From the Department of Pharmacology, School of
Medicine, Wayne State University, Detroit, Michigan 48201, the
§ School of Medical Technology, Chung Shan Medical and
Dental College, Taichung 402, Taiwan, and the ¶ BioScience Center
and
Laboratory of Molecular Microbiology, School of Agriculture,
Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
Received for publication, January 31, 2001, and in revised form, March 7, 2001
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ABSTRACT |
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The recently identified role of LeuO in the
regulation of transcription has prompted us to search for the specific
function(s) of LeuO in bacterial physiology. The cryptic nature of
expression of leuO has previously limited such analysis. A
conditional leuO expression was found when bacteria enter
stationary phase and was shown to be guanosine
3',5'-bispyrophosphate-dependent. Multiple physiological
events, including the stringent response, are induced upon the increase
of the bacterial stress signal, guanosine 3',5'-bispyrophosphate. In
this study, we tested whether LeuO was directly involved in the
bacterial stringent response. LeuO was shown to be indispensable for
growth resumption following a 2-h growth arrest caused by starvation
for branched-chain amino acids in an E. coli K-12
relA1 strain. This result supports a functional role for
LeuO in the bacterial stringent response.
The overexpression or underexpression of leuO
has been shown to affect a number of bacterial
hns In order to better understand the physiological impact of
leuO expression, we studied the LeuO function in the
ppGpp-dependent bacterial stringent response. Bacterial
stringent response is caused by a limited availability of amino acids
or by exhaustion of the primary carbon source (reviewed in Ref. 12).
Hence, the stringent response can be induced by limiting the
availability of a certain amino acid when such a strain that is
auxotrophic for that amino acid is growing in a chemically defined
medium. More interestingly, a stringent response can also be induced in an autotrophic strain due to the toxic effects of some amino acids. For
example, an overdose of valine was known to cause isoleucine starvation
in the Escherichia coli K-12 strain (13), while
supplementation with one-carbon amino acids, serine, methionine, and
glycine (SMG) in a chemically defined minimal medium was known to
deplete the endogenous branched-chain amino acids (14). The SMG-caused
toxic effect can be reversed by supplementing with branched-chain amino acids or the precursors of branched-chain amino acid biosynthesis (15).
These observed toxic effects reflect the complex metabolite-mediated feedback controls in the bacterial biosynthetic pathways for amino acids (13).
The relA gene encodes an enzyme responsible for the
synthesis of ppGpp, which is crucial in triggering the bacterial
stringent response. It was not surprising to find that SMG inhibited
the growth of relA mutants (13-15). Under such a
branched-chain amino acid-depleted condition, the growth of both the
relA wild-type (stringent) strains and the relA
mutant (relaxed) strains were dramatically interrupted. Upon the
nutrient downshift, the stringent strains resumed normal growth within
several hours, whereas the relaxed strains remained inhibited. Since
the addition of branched-chain amino acids could efficiently reverse
the inhibition (15), it suggested strongly that derepression of the
ilv (isoleucine-leucine-valine) operons are
impaired in the relA mutants while the derepression can be
induced in the relA+ strains within 1-4 h as a
stringent response (15). The ilv operons encode acetohydroxy
acid synthase isozymes that are responsible for the synthesis of
precursors for the biosynthesis of branched-chain amino acids,
isoleucine, leucine, and valine (reviewed in Ref. 16). The expression
of leuO was shown to be regulated via a promoter relay
mechanism (5) that is decisively controlled by the transcription
activity of ilvIH, which is one of the three ilv
operons in bacteria. We therefore tested whether the specific ilv derepression revealed the potential function of LeuO in
response to starvation for branched-chain amino acids. If the
expression of leuO is indeed induced upon the derepression
of the ilv operon via the promoter relay, then LeuO function
may be important for the bacterial stringent response. Clavo's group
has demonstrated that increase in cellular ppGpp level is crucial for
the expression of the leucine-responsive regulatory protein (Lrp),
which is the positive regulator for the expression of ilvIH
(17, 18). This result provides a reasonable explanation for why the
biosynthesis of ppGpp is required for the expression of leuO
(11). However, it was unclear whether the level of leuO
expression correlated with the cellular ppGpp level.
