Báo cáo Y học: The phosphotransferase system of Streptomyces coelicolor IIACrr exhibits properties that resemble transport and inducer exclusion function of enzyme IIAGlucose of Escherichia coli pptx

The phosphotransferase system of
Streptomyces coelicolor
IIA
Crr
exhibits properties that resemble transport and inducer exclusion function
of enzyme IIA
Glucose
of
Escherichia coli
Annette Kamionka
1
, Stephan Parche
1
, Harald Nothaft
1
,Jo¨ rg Siepelmeyer
2
, Knut Jahreis
2
and Fritz Titgemeyer
1
1
Friedrich-Alexander-Universita
¨
t Erlangen-Nu
¨
rnberg, Lehrstuhl fu
¨
r Mikrobiologie, Erlangen, Germany;
2
Universita

¨
t Osnabru
¨
ck,
Lehrstuhl fu
¨
r Genetik, Fachbereich Biologie/Chemie, Osnabru
¨
ck, Germany
We have investigated the crr gene of Streptomyces coelicolor
that encodes a homologue of enzyme IIA
Glucose
of Escheri-
chia coli, which, as a component of the phosphoenolpyru-
vate-dependent sugar phosphotransferase system (PTS)
plays a key role in carbon regulation by triggering glucose
transport, carbon catabolite repression, and inducer exclu-
sion. As in E. coli,thecrr gene of S. coelicolor is genetically
associated with the ptsI gene that encodes the general
phosphotransferase enzyme I. The gene product IIA
Crr
was
overproduced, purified, and polyclonal antibodies were
obtained. Western blot analysis revealed that IIA
Crr
is
expressed in vivo. The functionality of IIA
Crr
was demon-
strated by phosphoenolpyruvate-dependent phosphoryla-

tion via enzyme I and the histidine-containing phosphoryl
carrier protein HPr. Phosphorylation was abolished when
His72, which corresponds to the catalytic histidine of E. coli
IIA
Glucose
, was mutated. The capacity of IIA
Crr
to operate in
sugar transport was shown by complementation of the
E. coli glucose-PTS. The striking functional resemblance
between IIA
Crr
and IIA
Glucose
was further demonstrated by
its ability to confer inducer exclusion of maltose to E. coli.
A specific interaction of IIA
Crr
with the maltose permease
subunit MalK from Salmonella typhimurium was uncovered
by surface plasmon resonance. These data suggest that this
IIA
Glucose
-like protein may be involved in carbon meta-
bolism in S. coelicolor.
Keywords: inducer exclusion; protein phosphorylation;
protein–protein interaction; Streptomyces; surface plasmon
resonance.
Streptomycetes undergo global changes in gene expression
and enzyme activities in response to developmental stages,

secondary metabolite production (antibiotics), carbon util-
ization, and stress conditions [1–5]. The focus of our
research is the regulation of carbon source utilization
(C-regulation) and how this influences the other above-
mentioned processes.
Streptomyces coelicolor metabolizes a wide variety of
nutrients. Their utilization is subject to C-regulation, in
which glucose kinase appears to be of significant importance
[6,7]. However, the signal transduction pathways are poorly
understood. In many other bacteria, components of the
phosphoenolpyruvate-dependent sugar phosphotransferase
system (PTS) trigger C-regulation by mechanisms known as
carbon catabolite repression and inducer exclusion [8,9].
One key element in Escherichia coli is enzyme IIA
Glucose
(IIA
Glc
). IIA
Glc
becomes phosphorylated by the general
PTS proteins, which are histidine-containing phosphoryl
carrier protein (HPr) and enzyme I (EI). In turn, it
phosphorylates the sugar-specific PTS permeases that
catalyse the uptake of glucose, trehalose, and sucrose
[8,10,11]. Mutations in the respective gene crr exhibit a
pleiotropic catabolite repression resistant phenotype [12].
The underlying mechanisms are that unphosphorylated
IIA
Glc
inhibits a set of catabolic enzymes and sugar

