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Vol. 170, No. 11
JOURNAL OF BACTERIOLOGY, Nov. 1988, p. 5125-5133
Copyright © 1988, American Society for Microbiology
Reduced leu Operon Expression in a miaA Mutant of
Salmonella typhimurium
Department of Biochemistry, University of California, Berkeley, California 94720
Received 26 February 1988/Accepted 2 August 1988
Salmonella typhimurium tRNA contains 29 different
tRNA base modifications (9) whose syntheses require approximately 40 different enzymes and thus nearly 1% of the
genome of the cell (3). The number and diversity of tRNA
modifications increase with biological complexity, and many
of them are conserved across phylogenetic lines. The chemical identity of many modified nucleosides has been solved
(22), and some of their contributions to tRNA function have
been studied. Modification-deficient tRNAs are defective in
their interactions with ribosomes (18), during translation
elongation (16, 31), in codon-anticodon interactions (39), and
as tRNA nonsense suppressors (6, 7, 21, 33).
In S. typhimurium, the modified base cis-2-methylthioribosylzeatin [N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A)] is present only in tRNAs cognate to codons
beginning with uridine (tRNATrP, tRNAPhe, tRNATYr, and
some species of tRNALeU, tRNACYS, and tRNASe). Ms2io6A
does not occur in the tRNA of Escherichia coli; instead, the
unhydroxylated ms2io6A precursor ms2i6A is present (10).
Synthesis of the ms2io6A modification begins with isopentenylation at the N-6 position of adenine, followed by
2-methylthiolation and finally cis-hydroxylation of the isopentenyl group (8). Physiological conditions have been identified that regulate synthesis of ms2io6A and its precursor
ms2i6A. 2-Methylthiolation is dependent on the availability
of iron (34) and cysteine (1), and cis-hydroxylation occurs
only during aerobic respiration (8). It has been proposed that
ms2io6A and its precursors ms2i6A and i6A may be part of an
integrated signaling system for controlling genes related to
electron transport pathways essential for both aerobic and
anaerobic respiration (8).
A mutation has been isolated in S. typhimurium which
results in complete loss of this modification due to inactivation of a gene (miaA) encoding an isopentenyl transferase
(16). A mutation in a similar locus has been described
previously for E. coli (15, 42) and has numerous effects in
both species. In E. coli, the miaA mutation causes derepres-
sion of operons related to aromatic amino acid metabolism
(20, 42) and reduced tRNA suppressor efficiency (33). In S.
typhimurium this mutation (miaAl) is also highly pleiotropic
and reduces cellular yield and tRNA nonsense suppressor
efficiency (7, 16). In the work presented here, several new S.
typhimurium miaA mutant phenotypes are described which
are shown to result primarily from defective synthesis of
branched-chain amino acids.
Bacterial strains, phage, and media. Bacterial strains are
described in Table 1. Phage P22 HT105Iint-201 was used for
strain constructions in Salmonella typhimurium. Complex
media were nutrient broth (NB; Difco Laboratories), containing 0.5% (wt/vol) NaCl, or LB medium (29), and defined
medium was VB (40) or N- C- medium (2) containing 0.4%
(wt/vol) glucose as the carbon source and 10 mM NH4Cl as
a nitrogen source unless otherwise indicated. Amino acids
were added at 50 ,ug/ml as indicated except for D-leucine,
which was added at 200 jig/ml. Phage indicator plates (37)
were used to purify cells of phage P22. MacConkey base
medium (Difco) was used to screen the heat-sensitive phenotype of miaA mutants. Tetracycline and ampicillin were
added to complex medium at final concentrations of 10 and
50 jig/ml, respectively. Top agar contained 0.6% (wt/vol)
agar and 0.5% (wt/vol) NaCl in distilled water. Solid medium
contained 1.5% (wt/vol) agar.
Genetic methods. Unless otherwise noted, all manipulations of strains carrying miaA mutations were performed at
30°C in LB medium to avoid accumulation of miaA-compensatory mutations (P. Blum and M. Valencik, unpublished).
Crosses involving generalized transduction were performed
as described before with phage P22 (31). Transduction of S.
typhimurium miaA mutations was done as described before
The heat-sensitive miaA mutant phenotype was scored as
the ability to form colonies at 30°C but not at 43°C on
MacConkey medium plates after 24 h of incubation. An
otherwise isogenic wild-type strain was used as a heatresistant control. Analyses of strain sensitivities to chemical
t Present address: Department of Microbiology and Immunology,
Stanford University, Stanford, CA 94305.
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Salmonella typhimurium miaA mutants lacking the tRNA base modification cis-2-methylthioribosylzeatin
(ms2io6A) were examined and found to be sensitive to a variety of chemical oxidants and unable to grow
aerobically at 42°C in a defined medium. Leucine supplementation suppressed both of these phenotypes,
suggesting that leucine synthesis was defective. Intracellular levels of leucine decreased 40-fold in mutant
strains after a shift from 30 to 42°C during growth, and expression of a leu-lacZ transcriptional fusion ceased.
Steady-state levels of leu mRNA were also significantly reduced during growth at elevated temperatures.