To address these questions, we took advantage of the E. coli
relA1 strains that are "relaxed" but retain a residual
"stringent response capability" owing to a low level ppGpp
synthetic capacity (19). The relA1 mutation is due to an
insertion of an IS2 element between the codons 85 and 86 of the
relA gene (20). Owing to promoter activity originating from
the IS2 insert, the mutant gene retains a low level of ppGpp synthetic
activity. The residual stringent response capability of the
relA1 strain may be important for a "slow onset"
derepression of ilv operons that allows detection of
transient leuO induction upon the ilv
transcription activity. The ilv derepression is expected to
be efficient in a relA+ stringent strain, as are
other stringent responses due to the "rapid onset" of cellular
ppGpp in the relA+ strain. These phenotypes are
in contrast to that of a completely relaxed strain, such as a
relA/spoT double mutant, which will not cause the
derepression of ilv due to the absence of ppGpp and will not
survive the starvation of branched-chain amino acids either (21).
Bacteria--
Bacteria used in this study were derived from an
E. coli K-12 strain, MC4100 (22). The genotypes are listed
in Table I. TO2 is a
RH147 and RH12079 were gifts from Dr. Hengge-Aronis (Freie University
Berlin, Germany). The isogenic pair of
relA+ and relA1 strains was
constructed by P1 transduction of relA+ or
relA1 allele linked
fuc-3072::Tn10 into MC4100 (23).
RH12079 is essentially MC4100, and RH147 is the
relA+ derivative of MC4100.
Bacteria were grown aerobically at 37 °C in a synthetic (chemically
defined) medium base, SSA, which is a minimal salt buffer (K2HPO4 (Sigma), 10.5;
KH2PO4 (Sigma), 4.5;
(NH4)2SO4 (Sigma), 1.0; sodium
citrate dihydrate (Sigma), 0.97 (each component is in grams per liter
of deionized water)) plus 4 µg/ml thiamin, 0.2% glucose, 20 µg/ml
guanine, 40 µg/ml adenine, and 50 µg/ml MgSO4. The
no-aa SSA medium is the SSA medium base without any amino acid
supplement. The following amino acids were supplemented to the SSA
medium base for the various SSA media as described under "Results."
20 µg/ml each of L-histidine, L-lysine,
L-tyrosine, L-tryptophan,
L-threonine, L-cysteine,
L-methionine, L-serine, L-alanine,
and L-aspartic acid, and 40 µg/ml each of
L-phenylalanine, L-glutamic acid,
L-proline, L-arginine, L-valine,
L-isoleucine, L-leucine,
L-asparagine, L-glutamine, and
L-glycine were used when necessary. Ampicillin (50 µg/ml)
was added to cultures as needed.
Due to the possible selection of RNA polymerase mutants in the
synthetic media with various amino acid supplements (15), fresh
cultures inoculated from the frozen stocks were prepared for each
experiment. Prolonged exposure to the selection pressure was prevented.
As a precaution, at the end of each experiment, the relA1
strains were checked for the characteristics of the relA1
mutation using both the serine hydroxamate inhibition assay (24), and
the susceptibility assay for the toxic effect mediated by SMG, or by an
excessive amount of valine or leucine as described previously (14).
Plasmid--
pWU204 was made by a two-step cloning procedure.
First, a 1291-bp HindIII-NdeI fragment containing
galK coding region was deleted from pWU804 (10) to generate
pWU804S. Second, a 1919-bp fragment containing E. coli
chromosomal sequence from the ilvIH promoter (+53 of the
ilvIH operon; Ref. 25) to the leu promoter (+64
of the leu operon; Ref. 26) was generated by PCR. The
sequence of the 1.9-kilobase pair DNA has been determined and deposited to the GenBankTM data base (NCBI accession no. AF106955).
