Conversion of Temperature-sensitive to -resistant Gene Expression
Due to Mutations in the Promoter Region of the Melibiose Operon in
Escherichia coli*
Eiji
Tamai
,
Tadashi
Shimamoto§,
Masaaki
Tsuda
¶,
Tohru
Mizushima
, and
Tomofusa
Tsuchiya
§
From the
Department of Microbiology, Faculty of
Pharmaceutical Sciences, and § Gene Research Center,
Okayama University, Tsushima, Okayama, 700-8530, Japan
 |
ABSTRACT |
The melibiose utilization system of
Escherichia coli W3133, a derivative of K12, is
nonfunctional between 37 and 42 °C. The reason for this temperature
sensitivity was thought to be that the melibiose transporter (MelB) of
W3133 cells was temperature-sensitive. A mutant W3133-2 has been
isolated as a temperature-resistant strain that can utilize melibiose
between 37 and 42 °C. However, we found that the melibiose
transporter of the W3133-2 was still temperature-sensitive. Half-life
activities of the melibiose transporter at 37 °C (or 40 °C) in
both E. coli W3133 and W3133-2 were exactly the same.
Furthermore, we found that the nucleotide sequence of coding region of
the melB structural gene (the second gene of the melibiose
operon) of W3133-2 was exactly the same as that of W3133. Activity of
-galactosidase (product of the first gene, melA, of the
melibiose operon) of W3133 cells grown at 40 °C was very low,
although that of W3133-2 cells grown at 40 °C was high. These
observations suggested that expression of the melibiose operon in W3133
is also temperature-sensitive. In fact, we found that the expression in
W3133 cells was temperature-sensitive, while that in W3133-2 cells was
temperature-resistant, by analyzing mRNA levels using the Northern
blot method. Furthermore, we identified mutations in the promoter
region of the melibiose operon of W3133-2 that resulted in the
elongation of an 18 nucleotide inverted repeat sequence to a
28-nucleotide repeat sequence present immediately upstream of the
35
region. This may stabilize a possible stem structure due to the
inverted repeat at 37-42 °C.
 |
INTRODUCTION |
For utilization of melibiose by Escherichia coli, both
the melibiose transporter and
-galactosidase are necessary. The
melibiose transporter mediates uptake of melibiose into cells. The
-galactosidase catalyzes cleavage of melibiose into galactose and
glucose, which are metabolized via the glycolysis pathway. Genes
encoding these two proteins, melA (
-galactosidase) and
melB (melibiose transporter), constitute structural genes in
the melibiose operon (1-3). The melibiose operon is inducible by
either melibiose or garactinol (4), or melibiitol (2). A regulatory
gene for the melibiose operon, melR, is located in a region
upstream of the melAB genes in the opposite orientation (5).
The MelR is an activator necessary for expression of the
melAB genes. An inducer such as melibiose is required to
activate MelR (6).
The melibiose transporter in E. coli was first reported as
the TMG1 permease II, because
this transport system was found to be the second transport system for
TMG (4). The first TMG transport system (TMG permease I) is known as
the lactose transporter LacY (7). LacY is also able to transport
melibiose (7). Therefore there are two transport systems for melibiose
in E. coli cells. One of the unique properties of the
melibiose transporter in E. coli K12 and its derivatives is
that it is a temperature-sensitive system (4). When cells are grown at
30 °C, the melibiose transporter is active, but if cells are grown
at 37 °C or above, the transporter is inactive. We have previously
shown that the melibiose transporter, but not
-galactosidase, was
irreversibly inactivated by incubation at 37 °C (8).
It is necessary to use a LacY-defective mutant to investigate the
melibiose transporter. E. coli W3133 is a derivative of K12
and lacks the lactose transporter (9). The melibiose transporter of
W3133 is temperature-sensitive (9). A mutant W3133-2 was isolated as a
temperature-resistant strain derived from W3133 (9). Although cells of
W3133 showed either very poor growth or no growth on melibiose as a
sole source of carbon at 37 °C, cells of W3133-2 showed normal
growth at this temperature (9) or even at
40 °C.2 Furthermore, cells
of W3133 grown and induced at 37 °C showed either little or no
activity of the melibiose transporter. Cells of W3133-2 grown and
induced at the same temperature showed high activity (9). Thus, it
seemed very clear that the melibiose transporter of W3133 cells is
temperature-sensitive and that of W3133-2 cells is
temperature-resistant. We have cloned and sequenced the melB
gene of E. coli. The amino acid sequence of this melibiose transporter protein has been deduced from melB (10). Thus it became possible to identify the substituted amino acid residue(s) in
the melibiose transporter of the temperature-resistant W3133-2. However, we found no mutation in the nucleotide sequence of the melB gene from the mutant strain (W3133-2).2
These results suggest that the reason for the temperature-resistant growth of W3133-2 cells on melibiose is not due to changes in the
primary structure of the melibiose transporter but in other factor(s)
of W3133-2.