Failure of miaA mutant leu-lacZ expression to be fully derepressed during L-leucine limitation at 30°C and
suppression of the miaA mutation by a mutation in the S. typhimurium leu attenuator suggests that translational
control of the transcription termination mechanism regulating ku expression is defective. Since the S.
typhimurium miaA mutation was also suppressed by the Escherichia coli leu operon in trans, phenotypic
differences between E. coli and S. typhimurium miaA mutants may result from a difference between their
respective leu operons.
TABLE 1. Genotype and origin of bacterial strains
S. typhimurium LT2
Source or reference
41eu-1086::Mu dl(Apr lac)
Dilv-1466::Mu dl(Apr lac)
S. Artz
S. Artz
pyrF146 trp43 leu-500 recAl srl::TnJO F+ Mu
1Dleu-1086::Mu dl X::Tn9(Apr lac)
Dilv-1466::Mu dl X::Tn9(Ap' lac) miaAI
4Dilv-1466::Mu dl X::Tn9(Apr lac)
leuL2007 miaAl
miaAI leu-llSl::TnlO
Isogenic with TA4288 but F'101 leu+
hisO1242 hisD6580
E. coli K-12
J. Calvo
G. Bjork
Lab collection
Lab collection
Imprecise eductant of TA3841
Transduction of AZ3127 to Camr with AZ1430 donor
and transduction to Tetr with TA3840 donor;
eduction of TnJO
As for TA4288
Transduction of AZ3157 to Camr with AZ1430 donor
and transduction to Tetr with TA3840 donor;
eduction of TnlO
As for TA4290
Transduction of TA4296 to Leu+ with CV173 donor
Transduction of TA3842 to Tetr with TT206 donor
Mating of TA4288 with KL719
J. Roth
K. Sanderson
K. Sanderson
trpR tna (TnlO)
Isogenic with W3110-18 but miaA (trpX)
F'101 leu+lthr-i leuB6 hisG4 recA13 argE3 thi-I ara-14
lacYl galK2 xyl-S mtl-l rpsL31 tsx-33 supE44
thr-16 pyrF30 his-53 str A(ara-leu) purE41(X PC-0)
thr-J leu-6 thi-I lacYl galk2 ara-14 xyl-5 mtl-l proA2
his4 argE3 str-31 tsx-33 supE44
A(lac-pro) thi strA endA sbcB15 hsdR4 supEiF' traD36
C. Yanofsky; TnlO linked to miaA by phage P1
C. Yanofsky
B. Bachman
J. Calvo
A. McPartland
J. Messing
proAB lacIq
oxidants were performed by the soft agar overlay technique.
Cultures were grown at 300C to the stationary phase in VB
medium containing 0.4% glucose. Cells were pelleted by
centrifugation at room temperature and suspended in an
equal volume of 0.9% NaCl, and 0.1 ml was added to 2.5 ml
of molten (470C) top agar and poured onto VB-glucose
plates. Disks were placed on the surface of the plates, and
the chemicals in the amounts indicated were applied to the
disks. Sensitivity to chemical oxidants was scored after 24 h
of incubation at 300C by measuring the diameter of the zone
of growth inhibition on overlay plates (Table 2). Conditions
used to measure the effect of temperature on growth of the S.
typhimurium miaA mutant (Fig. 1) were identical to those
described for experiments measuring leu-lacZ fusion expression during shift-ups in growth temperature (see Fig. 3).
Operon fusions. Strains AZ3127 and AZ3157 were from a
collection of random phage Mu dl(Apr lac) insertions in S.
typhimurium shown to require leucine or isoleucine and
valine, respectively, for growth in defined medium (Table 1)
as well as the ability to utilize lactose (Lac') (4). The two
insertion mutations were mapped to the leuABCD and ilvGEDA operons, respectively, by demonstrating cotransduction between previously mapped TnJO insertions in leu and
ilv and the phage Mu dl insertion mutant ampicillin-resistant
(Apr) phenotype. The Mu dl(Apr lac) insertion in strain
AZ3127 was 100% linked to leu-JJSJ::TnJO by phage P22-
generalized transduction; 15 of 15 tetracycline-resistant
(Tetr) transductants lost Apr. Similar genetic linkage was
seen between ilvE: :TnJO and the Mu dl(Apr lac) insertion in
strain AZ3157. Transduction of both strains to prototrophy
resulted in 100% loss of the phage Mu dl-associated phenotypes, indicating that the insertion was present in single
Mu dl insertions are unstable at temperatures above 30°C;
therefore, Mu dl insertions were stabilized to allow cultivaTABLE 2. Sensitivity of S. typhimurium and E. coli miaA
mutants to oxidizing chemicalsa
Diam of zone of inhibition (mm)
Oxidizing chemical
S. typhimurium
t-Butyl hydroperoxide (70)
Cumene hydroperoxide (80)
Hydrogen peroxide (30)
Menadione (200)
Chlorodinitrobenzene (250)
Chloramphenicol (120)
E. coli
" S. typhimurium strains were TA3842 (miaAl) and LT2 (miaA+). E. coli
strains were W3110-16 (miaA) and W3110-18 (miaA+).
b Values in parentheses are the amounts applied to each 6-mm paper disk.
