Salmonella typhimurium are ( reviewed in refs .
In particular , the hisR multigene tRNA operon ( encoding the sole S. typhimurium tRNAHiS gene ) is transcribed efficiently in-vitro only when the DNA template is supercoiled ( Lionello Bossi , personal communication ) .
We suggest that the his constitutive phenotypes of the his W ( gyrA ) and his U ( gyrB ) mutations are a consequence of reduced transcription initiation at the hisR promoter in-vivo .
This implies that expression of the tRNA iS gene , and consequently the his structural gene operon , is controlled by DNA supercoiling .
ABSTRACT The hisW mutations of Salmonella typhimu-rium are highly pleiotropic mutations that elevate his operon expression , reduce ilv gene expression , alter stable RNA metabolism , and confer defective growth properties .
The hisW mutations are highly linked to a naladixic acid-resistant gyrA mutation of S. typhimurium .
Multicopy recombinant plasmids containing the Escherichia coli gyrA gene are able to complement both the growth defects and the elevated his operon expression associated with the hisW mutations .
We conclude that hisW mutations are alleles of the gyrA gene .
The hisUl820 mutant of S. typhimurium exhibits many of the same phenotypes as hisW mutants .
Several lines of evidence , including high transduction linkage to recF , suggest that hisUl820 is an allele of gyrB .
Finally , well-characterized gyrA and gyrB alleles of E. coli are also his regulatory mutations .
We propose that a wild-type degree of chromosomal superhelicity is required for maximal production of histidyl-tRNA and normal his operon regulation .
Regulatory mutations at several loci ( hisR , S , T , U , W ) that are unlinked to the his operon result in the increased expression of the his operon in Salmonella typhimurium and Escherichia coli ( 1-3 ) .
These mutations all affect histidyl-tRNA metabo-lism , resulting in either lowered levels of histidyl-tRNA or , in one case ( hisT ) , undermodified histidyl-tRNA ( 3-5 ) .
When the intracellular level of histidyl-tRNA is low , translation of the his leader peptide is inefficient , impeding formation ofthe his attenuator and allowing increased transcription of the his structural genes ( 6 ) .
Mutations hisW3333 ( Cs ) ( cold sensitive , lethal ) and hisU-1820 ( Ts ) ( thermosensitive , lethal ) of S. typhimurium are highly pleiotropic .
In addition to the his regulatory phenotype , these mutants have lowered levels of several tRNA species ( including histidyl-tRNA ) ( 4 ) , reduced levels of enzymes involved in isoleucine-valine biosynthesis ( 7 , 8 ) , and are defective in the normal control of stable RNA accumulation ( 9-11 ) .
We show that the S. typhimurium hisW mutations are alleles of gyrA , the structural gene for the A subunit of DNA gyrase .
In addition , we provide evidence that the his U1820 - ( Ts ) mutation is an allele of the gyrB gene , the structural gene for the B subunit of DNA gyrase .
Further , we were able to show that well-characterized gyrA and gyrB alleles of E. coli are his constitutive regulatory mutations causing phenotypes similar to those of the hisW3333 ( Cs ) and hisU1820 ( Ts ) mutations of S. typhimurium .
DNA gyrase is the enzyme responsible for maintaining the bacterial chromosome in a state of negative superhelicity ( 12 , 13 ) .
Chromosomal superhelicity has been implicated in the control of gene expression since the activities of many promoters are modulated by changes in the superhelical density of the template DNA both in-vivo and in-vitro The publication costs of this article were defrayed in part by page charge payment .
This article must therefore be hereby marked `` advertisement '' in accordance with 18 U.S.C. § 1734 solely to indicate this fact .
MATERIALS AND METHODS Bacteria , Media , and Growth Conditions .
The bacterial strains used in this study are listed in Table 1 and below .
The original strains used as sources of hisW mutations are TA800 ( hisW1821 ) , SB634 ( hisW1824 ) , TA805 ( hisW1825 ) , SB234 ( hisW1295 ) , SB230 ( hisW1291 ) , KRS2316 ( hisW1509 ) , and JL681 [ hisW3333 ( Cs ) ] .
JL681 was obtained from John Ingraham ; the remainder were obtained from Phil Hartman .
Minimal glucose and rich media used were described ( 19 ) .
Ampicillin , tetracycline , kanamycin , and naladixic acid ( Nal ) were used at concentrations of 100 , 25 , 30 , and 10 , ug/ml , respectively .