Here we report that gene expression of the melibiose operon in the
parental strain is temperature-sensitive and that in the mutant strain
is temperature-resistant. We found mutations in the promoter region of
the melibiose operon of the mutant.
 |
EXPERIMENTAL PROCEDURES |
Bacterium and Growth--
E. coli W3133, a derivative
of K12, is a lactose-deleted (
lacZY) strain containing
the wild type melibiose utilization system (9). Cells of W3133 are
unable to grow on melibiose as a sole source of carbon at 37 °C or
above. A mutant called W3133-2, a derivative of W3133, was isolated as
a temperature-resistant strain with regard to melibiose utilization and
can grow on melibiose at 37 °C (9). E. coli DW1
(
lacZY,
melAB) (11) was used as a host for
cloning of the melibiose operon from chromosomal DNA of W3133 and
W3133-2. Cells of W3133 and W3133-2 were first grown in the LB medium
(12) at 37 °C, diluted 200-fold into a modified Tanaka medium (13)
(Na+ salts were replaced with K+ salts)
supplemented with 1% tryptone, 0.0001% thiamine, and 10 mM melibiose (an inducer of the melibiose operon) and
shaken at 30, 34, 37, 40, or 42 °C under aerobic conditions. Cells
were harvested at logarithmic phase of growth.
For measurement of the growth on melibiose, cells were first grown in
the modified Tanaka medium supplemented with 10 mM
melibiose at 30 °C, diluted 200-fold into fresh medium, and shaken
at various temperatures under aerobic conditions. Cell growth was
monitored turbidimetrically at 650 nm.
Measurement of Transport--
TMG uptake was measured as
described previously (9) with minor modification using
[methyl-14C]TMG as a substrate. To test the
effect of temperature on the transport activity, cells suspended in the
modified Tanaka medium containing 10 µg/ml chloramphenicol were
incubated at 30, 37, 40, or 42 °C for the indicated time. The
transport assays were performed in the modified Tanaka medium
containing 20 mM potassium lactate, 20 mM NaCl,
10 µg/ml chloramphenicol, cells (0.15 mg of protein/ml), and 0.1 mM [14C]TMG at 25 °C.
-Galactosidase Assay--
-Galactosidase activity was
measured as described previously (14) using
p-nitrophenyl-
-D-galactopyranoside as a
substrate.
Preparation of RNA--
Total cellular RNA was prepared from
E. coli cells by the method of Aiba et al.
(15).
Northern Hybridization Analysis--
RNA was denatured with
formaldehyde and then electophoresed on 1% agarose, 2 M
formaldehyde gel with constant recirculation of a buffer solution (20 mM MOPS-NaOH, pH 7.0, 5 mM
CH3COONa, 1 mM EDTA). rRNA in the gel was
visualized by staining with ethidium bromide. Total RNA in the gel was
transferred to a nylon membrane, Hybond-N (Amersham Corp.), overnight
by the capillary transfer method as suggested by the manufacturer, and
then hybridization was performed as described previously (16). The DNA
probe was synthesized and labeled with [
-32P]CTP using
the MultiprimeTM DNA labeling system (Amersham Corp.) with
a melB-specific primer (5'-AGGCGATAGAAACGGAAG-3') and a
BamHI fragment derived from the melB gene as the
template. The synthesized labeled DNA probe covers 80% of the
melB gene. The filter was washed twice with 2× SSPE (180 mM NaCl, 1 mM Na2PO4
and 0.1 mM EDTA, pH 7.7) containing 0.5% SDS at 70 °C,
once with SSPE containing 0.5% SDS at 70 °C, and once with 10-fold
diluted SSPE containing 0.5% SDS at room temperature. The filter was
air-dried and exposed to an imaging plate (Fuji Film Co.) to determine
the amount of melAB mRNA and to x-ray film (Fuji Film
Co.) at
70 °C with an intensifying screen to visualize the
melAB mRNA. The amount of melAB mRNA was
determined by densitometric scanning using BAS 2000 (Fuji Film
Co.).
