JOURNAL OF BACTERIOLOGY, Dec. 2006, p. 8259–8271
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 188, No. 23
Experimental and Computational Assessment of Conditionally
Essential Genes in Escherichia coli?
Andrew R. Joyce,1,2† Jennifer L. Reed,2† Aprilfawn White,3,4Robert Edwards,5,6Andrei Osterman,6,7
Tomoya Baba,8Hirotada Mori,8,9Scott A. Lesely,3,4Bernhard Ø. Palsson,2* and Sanjay Agarwalla3,4*†
Program in Bioinformatics, University of California, San Diego, La Jolla, California 920931; Department of Bioengineering, University of
California, La Jolla, California 920932; The Joint Center for Structural Genomics3and The Genomics Institute of
the Novartis Research Foundation, San Diego, California 921214; Center for Microbial Sciences,
San Diego State University, San Diego, California 921825; Fellowship for the Interpretation of
Genomes, Burr Ridge, Illinois 605276; Burnham Institute for Medical Research, La Jolla,
California 92037;7Graduate School of Biological Sciences, Nara Institute of Science and
Technology (NAIST), Ikoma, Nara, Japan8; and Advanced Institute of
Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan9
Received 22 May 2006/Accepted 9 September 2006
Genome-wide gene essentiality data sets are becoming available for Escherichia coli, but these data sets have
yet to be analyzed in the context of a genome scale model. Here, we present an integrative model-driven analysis
of the Keio E. coli mutant collection screened in this study on glycerol-supplemented minimal medium. Out of
3,888 single-deletion mutants tested, 119 mutants were unable to grow on glycerol minimal medium. These
conditionally essential genes were then evaluated using a genome scale metabolic and transcriptional-regula-
tory model of E. coli, and it was found that the model made the correct prediction in ?91% of the cases. The
discrepancies between model predictions and experimental results were analyzed in detail to indicate where
model improvements could be made or where the current literature lacks an explanation for the observed
phenotypes. The identified set of essential genes and their model-based analysis indicates that our current
understanding of the roles these essential genes play is relatively clear and complete. Furthermore, by
analyzing the data set in terms of metabolic subsystems across multiple genomes, we can project which
metabolic pathways are likely to play equally important roles in other organisms. Overall, this work establishes
a paradigm that will drive model enhancement while simultaneously generating hypotheses that will ultimately
lead to a better understanding of the organism.
The advent of whole-genome sequencing and other high-
throughput experimental technologies provides system level
measurements that are driving efforts to develop computa-
tional models of the cell. The constraint-based reconstruction
and analysis (COBRA) approach (36) has emerged in recent
years as a successful approach to modeling systems on a ge-
nome scale. The COBRA approach begins with developing a
metabolic network reconstruction based on the annotated
genome sequence, known biochemistry, and other physio-
logical data (38). Known constraints, such as enzymatic-
reaction reversibility and maximum flux capacity, are then
imposed on the network reconstruction to generate a model
that defines all attainable network states (36). A current
metabolic and regulatory model of Escherichia coli contains
932 unique metabolic reactions and Boolean logic state-
ments for how 104 transcription factors regulate the expres-
sion of 479 out of the 906 metabolic genes (6). COBRA
methods are available to predict which metabolic and reg-
ulatory genes are required for growth under given environ-
mental conditions (7, 11, 43, 44).
Knowledge of which genes in an organism are essential and
under what conditions they are essential is of fundamental and
practical importance. This knowledge provides us with a
unique tool to refine the interpretation of cellular networks
and to map critical points in these networks. Examples of
applications in which this information may be useful include
engineering industrial microbial strains, as well as developing
novel anti-infective agents. The importance of this emerging
field devoted to investigations of gene essentiality is widely
accepted, as witnessed by the rapid accumulation of genome-
wide essentiality data, which are now available for several
model and pathogenic microbial species (1, 3, 16, 17, 19, 25, 27,
30, 42, 45, 48).
From a modeling perspective, a major limitation of the pre-
vious gene essentiality studies of E. coli was that they were
performed using only partial (18, 24, 52) (i.e., not all mutants
were evaluated) or heterogeneous (“historical” single-gene
studies of a variety of strains and conditions compiled in the
Profiling of E. coli Chromosome database [http://www.shigen
.nig.ac.jp/ecoli/pec/]) data. Data provided by the first published
genome scale genetic-footprinting study of E. coli (16) are
generally not amenable to immediate model-based interpreta-
tion, as they (i) captured a rather complex phenotype (fitness
* Corresponding author. Mailing address for Bernhard Palsson
(computational work): Department of Bioengineering, University of
California, San Diego, 9500 Gilman Dr. MC 0412, La Jolla, CA 92093-
0412. Phone: (858) 534-5668. Fax: (858) 822-3120. E-mail: palsson
@ucsd.edu. Mailing address for Sanjay Agarwalla (experimental
work): The Genomics Institute of the Novartis Research Foundation,
10675 John Jay Hopkins Drive, San Diego, CA 92121. Phone: (858)
812-1500. Fax: (858) 812-1502. E-mail: firstname.lastname@example.org.
† Andrew R. Joyce, Jennifer L. Reed, and Sanjay Agarwalla con-
tributed equally to this work.
?Published ahead of print on 29 September 2006.
within a competitive growth environment) and (ii) were ob-
tained in undefined rich medium.
The recent release of the first complete collection of vi-
able single-gene knockout E. coli strains (1) has opened an
opportunity for systemic, genome scale gene essentiality
studies in minimal and defined growth media. The group
responsible for generating this valuable resource also re-
ported the first genome scale conditional-essentiality screen
on rich medium and glucose-supplemented minimal me-
dium (1). In this study, we used this strain collection to
integrate high-throughput experimental data and computa-
tional modeling to assess E. coli gene essentiality for growth
on glycerol-supplemented minimal medium. The results of
this conditional-essentiality screen were analyzed in the con-
text of the most current genome scale metabolic and tran-
scriptional regulatory model (6).
