Reduced leu Operon Expression in a miaA Mutant of Salmonella typhimurium 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 .
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 tRNA ( 6 , 7 , 21 , 33 ) .
as nonsense suppressors In S. typhimurium , the modified base cis-2-methylthiori-bosylzeatin [ N6 - ( 4-hydroxyisopentenyl ) -2-methylthioadeno-sine ( ms2io6A ) ] is only in tRNAs codons present cognate to 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 ) .
modification begins with isopenSynthesis of the ms2io6A tenylation at the N-6 position of adenine , followed by 2-methylthiolation and finally cis-hydroxylation of the iso-pentenyl 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 essential for both aerobic and electron transport pathways respiration ( 8 ) .
anaerobic A mutation has been isolated in S. typhimurium which results in complete loss of this modification due inactivato tion 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-t Present address : Department of Microbiology and Immunology , Stanford University , Stanford , CA 94305 .
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 .
MATERIALS AND METHODS 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 for D-leucine , except which was added at 200 jig/ml .
Phage indicator plates ( 37 ) were used to purify cells of phage P22 .
MacConkey base was used to screen the heat-sensitive medium ( Difco ) phemutants .
and were notype of miaA Tetracycline ampicillin added to complex medium at final concentrations of 10 and 50 jig/ml , respectively .
Top agar contained 0.6 % ( wt/vol ) and 0.5 % NaCl in distilled water .
Solid medium agar ( wt/vol ) 1.5 % contained ( wt/vol ) agar .
otherwise all Genetic methods .
Unless noted , manipulations of strains carrying miaA mutations were performed at in LB-medium to avoid accumulation of 30 °C miaA-compen-satory mutations ( P. Blum and M. unpublished ) .
Valencik , Crosses involving generalized transduction were performed as described before with P22 ( 31 ) .
Transduction of S. phage miaA mutations was done as described before typhimurium ( 8 ) .
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 heat-resistant control .
Analyses of strain sensitivities to chemical 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 the effect of temperature growth of the S. to measure on 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 ) .
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 ilv-GEDA 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 copy .
Mu dl insertions are unstable at temperatures above 30 °C ; therefore , Mu dl insertions were stabilized to allow cultiva ¬ `` S. strains were TA3842 and LT2 ( miaA + ) .
E. coli typhimurium ( miaAl ) strains were W3110-16 ( miaA ) and W3110-18 ( miaA + ) .
b Values in parentheses are the amounts applied to each 6-mm paper disk 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 underpseudouri-dylation 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 strain .
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 .
, - Galacto-sidase 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 iso-leucine , 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-galac-tosidase 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 micro-fuge 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 chroma-tography .
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 followin 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 .
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 ) form-amide 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 .
RESULTS 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 oxy-gen-related stress such as chemical oxidants .
An S. typhi-murium 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 ms2i6A 2 ) .
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. typhi-murium 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 L-leucine 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. typhi-murium 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 .
Whe Decreased expression of the leu and ilv operons 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 L-leucine 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 th 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-galacto-sidase 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 dereof pression 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 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 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 ms2io6A-deficient 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 .
DISCUSSION 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 mechanism .
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 , 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 termina-tlon 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. typhimu-rium is an important metabolic target of oxidative damage .
In S. typhimurium , hyperbaric oxygen-mediated and para-quat-mediated growth inhibition in minimal-medium can be suppressed by addition of branched-chain amino-acids , and cumene hydroperoxide treatment of wild-type S. typhimu-rium 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 manner similar to that obtained with leu during cumene hydroperoxide exposure .
It is unclear to what extent the different forms of the base modification ( ms2io6A , ms2i6A , i6A , and ms2io6A tRNA 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 phenoclone the S. typhimurium miaA types .
It will be necessary to gene and perform complementation analysis of the mutant strains to test this possibility .
I thank G. Bjork and J. Calvo for bacterial strains and Bruce N. Ames , in whose laboratory these studies were performed .
P.H.B. was the recipient of a National Research Service Award ( ES05339 ) from the National Institutes of Health .
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