Cloning and Sequencing--
Chromosomal DNA was prepared from
W3133 cells or W3133-2 cells as described elsewhere (17). The DNA was
digested with the restriction endonuclease EcoRI. The
digested fragments were ligated to pBR322 which had been digested with
EcoRI and treated with shrimp alkaline phosphatase using a
ligation kit (TaKaRa Co.). After the ligation, competent cells of
E. coli DW1 (
melAB) were transformed with the
hybrid plasmids. Transformants that grew on agar (1.5%) plates
containing the modified Tanaka medium, 10 mM melibiose and
0.1 mg/ml ampicillin were picked. Candidate plasmids were digested with
several restriction endonucleases to test whether the plasmids carry
the melibiose operon. The plasmid carrying the EcoRI
fragment containing the melibiose operon from W3133 was designated as
pBM3133 and that from W3133-2 as pBM3133-2. For sequencing, DNA
fragments from the cloned region were subcloned into pBluescript II
KS(+). The subcloned fragments were sequenced by the dideoxy chain
termination method (18). A DNA sequencer (ABI PRISMTM 310)
and a DNA sequencing kit (dRhodemine terminator cycle sequencing ready
reaction) (Perkin-Elmer) were used.
Chemicals--
[methyl-14C]TMG (60 mCi/mmol) was purchased from NEN Life Science Products or American
Radiolabeled Chemicals Inc. [
-32P]CTP was from ICN
Inc. All other chemicals were reagent grade and obtained from
commercial sources.
 |
RESULTS |
Temperature Sensitivity of Melibiose Transporter--
The
melibiose transport system is the sole uptake system for melibiose (and
TMG) in E. coli W3133 and W3133-2 (9). W3133 cells were
unable to grow on melibiose at 37 °C or above, although cells were
able to grow on other carbon sources such as glucose or amino acid
mixtures at 42 °C. However, W3133-2 cells were able to grow on
melibiose at 42 °C (data not shown). As reported previously (9),
both types of cells grew well on melibiose at 30 °C. W3133 cells
were able to grow on melibiose at 35 °C. However, 37 °C is an
unreliable temperature for the measurement of growth of W3133 cells on
melibiose. On some occasions the cells grew and at other occasions they
did not. W3133 cells were unable to grow on melibiose at 40 °C.
Thus, 40 °C instead of 37 °C is a convenient temperature for
investigation of the temperature sensitivity and resistance of the
melibiose utilization system of W3133 and W3133-2.
Fig. 1 shows that activity of the
melibiose transporter (TMG uptake) in W3133 cells was very low when
cells were grown at 37 °C or above. Although the activity was fairly
high when cells were grown at 30 °C. The activity of the melibiose
transporter in W3133-2 was high when cells were grown at
30-34 °C, moderate when grown at 37-40 °C, and very low when
grown at 42 °C. These results suggest that the melibiose transporter
in W3133-2 cells are still temperature-sensitive to a certain
extent.

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Fig. 1.
Activity of melibiose transporter in cells
grown at various temperatures. Cells of E. coli W3133
( ) or W3133-2 ( ) were grown in the modified Tanaka medium
supplemented with 1% tryptone and 10 mM melibiose under
aerobic conditions at the indicated temperatures. Cells were harvested
at the exponential phase of growth. Initial velocity of TMG transport
was measured at 25 °C.
|
|
To compare temperature sensitivity of the melibiose transporter between
W3133 cells and W3133-2 cells, we tested the effect of temperature on
activity of the melibiose transporter. Fig. 2 shows decay lines of TMG transport
activity when cells were incubated at 30, 37, or 40 °C.
Surprisingly, W3133 cells and W3133-2 cells showed the same decay lines
at 37 °C. The half-life of the melibiose transport activity in the
two strains was exactly the same, 35 min. This half-life is very
similar to that we reported previously with another strain (GN22)
derived from K12 (8). When cells were incubated at 40 °C, again the
activity decay lines were not distinguishable between W3133 and
W3133-2. The half-life was 17 min. We also tested heat inactivation at
42 °C and obtained very similar decay profiles to that obtained at
40 °C in both strains (data not shown). No decrease of the transport
activity was observed with both W3133 and W3133-2 when cells were
incubated at 30 °C (Fig. 2). Thus, it became clear that there is no
difference in the temperature sensitivity of the melibiose transport
protein between W3133 cells and W3133-2 cells.

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Fig. 2.
