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Control of key metabolic intersections
in Bacillus subtilis
Abraham L. Sonenshein
Abstract | The remarkable ability of bacteria to adapt efficiently to a wide range of nutritional
environments reflects their use of overlapping regulatory systems that link gene expression
to intracellular pools of a small number of key metabolites. By integrating the activities of
global regulators, such as CcpA, CodY and TnrA, Bacillus subtilis manages traffic through two
metabolic intersections that determine the flow of carbon and nitrogen to and from crucial
metabolites, such as pyruvate, 2‑oxoglutarate and glutamate. Here, the latest knowledge on
the control of these key intersections in B. subtilis is reviewed.
Glycolysis
The metabolic pathway that
converts glucose into pyruvate,
with the concomitant
production of ATP and NADH.
Pentose-phosphate pathway
The metabolic pathway by
which glucose-6-phosphate is
oxidized to ribose-5-phosphate,
with the concomitant
production of NADPH.
Citric acid cycle
The metabolic pathway that
oxidizes acetyl CoA to carbon
dioxide.
2-oxoglutarate–glutamate–
glutamine cycle
The metabolic pathway that
connects carbon and nitrogen
metabolism and permits
ammonium ions to be
incorporated into organic
molecules.
Department of Molecular
Biology and Microbiology,
Tufts University School of
Medicine, 136 Harrison
Avenue, Boston,
Massachusetts 02111, USA.
e-mail:
[email protected]
doi:10.1038/nrmicro1772
Published online
5 November 2007
Most culturable bacteria thrive in the rich media that
are used to study their behaviour in the laboratory. In
the natural environment, however, nutrients are often
present in extremely dilute concentrations (for example, in the sea), inaccessible because of the paucity of
water (for example, in the desert) or only transiently
available (for example, in soil or the mammalian oral
cavity and gastrointestinal tract). Moreover, the quality
of the available nutrients can be highly variable. As a
result, bacteria have evolved remarkably sophisticated
adaptation systems that allow them to take advantage
of the wide range of sources of the essential elements
carbon, nitrogen, phosphorus and sulphur. This enables
them to feed the central metabolic pathways — glycolysis,
the pentose-phosphate pathway, the citric acid cycle and
the 2‑oxoglutarate–glutamate–glutamine cycle — from
which all of the precursors that are required for the
synthesis of the cell’s macromolecules (DNA, RNA,
proteins, peptidoglycan and lipid bilayers) are derived.
With the exception of peptidoglycan, these precursors
and polymers are universal in the biological world and
most of the biochemical pathways that are involved in
their synthesis are highly conserved.
The interactions between bacteria and eukaryotic
host organisms are often commensal or symbiotic, but
are sometimes parasitic, which causes serious harm
to the host. However, not all pathogenic bacteria are
constitutively virulent; many reserve the traits that we
perceive as virulent for conditions of stress, particularly
nutritional stress1. It is possible that these bacteria synthesize proteases, lipases and other virulence factors that
damage eukaryotic cells because they lack nutrients or,
in the case of intestinal pathogens, because they need to
be excreted into the environment in order to colonize
new hosts or niches.
nature reviews | microbiology
Rigorous biochemical, genetic and molecular analyses
carried out over the past 50 years have given us a detailed
view of the intricate reactions that generate the central
metabolites in bacterial cells, the genes that encode the
enzymes that carry out these reactions and many of the
mechanisms that regulate the expression of these genes2.
The genes that are required for the utilization of nutritional sources are typically regulated by the availability of
the substrate, just as the genes that are required for the
biosynthesis of a particular cellular constituent are typically regulated by the accumulation of the end-product.
As far as we know, virtually all bacteria have additional
layers of control that coordinate the use of nutrients of
the same type (for example, carbon or nitrogen) using
global regulators. Recent work has established that some
of these global regulators are also integrated into a larger
regulatory scheme by which the cell (Bacillus subtilis in
this Review) coordinates the flow through key metabolic
intersections in response to a small number of specific
signalling metabolites (TABLE 1). Here, the goal is to summarize our current knowledge about the regulation of the
genes that encode the enzymes that mediate the flow of
carbon from pyruvate to the citric acid cycle and carbonoverflow pathways, as well as the enzymes that interconvert 2‑oxoglutarate, glutamate, glutamine and certain
other amino acids in B. subtilis. It is beyond the scope of
this Review to consider the regulation of sugar transport,
glycolysis, gluconeogenesis and the pentose-phosphate
pathway. For detailed information about these pathways,
the reader is referred to excellent reviews by Deutscher
and collegues3,4 and Aymerich and colleagues5.
Carbon overflow in B. subtilis
B. subtilis is a Gram-positive, spore-forming bacterium
that has been the subject of intense investigation since
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Table 1 | Bacillus subtilis regulators of central metabolism genes
Regulatory Target genes or proteins Metabolites
protein
sensed
Direct or
indirect sensing
Mediator
Effect of metabolite
Citrate
Direct
None
Inactivation
Specific regulators
CcpC
citZCH, citB and ccpC
RocR
rocABC, rocDEF and rocG
Ornithine
Direct
None
Activation
GltC
gltAB and gltC
2-oxoglutarate
and glutamate
Direct
None
Activation by 2oxoglutarate and
inactivation by glutamate
RocG
GltC
None
Direct
None
Inactivation
GlnR
glnRA, ureABC and tnrA
Glutamine
Indirect
Glutamine synthetase by an
unknown mechanism
Activation
Global regulators
CcpA
Many carbon-metabolism
genes
FBP and
glucose-6phosphate
Direct and
indirect
FBP stimulates HPr kinase to
Activation
phosphorylate Hpr and Crh, which
interact with CcpA; the interaction
of FBP and glucose-6-phosphate
with the P-HPr–CcpA complex
CodY
Many carbon- and
nitrogen-metabolism
genes, transport genes,
sporulation genes and
competence genes.