A systematic cross-validation of genome scale gene essenti-
ality data with in silico predictions would play a critical role in
refining the current metabolic reconstruction and the underly-
ing model. At the same time, such an integrative analysis would
assist in data analysis and interpretation in the structured-
network context. For example, a recent study utilized the pre-
viously described integrated E. coli transcriptional-regulatory
and metabolic model to validate its predictive capability
against 13,750 growth phenotypes corresponding to 110 gene
knockout strains grown under 125 different defined conditions
(6). Discrepancies between the model predictions and experi-
mental results pointed to poorly understood metabolic or reg-
ulatory events requiring further experimental investigation.
The gene deletions evaluated in this previous study, however,
covered less than 11% of the genes included in the current
Here, we identify the set of genes needed for growth on
glycerol-supplemented minimal medium and analyze the re-
sults using a genome scale metabolic and regulatory model. We
show this approach to be useful for a rigorous global evalua-
tion of the genome scale modeling predictive power while
simultaneously identifying directions for model improvement.
The gene essentiality data obtained in this study were generally
in good agreement with the model predictions, as well as with
the results of the previously reported screen on glucose-sup-
plemented minimal medium (1). This work represents the
most thorough assessment on a gene-by-gene basis of the E.
coli constraint-based metabolic model and is the first model-
based evaluation of a truly genomewide gene essentiality
screen on a single defined minimal medium for E. coli.
MATERIALS AND METHODS
High-throughput phenotyping of the E. coli gene knockout collection. A re-
cently described collection of 3,888 E. coli single-gene deletion mutants was
constructed (1; http://ecoli.naist.jp/) by the method of Datsenko and Wanner (9).
To determine the phenotypes of deletion mutants in M9 minimal medium con-
taining glycerol as the carbon source, the mutants were inoculated in LB medium
in the presence of kanamycin (30 mg/liter) using a 96-pin tool and were grown
overnight at 37°C. The overnight cultures were washed twice with phosphate-
buffered saline and then inoculated in glycerol-supplemented M9 liquid medium
with kanamycin. The liquid culture was grown at 37°C with agitation for about
24 h, and the optical density (OD) was measured at 600 nm. The ODs from all
wells of a plate were averaged, and the mutants in the wells with less than
one-third of the average OD were considered nongrowers or slow growers. The
experiment was done in triplicate, and mutants that were below the one-third
average OD cutoff in at least two of three experiments were selected. This initial
screen yielded about 230 deletion mutants that had slow or no growth on
M9-glycerol medium. A secondary screen using the same procedure was re-
peated on this subset of mutants, using the same one-third of the average OD as
the cutoff and yielded a final set of 119 E. coli deletion mutants that represented
the conditionally essential complement of genes required for growth on glycerol.
This second round of screening confirmed the genuine hits and eliminated false
and nonreproducible hits. Each liter of M9 medium (Sigma catalog no. 6030)
contained Na2HPO4· 7H2O (6.8g), KH2PO4(3g), NaCl (0.5g), NH4Cl (1g),
MgSO4(2 mM), CaCl2(0.1 mM), glycerol (1%), and kanamycin (10 mg).
For comparison with the conditionally essential genes reported in the recently
published data for growth on glucose-supplemented minimal medium (1), we
selected the 119 slowest growers based on the observed OD at 24 h. This set
coincidently included nearly all of the strains with less than one-third of the
average OD at 24 h for all strains tested.
Computational predictions for essentiality. A previously developed metabolic
model of E. coli (6, 39) was used to predict the metabolic genes and reactions
essential for growth on glycerol minimal medium. The model was modified to
take into account genetic differences between MG1655 and BW25113 and recent
changes in the genome annotation (40). Five metabolic reactions were removed
(L-arabinose isomerase, L-ribulokinase, rhamnulokinase, L-rhamnose isomerase,
and rhamnulose 1-phosphate aldolase), since the associated genes (araBAD,
rhaBAD, and lacZ) are absent in the BW25113 strain that was the parental
background for the genetic manipulations. Based on recent updates to the E. coli
genome annotation (40), two additional metabolic genes (dfp and coaE) were
also included in the metabolic model by associating them with three reactions
involved in coenzyme A (CoA) biosynthesis that previously had no genes asso-
ciated with them. Furthermore, atpI was removed from the model, since evidence
suggested it did not participate in the ATP synthase complex (14). Additional
changes in the genome annotation (40) also have merged (tdcG, araH, and ytfR)
and split (dgoAD and glcEF) some genes included in the model. As a result, 899
metabolic genes are accounted for in the metabolic model and an additional 104
transcription factors are used in the combined metabolic and regulatory model.
Growth on glycerol minimal medium was simulated by maximizing flux
through a defined biomass objective function and allowing the uptake of glycerol,
NH4, SO4, O2, and Piand the free exchange of H?, H2O, and CO2(see reference
39 for further details). The biomass objective function is specified to define the
weighted consumption of metabolites required to generate the cellular biomass.
Simulations conducted in this manner represent approximations of the maximum
attainable growth rate under the given environmental conditions and model
The maximum growth rates of gene knockout strains were calculated with each
gene independently removed from the network. When simulating the deletion of
a gene, all associated reactions were removed from the network except for those
reactions with isozymes. Gene deletions where the predicted maximum growth
rate was zero were categorized as essential. To evaluate the effects of transcrip-
tion factor mutants, a combined metabolic and regulatory model was used to
evaluate whether the deletion of a transcription factor is lethal for growth on
glycerol minimal medium (6, 39). The regulatory model contains Boolean logic
statements describing the transcription factors and environmental conditions
needed for metabolic genes to be expressed (7, 8). All calculations with only the
metabolic model were done using SimPheny (Genomatica, San Diego, CA), and
LINDO (Lindo Systems, Inc., Chicago, IL) was used to calculate growth rates for
the combined metabolic and regulatory model.
Cross-genome comparison of conditionally essential genes. We used The
SEED genomic platform (http://theseed.uchicago.edu/FIG/index.cgi) for a cross-
genome comparison of metabolic subsystems implicated by the set of condition-
ally essential E. coli genes identified in this study. A subsystem is defined in The
SEED environment as a collection of functional roles (enzymes, transporters, or
regulators) known to be involved in a well-defined biological process, such as a
subnetwork (a cluster of pathways) associated with a particular aspect of metab-
olism (e.g., glycolysis) (34). A populated subsystem is defined as a table of
tentative role-to-gene connections asserted by curators for a broad range of
species containing a functional variant of this subsystem (51). In this study, we
used The SEED tools to generalize the data from the described essentiality
screen in a broader phylogenetic context. This approach circumvents certain
limitations of traditional gene-by-gene comparisons, as there are reported cases
where the same reaction or functional role can be implemented by nonortholo-
gous enzymes in different species (28).
