Genes aroA and serC of Salmonella t an Operon yphimurium Constitute SUSAN K. HOISETHt AND B. A. D. STOCKER * Department of Medical Microbiology , Stanford University School of Medicine , Stanford , California 94305 Received 29 October 1984/Accepted 26 April 1985 Genetic analysis of aroA554 : : TnlO derivatives of two mouse-virulent Salmonella typhimurium strains , `` FIRN '' and `` WRAY , '' and of a nonreverting derivative of each constructed for use as a live vaccine , showed the site of the insertion among mapped aroA point mutants .
The WRAY live-vaccine strain gave no aro + recombinants in crosses with aroA point mutations to one side of the insertion , indicating a deletion from TnlO through the sites of these point mutations .
The FIRN live-vaccine strain gave wild-type recombinants with all tested point mutants ; it probably has a deletion or inversion extending from TnlO into aroA but not as far as the nearest point mutation .
Some tetracycline-sensitive mutants of aroA554 : : TnlO strains required serine and pyridoxine , indicating loss of serC function , and some that were found to be SerC-did not produce gas from glucose , indicating a loss ofpfl function .
These results show the gene order pfl-serC-aroA , as in Escherichia coli .
Ampicillin enrichment applied to pools of tetracycline-sensitive mutants of strains with TnlO insertions near aroA ( i.e. , zbj : : TnlO strains ) yielded Aro-SerC-Pfli , Aro-SerC + Pfl + , and Aro-SerC-Pfl + mutants but none which were Aro + SerC - .
All of the mutants are explicable by deletions or inversions extending clockwise from zbj : : TnlO into or through an operon comprising serC ( promoter-proximal ) and ar9A .
Such an operon was also shown by the identification of two TnlO insertions causing phenotype Aro-SerC - , each able to revert to Aro + SerC + by precise excision .
serC corresponds to the open reading frame promoter-proximal to aroA that was identified elsewhere by base-sequencing of a cloned aroA segment of S. typhimurium ( Comai et al. , Science 221:370 -371 , 1983 ) .
Both serine and chorismate are precursors of enterochelin ; this may be why serC and aroA are in a single operon .
We later identified two TnJO insertions causing requirement both for aromatic metabolites and for serine and pyridoxine , i.e. , phenotype Aro-SerC - .
We describe these two insertion mutants first , because they prove the existence of the serC aroA operon and thus simplify the analysis of the deletion and inversion mutants .
In the Discussion section we speculate about a possible reason for the grouping of serC and aroA into an operon , so permitting their coregulation .
We have described the construction of strains of Salmo-nella sp. .
with nonleaky , nonreverting blocks in the common aromatic biosynthesis-pathway .
They were isolated as secondary deletion or deletion-inversion mutations in strains made aroA554 : : TnlO .
Such strains proved to be nonvirulent because , we believe , of their requirement for p-aminobenzoate and for 2,3-dihydroxybenzoate ( DHB ) ( which are not available in mammalian tissues ) ; some of them have been found effective as live vaccines in mice and calves ( 7 , 15 , 21 ) .
The safety of a live-vaccine strain depends on its inability to revert to virulence .
We therefore undertook genetic characterization of two nonreverting aro live-vaccine strains by crosses with 10 aroA mutants mapped by Nishioka et al. ( 12 ) .
One vaccine strain , SL3261 , proved to be deleted for one segment of gene aroA .
This gene , at 19 min on the linkage map ( 17 ) , specifies 3-enolpyruvateshikimate-5-phosphate synthetase , which catalyzes a reaction of the aromatic biosynthesis pathway .
In the course of our examination of tetracycline-sensitive mutants with TnIO-generated deletion or inversion mutations extending from aroA554 : : TnJO or from silent insertions of TnJO near aroA , we encountered mutants requiring serine and pyridoxine .
This is the phenotype of Escherichia coli with mutations of gene serC ( 3 , 20 ) , which is located at about 20 min ( thus near aroA ) and determines the enzyme phosphoserine aminotransferase ( EC 2.6.1.52 ) .
Genetic analysis of these and other mutants indicated that in Salmonella typhimurium , genes aroA and serC are in the same operon , with serC being t Present address : Division of Bacterial Products , Office of Biologics Research and Review , Center for Drugs and Biologics , Food and Drug Administration , Bethesda , MD 20205 .