permeases including the MalK subunit of the maltose
permease by protein–protein interaction (inducer exclu-
sion). At the same time the cellular cAMP level is low,
because dephosphorylated IIA
Glc
is unable to stimulate
adenylate cyclase. Under these conditions the cAMP-
dependent catabolite activator protein CAP, which serves
as a global activator of many catabolite-controlled genes,
remains in a switched off state [9]. IIA
Glc
further appears to
be involved in carbon catabolite repression exerted by non-
PTS substrates such as glucose 6-phosphate [13]. This could
be correlated with the variation of the phosphorylation state
of IIA
Glc
. Recently, another cellular function for IIA
Glc
has
been proposed that suggests that it may be involved in the
linkage between carbon metabolism and stress response
[14].
We have described that the PTS is operative in strepto-
mycetes [15,16]. Analysis of the S. coelicolor genome
revealed the presence of nine genes that may encode four
sugar-specific permeases, as well as the genes ptsH and ptsI
Correspondence to F. Titgemeyer, Friedrich-Alexander-Universita
¨
t

Erlangen-Nu
¨
rnberg, Lehrstuhl fu
¨
r Mikrobiologie, Staudtstrasse 5,
91058 Erlangen, Germany. Fax: + 49 91318528082,
Tel.: + 49 91318528095, E-mail:
Abbreviations: aMG, methyl a-glucoside; EI, enzyme I; HPr, histidine
containing phosphoryl carrier protein; II(ABC)
sugar
, enzyme II(ABC)
transporter protein; PTS, phosphoenolpyruvate-dependent sugar
phosphotransferase system; isopropyl, thio-b-
D
-galactose (IPTG);
Enzymes: enzyme I of the phosphoenolpyruvate-dependent sugar
phosphotransferase system (EC 2.7.3.9); enzyme II of the phos-
phoenolpyruvate-dependent sugar phosphotransferase system
(EC 2.7.1.69).
(Received 22 October 2001, revised 25 February 2002, accepted
4 March 2002)
Eur. J. Biochem. 269, 2143–2150 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02864.x
encoding HPr and EI [17]. Beside this, a crr-like gene was
found upstream of ptsI. In this communication we provide
evidence that this putative crr gene is expressed in vivo and
that it constitutes a functional equivalent of its homologue
in E. coli.
MATERIALS AND METHODS
Bacterial strains, growth conditions, and plasmid
construction

S. coelicolor A3(2) M145 (SCP1-, SCP2-, prototroph) was
used as wild-type strain [18]. E. coli DH5a was the host
strain for subcloning experiments [19]. E. coli FT1
DptsHIcrr Kan
r
(pLysS Cm
r
) was used to produce native
and hexa-histidine (His)-tagged S. coelicolor IIA
Crr
,His-
tagged S. coelicolor HPr, and His-tagged E. coli IIA
Glc
[16].
M15(pREP4, pAG3) was used to produce His-tagged
Bacillus subtilis EI [16,20]. The glucose-negative E. coli crr
mutant strain LM1 tonA galT nagE manAI kba
ts
rpsL xyl
metB thi his mglA-C argG crr was used for heterologous
complementation experiments [21].
S. coelicolor cultures were grown for 30–72 h with
vigorous shaking in complex medium (tryptic soy medium
without dextrose; Difco) at 37 °C or in mineral medium
supplemented with 0.1% casamino acids or 50 m
M
carbon
source at 28 °C [17]. E. coli cultures were grown in Luria–
Bertani medium at 37 °C.
Total DNA from S. coelicolor M145 was isolated as

