 |
INTRODUCTION |
KefB and KefC are independent, glutathione-gated potassium efflux
systems found in Escherichia coli (1). The efflux systems are maintained in a closed state by glutathione or by its non-sulfydryl analogue, ophthalmic acid (2, 3). The systems are fully activated by
adducts formed by reaction of glutathione with electrophilic compounds,
such as N-ethylmaleimide
(NEM),1 methylglyoxal (MG),
and chlorodinitrobenzene (3, 4). Activation of KefB and KefC provokes
rapid potassium efflux, accompanied by acidification of the cytoplasm
and influx of sodium ions (5, 6). The viability of mutants lacking KefB
and KefC is markedly reduced, but incubation with weak acids, which
mimics the fall in pH associated with channel activation, leads to
retention of viability (5, 6). Thus, a major determinant of the
sensitivity of E. coli cells to electrophiles is the
cytoplasmic pH, and this can be modulated by the controlled activation
of KefB and KefC by glutathione adducts.
The structural gene for KefC has been cloned and sequenced (7). The
protein has a distinct domain structure: an amino-terminal membrane
protein (residues 1-380) and an extremely hydrophilic linker that
connects the membrane domain to the carboxyl-terminal hydrophilic
domain (residues 401-620) (8). The carboxyl-terminal domain contains a
sequence highly similar to a Rossman fold (7, 8). A number of mutations
that cause increased spontaneous activity in KefC have been
characterized and fall in two regions: a region (the "HALESDIE"
sequence) predicted to lie at the cytoplasmic face of the membrane
domain and residues within, and adjacent to, the Rossman fold of the
carboxyl-terminal domain (7-9). One mutation at the latter site alters
the glutathione regulation of the KefC protein (9), but the specific
mechanism of activation by the other lesions is not known.
The two E. coli glutathione-gated K+ efflux
systems can be differentiated by their activation by MG (4).
Methylglyoxal only weakly activates KefC, whereas KefB achieves almost
maximum activity with this electrophile. In this study we sought to
characterize the structural gene for KefB to determine the relatedness
to KefC. The two proteins are similar at the sequence and
organizational levels. However, the creation of equivalent mutations at
a number of positions in KefB and KefC shows that the residues
controlling the activation of the two systems are different.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
All chemical reagents were purchased from Sigma or
BDH and were of analytical grade where possible. Chemicals used for
preparation of complex growth medium were supplied by Oxoid.
Restriction enzymes and Taq DNA polymerase were supplied by
Boehringer. Pfu polymerase was obtained from Stratagene. The
Qiagen Plasmid Preparation Kits were obtained from Qiagen. All primers
used in this study were purchased from Genosys Biotechnologies Inc. The
Wizard PCR Preps DNA Purification System was obtained from Promega. The
PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit was
obtained from Applied Biosystems Ltd.
Bacterial Strains--
Bacterial strains used in this study are
all derivatives of E. coli K-12 (Table
I). Strains MJF270 and MJF276 were
previously thought to carry an internal deletion in the kefB
gene, as they were isolated as suppressers of the kefB110
mutation.2 However, sequence
analysis during the current work revealed that the strain carries two
mutations, L75S and D157N, that together inactivate KefB.
Growth and Cell Viability--
The growth medium used throughout
was KX, where X is the concentration of
K+ (10). Strains were grown overnight at 37 °C in
K10 minimal medium supplemented with 0.04% (w/v) glucose
and 1 µg·ml
1 thiamine. Ampicillin (25 µg·ml
1) was included if the strain carried a plasmid.
Aliquots of 3 ml were washed in K1 buffer, suspended in 30 ml of K1 minimal medium containing 0.2% (w/v) glucose and
1 µg·ml
1 thiamine placed at 37 °C and the
OD650 monitored over time. For analysis of cell viability
the appropriate strains were grown as above and grown to early
exponential phase (OD650 = 0.4) before diluting 10-fold
into fresh prewarmed medium containing MG from a 540 mM
stock solution. Cell viability was determined exactly as described
previously (4).