MATERIALS AND METHODS Bacterial strains and phages .
The main strains used are listed in Table 1 .
Most of the aro : : TnlO strains were from the collection of auxotrophic TnJO insertion mutants of John Roth ( University of Utah ) .
The aroA point mutants ( 12 ) were obtained from K. Sanderson ( Salmonella Genetic Stock Centre , University of Calgary ) .
The general transducing phage used was an int ( integration-deficient ) mutant of a `` high-transducing '' variant , P22 HT105/1 ( 19 ) , of phage P22 .
Phage ANK55 ( 9 ) was used for TnJO mutagenesis .
Culture media and cultural methods .
The nutrient agar and nutrient broth used were Oxoid blood agar base ( code CM55 ) and Oxoid nutrient broth no. 2 ( code CM67 ) .
The defined medium used was that of Davis and Mingioli ( 5 ) with glycerol at 5 ml/liter as a carbon and energy source ; it was used either as liquid or solidified with either New Zealand agar ( 15 g/liter ; Davis Gelatin Co. , Christchurch , New Zealand ) for routine purposes , or Difco Noble agar ( 20 g/liter ) , for tests of requirement of minor aromatic metabolites .
Required amino-acids were added to ca. 20 jig/ml , usually by spreading ; p-aminobenzoic acid , DHB , and other vitamins were added to ca. 1/10 this concentration .
Except where otherwise stated , cultures were incubated at 37 °C , without shaking in the case of broth cultures .
Because of the accumulation of no .
phenotype ( reference ) TT472 LT2 aroA - ( serC ) 1121 : : TnlO TT1455f LT2 aroA554 : : TnlO , Gas-a All strains are S. typhimurium , derived from wild-type strain LT2 , strain S2337/65 ( WRAY ) ( 7,21 ) or M7471 ( FIRN ) ( 7,21 ) .
b TnJO-generated mutations causing loss of tetracycline resistance are indicated as CRR ( complex rearrangement , nature not known ) , DEL ( deletion ) , or INV ( inversion ) , numbered in a single series ; the original TnIO insertion is recorded within brackets , followed by notation , within parentheses , of new phenotypic traits .
All the listed tetracycline-sensitive ( Tc ' ) mutants of aroA554 : : TnlO strains were nonreverting ( to aro + ) .
c Bochner sel. , Selection for tetracycline sensitivity on Bochner medium ( 2 ) with DHB .
Bochnter/Amp sel. , Selection for tetracycline sensitivity , then ampicillin enrichment for ( new ) auxotrophy .
d Strain SL2454 gave a `` pale '' reaction in glucose-phenol red broth .
e Strain SL3261 was obtained by selection on Bochner hedium without added DHB ; it was later found to have a mutation causing partial resistance to fusaric acid ( Fus ) .
f Strain TT1455 fails to produce gas in glucose-phenol red broth , apparently from some cause other than its aroAS54 : : TnJO character .
aro + revertants in stock cultures held at room temperature , strains with aro : : TnJO mutations were stored at -70 °C with glycerol or reisolated after selection for tetracycline resist-ance , before use .
Gas production was tested in Difco glu-cose-phenol red broth with a Durham tube .
Phage and genetic methods .
Phage P22 HT105/1 int was propagated by overnight incubation of broth inoculated with the propagating strain and a phage inoculum , usually ca. 3 x 107 CFU and ca. 3 x 105 PFU per 10 ml of broth .
If the propagating strain carried TnJO , the broth used was supplemented with-tetracycline ( 25 , ug/ml ) .
Most lysates had titers of 5 x 109 to 10 x 109 PFU/ml .
Phage XNK55 was propagated on an E. coli amber-suppressor strain .
Transduction was effected by the `` drop-on-lawn '' method ( 6 ) with serial 10-fold dilutions of lysate for quantitation .
Tetra-cycline-resistant transductants were selected on nutrient agar with-tetracycline ( 25 , ug/ml ) ; aro + transductants were selected on defined medium .
Transductant colonies were streaked out for single-colony reisolation on selective me-dium before tests of phenotype , etc. .
The medium used to test for reversion of aro mutants was defined medium with a growth-limiting amount , usually 20 jig per plate , of L-tryptophan and an excess of all other required substances ; plates were inoculated by flooding with broth culture .