L-Amino acids and other nutritional supplements were added at the concentrations recommended by Davis et al. ( 20 ) .
All incubations were carried out at 37 °C unless otherwise noted .
Cold-resistant growth is defined as the ability to grow on rich-medium plates at room temperature ; thermoresistant growth is defined as the ability to grow on minimal-glucose plates at 42 °C .
Antibiotics , o-nitrophenyl-3-D-galactoside , amino-acids , 1,2,4-triazole-3-alanine ( triazole alanine ) , and 3-amino-1,2,4-triazole were obtained from Sigma .
Restriction enzymes , T4 DNA ligase , and mung bean exonuclease were obtained from either New England Biolabs or Bethesda Research Laboratories .
Genetic and Biochemical Techniques .
Histidinol dehydrogenase ( EC 1.1.1.23 ) was assayed using the toluenized cell method of Ciesla et al. ( 21 ) .
P-Galactosidase assays were performed as described by Miller ( 22 ) .
Triazole alanine plate tests were performed as described ( 19 ) except that 3-amino-1,2,4-triazole was used at 1.0 mM and only required amino-acids were added .
S. typhimurium transduction crosses were performed using bacteriophage P22 ( HT105/1 , int-201 ) as described by Maloy and Roth ( 23 ) .
E. coli transduction crosses using Plvir , genetic transformations , and plasmid DNA isolations were as described et performed by Silhavy al. .
S. typhimurium transformations performed using strains derived from the transformable strain TR1859 .
Abbreviations : Nal , naladixic acid ; Cs , cold sensitive lethal ; Ts , thermosensitive lethal .
tTo whom reprint requests should be addressed .
§ Present address : E. I. du Pont de Nemours and Co. , Experimental Station E328/155 , Wilmington , DE 19898 .
KNK453 KRE447 KRE449 K124 K1891 K2030 K1972 RM146 S. typhimurium KRS112 rnpAl ( Ts ) This study KRS1717 gyrAI ( Nal ) This study KRS2425 hisW3333 ( Cs ) t This study KRS2426 hisW + t This study hisD9953 : : MudJP § KRS2446 hisW3333 ( Cs ) This study hisD99S3 : : MudJP KRS2447 hisW + This study KRS2304 recF521 : : TnS This study TR6673 hisU1820 ( Ts ) John Roth TR5525 dnaAI ( Ts ) John Roth Additional strains used in this study are listed in Materials and Methods and in Table 3 .
* Isogenic pair constructed using zei-564 : : TnlO .
tIsolated as a himB mutation ( 17 , 18 ) .
tIsogenic pairs constructed using zeh-754 : : TnlO .
§ See Materials and Methods for definition of MudJ .
Genetic manipulations of gyrA and hisW mutations were facilitated by the use of transductionally linked TnlO elements zei-564 : : TnlO ( in RM101 , 15 % linked to E. coli gyrA ) and zeh-754 : : TnJO ( in TT5371 , 90 % linked to S. typhimurium hisW ) .
The S. typhimurium hisUl820 ( Ts ) , dnaAl ( Ts ) , and rnpAI ( Ts ) mutations were genetically manipulated using zia-748 : : TnlO ( in TT3920 , 15 % linked to rnpA ) .
MudI1734 is a stable , kanamycin-resistant , ampicillin-sensitive derivative of MudIl ( lac , Ap ) constructed by Malcolm Casadaban and coworkers ( 25 ) and is referred to as MudJ throughout this paper .
S. typhimurium his operon expression was monitored using the single copy , chromosomal hisD9953 : : MudJ operon fusion , which places the f-galactosidase gene under the control of the his promoter and attenuator .
Isolation of S. typhimurium gyrAl , rnpAl , and recF521 : : TnS Mutants .
KRS1717 [ gyrAl ( Nal ) ] was isolated as a spontaneous mutant resistant to Nal at 10 , ug/ml .
KRS2304 ( recF521 : : Tn5 ) was isolated by transducing TR6673 [ hisU-1820 ( Ts ) ] to temperature resistance using bacteriophage P22 grown on a pool of independent Tn5 insertion mutations .
Kanamycin-resistant , temperature-resistant transductants were screened for UV sensitivity .
The recF521 : : Tn5 insertion is unusually stable since 300 of 300 kanamycin-resistant transductants of LT2 also became UV sensitive .
KRS112 [ zia-748 : : TnlO , rnpA1 ( Ts ) ] was isolated by hydroxylamine-induced localized mutagenesis ( 20 ) .
