Altered Growth-Rate-Dependent Regulation of 6-Phosphogluconate Dehydrogenase Level in hisT Mutants of Salmonella typhimurium and Escherichia coli WILLIAM R. JONES , GERARD J. BARCAK , t AND RICHARD E. WOLF , JR. * Department ` of Biological Sciences , University of Maryland Baltimore County , Baltimore , Maryland 21228 In Escherichia coli , the level of 6-phosphogluconate dehydrogenase is directly proportional to the cellular growth-rate during-growth in minimal media .
This contrasts with the report by Winkler et al. ( M. E. Winkler , J. R. Roth , and P. E. Hartman , J. Bacteriol .
133:830 -843 , 1978 ) that the level of the enzyme in Salmonella typhimurium LT-2 strain SB3436 is invariant .
The basis for the difference in the growth-rate-dependent regulation between the two genera was investigated .
Expression of gnd , which encodes 6-phosphogluconate dehydrogenase , was growth-rate uninducible in strain SB3436 , as reported previously , but it was 1.4-fold growth-rate inducible in other S. typhimurium LT-2 strains , e.g. , SA535 .
Both the SB3436 and SA535 gnd genes were growth-rate inducible in E. coli K-12 .
Moreover , the nucleotide sequences of the regulatory regions of the two S. typhimurium genes were identical .
We concluded that a mutation unlinked to gnd is responsible for the altered growth-rate inducibility of 6-phosphogluconate dehydrogenase in strain SB3436 .
Transductional analysis showed that the altered regulation is due to the presence of a mutation in hisT , the gene for the tRNA modification enzyme pseudouridine synthetase I .
A complementation test showed that the regulatory defect conferred by the hisT mutation was recessive .
In E. coli , hisT mutations reduced the extent of growth-rate induction by the same factor as in S. typhimurium .
The altered regulation conferred by hisT mutations was not simply due to their general effect of reducing the polypeptide-chain elongation rate , because miaA mutants , which lack another tRNA modification and have a similarly reduced chain growth-rate , had higher rather than lower 6-phosphogluconate dehydrogenase levels .
Studies with genetic fusions suggested that hisT mutations lower the gnd mRNA level .
The data also indicated that hisT is involved in translational control of gnd expression , but not the aspect mediated by the internal complementary sequence .
Growth-rate-dependent regulation alters the synthesis rate and/or the relative amount of a given protein in response to changes in growth-rate ( 39 ; reviewed in reference 53 ) .
The accumulation rate of some proteins is proportional to growth-rate , such that the amount of the protein relative to total protein is constant .
For other proteins , the accumulation rate increases in proportion to the square of the growth-rate , and thus the relative amount of these proteins increases in proportion to the growth-rate .
The accumulation rate for a third class of proteins is constant , with the result that the level of these proteins is inversely proportional to the growth-rate .
The mechanism for this regulation is not yet completely understood for any gene ( for reviews , see references 17 , 24 , and 26 ) .
The gnd genes of Escherichia coli K-12 and Salmonella typhimurium LT-2 are among the few nonribosomal genes whose growth-rate-dependent regulation is currently under investigation .
The gnd gene encodes 6-phosphogluconate of the dehydrogenase ( 6PGD ; EC 1.1.1.44 ) , an enzyme of 6PGD shunt .
The relative hexose monophosphate amount threefold the fivefold of increases approximately over range obtained with cells growing in minimal-medium growth-rates on acetate and on glucose , and the level does not increase further when cells are growing faster ( 55 ) .
Because the relative amount of P-galactosidase is constant in strains carrying operon fusions of the E. coli K-12 gnd and lacZ t Present address : Department of Biological Chemistry , University of Maryland School of Medicine , Baltimore , MD 21201 .
genes ( 6 ) , the accumulation rate of gnd mRNA varies in proportion to growth-rate , and so does translational efficiency .
In other words , growth-rate-dependent regulation of 6PGD expression in E. coli K-12 is subject to translational control as well as to control of either gnd transcription or mRNA stability .
Regulation of translational efficiency requires a negative control site that lies in the 6PGD-coding sequence ( 7 ; P. Carter-Muenchau and R. E. Wolf , Jr. , manuscript in preparation ) .
Recently , the internal regulatory region has been shown to be the segment at codons 69 to 74 of the gnd structural gene ( 16 ) .
The region is complementary to the ribosome-binding site of gnd and includes the complement of the Shine-Dalgarno sequence .
Evidence has been presented that suggests that this internal complementary sequence ( ICS ) mediates the translational control by sequestering the ribosome-binding site into a secondary structure and therefore functions as a cis-acting antisense RNA ( 16 ) .
The ICS and its ability to sequester the ribosome-binding site into a secondary structure are conserved in the gnd genes of E. coli B/r and four other E. coli strains from natural populations , all of which are rate inducible growth ( 8 , 9 ) .
has been for the Although a mechanism proposed growth-rate-dependent regulation of translational efficiency ( 16 ) , the effector of this regulation is unknown .
Moreover , essentially the nothing is known of the mechanism that regulates growth-rate dependence of the accumulation rate of gnd mRNA .
In studies of the growth-rate-dependent regulation of the histidine operon in S. typhimurium LT-2 strain SB3436 , Winkler et al. used gnd as a control and found that the level of 6PGD does not vary with growth-rate ( 51 ) .
This was in accord with the preconceptions of the day about expression of `` constitutive '' enzymes .
However , Wolf et al. subsequently showed that the 6PGD level in E. coli K-12 increases in proportion to growth-rate during-growth in minimal media ( 55 ) .
The work described here followed up on this discrepancy , because it offered the possibility of opening a new route toward characterizing the mechanisms of growth-rate-dependent regulation of 6PGD level .