After incubation , aromatic-independent revertants were easily detected as colonies in the thin film of growth ( ca. 1010 CFU per plate ) of aromatic-dependent bacteria .
Reconstruction experiments have shown that reversion at a rate of 10-1 per bacterium per generation would be detected by this method , with use of 10 plates ( 21 ) .
Tetracycline-sensitive mutants of TnJO-containing strains were obtained by selection on the autoclaved chlortetracyline-fusaric acid medium devised by Bochner and his colleagues ( 2 ) , or on the modified medium with glucose omitted ( 10 ) .
( For brevity we call this procedure `` Bochner selection .
'' ) We found that aro strains of S. typhimurium , even if tetracycline sensitive , did not grow on Bochner medium , perhaps because of chelation of ferric ions by its fusaric acid component ; addition of DHB at ca. 2 p.g / ml ( allowing synthesis of enterochelin by aro bacteria ) permitted normal growth of aro tetracycline-sensitive bacteria but did not prevent the inhibition of growth of TnJO carrying bacteria , aro or aro + .
( Strain SL3261 , the `` WRAY '' live-vaccine strain , had been obtained as a rare Bochner-resistant mutant on medium without DHB ; it was later found to have a mutation causing partial resistance to fusaric acid .
) Bochner-resistant mutants were purified by single-colony reisolation on the selection medium .
For TnJO transposon mutagenesis we used a X : : TnJO phage , XNKS5 ( 9 ) , and the Palva ( 13 ) S. typhimurium strain TS736 , which has an F ' factor carrying lamB + of E. coli and is restriction negative for the LT and SA systems of S. typhimurium .
Strain TS736 was grown with shaking in liquid defined medium with maltose as energy source ( because without such selection the F ' was rapidly lost ) ; MgSO4 , to 10 mM , was added to the log-phase culture , and then phage XNK55 was added ( ca. 3 x 108 PFU/ml ) .
After 30 min of incubation at 30 °C to allow adsorption , broth was added , and the mixture was incubated at 37 °C for 75 min .
Plates inoculated with 0.1-ml volumes of lOx concentrated culture yielded ca. 500 tetracycline-resistant colonies per plate .
Such colonies were washed off in defined medium and subjected to ampicillin enrichment .
Ampicillin enrichment was used to select for auxotrophs among ( i ) tetracycline-sensitive mutants of Tn1O-containing strains , obtained by Bochner selection , and ( ii ) tetracycline-resistant TnJO insertion mutants obtained from strain TS736 by A : : TnJO mutagenesis .
Colonies washed from selection plates were diluted in defined medium with any required supplements to give a faintly turbid suspension .
This suspension was shaken at 37 °C for 1 to 2 h ; glucose was added to 5 g/liter , and shaking was continued until the culture was moderately turbid .
Ampicillin was then added to 2.5 mg/ml , and shaking at 37 °C was continued for 90 min .
Volumes of 1 ml were then membrane filtered , the deposited bacteria were washed on the filter with deionized water , and the membrane filter was then placed face down on an appropriate plate , either Bochner medium with DHB or nutrient agar with-tetracycline ( 25 , ug/ml ) .
After 20 min , the filter was removed and the bacteria were spread with a glass rod .
RESULTS Assignment of aro : : TnlO mutations to loci and identification of aroA ( serC ) : : TnlO insertion mutations .
The aro-554 : : TnJO insertion mutation used for construction of nonvirulent strains of S. typhimurium and Salmonella dublin ( 7 , 21 ) was shown by transductional crosses to be at aroA .
Testing of 14 TnWO insertion auxotrophs with unidentified ( because complex ) nutritional requirements showed that one of them , strain TT472 , required not only aromatic metabolites but also serine and pyridoxine , i.e. , was of Aro-SerC-phenotype .
Strain TT472 readily gave rise to pyridoxine-independent mutants which remained serine dependent ( and aromatic dependent ) ; frequent mutational loss of pyridoxine requirement has been reported for serC mutants of E. coli ( 20 ) .
Strain TT472 gave rise to prototrophic , tetracyclinesensitive revertants at about the frequency expected for precise excision of TnlO .
Lysates of TT472 applied to aro + recipients evoked tetracycline-resistant transductants ; all of 25 tested had the Aro-SerC-phenotype of the donor .