BamHI and AccI sites were introduced into the 5'
and 3' end of the PCR product, respectively, by incorporating mismatch
base pairs in the primers. The
BamHI/AccI-digested PCR product was cloned into pWU804S for replacing the 1.9-kilobase pair S. typhimurium
sequence flanked by BamHI and AccI sites and
resulted in pWU204. The parental plasmid, pWU800, from which pWU804 was
originally derived (9) was used as the vector control.
Western Blot Analysis--
Bacterial cells were harvested and
prepared for total protein lysates. Cell densities of the harvested
bacterial cultures were determined by the viable cell counts using LB
agar plates. The protein concentration of lysate was determined using
BCA protein assay kit (Pierce). Based on the obtained information,
lysate prepared from 5 × 106 viable cells contains
~25 µg of total protein. A fixed amount (25 µg) total protein
from each lysate was electrophoresed on a SDS-PAGE and transferred onto
a nitrocellulose (NC) paper for the subsequent immunoblotting.
Hence, the detached LeuO level was normalized to both total cellular
protein content and viable cell number.
A LeuO-specific antiserum was raised by injecting the purified
overexpressed S. typhimurium His-tagged LeuO into a rabbit. The affinity-purified IgG (1.4 mg/ml) from the antiserum was used at a
dilution factor of 1:5000 as the primary antibody to detect the
cellular LeuO protein. The secondary antibody was anti-rabbit IgG
conjugated to alkaline phosphatase. The blot was developed by ECF using
ECF Western blotting kit (Amersham Pharmacia Biotech). The
chemifluorescent signal was detected and quantified by a Storm 840 imaging system (Molecular Dynamics).
A relA1 Mutation-dependent 2-h Growth Arrest Was Found
during the Exponential Growth of MC4100 in the 17-aa SSA
Medium--
An E. coli K-12 relA1 strain,
MC4100, was used for testing the possible LeuO function in the
bacterial stringent response upon starvation for branched-chain amino
acids. The branched-chain amino acid starvation was achieved by growing
MC4100 in a chemically defined SSA medium base supplemented with all
amino acids except the three branched-chain amino acids: isoleucine,
leucine, and valine (the 17-aa SSA medium). The relA1
strains were shown to grow in such a medium normally without the
SMG-mediated toxic effect due to the reverting power of threonine in
the medium (15). However, the combination of the absence of the
exogenous branched-chain amino acids, the presence of SMG-mediated
branched-chain amino acid depletion, and the exhaustion of
intracellular pools of branched-chain amino acids due to rapid protein
synthesis is expected to cause a temporary branched-chain amino acid
shortage (starvation) when cells are exponentially dividing in the log
phase. The relA1 strain should be susceptible to such amino
acid starvation and thus to the derepression of ilv operons.
The promoter relay mechanism (5, 6) predicts that the expression of
leuO should be detectable upon ilv derepression.
In contrast to the rapid onset stringent response in the
relA+ strains, the process is expected to be
slow due to the low ppGpp level in the relA1 strains. This
slow response is expected to cause a distinct growth "phenotype"
compared with the growth of the relA+ strains in
the 17-aa SSA medium. This rationale provided the base of the study and
was tested experimentally.
Indeed, a 2-h growth arrest during the log phase was observed when
MC4100 was grown in the 17-aa SSA medium (Fig.
1). The growth resumed after the 2-h
arrest, suggesting that some major physiological events must have
occurred during that period. This was strikingly consistent with our
prediction that the branched-chain amino acids are most likely depleted
in the rapidly dividing cells under exponential growth in this medium.
Presumably due to the low ppGpp level in the relA1 strain, a
2-h period was necessary to allow the derepression of ilv
operons and to finally overcome the temporary starvation for
branched-chain amino acids during the exponential growth. Hence, this
growth arrest should be specific to the relA1 genetic
background.
