An experimentally-supported genome-scale metabolic network reconstruction for Yersinia pestis CO92.

Abstract

Yersinia pestis is a gram-negative bacterium that causes plague, a disease linked historically to the Black Death in Europe during the Middle Ages and to several outbreaks during the modern era. Metabolism in Y. pestis displays remarkable flexibility and robustness, allowing the bacterium to proliferate in both warm-blooded mammalian hosts and cold-blooded insect vectors such as fleas.
Here we report a genome-scale reconstruction and mathematical model of metabolism for Y. pestis CO92 and supporting experimental growth and metabolite measurements. The model contains 815 genes, 678 proteins, 963 unique metabolites and 1678 reactions, accurately simulates growth on a range of carbon sources both qualitatively and quantitatively, and identifies gaps in several key biosynthetic pathways and suggests how those gaps might be filled. Furthermore, our model presents hypotheses to explain certain known nutritional requirements characteristic of this strain.
Y. pestis continues to be a dangerous threat to human health during modern times. The Y. pestis genome-scale metabolic reconstruction presented here, which has been benchmarked against experimental data and correctly reproduces known phenotypes, provides an in silico platform with which to investigate the metabolism of this important human pathogen.

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RESEARCH ARTICLEOpen Access
An experimentally-supported genome-scale
metabolic network reconstruction for Yersinia
pestis CO92
Pep Charusanti1*, Sadhana Chauhan2, Kathleen McAteer3, Joshua A Lerman1, Daniel R Hyduke1,
Vladimir L Motin2,4, Charles Ansong3, Joshua N Adkins3and Bernhard O Palsson1
Abstract
Background: Yersinia pestis is a gram-negative bacterium that causes plague, a disease linked historically to the
Black Death in Europe during the Middle Ages and to several outbreaks during the modern era. Metabolism in Y.
pestis displays remarkable flexibility and robustness, allowing the bacterium to proliferate in both warm-blooded
mammalian hosts and cold-blooded insect vectors such as fleas.
Results: Here we report a genome-scale reconstruction and mathematical model of metabolism for Y. pestis CO92
and supporting experimental growth and metabolite measurements. The model contains 815 genes, 678 proteins,
963 unique metabolites and 1678 reactions, accurately simulates growth on a range of carbon sources both
qualitatively and quantitatively, and identifies gaps in several key biosynthetic pathways and suggests how those
gaps might be filled. Furthermore, our model presents hypotheses to explain certain known nutritional
requirements characteristic of this strain.
Conclusions: Y. pestis continues to be a dangerous threat to human health during modern times. The Y. pestis
genome-scale metabolic reconstruction presented here, which has been benchmarked against experimental data
and correctly reproduces known phenotypes, provides an in silico platform with which to investigate the
metabolism of this important human pathogen.
Background
Yersinia pestis is a gram-negative bacterium within the
family Enterobacteriaceae that, along with Yersinia pseu-
dotuberculosis and Yersinia enterocolitica, is one of
three members of its genus that can cause disease in
humans. Y. pestis diverged from Y. pseudotuberculosis
only 1,500 - 20,000 years ago, but Y. pestis and Y. pseu-
dotuberculosis diverged from Y. enterocolitica in the
more distant past [1]. Despite their close evolutionary
relationship, the diseases they cause differ markedly.
Whereas Y. pseudotuberculosis and Y. enterocolitica are
primarily gastrointestinal pathogens in humans, Y. pestis
infections lead to a systemic disease known as plague
that can become fatal rapidly. In the last 2000 years,
there have been three distinct outbreaks of plague that
have led to a large number of fatalities: the Justinian
plague between the 5thand 7thcenturies, the Black
Death in Europe between the 13thand 15thcenturies,
and the modern plague from the latter half of the 1800s
to the present. These outbreaks of fatal infections that
continue to occur [2,3], the recent isolation of a multi-
drug resistant strain [4], and the potential to develop
into a bioweapon all signify that Y. pestis remains a sig-
nificant threat to human health.