For example , the S. typhimurium and E. coli gnd genes might differ in the nucleotide sequence of a regulatory site , in which case determining the responsible nucleotide differences might identify a site for either type of regulation .
Alternatively , S. typhimurium SB3436 might be defective in a component of a system involved with the capacity to carry out growth-rate-dependent regulation of 6PGD level ; in this case , understanding the basis for the defect might shed light on the mechanism of either type of regulation or on the nature of an effector of regulation .
In this paper we confirm the observation of Winkler et al. ( 51 ) that the level of 6PGD is invariant in strain SB3436 , but we show that the level increases with increasing growth-rate in other S. typhimurium LT-2 strains .
By cloning the gnd gene of strain SB3436 into phage X we show that expression of this gene in E. coli K-12 is as growth-rate inducible as the E. coli K-12 gene itself .
Moreover , the nucleotide sequence of the regulatory region of the SB3436 gene is identical to that of the gene from a growth-rate-inducible S. typhimurium strain .
Thus , a mutation unlinked to gnd is responsible for the altered regulation .
The most obvious difference between strain SB3436 and the strains in which the 6PGD level is growth-rate inducible is that strain SB3436 has a hisT mutation .
hisT mutations were originally identified among mutants selected for having derepressed expression of the histidine operon ( 18 ) .
For their studies of the metabolic regulation of his operon expression , Winkler et al. used a hisT mutant because the mutation eliminated attenuation-specific control ( 51 ) .
hisT maps near 47 min on the chromosome of S. typhimurium ( 41 , 42 ) , about 5 min from his , and encodes pseudouridine synthetase I ( PSUI ) , a tRNA modification enzyme that introduces pseudouridine into the anticodon loop of many tRNA species ( 19 , 48 ) .
Although PSUI is not essential for growth , mutations in hisT are pleiotropic , with effects on growth-rate ( 14 , 25 ) , polypeptide-chain elongation rate ( 37 ) , amino-acid analog resistance ( 48 ) , and derepression of amino-acid biosynthetic operons ( 20 , 25 ) .
hisT has been cloned , sequenced , and found to be part of a complex multigene operon ( 3 , 4 , 29 , 36 ) .
Here we demonstrate that the altered growth-rate-depen-dent regulation of 6PGD level in S. typhimurium SB3436 is , indeed , due to its hisT mutation and that E. coli hisT mutations have a similar effect on regulation .
By physiological and genetic experiments we show that the altered regulation is not a trivial consequence of several of the known pleiotropic effects of hisT mutations .
With gnd-lac fusions we investigated the step in gnd expression that is affected by hisT mutations .
Finally , we report that the level of 6PGD is elevated in S. typhimurium and E. coli miaA mutants , which lack tRNA A2-isopentenylpyrophosphate transferase , the enzyme that catalyzes the first step in the modification of the adenosine residue adjacent to the antico-don of several tRNA species ( 10 , 13 ) .
119 MATERIALS AND METHODS Media and growth-conditions .
Physiological experiments were carried out by using MOPS ( morpholinepropanesulfonic acid ) - minimal-medium supplemented with acetate and glucose ( 55 ) .
Auxotrophic requirements of various strains were met by appropriate concentrations of the required nutrients ( 21 , 33 ) .
Where indicated , the media for the growth of hisT mutants were supplemented with 0.4 mM adenine , 0.4 mM isoleucine , and 80 , ug of uracil per ml ( 14 ) .
Growth rates are expressed as the specific growth-rate constant ( k = ln2 / , u ) .
The following nutrient media were used : LB broth ( 31 ) for routine subculture ; TB broth and top agar ( 47 ) for the propagation of strains during manipulations with bacteriophage X ; YT agar plates and broth ( 30 ) for the isolation , cloning , and propagation of M13 phage and M13 DNA ; TBYCM broth and MC buffer ( 31 ) for the preparation of phage P1 lysates from E. coli ; P22 broth ( 21 ) for the preparation of phage P22 lysates from S. typhimurium ; and F-top and H-top agars ( 31 ) for generalized transductions .
Antibiotics included ampicillin ( 50 jig/ml ) , chloramphenicol ( 15 Fg/ml for selection of P1 cml clr-100 lysogens ) , and tetracycline ( 25 , ug/ml ) .
Minimal medium 63 ( 31 ) with appropriate supplements was used for genetic selections .
Strains lysogenic for bacteriophage P1 cml clr-100 or for A NF1955 and its deiivatives were cultured at 30 °C .
All other strains were grown at 37 °C .
Table 1 shows the S. typhimurium LT-2 and the E. coli K-12 strains used in this study .
Lysogens carying the A gnd phages integrated at attA were prepared with strain GB1815 , which contained deletions of the lactose operon and gnd to prevent recombination between regions of sequence homology on the phage and the bacterial chromosome .
The strain also carried supF to permit the propagation of the K phages , which were derived from phage A NF1955 ( 47 ) .
Strain GB1815 was prepared as follows .
Strain RW181 , which contains a large deletion of the gnd locus and amber mutations in lacZ and trpA , was transduced to Lac ' Trp + on lactose-minimal-medium with a phage P1 generalized transducing lysate prepared on the amber-suppressor-containing strain LE392 .
Transductants were scored for SupF by their ability to support the formation of plaques of k NF1955 .
The lactose operon of one such transductant , GB1811 , was deleted by cotransduction of the mutation A ( argF-lac ) U169 with the proC : : TnJO mutation of strain SG47 .
Tetracycline-resistant transductants were selected and scored for Lac-Pro - .
The proC : : TnJO mutation of one of these transductants , strain GB1814 , was repaired by transduction to growth-on-glucose minimal-medium with a P1 lysate prepared on strain RW181 .
Strain GB1815 was a Pro ' , tetracycline-sensitive transductant that remained Lac - .
