Download Regulation of Galactoside Transport by the PTS

J. Mol. Microbiol. Biotechnol. (2001) 3(3): 381-384.
JMMB Symposium
PTS Regulation of Galactoside
Transport 381
Regulation of Galactoside Transport by the PTS
Masayuki Kuroda1,2, Thomas H. Wilson3, and Tomofusa
Tsuchiya1,*
Model for the Regulation of non-PTS Sugar Transport
System by the PTS
1
Since the PTS had been discovered (Kundig et al., 1964),
various mutants defective in sugar utilization have been
isolated and extensively characterized in enteric bacteria
such as E. coli and Salmonella typhimurium. Some of
mutations were mapped in genes for enzyme I and HPr,
common components of the PTS. Although it had been
suggested that the PTS is responsible for the uptake of
many carbohydrates, it has been later found that there are
several sugars (lactose, melibiose, maltose, and glycerol)
which are not transported by the PTS. These sugars are
now generally called non-PTS sugars. Interestingly, it has
been demonstrated that mutations in enzyme I and HPr
resulted in loss of utilization of not only PTS-sugars but
also non-PTS sugars. This finding directed researchers to
study the interaction between the utilization system of nonPTS sugars and the PTS. Regulation of catabolic enzyme
synthesis appeared to be through the inhibition of inducer
sugar uptake and cAMP synthesis. Based on numerous
observations, a mechanism for PTS-mediated regulation
has been proposed (Saier and Feucht, 1975; Saier and
Stiles, 1975; Saier, 1977; Figure 1.). In this model, IIAGlc is
a central regulatory protein whose phosphorylation state
is crucial for the regulatory functions. According to this
model, non-PTS sugar transport systems such as lactose
transport system and melibiose transport system are
negatively controlled by non-phosphorylated form of IIAGlc
(inducer exclusion), whereas adenylate cyclase is activated
by phosphorylated form of IIAGlc.
Gene Research Center, Okayama University, Tsushima,
Okayama 700-8530, Japan
2
Current address: Department of Microbiology, Kochi
Medical School, Kohasu, Okoh, Nankoku, Kochi 783-8505,
Japan
3
Department of Cell Biology, Harvard Medical School,
Boston, MA, 021152, USA
Abstract
Inducer exclusion, regulation of activity of transporter, is
mediated by phosphoenolpyruvate:carbohydrate
phosphotransferase system (PTS). To elucidate the
molecular mechanism of the inducer exclusion, numerous
biochemical and genetic studies have been performed. It
is now well known that non-phosphorylated IIAGlc inhibits
the transport via direct binding to the transporter. Analysis
of inducer exclusion resistant mutants of lactose transporter
and melibiose transporter in Escherichia coli and
Salmonella typhimurium revealed amino acid residues that
are involved in the interaction with IIAGlc. It is concluded
that there are multiple interaction sites for IIAGlc in these
transporters.
Introduction
Since the glucose effect and diauxic growth were
documented in 1940s (Monod, 1942; Gale, 1943),
numerous studies have been performed to understand the
molecular mechanisms behind those phenomena. It has
been demonstrated that uptake of various sugars was
inhibited in the presence of glucose in Escherichia coli. At
the same time, it has been shown that cAMP and its
receptor protein (CRP) are involved in the synthesis of
many inducible carbohydrate catabolic enzymes (Pastan
and Perlman, 1970). Gene expression of catabolic
enzymes seems to be regulated by two distinct
mechanisms: one is termed catabolite repression, which
is at the level of gene expression via cAMP and CRP; the
other is termed inducer exclusion, which is at the level of
transport activity. It is now well known that
phosphoenolpyruvate:carbohydrate phosphotransferase
system (PTS) is involved in the both mechanisms (Saier,
1989; Postma et al., 1993). In this review, the molecular
mechanisms of inducer exclusion, regulation of activity of
transporter, especially in the lactose transport system and
the melibiose transport system, are discussed.
Accepted September 29, 2000. *For correspondence. Email
[email protected]; Tel. +81-86-251-7957; Fax. +81-86-2517957.
© 2001 Horizon Scientific Press
Figure 1. Proposed model for the regulation of melibiose utilization. IIAGlc
of PTS is a central regulatory protein. Its phosphorylated form activates
adenylate cyclase whereas non-phosphorylated form inhibits the melibiose
transporter. aMG; methyl-a-glucoside, CRP; cAMP receptor protein, 2DG;
2-deoxyglucose, mel; melibiose.
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382 Kuroda et al.
by the presence of substrate of the lactose transporter.