GTP and BCAAs
Direct
None
Activation
TnrA
Many nitrogenmetabolism genes
Glutamine
Indirect
Glutamine synthetase–glutamine
complex interacts with TnrA
Inactivation
A summary of the regulatory proteins that are implicated in the control of some central metabolism genes. See main text for details and references. BCAA,
branched-chain amino acid; FBP, fructose-1,6-bisphosphate.
Global regulator
A protein that controls many
genes and operons in response
to a specific signal.
Sporulation
A developmental programme
in some microorganisms in
response to unfavourable
environmental conditions that
results in spores that are highly
resistant to environmental
stresses.
LacI protein family
The proteins that are related in
sequence and function to the
classical repressor of the E. coli
lac operon.
Phosphoenolpyruvatedependent
phosphotransferase
transport system
A multi-protein phosphorelay
system that couples the
phosphorylation of sugars to
their transport across the
cytoplasmic membrane.
the early 1950s. The attraction of sporulation as a prototypical system of cellular differentiation and the ease of
genetic manipulation made B. subtilis an early choice for
detailed investigation. At present, B. subtilis is the second
most intensively studied bacterium, after Escherichia coli,
and is a useful paradigm for most of the Gram-positive
bacterial world6.
If growing in a medium that contains an excess of
glucose (the preferred carbon source for many bacteria), B. subtilis metabolizes a large proportion of
the glucose only as far as pyruvate and acetyl CoA,
and subsequently converts these compounds to
by-products of metabolism (also known as fermentation products), including lactate, acetate and acetoin,
which are excreted into the extracellular environment
(FIG. 1). The enzymes of glycolysis depend on the cofactor NAD+ to take up electrons and hydrogen atoms
that are released by substrate oxidation; the conversion of pyruvate to lactate has the advantage of regenerating NAD+ from its reduced form, NADH, which
is a step that is essential for continued glycolysis. The
conversion of acetyl CoA to acetate is coupled to the
synthesis of ATP by the activities of the enzymes
phosphotransacetylase and acetate kinase. Thus,
these overflow pathways enable the cell to maintain
redox balance and generate ATP without using the
cytochrome system. When the glucose has been fully
consumed, the cells reintroduce the by-products into
central metabolism (using lactate dehydrogenase,
acetoin dehydrogenase and acetyl CoA synthetase)
and metabolize them further through the citric acid
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cycle, so generating additional ATP and reducing
power (FIG. 1).
Unsurprisingly, the genes that encode the enzymes
that are involved in overflow metabolism are regulated
by proteins that sense the nutritional status of the cell.
In B. subtilis, CcpA activates the expression of the genes
that are required for the synthesis of acetate, lactate and
acetoin7–12 (FIG. 1). CodY also contributes to the activation of the acetate- and lactate-synthesis pathways, as
discussed below (FIG. 1). Moreover, the re-utilization
enzymes for acetate and acetoin are repressed by CcpA
and CodY11–16 (FIG. 1). As described in more detail below,
these two regulators function together to integrate
the expression of carbon-overflow genes with overall
B. subtilis metabolism.
The global regulators CcpA and CodY
CcpA is a member of the LacI protein family and a
global regulator of carbon-metabolism pathways
in many Gram-positive bacteria 17. In B. subtilis,
CcpA contributes to the regulation of more than
100 genes, the products of which, in most cases, are
involved in carbon acquisition or metabolism10–12,18.
CcpA can be either a positive or negative regulator
of transcription, and its activity is determined by a
complex interaction with two other proteins and at
least one signalling metabolite4. HPr, a component of
the phosphoenolpyruvate-dependent phosphotransferasetransport system, and the closely related protein Crh,
are phosphorylated on a specific serine residue by HPr
kinase if cells are grown in media that contain glucose
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REVIEWS
as a DNA-binding protein is stimulated by interaction
with either of two ligands, GTP or a BCAA (branchedchain amino acid; particularly isoleucine or valine)40,41.
These two ligands have additive effects on CodY activity 41. Consensus sequences for CodY-binding sites (the
‘CodY box’) have been proposed, but adherence to
this consensus is variable42,43. Taking these properties
of CcpA and CodY into account, we can see that the
decision to convert pyruvate to excretable overflow
products is made by two regulatory proteins (CcpA
and CodY) that respond to three metabolites (FBP,
GTP and BCAAs). The same regulatory proteins and
metabolites control the re-use of the overflow products
if the supply of glucose is exhausted.
Glucose
GTP
BCAAs
CodY
CcpA
FBP
CcpA
Lactate
Fatty acids
Pyruvate
Acetolactate
Acetoin
Acetyl CoA
Acetyl~ P
Acetate
GTP
CcpA
CodY
BCAAs
Citric acid cycle
Figure 1 | Interactions of the global regulators CcpA and CodY with carbonoverflow metabolism in Bacillus subtilis. In the presenceNature
of excess
glucose,
B. subtilis
Reviews
| Microbiology
metabolizes a large proportion of the glucose only as far as pyruvate and acetyl CoA.