Strain EF2 was prepared by specialized transduction of strain EF1 to His ' Gnd + with 4 ) 80 dhis gnd phage isolated and purified from strain XX30 as described previously ( 54 ) .
S. typhimurium hisT alleles were transduced into other S. typhimurium strain backgrounds with phage P22 .
HisT + and HisT-cells were distinguished by the morphology of colo-nies growing on minimal agar plates with 2 % glucose , where wild-type colonies are smooth and hisT mutants are rough ( 18 ) .
The phenotypes were verified by assaying the activity of histidinol dehydrogenase ( 27 ) , which is derepressed in hisT mutants ( 18 , 41 ) .
In every case , cells that produced rough colonies had histidinol dehydrogenase levels about fivefold higher than those of cells that produced smooth colonies .
A P22 lysate of strain TT317 was used to introduce the hisT-linked selectable marker purF : : TnJO into strain SB3436 by infecting it with the lysate and selecting for tetracycline-resistant ( Tcr ) transductants .
A lysate of a hisT Tcr trans ductant , strain WJ167 , was subsequently used to introduce the hisT mutant allele into the S. typhimurium LT-2 prototroph ara-9 by selecting for Tcr transductants and scoring for HisT .
In a reciprocal transduction , a phage stock prepared on strain ara-9 was used to replace the hisT mutant allele in strain WJ167 with a hisT + allele by selecting for adenine independence and scoring for HisT .
For the complementation test , plasmid DNA carrying the E. coli hisT operon was isolated by the alkaline lysis method ( 12 ) , transformed into the restriction-deficient and modificationproficient S. typhimurium LT-2 strain LB5010 ( 15 ) in the presence of 100 mM calcium chloride ( 21 ) , and then reisolated and transformed into S. typhimurium LT-2 strain SB3436 by the same methods TABLE 1 .
b Brackets indicate that a gene was introduced by transduction .
C All strains are E. coli K-12 except MMS345 , which is E. coli C. 4 ) 80 dhis gnd x EF1 This study This study This study This study This study This study This study 8 8 This study 6 7 7 J. Parker ( 38 ) S. Bingham 0 .
Karlstrom L. W. Bergman M. Stahl ( 40 ) M. E. Winkler ( 4 ) M. E. Winkler ( 4 ) M. E. Winkler ( 4 ) 52 32 8 M. L. Berman This laboratory C. Yanofsky C. Yanofsky P1 ( JK334 ) x W3110 P1 ( JK334 ) x W3110 P1 ( JK334 ) x HB545 P1 ( JK334 ) x HB545 P1 ( JK334 ) x HB354 P1 ( JK334 ) x HB354 P1 ( JK334 ) x HB552 P1 ( JK334 ) x HB552 50 A hisT mutation was introduced into the E. coli W3110 background by using a phage P1 lysate of strain JK334 , which contains the hisT-linked selectable marker fadL : : TnJO .
Lysates of phage K NF1955 ( 47 ) were prepared by lytic infection of strain LE392 ( 28 ) .
Lysates of the A gnd phages were prepared by heat induction and specialized transductions performed as described previously ( 31 ) .
Lysates of K cI90 cJ7 were prepared by the plate method ( 31 ) .
Monolysogens carrying the K gnd phages were identified by a plaque test with K cI90 c17 ( 45 ) .
All manipulations of bacteriophage M13 were performed as described by Messing ( 30 ) , with KK2186 and JM109 as the host strains .
Phage P22 HT105/1 ( P22 ; 44 ) lysates were prepared , and generalized transduction was performed as described by Davis et al. ( 21 ) .
Phage P1 cml clr-100 ( P1 ) lysates were prepared by heat induction of lysogens , and P1 generalized transductions were performed as described by Miller ( 31 ) .
Plasmid DNA was isolated by the alkaline lysis method ( 12 ) and further purified as described previously ( 8 ) .
Restriction enzymes ( Bethesda Research Laboratories , Inc. , Gaithersburg , Md. , or New England BioLabs , Inc. , Beverly , Mass. ) , were used according to the recommendations of the suppliers .
Other standard recombinant DNA techniques were used ( 28 ) .
Plasmids , DNA cloning , and DNA sequencing .
To clone the gnd genes of S. typhimurium and E. coli K-12 into A , DNA of A NF1955 was isolated by the rapid method of Silhavy et al. ( 47 ) , and the cohesive ends were ligated for 6 h at 16 °C under conditions favoring intramolecular ligation .
The DNA was cleaved at the unique EcoRI restriction site .
The gnd gene of E. coli K-12 was obtained from plasmid pMN3 ( 32 ) as a 3.8-kilobase EcoRI fragment , and the gnd gene of S. typhimurium SB3436 was obtained from plasmid pGB3436 ( 8 ) as an 8.8-kilobase EcoRI fragment .
The fragments were purified from agarose gels by the method of Benson ( 11 ) and ligated to the EcoRI-cleaved X NF1955 DNA .
The recombinant DNAs were packaged in-vitro by the method of Rosenberg et al. ( 40 ) by using cell extracts prepared from strain MMS345 .
Strain GB1811 was infected with the resulting phage particles , and phage lysates were prepared by the plate method ( 31 ) .
Strain GB1811 was infected with the low-frequency transducing lysates , and Gnd + specialized transductants were selected on gluconate minimal-medium containing histidine at 30 °C .
High-frequency transducing lysates were prepared by heat induction of the transductants .
To verify the presence of the correct gnd gene and to determine the orientation of the gene in the respective A gnd phages , DNA was isolated from the lysates and subjected to restriction analysis .
Phages gndKl and X gndK6 carry the E. coli K-12 gene in the two possible orientations , and phages X gndS3 and X gndS4 carry the gene from S. typhimurium SB3436 .