The specificity was tested using an isogenic
relA+ and relA1 strain pair, RH147
versus RH12079, constructed by Dr. Hengge-Aronis's group
(23). This E. coli pair is appropriate for testing the phenotype caused by the relA1 mutation since they were
constructed by introducing either the wild-type
relA+ or relA1 into MC4100. A
phenotype caused by the relA1 mutant (RH12079) should be
eliminated in RH147 due to the restoration of the wild-type
relA without a possible interference from any second site
suppresser. In the 17-aa SSA medium, the growth curve of the
relA1 mutant, RH12079, again showed a 2-h growth arrest in
the log phase (Fig. 2A,
unfilled circles; time points 1-4). The 2-h
arrest was not seen in the growth curve of RH147 in which wild-type
relA was restored (Fig. 2A, filled
circles). This observation provided strong evidence that the
2-h growth arrest was indeed dependent on the relA1 genetic
background in MC4100 or RH12079.
The 2-h Growth Arrest Was Caused by the Starvation for
Branched-chain Amino Acids--
The addition of the three
branched-chain amino acids in the amino acid supplement of the growth
medium abolished the 2-h growth arrest (Fig.
3, unfilled
triangles). This result argued that the 2-h growth arrest is
most likely due to the starvation for branched-chain amino acids. Both
the well known SMG-mediated depletion of branched-chain amino acids and
the fast exhaustion of branched-chain amino acids due to rapid protein
synthesis may contribute to temporary amino acid starvation during
exponential growth. Indeed, by omitting all amino acids from the
supplement (and thus in the absence of the SMG-mediated toxic effect),
the relA1 strain grew normally without the 2-h growth arrest
(Fig. 3, unfilled circles). It was therefore
possible that the 2-h growth arrest in the log phase is due to
starvation for branched-chain amino acids, which is partially caused by
the SMG-mediated depletion. As expected, a short (approximate 4 h)
lag phase was observed in the chemically defined rich (20-aa) medium,
whereas a 14-h lag phase was required for the growth of the same
bacterial strain in the 17-aa SSA and no-aa SSA media. The
branched-chain amino acid starvation-specific 2-h growth arrest in the
relA1 strain was used to study the potential physiological
function of LeuO in bacterial stringent response.
A Transient Increase in LeuO Expression Correlated with the relA1
Genetic Background-dependent 2-h Growth Arrest--
Based
on the possibility that ilv was derepressed upon the
starvation for branched-chain amino acids (15), we hypothesized that
leuO expression may be associated with the relA1
genetic background-specific growth arrest. To address this issue, the cellular LeuO level was immunologically detected during the growth of
the isogenic relA+ and relA1 strains
(Fig. 2B). Western results clearly showed that the level of
LeuO was elevated during the growth slow-down in the relA1
strain, whereas a constant low LeuO level was detected in the isogenic
relA+ strain (time points 1-4 in
Fig. 2B). Quantitation of the Western blot indicated an
~5-fold increase of LeuO at the first two time points (Fig.
2C). The low but constant LeuO level in the
relA+ strain was likely due to the rapid onset
of stringent response when growing in the chemically defined medium
(limited nutrients), since LeuO was almost undetectable during the log
phase when a wild-type E. coli K-12 strain was grown in a
nutrient-rich broth, LB medium (11).
LeuO Is Essential for the Resumption of the Growth of relA1 Strain
after the 2-h Growth Arrest--
The transient LeuO peak in the
relA1 strain, MC4100, was confirmed in a more detailed
Western analysis (Fig. 4E).