The RnpA phenotype was determined by observing the accumulation of tRNA precursors ( ref .
26 ; Lionello Bossi and K.E.R. , unpublished results ) .
An li-kilobase ( kb ) BamHI DNA restriction fragment containing the E. coli gyrA gene was subcloned from pMK90 ( 27 ) into pKO100 ( 28 ) to obtain pRM378 ( see Fig. 1 ) .
The pRM386 and pRM389 subclones were obtained following the partial HindIII digestion of pRM378 .
The pRM387 plasmid was derived by inserting a blunt-ended chloramphenicol acetyltransferase cassette ( from pBR325 ) into pRM386 at the unique Sst I site present at the 19th codon of gyrA ( R.M. and M. Gellert , unpublished results ) .
Prior to ligation of the chloramphenicol acetyltransferase cassette with T4 DNA ligase , the Sst I restriction cut was blunted with mung bean nuclease , which removed = 200 base pairs .
RESULTS The hisW Mutations Are Highly Linked to Nal Resistance by P22 Transduction .
We have determined the genetic linkage between the gyrAl ( Nal ) and hisW3333 ( Cs ) mutations of S. typhimurium by P22 transduction .
Using P22 phage grown on KRS1717 [ gyrAI ( Nal ) ] , Nal-resistant transductants of JL681 - [ hisW3333 ( Cs ) ] were selected .
Nal-resistant transductants also inherited the hisW + ( cold resistance ) phenotype 598 of 600 ( 99.7 % ) .
To confirm that selective pressure for Nal resistance was not causing preferential survival of hisW + cotransductants , we also determined the gyrAl ( Nal ) hisW-3333 ( Cs ) genetic linkage using both alleles as unselected markers in a three factor cross .
In this experiment a nearby tetracycline-resistance transposable element ( zeh-754 : : TnJO , 92 % linked to hisW ) was used as the selected marker .
Tetracycline-resistant , Nal-resistant transductants of JL681 - [ hisW3333 ( Cs ) ] were cold resistant ( 374 of 375 ) , again indicating a gyrAl ( Nal ) hisW3333 ( Cs ) linkage of In 99.7 % .
addition , linkage of gyrAl ( Nal ) to all of the hisW alleles was determined .
The triazole alanine - ( a toxic histidine analogue ) resistant phenotypes associated with the hisW mutations ( hisW1509 , hisW1824 , hisW1825 , hisW1821 , hisW1291 , and hisW1295 ) ( 1-3 ) are between 92 % and 98 % cotransducible with gyrAl ( Nal ) .
Complementation of gyrA and hisW Phenotypes with Plasmids Containing theE .
Fig. 1 shows the E. coli DNA fragments containing either an intact gyrA gene or various deletion derivatives that were used to transform several E. coli and S. typhimurium gyrA and hisW mutants .
The gyrA gene has been sequenced , and its position and orientation relative to the restriction sites depicted are known ( R.M. and M. Gellert , unpublished results ; Stephen L. Swanberg and James C. Wang , personal communication ) .
The recombinant plasmids pRM378 and pRM386 contain 11 kb and 6 kb of E. coli DNA , respectively .
Both plasmids complement all of the gyrA and hisW phenotypes tested , including Nal resistance , thermosensitive-and cold-sensitive growth , small colony size , and constitutive his operon derepression ( see Tables 2 and 3 ) .
The DNA insert present in pRM386 contains the intact gyrA coding sequence , 0.5 kb of downstream DNA , and 2 kb of DNA that precedes gyrA .
The region distal to the carboxyl terminus of gyrA but upstream of the HindIII site ( see Fig. 1 ) has the potential to code for a peptide with a maximum molecular weight of 18,000 and is thus unlikely to code for complementing activity ( Stephen L. Swanberg and James C. Wang , personal communication ) .
The pRM389 plasmid contains the first half of the gyrA gene in addition to the 2 kb of DNA that precedes the gyrA coding sequence .
This plasmid has no gyrA and hisW complementing activity ( Table 2 ) , thus reducing the possibility that a gene other than gyrA is responsible for the complementation .
Additionally , pRM387 , in which a chloramphenicol acetyltransferase cassette has replaced -200 base pairs of gyrA DNA , also fails to show complementing activity ( Tables 2 and 3 ) .
The pRM387 plasmid has the same downstream present does pRM386 , the shortest complesequences as menting plasmid .