described [16]. Cloning of the crr gene of S. coelicolor was
performed as follows. A DNA fragment of 475 bp compri-
sing crr was amplified by PCR with Pfu DNA polymerase
using S. coelicolor M145 wild-type chromosomal DNA as
template together with oligonucleotides engineered to
introduce the restriction sites NdeIandBamHI, respectively
(Crr1, 5¢-GGAGGTTTCATATGACCACCGTTTCTTC
CCCGC-3¢ and Crr2, 5¢-GACGGATCCGACGTCAC
TTCCAGAGG-3¢, restriction sites are in italic type). The
amplified DNA was digested with NdeIandBamHI and
cloned into plasmids pET15b and pET3c (Novagen)
resulting in crr expression plasmids pFT41 and pFT42,
respectively [22]. A two-step PCR mutagenesis procedure as
described by Landt et al. was used to change the codon for
His72 to an alanine codon [23]. Chromosomal DNA of
S. coelicolor M145 served as template together with oligo-
nucleotide Crr3 (5¢-GCGTGCTGACC
GCTCTCGG
GATCGAC-3¢; altered positions are underlined) and the
two flanking primers as described above. The NdeI–BamHI
digested PCR fragment was cloned into pET3c digested
with the same enzymes giving pFT44. The expression
plasmid pCRL13 for the production of a C-terminal His-
tagged IIA
Glc
of E. coli was derived by cloning of an NdeI–
HindIII fragment into plasmid pET23a(+) (Novagen) [22].
The crr fragment was generated by PCR (primers: Crr4,
5¢-GGAGAAGCATATGGGTTTGTTCG-3¢ and Crr5,
5¢-TTAAAGCTTGATGCGGATAACCGG-3¢;restriction

sites are in italic type). All PCR-based constructs were
confirmed by DNA sequencing. For constitutive expression
of crr,thecrr alleles from plasmids pFT41 and pFT42 were
prepared by sequential treatment with XbaI, T4 DNA
polymerase, and HindIII. The fragments were cloned
into the pSU2718 derivative pFT76 (K. Mahr, unpublished
data) that was sequentially treated with KpnI, T4
DNA-polymerase, and HindIII giving plasmids pFT111
(his-tagged IIA
Crr
) and pFT112 (IIA
Crr
)[24].
Protein overproduction and purification
Recombinant His-tagged HPr from S. coelicolor,His-
tagged IIA
Crr
from S. coelicolor, His-tagged IIA
Glc
from
E. coli, and His-tagged EI from B. subtilis were overpro-
duced and purified as described previously [16]. Purification
of native IIA
Crr
was achieved in a single step by anion
exchange chromatography (HQ-column; 1.6 mL bed vol-
ume; Poros) in buffer (20 m
M
Tris/HCl pH 7.5, 3 m
M

dithiothreitol) with a linear gradient of 0–500 m
M
NaCl. Protein concentrations were determined with the
Bio-Rad protein assay. Proteins were stored at )20 °Cor
)70 °C.
Phospho
enol
pyruvate-dependent phosphorylation
Preparation of [
32
P]phosphoenolpyruvate and protein phos-
phorylation assays were carried out as described previously
[16]. Radiolabelled proteins were detected by radiolumi-
nography on a phosphoimager (Fuji).
Enzyme assays
IIA
Crr
activity was assayed by complementation of the
glucose-specific PTS of E. coli measuring phosphoenolpyru-
vate-dependent phosphorylation of methyl [a-
14
C]glucoside
([
14
C]aMG; Amersham) in the presence of E. coli LM1 cell
extract [16]. The assay was carried out at 30 °Cinareaction
volume of 0.1 mL containing rate-limiting amounts of
IIA protein (50 pmoles), 55 lgproteinofLM1extract,and
a final concentration of 12 l
M

[
14
C]aMG (1.4 mCiÆmmol
)1
).
Phosphorylation of aMG was linear within the first minute.
The initial phosphorylation rates were calculated from
triplicates by subtraction of the blank value (LM1 extract
without IIA protein) of 140 ± 8 nmol aMG-PÆmin
)1
.
Transport assays
Cells of E. coli FT1 bearing either plasmid pET23a(+),
pCRL13(crr
+
E. coli), pET3c, or pFT42(crr
+
S. coelicol-
or) were grown at 37 °C in 100 mL Luria–Bertani
medium supplemented with 25 m
M
maltose. At
D
600
¼ 0.8, 50 mL of FT1(pCRL13) or FT1(pFT42)
culture were harvested. The remaining 50 mL of the
cultures were supplemented with 1 m
M
isopropyl thio-b-
D