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leuL2007 ara-9 gal-205
hisO1242 hisD6404 leu414 supF30 zej-636::Tn5 miaAI
purA837: :Tnl O
Wild type
hisO1242 hisD6610
purA837::TnlO miaAI
AmiaA (purA miaA)51
'1leu-1086::Mu dl X::Tn9(Apr lac) miaAl
VOL. 170, 1988
Time (hr)
FIG. 1. Growth of an miaA mutant (TA3842) during shift-ups in
growth temperature. The arrow indicates the time and cell density at
which the culture was shifted.
tion at elevated temperatures. The ilv-1466::Mu dl insertion
and leu-1086::Mu dl insertion were stabilized by the introduction of a Tn9 insertion located in Mu phage DNA (4).
This insertion destroys phage functions necessary for transposition and host strain killing.
Fusion between the Mu dl lacZ gene and the promoters of
leu and ilv were determined by the ability of Mu dl ("X"
Tn9) insertion mutant derivatives of the leu-1086::Mu dl and
ilv-1466::Mu dl insertions to revert to prototrophy (4).
Results indicating that leu-lacZ fusion expression was from
the leuAp promoter were obtained from experiments indicating that the hisT1504 mutation increased leu-lacZ fusion
expression. The hisT1504 mutation causes underpseudouridylation of tRNA (36) and has been shown to increase
steady-state expression of leu (13), an effect presumed to
depend on the leuAp promoter. Isogenic hisTJ504 mutant
and wild-type strains containing the leu-lacZ fusion were
grown in VB glucose medium at 37°C and sampled to assay
the differential rate of 3-galactosidase expression. The specific activity of P-galactosidase expression in the hisT1504
mutant derivative was 7.4-fold higher than in the wild-type
I8-Galactosidase assays and cultivation conditions. Assays
for the differential rate of P-galactosidase activity were
performed essentially as described before (31). Samples (1
ml) were removed from log-phase cultures and transferred to
prechilled glass test tubes on ice, and their absorbance at 650
nm was recorded. After overnight storage at 4°C, cells were
permeabilized by addition of 10 ,ul of toluene and 20 s of
rapid mixing at room temperature. Toluene was evaporated
by incubation in a shaking waterbath at 37°C for 30 min.
Samples were stored at 4°C for not longer than 30 min before
being assayed for ,B-galactosidase activity. P-Galactosidase
specific activities were determined as differential rates of
enzyme synthesis from differential rate plots by using at least
four samples taken at intervals during growth. ,-Galactosidase units were calculated as the optical density of the
reaction at 420 nm divided by the reaction time in minutes
and the reaction volume in milliliters and then multiplied by
1,000. For measurements of leu-lacZ and ilv-lacZ fusion
expression during shift-ups in growth temperature, strains
were grown overnight at 30°C with glucose limitation (0.04%
[wt/vol] glucose) in VB medium containing leucine or isoleucine, valine, and leucine, respectively. Overnight cultures
were subcultured 1:20 into 50 ml of the same medium with
excess glucose (0.4%, wt/vol) at 30°C. To shift up the
temperature, cultures were subcultured approximately 1:10
into 50 ml of the same prewarmed medium. Portions were
removed at the optical density readings indicated and assayed for P-galactosidase activity. For measurement of
leu-lacZ fusion expression during growth on D-leucine,
cultures were grown overnight in VB medium with limiting
glucose (0.04%) and L-leucine (50 ,ug/ml) at 30°C. Cells were
collected by centrifugation, washed in VB medium, and
transferred to 50 ml of VB medium containing 0.4% glucose
and D-leucine (200 ,ug/ml) at 30°C. After the cultures entered
log phase, samples were removed and assayed for P-galactosidase activity.
Quantitation of free amino acid pools. Intracellular levels of
amino acids were determined by high-performance liquid
chromatography (HPLC) separation and fluorimetric detection of their o-phthaldialdehyde (OPT) derivatives as described before (25, 27). Cells were grown in N- C- medium
containing 0.4% glucose and 5 mM glutamine as a nitrogen
source. Glutamine was used instead of NH4Cl to reduce
peak interference between NH4Cl and branched-chain
amino acids. In this medium, the miaA mutant conditionalgrowth phenotype and hydroperoxide-sensitive phenotypes
were identical to those observed in VB medium. Samples (1
ml) were removed and transferred to polypropylene microfuge tubes on ice, and cells were pelleted by 30 s of
centrifugation at 4°C. Cells were resuspended in 1 ml of 0.9%
NaCl, and 4 ml of HPLC grade methanol was added.
Samples were vortexed briefly, and insoluble material was
removed by centrifugation in a Beckman microfuge at 10,000
rpm for 10 min at 4°C. Samples were lyophilized to dryness
on a spinning lyophilizer, suspended in 0.5 ml of deionized
distilled water, and frozen at -20°C until used for chromatography. To reduce fluorescent contaminants in the assay,
OPT derivatives were prepared by using previously unopened bottles of 100% ethanol and ,3-mercaptoethanol.