To demonstrate that all of the previously studied hisW mutations of S. typhimurium constitute one complementation group , we transformed strains containing the hisW mutations ( listed above ) with pRM386 and pRM387 DNA .
The small colony phenotypes of hisW1509 and hisWi824 and the his operon constitutive derepression phenotypes of all the mutants were completely reversed by pRM386 ( gyrA + ) but not by pRM387 ( gyrA : : CAT ) .
The genetic linkage data above , together with the observed complementation , indicate that all hisW mutations are alleles of gyrA .
The hisU1820 Mutation of S. typhimurium Is Probably an Allele of gyrB .
Although complementation of the hisU1820 - ( Ts ) mutation with cloned gyrB DNA has not been achieved , negative complementation results , phenotypic analysis , and genetic linkage studies indicate that his U1820 ( Ts ) is an allele of the gyrB gene of S. typhimurium .
A clone of the E. coli gyrB gene that is capable of complementing known gyrB mutations has not been constructed .
The pleiotropic hisW-3333 ( Cs ) and hisU1820 ( Ts ) mutations cause a variety of strikingly similar phenotypes ( see Discussion ) .
In E. coli , the genes near gyrB are ( in order ) gyrB , recF , dnaN , dnaA , rpmH , and rnpA ( 29 , 30 ) .
Several hisU mutations of S. typhimurium have been shown to be rnpA ( the structural gene for the protein subunit of RNase P ) mutations , but hisU1820 ( Ts ) is RnpA + ( 31 ) .
Using plasmids containing cloned DNA of S. typhimurium , which can complement recF , dnaN , dnaA , and rnpA mutations of both S. typhimurium and E. coli ( Russell Maurer and K.E.R. , unpublished results ) , we were unable to complement the his U1820 ( Ts ) mutation of S. typhimurium .
Several restriction sites in the region ( depicted in Fig. 2 ) are conserved between E. coli and S. typhimurium .
The gene orders of the two organisms in this region are identical , and the cloned S. typhimurium DNA should contain only half of the gyrB gene .
We also determined the P22 transductional linkages of recF521 : : TnS to rnpAl ( Ts ) , dnaAl ( Ts ) ( 32 ) , and his U1820 ( Ts ) .
The results shown in Fig .
Complementation of gyrA and hisW phenotypes with E. coli cloned DNA Plasmids gyrA204 gyrA43 gyrAl hisW3333 pRM378 ( gyrA + ) NalS Tr Nals Cr pRM386 ( gyrA + ) Nals Tr Nals Cr pRM389 ( AgyrA ) Nalr Ts Nalr Cs pRM387 ( gyrA : : CAT ) Nair Ts Nal Cs pKO100 ( vector ) Nalr Ts Nalr Cs Plasmid insert structures are depicted in Fig. 1 .
Strains used for transformation with various plasmids were RM146 , KRE449 , KRS2425 , and KRS1717 ( see Table 1 for genotypes ) .
Plasmids had no effect on the phenotypes of wild-type strains .
All transformants were selected and maintained on rich-medium plus ampicillin at permissive growth temperatures [ 32 °C for gyrA43 ( Ts ) ; 37 °C for his W3333 ( Cs ) ] .
Resistance ( r ) sensitivity ( s ) to Nal determined or was by the ability to form single colonies on Nal plates .
Cold-resistant growth ( Cr ) and thermoresistant growth ( Tr ) were assessed .
The small-colony phenotypes associated with both gyrA43 ( Ts ) and hisW3333 ( Cs ) at permissive temperatures were also complemented by the gyrA ' plasmids .
2 are consistent with the E. coli gene order in this region .
The gene assignments of recF521 : : TnS and rnpAI ( Ts ) were confirmed by complementation studies using cloned E. coli DNA ( K.E.R. , unpublished results ) .
The assignment of dnaAl ( Ts ) was confirmed by complementation and marker rescue using cloned S. typhimurium DNA ( Russell Maurer and K.E.R. , unpublished results ) .
We have also noted that growth of the hisU1820 mutant is 10-fold more sensitive than wild type to the gyrase B subunit-specific antibiotic coumermycin AL. .
These results strongly suggest that the hisU1820 ( Ts ) mutation of S. typhimurium is an allele of the gyrB gene .
