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FEMS Microbiology Letters 209 (2002) 141^148
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MiniReview
Carbon catabolite repression in bacteria: choice of the carbon source
and autoregulatory limitation of sugar utilization
Reinhold Bru«ckner
b
a;
, Fritz Titgemeyer
b
a
Mikrobiologie, Universita«t Kaiserslautern, Paul-Ehrlich-Strasse 23, D-67663 Kaiserslautern, Germany
Lehrstuhl fu«r Mikrobiologie, Friedrich-Alexander-Universita«t Erlangen-Nu«rnberg, Staudtstrasse 5, D-91058 Erlangen, Germany
Received 4 October 2001 ; received in revised form 12 February 2002; accepted 12 February 2002
First published online 12 March 2002
Abstract
Carbon catabolite repression (CCR) in bacteria is generally regarded as a regulatory mechanism to ensure sequential utilization of
carbohydrates. Selection of the carbon sources is mainly made at the level of carbohydrate-specific induction. Since virtually all
carbohydrate catabolic genes or operons are regulated by specific control proteins and require inducers for high level expression, direct
control of the activity of regulators or control of inducer formation is an efficient measure to keep them silent. By these mechanisms,
bacteria are able to establish a hierarchy of sugar utilization. In addition to the control of induction processes by CCR, bacteria have
developed global transcriptional regulation circuits, in which pleiotropic regulators are activated. These global control proteins, the
catabolite gene activator protein (CAP), also known as cAMP receptor protein, in Escherichia coli or the catabolite control protein
(CcpA) in Gram-positive bacteria with low GC content, act upon a large number of catabolic genes/operons. Since practically any carbon
source is able to trigger global transcriptional control, expression of sugar utilization genes is restricted even in the sole presence of their
cognate substrates. Consequently, CAP- or CcpA-dependent catabolite repression serves as an autoregulatory device to keep sugar
utilization at a certain level rather than to establish preferential utilization of certain carbon sources. Together with other autoregulatory
mechanisms that are not acting at the gene expression level, CCR helps bacteria to adjust sugar utilization to their metabolic capacities.
Therefore, catabolic/metabolic balance would perhaps better describe the physiological role of this regulatory network than the term
catabolite repression. 4 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Global transcriptional control ; Signal transduction; Metabolic balance ; Autoregulation ; Inducer exclusion
1. Introduction
The term carbon catabolite repression (CCR) is currently in use to describe the general phenomenon in microorganisms whereby the presence of a carbon source in the
medium can repress expression of certain genes and operons, whose gene products are often concerned with the
utilization of alternative carbon sources [1]. In the vast
majority of documented cases, the preferred carbon source
is glucose with the famous Escherichia coli glucose^lactose
diauxie as the classical example [2]. On the contrary, the
lactic acid bacterium Streptococcus thermophilus prefers
lactose over glucose [3], indicating that adaptation to special ecological niches may result in the choice of practi-
* Corresponding author. Tel. : +49 (631) 205 2199 ;
Fax : +49 (631) 205 3799.
E-mail address : [email protected] (R. Bru«ckner).
cally any carbohydrate as favored substrate. While the
¢nal outcome of CCR is uniform, reduced expression of
certain genes and operons, the mechanisms leading to repression may be quite diverse. The presence of a repressing
carbon source can result in lower concentrations of inducers speci¢c for alternate routes of catabolism, in altered activities of speci¢c regulators, or in the activation
of global control proteins, such as the catabolite gene activator protein (CAP) in enteric bacteria or the catabolite
control protein (CcpA) in low-GC Gram-positive bacteria.