The results of Osumi and Saier have been confirmed and
further quantified by Nelson et al. (1983; 1984). Since
phosphorylated IIAGlc did not bind to the lactose transporter,
it was concluded that only non-phosphorylated IIAGlc could
bind to the transporter and inhibit the activity. It has been
estimated that there were 1 to 1.5 molecules of IIAGlc bound
per a molecule of the lactose transporter.
Among four non-PTS sugar transport systems, direct
binding of IIAGlc to the transporter has been shown except
in the melibiose system. However, since it has been shown
that inclusion of IIAGlc in membrane vesicles inhibited the
melibiose transporter, interaction between melibiose
transport protein and IIAGlc is expected to be similar to that
in the other three systems.
Identification of Mutations Which Tolerate the
Regulation by IIAGlc
Figure 2. Vertical view of a putative a-helix of the carboxyl-terminal region
of the melibiose transporter of S. typhimurium. A region from Gly-437 to
Thr-454 is shown. The region from Gly-437 to Arg-452 is predicted to form
an a-helix structure. Amino acid residues which are involved in the interaction
with IIAGlc are circled.
Direct Evidence for the Interaction Between Sugar
Transporter and IIAGlc Component of Glucose-PTS
The role of IIAGlc in the inducer exclusion is now well
established based on various experimental observations.
In the study of the lactose transport system, considerable
biochemical evidence for the interaction between the
transport protein and IIAGlc have been obtained. The first
evidence for the involvement of IIAGlc in the regulation of
the lactose transport system has been reported by Dills et
al. (1982). They demonstrated that the partially purified
preparation of IIAGlc loaded into E. coli membrane vesicles
possessing high activity of lactose transport inhibited the
lactose uptake. In addition, in the presence of enzyme I
and HPr, intravesicular PEP effaced the inhibition. The
results have been later confirmed by Misko et al. (1987). It
has been reported that in the presence of methyl-aglucoside, a non-metabolizable analog of glucose, the
activity of lactose transport decreased in membrane
vesicles. When the vesicles were prepared from ptsI-, ptsH, or crr-negative strain, methyl-a-glucoside did not inhibit
the lactose transport. In this study, it has been also shown
that melibiose and galactose transport activities reduced
in the presence of intravesicular IIAGlc.
Further evidence for the involvement of IIAGlc in the
transport regulation of non-PTS sugar has been obtained
in the lactose transport system by Osumi and Saier (1982;
1982). They used lacY gene cloned into a multicopy
plasmid, overproduced the lactose transporter and studied
the binding of purified IIAGlc to the membrane fragment
from this strain. They demonstrated that IIAGlc directly
bound to the membrane containing the overproduced
lactose transporter, and the binding activity was enhanced
Although inducer exclusion is observed in wild-type ptsI+,
ptsH+ strains, the inhibition is much stronger in ptsI-leaky
strain. Using such inducer exclusion-hypersensitive strain,
Saier and coworkers isolated mutants of E. coli and S.
typhimurium in which individual non-PTS sugar transport
system was no longer sensitive to inducer exclusion (Saier
et al., 1978). These mutations were mapped in or closely
linked to the gene(s) responsible for the metabolism of each
non-PTS sugar. This report touched off the extensive
studies on the domains or amino acid residues of the
transporters that interact with IIAGlc.
With respect to the lactose transport system, Nelson
et al. (1983) first reported that a mutant lactose carrier with
triple amino acid substitutions in N-terminal region exhibited
no binding to IIAGlc. They concluded that the N-terminal
cytoplasmic portion of the LacY was probably a binding
site of IIAGlc . Wilson et al. reported the amino acid
substitutions in lactose transport mutants of E. coli resistant
to inducer exclusion (Wilson et al. , 1990). It was
demonstrated that Ala-198 and Ser-209 were replaced in
these mutants. The results suggested that cytoplasmic
central loop of the lactose carrier protein was also a binding
site of IIAGlc. Hoischen et al. (1996) later confirmed the
results of Wilson et al. by using direct binding assay.
Hoischen et al. reported that Pro-192 which is located in
cytoplasmic central loop of the protein was also important
for the regulation by the PTS. In contrast to the results of
Nelson et al. some deletion mutations in N-terminal region
had little effect on the binding. It might be due to the
difference between point mutation and deletion.
Seok et al. (1997) examined the binding activity of LacY
mutants in which 6 contiguous His residues were inserted
into each loop or N-terminus, or C-terminal deletion to IIAGlc.