These molecules are then converted to by-products of metabolism, such as lactate,
acetate and acetoin, using carbon-overflow pathways. CcpA activates the expression of
the genes that are required for the synthesis of these by-products, and CodY contributes
to the activation of the acetate- and lactate-synthesis pathways. Additionally, the
re-utilization enzymes for acetate and acetoin are repressed by both CcpA and CodY
(indicated by blunt arrows). In turn, CcpA is regulated by fructose-1,6-bisphosphate
(FBP). CodY is subject to regulation by GTP and branched-chain amino acids (BCAAs).
Competence
The ability of bacteria to take
up extracellular DNA.
or other rapidly metabolized carbon sources19–21. The
phosphorylated proteins can independently bind to
CcpA 22 and increase its affinity for cre sites — the
12-base pair sequences that are the preferred binding
site for CcpA23–25. The activity of HPr kinase is stimulated by ATP and fructose‑1,6-bisphosphate (FBP)26,27,
which links the activity of CcpA to the availability of a
glycolytic intermediate4. FBP and glucose‑6-phosphate
can also bind directly to CcpA in complex with phosphorylated HPr 24,28. Both mechanisms appear to contribute
to CcpA activity.
B. subtilis CodY, and its homologues in other Grampositive bacteria, defines a unique family of regulatory
proteins29. CodY was first discovered as a repressor of
the genes which, through their products, help cells to
adapt to poor nutritional availability30–36. That is, many
genes that are repressed by CodY encode proteins that
allow cells to move to new, potentially nutritionally
richer locations, to degrade extracellular macromolecules and transport the breakdown products into the
cell for degradation. CodY also represses the development of genetic competence and sporulation as adaptations to nutrient limitation37–40. The activity of CodY
nature reviews | microbiology
Global regulators and the citric acid cycle
The first three enzymes of the citric acid cycle — citrate
synthase, aconitase and isocitrate dehydrogenase
— form the tricarboxylic acid (TCA) branch (FIG. 2).
Together with pyruvate dehydrogenase, they drive the
conversion of pyruvate to 2‑oxoglutarate; although
citrate synthase is generally regarded as the ratelimiting enzyme of the pathway, the irreversibility of
isocitrate dehydrogenase forces the pathway towards
2‑oxoglutarate. The regulation of the TCA-branch
enzymes is intricate and they respond to multiple
metabolic signals at the levels of gene expression and
enzyme activity. The genes that encode citrate synthase (citZ) and isocitrate dehydrogenase (citC) are
joined in an operon by citH (also called mdh), which
encodes malate dehydrogenase44,45. The promoter of
the citZ gene drives the expression of all three genes,
but, as citC and citH also have gene-specific promoters, the control of citC and citH by repression at the
citZ promoter is incomplete 46. The aconitase gene
(citB) is encoded elsewhere on the chromosome in a
monocistronic transcription unit47.
Transcription from the citZ and citB promoters is
repressed by CcpC, a member of the LysR family of regulatory proteins48,49 (FIG. 2). CcpC has only one other known
target in the B. subtilis chromosome: its own promoter50.
In Listeria monocytogenes, CcpC has been implicated in
the regulation of a glutamine-transport gene as well as
citB51.
CcpC is inactivated as a repressor of citZ and citB by
interaction with citrate48,52. Although it is logical for the
synthesis of aconitase to increase as its substrate, citrate,
accumulates, it seems bizarre that citrate synthase would
be induced by the accumulation of its end-product. It
is worth noting, however, that such a phenomenon is
as much the rule as the exception. The lac operon of
E. coli is induced by allolactose, the synthesis of which
depends on β‑galactosidase activity53, and the histidine-utilization (hut) operon of Klebsiella aerogenes is
induced by urocanate, the product of the first enzyme
of the pathway54.
Although CcpC is a specific regulator of the TCAbranch genes, two other modes of regulation are layered
on top of this primary regulatory mechanism. First, the
citZCH operon is repressed directly by CcpA12,49 and,
second, CodY is also a repressor of the citB gene55.
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Glucose
FBP
Valine
CcpA
Pyruvate
Acetyl CoA
Aspartate
Oxaloacetate
Citrate synthase
citZ
Citrate
CcpC
Aconitase
citB
Isocitrate
Isoleucine
GTP
CodY
2-oxoglutarate
Nature
Figure 2 | Interactions of global regulators with the citric
acidReviews
cycle in| Microbiology
Bacillus
subtilis. In B. subtilis, three regulatory proteins (CcpC, CcpA and CodY) and four
metabolites (citrate, fructose-1,6-bisphosphate (FBP), GTP and isoleucine or valine)
function together to determine the extent to which pyruvate and acetyl CoA enter the
tricarboxylic acid branch of the citric acid cycle.
CcpA is an indirect repressor of citB by its control
of citrate synthesis49,52. Therefore, the full expression of
citZ, citB and citC requires the antagonism of at least
three repressor proteins (FIG. 2). This result is achieved
for CcpC by the accumulation of citrate; for CodY, the
intracellular concentrations of GTP, isoleucine or both
must drop below a critical level; and for CcpA, the FBP
pool must decrease. These compounds do in fact change
in concentration if cells experience general nutrient
limitation56.