Briefly, a table was constructed that relates conditionally essential genes (both
identified by the experiment and predicted by computational modeling) to The
SEED collection of metabolic subsystems. For further analysis, this table was
simplified to a set of binary associations (one gene to one “primary” subsystem)
and limited to the approximately 20 key subsystems that contained more than
8260 JOYCE ET AL.J. BACTERIOL.
TABLE 1. Essential genes for growth on glycerol minimal mediuma
Gene (Blattner no.)
Gene (Blattner no.)
Amino acid metabolism....................................... argA (b2818)guaA (b2507)
Cofactor production..................................................... folB (b3058)
Regulatory proteins...................................................... cysB (b1275)
Transport....................................................................... crr (b2417)
Others............................................................................ atpA (b3734)
Purine and pyrimidine biosynthesis.................... carA (b0032)
aThe 119 genes identified as being required for growth on glycerol M9 minimal medium are divided into six groups based on shared annotations and/or properties.
The 26 genes that appear in bold-face represent genes that were not predicted to be essential by the model and were not associated with essential reactions (described
in further detail in the text and Table 2). The seven genes in brackets are the essential genes identified in this study that do not appear in the model.
VOL. 188, 2006 COMPUTATIONAL ANALYSIS OF ESSENTIAL GENES IN E. COLI 8261
two experimentally defined essential genes. We then examined operational vari-
ants of these subsystems (as defined by a subsystem curator) over a diagnostic set
of 31 species with available completely sequenced genomes spanning much of the
known bacterial phylogeny. For illustrative purposes, we used the same set of
genomes as in the previous analysis of genetic-footprinting data (16) (see sup-
plementary Table 4 [http://systemsbiology.ucsd.edu/publications/supplemental
_material/JBact2006/]). For this simplified analysis, we monitored only the pres-
ence or absence of at least a minimal functional variant for each subsystem and
each genome in the set. The results were hierarchically clustered for visualization
and analysis purposes (see Fig. 6) using the Hamming distance metric and
Quantitative RT-PCR measurements of gene expression. Real-time RT-PCR
was used to quantify gene expression levels for genes related to glycerol metab-
olism (glpK, glpD, glpB, gpsA, gldA, and dhaM). Total RNA was extracted from
cells harvested from mid-log-phase cultures of E. coli strain BW25113 (9) grown
on glucose-supplemented (A600? 0.5) and glycerol-supplemented (A600? 0.3)
M9 minimal medium (2 g/liter). Triplicate RNA samples (biological replicates)
were stabilized using RNAProtect Bacterial Reagent (QIAGEN) and isolated
using the RNeasy mini kit (QIAGEN). Synthesis of cDNA was performed using
SuperScript III (Invitrogen) and purified using the QIAquick PCR Purification
The resulting cDNA samples were used in subsequent real-time reverse tran-
scription (RT)-PCR assays using the QuantiTect SYBR Green PCR kit
(QIAGEN) and iCycler iQ system (Bio-Rad). Nine replicate measurements
(three technical replicates for each biological replicate) were performed for each
assayed gene under both growth conditions. The acyl carrier protein (ACP)-
encoding gene acpP was used as a reference for each assay. A standard curve was
generated by varying amounts of genomic DNA with fixed primer concentrations
and was used to calculate primer efficiencies. The reported relative expression
levels for each gene were determined by normalizing the amount of cDNA
product to acpP cDNA quantified from the same cDNA sample.
By evaluating single-gene deletion strains for growth on glyc-
erol-supplemented minimal medium, we identified genes es-
sential for growth in a minimal-medium environment that are
not essential in a rich-medium environment. A genome scale
metabolic and regulatory model was used to evaluate the data
and to identify any discrepancies between the model and the
experimental data. In addition, the essential genes identified in
this study were compared to gene essentiality data for growth
on glucose-supplemented minimal medium (1), and their phy-
logenetic distribution across multiple genomes was evaluated.
Experimentally essential genes for growth on glycerol min-
imal medium. Of the 3,888 single-gene deletion E. coli mutants
viable on rich medium and screened in this study, 119 were
reproducibly incapable of growth on glycerol minimal medium
(Table 1; for complete results, see supplementary Tables 1
_material/JBact2006/]). Most of these conditionally essential
genes are involved in core metabolic processes: amino acid
metabolism (59 genes), nucleotide metabolism (19 genes), co-
factor metabolism (15 genes), and transport (5 genes). Seven-
teen genes are involved in other miscellaneous processes, and
four regulatory genes were also found to be conditionally es-
Only seven (cysQ, fes, leuL, prfB, rpsU, yhhK, and yjhS) of the
119 identified essential genes are not accounted for in the
current metabolic and regulatory model, since the genes do not
encode metabolic enzymes or transcription factors with known
functions. While the specific role of cysQ in sulfate assimilation
is unknown (33), it is an important component of cysteine
biosynthesis. fes is important for iron transport and utilization
of ions in low-concentration environments, such as that used in
this minimal-medium study (10). Alteration of transcriptional
attenuation (32) mediated regulation of the leuLABCD operon
(50), which encodes the proteins critical for leucine biosynthe-
sis, and likely explains the essentiality of the leader peptide
encoded by leuL. Several other nonmodel genes encoding PrfB
(a peptide chain release factor) and RpsU (30S ribosomal
subunit S21), as well as the observed conditional essentiality of
the uncharacterized genes yhhK (a putative acyltransferase)
and yjhS, cannot be readily interpreted without further exper-
FIG. 1. Comparisons between experimental data and model predictions for gene essentiality under minimal-medium conditions. In silico gene
deletion studies were carried out by evaluating the effect of deleting a gene from the model. If the model was unable to generate all of the biomass
components due to a gene deletion, as determined by flux balance analysis, then the mutant was considered lethal. Experiments were also
conducted by testing for growth in glycerol-supplemented minimal medium using the 3,888 mutants that were viable when grown in rich medium.
In total, 819 out of the 896 mutants (91%) showed growth behaviors in glycerol minimal medium in agreement with computational predictions.