Inactivation of melibiose transporter by heat
treatment. Cells of E. coli W3133 (open
symbols) or W3133-2 (closed symbols) were grown in the
modified Tanaka medium supplemented with 1% tryptone and 10 mM melibiose under aerobic conditions at 30 °C. Cells
were harvested at the exponential phase of growth and suspended in the
modified Tanaka medium containing 10 µg/ml chloramphenicol. Cells
were incubated at 30 °C (circles), 37 °C
(squares), or 40 °C (triangles). At the
indicated time points, samples were removed and cooled in an ice bath
until they were assayed. The samples were assayed for TMG transport at
25 °C. Initial values (100%) were: 35 nmol/min per mg of cell
protein for W3133 and 47 nmol/min per mg of cell protein for
W3133-2.
|
|
Previously we reported the nucleotide sequence of the melB
gene and the deduced amino acid sequence of the melibiose transporter (10). Thus, it became possible to identify substitution of amino acid
residue(s) in the melibiose transporter in mutant cells. We cloned and
sequenced the melB gene from W3133 and from W3133-2 and
found that there was no difference in the nucleotide sequence in their
melB structural genes.2 Thus, we conclude that
the primary structure of the melibiose transport protein in the two
strains is identical.
Thus, the question arose why W3133-2 cells, but not W3133 cells, were
able to utilize melibiose at 40 or 42 °C.
Effect of Temperature on Gene Expression--
As we reported
previously, the W3133-2 cells showed partially constitutive expression
of the melibiose operon, although expression in the parental W3133
cells was completely inducible (9). This indicates that there is a
difference in the expression of the operon between the parent and the
mutant, and suggests that the reason for temperature sensitivity is the
expression of the melibiose operon. We therefore tested the effect of
temperature on gene expression of the melibiose operon with the two
strains. We have shown previously that
-galactosidase of E. coli was not temperature-sensitive (8). Thus, the level of
-galactosidase activity reflects the level of expression of the
melibiose operon. We measured
-galactosidase activity in cells grown
at various temperatures. In the experiments, melibiose was added to the
growth medium to fully induce the operon. The
-galactosidase
activity in W3133 cells grown at 30 °C was high, moderate in cells
grown at 37 °C, and very low in cells grown at 40 or 42 °C (Fig.
3). The activity in W3133-2 cells was much higher than that in W3133 cells (Fig. 3). The enzyme activity in
W3133-2 cells was very high even if cells were grown at 40 or 42 °C.
These results support the idea that expression of the melibiose operon
in W3133 cells is temperature-sensitive while that in W3133-2 cells is
not.

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Fig. 3.
-Galactosidase activity in cells grown at
various temperatures. Cells of E. coli W3133 ( ) or
W3133-2 ( ) were grown in the modified Tanaka medium supplemented
with 1% tryptone and 10 mM melibiose under aerobic
conditions at the indicated temperatures. Cells were harvested at the
exponential phase of growth. Activity of the -galactosidase was
measured at 37 °C. One unit of enzyme activity is defined as the
release of 1 nmol of p-nitrophenol per min.
|
|
We tested this possibility by Northern blot analysis. The probe used
was a BamHI fragment derived from the melB gene,
which is located downstream from the melA gene (3, 10).
Therefore the mRNA detected in our assay is melAB
mRNA (19). The mRNA level in W3133-2 cells induced with
melibiose was about two times higher than that in W3133 cells when
grown at 30 °C (Fig. 4). The ratio of
the mRNA level in W3133-2 cells to that of W3133-2 cells grown at
30 °C was comparable to the ratio of the
-galactosidase activity
of these cells (Table I). The levels of
mRNA in both W3133-2 and W3133 increased when cells were grown at
37 °C, and the level in W3133-2 was 2.7 times higher than that in
W3133. As expected, we detected a faint band of the melAB
mRNA with W3133 cells grown at 40 °C, although a dense band was
detected with W3133-2 cells (Fig. 4). The mRNA level in W3133-2
cells was 7.9 times higher than that in W3133 cells. A very low level
of melAB mRNA was detected in W3133 cells grown at
42 °C, but a considerable level of the mRNA was detected in
W3133-2 cells. The ratio of the mRNA level in W3133-2 cells to that
of W3133 cells is comparable to the ratio of the
-galactosidase
activity in these cells for all temperatures tested except at 42 °C
(Table I). The experiments were repeated four times and very similar
results were obtained. Thus, levels of
-galactosidase activity
accurately reflect the levels of melAB mRNA at
30-40 °C. The reason why the mRNA level is very low at 42 °C
irrespective of the relatively high
-galactosidase activity is not
yet clear. In any case, our results indicate that expression of the
melibiose operon in W3133 cells is temperature-sensitive while that in
W3133-2 cells is fairly temperature-resistant.