Chromatography was performed on a Supelcosil LC-18,
5-jim bead column with model 510 and model 6000 pumps
(Waters Associates). Fluorescence emission above 418 nm
was monitored with a Kratos FS950 Fluromat following
excitation of the sample at 360 nm. Fluromat settings used
were: range, 1.0; sensitivity, 8.1; time constant, 0.5; suppression, 1.0; auto range. Data were collected by a Nelson
Analytical Interface 760 series and processed on a Hewlett
Packard 9816 computer with Nelson Analytical Xtrachrom
software (version 7.1). Mobile phases were 80% buffer A (50
mM sodium acetate, pH 6.2)-20% buffer B (100% methanol)
to 40% buffer A-60% buffer B in 10 min, followed by 27 min
of isocratic elution with 40% buffer A-60% buffer B. A
Waters system controller was used to control the gradient by
using a shallow concave gradient (Water's gradient curve no.
5). The presence of two highly fluorescent compounds with
retention times of approximately 12 and 14 min required
manual resetting of the Fluromat to avoid photocell damage,
although data collection was continuous throughout the
experiment. Retention times in minutes for the following
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Chemical oxidant sensitivity of an S. typhimurium miaA
mutant. Ms2io6A has been suggested to act as a cellular
indicator of oxygen availability (8); therefore, ms2io6A deficiency might perturb cellular responses to oxygen or oxygen-related stress such as chemical oxidants. An S. typhimurium mutant lacking the ms2io6A modification (miaA) was
compared with an otherwise isogenic wild-type strain for
altered sensitivity to chemical oxidants (Table 2). miaA
mutants of E. coli have been described previously (15, 42),
and therefore similar experiments were performed with an E.
coli miaA mutant (Table 2). ms2i6A deficiency in the E. coli
miaA mutant results from a UAA mutation in the miaA gene
(33), which reduces ms2i6A cellular content to between 5 and
16% of wild-type levels (41). Chemical oxidant toxicities
were examined by the soft agar overlay technique, and the
results are expressed as the diameter of the zone of growth
inhibition due to diffusion of the test compound from a paper
disk placed on the surface of the agar plate. The S. typhimurium miaA mutant (TA3842), relative to its wild-type
congenic pair, was unusually sensitive to the hydroperoxides
cumene, tert-butyl, and hydrogen peroxide, the quinone
menadione, and the glutathione depleter chlorodinitrobenzene. Similar results were obtained with another miaA
mutant allele in strain TA4284. The E. coli miaA mutant was
not differentially sensitive to these chemical oxidants relative to its wild-type congenic pair. The S. typhimurium and
E. coli miaA mutants were not differentially sensitive to the
translation inhibitor chloramphenicol (Table 2), deoxycholic
acid, or the dye methylene blue (data not shown), indicating
that the mutants did not have a generalized defect in permeability.
Leucine counteracts heat stress and oxidative stress in the
miaA mutant. Growth (measured as single colony size) of the
S. typhimurium miaA mutant (TA3842) on VB glucose plates
at 37°C was slow relative to that of an isogenic wild-type
control strain (LT2). At 42°C the mutant failed to form
colonies at all. Supplementation of the medium with Lleucine but no other amino acid or vitamin strongly suppressed the growth defect. Growth of the miaA mutant at
42°C was normal on anaerobic incubation. Identical results
were obtained with the miaA mutation in strain TA4284.
At 30°C in VB glucose liquid medium, the miaA mutant
(TA3842) had a 60-min generation time (Fig. 1, open circles).
Shifting the culture from 30°C to higher temperatures resulted in reduced growth rates. Heat-dependent growth
inhibition displayed a marked dependence on the absolute
final temperature of growth; at a postshift temperature of
39°C (solid circles), the mutant maintained preshift growth
rates for at least two divisions before growth ceased, while
postshift temperatures only 3°C higher, 42°C (crosses), resulted in an immediate cessation of growth. Postshift temperatures intermediate between these values gave intermediate amounts of growth inhibition. At 42°C, addition of
L-leucine restored growth to 50% of the normal growth rate,
while addition of all three branched-chain amino acids
increased growth only slightly more than did L-leucine alone.
Unlike the S. typhimurium miaA mutant, the wild-type strain
grew well at 42°C and somewhat faster than at 30°C. Growth
rates for the wild-type strain at 30 and 42°C were 60 and 55
min, respectively. These results indicate that the S. typhimurium miaA mutant is a leucine auxotroph at 42°C and not
a slow-growing leucine bradytroph.
L-Leucine addition also suppressed sensitivity of the S.
typhimurium miaA mutant to chemical oxidants. When
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amino acids were: leucine, 35.8; isoleucine, 23.1; valine,
31.6; phenylalanine, 24.7. Variation between duplicate samples was never more than 10% of total peak areas. The
identities of the cellular OPT amino acid derivatives were
confirmed by comigration with standards prepared in the
laboratory and with commercial standards. Intracellular
amino acid levels were determined by interpolation from
standard curves performed with cell extracts.