The 8.8-kilobase EcoRI fragment containing the gnd gene of S. typhimurium SA535 was cloned from chromosomal DNA into plasmid pBR322 to form plasmid pGB535 as described previously for the cloning of the gene from strain SB3436 ( 8 ) .
For DNA sequencing of the control region of the SA535 gene , the 760-base-pair HindlIl fragment of pGB535 was cloned in both orientations into phage M13mpl8 to form phages mGB535H182 and mGB535H184 .
The HindlIl fragment extends from position -165 with respect to the start of the coding sequence to codon 200 .
DNA sequencing was by the chain termination method ( 43 ) .
Measurements of enzyme specific activity .
The activities of 6PGD and glucose 6-phosphate dehydrogenase in sonic extracts were assayed spectrophotometrically as previously described ( 55 ) ; specific activity is expressed as nanomoles of NADPH formed per minute per milligram of protein .
Cultures in exponential-growth were collected at -108 cells per ml , concentrated fivefold by centrifugation , and sonically disrupted .
When HisT + and HisT-transductants of S. typhimurium were being scored for the growth-rate induc-ibility or uninducibility of 6PGD level , individual transductants were grown in 8 ml of MOPS-minimal-medium containing a limiting concentration of glucose ( 12.5 mM ) , such that bacteria were arrested in exponential-growth at -109 cells per ml .
Samples ( 1 ml ) of these cultures were sonically disrupted without prior concentration and assayed for 6PGD activity by the standard method ( 55 ) .
The activity of histid-inol dehydrogenase in sonic extracts was assayed spectro-photometrically as described by Loper and Adams ( 27 ) .
1-Galactosidase activity was assayed in cells permeabilized by treatment with chloroform and sodium dodecyl sulfate and is expressed in Miller units ( 31 ) .
All values represent the average of duplicate assays of at least two samples from each of two independent cultures and are followed by their standard deviations , expressed as the percentage of the mean value .
Most of the experiments were repeated , and day to day variations were less than 10 % .
The growth-rate induction ratio for 6PGD , glucose 6-phosphate dehydrogenase , or , B-galactosidase is the specific activity of the enzyme during-growth-on-glucose divided by the specific activity on acetate .
Propagated standard deviations for the induction ratios are given as the square root of the sum of the square of the percent standard deviation of the respective specific activities .
RESULTS Growth rate dependence of 6PGD level in S. typhimurium .
To determine whether the growth-rate-invariant level of 6PGD in strain SB3436 was a general property of S. typhi-murium LT-2 strains , we measured the effect of growth-rate on the level of the enzyme in the parental ara-9 prototroph and in strain SA535 .
Strain SA535 was used because it is the strain from which a 480 transducing phage carrying S. typhimurium gnd , used later in this study , was isolated ( 50 ) .
Table 2 shows the specific activity of 6PGD during steady-state growth for the three strains growing in acetate-and glucose-MOPS-minimal media .
As reported by Winkler et al. ( 51 ) , 6PGD level was growth-rate invariant in strain SB3436 , but it was about 1.4-fold growth-rate inducible in strains ara-9 and SA535 .
Although the extent of the growth-rate induction with strains ara-9 and SA535 was small , only about half as large as that in E. coli K-12 ( 55 ) , it was reproducible .
Moreover , whereas the level of 6PGD under slow-growth-conditions , with acetate as the sole carbon source , was about the same in the three S. typhimurium LT-2 strains , the enzyme level in cells grown on glucose was always about TABLE 2 .
Growth-rate-dependent regulation of 6PGD levels in hisT mutants of S. typhimurium Acetate Glucose Strain hisT Induction ratio + % allele k Sp act ± % k Sp act ± % ara-9 SA535 + + 0.30 66 8 0.28 63 2 0.20 55 ± 7 0.29 41 ± 5 0.89 87 ± 41 0.81 91 7 0.81 51 ± 6 0.93 60 ± 7 1.3 ± 8 1.4 ± 7 0.9 + 9 1.5 ± SB3436 SB3436 ( p * 300 ) ¬ - / + 1.4-fold higher in strains ara-9 and SA535 than in strain SB3436 .
Thus , S. typhimuriuim LT-2 and E. coli K-12 do not differ in their capacity for growth-rate induction of gnd expression .
Rather , strain SB3436 appears to be defective in growth-rate induction of 6PGD .
Growth-rate-inducible expression in E. coli K-12 of the gnd gene of strain SB3436 .
There are two general ways in which growth-rate-dependent regulation of 6PGD level could be defective in strain SB3436 .
One is that the gnd gene of strain SB3436 carries a mutation that prevents growth-rate induction .
Alternatively , strain SB3436 might carry a mutation in a gene for a trans-acting factor involved in growth-rate-dependent regulation of gnd expression or in a gene whose product is involved in sensing differences in growth-rate .
To address the first possibility we cloned ( as described in Materials and Methods ) the gnd gene of strain SB3436 in both possible orientations into K NF1955 , an integration-proficient cloning vector .
As a control we also prepared k phages carrying the E. coli K-12 gene in both possible orientations .
The effect of growth-rate on the specific activity of 6PGD was determined in strains monolysogenic for the four k gnd phages .
In all four strains the level of the enzyme was about 1.9-fold higher in glucose-grown cells than in acetate-grown cells ( data not shown ) .
Moreover , 6PGD was also growth-rate inducible in strain EF2 , which is lysogenic for a +80 specialized transducing phage that carries the gnd gene of strain SA535 ( data not shown ) .
Thus , the two S. typhimurium gnd genes that are regulated differently in their native backgrounds are similarly regulated in E. coli .
Moreover , the gnd gene of strain SB3436 has all sequences necessary and sufficient for growth-rate-dependent regulation in E. coli K-12 .