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Fig. 4.
Northern blot analysis of melAB
mRNA. Cells of E. coli W3133 or W3133-2 were
grown in the modified Tanaka medium supplemented with 1% tryptone and
10 mM melibiose under aerobic conditions at the indicated
temperatures. 5 µg of total RNA prepared from cells were loaded onto
each lane of gel and subjected to Northern blot analysis using the
radiolabeled BamHI fragment derived from melB
gene as a probe. Densitometric scanning of the autoradiograph was
performed to determine the relative levels of melAB mRNA
in the samples. Positions of 23 S rRNA, 16 S rRNA, and 5 S rRNA are
shown.
|
|
Identification of a Promoter Mutation--
The
temperature-sensitive expression of the melibiose operon in W3133 cells
and the temperature-resistant expression in W3133-2 cells suggested
that mutation(s) may be present in the regulatory gene
(melR) for the operon or in the promoter region of the
melAB genes. We cloned the entire melibiose operon from both
W3133 and W3133-2 cells. Plasmids carrying the melibiose operon were
introduced into cells of E. coli DW1, which contains a
deletion through the melA and melB genes (11).
The melAB mRNA was not detected with uninduced cells of
DW1 harboring the plasmid pBM3133 carrying the melibiose operon from
W3133 (Fig. 5). However, we detected dense band of melAB mRNA with uninduced DW1 cells
harboring a plasmid pBM3133-2 carrying the melibiose operon from
W3133-2 (Fig. 5). A faint mRNA band was detected in DW1/pBM3133
cells induced with melibiose at 40 °C and a more dense band was
detected with melibiose-induced cells of DW1/pBM3133-2 (Fig. 5). Thus,
mutation(s) responsible for the temperature-resistant expression of the
melibiose operon in W3133-2 must be present in the cloned region.
Again, the melibiose operon derived from W3133-2 was partially
constitutive. Therefore mutation(s) responsible for the partial
constitutive phenotype must be also in the cloned region.

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Fig. 5.
Northern analysis of melAB
mRNA expressed from cloned melibiose operon. Cells of
E. coli DW1/pBM3133 or DW1/pBM3133-2 were grown in the
modified Tanaka medium supplemented with 1% tryptone and 0.1 mg/ml
ampicillin either in the absence (uninduced) or presence
(induced) of 10 mM melibiose under aerobic
conditions at 40 °C. 3.3 µg of total RNA prepared from cells were
loaded onto each lane of gel and subjected to Northern blot analysis
using the radiolabeled BamHI fragment derived from
melB gene as a probe. Densitometric scanning of the
autoradiograph was performed to determine the relative levels of
melAB mRNA in the samples. Positions of 23 S rRNA, 16 S
rRNA, and 5 S rRNA are shown.
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|
We sequenced the entire melibiose region. We found no difference in the
nucleotide sequence of the melR coding region, in the
promoter region for the melR gene, and in the
melA to melB region. Instead, we found a
replacement of 5 consecutive nucleotides in the promoter region for the
melAB genes (Fig. 6). The
transcription initiation site for the melAB genes in Fig. 6
has been reported (5). The translation initiation site for the
melA gene was confirmed by amino acid sequencing of the
NH2 terminus of purified
-galactosidase (20). The
sequence of the melAB promoter region from W3133 was
identical with that from E. coli CS520 (3, 21), a derivative
of K12, of which we were the first to report the promoter region
sequence (22). There is one long inverted repeat and several short
inverted repeats in the promoter region (22). The replacement is
immediately downstream from the long inverted repeat which has been
reported as one of the binding sites for MelR (23, 24), and lies
between the long inverted repeat and the short inverted repeat (Fig.
6).

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Fig. 6.
Mutations in the promoter region of the
melibiose operon. Nucleotide sequence of the promoter regions of
the melibiose operon from E. coli W3133 or from W3133-2 are
shown. Both the top and bottom sequences shown are common in W3133 and
W3133-2. Position of the transcription initiation for melAB
mRNA is 1. The 35 regions and 10 region are boxed.
The initiation codon ATG for the melA gene is
underlined. Replacement of consecutive 5 nucleotides is
shown in bold letters. Original inverted repeats in the
parental W3133 are shown by arrows with broken
lines. The longer inverted repeat which appeared in the mutant
W3133-2 is shown by arrows with solid
lines.