-
galactose (IPTG) to induce crr expression. Incubation was
continued for 45 min. FT1(pET23a(+)) and FT1(pET3c)
were grown to a final D
600
¼ 1.0. All cells were
harvested and washed twice in chilled transport buffer
(50 m
M
Tris/HClpH7.5,50m
M
NaCl, 10 m
M
KCl).
Cells were resuspended in transport buffer, adjusted to
D
600
¼ 1.0 and kept on ice. For transport analysis an
aliquot of cells was preincubated for 5 min at 37 °C.
Uptake was initiated by addition of [
14
C]maltose to a final
concentration of 20 l
M
(5 mCiÆmmol
)1
). Samples of
0.5 mL were taken between 0.5 and 5 min, rapidly filtered
(1 mLÆs
)1

) through nitrocellulose filters (NC45), and
washed three times with 2 mL ice-cold 0.1
M
LiCl.
Radioactivity was determined by liquid scintillation
counting.
2144 A. Kamionka et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Western blot analysis
Western blot analyses were carried out as described by
Parche et al. [16]. Rabbit polyclonal antibodies were raised
against His-tagged IIA
Crr
of S. coelicolor (Eurogentec). A
dilution of 1 : 3000 yielded specific signals against 10 ng
His-tagged IIA
Crr
and against 5 lgofS. coelicolor cell
extract that corresponded to a molecular size of 19 kDa and
17 kDa, respectively.
Surface plasmon resonance analysis
Interactions of proteins were detected by surface plasmon
resonance analysis using a BIAcore X optical biosensor
(Biacore AB). Three micrograms of S. coelicolor His-
tagged IIA
Crr
(180 pmoles), of E. coli His-tagged IIA
Glc
(150 pmoles), and of E. coli His-tagged tetracycline repres-
sor TetR (125 pmoles; control for nonspecific binding) were
applied for the immobilization on an NTA sensor chip. The

efficiency for the coupling reaction was 1200 resonance units
(71 fmoles) for His-tagged IIA
Crr
, 450 resonance units
(24 fmoles) for his-tagged IIA
Glc
, and 1500 resonance units
(60 fmoles) for His-tagged TetR. For binding analysis, 5 lg
(120 pmoles) of purified MalK protein of Salmonella
typhimurium was dialysed twice against eluent buffer
(10 m
M
Hepes pH 7.4, 150 m
M
NaCl, 50 l
M
EDTA,
0.005% (v/v) polysorbate 20) and introduced at a flow rate
of 5 lLÆmin
)1
for 10 min. Three micrograms of purified
glucose kinase protein (91 pmoles) from S. coelicolor were
used as a control for unspecific ligand binding.
Computer analyses
The program
DNA STRIDER
TM
1.2 and the Lasergene
workstation software (
DNASTAR

) were used to process
DNA sequence data [25]. DNA databank and protein
databank searches were performed using the
BLAST
server of
the National Center for Biotechnology Information at the
National Institutes of Health Bethesda, MD, USA (http://
www.ncbi.nlm.nih.gov). Binary sequence comparisons were
computed with the
FASTA
software [26].
RESULTS
Identification of the
crr
gene
Figure 1A depicts a detailed genetic map of the crr ptsI
genes that we had identified previously by in silico analysis
[17]. Both genes encode putative PTS phosphotransferase
components that constitute homologues of E. coli enzyme
IIA
Glc
and EI, respectively. They are flanked upstream by
rrnC, which encodes ribosomal RNA and downstream by
an ORF of unknown function. The sequence of the crr
region contains two possible start codons. Analysis of the
Fig. 1. Genetic organization of the S. coelicolor crr gene and protein alignment. (A)Thegeneticarrangementofthecrr and ptsI gene is shown.
Arrows indicate transcriptional orientation of genes. Numbers of base pairs and length of proteins (aa, amino acids) are denoted below coding
regions. Numbers in square brackets show the lengths of intergenic regions in bp. (B) The IIA
Crr
sequence (above) is shown together with the

