli and subsequently purified by nickel-nitrilotria-
cetic acid chromatography. Imidazole (100 mM for iPGM;
200 mM for dPGM) in the elution buffer resulted in release of
the proteins from the nickel resin with a high degree of purity.
The yield of each protein was in excess of 300 mg per liter. The
sizes of dPGM and iPGM bearing vector-encoded N-terminal
T7 and C-terminal His6 tags were consistent with their
calculated molecular masses of 31 kD and 58.6 kD, respectively
(Fig. S3A and B). Both E. coli enzymes exhibited PGM activity as
evidenced by the consumption of NADH by the coupling
enzymes used in the assay (Fig. S3C). The slopes of the curves in
the figure were used to calculate PGM specific activities of ,1.8
units/mg and 229 units/mg for iPGM and dPGM, respectively.
This result is in agreement with an earlier report of the
significantly higher specific activity of E. coli dPGM compared to
iPGM . However, in both studies, iPGM activity was
determined in buffer containing magnesium, yet manganese
appears to be the preferred ion for bacterial iPGM enzymes that
have been characterized (see  for review). Addition of 1 mM
manganese to the assay buffer resulted in more than a 4-fold
increase in iPGM activity (Fig. S3C) yielding a specific activity of
,8 units/mg. Somewhat surprisingly, the activity was also
enhanced when assayed in the presence of cobalt (data not
shown). Clostridium perfringens iPGM has higher activity with
cobalt than with manganese although biochemical evidence
suggests that the latter ion is used in vivo . Similarly,
manganese, rather than cobalt, is likely the physiologically
relevant ion for E. coli iPGM also since it has been found
integrally bound in this enzyme  and is the more abundant
ion in the cell . Although we demonstrated that certain ions
enhanced iPGM activity, the
significantly lower than that of dPGM. This relatively low
specific activity of E. coli iPGM may not result directly from the
coexistence of dPGM since bacterial iPGM enzymes can be of
low activity (,1 unit/mg or less) even in species that lack dPGM
[46,47,48,49]. This is in contrast to eukaryotic iPGMs where
specific activities are typically in the range of 50 to 400 units/mg
[13,50,51]. The activity of dPGM was unaffected by the addition
of manganese as expected (data not shown) since dPGM enzymes
are not metalloenzymes . However it was sensitive to
vanadate, a known inhibitor of dPGM , with an IC50 of
0.65 mM (data not shown).
level of activity was still
Evaluation of phosphatase activity
Bioinformatic analyses originally suggested the presence of both
dPGM and iPGM in Bacillus subtilis [6,9,53]. However, unlike the
situation in E. coli, it appeared that iPGM accounted for the major
PGM activity while the predicted dPGM had little or no activity
[46,54]. Further studies determined that the predicted dPGM was
a broad specificity phosphatase , a member of the acid
phosphatase superfamily to which dPGM belongs. Deletion of B.
subtilis iPGM resulted in a severe growth phenotype and
asporulation  while deletion of the phosphatase had no effect
. We explored the possibility that iPGM, the less active PGM
in E.coli, might similarly function as a phosphatase as suggested
previously . However, we could not detect any phosphatase
activity when the protein was assayed against the general
phosphatase substrate, p-nitrophenyl phosphate, using buffers
and metal ions (Mg2+, Co2+or Zn2+) preferred by bacterial
alkaline phosphatases . Our alkaline phosphatase positive
control, calf intestinal phosphatase, was active under all conditions
tested (data not shown). The finding of manganese, rather than
Mg2+, Co2+or Zn2+, bound to E. coli iPGM  is also consistent
with its function as a PGM  rather than an alkaline
phosphatase. We note that although both E. coli iPGM and
dPGM function as PGMs, additional cellular functions cannot be
Characterization of DiPGM and DdPGM mutant strains
We prepared strains deleted for each of the predicted PGM
genes in the wild-type E. coli K-12 strain, MG1655, using
established methodology . Repeated attempts to create a
DiPGM, DdPGM double deletion by targeting the remaining
locus in each of the mutant strains were unsuccessful. Although
we did not attempt creation of the double deletion by alternative
methods, we interpret this result as indicative of an absolute
requirement for some form of PGM. Both mutants were healthy
when grown in LB medium, but a growth lag was identified
using minimal medium for DdPGM. (Fig. 5A), consistent with
the higher enzyme activity of dPGM. This growth lag was seen
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as a delay in exiting stationary phase in the DdPGM strain
relative to DiPGM and MG1655. Doubling times for both
mutants and the MG1655 parent were similar during logarith-
mic growth in this medium. Similar results were obtained using
iPGM and dPGM transposon insertion mutants (data not shown)
supplied by Dr F. Blattner, University of Wisconsin. A clearer
phenotype emerged when overnight cultures in minimal
medium were serially diluted then plated to LB agar (Fig. 5B
and C): DdPGM failed to form colonies after 24 h growth.