The cellular LeuO level was almost undetectable (Fig. 4E,
lane 1), gradually increased (Fig. 4E,
lane 2), peaked during the 2-h growth arrest (Fig. 4E, lanes 3 and 4),
and was reduced when cells resumed their growth (Fig. 4E,
lane 5). The LeuO peak showed an exact
correlation with the 2-h growth arrest in MC4100 grown in the 17-aa SSA
medium (Fig. 4, A and E). This correlation
suggested that, rather than just a consequence of the growth slow-down,
LeuO may play an important role during the 2-h growth arrest. If so,
the growth resumption after the 2-h growth arrest may be dependent on
the expression of leuO. This possibility was examined by
knocking out the leuO in MC4100. The
leuO::cam in TO2 (the donor strain) was
introduced to MC4100 (the recipient strain) using P1 transduction to
form an isogenic knock-out strain, MF1. Immunoblotting confirmed that LeuO was absent from MF1, whereas it was present in the parental leuO+ strain, MC4100 (Fig. 4, compare
F and G), when expression reached maximum during
the growth arrest at A600 = 1.2 (Fig.
4E).
Comparing the growth curves of the isogenic
leuO
The growth resumption of MF1 after the 2-h growth arrest was largely
reversed by harboring pWU204 but not the control vector, pWU800
(compare the filled and unfilled
triangles in Fig. 4C). pWU204 contains the
wild-type leuO and its upstream and downstream regulatory
regions. Otherwise it is identical to the vector, pWU800. The growth
restoration by the exogenous LeuO provided in trans clearly
indicated that the growth blockade was indeed due to the deficiency of
leuO expression in MF1. Therefore, it is clear that the
transient LeuO increase during the 2-h growth arrest (Fig. 4E) is responsible for the subsequent growth resumption of
MC4100 in the 17-aa SSA medium. In the absence of the growth stress
(branched-chain amino acid starvation), both the
leuO+ and leuO LeuO-dependent Growth Resumption (Optical Density
Increase) Is Due to Cell Division Subsequent to the 2-h Growth
Arrest--
So far, the observed LeuO effects on cell growth have been
based on the measurement of optical density. The difference in optical
density readings could, however, be due to some nonspecific causes such
as alteration of light scattering properties of the bacterial cells. In
order to address the LeuO-dependent effect on the cell
growth more directly, viable cell counts were carried out (Fig.
5B) during the growth of
MC4100 and MF1 cells in 17-aa SSA medium (Fig. 5A). The
result clearly indicated that the growth of leuO-deficient
cell, MF1, was arrested but that the arrested cells remained viable up
to 24 h after the inoculation (unfilled circles in Fig. 5B). More importantly, the
relA1 MC4100 resumed its cell division (cell number doubled)
after the transient leuO expression during the 2-h growth
arrest (filled circles in Fig. 5B,
compare the 18-20-h period and the 20-22-h period). The cell density
during the arrested period was ~5.8 × 108 cells/ml
of culture (filled arrow in Fig. 5B).
At the end of the stationary phase (24 h after inoculation), the cell
density of the same culture had reached 4.8 × 109
cells/ml of culture. The 8-fold increase in cell density should be the result of at least three rounds of cell division. Hence, the
transient leuO expression during the 2-h growth arrest
period is likely to be crucial for triggering the subsequent cell
divisions.
Our results can best be explained by the promoter relay mechanism
(5, 6) whereby ilvIH transcription activity (ilv
derepression) triggers the activation of the intermediate
leuO and subsequently enhances the transcriptional activity
of the leuABCD operon. Upon the turn on of ilvIH,
a 3-4-fold enhancement of the transcription activity of
leuABCD was documented (5). Increased leuABCD
transcriptional activity may provide additional leucine biosynthesis
for cells to overcome the temporary but severe branched-chain amino
acid starvation during the exponential growth in the testing condition. This explanation is also consistent with the observed transient LeuO
peak during the 2-h growth arrest, since leucine deprivation is
required for the activation of ilvIH by Lrp (18, 25). Once the cellular leucine level increased due to the 3-4-fold enhancement of leuABCD activity, ilvIH activity was repressed
and so was the expression of leuO.