Thus, in B. subtilis, three regulatory proteins
(CcpC, CcpA and CodY) and four metabolites (citrate, FBP, GTP and isoleucine or valine) function
together to determine the extent to which pyruvate
and acetyl CoA enter the TCA branch of the citric acid
cycle (FIG. 2). A fifth metabolite also plays a part posttranslationally. Citrate synthase is subject to feedback
inhibition by 2‑oxoglutarate, which acts competitively with respect to the substrate oxaloacetate (H.J.
Kim and A.L.S., unpublished observations). This
observation helps to explain the inverse correlation
between aconitase synthesis and the 2‑oxoglutarate
pool57 and the discovery that the repression of citB
expression by arginine depends on the conversion
of arginine to 2‑oxoglutarate52. Based on these facts,
it is now possible to understand why the TCA branch
is strongly repressed during the growth of cells in a
rich medium that contains glucose and a mixture of
amino acids. Glucose generates FBP, which activates
CcpA, and therefore contributes to repression of the
920 | december 2007 | volume 5
citZCH operon. Any synthesis of citrate synthase will
be negated by the presence of a pool of 2‑oxoglutarate,
which is produced by the catabolism of amino acids.
As a result, the pool of citrate will be low, CcpC will
be highly active as a repressor and citZ transcription will be severely limited. Moreover, citB will be
repressed strongly by the combined activities of CcpC
and CodY; in this case the richness of the medium will
allow efficient synthesis of GTP, and the presence of
isoleucine and valine in the medium will help to keep
CodY highly active.
The complexity of this system of regulation can
be exacerbated by the repression of the ccpC gene
by both CcpA and CcpC 50,52, but the regulation of
CcpC synthesis by CcpA does not seem to be a major
contributor to the control of the gene expression of
TCA-branch enzymes52. In addition, two other global
regulators have been implicated in the regulation of
citB transcription. AbrB, a protein that is best known
as a repressor of genes that are expressed early in stationary phase, is a direct positive regulator of citB55
and TnrA — the global nitrogen metabolism regulatory protein (discussed below) — is also a positive
regulator that, perhaps, acts indirectly52.
The link between carbon and nitrogen metabolism
The metabolite 2‑oxoglutarate stands at the crossroads
between carbon metabolism and nitrogen metabolism
(FIG. 3). As one of the substrates of glutamate synthase
(GOGAT), 2‑oxoglutarate provides the de novo carbon
skeleton for the two most important nitrogen­‑containing
compounds in the cell, glutamate and glutamine.
Moreover, as the product of glutamate metabolism by
glutamate dehydrogenase, 2‑oxoglutarate can be the
entry point into central metabolism for the carbon
skeletons of several amino acids (including arginine,
ornithine, proline and histidine), which are catabolized
to glutamate. In addition, 2‑oxoglutarate is a substrate
(or product) of most of the amino-transferases that
interconvert amino acids and their keto acids. Each of
the pathways to and from 2‑oxoglutarate is subject to
operon-specific and global regulation (FIG. 3).
In B. subtilis, the de novo synthesis of glutamate
from 2‑oxoglutarate is catalysed uniquely by GOGAT,
the heterodimeric product of the gltAB operon (FIG. 3).
The operon-specific regulator of gltAB is GltC58,59 — a
LysR family member, the in vitro activity of which is
determined by its interaction with two different ligands; 2‑oxoglutarate activates GltC, whereas glutamate
acts as a competitive inhibitor with 2‑oxoglutarate60.
The two ligands are thought to cause GltC to adopt different conformations that lead to binding to the gltAB
promoter at different positions, so that transcription is
either activated or repressed60. Recent work has shown
that GltC activity is also regulated by direct interaction
with glutamate dehydrogenase (discussed below)61.
The synthesis of GOGAT and glutamine synthetase
is repressed by TnrA, the principal global regulator of
nitrogen-metabolism genes in B. subtilis62 (FIG. 3). TnrA is
only active if cells are trying to grow with slowly metabolizable nitrogen sources. Under such conditions, TnrA
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Glucose
Arginine uptake
Intracellular arginine
FBP
RocR
Ornithine
CcpA
GTP
Other
amino
acids
Glutamate
dehydrogenase
CodY
CcpA
rocG
GltC
2-oxoglutarate
Glutamate
gltAB
CodY
Glutamate
synthase
TnrA
GTP
BCAAs
NH4+
Histidine
Glutamine
synthetase
Glutamine
BCAAs
Figure 3 | Bacillus subtilis global regulators at the intersection between carbon and nitrogen metabolism. The
intersection between carbon and nitrogen metabolism is regulated by at least six proteins (GltC,
TnrA,
RocG,| Microbiology
RocR, CcpA
Nature
Reviews
and CodY) that respond to 2‑oxoglutarate, glutamine, ornithine, fructose-1,6-bisphosphate (FBP), GTP and branchedchain amino acids (BCAAs).
represses some genes, but also activates operons such
as nrgAB, nasBC and nasDEF, the products of which
mediate the uptake of ammonium ion and the use of
secondary nitrogen sources such as nitrate and nitrite62‑64.
If the favoured nitrogen source, glutamine, is available,
TnrA is inactivated, as it is pulled into a complex with
glutamine synthetase and glutamine65. The GOGAT
genes, gltAB, are also repressed by CodY36, probably by
indirect mechanisms.
The synthesis of 2‑oxoglutarate from glutamate
by glutamate dehydrogenase depends on the operonspecific activator RocR66. Other amino acids that are
converted to glutamate, such as arginine, proline and
histidine, can also be sources of 2‑oxoglutarate. The
glutamate dehydrogenase (rocG) gene lies upstream
of an operon (rocABC) that is involved in arginine–
ornithine–proline metabolism67, but is co-regulated with
rocABC through binding of RocR to an enhancer element.