RNA blot analysis. leu operon mRNA was isolated and
quantitated as follows. Cells were grown in VB glucose
medium at the temperature indicated to an A650 of 0.8, and
10-ml volumes were removed and added to 30-ml glass
centrifuge tubes containing 5 ml of VB medium frozen at
-20°C. Cells were pelleted by centrifugation at 4°C, and the
cell pellet was rapidly suspended in 0.5 ml of lysis buffer (30
mM Tris hydrochloride [pH 7.4], 100 mM NaCl, 5 mM
EDTA, 1% [wt/vol] sodium dodecyl sulfate [SDS], 20 mM
vanadium ribonucleoside complex [Pharmacia]) and then
extracted twice with equal volumes of phenol-chloroform
(1:1) and once with chloroform-isoamyl alcohol (24:1), precipitated with 2 volumes of ethanol and 0.1 volume of 3 M
sodium acetate, and centrifuged at 4°C for 30 min. The
nucleic acid pellet was redissolved in 0.1 ml of DNase I
buffer (50 mM Tris hydrochloride [pH 7.5], 1 mM EDTA [pH
8.0], 10 mM MgCl2), 40 p,g of RNase-free DNase I (Worthington Biochemicals) was added, and the sample was
incubated for 30 min at 37°C. The reaction was stopped by
adjusting the solution to 1% (wt/vol) SDS and 50 mM EDTA
(pH 8.0), and the RNA was purified by phenol-chloroform
extraction and ethanol precipitation.
A 1.2-kilobase-pair (kbp) restriction fragment containing
the promoter-proximal coding region of the leu operon was
used as a hybridization probe to identify leu mRNA in the
RNA blot analysis. The S. typhimurium leu operon DNA
was obtained from the lambda prophage in strain CV753 as
follows. Lambda PC-0 was induced by UV irradiation, and
the resulting phage were purified as described before (28).
The phage DNA was digested with EcoRI and Sall, and the
2.35-kbp piece (19) was subcloned into M13mp9 previously
digested with the same restriction enzymes. This phage was
designated M13mp9-leul, and the identity of its insert was
confirmed by dideoxy sequence analysis and comparison
with published sequences (19). M13mp9-leul was digested
with PstI, and the 1.2-kbp fragment was resolved on agarose
gels and purified by using Gene Clean (Bio 101). The leu
operon coding sequence in the 1.2-kbp PstI fragment begins
at the position corresponding to amino acid number 30 of the
leuA gene. For hybridization analysis, this fragment was
radiolabeled by random priming with [32P]dCTP and a hexamer labeling kit (Boehringer). Unincorporated [32P]dCTP
was removed by two successive passages through 1-ml spun
columns of Sephadex 50 (Pharmacia).
Whole-cell RNA was diluted in water and dotted onto
nitrocellulose membranes (Schleicher & Schuell) with a
Bio-Rad Laboratories dot blot apparatus as described by the
manufacturer. RNA blots from a culture of TA3842 grown at
30°C bracket the RNA blot derived from a culture grown at
41°C to ensure that probe hybridization occurred consistently over the entire surface of the nitrocellulose membrane. Hybridizations were performed in 50% (vol/vol) formamide at 42°C for 24 h as described before (28). Membranes
were baked for 2 h at 80°C in vacuo prior to hybridization.
Autoradiography of washed membranes was done for 48 h at
-80°C with Kodak XAR film.
VOL. 170, 1988
28 I
FIG. 3. Differential rate of p-galactosidase activity of a leu-lacZ
fusion in the miaAl mutant (TA4288; open circles) and mia+ strain
(TA4289; solid circles) at 30°C (A) and 41.5°C (B). Units are 103
OD420 units per minute per milliliter.
Time at 41.5°C (min)
Decreased expression of the leu and ilv
FIG. 2. Levels of leucine (solid circles) and phenylalanine (open
circles) in the miaA mutant (TA3842) after a shift-up in growth
temperature. The inset shows the corresponding growth curves for
the miaAl mutant (TA3842; solid circles) and mia+ strain (LT2;
open circles). The units (picomoles of amino acid per milliliter per
OD650 unit) are equivalent to the units of picomoles of amino acid
per 2 x 109 cells.
leucine was added to VB glucose plates at a final concentration of 50 ,uglml, all chemical oxidant zones of growth
inhibition for the miaA mutants were reduced to wild-type
levels. Simultaneous addition of the other two branchedchain amino acids, valine and isoleucine, without leucine did
not alter the sensitivity of the miaA mutant to these chemicals.
Heat stress reduces the free leucine pool in an miaA mutant.
Suppression of the S. typhimurium miaA mutant's conditional growth defect by L-leucine (Fig. 1) suggested that
intracellular leucine levels were depleted during restrictive
growth conditions. Intracellular levels of free leucine,
valine, isoleucine, and phenylalanine were measured directly during a temperature shift up in growth from 30 to
41.5°C by using HPLC separation and fluorescence detection
of the OPT derivatives of the amino acid pools (Fig. 2). The
initial sample shown (0 min) was taken immediately prior to
the shift-up in culture temperature; thereafter, data are
shown for samples assayed at 30-min intervals. At 150 min
post-temperature shift, the free leucine pool decreased over
40-fold (solid circles). Leucine pools were approximately the
same in miaA mutant and wild-type cultures at 30°C, and no
change was seen in leucine pools in temperature-shifted
wild-type cultures. Phenylalanine, an amino acid thought to
be uninvolved in the S. typhimurium miaA mutant conditional-growth phenotype, decreased only twofold within the
same time interval (open circles). Valine decreased 2.7-fold
from 1,488 to 542 pmol/ml per OD650 unit, and isoleucine
decreased 4.9-fold from 1,130 to 230 pmol/ml per OD650 unit
(Fig. 2, legend).