E. coli gyrA and gyrB Mutations with Known Defects in Supercoiling Are his Regulatory Mutations .
If the his constitutive phenotype of hisW ( gyrA ) mutants is a result of reduced chromosomal superhelicity caused by a reduction in DNA gyrase activity , then the well-characterized DNA gyrase mutants of E. coli should also be constitutive for the expression of the his operon .
We constructed an isogenic pair of strains containing either the gyrA + or the gyrA43 ( Ts ) ( 16 ) alleles ( see Table 1 ) .
These strains were tested for the triazole alanine resistance phenotype that is associated with his constitutive mutations ( 1 , 2 ) , and we assayed their histidinol dehydrogenase activity .
In a similar fashion , we examined his operon expression in four isogenic strains containing either wild-type or mutant alleles of himA ( encoding the A subunit of integration host factor ) and gyrB .
These strains have been examined for their ability to introduce supercoils into superinfecting X DNA ( 17 , 18 ) .
Table 4 shows that the presence of either the gyrA43 ( Ts ) or the gyrB230 alleles confers resistance to triazole alanine and causes a substantial elevation of histidinol dehydrogenase activity .
This indicates that the gyr alleles of E. coli are his constitutive regulatory mutations .
The presence of the himA82 mutation causes a 2-fold elevation in his expression in either the presence or the absence of the gyrB230 mutation ; however the himA allele alone does not confer triazole alanine resistance ( Table 4 ) .
This is consistent with the exacerbation of the gyrB super-coiling-defective phenotype by the himA mutation that was observed in-vivo ( 17 , 18 ) , thus demonstrating a link between a defect in supercoiling and an elevation in the level of his expression .
DISCUSSION We report that the S. typhimurium hisW mutations are highly linked to gyrA , are complemented by a gyrA + plasmid , and are , therefore , allelic to gyrA .
Additionally , the S. typhimu-rium his U1820 ( Ts ) mutation is highly linked to recF and can not be complemented by cloned dnaA ' , dnaN + , recF + , rpmH + , or rnpA ' DNA .
Phenotypically , hisW3333 ( Cs ) and hisUl820 ( Ts ) are similar to each other ( 3 , 7-11 ) and to E. coli gyr mutants ( see below ) .
Finally , E. coli gyrA and gyrB allele are shown to be his regulatory mutations .
Thus , it appears likely that the degree of chromosomal superhelicity regulates expression of the his operons of both E. coli and S. typhi-murium .
We believe that this is due to a dependence of the hisR ( tRNAHiS gene ) promoter on supercoiling for maximal activity .
This model is supported by the demonstration of a very strong supercoiling requirement for the in-vitro-transcription of the hisR promoter ( Lionello Bossi , personal communication ) .
Reduced levels of histidyl-tRNA allow readthrough of the his attenuator and cause elevated his expression ( 4 , 6 ) .
The hisW and hisU1820 ( Ts ) mutations reduce total ( aminoacylated and nonaminoacylated ) tRNAHiS levels by -50 % , resulting in a severalfold increase in his operon expression ( 4 ) .
Thus , expression of the his operon is a sensitive indicator of defects in tRNAHiS production due to defective gyrase function .
Identification of gyr mutations as his regulatory mutations will facilitate genetic analysis of gyr mutations , particularly in the isolation of mutations with supercoiling defects manifested during exponential-growth .
It should be possible to correlate his expression to the extent of chromosomal DNA supercoiling , at least in the domain containing the hisR gene .
This may provide a method to easily quantitate the degree of supercoiling defect in-vivo .
The E. coli gyrB230 mutant is defective in its ability to supercoil superinfecting X DNA , and this defect is exacerbated by himA82 ( 17 , 18 ) .
The himA82 mutation alone did not detectably impair in-vivo supercoiling function in these experiments .
In contrast to this synergistic effect , we observed a 2-fold effect of himA82 on his expression in both the presence and absence of gyrB230 ( Table 4 ) .
The himA + function responsible for the his phenotype could be positive control of gyrA expression ( 17 ) , facilitation of DNA gyrase action ( 18 ) , or gyrase-independent regulation of his expression .
Nonetheless the himA gyrB double mutant is more defective in both supercoiling function and his regulation than with model that his the gyrB single mutant , consistent the Table 4 .
The his operon derepression phenotypes of E. coli gyr mutations E. coli strains derepression ratio phenotype KRE447 ( gyrA + ) 1.0 Sensitive KRE449 ( gyrA43 ) 3.6 Resistant K124 ( himA ` gyrB + ) 1.0 Sensitive K2030 ( himA82 gyrB + ) 1.9 Sensitive K1891 ( himA + gyrB230 ) 6.8 Resistant K1972 ( himA82 gyrB230 ) 16.2 Resistant E. coli cells were grown in minimal glucose medium at 37 °C and assayed for histidinol dehydrogenase activity .