8262 JOYCE ET AL.J. BACTERIOL.
imental investigation. The remaining 112 essential genes and
nonessential genes can be compared to predictions made with
the current metabolic and regulatory model.
Model-based evaluation of essential genes. Given that most
of the essential genes involve metabolic genes and metabolic
regulators, we conducted a detailed comparison of the exper-
imentally observed and computationally predicted essential
genes (Fig. 1 and Table 1). Computational analysis of single-
gene deletion events predicted 182 genes (177 metabolic and 5
regulatory genes) to be lethal and thus required for growth in
glycerol minimal medium. Nearly half of these genes were still
predicted to be essential by the model even if all transportable
metabolites were allowed to be taken up by the cell simulta-
neously, so they are likely to be essential for growth on rich
medium, as well. Among the 182 model-predicted lethal mu-
tants, 63 were not present in the analyzed collection. Although
a fraction of these missing mutants may reflect technical fail-
ures, most of them are associated with genes expected to be
essential under any environmental conditions. Such genes are
typically responsible for producing essential metabolites that
cannot be salvaged even from rich medium.
As shown in Fig. 1, ?69% of experimentally identified con-
ditionally essential genes covered by the model (77 of 112)
were predicted to be essential by evaluating in silico single-
gene deletions. An additional 8% of experimentally essential
genes (9 of 112) would be correctly predicted by the model to
be essential if additional isozymes were not present, possibly
indicating that the expression of alternative isozyme-encoding
genes is not sufficient to compensate for growth on glycerol
minimal medium. Alternatively, these nine cases may point to
incorrect functional assignment of some paralogs.
This leaves 26 essential genes unexplained by the model, in
which the experimentally observed essential genes are associated
with predicted nonessential model genes (Table 2 and supple-
mentary Table 3 [http://systemsbiology.ucsd.edu/publications
/supplemental_material/JBact2006/]). Six genes out of these 26
discrepancies (atpA, atpB, atpC, atpF, atpG, and atpH) are part
of the ATP synthase complex. According to the model, the
deletion of the ATP synthase reaction should not be lethal but
it should reduce the maximum growth rate by ?75%, which
may be close to the viability threshold used in this study.
Interestingly, two other components of the ATP synthase
complex (atpD and atpE) were deemed nonessential in our
An additional large subset of these discrepancies (9 of 26)
appear to be caused by the existence of alternative pathways
available within the metabolic model but whose genes are
probably not expressed in vivo under the conditions of this
screen. For example, proA and proB can be functionally re-
placed in the model by the combined action of argA, argB,
argC, and argE gene products in proline biosynthesis, since
both result in the production of glutamate-5-semialdehyde
(Fig. 2). However, this alternate pathway is observed experi-
mentally only in double-deletion strains, where an argD dele-
TABLE 2. Discrepancies between experimental identification and model prediction for essential and nonessential genesa
RationaleSubsystem Gene(s) (Blattner no.)
Molecules not included in biomassUbiquinone biosynthesis
Amino acid biosynthesis
ubiG (b2232); ubiH (b2907)
pdxA (b0052); pdxB (b2320); pdxH (b1638); pdxJ (b2564)
carA (b0032); carB (b0033); glpD (b3426); glyA (b2551);
proA (b0243); proB (b0242); thrB (b0003); thrC (b0004)
atpA (b3734); atpB (b3738); atpC (b3731); atpF (b3736);
atpG (b3733); atpH (b3735)
crr (b2417); glpK (b3926); ppc (b3956); ptsI (b2416); fruR
Model includes alternative pathways/
Model predicts impaired but not lethal
Regulatory effect on glpK
Model biomass components which
might not be essential components
Fatty acid and lipid biosynthesis
cls (b1249); fabF (b1095)
glgA (b3429); glgC (b3430)
dgkA (b4042); gmhA (b0222); gmhB (b0200); lpxL
(b1054); msbB (b1855); rfaC (b3621); rfaD (b3619);
rfaE (b3052); rfaF (b3620); rfaG (b3631); rfaI (b3627);
rfaJ (b3626); rfaL (b3622)
pfs (b0159); speD (b0120); speE (b0121)
argB (b3959); argC (b3958); argD (b3359); argG (b3172)
ilvY (b3773); ilvE (b3770); lrp (b0889)
coaA (b3974); coaE (b0103); pabC (b1096)
dapF (b3809); ushA (b0480); lysR (b2839)
pyrI (b4244); trxB (b0888); ndk (b2518)
Branced amino acid biosynthesis
Nucleotide biosynthesis and salvage
Unaccounted-for transport mechanisms
Unaccounted-for metabolic enzymes
aTwenty-six false-negative cases in which the model incorrectly predicted growth of the gene deletion strain were identified, in addition to 42 false-positive cases in
which the model incorrectly predicted that genes were essential. Each case is grouped based on the likely rationale for the discrepancy and the gene functional
VOL. 188, 2006COMPUTATIONAL ANALYSIS OF ESSENTIAL GENES IN E. COLI8263
tion leads to increased levels of N-acetylglutamic ?-semialde-
hyde, which is then converted into glutamate 5-semialdehyde
by argE, thereby allowing compensation for the second dele-
tion, either proA or proB (23).
Another subset of these discrepancies (7 of 26) are associ-
ated with the biosynthesis of vitamins and cofactors: pyridoxal
5-phosphate (pdxABHJ), thiamine (iscS), and ubiquinone
(ubiGH) (Fig. 3), largely reflecting the fact that the need to
produce these cofactors was not duly accounted for in the
biomass objective function. The ubiG and ubiH gene products
are essential for growth on glycerol minimal medium, while
other gene products involved in the ubiquinone biosynthesis
pathway are essential during growth on rich medium (1) (ubiA,
ubiB, and ubiD) and still others are not essential under either
condition (ubiC, ubiX, and ubiF).
(PEP) metabolism and the PEP-carbohydrate phosphotrans-
ferase systems (PTS) likely resulted from posttranscriptional
regulation of GlpK (glycerol kinase) that is not accounted for
in the metabolic or regulatory model. It is known that deletion
of ppc (encoding the enzyme PEP carboxykinase) leads to the
accumulation of PEP, which allosterically inhibits glycolytic
enzymes, such as Pgi and Pfk (12). This inhibition would
lead to an increase in Pgi and Pfk metabolic intermediates,
including fructose 1,6-bisphosphate, a potent allosteric in-
hibitor of GlpK (22) (Fig. 4).