This element lies upstream of rocA but downstream of
rocG68,69. Ornithine is the co-activator; without ornithine,
the affinity of RocR for its binding sites is greatly reduced67.
A second, weaker promoter for rocG provides a low level
of rocG expression in the absence of ornithine70.
Commichau and colleagues61 have recently shown
that RocG has a second activity as a regulatory protein
— it directly contacts and inhibits GltC. This discovery
sheds light on two previously mysterious physiological
observations. The transcription of gltAB is stimulated by
glucose and requires CcpA; in other words, a ccpA mutant
is a glutamate auxotroph70–72 and is repressed if cells are
grown in a medium that contains arginine or ornithine73.
Consequently, the induction of the rocG gene by ornithine
(owing to its effect on RocR) leads to inhibition of gltAB
transcription by RocG; the repression of rocG by CcpA,
nature reviews | microbiology
if glucose is present, can explain the dependence of gltAB
transcription on CcpA and glucose.
The CcpA-dependent repression of rocG when cells
are exposed to glucose70 (FIG. 3) makes sense because
glutamate, and all other amino acids that can be converted to glutamate, are alternative carbon sources.
CcpA also represses the hut operon directly74. CodY collaborates with CcpA to repress the hut operon33 and also
represses the uptake of arginine (B. Belitsky, personal
communication), γ‑aminobutyrate32 and other amino
acids and peptides36.
Glutamine is essential for cell growth, is the preferred nitrogen source of B. subtilis and is the second
substrate of GOGAT (FIG. 3). In addition, in B. subtilis,
glutamine synthetase is virtually the only enzyme
that assimilates ammonium ions into organic compounds. The glutamine synthetase gene (glnA) is
the second gene of the glnRA operon75. GlnR is both
an autorepressor76,77 and a negative regulator of the
urease31,78 and tnrA79 genes. The activity of GlnR is
stimulated by glutamine synthetase and glutamine80–82.
In Streptococcus pneumoniae, which lacks TnrA,
GlnR represses the synthesis of glutamine and glutamate (glutamate is synthesized by anabolic glutamate
dehydrogenase), the enzyme glucose‑6-phosphate
dehydrogenase and the uptake of glutamine 83 .
Glutamine synthetase is necessary for the repressing
activity of S. pneumoniae GlnR83. The glutamate dehydrogenase gene is also repressed by CodY in this bacterium83. Gram-positive bacteria that lack TnrA might
generally use GlnR to carry out some of the global roles
that are assumed by TnrA in B. subtilis. In summary, the
intersection between carbon and nitrogen metabolism
is regulated by at least six proteins — GltC, TnrA, RocG,
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Aspartate
Glucose
Threonine
α-Ketobutyrate
Pyruvate
TnrA
CcpA
CodY
α-Ketoisocaproate
α-Ketoisovalerate
Leucine
Valine
α-Keto-β-methylvalerate
Isoleucine
Figure 4 | The metabolic context of the Bacillus subtilis branched-chain amino
| Microbiology
acid biosynthetic pathway. CcpA is a positive regulator ofNature
the ilvReviews
genes; TnrA
and
CodY are negative regulators.
RocR, CcpA and CodY — that respond to 2‑oxoglutarate,
glutamine, ornithine, FBP, GTP and BCAAs.
A regulatory network for metabolic intersections
As discussed, CcpA, TnrA and CodY are global regulators that sense diverse intracellular metabolites (FBP,
glutamine, GTP and BCAAs) and provide a top layer of
general nutritional regulation that determines the rate
of expression of the central metabolic genes, the products of which determine the flux through two crucial
metabolic intersections. This integrates these pathways
with each other and with the general state of the cell.
Thus, this information provides at least a rudimentary
idea of how B. subtilis has evolved to regulate crucial
decision points in central metabolism by taking into
account several different metabolic signals.
One interpretation of this is that the compounds
that are particularly important to the cell include pyruvate and 2‑oxoglutarate. Each molecule of pyruvate
that the cell generates has several potential fates: it can
be used directly for the synthesis of alanine, valine and
leucine; it can be converted to oxaloacetate to initiate
the pathway for the synthesis of aspartate, asparagine,
methionine, diaminopimelate, lysine, threonine and
isoleucine; it can be reduced to lactate; or it can be
converted to acetyl CoA for metabolism through
the citric acid cycle, biosynthesis of fatty acids or by
catabolism to acetate. It is clearly in the interest of the
cell that pyruvate is distributed to as many potential
fates as are appropriate to meet the cell’s specific and
overall metabolic needs84.
Similarly, the distribution of 2‑oxoglutarate to several potential pathways must be balanced. On the one
hand, if the cell is in carbon excess and needs to generate
glutamate, glutamine and other amino acids, molecules
of 2‑oxoglutarate, which are produced by the citric acid
cycle, must be siphoned off by GOGAT, whereas catabolism of the existing amino-acid pools must be avoided.
On the other hand, if the cell finds itself in nitrogen
excess and, therefore, needs to send carbon skeletons to
the citric acid cycle for the biosynthesis of other compounds, or for gluconeogenesis, the activities of GOGAT
and glutamine synthetase must be reduced.