in the S.
typhimurium miaA mutant. Reduction of the free leucine pool
in the S. typhimurium miaA mutant following a shift-up in
growth temperature could result from reduced expression of
the leucine-biosynthetic (leu) operon. In vivo expression of
leu was examined in wild-type and miaA mutant strains by
using a stabilized leu-lacZ operon fusion during growth at 30
and 41.5°C (Fig. 3; see Materials and Methods for isolation
and characterization of the fusion). Since the miaA mutant is
a leucine auxotroph at 41.5°C, high-temperature cultivation
of the mutant in a defined medium required addition of
exogenous L-leucine to the medium, and consequently Lleucine was added as a control to cultures of both strains at
both growth temperatures. Expression of the leu-lacZ fusion
was approximately the same in wild-type and miaA mutant
strains at 30°C; ,-galactosidase specific activity in the wildtype strain and in the miaA mutant was 172 and 228 x 103
OD420 units per min per ml, (Fig. 3A), consistent with the
prototrophic phenotype of the mutant at low temperatures.
Within half a generation after the cultures were shifted to
41.5°C (Fig. 3B), expression of the leu-lacZ fusion ceased in
the miaA mutant, while expression decreased slightly in the
wild-type strain. The specific activity of P-galactosidase in
the wild-type strain at the elevated temperature was 84.
Cultures were sampled for 3-galactosidase assays only during exponential growth; therefore, following the temperature
shift and prior to cessation of growth, samples were removed
at short time intervals (Fig. 3). In vivo expression of the ilv
operon was also measured by using an ilv-lacZ operon fusion
(see Materials and Methods for isolation and construction of
the fusion). Expression of the ilv-lacZ fusion was reduced in
the miaAl mutant at 42°C, paralleling the reduction in pool
size of valine and isoleucine. At 42°C, P-galactosidase specific activity in the wild-type strain was 1,040, while in the
miaA mutant it was 170. Therefore, expression of the
ilv-lacZ fusion in the miaA mutant at 42°C was about sixfold
less than that in the wild-type strain.
Heat stress reduces leu mRNA in the S. typhimurium miaA
mutant. lacZ expression in the leu-lacZ and ilv-lacZ fusions
should result from transcriptional fusion between the promoters of these respective operons and the lacZ gene of the
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_ 1200
c 10ooo
800 _
FIG. 4. RNA blot analysis of leu mRNA levels in S. typhimurium
LT2 grown at 30°C and an miaA mutant (TA3842) grown at 30 and
41°C in the presence of added L-leucine.
o 400 _
200 I-
FIG. 5. Differential rate of ,B-galactosidase activity of a leu-lacZ
fusion during L-leucine limitation (growth on D-leucine) at 30°C in an
miaA mutant (TA4288; open circles) and mia+ strain (TA4289; solid
circles). See Fig. 3 legend for P-galactosidase units.
Ieu-lacZ in the miaA mutant, indicating that leu derepression
was defective during L-leucine limitation.
Suppression of S. typhimurium miaA mutant phenotypes.
The leuL2007 mutation is a substitution mutation in the leu
attenuator and constitutively derepresses leu transcription
12-fold (35). This mutation was used to test whether its
effects on leu expression were epistatic to those of the miaAl
mutation. Ieu-J 151::TnJO insertion mutant derivatives of
strains TA3842 (miaAl) and LT2 (mia+) were constructed
and used as recipients to introduce the leuL2007 mutation
from strain CV173 by selection for prototrophy (Table 1).
Transductants containing the leuL2007 allele were identified
by auxonaugraphy as described before (11). The sensitivity
of these strains to hydroperoxides and their ability to grow at
high temperatures are shown in Table 3. Constitutive expression of leu suppressed sensitivity of the miaA mutant to
cumene hydroperoxide and stimulated growth at 42°C. miaA
mutant sensitivity to t-butyl hydroperoxide was unaffected
by increased expression of leu.
The E. coli miaA mutant lacks all of the distinguishing
phenotypes of S. typhimurium miaA mutants. Since the
miaA mutations in both species result in undermodification
TABLE 3. Suppression of S. typhimurium miaA mutant
phenotypes by a leu attenuator mutation or the E. coli leu operon
Zone of inhibitionsa
TA3842 miaAl
TA4295 miaAl leuL2007
TA4297 miaAl leu::Mu dl/F'101 leu
mia+ leu+
at 420C
hydroperoxide peroxide
a Diameters of the zones of growth inhibition were measured at 30°C. Paper
disks (6 mm) were used in the inhibition studies and contained 52 ,ug of
cumene hydroperoxide or 41 ,ug of t-butyl hydroperoxide.
b Growth at 42°C indicates the ability to form single colonies on VB glucose
plates after 24 h of incubation.