The growth-rate induction ratio was less for the lysogens carrying the E. coli K-12 gnd gene on a A prophage than the threefold induction obtained when gnd is at its normal chromosomal position ( 55 ) .
A similar effect of chromosomal position has also been observed recently with gnd-lacZ-fusions ( 16 ) .
Identical DNA sequence of the regulatory regions of the gnd genes of strains SA535 and SB3436 .
The fact that the gnd gene of strain SB3436 is as growth-rate inducible in E. coli K-12 as the native E. coli gene suggested that the growth-rate uninducibility of the gene in S. typhimurium SB3436 was due to a mutation located elsewhere in the genome and not to a mutation in a gnd regulatory site .
To confirm this conclusion , we cloned the gnd gene of strain SA535 as described in Materials and Methods , determined the nucleotide sequence of the regulatory regions , and compared the sequence to that of the gene from strain SB3436 .
The DNA sequence was identical to the sequence previously determined for the gnd gene of strain SB3436 ( data not shown ) ( 9 ) .
Therefore , the altered growth-rate inducibility of 6PGD in strain SB3436 is not due to a regulatory mutation in gnd but rather to a mutation located elsewhere .
The most apparent candidate was the hisT mutation .
Growth rate invariance of 6PGD level in hisT mutants of S. typhimurium .
To determine whether the apparent growth-rate uninducibility of strain SB3436 is due to its hisT mutation , the levels of 6PGD during-growth-on-glucose and acetate were determined in strain hisT1504 , which is the parent of strain SB3436 , and in strains TR36 and TT5866 , which contain different hisT mutations .
The level of 6PGD was growth-rate invariant in these hisT mutants , just as it was in strain SB3436 ( data not shown ) .
Thus , the growth-rate-invariant 6PGD level is a general property of S. typhimurium hisT mutants .
trans complementation of altered regulation in strain SB3436 by a hisT + plasmid .
A plasmid carrying the entire E. coli hisT operon , pqj3O0 , was transformed into strain LB5010 and subsequently into strain SB3436 by selection for ampicillin resistance ( Apr ) .
The plasmid complemented the his operon regulatory defect of the strain , in that strain SB3436 ( piP300 ) formed smooth colonies on 2 % glucose plates and had repressed levels of histidinol dehydrogenase .
The level of 6PGD was growth-rate inducible in this transformant ( Table 2 ) but not in a transformant carrying a control plasmid derived from pq30O that contained only part of the hisT gene ( data not shown ) .
These results show that the growth-rate-uninducible expression of 6PGD in strain SB3436 is recessive to the wild-type hisT allele .
Moreover , they also rule out the possibility that a mutation unlinked to hisT , putatively present as a compensatory mutation in all hisT mutants , is solely responsible for the regulatory defect .
Cotransduction of hisT mutation and growth-rate invariance of 6PGD level .
Because hisT mutations are highly pleiotro-pic , we next considered the possibility ( similar to the one above ) that the altered regulation of 6PGD level is due to two mutations , one in hisT and the other a compensatory mutation unlinked to hisT that is present in all hisT mutants .
We tested this possibility by examining the growth-rate dependence of 6PGD level in two pairs of isogenic strains made by reciprocal transduction crosses .
The strain constructions were facilitated by the availability of the closely linked marker purF : : Tnl O .
In one cross , the hisT mutation of strain WJ167 was replaced with the hisT + allele of strain ara-9 .
This was accomplished by using a phage P22 lysate prepared on strain ara-9 to transduce strain WJ167 , a purF : : TnJO derivative of strain SB3436 , to adenine independence and scoring for HisT + .
In the other cross , the hisT mutation of strain WJ167 was transduced into the wild-type genetic background of strain ara-9 by selecting Tcr transductants and scoring for HisT - .
A HisT + and a HisT-transductant were chosen from each cross , and the growth-rate dependence of the 6PGD level was determined for each pair .
Growth rate induction was regained when the hisT mutant was transduced to hisT + , and growth-rate uninducibility was obtained when a hisT mutation was transduced into the wild-type genetic background ( data not shown ) .
From these results we conclude that the altered regulation is due solely to a mutation in the hisT region of the chromosome , presumably in hisT itself .
Moreover , the altered regulation does not depend on the presence of a compensatory mutation unlinked to hisT .
These conclusions were confirmed by construction of additional strains , where the hisT1529 and hisT290 : : TnS mutations were introduced by transduction into strain ara-9 , and by transduction of strain TR36 to hisT + .
Again , the 6PGD level was growth-rate independent in the hisT mutants and growth-rate dependent in the hisT + strain ( data not shown ) .
Despite these results , it was still possible that the altered growth-rate inducibility of 6PGD was not due solely to a mutation in hisT but rather to a second mutation closely linked to hisT , either alone or in combination with it .
Testing this possibility required a rapid method of screening hisT + and hisT mutant transductants for growth-rate-inducible and-uninducible phenotypes of gnd .
This was accomplished by assaying 6PGD activity in cultures of transductants growing on glucose , since under these conditions the enzyme level of a hisT mutant is always lower than that of a hisT + strain ( Table 2 ) .
Cells were grown in minimal-medium containing a limiting concentration of glucose , which arrests cells in exponential-growth at a density high enough for assay o enzyme activity without prior concentration of the cultures .
Control experiments with strains ara-9 and SB3436 showed that the difference in enzyme level obtained with the two strains in exponential-growth was also evident for glucoselimited cultures .
Thirty-two isolated transductants from each of the reciprocal crosses between strains ara-9 and WJ167 were scored for colony morphology on 2 % glucose plates , grown in small volumes , and assayed for 6PGD level .
The results demonstrated 100 % linkage of hisT + and inducibility and of hisT-and uninducibility ( data not shown ) .
The conclusion drawn from these experiments is that altered growth-rate-depen-dent regulation of gnd expression in strain SB3436 is due to the hisT mutation , not to some other mutation in the strain background .