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The unexpected role of BCAA biosynthesis
A key element that balances the distribution of pyruvate
and 2‑oxoglutarate is the pathway for the biosynthesis
of BCAAs. B. subtilis has four transcription units that
are devoted to BCAA biosynthesis85. The ilvA, ilvD and
ybgE genes are present in monocistronic transcription
units. The other seven genes (ilvBHI and leuABCD) are
found in one large operon. All of these transcription
units are induced by the presence of glucose in a CcpAdependent manner11,86–88; all but ilvA are repressed by
CodY36,41. As a result, even though CcpA and CodY
cooperate to repress or activate genes of the carbonoverflow, amino-acid-degradation and citric acid cycle
pathways, they regulate BCAA biosynthesis antagonistically (FIG. 4). Moreover, TnrA is also involved in BCAA
regulation, serving as a repressor during conditions of
nitrogen limitation89 (FIG. 4).
The mechanism by which CcpA and CodY have
opposing effects on ilv gene expression has been
explored in detail. The two proteins bind to partially
overlapping sites just upstream of the –35 region of
the ilvB promoter. CcpA acts partly as an indirect
positive regulator by competing with CodY for binding and partly as a direct positive regulator of the ilvB
operon87,88. Both mechanisms are consistent with the
finding that a ccpA mutant is a partial BCAA auxotroph86, in which auxotrophy is suppressed by a codY
mutation87. The net result of these antagonistic effects
is that when cells are shifted to a glucose-containing
medium, CcpA stimulates the production of BCAAs,
making CodY a more avid DNA-binding protein.
Therefore, under such conditions, CodY is a stronger
repressor of the genes that it represses (such as the aconitase and acetyl CoA synthetase genes) and a stronger
activator of the acetate kinase and lactate dehydrogenase genes (FIG. 5). Of course, the greater production
of BCAAs in a glucose medium also means that CodY
will be a more effective competitor with CcpA for binding to the ilvB promoter; presumably the two proteins
quickly create a steady-state condition, in which the
rate of ilvB-operon expression reflects this competition.
By mediating the creation of different steady-state levels
of BCAA biosynthesis, under different conditions of
nutrient availability, CcpA and CodY determine the
extent to which CodY is active, thereby determining
the impact of CodY on the many genes that it controls.
In other words, if cells are exposed to glucose, CcpA
regulates not only its own target genes but also those
genes that are targets of CodY.
The impact of TnrA on ilv gene regulation has not
been studied in as much detail. Nonetheless, it can
be concluded that when nitrogen is limiting TnrA is
active, binds far upstream of the ilvB operon promoter
and represses transcription by twofold to threefold
through an unknown mechanism89. Thus, if carbon is
in excess and nitrogen is limiting, TnrA partially counterbalances the effect of CcpA on BCAA biosynthesis,
making CodY less active as a repressor of amino-acid
utilization pathways, while itself turning on the same,
and other, utilization pathways for alternative nitrogen
sources (FIGS 3,4).
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these factors explains why the BCAAs seem to be widely
used by bacteria to monitor their physiological state.
aa
Glucose
Uncharged
tRNA
CcpA
aa
CodY
ilvB
BCAAs
GTP + ATP
aa
RelA
pppGpp
ppGpp
tRNA and rRNA
synthesis
Nature Reviews
| Microbiology
Figure 5 | Intersection of RelA, CcpA
and CodY in
the
regulation of the Bacillus subtilis ilvB operon. If bacteria
are limited for amino acids, the stringent response is
induced. Uncharged tRNAs bind to the A site of the
ribosome and activate the ribosome-associated enzyme
RelA, which catalyses the formation of pppGpp from GTP
and ATP. The breakdown product of pppGpp, ppGpp,
reduces the rate of transcription of ribosomal RNA (rRNA)
and tRNA genes and also affects the transcription of many
other genes. In a relA mutant strain, GTP accumulates to a
higher-than-normal level, so increasing the activity of
CodY. If cells are growing in a glucose-containing medium,
CcpA stimulates the expression of ilvB, which leads to an
increase in the branched-chain amino acid (BCAA) pool
and greater activation of CodY (shown in the shaded area).
Homolactic fermentation
The production of lactic acid
(lactate) as the sole pyruvatederived product during growth
on sugars, such as glucose.
Why are BCAAs useful intracellular signals?
BCAAs seem to have a key regulatory role, by different mechanisms, in both Gram-positive and Gramnegative bacteria. Even though CodY, in Gram-positive
bacteria, and the leucine-responsive protein (Lrp) in
Gram-negative bacteria, are dissimilar in sequence and
structure, they have many of the same functional roles. Lrp
regulates a large number of functionally diverse operons,
responds to the presence of a BCAA (mainly leucine, but
also responds to serine and alanine), controls both stationary-phase genes and virulence and interacts with other
regulatory proteins90–92. Does it make sense for the cell to
monitor intracellular BCAA pools as opposed to pools
of other amino acids? Isoleucine, leucine and valine
are among the most abundant amino acids in proteins;
maintaining their pools is essential for high-level protein
synthesis. Moreover, cells should be able to monitor their
ability to synthesize BCAAs because one of the immediate precursors of the BCAAs, α‑ketoisovaleric acid, is
also a precursor of pantothenic acid and coenzyme A
(FIG. 4). In addition, the branched chain α‑keto acids
are precursors of the branched-chain fatty acids, which
in B. subtilis constitute ~90% of membrane fatty acids
during growth at 37°C93. Perhaps, some combination of
nature reviews | microbiology
How does the stringency factor RelA fit in?