The stress signal, ppGpp, may be required for activation of
ilvIH via the increase of cellular Lrp level (17) and
subsequent activation of leuO expression. However, this
event does not apparently correlate with the cellular ppGpp level. As
the 2-h growth arrest proceeds (Fig. 2, B and C)
in the relA1 strain, the LeuO levels begin to decrease
slowly. Thus, LeuO levels change inversely to the presumed slow rise in
ppGpp levels in the relA1 strain. More importantly, constant
low LeuO levels were detected (Fig. 2, B and C)
in the relA+ strain, where cellular ppGpp level
is expected to be high under the experimental condition. Since such a
background LeuO level during exponential growth was not observed when
relA+ strains were grown in rich broth/LB (11),
the low but constant LeuO level in the relA+
strain was likely due to the rapid onset of the stringent response when
bacteria were growing in the chemically defined medium. The well known
leucine-mediated feedback control on the expression of ilvIH
(18, 25) may provide the explanation for the observation of the low but
constant LeuO level in the relA+ strain. The
rapid onset of cellular ppGpp level in the relA+
strains is expected to efficiently activate the
ilvIH-leuO-leuABCD regulatory cassette via increasing the
cellular level of Lrp, the positive transcription regulator for the
expression of ilvIH (17), and result in the increase of
cellular leucine level. Then the increased cellular leucine negatively
regulates the regulatory cassette by repressing the transcriptional
activity of ilvIH since leucine is known to weaken the
binding of Lrp with its binding sites located upstream of
ilvIH (18, 25). The equilibrium of these two counteracting
regulatory activities is likely to be responsible for the low but
constant LeuO expression in the relA+ strains.
At the equilibrium, the changes of cellular LeuO levels were too
transient to be detected. If so, the failure of detecting LeuO level
changes does not suggest the absence of the similar stress-responsive
event in the relA+ strains.
This argument is reasonable since the enzyme activities required for
the stringent response should be readily turned on (derepressed) in a
stringent strain due to the rapid onset of cellular ppGpp accumulation,
whereas the enzyme activities remain repressed in the relaxed
relA1 strain due to the slow onset of cellular ppGpp accumulation. This rationale also explains why the relaxed
relA1 strain was in a physiological impasse (the 2-h growth
arrest) from which escape by derepression of the repressed genes (the ilv operons and hence leuO) was necessary for the
resumption of growth. Presumably due to the already derepressed
leuO and ilv operons in the stringent
relA+ strain, the RH147 surpassed the temporary
branched-chain amino acid starvation without a clear delay (arrest)
during the log phase growth (Fig. 2A). In contrast, the
ilv operons (and hence the leuO gene) remain
repressed in relA1 strain prior to the 2-h growth arrest
(Fig. 4). The LeuO level was almost undetectable (Fig. 4E,
lane 1) and gradually increased (Fig.
4E, lane 2) and peaked during the 2-h
growth lag (Fig. 4E, lanes 3 and
4).
Apparently, ppGpp is required for triggering the event including the
leuO expression. Subsequently, a ppGpp level-independent regulatory mechanism must determine the transient leuO
expression. The leucine-mediated feedback control mechanism discussed
above is almost certain to be one of such ppGpp-independent regulatory mechanisms. In addition, a possible positive feedback mechanism effect
directly on the expression of leuO has been suggested in our
recent study (4). It appears that leuO expression is
regulated in a complex manner. Additional studies are required to fully understand the auto-regulation of leuO expression.
Although our results report on a very specific condition for LeuO
expression in a specific strain (relA1 strain), there is, however, a clear-cut leuO The present study suggested a role of LeuO in the cell physiology,
which is essential for the growth (cell division) resumption after the
2-h growth arrest in relA1 strain (Figs. 4C and
5B). What could be the physiological function of LeuO?
Apparently, LeuO does not cause the growth inhibition (the 2-h growth
arrest) since the leuO- relA1 strain,
MF1, also stopped growing under the testing condition (Fig.