Colonies appeared only between 48 and 72 hrs. This phenotype
of DdPGM on solid medium confirms that observed in liquid
culture, suggesting a general problem in exiting stationary phase
in DdPGM cells. In contrast, when logarithmic phase cultures
were diluted and plated on solid medium, colony formation was
normal (data not shown). During stationary phase, energy
metabolism is limited and primarily consists of pathways that
scavenge potential nutrients from the medium and from within
the cell . However, upon a return to low density in glucose-
containing medium the pathways of central metabolism need to
be upregulated to permit rapid growth. This lag phase during
which the cell adjusts to the new conditions is extended in
DdPGM cells, presumably because they also have to compensate
for the absence of the major PGM activity in their glycolytic
pathway. No phenotype was observed for the DiPGM mutant
strain in these studies. It is possible that growth of the mutant
strains in the presence of alternative carbon sources could reveal
a phenotype for the DiPGM strain. However, our main goal was
to develop a system to examine whether the two PGM enzyme
forms do indeed have overlapping functional roles within E. coli.
This growth phenotype in E.coli lacking dPGM is consistent with
essentiality of PGM in Pseudomonas syringae, Bacillus subtilis,
Francisella novicida and Mycoplasma genitalium [49,57,58,59].
Studies of PGM null mutants or gene transcript reduction by
RNAi in eukaryotes such as yeast, protozoa and nematodes lend
further support to the essentiality of PGM in these organisms
Complementation of DdPGM by dPGM and iPGM
The observed colony delay phenotype of DdPGM provided a
system for complementation experiments using expression
pKKiPGM and pKKdPGM were introduced into the DdPGM
strain and plated on LB agar after overnight growth in MOPS
minimal medium. Strains MG1655, DdPGM and DdPGM
harboring empty plasmid (pKK) were grown in parallel. The
observed DdPGM growth phenotype could be restored to wild
type by dPGM expressed from the plasmid pKKdPGM as
expected. Interestingly, plasmid pKKiPGM also complemented
the DdPGM deletion. Both expression constructs, pKKiPGM and
pKKdPGM, complemented the DdPGM mutation such that the
colony formation at 24 hr was similar to the parental MG1655
(Fig. 6). No colonies were evident when DdPGM was transformed
with the empty vector, pKK (data not shown). These results
indicate that while expression of the chromosomal copy of iPGM
alone is not sufficient to fully compensate for the lack of dPGM
activity in the DdPGM mutant, the expression of additional
iPGM from a medium copy plasmid can restore the mutant cells
to normal growth characteristics. It further confirms that iPGM
and dPGM can function in the same metabolic pathways. Our
biochemical and genetic evidence unequivocally establishing
Figure 5. Phenotypes of DdPGM and DiPGM mutant strains.
Panel A: Parental wild-type MG1655 E. coli (¤) and DdPGM (&) and
DiPGM (s) mutant strains grown in minimal medium overnight were
inoculated into 10 ml fresh minimal medium to give initial OD600values
of 0.03. Growth was monitored by determining turbidity (Klett units)
during incubation at 37uC. Each data point represents the mean Klett
value of triplicate cultures (6 S.D.). Panels B and C: Overnight MOPS
minimal medium cultures of parental wild-type MG1655 E. coli and the
DdPGM and DiPGM mutant strains were serially diluted in minimal
medium and 100 ml of each dilution plated to LB agar. Cells were grown
at 37uC and the number of colonies counted. Each dilution of each
strain was plated in quadruplicate. Representative plates at 161025
dilution are shown (B) and the mean numbers of colonies (6 S.D.) per
plate at 161026dilution are plotted (C).