consensus sequence (below) derived from an alignment with 11 amino acid sequences of IIA
Crr
and IIA
Crr
-like proteins found in the current
databank. These are: S. coelicolor (AL353861), Bacillus stearothermophilus (P42015), Haemophilus influenzae (P45338), Corynebacterium glutam-
icum (Q45298), Bacillus subtilis (P39816), Escherichia coli (P08837), Klebsiella pneumonieae (P45604), S. mutans (P12655), Corynebacterium
ammoniagenes (3098512), Lactobacillus delbrueckii (P22733), S. thermophilus (P23926). Residues conserved in > 80% of all proteins are displayed
in upper case letters while residues conserved in 50–80% of all proteins are shown in lower case letters. Insertions/deletions are indicated by a dash.
Two conserved histidines are highlighted as putative phosphorylation (*) and active centre sites (!).
3
Ó FEBS 2002 IIACrr of Streptomyces coelicolor (Eur. J. Biochem. 269) 2145
codon usage, of potential ribosome binding sites, and
sequence alignments favoured an ORF of 449 bp beginning
at the second start codon that encodes a gene product with a
calculated mass of M
r
15 236. The protein sequence of
IIA
Crr
was aligned with 11 homologues (Fig. 1B). The
derived consensus sequence revealed two well-conserved
histidines in IIA
Crr
(57 and 72) that matched the active
centre residue histidine 75 and the experimentally proven
phosphorylation site histidine 90 of E. coli IIA
Glc
[27,28].
Overexpression and purification of

S. coelicolor
IIA
Crr
and IIA
Crr
(H72A)
To study the function of IIA
Crr
, we overexpressed three crr
alleles in E. coli encoding His-tagged IIA
Crr
,nativeIIA
Crr
,
and native IIA
Crr
(H72A). Therefore, plasmids pFT41,
pFT42, and pFT44 were transformed into the
1
DptsHIcrr
deletion mutant FT1(pLysS). Recombinant proteins were
produced and purified as outlined in Materials and meth-
ods. As depicted in Fig. 2, His-tagged IIA
Crr
, IIA
Crr
,and
IIA
Crr
(H72A) showed overexpression characteristics reveal-

ing prominent protein bands that migrated corresponding
to a size of 19 kDa for the His-tagged protein and 17 kDa
for the native protein (Fig. 2, lanes 2, 4, 6). His-tagged
IIA
Crr
was purified yielding  20 mg proteinÆL
)1
E. coli
culture, and IIA
Crr
and IIA
Crr
(H72A) were purified yielding
 9mgand 10 mg proteinÆL
)1
E. coli culture, respect-
ively (Fig. 2, lanes 3, 5, 7). His-tagged IIA
Crr
was used to
raise polyclonal antibodies.
Is the putative
crr
gene expressed in
S. coelicolor
?
To address this question, we monitored the presence of
IIA
Crr
protein in S. coelicolor. A Western blot analysis
showed IIA

Crr
-specific immunosignals in extracts of wild-
type mycelia (Fig. 3). IIA
Crr
protein was detectable under all
conditions tested and showed the highest levels in glucose-
grown mycelia, intermediate levels when fructose and
glycerol served as the carbon source, and lower levels in
mycelia grown on casamino acids or glutamate.
Is IIA
Crr
phosphorylated by HPr?
In vitro phosphorylation assays were performed to demon-
strate phosphoenolpyruvate-dependent phosphorylation of
IIA
Crr
in the presence of the general PTS phosphotrans-
ferases EI and HPr (Fig. 4). As shown in lane 1 of Fig. 4,
IIA
Crr
of S. coelicolor became phosphorylated upon incu-
bation with radiolabelled phosphoenolpyruvate, EI of
B. subtilis,andHProfS. coelicolor, while IIA
Crr
incubated
Fig. 2. Overexpression and purification of IIA
Crr
proteins. An
SDS/12% polyacrylamide gel stained with Coomassie brilliant blue is
shown. Lane 1, protein marker; lane 2, 30 lgcrudecellextractof