4A). Instead, a regulatory role in transcription has been documented for LeuO (4). A direct and global transcription regulatory
function for LeuO is currently being considered and studied in the
laboratory. The conditional leuO expression identified in
the study provides the initial step toward elucidating the physiological function of LeuO in the interesting but largely unknown
bacterial stringent response (reviewed in Ref. 12).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phenotypes (1-3). LeuO may effect these
unrelated genes via its transcription regulatory role, based on
evidence reported in our recent study (4). By studying the
leu-500 activation phenomenon in the Salmonella
typhimurium topA
strains (5-10), we have identified
a 72-bp1 bacterial gene
silencer, AT4, located upstream of leuO. LeuO appeared to
negate AT4-mediated gene silencing (4). This provided a molecular basis
for the speculated transcription regulatory role of LeuO. However,
owing to the cryptic nature of the expression of leuO in
rapidly growing bacterial cells under standard growth condition, the
physiological impact of the leuO expression was unclear. We
have recently found a conditional leuO expression when
bacterial cells enter the stationary phase grown in rich media/LB (11).
Such a conditional leuO expression was demonstrated to be
guanosine 3',5'-bispyrophosphate (ppGpp)-dependent. Since ppGpp is the stress signal in the bacterial stringent response pathway
(reviewed in Ref. 12), leuO may be one of those genes that
are not essential during exponentially growth of laboratory cultures
but important for cell survival in more stressful natural environments.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
leuO strain due to the replacement of the
BstXI-ApaI fragment of the putative
leuO coding region with a DNA fragment carrying the
camr gene (3). MF1 was prepared by introducing
the leuO::cam mutation into MC4100
(recipient strain) by P1 transduction using TO2 as a donor strain.
Hence, MC4100 and MF1 was a leuO+ and
leuO
isogenic pair. Disruption of chromosomal
leuO in MF1 was confirmed by the restriction enzyme cleavage
pattern and the size of the DNA product generated by polymerase chain
reaction (PCR) using primers flanking the camr
insert. Western blotting (Fig. 4G) further confirmed the
LeuO deficiency of MF1.
Bacterial strains used in this study
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A 2-h growth arrest during the log phase of
MC4100 growth in the 17-aa SSA medium. Optical density
was plotted against postinoculation time. The two
arrows mark the start and the end points of the 2-h growth
arrest period. The reported growth curve was one of the seven
experiments that all reproduced the 2-h growth arrest.
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Fig. 2.
LeuO expression correlates with the
relA1 genetic background-dependent 2-h growth
arrest. The isogenic relA+ and
relA1 pair, RH147 and RH12079, were grown in the 17-aa SSA
medium. Panel A, optical density was plotted
against postinoculation time. The difference between the growth curves
of RH147 and RH12079 was reproducible in three independent experiments.
At the marked time points (1-5), cells were harvested.
Total protein extracts were prepared from the harvested cells and an
equal amount (25 µg) total protein from each protein extract was
loaded onto a SDS-PAGE for the subsequent immunoblotting assay, shown
in panel B. The lane
numbers correspond to the time points marked on the growth
curves in panel A. Panel C,
the LeuO band was quantified and plotted against the lane
number.
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Fig. 3.
The 2-h growth arrest is due to the
branched-chain amino acid starvation during the exponential
growth. RH12079 was grown in the SSA medium base with various
amino acid supplements as indicated. Optical densities of the various
RH12079 cultures were plotted against postinoculation time. The
arrow indicates the 2-h growth arrest. The reported growth
curve was one of the four repeated experiments that all reproduced the
demonstrated difference.
View larger version (54K):
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Fig. 4.