PLoS ONE | www.plosone.org 12October 2010 | Volume 5 | Issue 10 | e13576
dPGM and iPGM as analogous enzymes (NISE) in E. coli is likely
applicable to other bacteria that also encode both forms. We
determined that generally such bacteria have genomes in excess
of 4Mb and can presumably accommodate this apparent
Since mammalian genomes encode only dPGM while many
pathogenic bacteria, fungi, protozoans and nematodes use only
iPGM, the latter has been proposed as a candidate drug target for
novel treatments for various infectious diseases [6,13,25,47]. The
development of null mutants of both dPGM and iPGM in E. coli
makes possible a whole organism screen for identification of
potential inhibitors with specificity for iPGM. Similarly, com-
pounds identified in high throughput screens against any
recombinant iPGM can now be tested for specificity in a well-
characterized bacterial system.
The widespread occurrence of NISE is becoming increasingly
apparent as more genome sequences are reported [1,3,62,63,64].
The phenomenon is attracting attention not only from an
evolutionary perspective, but also because of its confounding
implications for accurate genome annotation and metabolic
pathway reconstruction, and for its potential in highlighting drug
targeting opportunities against various pathogenic organisms
[1,3,4,64,65,66,67]. For example, a web-based tool AnEnPi
(Analogous Enzyme Pipeline) has been developed that enables
researchers to identify NISE in pathogen and host genomes .
Since vertebrates only contain dPGM , the iPGM protein of
any pathogen encoding only that form represents a candidate drug
target. In our analysis, we identified 243 bacterial genomes (,35%
of genomes examined) that encode only iPGM. These include
pathogenic representatives from a variety of genera such as
Mycoplasma, Campylobacter, Coxiella, Vibrio, Helicobacter, Pseudomonas,
Leptospira, Legionella amongst others. Thus iPGM represents a
potential drug target in diverse bacterial groups.
Glycolysis is an essential component of central metabolism and
is conserved in almost all prokaryotes and eukaryotes. However,
several glycolytic enzymes such as PGM, phosphofructokinase,
and lactate dehydrogenase have truly analogous forms (NISE),
while others such as glucokinase, aldolase, FBPase and phospho-
glucoisomerase, have highly variant, albeit structurally similar,
forms [3,68]. These enzymes, encoded by multiple gene
sequences, almost exclusively function in the early stages of
glycolysis or in associated areas of hexose metabolism. PGM is
unusual since it is the only variant enzyme found in the so-called
trunk pathway from glyceraldehyde-3-phosphate to pyruvate
which is otherwise highly conserved and indicative that the
ancestral function of the glycolytic pathway was biosynthetic
rather than glycolytic [3,68].
E. coli dPGM and iPGM have no sequence or structural
similarities and use dissimilar catalytic mechanisms. Their PGM
activities, shown both in this study and previously , coupled
with our mutant analyses demonstrating overlapping and
supplementary functions in the cell unequivocally establish the
two forms as NISE. Furthermore, enhancement of iPGM activity
by manganese agrees with earlier data reporting this ion bound in
the E. coli enzyme , and supports the lack of phosphatase
activity we report since known alkaline phosphatases require ions
other than manganese. Although our experimental data derives
from the model organism, E. coli, we anticipate it is valid for
diverse bacteria that contain both predicted PGM forms. Our
finding that bacteria that encode both PGM NISE predominantly
have larger genomes is consistent with their individual functions
being supplementary. Presumably smaller, compact genomes are
less able to accommodate and maintain genes encoding function-
ally equivalent proteins.
The presence of both PGM NISE forms in the same organism is
found in diverse bacterial groups (Table 1), but is particularly
prevalent in the Bacilli and Enterobacteriaceae (c-proteobacteria).