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Mu dl insertion. Therefore, decreased expression of these
fusions should result from events occurring at the level of
transcription. To confirm this supposition, levels of leu
mRNA were quantitated directly by RNA dot blot analysis
of total cellular RNA in an miaA mutant during growth at 30
and 41°C (Fig. 4). leu mRNA levels in the isogenic wild-type
strain were also determined at 30°C. RNA was probed with
a purified and radiolabeled 1.2-kbp fragment of the S.
typhimurium leu operon derived by PstI digestion of
M13mp9::leu-1 (Materials and Methods). The 5' end of this
fragment contains the leuA coding region and begins at
codon number 30 of the leuA gene (19). After three generations of growth of the miaA mutant at 41°C in the presence of
exogenous L-leucine, leu mRNA was nearly undetectable in
5 ,ug of total cellular RNA. In contrast, leu mRNA was
present in large quantities during growth of the mutant at
30°C in the presence of added L-leucine in 1 ,ug of total RNA.
Although L-leucine addition was only necessary to support
growth of the mutant at 41°C, L-leucine was added as a
control to cultures of the miaA mutant grown at both 30 and
41°C. In separate experiments, leu mRNA was detected in
approximately equal amounts in RNA preparations from
cultures of the wild-type grown at 30 and 41°C.
Expression of a leu-lacZ fusion during leucine limitation in
the miaA mutant. The involvement of ms2io6A-deficient
tRNA in leucine-specific control of leu was examined by
comparing expression of a leu-lacZ fusion in otherwise
isogenic wild-type and miaA mutant leucine auxotrophs
during growth on D-leucine at 30°C (Fig. 5). Generation
times for both strains on L-leucine were 66 min, but during
growth on D-leucine, generation times were biphasic with an
initial generation time identical to that during growth on
L-leucine (65 min) and a final much longer generation time
(336 min). The change in growth rate for the miaA mutant
culture occurred at an A650 of 0.1, and for the wild-type
culture the change occurred at an A650 of 0.12. These
biphasic generation times are thought to reflect the presence
of trace amounts of contaminating L-leucine in commercial
stocks of D-leucine. The addition of small amounts of
L-leucine to cultures containing D-leucine prolonged the
early and shorter generation time, supporting this explanation, and addition of L-leucine during the second and slower
phase of growth caused a resumption of the initial and more
rapid phase of growth. The specific activity of 3-galactosidase corresponding to the early generation times was 895
for the wild-type strain and 433 for the miaA mutant. Both of
these values are severalfold higher than those seen during
growth on L-leucine alone (Fig. 3), indicating partial derepression of leu under these conditions. As the wild-type
strain entered the second and slower phase of growth,
expression of the leu-lacZ fusion was derepressed further;
the specific activity of ,-galactosidase increased 4.2-fold to
3,770. In contrast, no change was seen in the expression of
VOL. 170, 1988
of adenosine-37 residues in tRNA, phenotypic differences
between the two mutants might result from a difference in
the target of modification-deficient tRNA such as the leu
operon. To test this possibility, an E. coli F' factor carrying
the leu operon was mated into the S. typhimurium miaA
mutant and used to test whether phenotypic differences
between the S. typhimurium and E. coli miaA mutants might
be due to some difference in this operon (Table 3). The S.
typhimurium leu operon in these strains was inactivated by
the presence of a Mu dl insertion. The E. coli leu operon
suppressed the S. typhimurium miaA mutant's sensitivity to
cumene hydroperoxide and its inability to grow at 42°C.
These results indicate that a major target of ms2io6Adeficient tRNA in S. typhimurium is the biosynthetic leu
operon and that some element of this operon that differs
from its E. coli counterpart may be responsible for several of
the unique phenotypes of S. typhimurium miaA mutants.
since cell growth rates and therefore rates of protein synthesis are normal, but at 42°C undermodified tRNA may become defective and alter leu expression via translational
control of the transcription termination mechanism. Since
the E. coli miaA mutant grew well at 42°C, ms2io6A-deficient
tRNA (or ms2i6A in E. coli) may be defective, translating
codons in only certain contexts, which may be absent in E.
coli but present in S. typhimurium. It is also possible that the
small amounts of ms2i6A remaining in the E. coli miaA
mutant (41) may contribute to the less pleiotropic E. coli
miaA mutant phenotype.
Reduced leu induction in the miaA mutant at permissive
growth temperatures during L-leucine deprivation also suggests that translational control of the transcription terminatlon mechanism regulating leu expression is defective. Failure to initiate leader RNA translation or translational
pausing at or near the initiation codon is thought to enhance
transcription termination of attenuator-regulated operons, a
process termed superattenuation (24, 38). A similar process
may occur in the miaA mutant. Consistent with this possibility is the observation that the translation elongation rate
for lacZ mRNA was reduced in the miaA mutant at 37°C
(16). A reduced rate of translation of leader codons was
proposed previously as the cause of leu derepression in hisT
mutants, which are deficient in the pseudouridine tRNA
modification and translate lacZ mRNA more slowly than a
wild-type strain (31). The contrasting effects of a reduced
rate of translation elongation on expression of leu in miaA
and hisT mutants may result from the translational context of
affected codons in the leader sequence as well as the pause
time per codon caused by these two types of tRNA undermodifications. Since the E. coli leu operon suppressed the S.
typhimurium miaA mutant phenotypes, only codons unique
to the S. typhimurium leu leader and cognate to tRNAs
containing ms2io6A could be involved, such as the unique
serine codon adjacent to the AUG initiator codon (19).