Moreover , the hisT mutation alone is sufficient for the growth-rate uninducibility .
Growth rate dependence of 6PGD level in E. coli hisT mutants .
Because of the availability of operon and protein fusions of gnd to lacZ ( 6 , 7 ) , further analysis of hisT effects on growth-rate-dependent regulation was done in E. coli .
Moreover , the magnitude of growth-rate induction of 6PGD level is greater in E. coli K-12 than it is in S. typhimurium LT-2 ( 2.8-fold versus 1.4-fold ) .
Accordingly , the growth-rate dependence of the 6PGD level was determined in two isogenic pairs of strains .
One hisT mutant , WJ301 , carried the hisT76 allele , whereas the other , NU611 , carried an insertion mutation .
The induction ratio was about 2.0 for the hisT mutants and about 2.8 for the respective hisT + strains ( Table 3 ) .
Thus , the induction ratio of the E. coli wild-type strains is about 1.4-fold higher than that of the hisT mutant , a difference of the same magnitude as that seen when comparing S. typhimurium hisT + and hisT strains ( Table 2 ) .
This indicates that E. coli hisT mutations reduce the extent of growth-rate induction by one-third , just as S. typhimurium mutations .
In other words , reduced expression of 6PGD during-growth-on-glucose is common to S. typhimurium and E. coli hisT mutants .
Pleiotropic effects of hisT mutations .
Bruni et al. reported that the E. coli hisT mutant FB105 grows at half the rate of the wild type on glucose ( 14 ) .
Under our growth-conditions , the mutant grew on glucose at about the same rate as the wild type and on acetate at about 75 % of the wild-type rate .
They also reported that the growth-rate in glucose-minimal-medium is enhanced to the wild-type rate by supplementation with adenine , isoleucine , and uracil .
The same effect was observed when strains WJ300 and WJ301 were grown in acetate-and glucose-minimal media containing these supplements .
However , when hisT + and hisT strains were growing at the same rate on acetate and almost the same rate on glucose , the induction ratio of the hisT mutant was significantly lower than that of the wild type ( data not shown ) .
To minimize the difference in growth-rates on acetate between hisT + and hisT strains , all subsequent experiments involving E. coli hisT mutants were performed in the presence of these supplements .
In any event , the growth-rate differences we observed between hisT + and hisT strains were not large enough to account for the differences in 6PGD levels .
To test the possibility that the reduced growth-rate induction is due to the 20 to 25 % reduction in polypeptide-chain elongation rate caused by hisT mutations ( 37 ) , the growth-rate dependence of 6PGD level was examined in miaA mutants , which are defective in another tRNA modification enzyme and have a polypeptide-chain elongation rate that is approximately 30 % lower than that of the wild type ( 22 ) .
Table 4 shows the effect of miaA mutations in S. typhimu-rium and E. coli on growth-rate induction of 6PGD level .
Unlike hisT mutations , the miaA mutations did not reduce 6PGD levels ; rather they increased them .
Thus , the reduced extent of growth-rate induction in hisT mutants is not an indirect effect of decreased polypeptide-chain growth-rate , nor is it due to a general deficiency in tRNA modification .
Effects of hisT and miaA mutations on zwf expression .
To determine whether hisT mutations exert a general effect on growth-rate-dependent regulation of central metabolism genes , we examined the expression of glucose 6-phosphate dehydrogenase in a hisT mutant .
This enzyme , encoded by zwf , is also an NADP-dependent dehydrogenase of the hexose monophosphate shunt , and growth-rate-dependent regulation of its level is similar to that of 6PGD in steady-state growth and during nutritional upshifts ( 23 , 55 ) .
However , growth-rate induction of glucose 6-phosphate dehydrogenase in E. coli was unaffected by a hisT mutation ( data not shown ) .
Also , a miaA mutation had no effect on glucose 6-phosphate dehydrogenase expression ( data not shown ) .
Thus , the effects of hisT and miaA mutations on growth-rate-dependent regulation are not common to central metab-olism genes .
Effect of hisT mutation on ( 3-galactosidase levels in operon fusions .
It is now well established that measurement of , B-galactosidase activity in strains carrying operon fusions of a target gene to the lac operon can be used as an indirect assay of the effect of a variety of physiological and genetic conditions on the mRNA level of the target gene ( 46 ) .
Thus , to determine whether the effect of hisT mutations on growth-rate inducibility of 6PGD is due to an effect on gnd mRNA level , we introduced a hisT mutation by transduction into an E. coli gnd-lac operon fusion strain .
If hisT mutations affect the relative amount of gnd mRNA , the level of P-galactosi-dase during-growth-on-glucose should be reduced by the same factor as 6PGD level is in a normal strain .
On the other hand , if hisT mutations affect the translational efficiency of gnd mRNA , the P-galactosidase level should be unaffected .
The level of P-galactosidase in the hisT + control strain , WJ324 , did not increase with increasing growth-rate ( Table 5 ) , as expected ( 6 ) .
The growth-rate induction ratio fo VOL .
Growth-rate-dependent regulation of 6PGD levels in miaA mutants of S. typhimurium and E. coli Acetate Glucose miaA allele Sp Sp rIantdiu k k oct + ion act % act % TABLE 3 .