Of these mechanisms, global regulatory circuits mediated
by CAP or CcpA were in the focus of interest, implicating
their outstanding importance. In a relatively recent controversy over the contribution of di¡erent CCR mechanisms to E. coli glucose^lactose diauxie, it has been demonstrated that inhibition of lactose permease and therefore
prevention of induction is the cause for lac operon repression in the presence of both sugars rather than CAP-mediated control [2]. This example from the best studied bac-
0378-1097 / 02 / $22.00 4 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 5 5 9 - 1
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terial species E. coli demonstrates that many aspects of
CCR remain to be uncovered, especially in less analyzed
bacteria.
In this review, we will discuss major mechanisms of
CCR and emphasize the importance of autoregulatory
processes in CCR to balance carbohydrate uptake and
metabolic capacities. Since a wealth of information is
available from enteric bacteria, especially E. coli, and
Gram-positive bacteria with low GC content, most of
the data will be from these groups of organisms.
2. Mechanisms preventing carbohydrate-speci¢c induction
The mode of carbohydrate uptake in bacteria may be
quite diverse depending on the proteins involved in transport. The most striking di¡erence in transport processes is
the entry of carbohydrates either in phosphorylated or
non-phosphorylated form, a distinction that is especially
important for CCR. Carbohydrate transport and concomitant phosphorylation is achieved by components of
the phosphoenolpyruvate (PEP)-dependent carbohydrate
phosphotransferase system (PTS) [6]. The system consists
of sugar-speci¢c PTS permeases, also referred to as enzymes II (EII), and two general PTS proteins, enzyme I
(EI) and histidine-containing protein (HPr), that participate in the phosphorylation of all PTS-transported carbohydrates. The speci¢c permeases are composed of up to
four protein domains (EIIA, B, C, D), at least one of
which is membrane-bound. These protein domains may
be detached or fused as a single polypeptide chain. E.
coli glucose permease consisting of the membrane protein
EIICBglc and the cytoplasmic EIIAglc is perhaps the best
known example for a composite PTS permease. The PTS
phosphoryl transfer chain starts with EI and PEP, proceeds via HPr, EIIA, and EIIB to the sugar, which is
transported by EIIC, and results in sugar phosphates
that may be metabolized immediately. On the contrary,
carbohydrates that are internalized in non-phosphorylated
form independently from the PTS need phosphorylation
by kinases prior to metabolism. We will in the following
refer to the proteins responsible for these transport processes as non-PTS permeases.
If more than one carbohydrate is present in the growth
medium, bacteria normally take up and utilize only one
carbon source at the same time and leave the other substrate in the medium for later use. Prevention of the substrate-speci¢c induction of catabolic genes has been identi¢ed as the major cause of preferential sugar utilization.
By processes like inducer exclusion, inducer expulsion [4],
or the control of the activity of regulators by phosphorylation [5], the presence of preferred substrates results in the
lack of expression of alternative pathways.
Inducer exclusion is a regulatory phenomenon whereby
a carbohydrate inhibits uptake of another carbon source.
In E. coli, inducer exclusion is mediated by the glucose-
speci¢c enzyme IIA (EIIAglc ) of the PTS (Table 1; Fig.
1A) [6]. When a PTS substrate, for example glucose, is
present, the phosphate group of PTS proteins is drained
to the incoming sugar. Consequently, EIIAglc exists predominantly in its unphosphorylated form. This form of
EIIAglc binds to non-PTS sugar permeases, that are speci¢c for lactose, maltose, melibiose, and ra⁄nose. As a
result, transport of these sugars is inhibited and formation
of inducers, e.g., allolactose for lac induction, is prevented.
Due to the lack of speci¢c intracellular inducers, the respective operons remain poorly expressed. Interestingly,
EIIAglc ^permease interactions are enhanced in the presence of the cognate substrates, ensuring economical use
of the regulatory protein [6]. In addition, EIIAglc is able
to bind to glycerol kinase preventing the production of
glycerol 3-phosphate, the inducer of the glycerol catabolic
genes. Since glycerol enters E. coli by facilitated di¡usion
and glycerol 3-phosphate is not a substrate for the glycerol
facilitator, phosphorylation is required for entrapping
glycerol inside the cell. Consequently, repression of glycerol kinase activity by EIIAglc also reduces glycerol uptake. A recent genome-wide survey of E. coli proteins to
detect a consensus sequence for EIIAglc binding revealed
several novel candidates [7]. Therefore, EIIAglc may carry
out more regulatory interactions than recognized so far.