FT1(pFT41); lane 3, 8 lg purified His-tagged IIA
Crr
;lane4,30lgcell
extract of FT1(pFT42); lane 5, 5 lg purified IIA
Crr
;lane6,30lgcell
extract of FT1(pFT44); lane 7, 5 lg purified IIA
Crr
(H72A).
Fig. 3. Western Blot analysis. A Western Blot of an SDS/12% poly-
acrylamide gel shows the immunoreactive signal of IIA
Crr
.Ineachlane
10 lg protein of crude cell extract were subjected to gel electrophoresis.
Extracts were prepared from cells grown in mineral medium contain-
ing 0.1% casamino acids (CAA; lane 1), or in minimal medium con-
taining 50 m
M
of either fructose (lane 2), glucose (lane 3), glycerol (lane
4), or glutamate (lane 5). The figure is representative for several simi-
larly performed Western blot experiments.
Fig. 4. Phosphorylation of EI, HPr, and IIA
Crr
. The phospholumino-
gram of an SDS/12% polyacrylamide gel shows [
32
P]phos-
phoenolpyruvate-dependent phosphorylation of purified B. subtilis
His-tagged EI (16 pmol), S. coelicolor His-tagged HPr (67 pmol), and
of S. coelicolor IIA

Crr
and IIA
Crr
(H72A) (235 pmol). The following
combinations were examined: lane 1: EI, HPr, and IIA
Crr
;lane2:EI
and HPr; lane 3: EI and IIA
Crr
;lane4:HPr;lane5:EI,HPr,andIIA
Crr
boiled for 10 min prior to protein gel loading; lane 6: EI, HPr, and
IIA
Crr
(H72A); lane 7: EI and IIA
Crr
(H72A). The migration of proteins
is indicated. Note that phosphorylated EI is not or barely visible due to
the low protein amounts used.
2146 A. Kamionka et al. (Eur. J. Biochem. 269) Ó FEBS 2002
only with EI was not phosphorylated (lane 3). After boiling,
IIA
Crr
-phosphate became dephosphorylated indicating a
heat-labile aminoacyl phosphorylation of IIA
Crr
(lane 5) as
occurs by histidine phosphorylation. When histidine 72 was
replaced by an alanine, the resulting product IIA
Crr

(H72A)
could not be phosphorylated (lane 6).
Can IIA
Crr
function in sugar transport?
AfteritwasshownthatIIA
Crr
is phosphorylated by HPr,
we investigated whether it could interact with an enzyme II
permease. As no such enzyme II has been characterized so
far in S. coelicolor, we asked whether IIA
Crr
can replace
IIA
Glc
of E. coli with respect to glucose transport. We
constructed plasmids pFT111 (His-tagged IIA
Crr
)and
pFT112 (IIA
Crr
), in which the crr genes should be expressed
constitutively. When pFT111 and pFT112 were trans-
formed into the crr mutant LM1, fermentation of glucose
was restored as indicated by red glucose-fermenting colonies
on MacConkey agar supplemented with glucose (Fig. 5).
This showed that IIA
Crr
could interact with the E. coli
components of the glucose-specific PTS, HPr, and enzyme

IIBC
Glc
. The complementation was quantified by a glucose-
PTS assay, in which cell extracts of LM1 were combined
with rate-limiting amounts of purified His-tagged IIA
Crr
of
S. coelicolor and His-tagged IIA
Glc
of E. coli. The initial
phosphorylation rates of methyl a-glucoside were
152 ± 22 nmol aMG-PÆmin
)1
when IIA
Crr
was added
and 429 ± 39 nmol aMG-PÆmin
)1
when IIA
Glc
was added.
This indicated that under these conditions the heterologous
IIA
Crr
protein could compensate to about 35% the function
of E. coli IIA
Glc
.
Can IIA
Crr

function in inducer exclusion?
We then studied whether IIA
Crr
could replace its E. coli
counterpart in a C-regulatory capacity. The DptsHIcrr
deletion strain FT1 provided the possibility to monitor
inducer exclusion of maltose uptake. If crr is expressed in
such a genetic background, the product should not be
phosphorylated due to the lack of EI and HPr. Non-
phosphorylated IIA
Glc
will block the activity of the maltose-
specific ABC transport complex by interaction with MalK.
This effect is shown in Fig. 6A, where maltose uptake was
severely reduced when IIA
Glc
of E. coli was expressed in
strain FT1(pCRL13). The same result, although less
pronounced, was observed when IIA
Crr
was expressed in
strain FT1(pFT42) (Fig. 6B). It should be noted that both
strains overproduced similar amounts of IIA protein as
judged by comparison of protein band intensities of a
CBB-stained SDS/polyacrylamide gel (data not shown).
To corroborate this finding, we performed protein–
protein interaction analysis of IIA
Crr
with purified MalK
from Salmonella typhimurium, which exhibits 95% amino