Most Y. pestis strains can be divided into three bio-
vars: Antigua, Mediavalis, and Orientalis, based on their
ability to ferment glycerol and reduce nitrate [5]. Biovar
Antigua strains can do both; biovar Mediavalis strains
can ferment glycerol but cannot reduce nitrate; and bio-
var Orientalis strains cannot ferment glycerol but can
reduce nitrate [5]. Moreover, each biovar has been
linked to one of the three pandemics. Biovar Antigua is
associated with the Justinian plague; biovar Mediavalis is
associated with the Black Death; and biovar Orientalis is
* Correspondence: pcharusanti@ucsd.edu
1Department of Bioengineering, University of California, San Diego, La Jolla,
California, USA
Full list of author information is available at the end of the article
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© 2011 Charusanti et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
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associated with modern plague [5]. Debate surrounding
these associations continues, however, as recent data
suggest that the Black Death might not have been
caused by strains belonging to Biovar Mediavalis but
rather by at least two strains that pre-date the emer-
gence of and are distinct from both biovar Mediavalis
and biovar Orientalis [6].
Here we present a metabolic network reconstruction
and corresponding mathematical model of metabolism
in Y. pestis CO92 (abbreviated YP CO92). This strain is
classified within Biovar Orientalis and is virulent to
humans, a feature which distinguishes the model pre-
sented here from one published previously for Y. pestis
91001 [7], which is avirulent to humans. We also pre-
sent a comprehensive analysis of pathways in the meta-
bolic network related to the biosynthesis and utilization
of key metabolites, acquire experimental data from
growth on several different carbon sources and compare
them to simulation results, and identify essential genes
that might constitute possible targets for antibiotic
development.
Results
Characteristics of the reconstruction
The Y. pestis CO92 metabolic reconstruction presented
here, iPC815, contains 815 genes, 678 proteins, 963
unique metabolites, and 1678 reactions. iPC815 was
assembled by comparison to existing E. coli [8], Salmo-
nella enterica serovar Typhimurium (hereafter referred
to as S. Typhimurium) [9], and Y. pestis strain 91001 [7]
metabolic reconstructions; genetic, protein, metabolite,
and biochemical data contained in databases such as
KEGG [10], BRENDA [11], MetaCyc [12], and PATRIC
[13]; and genus-, species-, and strain-specific informa-
tion gleaned from the literature. The model contains
distinct compartments for the cytoplasm and periplasm,
and inner and outer membrane transporters and their
associated transport reactions model the movement of
metabolites between the compartments. Of the 1678
reactions, 1539 (92%) are linked to their corresponding
genes and proteins through gene-protein-reaction asso-
ciations (GPRs); the majority of the 139 non-linked
reactions are transport reactions. Table 1 summarizes
the general features of iPC815, while Figure 1 depicts
the functional classification of the 815 genes according
to their COG function and presents a comparison to the
E. coli, S. Typhimurium LT2, and Y. pestis 91001 meta-
bolic reconstructions. The full list of all genes, metabo-
lites, reactions, and GPRs in iPC815 can be found in
Additional File 1.
The iPC815 model contains two biomass objective
functions (BOFs) [14] that were derived from the two
BOFs developed for the existing Y. pestis 91001 recon-
struction [7] and modified as follows. First, iPC815
contains pathways for the biosynthesis of four Y. pestis
fatty acid acyl chains that are not explicitly modeled in
the Y. pestis 91001 reconstruction, specifically 14:0, 16:1,
16:0, and 18:1. These four comprise approximately 86%
of the total fatty acid composition in 29 Y. pestis isolates
during growth at 28°C [15]. Second, the lipopolysacchar-
ide (LPS) core oligosaccharide contains an uncommon
D-glycero-D-talo-oct-2-ulosonic acid (Ko) analog not
normally found in Gram-negative bacteria [16,17]. Both
of these features have been incorporated into the two
BOFs in iPC815.