If bacteria are limited for amino acids, uncharged
tRNAs bind to the A site of the ribosome and activate the
ribosome-associated enzyme RelA, which catalyses
the formation of pppGpp from GTP and ATP94. The
breakdown product of pppGpp, ppGpp, is a global
signalling compound that reduces the rate of transcription of ribosomal RNA and tRNA genes and also
activates or inhibits the transcription of many other
genes94. In E. coli, two proteins, RelA and SpoT, contribute to pppGpp synthesis; SpoT also has pppGpp
hydrolase activity94. However, in B. subtilis, and some
other Gram-positive bacteria, a single protein, RelA,
has both RelA and SpoT activities95. Induction of the
stringent response in B. subtilis leads to a dramatic
decrease in the expression of genes that are involved
in RNA and protein synthesis, and an increase in the
expression of the ilvB operon, gabP, ureABC, appDAB,
several rap-phr operons and spo0A96, all of which are
members of the CodY regulon31,36. Interestingly, relA
mutants of B. subtilis are also defective in sporulation97
and the development of genetic competence38. Part of
the explanation for this phenotype could be the hyperaccumulation of GTP. Three decades ago, Freese and
co-workers98 showed that for B. subtilis sporulation to
be initiated the intracellular pool of GTP must decrease
by a factor of 3–5. More recently, Ochi and colleagues38
showed that a B. subtilis relA mutant is incapable of
mediating the decrease in the intracellular GTP pool
that normally occurs at the transition from exponential
growth to stationary phase; an artificial reduction in the
GTP pool or the introduction of a codY mutation into
the relA mutant strain restores sporulation and competence. As a crucial factor in sporulation and at least two
key competence genes are repressed by CodY37,40, the
simplest explanation for these results is that, in a relA
mutant, the GTP pool remains high, even under the
conditions of amino-acid limitation that would normally lead to competence and sporulation, so keeping
CodY in its active state and competence and sporulation
repressed. The effects of ppGpp synthesis on the GTP
pool might also explain the role of ppGpp in B. subtilis
ribosomal-RNA synthesis99. In addition, relA mutants
are partial BCAA auxotrophs95. The hyperactivity of
CodY in a relA mutant might explain this auxotrophy
by causing the CodY-dependent repression of BCAA
biosynthesis to be unusually strong (FIG. 5).
CcpA and CodY in other Gram-positive bacteria
CcpA. CcpA is found in virtually all low-G+C Grampositive bacteria and has a similar role to B. subtilis
CcpA in controlling metabolism in many of these species. Typically, a ccpA mutant grows slowly in glucose
medium, overexpresses utilization genes for alternative
carbon sources and is defective in the expression of
lactate dehydrogenase and other carbon-overflow pathways100–106. The non-pathogenic bacterium Lactococcus
lactis normally carries out homolactic fermentation; a
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REVIEWS
Two-component regulatory
system
A system that responds to an
environmental stimulus and
regulates gene expression
accordingly. Composed of a
histidine-kinase sensor, usually
situated in the outer
membrane, that
phosphorylates a response
regulator in the cytoplasm,
which, in turn, activates
transcription from selected
promoters.
ccpA mutant underexpresses the lactate dehydrogenase gene and produces a mixed acid fermentation
pattern instead103. Purified Streptococcus bovis CcpA
binds in vitro to a cre site, but only in the presence of
the serine-phosphorylated form of HPr102. The control
of CcpA activity by serine-phosphorylated HPr is also
highly conserved in Gram-positive bacteria.
In at least two strains of S. pneumoniae, a ccpA (regM)
mutant is attenuated for virulence in three models of
infection — bacteraemia100, nasopharyngeal carriage101
and pneumonia101. In the D39 strain, disruption of
ccpA results in a reduction in the synthesis of capsular
polysaccharide100. However, a direct mechanistic connection between CcpA activity and virulence has not
yet been discovered.
The CcpA protein of L. monocytogenes controls carbon use, but is not the mediator of the carbon catabolite
repression of most virulence-gene expression104,105. One
exception might be the internalin genes (inlA and inlB),
which are transcribed from several promoters, one
of which appears to be under the control of CcpA105. The
major regulator of L. monocytogenes virulence is PrfA,
a positive regulator of virtually all virulence genes107.
Although CcpA is not a factor in the regulation of PrfA
synthesis or activity104, the common components of the
phosphotransferase system — HPr and enzyme I — do
seem to have an important role in modulating PrfA
activity108, perhaps owing to their ability to determine
the phosphorylation state of phosphotransferasesystem permeases and other transporters 105,108. All
PrfA-dependent genes are overexpressed in a mutant
that is defective in HPr kinase105.
In Staphylococcus aureus, the role of CcpA in metabolism and virulence-gene expression is only beginning to
be explored. However, considerable work in the related
bacterium Staphylococcus xylosus has established that
CcpA is the primary regulator of sugar use and is activated by the serine-phosphorylated form of HPr109. In
an S. aureus ccpA mutant, growth in glucose medium
is slightly impaired and lactate excretion is delayed106.
The mutant expresses lower levels of the virulenceregulating RNA molecule RNAIII, but higher levels of
the virulence factors α‑toxin, protein A and serotype-5
capsule106. Canonical cre sites can be discerned upstream
of the α‑toxin and protein-A coding regions, but not near
the agr (RNAIII-encoding) or capsule genes106. Given the
complex, overlapping regulatory systems that control
S. aureus virulence, it is not surprising that some of these
effects are small; it might prove difficult to determine
the contribution of CcpA to the regulation of many of the
virulence genes in this species.