Fig. 5. Complementation of an E. coli crr mutant. The figure shows a
MacConkey agar plate supplemented with 25 m
M
glucose. While
E. coli LM1 crr bearing plasmid pSU2718 (control) formed white
colonies (no glucose fermentation), LM1(pFT111) producing His-
tagged IIA
Crr
of S. coelicolor or LM1(pFT112) producing native
IIA
Crr
of S. coelicolor yielded red (dark grey) colonies indicating aci-
dification of the medium as a result of glucose fermentation.
Fig. 6. Time-course of maltose uptake. (A) Maltose uptake of E. coli
FT1 bearing either pET23a(+) (control, d), pCRL13 (E. coli His-
tagged IIA
Glc
) after induction with IPTG (.). (B) Maltose uptake of
E. coli FT1 bearing either pET3c (control, d)orpFT42(S. coelicolor
his-tagged IIA
Crr
) after induction with IPTG (.). Values were deter-
mined in triplicate and experiments were performed at least three
times. Standard deviations are displayed by error bars.
Ó FEBS 2002 IIACrr of Streptomyces coelicolor (Eur. J. Biochem. 269) 2147
acid identity with MalK of E. coli (Fig. 7). Therefore, his-
tagged IIA
Crr
was coupled to an NTA-sensor chip and a
solution of MalK was allowed to flow over the immobilized

protein. A binding signal of 400 resonance units was
detected, while no interaction was observed when MalK
solution was passed over immobilized His-tagged TetR
protein (negative control). Immobilized His-tagged IIA
Glc
yielded a response of 500 resonance units with the MalK
protein (positive control). Therefore, the observed reduction
of maltose uptake in E. coli by IIA
Crr
could be confirmed by
the demonstration of its interaction with the MalK subunit
of the maltose permease complex.
DISCUSSION
In this study, we report on the analysis of an S. coelicolor
ORF that encodes a protein, IIA
Crr
, with significant
similarity to enzyme IIA
Glc
of E. coli, a global-acting factor
of carbon metabolism. We provided evidence that the gene
is expressed in vivo and that IIA
Crr
is phosphorylated in vitro
by the general PTS phosphotransferases EI and HPr. IIA
Crr
could replace the functions of E. coli IIA
Glc
in glucose
transport and inducer exclusion. These findings suggest that

IIA
Crr
might be involved in carbohydrate transport and
C-regulation in S. coelicolor.
The crr gene of S. coelicolor shares the highest similarity
to a putative crr gene of S. griseus (accession AB030569),
which indicated that crr is also present in other strepto-
mycetes. crr genes are further found in Gram-negative
bacteria such as E. coli and Haemophilus influenzae,andin
some mycoplasma species [8,29]. In contrast, many other
microorganisms including the actinomycetes Corynebacte-
rium diphtheriae and Mycobacterium smegmatis,some
mycoplasmae, and low-GC Gram-positive bacteria such
as Bacillus subtilis possess no crr gene. These have crr
homologues as part of sugar-specific enzyme IIABC
permeases that solely appear to fulfil transport function
(F. Titgemeyer, unpublished data; [30–32]). For Gram-
negative species a multiple role of IIA
Glc
has been
documented and proposed [9,13,14,29].
The reported data demonstrate that IIA
Crr
could effi-
ciently cross-communicate with the proteins HPr, enzyme
IIBC
Glc
, and MalK from enteric bacteria. This striking
functional resemblance to E. coli IIA
Glc

and the observation
that S. coelicolor IIA
Crr
is present under all nutritional
conditions tested may provide good indications that IIA
Crr
functions in a similar way in S. coelicolor. The amount of
IIA
Crr
washigherwhenS. coelicolor was grown on
carbohydrates than it was when the organism was grown
on amino acids. Thus, further investigation should be
carried out to determine in more detail which carbon
sources induce expression of crr.
What are the targets of IIA
Crr
? We could demonstrate
that IIA
Crr
is phosphorylated by S. coelicolor HPr. There-
fore, it should act as a PTS phosphotransferase. With
respect to carbon source transport, it seems to be clear that
IIA
Crr
is not an enzyme IIA
Glc
as streptomycetes appear to
lack the glucose-specific PTS [15,17]. An analysis of the
S. coelicolor genome revealed two loci, malX2-nagE1-nagE2
and malX1