Potassium Efflux Experiments and Determination of Cytoplasmic
pH--
Potassium efflux and cytoplasmic pH determinations were
carried out as described previously (3, 5, 6) with cells grown at
37 °C in K120 minimal medium (10) supplemented with 0.2% (w/v) glucose and 1 µg·ml
1 thiamine. For the
assay cells were washed and suspended in K0 buffer, which
lacks ammonium sulfate and MgSO4. To determine the intracellular K+ content of cells during growth, samples
were incubated in K120 minimal medium to an
OD650 of 0.8-1.0 and 6 × 1-ml samples were centrifuged through 200 µl of bromodecane oil and the supernatant and
oil removed. The intracellular potassium concentration was then
determined as for potassium efflux (3).
Cloning of kefB--
A 2.6-kb fragment encompassing the
yheR and kefB genes was amplified by PCR from
strain MJF277 using primers KefB3 and KefB4 (Table
II), both of which had BamHI
restriction sites incorporated at their 5' ends. The PCR products
obtained were end-filled by treating with the Klenow enzyme, restricted
with BamHI, ligated into similarly restricted plasmid pHG165
(11) to create plasmid pKefB, and transformed into strain JM109. Klenow
treatment, restriction enzyme digestion, ligation, and transformation
procedures were carried out following standard protocols (12).
DNA Sequencing--
For DNA sequencing, the cloned
yheR and kefB genes from plasmid pKefB and mutant
plasmids were amplified in 450 ± 50 base pairs overlapping
fragments, using primers designed specifically to complement the
available yheR and kefB gene sequences from the
E. coli genome project (13). The PCR products obtained were cleaned using Promega PCR DNA clean-up kit. A cycle sequencing reaction
with each one of the primer pair used for amplification was performed
using Applied Biosystems sequencing premix. The products were cleaned
by ethanol precipitation and run on the Applied Biosystems 373A
sequencer before being analyzed using the Applied Biosystems
"Sequence Editor" program.
Site-directed Mutagenesis--
To create plasmid pKefB-2, which
carries an aspartate residue at position 262 of the KefB protein in
place of the wild-type alanine residue (Table I), site-directed
mutagenesis was performed. Primer KefB8 (Table II) was designed such
that it encompassed bases 774-792 of the kefB gene (amino
acids 258-264 of the KefB protein) and contained one mismatch base
T781A, which created A262D. Used in conjunction with primer KefB3,
KefB8 amplified, by PCR, a product of 1.6 kb in length. This product
was Klenow-treated, cleaned, and restricted (as above) at two internal
restriction sites, ClaI and DraIII. The resulting
1.15-kb restricted product was ligated into similarly restricted
plasmid pKefB to create plasmid pKefB-2. The A262D mutation was
confirmed by DNA sequencing of the 1.15-kb insert (as above).
All other mutant plasmids were obtained using the following method,
which is based on a technique developed by Stratagene. Parental or
wild-type plasmid DNA was purified from a strain that methylates its
DNA (JM109 was used for this purpose), and this was used as template
for 18 rounds of PCR using the appropriate mutagenic primers (Table II)
and Pfu polymerase. Restriction with DpnI, an
enzyme that restricts methylated DNA only, digests template DNA, while
leaving amplified and, therefore, mutated DNA undigested. After
transformation of the restricted PCR reactions into JM109, the majority
of colonies obtained, therefore, should contain the desired mutant
plasmid. Analysis of the putative mutants was by restriction enzyme
digestion followed by DNA sequencing (see above).
 |
RESULTS |
Cloning of the kefB Gene from E. coli--
The kefB
locus at 75 min on the E. coli genetic map is required for
K+ efflux elicited by MG (4). Analysis of open reading
frames in this region of the E. coli genetic map identified
a sequence, ECOUW67_274 (P45522) with strong sequence similarity to
KefC from E. coli and KefX from Hemophilus
influenzae and Myxococcus xanthus. The predicted open
reading frame is 601 amino acids (compared with 620 residues for KefC)
shows 42% identity and 70% similarity at the amino acid sequence and
exhibits similar domain organization to KefC. Another open reading
frame ECOUW67_275 (P42621), yheR, overlapped kefB
by a single base the 5' end. This gene arrangement is similar to that
found for kefC, in which an upstream open reading frame,
yabF, is required for the activity of the KefC protein (14).
The putative yheR-kefB region was amplified and cloned into
plasmid pHG165 to create pKefB (see "Experimental Procedures") and
transformed into strain MJF276 (KefB
KefC
).