When aroA strains ( in line LT2 ) were crossed , as transductional donors , to strains given the Aro-SerC-tetracycline resist-ance character of TT472 , some transductants selected as requiring neither serine nor pyridoxine were aromatic independent and tetracycline sensitive and others were Aro-and tetracycline sensitive .
This shows that the mutation of TT472 is closely linked to aroA ; we indicate its insertion mutation as aroA ( serC ) 1121 : : TnJO .
Main bacterial strainsa TABLE 1-Continued Strain Relevant genotype ' and/or How obtained ' or source Relevant genotypeb and/or How obtained ' or source phenotype ( reference ) LT2 metA22 trpD2 , etc. ( 22 ) S2337/65 ( WRAY ) ( 7,21 ) hisG46 aroA554 : : TnlO As SL1346 but DEL409 From SL1346 by Bochner sel .
[ aroA554 : : TnlO ( Tcs SerC - ) ] SL1346 INV411 As but From SL1346 by Bochner sel .
[ aroA554 : : TnlO ( Tcs SerC - ) ] As SL1346 but CRR412 From SL1346 by Bochner sel .
[ aroA554 : : TnlO ( Tcs ) ] As SL1346 but DEL415 From SL1346 by Bochner sel .
[ aroA554 : : TnlO ( Tcs SerC-Pfl - ) ] As SL1346 but CRR416 From SL1346 by Bochner sel .
[ aroA554 : : TnlO ( Tcs ) ] As SL1346 but DEL417 From SL1346 by Bochner sel .
[ aroA554 : : TnlO ( Tcs SerC-Pfl - ) ] As SL1346 but CRR418 From!SL1346 by Bochner sel .
[ aroA554 : : TnlO ( Tcs ) ] As SL1346 but INV421 From SL1346 by Bochner sel .
[ aroA554 : : TnlO ( Tcs SerC - ) ] AS SL1346 but CRR422 From ` SL1346 by Bochner sel .
[ aroA554 : : TnlO ( Tcs ) ] As SL1027 but zbj-From SL1027 by transduction 903 : : TnlO ( P22 ) As SL1027 but zbj-From SL1027 by transduction 904 : : TnlO ( P22 ) As SL1027 but CRR436 From SL2439 by Bochner / [ zbj-904 : : TnlO ( Tcs Ampp .
Aro-SerC-Pf - ) ] As SL1027 but CRR437 From SL2439 by Bochner / [ zbj-904 : : TnlO ( Tcs Ampp .
Aro - ) ] As SL1027 but zbj-From !
SL1027 by transduction 903 : : TnlO As SL1027 but zbj-From SL1027 by transduction : TnlO 904 : LT2 aro + zbj-905 : : TnlO From aroA102 by transduc-i tion As SL1027 but CRR442 From $ L2444 by Bochner / [ zbj-903 : : TnlO ( Tcs Ampp .
Aro-SerC - ) ] As SL1027 but DEL444 From!SL2444 by Bochner / [ zbj-903 : : TnlO ( Tcs Ampp .
Pfl - ) ] Aro-SerC-As TS736 but aroA-From ' TS736 by X : : TnlO muta - ( serC ) 1123 : : TnlO gene 51is As SL1027 but CRR446 From!SL2445 by Bochner / [ zbj-904 : : TnlO ( Tcs Ampp sel .
Aro-SerC-Pfl - ) ] M7471 ( FIRN ) ( ColEl - ( 7,21 ) K30 ) leu-1051 malB479 cysll173 hisC527 aroA554 : : TnJO SL3218 As but CRR401 ( 7,21 ) [ aroA554 : : TnJO ( Tcs ) ] As SL1346 but DEL407 From SL1346 by Bochner sel .
[ aroA554 : : TnlO ( Tcs ) I ( 7,211 ) Fusr e As SL1027 but hspLT6 P. Lilj jestrom ( 16 ) hspS29 ilv-452 galE496 his-6165 AmalB/F ' 112 ( lamB + ) Strain no .
SL1027 SL1346 J. Roth J. Roth SL2409 SL2411 SL2412 SL2415 SL2416 SL2417 SL2418 SL2421 SL2422 SL2438 SL2439 SL2440 SL2441 SL2444 SL2445 SL2447 SL2454d SL2456 SL2459 SL2462 SL3218 SL3235 SL3261 TS736 Continued next column Another TnlO insertion mutant of Aro-SerC-phenotype was obtained by X : : TnlO mutagenesis .