Two BOFs are needed because the Y. pestis biomass
composition differs at 25-28°C versus 37°C. The signifi-
cance of these two temperatures stems from the two
types of hosts that Y. pestis infects in the natural envir-
onment: insect vectors at ambient temperature and
mammalian hosts with regulated body temperatures of
about 37°C. Accordingly, most laboratory studies of Y.
pestis are carried out at one or both of these tempera-
tures, and data from such studies have revealed two
principal differences in biomass composition at 25-28°C
versus 37°C. The first involves the lipopolysaccharide
(LPS) structure: at 37°C the LPS is composed of one
predominant core oligosaccharide and one predominant
lipid A isoform (tetraacyl 14:0), whereas at 25°C the LPS
is composed of multiple structures for both [17]. The
second involves the fatty acid composition. Lipid
Table 1 Overview of iPC815
CategorySubsystemNumber
Genes
Proteins
Metabolites
815
678
Cytoplasm
Periplasm
External
Unique
Total
873
398
281
963
1552
Metabolic Reactions
Cytoplasm
Periplasm
GPR-associated
Non-GPR-associated
Unique
932
133
1539
139
1678
Transport Reactions
Periplasm <=> External
Cytoplasm <=> Periplasm
281
332
281
2
1961
Exchange Reactions
Biomass Objective Functions
Total Reactions
To compile the number of unique metabolites, identical compounds
appearing in more than one compartment were only counted once. Unique
metabolic reactions encompass all reactions except exchange reactions and
biomass objective functions.
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Figure 1 A. Breakdown of the genes in iPC815 by COG classification. The COG classifications that make up the Miscellaneous category are:
K, L, Q, S, T, U, and V. B. Venn diagrams showing the overlap between iPC815 and published E. coli, S. Typhimurium, and Y. pestis 91001
reconstructions based on shared versus organism-specific reactions (E. coli and S. Typhimurium) and E.C. numbers (Y. pestis 91001). In the latter
case, an E.C. number associated with more than one reaction was only counted once. The low overlap between iPC815 and the Y. pestis 91001
reconstruction arises from the use of E.C. numbers to compare the two models rather than true biological differences between the two strains.
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measurements from Y. enterocolitica indicate that the
abundance of various fatty acids varies with growth tem-
perature [18], and we assume that this characteristic
holds for Y. pestis as well. The coefficients of the terms
representing the four fatty acids consequently differ
between the two BOFs. The 25-28°C BOF covers a tem-
perature range rather than a single value since the
experimental data on which this BOF was constructed
were taken over this range. Thus, the model also
assumes that the BOF composition does not change
appreciably between 25°C and 28°C.
Gap analysis of biosynthetic pathways
There were a number of gaps in iPC815 that initially
prevented the simulation of biomass formation. These
gaps consisted of reactions that were essential for simu-
lating growth but for which we could not identify
homologs in YP CO92 to the gene products in E. coli, S.
Typhimurium, or Y. pestis 91001 that catalyze the same
reactions. Each of these gaps constitutes testable experi-
mental hypotheses.
One gap occurs in the fatty acid biosynthesis pathway
(Figure 2A). The current annotation of the YP CO92
genome indicates that this strain contains the same full
suite of genes for fatty acid biosynthesis that E. coli and
S. Typhimurium have except one, the enoyl acyl-carrier
protein (ACP) reductase fabI. The enzyme encoded by
fabI catalyzes the reduction of the 2,3 double bond in
diverse acyl units during chain elongation [19], which is
an essential step in fatty acid biosynthesis. Consistent
with this data, the model cannot simulate growth with-
out this key enzyme. We have therefore included this
reaction in the model but it is not associated with a
gene or protein.