In low-GC Gram-positive bacteria, another PTS component, the phosphocarrier protein HPr, is involved in
inducer exclusion (Table 1; Fig. 1B). A form of HPr (PSer-HPr), phosphorylated at serine at position 46 by HPr
kinase [8,9], was shown in vivo to be essential for repression of non-PTS sugar transport in Lactobacillus casei [10]
and Lactococcus lactis [11]. Although these data clearly
implicate P-Ser-HPr in inducer exclusion, direct biochemical evidence for the proposed inhibition of permeases is
still missing. Another form of inducer control involving
glycerol kinase has recently been uncovered in Bacillus
subtilis [12]. Glycerol kinase activity is stimulated upon
PEP-dependent phosphorylation by phospho-histidine
HPr (P-His-HPr) and lack of this phosphorylation, for
instance in the presence of PTS substrates, results in prevention of glycerol uptake and repression of the glycerol
utilization operon.
Besides inducer exclusion, inducer expulsion is believed
to contribute to CCR in some Gram-positive bacteria [4].
The phenomenon was detected during sugar uptake studies using non-metabolizable carbohydrates. Addition of
rapidly metabolizable carbon sources led to a fast expulsion of the accumulated sugars. Genetic evidence in L.
lactis and in L. casei showed that P-Ser-HPr does not
mediate inducer expulsion in these organisms [10,11].
Therefore, the mechanism of inducer expulsion remains
to be elucidated and its physiological relevance for CCR
remains elusive.
Another mode to control carbohydrate-speci¢c induction, whose physiological signi¢cance is documented, depends on a specialized duplicated protein domain, the PTS
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Table 1
Proteins involved in carbon catabolite repression
Proteina
Function
Target(s)
Process
Organismsb
CAP
CcpA
Cya
HPrK/P
EIIAglc
activation
repression/activation
production of cAMP
phosphorylation of HPr at serine-46
allosteric inhibition
global transcriptional control
global transcriptional control
global signal transduction
global signal transduction
inducer exclusion
E
lGþ
E
lGþ
E
P-EIIAglc
P-Ser-HPrc
stimulation
transcriptional corepressor
CAP sites
CcpA sites (cre)
^
HPr
sugar permeases, glycerol
kinase
Cya
CcpA, sugar permeases
E
lGþ
P-His-HPr
control of protein activities
global signal transduction
global transcriptional control and inducer
exclusion
speci¢c transcriptional control
E, lGþ
GlkA
glucose phosphorylation, interaction with
regulator(s) ?
global signal transduction
hGþ
PRD regulators, glycerol
kinase
unknown
CAP, catabolite activator protein ; CcpA, carbon catabolite protein ; Cya, adenylate cyclase; HPrK/P, HPr kinase/phosphatase ; EIIAglc , glucose-speci¢c
enzyme IIA; HPr, histidine-containing phosphocarrier protein ; GlkA, glucose kinase ; cre, catabolite responsive element; PRD, PTS regulation domain ;
E, enteric bacteria ; lGþ and hGþ , Gram-positive bacteria with low and high GC content, respectively.
a
Only proteins that act globally and whose contribution to CCR has been de¢ned in vivo are included in the list. The phosphoryl group of EIIAglc and
P-His-HPr originate from PEP via the PTS phosphotransfer chain EI-HPr-EIIA. In the presence of PTS sugars these proteins exist predominantly in
non-phosphorylated form. In the absence of PTS sugar transport, they remain phosphorylated. On the contrary, P-Ser-HPr is produced at the expense
of ATP by HPrK/P in the presence of glycolytic substrates, regardless whether these were transported by the PTS or by non-PTS permeases.