Tetracycline-resistant clones were elicited by exposure of the X-sensitive S. typhimurium LT2 derivative TS736 of Palva et al. ( 13 ) to phage ANK55 : : TnlO .
These clones were subjected to ampicillin treatment to enrich for mutants with new auxotrophic characters .
One pool of about 1,500 tetracycline-resistant clones was subjected to ampicillin selection , allowing survival of about 10-4 .
Of 54 tested survivor colonies , 50 required aromatic compounds , serine , and pyridoxine .
A representative clone was saved as SL2459 ; transductants given the tetracycline resistance of SL2459 were Aro-SerC - .
We indicate this insertion mutation as aroA ( serC ) 1 123 : : TnJO .
Thus insertion of TnlO in or near aroA can cause either Aro-or Aro-SerC-phenotype .
We think this is because aroA is promoter distal to serC in a serC aroA operon .
Isolation and characterization of tetracycline-sensitive mutants of aroA554 : : TnlO strains .
Two tetracycline-sensitive mutants of aro-554 : : TnJO strains of S. typhimurium , chosen for trial as live vaccines , had been shown not to revert to aromatic independence in tests able to detect reversion at 10-11 per bacterium per generation ( 21 ) .
These are strain SL3235 , obtained by ampicillin selection in the presence of tetracycline from strain SL3218 , an aroAS54 : : TnlO derivative in the S. typhimurium FIRN line , and SL3261 , obtained by selection on Bochner medium from strain SL1346 , an aroA554 : : TnJO derivative in the WRAY line .
The secondary mutations in these two strains did not cause any new nutritional requirement .
Twenty additional tetracyclinesensitive mutants obtained by selection on Bochner medium ( + DHB ) from strain SL1346 ( WRAY aroA554 : : Tn1O ) were investigated .
Five of them , of independent origin , were of nutritional phenotype Aro-SerC - .
Of eight tested tetra-cycline-sensitive mutants without additional nutritional requirements , one reverted to Aro + at much higher frequency than its parent , indicating its origin by `` nearly precise excision '' of TnlO ( 16 ) ; the other seven did not revert to Aro + at detectable frequency .
The two live-vaccine strains , the five Aro-SerC-mutants , and the seven nonreverting Aro-SerC + mutants were further investigated .
Each was tested for gas production in glucose-phenol red broth as a test of function of gene pfl , which determines pyruvate formate lyase and lies close to aroA on the map ( 14 , 17 ) .
Each of the strains was also crossed , as transductional recipient , to several aroA point mutants the order of whose mutation within aroA had been examined by Nishioka et al. ( 12 ) .
The two aroA554 : : TnJO parent strains , SL3218 in the FIRN line and SL1346 in the WRAY line , were similarly tested , for comparison .
The results are summarized in Table 2 , in which the point mutations are shown in the order previously inferred ( 12 ) ; mutations whose relative order is not clear are indicated in footnote d .
In crosses of the two aroA554 : : TnlO parent strains to the point mutants the yield of aro + recombinants was low , as expected , and lowest for donors with mutations in the central ( 70 , 71 , 64 ) - ( 1 , 89 ) part of the map .
This suggests that the aroA544 insertion is in or close to this segment .
The 14 tetracycline-sensitive mutants tested fell into six classes whose behavior and inferred genetic constitution are summarized in Fig. 1 .
Class `` a , '' made up of strain SL3235 ( the FIRN live-vaccine strain ) and five of the nonreverting SerC + mutants , were Pfl + and gave aro + recombinants in crosses with all the point mutants at about the same frequencies as did the aroAS54 : : TnlO parent strains .
They probabl have deletions or inversions extending from within TnlO into aroA but not far enough to overlap the nearest of the tested point mutations .
( The data for the live-vaccine strain SL3235 and two of the other mutants of this class are included in Table 2 ) .
Class `` b '' comprised only SL3261 , the live-vaccine strain in the WRAY line .
It was Se ` rC ' and Gas ' and recombined at about the same frequency as its parent strain with the four point mutants inferred to be to the left of aroA554 , but gave no aro + recombinants in crosses to the six point mutants inferred to be to the right of aroA554 .
This indicates a deletion extending from within aroA554 : : TnJO rightwards into and perhaps through aroA .
Class `` c '' consists of two mutants which retained pfl and serC functions and gave no aro + recombinants in crosses with the point aroA mutants .