Growth-rate-dependent regulation of 6PGD levels in hisT mutants of E. coli Acetate Glucose Strain allele hisT rIantdiuoct ± io % n Strain Sp Sp k k act % 42 2 50 4 59 7 60 3 act % 114 4 98 2 167 1 134 2 + WJ300 WJ301 NU426 NU611 S. typhimurium GT522 GT523 2.7 2.0 2.8 2.2 5 4 7 4 0.26 0.79 0.68 0.69 0.46 ¬ + 0.34 83 5 0.92 110 3 1.3 ± 6-0.25 162 7 0.71 201 7 1.2 ± 10 0.18 0.14 0.09 + ¬ E. coli + 0.14 65 + 5 0.57 158 ± 4 2.4 + 6-0.10 133 ± 8 0.55 259 ± 1 1.9 ± 8 W3110-18 W3110-16 Acetate Glucose Induc ¬ Strain ICSb hisT tion ratio allele k l3-gal ' k 03-gal a Grown in the presence of adenine , isoleucine , and uracil .
b Presence ( + ) or absence ( - ) of the ICS regulatory element in the fusion .
NA , Not applicable , because the strains carry operon fusions and the ICS has no effect on expression of gnd-lac operon fusions ( 6 ; Carter-Muenchau and Wolf , in preparation ) .
c P-Galactosidase ( P-gal ) activity is expressed in Miller units ( MU ; 31 ) .
WJ324 WJ325 WJ328 WJ329 WJ320 WJ321 NA NA + + ¬ ¬ + - + ¬ + ¬ 0.33 0.15 0.16 0.16 0.17 0.18 623 ± 3 414 ± 4 554 ± 2 618 ± 1 1,859 ± 1 2,067 ± 3 0.92 0.69 0.69 0.67 0.69 0.65 460 ± 2 343 ± 2 1,496 ± 3 1,269 ± 1 2,456 ± 3 2,006 ± 4 0.7 ± 4 0.8 ± 2 2.7 ± 4 2.0 ± 1 1.3 ± 3 1.0 ± 5 3-galactosidase in the hisT mutant operon fusion strain , WJ325 , was the same as that of its isogenic partner , but the level of the enzyme was about one-third lower during-growth on both glucose and acetate .
Interaction between hisT and the internal regulatory sequence of gnd .
The ICS is a negative control element lying within the gnd structural gene that is necessary for growth-rate-dependent regulation of 6PGD level ( 7 ) .
It is highly complementary to the ribosome-binding site of gnd mRNA and is proposed to mediate regulation of gnd translational efficiency by forming an mRNA secondary structure that sequesters the Shine-Dalgarno sequence ( 16 ) .
In searching for a possible role of PSUI in gnd regulation we noticed that the core of the ICS , which contains the anti-Shine-Dalgarno sequence , matches six of seven bases at positions 40 to 46 in tRNAHis , the region modified by PSUI in many tRNAs ( 2 ) .
Moreover , the putative secondary structure of this segment of gnd mRNA ( 16 ) is similar overall to the PSUI-modified segment of tRNA , with a region of base pairing adjacent to unpaired bases .
These observations suggested that PSUI might be directly involved in regulating gnd expression by binding to and unwinding the ICS structure .
Indeed , Ames et al. have proposed that enzymes that bind tRNA , in particular modification enzymes , might play a regulatory role by binding to secondary structures in mRNA similar to those in tRNA ( 1 ) .
The recently demonstrated translational autoregulation of threonyl-tRNA synthetase expression lends support to this general idea ( 49 ) .
To test the hypothesis , we determined the epistatic relationship between hisT and the ICS with strains carrying gnd-lacZ protein fusions ( Table 5 ) .
A hisT mutation was introduced by transduction into strain HB552 , which has an intact internal regulatory region , and into isogenic strain HB545 , which contains an ICS-protein fusion .
In the control strain with an ICS + protein fusion , the hisT mutation reduced the extent of growth-rate induction of 3-galactosi-dase ( Table 5 , lines 3 and 4 ) by the same factor as it reduced induction of 6PGD expression from gnd itself ( Table 3 ) .
The hisT mutation had the same effect on expression in the ICS-fusion strain ( Table 5 , lines 5 and 6 ) .
Thus , the hisT mutation is epistatic to the absence of the ICS .
DISCUSSION The growth-rate invariance of 6PGD level observed by Winkler et al. ( 51 ) with S. typhimurium LT-2 strain SB3436 was the result expected at the time for a so-called constitutive gene .
We had not yet reported that the level of 6PGD in E. coli K-12 is directly proportional to the cellular growth-rate during-growth in minimal media ( 55 ) .
In the present work we showed that the discrepancy was not due to a general difference between the two genera nor to a gnd regulatory mutation .
We pursued the regulatory defect in strain SB3436 because few mutations unlinked to a growth-rate regulated gene are known which affect the growth-rate-dependent regulation of the gene .
In fact , aside from guanosine 3 ' - diphosphate , 5 ' - diphosphate ( 17 ) , and as yet incompletely characterized mRNases ( 35 ) , the effectors of growth-rate-dependent regulation of nonribosomal genes are unknown .
Thus , identifying and characterizing the mutation in S. typhimurium SB3436 responsible for the altered growth-rate regulation of 6PGD level was important because understanding the basis for the defect might provide insight into the mechanism of gnd regulation .
Transductional analyses and a complementation test showed conclusively that the altered regulation of strain SB3436 is due solely to its hisT mutation , which is recessive .
Moreover , regulation was similarly affected by two other S. typhimurium hisT mutations , including an insertion mutation .
Although hisT is the third gene of a complex four-gene operon ( 4 , 29 ) , it is unlikely that the altered regulation is due to deficiency of the product of the downstream gene , because neither the hisT1504 nor the hisT1529 mutation used in this study is suppressed by nonsense suppressors ( 18 ) , and hisT does not appear to be translationally coupled to the downstream gene ( 4 ) .
The availability of gnd-lacZ-fusions prompted us to determine the growth-rate dependence of 6PGD level in E. coli hisT mutants .
Also , with the growth-rate induction ratio of wild-type E. coli being about twice that of wild-type S. typhimurium , we anticipated a quantitatively larger effect of the E. coli hisT mutations .