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|
 |
DISCUSSION |
The cause of the temperature sensitivity of the melibiose
utilization system in E. coli K12 and its derivatives has
long been thought to be that the melibiose transporter is
temperature-sensitive (4, 8, 9). We found that expression of the
melibiose operon, in addition to the melibiose transporter, in E. coli W3133 (a derivative of K12) was temperature-sensitive. We
identified a replacement of 5 consecutive nucleotides in the promoter
region of the melibiose operon from the mutant strain. The 5 replaced nucleotides were located just between a long inverted repeat (18 nucleotides) and the following a short inverted repeat (5 nucleotides). In short, the replacement resulted in appearance of a huge inverted repeat (28 nucleotides). It has been reported that the original long
inverted repeat is the site for binding of MelR (23, 24). Thus, it is
likely that complex of MelR and melibiose (an inducer) can bind to the
huge inverted repeat region even if the temperature is 40-42 °C. On
the other hand, perhaps the MelR-inducer complex is unable to bind to
the original inverted repeat (18 nucleotides repeat) at such high
temperatures, although the complex can bind at lower temperature such
as 30 °C. Although it is not clear whether the stem-loop structure
of DNA is really formed in vivo, a stem structure formed
with a 28-nucleotide repeat would be more stable than that formed with
a 18-nucleotide repeat at 40 or 42 °C. Also it may be possible that
the 18-nucleotide inverted repeat forms a stem structure at 30 °C
but not at 40 °C. Although we do not have direct evidence for
in vivo formation of the stem-loop structure, our results
are consistent with the idea that MelR binds to the hypothetical stem
structure and formation of the stem structure is temperature-sensitive.
In any case, the temperature sensitivity and resistance of gene
expression due to the alteration of the structure (sequence) in the
promoter region of the melibiose operon of E. coli (but not
due to the regulatory protein) is a very unique case. Usually protein
is responsible for temperature-sensitive cellular processes (25).
The melibiose operon in the parental W3133 cells or other K12-derived
cells is inducible. An inducer such as melibiose is necessary for this
induction (4). The melibiose operon in the mutant W3133-2 cells,
however, is partially constitutive (9). Thus, it seems that the MelR
can bind to the 28-nucleotide inverted repeat structure, but not to the
18-nucleotide inverted repeat structure, to some extent without the
inducer. In the presence of inducer, perhaps the MelR-inducer complex
can bind to the longer inverted repeat easily in W3133-2 cells and
perhaps transcription increases even at 30 °C compared with W3133
cells. Therefore, it seems likely that the induction level in W3133-2
cells is higher than that in W3133 cells.
The melibiose transport proteins in both W3133 and W3133-2 cells are
equally temperature-sensitive (equally inactivated at 40 °C).
Nevertheless, cells of W3133-2 are able to grow on melibiose at
40 °C and cells of W3133 are not. One possible explanation for this
is as follows. Perhaps the rate of production of the melibiose
transporter exceeds the rate of inactivation of the protein in W3133-2
cells at 40 °C. The efficient production of the melibiose
transporter protein would be due to elevated mRNA synthesis in this
strain. On the other hand, the rate of the protein inactivation exceeds
the rate of the protein production in W3133 cells at 40 °C. The
half-lives of the melibiose transporter inactivation rates were 35 min
at 37 °C and 17 min at 40 °C. Thus, the production rate of the
melibiose transport protein must exceed the inactivation rate for cells
to grow on melibiose at such temperatures.
 |
ACKNOWLEDGEMENT |
We thank Dr. Manuel F. Varela of Eastern New
Mexico University for critically reading the manuscript.
 |
FOOTNOTES |
*
This research was supported in part by grant from the
Ministry of Education, Science and Culture of Japan.
¶
Present address: Faculty of Pharmaceutical Sciences, Toyama
Medical and Pharmaceutical University, Toyama, 930-01, Japan.
To whom correspondence should be addressed: Dept. of
Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama, 700-8530, Japan. Tel. and Fax:
81-86-251-7957; E-mail:
tsuchiya{at}pheasant.pharm.okayama-u.ac.jp.
1
The abbreviations used are: TMG,
methyl-
-D-thiogalactopyranoside; MOPS,
morpholinepropanesulfonic acid.
2
E. Tamai, T. Mizushima, and T. Tsuchiya,
unpublished results.
 |
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