However , in control crosses with an aroA + donor they gave only ca. 0.1 % of the number of recombinants obtained in a cross with an aroA554 : : TnJO recipient .
As these two mutants gave normal yields of his ' transductants , the low yield of aro + can not result from any defect in phage adsorption or recombination capacity .
We interpret this class as having deletions or inversions of chromosomal segments nearly as long as a P22 headful , so that transducing particles able to correct their defects are very uncommon ; or they may have deleted or inverted segments longer than a headful length of DNA , therefore repairable only by the rare coincidental adsorption of two transducing particles of appropriate DNA content ( 18 ) .
Phenotypes and transductional analysis of aroA554 : : TnJO strains and their tetracycline-sensitive derivatives used as live vaccines ; and additional tetracycline-sensitive mutants of strain SL1346 ' Phenotype No .
of aro + transductants per 108 PFU with aro donor : SerC 71d 64 554e If 89f 102 55R 469 4 35 Strain no .
and description 67 70d aro + aroA554 : : TnlO strains and livevacine mutantsb + + 6 4 4 8 SL3218 ( FIRN aroA : : TnlO ) SL3235 ( FIRN live vaccine ) class `` a '' CRR SL1346 ( WRAY aroA : : TnlO ) + SL3261 ( WRAY live vaccine ) class `` b '' DEL SL1346 Tcs mutants ' SL2416 , class `` a '' CRR + + 6 2 4 2 ISIO 2 0.5 SL2422 , class `` a '' CRR + + 0.8 0.3 0.1 0.2 ISIO 1 0.6 SL2412 , class `` c '' CRR + + 0 0 0 0 ISIO 0 0 SL2418 , class `` c '' CRR + + 0 0 0 0 ISIO 0 0 SL2411 , class `` d' ' INV + -5 0.8 0.3 0.8 ISIO 8 4 SL2421 , class `` d' ' INV + -5 0.2 0 0 ISIO 5 0.7 SL2409 , class `` e '' DEL + -0 0 0 0 ISIO 2 0.8 SL2415 , class `` f '' DEL -- 0 0 0 0 ISIO 4 2 SL2417 , class `` f ' DEL -- 0 0 0 0 ISIO 2 2 a The segments inferred to be deleted or inverted in mutants of classes `` a '' through `` f ' are illustrated in Fig. 1 .
b aroAS54 : : TnlO strains and their tetracycline-sensitive mutants used as live vaccines .
c Additional tetracycline-sensitive mutants of strain SL1346 .
df.g The relative order of the point mutations in each of these clusters is uncertain : 70 , 71 , and 64 ; 1 and 89 ; and 55 , 46 , and 43 .
eInferred position of the TnlO insertion in aroA554 : : TnIO strains and of a residual ISIO element in tetracycline-sensitive mutants of such strains .
TnJO 18 ISIO 17 38 5,000 59 6,100 23 8 21 19 55 56 57 31 + + 3 3 1 9 + 6 3 4 2 TnIO 7 IS10 0 30 3,700 0 3,800 11 10 38 0 8 40 0 + + 5 4 6 0 0 0.8 6 0 0 14 3 2 2 9 6 2 18 1 12 3,500 2,650 2.5 2.5 2,300 2,600 3,400 700 1,500 19 0 0 44 22 24 20 27 0 0 22 17 22 14 10 0 0 33 11 21 9 22 pfl I I Pr serC .
.55 67 70 554 1 55 class a , CRR , SerC Pfl + class b , DEL , SerC + Pfl + cla c , CRR , ScrC + l or npr + O r A r o 4 .
J. l clas d , INV , SsrC-Pfl + clas e , DEL , SerC Pfl + A    C-IAU-S .
S 4t , DMELN , S9rC Prn vrTci-sa - I I I I FIG. 1 .
Inferred deletions or inversions in tetracycline-sensitive , nonreverting mutants of aroA554 : TnlO strains ( distances not to scale ) .
( ) Segment either deleted or inverted .
Interrupted lines indicate possible extension of deleted or inverted segment .
The parent strains have TnlO inserted at aroA544 .
Both the deletion and the inversion mutants are inferred to have a residual copy of ISIO at position aroA554 , and the inversion mutants are inferred to have a second ISIO at the other end of the inverted segment .