b
For clarity, individual species within the group of Gram-positive bacteria are not listed. Inducer control mechanisms may vary, but CcpA and HPrK/P
appear to be uniformly present.
c
In B. subtilis and, most likely, in other bacilli, the HPr homologue Crh participates as P-Ser-Crh in CcpA corepression [48].
regulation domain (PRD), which is found in antiterminators and activators [5]. PRD proteins mediate sugar-speci¢c induction mostly of PTS carbohydrate catabolic operons. The RNA- or DNA-binding activity of their Nterminal e¡ector domains is controlled through two
PRDs by multiple PTS-mediated phosphorylations in a
complex manner. The cognate PTS permeases, acting negatively upon the regulators, are needed for sugar-speci¢c
induction. Most but not all PRD-containing regulators
require P-His-HPr-mediated phosphorylation for activity.
This positive regulation provides the opportunity to control the induction process by CCR. If phosphorylation of
PRD is prevented, for example by the transport of another
PTS substrate, induction does not occur. Therefore, the
¢nal result of PRD-mediated regulation is the same as
for inducer exclusion, prevention of sugar-speci¢c induction. In addition to the mechanisms of induction prevention presented above, competition among PTS permeases
for phosphoryl transfer from P-His-HPr would be another
way to govern hierarchical sugar utilization [6].
A specialized PTS-mediated regulation through a PTS
EIIA-like protein domain that is fused to a non-PTS lactose transporter is found in S. thermophilus [13]. Phosphorylation of this PTS domain by P-His-HPr is not involved in establishing a hierarchy of sugar utilization, but
it modulates lactose transport in an autoregulatory control
circuit [13].
3. Transcriptional control by global regulators
One of the best studied consequences of the availability
of carbohydrates is the activation of global transcriptional
control systems, which are mechanistically di¡erent in enteric bacteria and low-GC Gram-positive bacteria. While
in enteric bacteria an activation mechanism that uses the
CAP protein is realized [14], and CCR is actually the
result of diminished activation of promoters for catabolic
genes or operons, a negative regulation mechanism by
CcpA is operative in low-GC Gram-positive bacteria [15].
In E. coli, CAP activates transcription at more than 100
promoters and is in some cases also involved in repression.
The activation process is well understood in structural and
mechanistic detail, at least at simple CAP-dependent promoters [14]. CAP needs the allosteric e¡ector cAMP in
order to bind e⁄ciently to DNA. Global regulation by
CAP responds to the intracellular amount of cAMP
(Fig. 1A) and by means of autoregulation to CAP levels
[16]. The intracellular cAMP level, in turn, is adjusted by
adenylate cyclase, whose activity depends on the phosphorylated form of EIIAglc (P-EIIAglc ) [6]. Besides controlling carbohydrate catabolic genes, CAP is directly involved in the modulation of a large number of other
cellular processes. It exerts also indirect control by in£uencing expression of global regulators such as FIS [17], or
by contributing to c factor selectivity [18]. Therefore, the
role of CAP goes far beyond regulation of sugar utilization. On the other hand, sugar utilization is controlled by
further pleiotropic regulators. Several glycolytic, gluconeogenic and glucose-related genes are controlled by Cra (formerly FruR) and Mlc [19,20]. Control of mRNA stability
of some of these genes by CsrA adds another level of
regulatory complexity [21]. A striking example of the multi-level regulation is the central ptsHIcrr operon, encoding
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HPr, EI, and EIIAglc , which is controlled by CAP, Cra,
and Mlc.
In low-GC Gram-positive bacteria, CcpA is of central
importance for global transcription control in CCR (Table
1; Fig. 1B) [15]. Genome-wide analysis of CCR in B. subtilis estimated about 300 genes to be regulated by CcpA
[22]. CcpA functions mainly as a repressor of transcription, but activation is also documented. The regulator requires the co-repressor P-Ser-HPr to bind e⁄ciently to its
operator sequence cre (catabolite responsive element).