In most bacterial taxa that have several representative sequenced
genomes, the PGM profile is non-uniform. Different genomes may
have both forms, as E. coli does, or only dPGM or iPGM, or, in a
few cases, neither. Further complexity results from the presence of
two or more bacterial-type dPGM genes in many genomes and
from the occurrence of archaeal iPGM in over 40 genomes
(Table 1). The patchy distribution of the NISE forms appears to be
Figure 6. Complementation of the DdPGM phenotype by dPGM
and iPGM. Overnight minimal medium cultures of parental wild-type
MG1655 E. coli, the DdPGM mutant, and the DdPGM mutant carrying
either the plasmid pKKiPGM or pKKdPGM were serially diluted in MOPS
minimal medium. Triplicate aliquots of 100 ml of each dilution were
plated to LB agar and the number of colonies counted after incubation
at 37uC. Strains harboring plasmid constructs were grown in the
presence of 100 mg/ml ampicillin. Representative plates at 161026
dilution are shown (A) and the mean number of colonies per plate
(6 S.D.) are plotted (B).
PLoS ONE | www.plosone.org13 October 2010 | Volume 5 | Issue 10 | e13576
partly due to LGT but is undoubtedly due to gene losses in specific
lineages. The PGM NISE forms are a clear case of a phenomenon
coined non-orthologous gene displacement [69,70]. In its simplest
form this is represented by the presence of non-orthologous genes
that encode enzymes capable of carrying out the same reaction
being present in an ancestral genome followed by lineage-specific
gene losses . Non-orthologous gene displacement could also
follow events such as LGT or enzyme recruitment that lead to the
presence of both NISE forms in the same genome , again
followed by losses in some lineages. Such mechanisms seem most
likely in the case of PGM. Firstly, both iPGM and dPGM are
members of the larger alkaline phosphatase and acid phosphatase
enzyme superfamilies, respectively [7,8,9] and could evolve by
recruitment by shifting the substrate specificity of a related but
different enzyme. Secondly, PGM genes appear to have moved
frequently between different bacteria and between the domains of
life (this study, and [15,35,37,40]) and introduced new PGM
coding potential into recipient genomes. Regardless of the origins
of the two enzyme forms, functional redundancy, where a
bacterium contains both NISE, must be a prerequsite for non-
orthologous gene displacement, where essential genes are
concerned, and must precede any subsequent selective loss of
one gene. Whether any of the bacteria we report as containing
both PGM NISE are undergoing the early stages of non-
orthologous PGM gene displacement or reflect a retained
ancestral condition is an open question. Nonetheless, it appears
that enzyme recruitment, gene duplications, gene losses, LGT
events and non-orthologous gene displacement have contributed
to the intriguing non-uniform distribution of analogous PGM
enzymes (NISE) across the bacterial domain that we see today.
genome sequences from the Class c-proteobactria. Taxonomic
nodes (left to right) are Class, Order, Family, Genus (or species in
the case of 3 incompletely classified bacteria at the bottom of the
Figure). Taxa with genomes containing only iPGM are shaded
yellow, those with only dPGM are shaded blue, those with both
iPGM and dPGM are shaded green, while taxa with non-uniform
PGM profiles are shaded pink. Taxa with no PGM are unshaded.
The numbers in boxes accompanying each taxon identifier
correspond to (left to right) number of genomes with only dPGM,
only iPGM, both dPGM and iPGM, and no PGM.
Found at: doi:10.1371/journal.pone.0013576.s001 (0.05 MB
Distribution of PGM types across 184 completed
iPGM sequences from bacterial genomes queried against com-
pleted archaeal genomes. The 50 archaeal type sequences
identified in 43 bacterial genomes were compared to the
completed genomes of 48 archaeal species. The identities of the
archaeal species, numbered 1 to 48 on the y-axis, are provided in
Table S3. Different phyla within the kingdom archaea are
differentially shaded. Classes of archaea having multiple repre-
sentative genome sequences are indicated above the shaded boxes
along with the average bit score for that entire class.