CcpA has a complex role in regulating the synthesis of Clostridium perfringens enterotoxin. On the one
hand, a ccpA mutant is derepressed for enterotoxin
gene expression during exponential growth phase;
on the other hand, the mutant is highly defective in
sporulation, which is normally required for the induction of enterotoxin expression110. In addition, CcpA
is needed for the glucose-dependent repression of
capsule synthesis and for the high-level expression
of the collagenase gene110.
924 | december 2007 | volume 5
CodY. CodY is also a highly conserved protein in lowG+C Gram-positive bacteria. Global analysis — predominantly using transcription microarrays — has revealed
that CodY regulons in a wide range of Gram-positive
bacteria have much in common. Typically, CodY represses
the synthesis of the BCAAs, histidine and arginine
and transporters for amino acids, peptides and
sugars, and activates the transcription of carbon-overflow
pathways and guanine nucleotide synthesis36,43,111,112. In
L. lactis, a staple of the dairy industry, CodY represses
the synthesis of amino acids, peptide transporters,
peptidases and amino-acid catabolism enzymes42,43,113–115
in response to BCAAs, but not to GTP116,117. This selective
response to BCAAs seems to be unique to the CodY proteins from the Streptococcus–Lactococcus–Enterococcus
family of bacteria.
In the past few years, a number of studies have sought
to link the activity of CodY to the control of virulence
in pathogenic species. In the first such study, Malke,
Ferretti and colleagues112 showed that a large number of
Streptococcus pyogenes genes are regulated, directly or
indirectly, by CodY. Some of these genes, such as fasX
and mga, encode positive regulators of virulence and
are themselves positively regulated by CodY112,118. The
genes that depend on FasX and Mga for their expression
are therefore part of the CodY regulon. Others, such as
the CovRS two-component regulatory system, are negative regulators of virulence genes and are repressed by
CodY112,118. It is unclear which of the virulence-associated
genes are direct targets of CodY, although it is intriguing
that the gene for a magnesium transporter is preceded
by a CodY box, as CovS, the sensor protein of the CovRS
two-component system, is activated by magnesium118.
Clostridium difficile, the primary causative agent
of antibiotic-associated diarrhoea, secretes two toxins
that are the primary virulence factors. Expression of the
toxin genes depends on an alternative RNA-polymerase
sigma factor, TcdR119, the synthesis of which, as well
as the synthesis of the toxin proteins, is restricted to
stationary phase if cells are grown in rich medium120–122.
The tcdR gene is strongly repressed by CodY, which
links virulence-factor synthesis to conditions of nutrient
limitation123. C. difficile CodY binds tightly to the tcdR
promoter region in the presence of GTP and BCAAs.
In two clinical isolates of S. aureus, a large number
of virulence genes, including regulatory genes and genes
that encode toxins, proteases, capsule biosynthesis and
biofilm matrix polysaccharide (polysaccharide intercellular antigen), have proved to be derepressed in a codY
mutant (C. Majerczyk et al., unpublished observations).
Surprisingly, in a third isolate, the absence of CodY led
to the failure to produce a biofilm124.
More complex roles for CodY have been suggested
by recent studies that used L. monocytogenes and
Bacillus anthracis. In a mouse model, the growth of
L. monocytogenes biofilms and its virulence are both
dependent on the RelA protein125. Adherence induces
ppGpp accumulation; in the absence of RelA, the
bacteria can adhere but cannot multiply125. The severe
virulence defect seems to be attributable, at least in
part, to the higher GTP pool in relA mutants, as the
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introduction of a codY mutation partially suppresses the
virulence defect of the relA single mutant111. However,
CodY might also be a positive regulator of virulence, as
a L. monocytogenes codY mutant is not as virulent as its
wild-type parent111 and a codY mutation augments the virulence defect that is caused by a ccpC mutation (M. Mittal
et al., unpublished observations). Perhaps some aspects of
pathogenesis (such as growth in a host) are activated by
CodY and others (such as toxin synthesis) are repressed.
In B. anthracis, CodY is essential for the synthesis of the
three major toxin proteins and for virulence in a mouse
model of anthrax (W. van Schaik et al., unpublished
observations). It remains to be seen whether this effect is
direct or indirect.
Conclusions
Why would a pathogenic bacterium evolve to have some
or all of its virulence genes under the control of CcpA
and CodY? The answer to this question depends on our
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Acknowledgements
The author thanks the anonymous reviewers for many helpful
suggestions. The unpublished work cited in this Review was
supported by research grants from the US Public Health
Service (GM042219, GM036718 and AI057637).
DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
citB | citC | citZ | mdh
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
entrez/query.fcgi?db=genomeprj
Bacillus anthracis | Bacillus subtilis | Clostridium difficile |
Clostridium perfringens | Escherichia coli | Lactococcus lactis |
Listeria monocytogenes | Staphylococcus aureus |
Staphylococcus xylosus | Streptococcus pneumoniae |
Streptococcus pyogenes
Entrez Protein: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=protein
CcpA | CcpC | CodY | Crh | GltC | HPr | pppGpp | RocG | RocR |
TnrA
FURTHER INFORMATION
Abraham L. Sonenshein’s homepage: http://www.tufts.edu/
sackler/microbiology/faculty/sonenshein/index.html
All links are active in the online pdf
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