Consequently, CcpA activity responds to the level of
P-Ser-HPr. In some organisms, the ccpA gene is subject
to autoregulation [23,24]. Compared to the profound
knowledge on CAP activity, relatively few mechanistic details of CcpA action have been worked out. It appears that
further work is needed to fully understand this important
regulator. Two other proteins, ccpB and ccpC, participate
in glucose repression in B. subtilis [25,26]. Understanding
their contributions to global regulation and their interplay
with CcpA will be required to get a comprehensive view
on CCR in this organism.
4. Signal transduction leading to CCR
Fig. 1. Models of carbon catabolite repression. The ¢gure shows regulatory circuits in enteric and low-GC Gram-positive bacteria. The schemes
highlight the equivalent roles of two PTS proteins, EIIAglc in enteric
bacteria and HPr in Gram-positive bacteria. Their state of phosphorylation and di¡erent phosphorylated forms trigger and coordinate the major responses of carbon regulation. Solid lines indicate catalytic interactions/activities and carbon £ow, while dashed lines show information
pathways. A: CCR in enteric bacteria. Incoming carbon sources generate speci¢c signals by which the activity of speci¢c regulators is modulated. Concomitantly, metabolism of the internalized carbon sources
determines the ratio of phosphoenolpyruvate to pyruvate, which
in£uences, via EI and HPr, the phosphorylation state of the major signal distribution factor EIIAglc . Non-phosphorylated EIIAglc exerts inducer exclusion of non-PTS permeases by allosteric regulation (inhibition), while phosphorylated EIIAglc stimulates adenylate cyclase, thereby
triggering global transcriptional control by CAP. B: CCR in low-GC
Gram-positive bacteria. Besides carbohydrate-speci¢c induction processes, incoming carbon sources generate glycolytic intermediates that stimulate HPrK/P leading to the phosphorylation of HPr at serine-46. An
elevated amount of P-Ser-HPr has three consequences: (i) global
transcriptional control by CcpA, (ii) inducer exclusion of non-PTS permeases, and (iii) feedback inhibition of EI-dependent phosphorylation of
HPr resulting in reduced PTS transport activity and diminished activity
of PRD-containing activators.
From the descriptions of major mechanisms of CCR it
is apparent that the phosphorylation state of two PTS
components, EIIAglc and HPr, determines regulatory responses in E. coli as well as in low-GC Gram-positive
bacteria (Fig. 1). However, there is an important di¡erence
concerning mutations in the general PTS for both types of
regulations. Since EIIAglc /P-EIIAglc is part of the regular
PEP-PTS phosphoryl chain, defects in either of the two
general PTS components will abolish CCR in E. coli. On
the other hand, the regulatory P-Ser-HPr is produced by
HPr kinase in an ATP-dependent manner and PTS phosphoryl transfer is not required to trigger P-Ser-HPr-dependent CCR. P-His-HPr-dependent processes, however, require a functional PTS. Consequently, only mutations in
ptsH, the gene encoding HPr, are able to block CCR completely in low-GC Gram-positive bacteria. EI de¢ciency
leaves P-Ser-HPr-dependent regulation intact, provided a
sugar is able to enter the cell by non-PTS transport [27].
How the ratio of P-EIIAglc to EIIAglc could be adjusted
according to sugar availability is immediately apparent
considering PTS-mediated glucose transport. If glucose is
transported, the phosphoryl group of P-EIIAglc is delivered to incoming glucose via EIICBglc , the major PTS
glucose transporter. Dephosphorylation of EIIAglc would
be the consequence. Similarly, any PTS sugar could indirectly e¡ect the phosphorylation state of EIIAglc by draining phosphate groups to the incoming sugar. This scenario, however, provides no ready explanation for strong
CCR by non-PTS carbohydrates such as glucose 6-phosphate. Surprisingly, glucose 6-phosphate and several other
non-PTS substrates caused dephosphorylation of EIIAglc
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to various extents [28]. To explain these unexpected results, the authors proposed the PEP to pyruvate ratio to
be involved in the control of EIIAglc phosphorylation
state. The PEP/pyruvate ratio in turn is in£uenced by metabolism of carbohydrates.