Two PTS genes, ptsI and crr, were also detected as discrep-
ancies in this study, in which the model predicts an observed
essential gene to be nonessential. PTS enzyme I, encoded by
ptsI, is phosphorylated in a reaction with PEP in the first step
of the PTS, and crr encodes PTS glucose-specific enzyme IIA
(EIIAGlc), which is another intermediate that transfers the
PTS phosphate to glucose. EIIAGlcis also a central regulatory
molecule in E. coli metabolism (35), and in its unphosphory-
lated form, EIIAGlcbinds and allosterically inhibits GlpK, thus
ultimately impeding glycerol uptake and metabolism (21, 22).
Phosphorylation of EIIAGlcreleases GlpK, however, and fa-
cilitates normal glycerol uptake and metabolism. Therefore, a
ptsI deletion would interfere with the transfer of a phosphate
to EIIAGlcand block the release of GlpK inhibition. The
deletion of crr is more difficult to explain in this context, as one
might expect that the resultant constitutive relief of EIIAGlc
inhibition would lead to enhanced glycerol uptake and metab-
olism. The observed essentiality of crr likely stems from the
general disruption of its other critical cellular roles. For exam-
ple, phosphorylation of EIIAGlcactivates adenylate cyclase,
and accordingly, the crr mutant has reduced cyclic AMP levels
(29), likely resulting in potentially harmful pleiotropic effects
due to improper global gene regulation by crp. Despite these
readily explained results, we do not yet have a rationale for the
observed nonessentiality of ptsH and cyaA, which encode the
PTS protein HPr and adenylate cyclase, respectively.
Model-based evaluation of nonessential genes. In addition
to the strong correlation between conditionally essential genes,
there is also good agreement between the predicted and ob-
served nonessential genes. Of the 3,769 observed nonessential
genes, 784 are represented in the model, and ?95% (742 of
784) of these are correctly predicted to be nonessential by the
model (Fig. 1). This leaves 42 discrepancies (listed in Table 2
and supplementary Table 3 [http://systemsbiology.ucsd.edu
/publications/supplemental_material/JBact2006/]) where the
model incorrectly predicts genes to be essential. Some of these
42 predicted essential genes not identified in the experimental
screen are involved in the biosynthesis of biomass components,
such as lipopolysaccharide (LPS), spermidine, and glycogen,
which in fact may not be essential biomass components. For
example, it is known that a complete LPS is not required for
For other biomass components like arginine and lysine, a ra-
tionale for the observed discrepancies may be related to the ex-
istence of alternative reactions and/or isozymes that are
unaccounted for in the model. For example, argD encodes
an enzyme with dual activity as both acetylornithine amino-
transferase (EC 184.108.40.206; required for arginine biosynthesis)
and N-succinyl-L,L-diaminopimelate aminotransferase (EC
220.127.116.11; required for lysine biosynthesis) and is predicted to be
essential by the model. The astC gene (also known as argM)
encodes an enzyme with succinyl- and acetylornithine amino-
transferase activities and has been speculated to have N-suc-
cinyl-L,L-diaminopimelate aminotransferase activity, as well
(16). As another example, both coaA and coaE gene products
FIG. 2. Essential genes involved in arginine and proline biosynthe-
sis. The map shows the biosynthesis pathway for the conversion of
glutamate (upper left) into arginine and proline. Red arrows are used
to indicate reactions associated with an experimentally identified es-
sential gene in glycerol-supplemented minimal medium, while gray
arrows indicate reactions in which the associated gene(s) is experimen-
tally nonessential. Associated genes are listed next to their correspond-
ing reactions (“or” indicates that the gene encodes isozymes). Gene
names listed in black indicate that the model makes an incorrect
prediction regarding essentiality, while those in gray indicate the
model and data are in agreement. The genes proB and proA are both
essential experimentally during growth on minimal medium, but the
model predicts that argA, argB, argC, and argE can be used instead to
produce glutamate 5-semialdehyde for proline biosynthesis.
8264 JOYCE ET AL. J. BACTERIOL.
are required to produce CoA; however, neither gene was es-
sential in rich medium or glycerol minimal medium, while the
remaining genes involved in the pathway were essential (Fig.
3). Other enzymes may be present which can carry out these
essential reactions, although it is likely that the apparent via-
bility of at least one of these strains (coaE) was due to a
yet-unknown artifact, since the coaE gene (formerly yacE) was
shown to be essential in a number of mutant studies (16, 20).
Two transporters were also computationally predicted to be
essential, glpF and amtB. Although in the model these are the
only transporters for glycerol and ammonia, respectively, both
compounds freely diffuse through membrane vesicles (13, 26)
and their transporters are likely essential only at very low
solute concentrations. We subsequently tested the growth ca-
pabilities of the ?glpF mutant (after removal of the kan gene as
previously described ) on different concentrations of glyc-
FIG. 3. Model-based evaluation of experimentally essential genes involved in cofactor biosynthesis. The map shows the metabolic pathways
involved in vitamin and cofactor metabolism, where biosynthesis proceeds in the downward direction and the end products are indicated below
each pathway. Red and blue arrows are used to indicate reactions associated with experimentally identified essential genes in glycerol minimal
medium and rich medium (1), respectively. Gray lines indicate cases in which the gene(s) associated with the corresponding reaction was
experimentally nonessential. Only the experimentally essential genes are listed next to the corresponding reactions. “&” indicates that the genes
form a complex. For pathways containing essential genes, a “No Gene” designation is placed next to reactions that lack a corresponding gene
assignment. Gene names listed in black indicate discrepancies between the model predictions and experimental data (i.e., the model predicts a
nonlethal phenotype for growth on glycerol minimal medium but the gene deletion is lethal experimentally), and genes in gray indicate agreement
(both model and data predict essentiality). Genes in black next to gray reactions (coaA, coaE, aldA, and pabC) indicate that the model predicts
that a gene deletion is essential whereas the corresponding mutant strain is viable. Note that unlabeled gray reactions also represent agreement
of the model prediction and experimental data, as both find the associated gene(s) to be nonessential.