The cloned fragment was sequenced and confirmed to carry the same
sequence as that deposited in the data base (13). The transformants were analyzed for electrophile-elicited K+ efflux activity
and for restoration of protection against MG.
Strain MJF276/pKefB rapidly lost 25% of the cell K+ pool
on suspension into K0 buffer (first time point 40 s
after suspension in K0), and the pool declined to less than
50% of the control over a 25-min incubation (Fig.
1A). The K+ pool
of MJF276/pKefB was equal to, or greater than, that of MJF276 prior to
suspension in K0 (705 ± 21 and 598 ± 51 µmol·g
1 dry cell mass, respectively). Addition of MG
caused more than 85% of the K+ pool to be lost in the
first 7 min of the incubation with the electrophile. The MG-elicited
rate of efflux was considerably faster than that observed with strain
MJF276 (KefB
KefC
) and MJF274
(KefB+ KefC+), which carries a single
chromosomal copy of the kefB gene (note that KefC makes
little contribution to MG-elicited efflux) (Fig. 1B). It is
notable that the initial rate of K+ loss after addition of
MG is slower than the maximum activity, which was achieved
approximately 3-5 min after addition of the electrophile. Activation
by NEM, which reacts spontaneously with glutathione to form the
activator N-ethylsuccinimido-S-glutathione elicited high rates of K+ efflux from MJF276/pKefB (Fig.
1C). The rate of K+ efflux declined steadily as
the K+ pool declined. When compared with data for strains
MJF274 (KefB+ KefC+) and MJF277
(KefB+ KefC
), which possess single copies of
KefB, these data suggest a 10-12-fold increased expression of the KefB
protein in MJF276/pKefB.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 1.
The cloned kefB gene
restores MG-elicited potassium efflux to cells of strain
MJF276. A, spontaneous potassium efflux;
B, potassium efflux elicited by MG (3 mM);
C, potassium efflux elicited by NEM (0.5 mM).
Time 0 indicates suspension of the cells in K0 buffer;
after 3 min (arrow) either MG (B) or NEM
(C) was added. Symbols: and , MJF276
(KefB KefC ); and , MJF274
(KefB+ KefC+); ×, MJF277 (KefB+
KefC ); and and , MJF276/pKefB
(KefB+).
|
|
The cloned kefB gene provided full protection against MG.
When incubated with 0.4 mM MG growth of E. coli
cells was inhibited but strain MJF274 (KefB+
KefC+) and MJF276/pKefB recovered and subsequent growth
occurred at the same rate. Strain MJF276, which lacks functional KefB
and KefC systems, also recovered but at a much slower rate (data not shown). When exposed to higher concentrations of MG cell death ensued
and the degree of survival was greater in MJF276/pKefB than in MJF274
(Fig. 2A). The enhanced
protection afforded by the higher activity of KefB in strain
MJF276/pKefB correlated with the rate and magnitude of the lowering of
cytoplasmic pH (pHi). Thus, on addition of MG, the cytoplasmic
pH of MJF276/pKefB fell rapidly to a level lower than in either MJF276 or MJF274 (Fig. 2B), and these observations are
consistent with our previously published model (5, 6).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2.
The cloned kefB gene
restores viability to cells of strain MJF276 due to a decrease in
pHi. Viability (A) and pHi
(B) in the presence ( , , ) and absence ( ) of MG
(0.6 mM). The arrow indicates the time at which
MG was added. Symbols: and , MJF276 (KefB
KefC ); , MJF274 (KefB+ KefC+);
and , MJF276/pKefB (KefB+). Closed symbols
represent addition of MG and open symbols no addition.
|
|
Mutations Affecting the Regulation of KefB
Activity--
UV-induced chromosomal kefB mutants, which
exhibit a rapid K+ leak, have been isolated previously (1,
10). Five independent mutants, MJF110, MJF111, MJF113, MJF115, and
MJF117, were analyzed by PCR amplification of gene fragments from the
mutant kefB genes. In each case the same single amino acid
change was observed, L75S. This residue is strongly conserved in
members of the KefB/C family for which the gene sequence is available
(Fig. 3). The amino acid change causes
rapid spontaneous K+ efflux via the chromosomally encoded
KefB system (Fig. 4A). The addition of either MG or NEM did not greatly amplify the rate of
K+ efflux, which may indicate that the L75S mutation causes
the protein to achieve almost maximum activity.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 3.