Experiments are under way to test this hypothesis.
S. typhimurium miaA mutant hypersensitivity to oxidative
stress may result from an inability to restore leucine levels
depleted by oxidative damage to branched-chain amino
acid-biosynthetic enzymes. Several observations indicate
that branched-chain amino acid biosynthesis in S. typhimurium is an important metabolic target of oxidative damage.
In S. typhimurium, hyperbaric oxygen-mediated and paraquat-mediated growth inhibition in minimal medium can be
suppressed by addition of branched-chain amino acids, and
cumene hydroperoxide treatment of wild-type S. typhimurium cultures depletes the free leucine pool (P. Blum,
unpublished). Numerous reports indicate that branchedchain amino acid synthesis is a target of oxidative stress in E.
coli (5, 12, 14, 17, 23). Recent work indicates that E. coli
dihydroxyacid dehydrase (isoleucine and valine biosynthesis) and isopropylmalate dehydrogenase (leucine biosynthesis) are iron-sulfur cluster proteins and their activities are
unusually sensitive to inactivation by oxidation (J. Schloss,
personal communication). Although leucine supplementation suppressed the miaA mutant's hypersensitivity to tbutyl hydroperoxide, the leuL2007 mutation and E. coli leu
operon in trans did not. This result suggests that t-butyl
hydroperoxide and cumene hydroperoxide have different
mechanisms of toxicity and is consistent with the observation that the former is bacteriostatic and the latter is bactericidal. It is possible that t-butyl hydroperoxide is more
specific to isoleucine and valine biosynthesis (P. Blum,
unpublished) and that genetic alterations in ilv would act in a
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Loss of ms2io6A tRNA modification in S. typhimurium
miaA mutants has been shown to cause pleiotropic changes,
including a generalized reduction in cellular yield as a
consequence of a reduction in tRNA coding capacity (7).
The work presented here identifies several new miaA phenotypes and indicates that a defect in leucine biosynthesis is
the major cause of these phenotypes. At 42°C, miaA mutant
cells are rapidly depleted of free leucine, while production of
leu mRNA is reduced and expression of a leu-lacZ operon
fusion ceases. Suppression of miaA mutant phenotypes by
L-leucine supplementation, by increased leu transcription,
and by the presence of the E. coli leu operon in trans all
suggest that leu expression is dysfunctional. Changes in the
levels of isoleucine and valine and in the expression of the ilv
operon were also observed in the miaA mutant at 42°C.
These changes contributed little to the miaA mutant phenotypes; therefore, this work focused on the basis of defective
leucine synthesis.
Transcription of the leu operon in S. typhimurium and E.
coli is controlled by translation of four contiguous leucine
codons in the leu leader RNA (19). High levels of charged
leucyl tRNA permit efficient leucine codon translation and
cause premature transcription termination at the leu attenuator. Low levels of charged leucyl tRNA inhibit leucine codon
translation and promote continued transcription elongation
through the attenuator. Attenuation of transcription has
been suggested to be the major form of control of this operon
during growth in minimal medium (35). A similar mechanism
has been proposed for the regulation of the ilvGEDA operon
(26, 30). Destabilization of the leu attenuator by the leuL2007
mutation overcame the miaA mutant's conditional-growth
defect and cumene hydroperoxide sensitivity. Therefore,
defective leu expression in an miaA mutant may occur
through the translational control of transcription termination
Ms2i6A-deficient tRNA from E. coli defectively interacts
with ribosomes (18), peptidyl hydrolase (32), and cognate
anticodons (39). It is likely that loss of the hydroxylated form
of this modification (ms2io6A) in S. typhimurium miaA
mutant tRNA would alter tRNA function in similar ways. In
S. typhimurium miaA mutants, steady-state expression of a
leu-lacZ operon fusion was normal at 30°C but ceased at
42°C. leu mRNA levels were reduced in the miaA mutant at
similar temperatures, indicating that reduced leu expression
occurs at the level of mRNA synthesis or degradation.
ms2io6A-deficient tRNA must retain most functions at 30°C,
manner similar to that obtained with leu during cumene
hydroperoxide exposure.
It is unclear to what extent the different forms of the
ms2io6A tRNA base modification (ms2io6A, ms2i6A, i6A, and
A) are involved in the miaA mutant defects. Answering this
question will require the isolation of other mutations in the
pathway to ms2io6A synthesis. It is also possible that the S.
typhimurium miaA mutations examined in this study alter
expression of additional genes as well; deletions removing
miaA and surrounding regions or miaA mutations that are
polar on the expression of neighboring genes in an operon
could contribute to the S. typhimurium miaA mutant phenotypes. It will be necessary to clone the S. typhimurium miaA
gene and perform complementation analysis of the mutant
strains to test this possibility.
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I thank G. Bjork and J. Calvo for bacterial strains and Bruce N.
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P.H.B. was the recipient of a National Research Service Award
(ES05339) from the National Institutes of Health.
VOL. 170, 1988
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