VOL. 188, 2006 COMPUTATIONAL ANALYSIS OF ESSENTIAL GENES IN E. COLI8265
erol to confirm this hypothesis. As the glycerol concentrations
were reduced (from 2 g/liter to 0.25 g/liter), the ?glpF mutant
strain had increasingly lower growth rates than the BW25113
parental strain (seethe
/JBact2006/]). At a glycerol concentration of 0.125 g/liter, the
parental strain was able to grow at a lower rate, whereas
growth for the ?glpF mutant strain was abolished. Similar
observations have been made in previous ammonium-limited
growth experiments for amtB mutants, and it was speculated
that 10 ?M NH4
growth defects in ?amtB strains (46).
Combined analysis of both essential and nonessential genes
indicated a total of 68 discrepancies (only ?8% of total pre-
dictions) between experimental and computational essentiality
assignments (Table 2). These discrepancies can be grouped
into three types, pointing to possible model improvements with
respect to boundary conditions (a formula for essential bio-
mass components), gene-reaction associations (annotations),
and quantitative constraints for the passive uptake of nutrients
supplementary figure [http:
?concentrations would be needed to see
Comparison of genome scale conditional-essentiality data
sets. In the recently published description of the “Keio collec-
tion” (1), the authors described the conditional essentiality of
the single-gene knockout strains when grown on glucose-sup-
plemented minimal 3-N-(morpholino) propane sulfonate
(MOPS) medium. Using this data set, we identified the 119
slowest growers on glucose-supplemented minimal medium by
ranking the ODs measured at 24 h. For the purposes of this
analysis, this subset represents the conditionally essential genes
required for growth on glucose minimal medium. The collec-
tion of conditionally essential genes largely overlaps the glu-
cose-specific and glycerol-specific data sets (Fig. 5). The genes
found in this overlapping group primarily include those re-
quired to form biomass components in the absence of rich
medium, such as nucleotides and amino acids, as well as those
needed to generate required cofactors, such as NAD(P), CoA,
folates, and pyridoxal 5-phosphate. Accordingly, these genes
represent a conserved conditionally essential core that is re-
quired for E. coli to grow under minimally supplemented
growth conditions and is not required for growth under rich
(i.e., LB medium) conditions.
FIG. 4. Essential genes involved in glycerol metabolism. The pathway map in the center shows the metabolic pathways involved in the first steps
of glycerol metabolism. Red and blue arrows are used to indicate reactions associated with experimentally identified essential genes in glycerol
minimal medium and rich medium (1), respectively. Gray lines indicate cases in which the gene(s) associated with the corresponding reaction was
experimentally nonessential. Associated genes are listed next to their corresponding reactions (“or” indicates that the genes encode isozymes, and
“&” indicates that the genes form a complex). Gene names listed in black indicate that the model makes an incorrect prediction regarding
essentiality, while those in gray indicate that the model and data are in agreement. The phosphorylation of dihydroxyacetone is carried out by
DhaKLM coupled with PtsHI; of these five genes, only ptsI is essential during growth on minimal medium. Three enzymes can convert glycerol
3-phosphate into dihydroxyacetone phosphate, but the data indicate that glpD is the only essential gene. The model predicts that the only gene that
is essential on this map is glpF, the glycerol transporter, since the conversion of glycerol into dihydroxyacetone phosphate can proceed through
either of two routes. The four plots show results from quantitative RT-PCR performed to assess the expression levels of genes that encode enzymes
associated with reactions on the map. Gene expression levels during growth on glucose-supplemented and glycerol-supplemented M9 minimal
medium are reported relative to levels of the control gene acpP (mean ? standard error). The gene expression data indicate that the GlpK-GlpD
branch is dominantly active during growth on glycerol-supplemented minimal medium, in agreement with the conditional-essentiality data.
8266 JOYCE ET AL. J. BACTERIOL.
Relatively few genes are conditionally essential for growth
on glucose relative to growth on glycerol (Fig. 5). Among the
glucose-specific conditionally essential genes are 10 that may
simply be slow growers, as their ODs after 48 h were substan-
tially increased. Furthermore, three (argB, argC, and metE)
were likely false positives, given their nonessentiality in inde-
pendent phenotype microarray screens (18, 24), while one
(argG) agrees with prior studies (24). Perhaps more interesting
are the six biotin biosynthesis-related genes that are essential
in glucose- but not glycerol-supplemented growth on minimal
medium. This discrepancy involving all biotin biosynthesis
genes may indicate an unidentified source of biotin in the
glycerol essentiality screens. Five additional genes (ilvE, cysG,
ubiE, exoX, and hflD) are also glucose-specific essential genes,
although the rationale for their conditional essentiality re-
An equal number of genes have been observed to be essen-
tial for growth on glycerol as opposed to specific growth on
glucose. Four genes in this set of glycerol-specific conditionally
essential genes are directly related to glycerol metabolism or
its regulation. As previously described, glpK and glpD are in-
volved in the initial steps of glycerol catabolism, while crr and
cra (also known as fruR) are key components of the PTS and
mediators of catabolite repression. The differential essentiality
of ubiG and ubiH can be explained by the requirement for an
electron acceptor for growth on glycerol and the utilization of
ubiquinone in oxygen respiration (15). This suggests that ubiC,
ubiE, and ubiF should also be essential for aerobic growth on
glycerol; however, this conflicts with the observed experimental
Another six genes in this glycerol-specific set are involved in
sulfate transport and assimilation (cysADKPQU). This result
likely stems from the fact that the medium used in the glucose
essentiality screen contains MOPS, which can be utilized as a
sulfur source under sulfate-limited conditions (4), whereas the
M9 minimal medium used in this glycerol-specific screen does
not contain an alternative sulfur source besides sulfate. M9
minimal medium does not include iron, whereas MOPS min-
imal medium contains 10 mM of iron; this difference in me-
dium formulations accounts for the fact that fes (encoding an
iron-scavenging protein) is essential in glycerol-supplemented
M9 medium and not in MOPS-supplemented glucose medium.