The activities ( in units , 1 unit = nmol/hr per ml ) for gyr wild-type strains ( defined as 1.0 ) were : KRE447 , 3.9 units/OD65o ; K124 , 4.8 units/OD6M , .
The value for wild-type S. typhimurium in this assay was 3.1 units/OD650 .
The triazole alanine ( a toxic histidine analogue ) phenotype was determined .
expression is controlled by the degree of chromosomal superhelicity at the hisR locus .
It has been repeatedly suggested ( 3-5 , 33 ) that the hisWand hisUJ820 ( Ts ) mutations confer defects in tRNA modification ; however , no evidence of this has ever been presented .
In fact , this interpretation has been discounted due to the fact that rRNA accumulation is also affected ( 10 ) .
Ames has alternatively suggested ( 33 ) a defect in tRNA biosynthesis as a possible explanation for the observation that several tRNAs are affected by the hisW mutations .
The levels of several specific tRNAs ( in addition to tRNAHis ) are low and the overall accumulation of 4S RNA is reduced in the hisW-3333 ( Cs ) and hisU1820 ( Ts ) mutants ( 10 , 11 , 26 ) .
A definitive analysis ofthe in-vivo effects of supercoiling on stable RNA synthesis has been elusive .
In vitro , both tRNA and rRNA transcription can be stimulated by template supercoiling ( 34 , 35 ) .
However , in-vivo analyses have been contradictory and inconclusive ( 34 , 36 ) .
Analysis of a variety of gyr mutants whose exponential-growth and temperature-shifted gyrase defects can be graded on the basis of quantitative his operon expression will help identify problems with allele specificity or phenotypic leakiness of specific gyr mutations .
To definitively sort out primary and secondary effects of gyr mutations will require very thorough investigations .
We have not yet concluded our analysis of the other RnpA + alleles of his U , and it is possible that some of them could be dnaA mutations since the dnaA protein has been shown to be a transcriptional regulatory protein ( 37 , 38 ) .
The failure of dnaA + plasmids to complement hisU1820 ( Ts ) makes it unlikely that it is a dnaA allele .
The phenotypes of the hisW mutants , particularly the well-characterized hisW3333 ( Cs ) , can now be attributed to defects in DNA gyrase function .
The defects in isoleu-cine-valine biosynthesis caused by the hisW3333 ( Cs ) and hisU1820 ( Ts ) mutations ( 7 , 8 ) are consistent with the observation that the gyrB230 and gyrB231 alleles of E. coli confer isoleucine auxotrophy in the presence of the himA82 mutation ( 18 ) .
The failure of the hisU1820 ( Ts ) mutant to control stable RNA accumulation during carbon and energy starvation was attributed to a problem with RNA degradation , not synthesis ( 11 ) .
This may reflect the highly pleiotropic nature of the gyr mutations ; the expression of certain RNase genes may be controlled by supercoiling .
The effects of the E. coli gyr mutations on DNA synthesis are consistent with the observation that both hisW3333 ( Cs ) and hisU1820 ( Ts ) form filamentous cells , a phenotype associated with a cell division defect ( refs .
Another interesting observation taken from the his literature , which we have corroborated , is that the his constitutive phenotype of hisW ( gyrA ) mutants is substantially suppressed by growth in rich media ( ref .
Reevaluation of the published characteristics of the hisW and hisU1820 ( Ts ) mutations should enrich our understanding of the cellular effects of DNA supercoiling .
It is possible that tRNA gene expression , and consequently attenuator-regulated amino-acid biosynthetic operon expres sion , is controlled by the degree of chromosomal superhelicity as part of a global regulatory system .
This putative global control system must be quite complex in light of the opposite of the ilv and his gyr responses operons to mutations .
The function of such global control system would a depend on the ability of the cell to modulate its level of supercoiling in response to environmental conditions or possibly timing within the cell cycle .
We thank L. Bossi , J. E. Brenchley , M. Cashel , M. Gellert , F. G. Hansen , P. E. Hartman , J. L. Ingraham , R. Maurer , A. Riccio , J. R. Roth , S. L. Swanberg , and J. C. Wang for materials , advice , and communication of unpublished results .
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