Another surprise came, when cAMP and P-EIIAglc levels were compared [28]. As P-EIIAglc is an e¡ector of adenylate cyclase, dephosphorylation of EIIAglc should result
in low cAMP production. However, P-EIIAglc levels and
the amount of cAMP did not correlate. These results are
consistent with previous studies questioning this simpli¢ed
regulatory model [29]. Despite long lasting e¡orts to clarify signal transduction leading to CAP-dependent CCR in
E. coli, it appears that a major factor contributing to regulation of adenylate cyclase escaped detection.
Recently, the glycolytic pathway and its intermediates
were implicated in additional regulatory events [30,31].
The dominant mechanism of glycerol kinase repression
was found to be the allosteric inhibition by fructose 1,6bisphosphate (FBP) rather than inhibition by EIIAglc .
Blocking glycolysis resulted in a markedly reduced level
of the main glucose transporter EIICBglc caused by rapid
decay of the EIICBglc encoding ptsG mRNA. Hence, glycolysis and glycolytic intermediates could be more important for signal transduction in E. coli than anticipated.
Glycolytic intermediates such as FBP are of central importance for signal transduction in low-GC Gram-positive
bacteria [9]. A protein kinase, HPr kinase, is activated by
elevated levels of FBP to carry out ATP-dependent phosphorylation of HPr at the serine at position 46. High concentrations of inorganic phosphate (Pi ) stimulate the reverse reaction, dephosphorylation of Ser-46, catalyzed by
the same enzyme [32]. Therefore, the enzyme is actually
bifunctional, an HPr kinase/phosphatase (HPrK/P). How
kinase/phosphatase activities are balanced is not yet
known. However, recent determination of the X-ray structure of an active fragment of L. casei HPrK/P suggested
direct competition of Pi and ATP for binding to HPrK/P
[33]. It has been shown in L. lactis that cells taking up
rapid metabolizable sugars have a high level of FBP and a
low amount of Pi [34]. This situation would therefore favor kinase activity. In addition ATP levels may be involved in regulation [32]. Due to the involvement of glycolysis in the stimulation of HPrK/P kinase activity, it is
immediately apparent that PTS and non-PTS sugars are
able to cause CCR in this group of organisms (Fig. 1B).
Another pathway of signal transduction leading to CCR
appears to be operative in Streptomyces coelicolor, a
Gram-positive bacterium with high GC content, that lacks
the HPrK/P-dependent regulatory system. A central function has been attributed to glucose kinase GlkA, that is
also essential for glucose metabolism [35,36]. A global regulatory role for GlkA, distinct from its catalytic function,
was proposed, since complementation of a glkA mutant
with a heterologous glucose kinase or activation of a cryptic glucose kinase could restore glucose fermentation but
145
not CCR. Moreover, it was reported that CCR exerted by
carbon sources that are not metabolized via GlkA was
abolished in the glkA mutant. The ¢nding that GlkA belongs to the ROK protein family [37], which consists of
transcriptional regulators, open reading frames of unknown function and sugar kinases, supported further the
idea of a general regulatory role for GlkA. It had been
suggested that kinases of this family would exert their
regulatory function interacting with transcription factors.