The point mutation sites shown are representatives of those closest to and farthest from aroA544 on each side ( Transductional crosses to point-mutant recipients would have shown whether the defects were deletions or inversions .
However , the WRAY strain , though susceptible to P22-mediated transduction and not lysogenic for any P22-like phage , does not support growth of phage P22 or related general transducing phages , so derivatives in this line could not be tested as donors ) .
Class `` d' ' comprised two mutants which were Pfl + SerC-and able to recombine with point mutants with sites to the right of aroA554 and with several of those to its left .
Since they gave aroe in crosses with point mutants on the left , their loss of serC function can not result from a deletion leftwards from aroA554 into through serC ; it must thereor fore result from inversion of a segment with a left endpoint within serC or the promoter region of the serC aroA operon .
Class `` e '' consisted of a single Pfl + SerC-mutant giving aro + recombinants in no crosses to the point mutants with affected sites to the left of aroA554 ; this is as expected for a strain with a deletion from aroA leftwards to an endpoint either within serC or in the promoter of the serC aroA operon or beyond it , but not so far as to involve gene pfl .
Class `` f ' comprised two Pfl-SerC-mutants giving no aro + recombinants with point mutants to the left of aroAS54 ; we attribute them to deletions extending leftwards from aroA554 through serC and the serC aroA promoter and into or through pfl .
TnlO insertions near aroA .
P22 lysates of pools of LT2 sublines made tetracycline resistant by random TnJO insertions ( 9 ) were screened for ability to evoke aro + tetra-cycline-resistant transductants from an aroA recipient .
Four independent TnJO insertions at sites cotransducible with aroA , labeled zbj-903 through zbj-906 , did not cause auxotrophy or obvious alteration in phenotype other than tetracycline resistance .
Each was crossed by transduction to recipients with aroAS54 : : TnJO-derived deletions : SL3261 ( rightward deletion ) and SL2415 ( leftward deletion extending through serC into or through pfl ) .
Transductants selected as tetracycline resistant were scored for aro .
The results ( Table 3 ) suggest that each of the silent TnJO insertions is to the left of aroA , close to the left end of the aroA serC pfl leftward deletion , but with at least zbj-904 and zbj-906 not overlapped by the deletion .
None of the tested Bochner derivatives of zbj-903 ( 87 isolates ) or zbj-904 : : TnJO ( 18 isolates ) was auxotrophic .
Mutants affected in aroA or serC function were therefore sought by Bochner selection followed by ampicillin enrichment for auxotrophy ( see Materials and Methods ) .
Table 4 records the Aro , Ser , and Pfl ( gas production ) phenotypes of clones of survivors of ampicillin killing of pooled Bochner-resistant mutants of zbj-903 , zbj-904 , and zbj-905 : : TnJO strains .
The results were qualitatively similar in all the experiments , except that no gas-negative mutants were isolated from the zbj-905 : : TnIO strain .
( Given the order pfl-serC-aroA and that the four zbj insertion sites are to the left of aroA [ Table 3 ] , the isolation of gas-negative mutants '' Gas production was tested in glucose-phenol red broth with Durham tube .
`` Pale '' indicates pale yellow-brown color after overnight incubation , attributed to decolorization of the acid form ( yellow ) of the phenol red indicator .
b Because of the way selection was applied , some phenotypically similar clones in any one experiment may be members of a single mutant clone .
' Parent strain SL2447 is LT2 aroA102 , made aro ' zbj-905 : : TnlO by transduction , and therefore prototrophic .
The other parent strains are from strain SL1027 ( which is Met-Trp - , etc. , see Table 1 ) made zbj : : TnIO by transduction .
d These clones , not having any new nutritional requirements , were not tested for gas production .
from zbj-903 : : TnJO and zbj-904 : : TnlO but not from zbj-905 : : TnJO strains suggests the gene order [ zbj-903 zbj ¬ 904 ] - pfl-zbj-905-serC-aroA .
) All the Bochner selection-ampicillin enrichment experiments except that involving the zbj-905 : : TnJO parent gave some Aro-SerC-Pfl-clones .
These may be rightward deletions from zbj-903 or -904 through pfl and serC into or through aroA , or through pfl into the promoter or proximal gene , serC , of the serC aroA operon .
Three such clones were crossed , as recipients , to the aroA point mutants ( Table 5 ) .