Sequence alignment of the regions surrounding
L75S (A), the HELETAID motif (B), and
the Rossman fold motif of KefB from known homologues
(C). KefB and KefC from E. coli
(KefB E. coli and KefC E. coli), KefX from H. influenzae (KefX Haem),
and KefX from M. xanthus (KefX Myx). Residues
shaded in black are conserved. Leu75,
Ala262, and Val428 in KefB are indicated
(arrow).
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
L75S in KefB is important for channel
regulation and activity. A, potassium efflux from the
KefB leaky mutant, MJF111 (kefC::Tn10;
kefB111; L75S). Symbols: , MJF111; , MJF111 plus 3 mM MG; and , MJF111 plus 0.5 mM NEM. The
electrophile was added after 3 min (arrow). B,
partial suppression of K+ efflux in strain MJF111/pKefB.
Symbols: , spontaneous K+ efflux from Frag5
(KefB+ KefC+); , Frag5/pKefB; , MJF111
(kefC::Tn10, kefB111; L75S);
, MJF111/pKefB; and , MJF111/pkC11. Plasmids pKefB and pkC11
carry the yheR-kefB and yabF-kefC genes,
respectively, and their upstream regulatory regions. Time 0 indicates
suspension of the cells in K0 buffer.
|
|
We have shown previously that the wild-type kefC gene can
suppress mutations that cause partial spontaneous activation of KefC.
The rapid K+ leak seen in strain MJF276/pKefB was not
observed in strain Frag5/pKefB; there was no immediate loss of
K+ on suspension in K0 medium, and the cells
retained a similar K+ pool to Frag5 throughout the
incubation (Fig. 4B). Since Frag5 is the isogenic parent of
MJF111, this enabled the potential suppression of the KefBL75S mutant
by the wild type gene to be analyzed (Fig. 4B). Potassium
loss was consistently observed to be slower from MJF111/pKefB than from
MJF111. However, the effect was small relative to the suppression seen
previously with the cloned kefC gene and kefC
missense mutants (9, 15). Introduction of pkC11, which carries the
kefC gene in the same plasmid vector as pKefB, did not alter
the rate or extent of K+ loss. These data suggest that the
small effect seen with pKefB is specific and is not due to a general
change in membrane organization consequent upon the higher level of
expression of the KefB system in MJF111/pKefB. Thus, the L75S mutation
has a profound effect on the regulation of the activity of the KefB
system and is dominant over the wild-type allele.
Regulatory Mutations in KefB and KefC--
We have described
previously the effects of a number of mutations that increase the
spontaneous activity of the KefC system (9). Since KefB and KefC
display significant similarity of sequence and organization, we sought
to determine whether each was affected by mutations that affect the
spontaneous activity of the other, i.e. do they share common
control points.
A262D--
We have established previously that the KefC mutation
D264A causes high rates of spontaneous K+ efflux (9). The
KefB protein carries an alanine at the equivalent position (A262) as
the wild type sequence (Fig. 3). Since pKefB causes a spontaneous leak
we generated the mutation A262D, predicting that it would reduce the
leak, but strain MJF276/pKefB-2, which carries the A262D mutation,
exhibited only a slightly reduced rate of spontaneous K+
efflux (data not shown). The initial rate of MG-induced K+
efflux was significantly inhibited and there was a slight reduction in
NEM-elicited efflux (data not shown). Therefore, it is clear that this
residue plays a less significant role than D264 in KefC.
V427A--
Mutations in the Rossman fold of KefC (R416S and V427A)
result in a similar phenotype to that seen with the L75S mutation in
KefB (9). Val427 is conserved in the KefC family of
proteins (Fig. 3) and the E. coli KefC mutant V427A exhibits
rapid K+ efflux when present in single or low copy number
(9). Strain MJF276/pkC11-4 (V427A), which is a multicopy plasmid based
on pkC11 (Table I), failed to grow even in K120 medium
suggesting that the K+ leak is too severe to allow growth.