A glmM deletion has previously been reported to be essential
(31), which agrees with the essentiality of glmM reported in this
glycerol lethal data set and may represent a false-negative
result in the glucose conditional-essentiality data set. ATP
synthase components were also found to have different essen-
tiality results, with atpABCFGH being essential for growth on
glycerol and only atpBC being essential for growth on glucose.
For both minimal-medium conditions, another ATP synthase
component, atpD, was not essential. Finally, seven additional
genes conditionally essential for growth on only glycerol-sup-
plemented medium remain difficult to explain.
Phylogenetic distribution of conditionally essential sub-
systems. The analysis of conditionally essential genes in the
context of metabolic subsystems described in The SEED pro-
jected over a diagnostic set of 31 diverse bacterial genomes is
illustrated in Fig. 6. Only those subsystems that contained
more than two experimentally defined genes conditionally es-
sential for growth on glycerol minimal medium are shown.
Overall, 103 out of 119 experimentally essential genes (as well
as 11 additional genes predicted by the model to be essential)
are covered by a rather small set of 18 subsystems (a complete
list of gene-to-subsystem correspondences is provided in sup-
plementary Table 4 [http://systemsbiology.ucsd.edu/publications
Although this deliberately simplified analysis masks substan-
tial differences between the specific variants of subsystems (or
pathways) implemented in different species, it reveals some
important trends. First, the majority of organisms possess an
operational variant of most of these conditionally essential
subsystems. Not surprisingly, the group of organisms that lack
functional versions of many of these essential subsystems, al-
beit phylogenetically quite diverse, are all obligate pathogens
or symbionts, many of them intracellular. In particular, five
species (Borrelia burgdorferi, Chlamydia trachomatis, Myco-
plasma pneumoniae, Rickettsia prowazekii, and Treponema pal-
lidum) lack functional variants in all but two to four sub-
systems. Moreover, the most conserved subsystem across all
organisms examined (glycine, serine, and threonine synthesis)
is represented in these species by only a single-enzyme pathway
(serine hydroxymethyltransferase [EC 18.104.22.168]). In stark con-
trast, 15 organisms share each of the 18 identified conditionally
essential subsystems with E. coli. This observed dichotomy
reflects two drastically different lifestyles, as these 15 organ-
isms are able to thrive outside of a host. This analysis confirms
that nearly all subsystems implicated by this conditional-essen-
tiality study in a single model organism are universally impor-
tant for a broad range of phylogenetically distant free-living
The screening of single-gene deletion mutants on glycerol
minimal medium provides a meaningful addition to the collec-
tion of data regarding essential genes for E. coli. With the
combination of other such genome scale gene essentiality stud-
ies, we continue to refine our notion of what genes are
required for growth on rich and minimal media. From a com-
FIG. 5. Glycerol- versus glucose-supplemented minimal medium
essential genes. The overlap between the data set presented in this
work and the recently published data for conditionally essential genes
for growth on glucose-supplemented minimal medium (1) is shown. A
large majority of conditionally essential genes are consistent between
the glycerol and glucose data sets, with a relatively small fraction of
conditionally essential genes apparently specific to one growth condi-
tion or the other. Brief descriptions of the condition-specific essential
genes are provided on the left (glycerol specific) and right (glucose
specific) of the Venn diagram, and a more detailed treatment is pro-
vided in the text.
VOL. 188, 2006 COMPUTATIONAL ANALYSIS OF ESSENTIAL GENES IN E. COLI 8267
parison of genes required for growth under rich- and minimal-
medium conditions, a toolkit of genes enabling growth in lim-
iting environments can be identified. By studying the genes
required for growth on glycerol minimal medium, we showed
that (i) our understanding of the roles that these essential
genes play in this toolkit is clear and relatively complete, as
only two putative genes of unknown function (yjhS and yhhK)
were identified as essential in this phenotyping screen; (ii) the
current metabolic and regulatory model is highly accurate in its
essentiality predictions; and (iii) comparisons of model predic-
tions and high-throughput phenotyping data represent a pow-
erful approach to rapidly generate model refinements and hy-
potheses likely to lead to an enhanced understanding of the
Remarkably, 112 of the identified 119 conditionally essential
genes are included in the current metabolic model. This ob-
servation suggests that the applied experimental approach has
a very low rate of incorrectly identifying essential genes. Oth-
erwise, nonmetabolic and uncharacterized genes (at least 40%
of E. coli genes) would comprise a substantially larger fraction
of the identified set. At the same time, it indicates that an
inventory of E. coli metabolic genes captured in the current
model (1,003 out of ?4,400 genes in the E. coli genome) is
rather comprehensive, at least with respect to the pathways
required to support growth on minimal medium. The fact that
the identified conditionally essential gene set contained only
two genes of unknown function is notable but not surprising,
since our screening protocol is conceptually equivalent to the
identification of auxotrophs, a historical standard in the study
of E. coli genetics.
These experimentally essential genes can be mapped to met-
abolic subsystems, which allows a level of generalization en-
abling us to detect tendencies across multiple organisms that
may be obscured by details of functional variants. This type of
analysis readily facilitates the identification of metabolic func-
tions that are required by different organisms without the po-
tentially complicating details regarding how the molecules are
synthesized. For example, Bacillus subtilis, E. coli, and Coryne-
bacteria use three different chemistries in the lysine biosynthe-
sis DAP (meso-diaminopimelate) pathway, but their purposes
remain the same. It should be noted that these subsystem
projections were made only for conditionally essential genes
and not for genes that are essential for growth on rich medium
(and likely essential in minimal-medium environments, as
well). For example, only the portions of the pathways that are
required for NAD and CoA biosynthesis on minimal and not
rich medium are represented. Otherwise, these fundamentally
essential subsystems would be present in all analyzed genomes.
The set of conditionally essential subsystems (and genes
therein) identified in this study may also be used to assess the
metabolic potentials of organisms present in environmental
samples as captured by emerging metagenomics data (49).