A search for GlkA-binding factors and attempts to demonstrate a regulatory modi¢cation of GlkA did not give
conclusive results [38]. It should be noted that the PTS
components HPr and an EIIAglc -like protein are present
in S. coelicolor [39]. The EIIAglc -like protein could complement EIIAglc of E. coli in glucose uptake and inducer
exclusion demonstrating its potential in CCR (Titgemeyer,
unpublished). Mutation in ptsH, however, showed no effect on glucose repression [40]. Therefore, the true role of
the PTS and GlkA and their interplay in CCR of streptomycetes as well as the nature of the central transcriptional
regulator remain to be elucidated.
A special regulatory role for a glucose kinase of the
ROK protein family was also proposed in the low-GC
Gram-positive bacteria Staphylococcus xylosus and Bacillus megaterium [41,42]. Further analysis of GlkA in S.
xylosus showed that the enzyme contributes to CCR by
its catalytic function as part of a non-PTS glucose utilization system [43,44].
5. Autoregulatory aspects of CCR
The concept of preferred and, therefore, repressing carbon sources seems to implicate that other carbohydrates,
whose utilization genes are repressed in the presence of the
favored substrate, would not be able to trigger CCR.
However, any carbohydrate entering glycolysis in Grampositive bacteria will inevitably activate HPrK/P and turn
on CcpA-dependent regulation and inducer exclusion. In
E. coli, carbohydrates provoke an equivalent response by
modulating cAMP and P-EIIAglc levels. Therefore, CCR
results in autoregulatory restriction of sugar utilization, an
aspect which has received relatively little attention.
The series of autoregulatory events in CCR of E. coli is
best illustrated discussing the re¢ned model of lac regulation. As already mentioned, repression of the lac operon in
the presence of glucose is due to inducer exclusion and
dependent on the lac repressor [2]. When E. coli cells
grow on lactose, one could assume that lac operon expression would be at its maximum. Two autoregulatory circuits, however, restrict lac expression under these conditions. First, in contrast to a widespread belief, the level of
cAMP in lactose-consuming E. coli remains even slightly
lower than in glucose-grown cells, leading to an only moderate activation of the lac promoter by CAP [2]. Secondly,
since about half of EIIAglc exists in non-phosphorylated
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form in lactose-consuming cells, inducer exclusion is operative and reduced lactose permease activity is the consequence [45]. Taken together, the apparent induction of the
lac operon by lactose is the result of enhanced transcription due to the relief from repression, which is partially
counteracted by moderate CAP activation and restricted
inducer uptake. The ¢nal lac expression level is de¢ned by
global autoregulatory CCR.
In low-GC Gram-positive bacteria, no complete view on
autoregulatory aspects of one operon is available, but
some cases of autoregulatory CCR are documented
[12,13]. Since CcpA-dependent regulation is triggered by
glycolytic intermediates, all catabolic genes for glycolytic
sugars harboring cre for CcpA regulation are subject to
CcpA-mediated autoregulation. In addition, inducer control may be exerted, provided the cognate sugar permease
is sensitive to inhibition by P-Ser-HPr. A third autoregulatory consequence of HPrK/P-mediated HPr phosphorylation has recently been documented [46]. When a mutant
allele of the L. casei hprK/P gene encoding an HPrK/P
enzyme with strongly reduced phosphatase was expressed
in B. subtilis, growth on PTS sugars was abolished. The
elevated level of P-Ser-HPr in the mutant strain apparently
blocked PTS phosphate transfer. Since P-Ser-HPr was
found to be a poor substrate for in vitro PEP-dependent
phosphorylation by EI, this inhibition was anticipated, but
in vivo evidence was lacking. These results obtained with
mutant HPrK/P strongly suggest that phosphorylation of
HPr by HPrK/P reduces PTS phosphotransfer also in the
wild-type situation, thereby slowing down PTS-dependent
sugar transport. In addition, regulatory phosphorylations
by P-His-HPr enhancing the activity of glycerol kinase and
PRD-containing regulators are reduced.