A similar gap appears in the lysine biosynthesis path-
way. Lysine is synthesized from aspartate in both E. coli
and S. Typhimurium through a series of nine enzyme-
catalyzed reactions [20], and YP CO92 has homologs to
each of the nine enzymes except one, ArgD (YPO0170)
(Figure 2B). In YP CO92, an IS element (IS100) is
inserted directly into the ORF of YPO0170 [21], splitting
it into two and likely rendering it nonfunctional. Despite
this disruption, we experimentally tested and found no
evidence for lysine auxotrophy in YP CO92, a phenoty-
pic observation that suggests additional enzyme(s) can
catalyze the same reaction as YPO0170 during lysine
biosynthesis. We consequently searched for alternative
enzymes within the YP CO92 genome that can accept
both N-succinyl-L-2-amino-6-oxopimelate and any
molecule capable of donating an amino group as reac-
tants (Figure 2B), but found no support for such an
enzyme. Subsequently, we searched for paralogs within
the YP CO92 genome that have homology to both
YPO0170 fragments. The gene with the greatest
homology was YPO1962 with 59% nucleotide identity,
and we have assumed in the model that YPO1962 can
replace YPO0170 in the lysine biosynthetic pathway
(Figure 2B).
Phenotype simulations
Once all critical gaps necessary for in silico biomass for-
mation were filled, we deployed the model to compute
growth versus no-growth on different carbon, nitrogen,
phosphorus, and sulfur sources at both 25-28°C and 37°
C (Additional File 1). We focused particular attention
on rhamnose and melibiose since the ability or inability
to ferment these two sugars is one of several phenotypes
commonly used to classify different strains of Y. pestis.
For example, two of the best predictors for virulence in
humans are the absence of rhamnose fermentation and
virulence in guinea pigs [22]. YP CO92 is known to be
lethal to humans, and consistent with this phenotype
there is no growth in silico if rhamnose is the sole car-
bon source. The model predicts that this outcome is
due to the overaccumulation of L-lactaldehyde. Specifi-
cally, the uptake pathway that connects periplasmic
rhamnose with central metabolism (Figure 3) leads to
the synthesis of a byproduct, L-lactaldehyde, that is not
removed through secretion, degradation, incorporation
into metabolism, or by any other means. Its production
via this pathway would therefore result in an infinite
accumulation of the compound, which is a thermodyna-
mically impossible outcome. On the other hand, the
melibiose utilization pathway appears to be intact and
thermodynamically consistent. We assume that a general
outer membrane porin allows passage of melibiose from
the extracellular space into the periplasm, after which
an inner membrane sodium:galactoside symporter
(YPO0995) appears capable of transporting melibiose
into the cytosol. Subsequently, the alpha-galactosidase
RafA (YPO1581) [23] connects imported melibiose with
central metabolism (Figure 3). This analysis implies that
regulation of these genes in the melibiose utilization
pathway, rather than the genes themselves, underlies the
inability of YP CO92 to utilize melibiose. We accounted
for this observation by retaining this set of reactions in
the model but deactivating the melibiose extracellular to
periplasm uptake reaction.
Using the individual components of the BCS chemical
medium to constrain the model [24], we next assessed
whether the model could recapitulate common amino
acid auxotrophies seen in epidemic strains of Y. pestis
such as CO92. These are methionine, phenylalanine,
and the combination of glycine and threonine [25,26].
Consistent with these data, the model exhibits auxotro-
phy for methionine and phenylalanine but growth still
occurs at a slow rate (~0.08 hr-1) in silico in the absence
of both glycine and threonine as long as all other amino
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Figure 2 Essential gaps in key Y. pestis CO92 metabolic pathways. A. The last step during each fatty acid elongation cycle involves the
reduction of an enoyl-ACP intermediate. In other Enterobacteria such as E. coli and S. Typhimurium, FabI catalyzes this step; however, the
current annotation of the YP CO92 genome does not contain fabI. B. The enzyme ArgD catalyzes the sixth step in the nine-step lysine
biosynthesis pathway, but argD (YPO0170) is disrupted in YP CO92. YPO1962 has the greatest homology to YPO0170 within the Y. pestis CO92
genome, and we hypothesize here that YPO1962 can replace the function of YPO0170. Note: although each reaction in each pathway is
depicted as unidirectional, some are reversible.
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