Found at: doi:10.1371/journal.pone.0013576.s002 (0.02 MB
Average best TBLASTN bit score of archaeal type
activity (Panel C) of recombinant dPGM and iPGM. Panels A
(dPGM) and B (iPGM): Lanes: 1, E. coli total protein without
induction with IPTG; 2, E.coli total protein following induction
with IPTG; 3, soluble E. coli proteins after cell disruption; 4, flow-
Overexpression and purification (Panels A, B) and
through from the nickel column; 5, Wash of nickel column prior to
elution; 6 and 7, elution fractions from nickel column using
imidazole (200 mM for dPGM, 100 mM for iPGM). Panel C:
PGM activity of recombinant dPGM and iPGM. Conversion of 3-
PG to 2-PG by 0.25 mg dPGM (&) and 10 mg iPGM (m) assayed
in standard, magnesium-containing buffer. Conversion of 3-PG to
2-PG by iPGM in buffer supplemented with 1 mM manganese
chloride is shown for comparison (*). A control lacking any
recombinant protein is also shown (¤). Conversion of 3-PG to 2-
PG is determined indirectly by a decrease in NADH concentration
as measured by its absorbance at 340 nm. Consumption of NADH
is directly proportional to PGM activity.
Found at: doi:10.1371/journal.pone.0013576.s003 (8.63 MB TIF)
related superfamily member proteins across 702 complete
bacterial genomes. The protein queries are as described in
Materials and Methods. The number of predicted proteins in
each genome sequence that match the query protein above a
TBLASTN bit score of 100 are provided. Based on these hits, each
genome is assigned a status of either ‘‘I’’ (iPGM) ‘‘D’’ (dPGM),
‘‘D+I’’ (dPGM plus iPGM) or ‘‘None’’ (No PGM). The
assignments were made automatically or manually as described
in Materials and Methods. A plus sign (+) in the column headed
‘‘Multiple Molecules’’ indicates that the queried genome has more
than one molecule and that the multiple hits are to different
chromosomes or extrachromosomal plasmids. [However, Coryne-
bacterium glutamicum ATCC 13032 and Bacillus licheniformis ATCC
14580 have been sequenced twice resulting in duplicate molecules
and are also marked ‘‘+’’; Burkholderia multivorans ATCC 17616,
also sequenced twice, has 3 chromosomes and one plasmid and
has 2 dPGM hits on chromosome 1 and is marked ‘‘+’’. Similarly,
Ehrlichia ruminantium str. Welgevonden has been sequenced twice
(+) and has one iPGM hit on each sequence. However, the
assignment of different origins in the E. ruminantium genome
sequences results in the hits not overlapping so being scored twice].
Found at: doi:10.1371/journal.pone.0013576.s004 (0.32 MB
Identification of orthologs of iPGM, dPGM and
Found at: doi:10.1371/journal.pone.0013576.s005 (1.29 MB
PCR primers for amplification of E. coli dPGM and
sequences that served as subjects for TBLASTN analysis. The
archaeal genomes were queried using the 50 archaeal type iPGM
sequences identified in the completed bacterial genomes. The
number accompanying each species corresponds to the y-axis
numbering in Fig. S2. The multiple GI numbers associated with
each species represent genome sequences of different strains and/
or multiple molecules (eg. plasmids) of certain species/strains.
Found at: doi:10.1371/journal.pone.0013576.s006 (0.03 MB
The 48 archaeal species with complete genome
We thank Dr. Don Comb for continued encouragement and support, Scott
Zimmer for initial cloning of PGM, and Barton Slatko for critical reading
of the manuscript.
Conceived and designed the experiments: JMF PJD SR MHS EAR SK
CKSC. Performed the experiments: JMF SR MHS. Analyzed the data:
JMF PJD SK. Contributed reagents/materials/analysis tools: MHS. Wrote
the paper: JMF PJD SK CKSC.
PLoS ONE | www.plosone.org14 October 2010 | Volume 5 | Issue 10 | e13576
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PLoS ONE | www.plosone.org15 October 2010 | Volume 5 | Issue 10 | e13576