2
, that encode PTS permeases of the glucose/
sucrose family [17,33]. The fact that all lack a IIA domain
may support the speculation that IIA
Crr
serves as the
corresponding phosphotransferase.
A fascinating issue to investigate is whether the mechan-
ism of inducer exclusion is realized in S. coelicolor.Our
observation that IIA
Crr
could replace the inducer exclusion
function of E. coli IIA
Glc
by inhibition of maltose uptake
might be a good indication for this hypothesis. The
demonstration of the IIA
Crr
–MalK interaction suggests
that IIA
Crr
may regulate the function of some of the
> 140 MalK homologues found in the S. coelicolor
genome. The one with the highest similarity of 46%
identicalaminoacidsisMsiK,whichservesastheATPase
subunit for ABC transporters specific for maltose, cellobi-
ose, xylobiose, and trehalose [34–36]. MsiK could therefore
be a potential candidate for regulation by IIA
Crr
.Initial

attempts to demonstrate IIA
Crr
-MsiK binding by surface
plasmon resonance failed probably because overproduced
MsiK forms inclusion bodies yielding incorrectly folded
protein (unpublished data) [37].
IIA
Crr
could also play a role in carbon catabolite
repression. The mechanism of this phenomenon is not
solved in streptomycetes [6,7,38–40]. It appears that glucose
kinase serves a global regulatory function, but how it senses
and transmits carbon source signals is unclear. It has been
demonstrated that IIA
Glc
of E. coli senses C-regulatory
signals from both PTS and non-PTS carbon sources and
responds via its phosphorylation state [9,13]. It would be of
great interest to examine whether IIA
Crr
of S. coelicolor
operatesinasimilarway.
Finally, another hint as to a possible function of IIA
Crr
should be mentioned here. Ueguchi and coworkers have
reported that E. coli IIA
Glc
controls the sigma factor of the
general stress response RpoS [14]. They suggested that this
could be a linkage between carbon metabolism and stress

response upon nutrient starvation. Thus, IIA
Crr
could be
involved in controlling some of the many sigma factors that
S. coelicolor possesses [4,41].
Fig. 7. Surface plasmon resonance analysis. A real-time interaction
analysis of his-tagged IIA
Crr
(broken line) and His-tagged IIA
Glc
(dotted line) with MalK is shown. The control with tetracycline
repressor (TetR) is depicted by a solid line. The sensorgram represents
the binding responses of MalK in resonance units (RU)
4
as a function
of time. MalK solution was passed for 10 min over immobilized
protein resulting in an increase of RU caused by buffer components
and protein binding. Removal of MalK by application of washing
buffer revealed an RU-increase of the baseline (dotted line) indicating
solely the binding of MalK to immobilized IIA protein (arrows).
The experiment was repeated three times with almost identical results.
When purified glucose kinase from S. coelicolor was applied as a
ligand, no binding was observed (negative control).
2148 A. Kamionka et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Further analyses are required to address the ideas
mentioned above. These should cover phenotype analysis
of a crr mutant, protein–protein interaction studies with
candidate proteins, and the determination of the levels of
nonphosphorylated/phosphorylated IIA
Crr

in relation to
the nutritional state of the streptomycetes mycelium.
ACKNOWLEDGEMENTS
These studies were carried out in the laboratories of W. Hillen. His
support is greatly appreciated. We thank E. Schneider for providing
MalK protein and O. Scholz for a gift of TetR protein. We are grateful
to K. Mahr for critical reading of the manuscript. The work was
funded by SFB171 and SFB473 of the Deutsche Forschungsgemein-
schaft. J. S. was supported through SFB431 grant given to J. W.
Lengeler.
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