FIG. 6. Phylogenetic distribution of essential genes by subsystem. The phylogenetic distribution of essential subsystems (see Materials and
Methods for further details) across a diverse set of bacterial species is shown. Each row of the heat map corresponds to one of the 18 essential
subsystems that were considered; next to each subsystem name, in parentheses, is the number of essential genes (both experiment and model
assessed) found in the subsystem in E. coli over the total number of genes found in the subsystem in E. coli. Each column represents one of the
31 representative organisms used in this analysis. The dark-gray elements indicate the presence of the subsystem within the organism, whereas light
gray indicates its absence. The dendrograms display the result of hierarchical clustering using the Hamming distance metric and average linkage
for subsystems (left) and organisms (top), respectively. Organism abbreviations are as follows: ATU, Agrobacterium tumefaciens; AQA, Aquifex
aeolicus; BAS, B. subtilis; BOB, B. burgdorferi; BRJ, Bradyrhizobium japonicum; BRM, Brucella melitensis; BUA, Buchnera aphidicola; CAJ,
Campylobacter jejuni; CAC, Campylobacter crescentus; CHT, C. trachomatis; CLA, Clostridium acetobutylicum; COG, Corynebacterium glutamicum;
DER, Deinococcus radiodurans; FUN, Fusobacterium nucleatum; HAI, Haemophilus influenzae; HEP, Helicobacter pylori; LIM, Listeria monocy-
togenes; MYT, Mycobacterium tuberculosis; MYP, M. pneumoniae; NEG, Neisseria gonorrhoeae; NOS, Nostoc; PSA, Pseudomonas aeruginosa; RAE,
Ralstonia eutropha; RIP, R. prowazekii; SHO, Shewanella oneidensis; STA, Staphylococcus aureus; STP, Streptococcus pneumoniae; SYN, Synecho-
cystis; THM, Thermotoga maritima; TRP, T. pallidum; XYF, Xylella fastidiosa.
8268 JOYCE ET AL.J. BACTERIOL.
Researchers will be able to rapidly assess the pathways present
within an environmental sample and use the essentiality infor-
mation to develop potential laboratory medium formulations
to facilitate further controlled study in the laboratory (47).
Furthermore, the presence of certain pathways and the ab-
sence of others may provide insights into the microenviron-
ment from which the sample was taken and also indicate local
intracommunity relationships between species that are present
in the sample. This subsystem-based essentiality analysis ap-
proach could be a useful tool to add to the growing compen-
dium of methods (5, 41) being developed to analyze and in-
terpret these complex data.
Further analysis of the generated gene essentiality data set
was made using a metabolic and regulatory model allowing the
data to be easily placed into biological context. Discrepancies
between model and experiment can be used to improve the
predictive capabilities of the model by indicating regions that
are not captured accurately by the models or, more impor-
tantly, can point to areas in metabolism or regulation that
require further experimental interrogation. For example, a
number of independent gene deletion studies have shown that
some genes involved in arginine biosynthesis are not essential
(18, 24), but without these enzymes, the current literature
cannot explain how this essential amino acid is synthesized.
Therefore, further experiments need to be conducted to either
identify novel arginine biosynthetic genes or determine which
multifunctional enzymes can compensate for any perturbation
of the genes.
Additionally, based on the experimental results, several
model improvements are suggested. Since a number of exper-
imentally essential genes are involved in cofactor biosynthesis,
a number of cofactors should be included in the biomass ob-
jective function used to conduct the growth prediction simula-
tion. These cofactors include pyridoxal-5-phosphate, isopre-
noids, hemes, ACP, and ubiquinone. These will help correct
for the false negatives (lethal phenotypes with nonlethal model
predictions) that account for a large number of discrepancies
in both minimal- and rich-medium phenotypes (data not shown
for rich medium). A wild-type biomass composition does not
always correlate with an essential biomass composition; for
example, only a core and not a complete LPS is required for
cell survival (37). Accordingly, the essentiality of these and
other biomass components can be refined or relaxed based on
the nonessentiality of the corresponding biosynthetic-pathway
genes. These issues are being addressed in a forthcoming up-
dated metabolic reconstruction of E. coli (A. Feist and B. O.
Palsson, personal communication) and represent a significant
Model improvements are also suggested with regard to
the first steps of glycerol metabolism (Fig. 4). As previously
noted, analysis of the false positives suggests that glycerol
import can occur by passive transport across the cell mem-
brane in the absence of the glpF-encoded transporter. Ad-
ditionally, the initial enzymatic steps required to convert
glycerol to dihydroxyacetone phosphate appear to be exclu-
sively mediated by GlpK and GlpD rather than by GldA and
the DhaKLM-PtsHI complex. This pathway bias is likely due
to transcriptional regulatory effects. Indeed, the elevated
expression of glpK and glpD during growth on glycerol re-
vealed by quantitative RT-PCR (Fig. 4) further supports the
notion that the GlpK-GlpD branch is dominant under these
conditions. Furthermore, a recent study showed that the
DhaR transcriptional regulator specifically upregulates the
genes encoding DhaKLM in the presence of dihydroxy-
acetone, but not glycerol (2). Under the conditions utilized
in this study, quantitative RT-PCR of dhaM (Fig. 4) showed
that the dhaKLM genes are only minimally expressed, leav-
ing the alternative glycerol metabolic pathway dormant.
Including the recently characterized DhaR regulatory inter-
action (2) in the integrated regulatory-metabolic model will
readily correct this discrepancy.
In summary, this high-throughput phenotyping screen pro-
vides a significantly enhanced view of the conditionally essen-
tial gene set required for growth under minimally supple-
mented growth conditions and additionally represents the most
comprehensive assessment of the constraint-based metabolic
model of E. coli conducted to date. Moreover, this study fur-
ther highlights the utility of using genome scale models as a
context for content in interpreting and analyzing complex high-
throughput data sets. This powerful synergistic approach of not
only using models as data analysis tools, but also using high-
throughput data as feedback for model improvement, is be-
coming a paradigm that will continue to drive systems biology
We thank Adam Feist for his critical reading of the manuscript;
Trina Patel, Vasiliy Portnoy, and Eric Knight for technical assistance;
and Christian Barrett and other members of the Palsson laboratory for
insightful discussions and suggestions.
We gratefully acknowledge the support of the NIH Protein Struc-
ture Initiative, grant numbers P50 GM62411 and U54 GM074898, and
also grant no. NIH R01 GM5708.
Bernhard Palsson has a financial interest in Genomatica, Inc. Al-
though the NIH R01 GM5708 grant has been identified for conflict of
interest management based on the overall scope of the project and its
potential to benefit Genomatica, Inc., the research findings included in
this publication do not necessarily directly relate to the interests of
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