However , the E. coli hisT mutations decreased the induction ratio from about 3 in the wild-type strain to about 2 in the mutant , an effect of the same magnitude as the decrease in ratio from 1.4 to 1.0 brought about by the S. typhimurium mutations .
Thus , rather than having an all-or-none effect , i.e. , conferring total growth-rate uninducibility , the hisT mutations of both S. typhimurium and E. coli may only reduce the extent of induction by about one-third .
The possibility that the altered regulation might be due to the decrease in polypeptide-chain elongation rate brought about by hisT mutations ( 37 ) was considered , especially because uncoupling of transcription and translation has been proposed as an essential step in regulating the translational efficiency of gnd mRNA ( 16 ) .
However , this is not the case , since the level of 6PGD is higher in miaA mutants , whose chain growth-rate is also about 25 % lower than that of the wild type ( 22 ) .
Also , since growth-rate-dependent regulation of the level of another hexose monophosphate shunt enzyme , glucose 6-phosphate dehydrogenase , is normal in hisT and miaA mutants , deficiency in tRNA modification enzymes does not have a global effect on expression of central metabolism genes .
Operon and protein fusions were used as a way of determining whether hisT mutations affect regulation of gnd mRNA level or translational efficiency and in particular whether ICS function is altered .
The hisT mutation had the same effect on the growth-rate dependence of , B-galactosi-dase level from both the ICS-and the ICS + protein fusions , showing that the mutation does not act on the known regulatory element of translational efficiency .
Thus , the similarity described above between the sequence of a por tion of the ICS and a segment of tRNAHiS that includes the site for PSUI modification has no functional significance .
Interpretation of the results obtained with the operon fusion strains is more complex ( Table 5 ) .
The hisT76 mutation did not change the growth-rate induction ratio for , B-galactosidase in the operon fusion strain , but the overall level of the enzyme was about one-third lower in both acetate-and glucose-grown cells .
This indicates that hisT mutations reduce the level of gnd mRNA and hence its accumulation rate by the same one-third factor and that this reduction is growth-rate independent .
Thus the data argue that hisT + has a positive effect on gnd mRNA level .
The effect could be on the rate of gnd transcription or on the decay rate of gnd mRNA .
The fusion operon data also indicate that hisT + has a negative effect on translational efficiency under slow-growth-conditions .
During growth on acetate hisT mutations have no effect on the level of 6PGD ( Tables 2 and 3 ) , but hisT mutation reduces,-galactosidase level in an operon fusion strain by one-third .
In other words , during-growth on acetate the reduction in gnd mRNA level brought about by the hisT mutation is offset by increased translational efficiency , whereas during-growth-on-glucose only the gnd mRNA level is affected .
From the protein fusion data discussed above , it appears that an aspect of translational efficiency that does not involve the ICS is negatively regulated .
The interpretation presented above of the results obtained with the operon fusion strains rests on the assumptions that the effect of the hisT76 mutation on the 3-galactosidase level in the operon fusion strain is due to an effect on the level of the fusion mRNA and that the mutation has the same effect on the gnd mRNA level .
When the properties of the operon fusion strains were initially reported , we pointed out that , because of translational coupling , polarity , or other unknown phenomena , the effect of growth-rate on level of , B-galactosidase might accurately reflect the level of gnd not mRNA ( 6 ) .
To test this possibility we subsequently determined the growth-rate dependence of galactoside transace-tylase in gnd : : Mu dlI strains , since their gnd-lacZ protein fusions were also gnd-lacA operon fusions ( 7 ) .
The level of the transacetylase was growth-rate invariant , just like the level of 3-galactosidase in the gnd : : Mu dI strains .
Thus , since the same conclusion concerning the effect of growth-rate on gnd mRNA level could be drawn from data uncomplicated by the possibility of polarity , etc. , as from measurement of,-galactosidase in the gnd : : Mu dl strains , we believe that the above-stated assumptions and the interpretations based on them are probably valid .
Although speculation about possible mechanisms for the putative positive and negative regulatory roles of hisT + would be premature , it is worth considering the physiological significance of the effect .
The altered gnd regulation in hisT mutants could be evidence of a global regulatory circuit that coordinates the roles of protein synthesis and central metabolism , both of which have established links to nucle-otide modification ( 13 ) .
Although zwf expression was not affected , expression of other central metabolism genes might be .
The fact that the effect of hisT mutations is relatively small might be because the primary role of the affected regulation is not for steady-state growth but rather for some other physiological condition , e.g. , adaptation to a different nutrient environment .
The rationale for coordination of macromolecular synthesis and metablism has been discussed ( 34 ) .
It is also interesting that gnd expression was altered by hisT and miaA mutations , whereas expression of zwf , which responds like gnd to changes in growth-rate , was unaffected .
Perhaps there is a fundamental difference between the basic mechanisms regulating these two central metabolism genes .
In conclusion , the research reported here has uncovered a new layer of gnd regulation .
It will be interesting to determine whether the opposing effects of hisT and miaA mutations are direct or indirect and whether they are mechanistically related .
0.7 ± 4 0.8 ± 2 2.7 ± 4 2.0 ± 1 1.3 ± 3 1.0 ± 5 We gratefully acknowledge P. E. Hartman , G. R. Bjork , L. B. Bullas , M. J. Voll , J. R. Roth , S. Bingham , 0 .
Karlstrom , L. W. Bergman , M. L. Berman , T. J. Silvavy , M. Stahl , J. Parker , M. E. Winkler , and C. Yanofsky for providing us with the bacterial strains and phages used in this study .
We thank D. Cheyney and E. Ozgun for help with the cloning and DNA sequencing of gnd ( 535 ) and E. Farrish for experiments with strain EF2 .
This research was supported by Public Health Service grant GM27113 from the National Institute of General Medical Sciences .
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