In contrast, only a small increase in spontaneous K+ leak
was observed when the equivalent V428A mutation was introduced into
KefB (Fig. 5; cf. pKefB and
pKefB-4). Rates of MG-elicited efflux were rapid but showed no
significant difference between pKefB and pKefB-4 (V428A) (data not
shown).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
The effect of HALESDIE on KefB
regulation. Spontaneous K+ efflux was measured after
suspension of the cells in K0 buffer. Symbols: MJF276
(KefB KefC ) transformed with: pKefB
(wild-type KefB) ( ), pKefB-3 (HALESDIE) ( ), pKefB-4
(V428A) ( ), and pKefB-5 (V428A/HALESDIE) ( ). All pKefB constructs
carry the upstream transcription and translation sequences, the
yheR gene, and the kefB gene.
|
|
The HALESDIE Sequence--
Located between two highly conserved
regions of KefC is a variable sequence HALESDIE that contains three
acidic residues in all four known sequences (Fig. 3). Two UV-induced
mutations in this region in E. coli KefC, D264A and E262K,
enhance spontaneous K+ efflux (9). The KefB protein also
has three acidic residues in the equivalent sequence (HELETAID), but
also carries an alanine residue at position 262, echoing the D264A
mutation in KefC. Therefore, we determined whether it was the presence
of three acidic residues or their location at specific positions that
controlled the activity of the KefC system. We exchanged the equivalent
regions from KefB and KefC, namely the HELETAID and HALESDIE motifs,
respectively, and measured the spontaneous and electrophile-induced
rates of K+ efflux (Figs. 5 and
6, A and B).
Replacement of the KefB HELETAID with KefC HALESDIE in plasmid pKefB-3
(Table I) had only a small effect on spontaneous efflux, enhancing the
initial rate approximately 2-fold (Fig. 5). The mutation did not
significantly affect the rate of electrophile-elicited efflux, which
was faster than the spontaneous rate of K+ loss (data not
shown). Combinations of the HALESDIE motif and V428A in KefB (pKefB-5)
also led to higher spontaneous rates of K+ efflux, but the
double change did not emulate the severity of the combination in KefC.
Electrophile-elicited efflux was not significantly affected in the KefB
mutant (data not shown). In contrast, in KefC, replacement of the
HALESDIE sequence with HELETAID (plasmid pkC11-3) significantly
enhanced the spontaneous K+ loss (Fig. 6A). This
multiple change creates in KefC the D264A mutation but leaves three
acidic amino acids in the motif. As a control, an equivalent plasmid
pkC11-2 (KefC D264A) was created. Strain MJF276/pkC11-2 failed to grow
in K120 medium, suggesting that the K+ leak
overwhelms the uptake capacity of the strain. In contrast, MJF276/pSM19
(D264A), which has reduced expression of KefC due to a deletion 5' to
the structural gene, was able to grow normally in K120
medium (9). Strain MJF276/pkC11-3 (HELETAID), which recreates the D264A
mutation but in a different context to pkC11-2 (KefC D264A) (Table I),
grew normally in K120 medium and exhibited only a moderate
K+ leak. These data suggest that the D264A mutation in
plasmid pkC11-3 (HELETAID) is partially compensated by the presence of
the three acidic residues in the motif.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 6.
The effect of HELETAID on KefC regulation and
activation. K+ efflux was measured after suspension of
the cells in K0 buffer. The electrophile was added
(arrow) after 3-min incubation of the cells in
K0 buffer. A, spontaneous; B, 3 mM MG-elicited potassium efflux. Symbols: MJF276
(KefB KefC ) transformed with: pkC11
(wild-type KefC) ( ), pkC11-3 (KefC HELETAID) ( ), pkC11-1 (KefC
L74S) ( ), pkC11-5 (KefC HELETAID/L74S) ( ), and pKefB
(KefB+) ( ). All pkC11 constructs carry the upstream
transcription and translation sequences, the yabF gene, and
the kefC gene.
|
|
L75S--
The KefB L75S mutants exhibited rapid spontaneous efflux
(Fig. 4). The importance of this residue in regulating KefC activity was therefore investigated. Strain MJF276/pkC11-1 (L74S) exhibited spontaneous K+ efflux, such that there was a rapid initial
loss of K+ (approximately 16% of the K+ pool)
followed by a slower loss of 45% of the K+ pool over 25 min (Fig. 6A). However, given that the mutation in the
kefC gene is carried on a multicopy plasmid, which leads to
an approximately 20-fold increase in KefC protein (4), this rate of
K+ loss is slow compared with the rate of spontaneous
K+ efflux observed from the chromosomal KefBL75S mutant
(MJF111). This observation applies to both spontaneous (Fig.