Insertion mutations of cytoplasmic loops IV/V and VI/VII
(which is called cytoplasmic central loop as indicated
above) resulted in retention of sugar transport (i.e. binding)
activity, but loss of IIAGlc binding. Interestingly, a mutant
with insertion in periplasmic loop VII/VIII also exhibited
similar properties. Since IIAGlc is a cytoplasmic soluble
protein and binds to the LacY from cytoplasmic side,
involvement of this periplasmic loop in IIAGlc binding
remains to be examined. Sondej et al. (1999) further
examined the cytoplasmic loops IV/V and VI/VII by Cys-
PTS Regulation of Galactoside Transport 383
scanning mutagenesis. It has been shown that replacement
of either Val-132, Arg-135, or Arg-142 in or adjacent to loop
IV/V by Cys resulted in decreased binding with no effect
on the transport activity. In loop VI/VII, mutation of either
Thr-196, Val-197, Asn-199, Gly-202, Asn-204 or Ser-206
had similar effects.
With respect to the melibiose transport system, we
isolated inducer exclusion resistant mutants in MelB of S.
typhimurium and identified the following substitutions of
amino acid residues: Asp-438 with Tyr, Arg-441 with Ser,
and Ile-445 with Asn (Kuroda et al., 1992). Recently, we
have identified different amino acid substitution (Arg-441
with Gly) in another mutant (unpublished result). The
replacement of Ile-445 by Asn was identified in more than
half of independently isolated mutants, suggesting that Ile445 was the most important residue for interaction with
the IIAGlc. Amino acid residues substituted in the mutants
were located in carboxyl-terminal cytoplasmic region and
the region containing these residues was predicted to form
an α-helix structure. According to the helical wheel analysis,
these residues were on the same side of the α-helix (Figure
2) To further evaluate the role of the C-terminal region of
the melibiose transporter, site-directed mutants were
constructed and characterized. Mutants of Asp-449 and
Arg-452, which are on the same side of the α-helix,
exhibited the transport activity fairly resistant to inducer
exclusion (unpublished result). Surprisingly, any amino acid
replacements of Ile-445 (including replacements by Val or
Leu residues) tested resulted in resistance to the PTS
regulation. Since prediction of the secondary structure by
the method of Chou and Fasman revealed that these
substitutions did not significantly affect the secondary
structure of the C-terminal region, this result suggests that
side chain of Ile-445 is critical for the binding of IIAGlc
(unpublished result).
We introduced a series of C-terminal truncations in
the E. coli MelB (each mutants lacked 16, 19, 20, 21, 22,
23, or 24 amino acid residues from the C-terminus) and
examined regulation by the PTS. The inhibition was 65,
65, 63, 46, and 19% in the MelB mutants lacking 16, 19,
20, 21, 22 amino acid residues, respectively, whereas
inhibition in wild type was 84 %. Mutants lacking 23 residues
and lacking 24 residues exhibited very little transport
activity. These results suggest that the C-terminal region
of MelB is essential not only for the regulation of activity by
the IIA Glc but also for the transport activity itself
(unpublished result).
Deduction of Interaction Sites in Target Protein of IIAGlc
In the maltose transport system, analysis of inducer
exclusion resistant mutants has identified amino acid
residues in MalK, which were involved in IIAGlc interaction
(Dean et al., 1990; Kühnau et al., 1991). Additionally,
analysis of three-dimensional structure of E. coli glycerol
kinase-IIAGlc complex revealed amino acid residues at the
contact sites (Hurley et al., 1993).
Based on studies summarized above, some reports
postulated the consensus sequences of proteins that
interact with IIAGlc (Dean et al., 1990; Titgemeyer et al.,
1994; Sondej et al., 1999). Especially, REGION I and II
proposed by Sondej et al. are also present in HPr, which
contacts with IIAGlc in phospho-transfer reaction of the PTS.
Among four proteins (LacY, MelB, MalK, and GlpK) that
interact with IIAGlc, MelB is considered to be a little different
one, since some of the conserved residues in REGION II
are absent. In fact, our recent experiments demonstrated
that deletion of Arg-215 to Leu-220, which corresponds to
the latter half of the REGION II, did not affect the PTS
regulation and transport activity in the melibiose carrier of
S. typhimurium. In agreement with this result, REGION II
is not conserved well in four MelBs of enteric bacteria,
sequences of which have been reported so far.
Furthermore, E. coli PTS can regulate MelBs from S.
typhimurium and K. pneumoniae (unpublished result).
Therefore, we proposed that C-terminal region of the MelB
protein is at least one of the interaction sites with IIAGlc,
instead of the REGION II.
Discussion
Although several reports revealed amino acid residues that
were involved in IIAGlc binding, consensus sequences with
complete match seems not to be completely deduced so
far. As shown in these studies, several portions of target
proteins are considered to be required to form regulatory
complex with IIAGlc. To solve the contact sites, threedimensional structure of target protein-IIAGlc complex
remains to be studied. Through these studies, it has been
demonstrated that IIAGlc is one of the most important
proteins that regulates various events occurring in
Enterobacteriae.
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