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

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,

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

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

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

[
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

) 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

) 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

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-

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

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

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

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

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

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

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

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