6A) and MG-elicited (Fig. 6B) efflux.
Transformants carrying pKefB-1 (L75S), a construct equivalent to
pkC11-1 (KefC L74S), grew poorly in K120 medium and could
not be assayed for K+ efflux. Therefore, the L75S mutation
has a much greater effect on the activity of KefB than on KefC.
When the L74S and HELETAID mutations were combined in KefC (plasmid
pkC11-5) spontaneous K+ efflux was so rapid that at the
first time point (approximately 40 s) the cells were completely
depleted of K+ (Fig. 6A). These cells grew
poorly and even in K120 medium achieved a rate that was
only 78% of that of MJF276/pkC11 (µ = 0.6 h
1 and 0.47 h
1, for MJF276/pkC11 and MJF276/pkC11-5 (HELETAID + L74S), respectively. Thus, L74S acted synergistically with the HELETAID
mutation. These data are consistent with the effect of the L75S
mutation on KefB, which naturally possesses the HELETAID motif, and
suggest that these two regions are critical to maintenance of the
closed state of KefB.
 |
DISCUSSION |
These studies were undertaken to ascertain whether the amino acid
residues critical to the regulation of two homologous K+
efflux systems were the same. KefB and KefC are 601 and 620 amino acid
proteins, respectively, and are 42% identical and 70% similar in
their sequences. The linker regions (amino acids 380-400 in KefB) are
quite diverse and the major points of sequence deviation lie in the
extreme carboxyl-terminal region. In view of their overall similarity,
it was reasonable to expect that they might possess common regions
responsible for the regulation of their activity. KefC is maintained in
an inactive state even when present on a multi-copy plasmid, except in
the presence of an activating electrophile (7). We have documented
previously a number of KefC mutations that increase the spontaneous
K+ efflux via this protein (9). The mutations substantially
increased the rate of K+ loss from cells such that they
could not grow in media low in K+ (K0.2) (1, 9,
15). The mutations with the greatest effect on activity clustered to
two sequences, the Rossman fold and HALESDIE, suggesting that these
might be significant controlling regions in the protein. However, this
study suggests that KefB and KefC have evolved different critical
residues and that sequence conservation alone is not a guide to the
identification of important sequences.
The HALESDIE region is different in KefB and KefC despite strong
conservation in the flanking sequences (Fig. 3). Both proteins, and the
KefX proteins of H. influenzae and M. xanthus,
contain three acidic residues in this sequence, but it is noteworthy
that their positions are not conserved. This study aimed to analyze the
relative importance of position and sequence. Cells overexpressing the
KefC D264A mutation in the HALESDIE context exhibit a much more
profound growth defect in K120 medium than those where the mutation is surrounded by HELETAID, which retains the three acidic residues. The rate of spontaneous K+ loss in MJF276/pkC11-3
(KefC HELETAID) is similar to that observed previously in MJF276/pSM26
(9), which carries the kefC D264A mutation but expresses the
KefC protein at an approximately 20-fold lower level. Consistent with
this observation, MJF276/pkC11-2 (KefC D264A), which has high level
expression of the KefC system, cannot grow in K120 medium.
These data suggest that the context of the D264A mutation is a
significant determinant of its impact on KefC activity and is
consistent with the hypothesis that the number of acidic residues in
the HALESDIE region of KefC is more important than their absolute position.
Five independent UV-induced mutations causing fast spontaneous
K+ leak via KefB were found to be L75S. The importance of
Leu75 is consistent with the observations on the
L74S/HELETAID double mutant of KefC. Combination of L74S and HELETAID
in KefC resulted in spontaneous efflux characteristics, resembling
those of KefB (L75S). The KefB mutation is more severe than the change
in KefC, since strain MJF276/pKefB-1 (L75S) could not grow in
K120 medium, whereas MJF276/pkC11-5 (KefC L74S/HELETAID)
grew, albeit with a reduced growth rate. The combination of the two
mutations had a synergistic effect on spontaneous K+ loss
via KefC (Fig. 6A). These data strongly suggest a possible interaction between the region surrounding L75S and the HELETAID motif
that leads to the maintenance of the protein in the closed state.