6. Importance of autoregulatory restriction of sugar
utilization
To assess the signi¢cance of autoregulation in CCR, it is
again instructive to look at certain mutants. Inactivation
of the lactose repressor abolishes speci¢c regulation as well
as glucose^lactose diauxie, but the cells grow well in the
presence of lactose [2]. In a mutant strain with a lactose
permease that is insensitive to EIIAglc -mediated inhibition,
lactose became inhibitory for growth, when lac gene expression was additionally stimulated by exogenous cAMP
[45]. Thus, loss of speci¢c lac regulation is easily tolerated
by E. coli, but the absence of CCR is detrimental. Likewise, growth of an HPrK/P-de¢cient strain of S. xylosus
was inhibited by glucose in a concentration-dependent
manner [47]. In addition, the mutant strain produced elevated levels of methylglyoxal, indicating unbalanced metabolism. The amount of methylglyoxal, however, was not
high enough to explain the inhibiting e¡ect of glucose on
growth [47]. Thus, the reason for the severe growth retardation of S. xylosus is not clear at the moment, but accu-
mulation of toxic metabolites would be a conceivable consequence of unrestricted sugar uptake. Under the same
conditions, growth of a CcpA-de¢cient S. xylosus mutant
was only slightly impaired [23]. Therefore, loss of a regulatory mechanism other than CcpA control must be responsible for glucose sensitivity in the S. xylosus HPrK/P
mutant strain. Further analysis of this phenomenon indicated that the HPrK/P-mediated regulatory events described above are not involved [27].
Interestingly, mycoplasmas, cell wall-less organisms related to low-GC Gram-positive bacteria, retained the gene
encoding HPrK/P in their small genomes [9], but have no
ccpA gene. This observation may suggest that a common
HPrK/P-dependent regulation exists in low-GC Grampositive bacteria, whose nature has not yet been recognized. In conclusion, autoregulatory CCR seems to be
important to protect cells from adverse e¡ects caused by
the uptake of excess carbohydrate. It will be interesting to
analyze further examples of this aspect of CCR.
7. Conclusions
The complex regulatory network responsible for CCR
serves bacteria to achieve two distinct goals: (i) to utilize
one carbon source preferentially from a mixture in the
growth medium, and (ii) to limit carbohydrate uptake
and utilization according to the cell’s metabolic capacities.
In both cases the same control mechanisms are operative,
but the strength of the regulatory response di¡ers greatly.
The choice of the carbohydrate is made at the level of
sugar-speci¢c induction. Repression of alternate carbohydrate utilization genes ensures that only a minimal set of
genes is expressed to obtain a certain amount of a carbon
source. Thus, hierarchical sugar utilization is meant to use
carbon and energy sources economically. If only one carbon source is available, CCR is not absent, but is the
inevitable consequence of carbohydrate metabolism acting
as a built-in autoregulatory device to limit carbohydrate
consumption. It is apparent that this form of regulation
should not result in complete repression, otherwise carbon
supply would stop. Considering the growth defects of mutants altered in CCR, especially the glucose sensitivity of
an S. xylosus HPrK/P-de¢cient strain, autoregulatory
CCR appears to be devoted to balance carbohydrate uptake and catabolic/metabolic capacities of the cell. Therefore, autoregulatory limitation of sugar consumption by
CCR is indispensable when carbon sources are plentiful
and by far more important for bacteria than the ability
to utilize certain substrates preferentially.
Acknowledgements
We would like to thank J. Deutscher for providing information prior to publication. Work in our laboratories
FEMSLE 10407 3-5-02
R. Bru«ckner, F. Titgemeyer / FEMS Microbiology Letters 209 (2002) 141^148
was supported by the Deutsche Forschungsgemeinschaft
(DFG) within the priority program, Molecular Analysis
of Regulatory Networks in Bacteria (to R.B. ; BR947/41), by an individual grant (to R.B. ; BR947/3), and to F.T.
by grant SFB473, Signal Mechanisms of C-Regulation.
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