LeuO is functionally important for the
resumption of growth after the 2-h growth arrest. Panel
A, the growth curves of MC4100 (filled
circles) and MF1 (unfilled circles)
cultures in the 17-aa SSA medium. Panel B, the
growth curves of MC4100 (filled circles) and MF1
(unfilled circles) cultures in the 20-aa SSA
medium. Panel C, the growth curve of MF1
harboring either pWU204 (filled triangles) or
pWU800 (unfilled triangles) were superimposed
with the growth curve of MC4100 (gray filled
circles) and MF1 (gray unfilled
circles). All four strains were grown in the 17-aa SSA
medium. The reported growth curves were reproducible in four
independent experiments. Panel D, aliquots of
MC4100 cultures in 17-aa SSA medium were harvested and total protein
extracts prepared for a SDS-PAGE. Equal amount (25 µg) of total
protein for each sample was loaded. Both the
A600 readings and the elapsed incubation time of
the MC4100 culture at the harvest are shown above each sample in the
Coomassie Blue-stained gel (panel D). The gel was
transferred on an NC paper for immunological detection of LeuO protein
(panel E). Total protein extracts were also
prepared from MC4100 and MF1 cultures in the 17-aa SSA medium 17 h
after inoculation when LeuO expression was induced in MC4100 (refer to
panel E, lane 4). Various
amounts (from high to low, lanes 1-5) of the
total protein extracts of MC4100 and MF1 were subject to
electrophoresis (panel F) and transferred to an
NC paper and subsequently immunoblotted with LeuO-specific antibody
(panel G). Lane M shows the
high range protein molecular size standards (Life Technologies,
Inc.).
and leuO+ pair in
the 17-aa SSA (Fig. 4A), both MF1 and MC4100 grew
exponentially without a significant difference until the growth slowed
down at A600 = 1.2. Although MC4100 resumed
exponential growth 2 h later and entered the stationary phase at
A600 = 2.1 (filled circles in Fig. 4A), the leuO knock-out strain, MF1,
failed to resume growth and entered directly into a resting state
(optical density remained constant) at A600 = 1.2 (open circles in Fig. 4A). This result confirmed an essential role of LeuO for the growth resumption of
the relA1 strain after the 2-h growth arrest.
relA1 strains grew normally in the chemically
defined rich (20-aa SSA) medium (Fig. 4B). It is, therefore,
clear that leuO expression is a specific cellular response
upon the growth stress caused by the branched-chain amino acid starvation.
View larger version (24K):
[in a new window]
Fig. 5.
Viable cell counts during the growth of
MC4100 and MF1 in 17-aa SSA medium. During the growth of MC4100
and MF1 in the 17-aa SSA medium (panel A), viable
cells were counted by plating diluted cultures on LB agar plates at
various time points. The obtained colony formation units were plotted
against postinoculation time (panel B). The
two arrows mark the time points when LeuO level
in MC4100 peaked (Fig. 4E, lane 3 (unfilled arrow) and lane 4 (filled arrow)).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phenotype (cell
growth is interrupted at A600 = 1.2 in the
absence of the functional leuO gene). Although this
particular experimental condition will provide an excellent opportunity
for evaluating the more complete physiological functions of LeuO in the
relA1 strain, we believe that LeuO is likely to be also
important for normal bacterial physiology in all strains. This is a
typical difficulty of identifying the functions of genes that are not essential for exponentially growing cells. Lack of phenotypes under
standard laboratory testing conditions does not, however, mean the lack
of function physiologically. In fact, due to cryptic phenotypes in
laboratory conditions, the regulation and functions of many genes have
been overlooked. We believe that LeuO is one of those underappreciated
genes in bacteria.
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ACKNOWLEDGEMENT |
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We are in debt to Dr. Heggna-Aronis for providing crucial bacterial strains and to Drs. Ray Mattingly and Ellen Tisdale for their critical readings of the manuscript. We also appreciate comments from the anonymous reviewers who reviewed the earlier submissions of this work.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM53617 (to H.-Y. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF106955.
** To whom correspondence should be addressed: Dept. of Pharmacology, Wayne State University, School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201. Tel.: 313-577-1584; Fax: 313-577-6739; E-mail: haiwu@med.wayne.edu.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M100945200
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ABBREVIATIONS |
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The abbreviations used are: bp, base pair(s); Lrp, leucine-responsive regulatory protein; ppGpp, guanosine 3',5'-bispyrophosphate; aa, amino acid(s); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; NC, nitrocellulose; SMG, serine, methionine, and glycine.
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REFERENCES |
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