One of them , SL2456 , gave no wild-type recombinants with any of the point mutants , as expected if it has a deletion extending from zbj-903 through pfl , serC , and aroA .
The other two , SL2440 and SL2462 , derived from zbj-904 parents , gave wild-type recombinants with all the point mutants .
We think they have deletions rightward from zbj-904 through pfl into the serC aroA operon and with a right endpoint either in this operon 's promoter or proximal structure gene , serC , or in the left extremity of aroA ; a deletion with its right end at any of these points would cause loss of aroA function , even though the structural gene , or most of it , would be intact .
At least one Aro-SerC + Pfl + clone was obtained from each of the three zbj : : TnJO parents tested ( Table 4 ) .
We attribute such clones to inversion of a segment extending from the zbj : : TnJO site through serC to an endpoint within aroA ; gene serC ( and pfl , for mutants of zbj-903 or zbj-904 : : TnJO parents ) , with promoter intact , would be expected to function normally despite inversion .
One of these clones , SL2441 , derived from a zbj-904 : : TnJO parent , was crossed to the aroA point mutants as donors ; the low yield of aro + recombinants ( Table 5 ) is as expected for a recipient with an inversion of the postulated type ( 8 ) .
Mutants of nutritional character Aro-SerC-but producing gas in glucose-phenol red broth were obtained from zbj-903 , -904 , and -905 parents ( Table 4 ) .
All 32 isolates of this type ( not necessarily all of independent origin ) differed from the 34 tetracycline-sensitive isolates found to have other combinations of Aro , SerC , and gas production character in that the glucose-phenol red broth cultures after overnight incubation were a pale brown color , instead of the bright yellow , apparently because of decolorization of acid form of phenol red .
The cause of the `` pale '' phenotyp is not known , but we feel reasonably sure that this class are pfl + .
We infer them to have inversions of segments extending from their zbj : : TnJO insertions to a right endpoint in the promoter of the serC aroA operon or in serC .
Several of this class , including SL2454 ( Table 5 ) , when crossed to the aroA point mutants as donors gave aro + recombinants with all of them,-as expected if they have the postulated type of inversion .
New phenotypic traits of mutants isolated from zbj : : TnlO strains by Bochner selection followed by ampicillin enrichment for ( new ) auxotrophic character No .
'' among clones derived from parent ' : Phenotypic class ( new characters ) a SL2438 , SL2444 , SL2439 , SL2445 , SL2447 , zbj-903 zbj-903 zbj-904 zbj-904 zbj-905 14d 3 8d None 4 8 Gas-0 1 0 Aro-SerC-Gas-13 1 1 5 0 Aro-1 2 1 3 1 Aro-SerC - , pale 10 8 0 8 6 TABLE 3 .
Proportion of tetracycline-resistant transductants acquiring donor aroA + in crosses of zbj : : TnlO donors to recipients with leftward or rightward deletions derived from aroA554 : : TnlO No .
of transductants in cross with donor : zbj-903 zbj-904 zbj-905 zbj-906 SL2415 , leftward deletion 29/29 26/28 28/28 28/30 SL3261 , rightward deletion 40/48 19/30 28/30 13/24 Recipient DISCUSSION The identification of two mutants with deletions leftwards from aroAS54 : : TnJO through serC and into or through pfl ( class `` f , '' Table 2 and Fig. 1 ) , and of another with a deletion extending into or through serC but not affecting pfl ( class `` e , '' Table 2 and Fig. 1 ) , showed the gene order pfl-serC-aroA , as in E. coli ( 1 , 3 ) .
The properties of all the tetra-cycline-sensitive mutants isolated from aroASS4 : : TnJO strains ( Table 2 ) or from zbj : : TnJO strains ( Table 5 ) can be accounted for by deletions or inversions extending for various distances to one side or the other from the site of their TnJO insertions .
One result at least seemed to indicate that serC and aroA made up an operon , with serC being promoter proximal .
Strain SL2454 , a tetracycline-sensitive mutant of a zbj-903 : : TnJO parent , was Pfl + SerC-Aro - ( Table 5 ) .
If , as inferred , pfl lies between zbj-903 and serC , the Pfl + character of this mutant indicates inversion of a segment extending from zbj-903 , not its deletion .
Such an inversion would inactivate only the gene or operon disrupted by its right endpoint , and thus only serC or only aroA , if these two genes were in separate operons .
An inversion with its right end in the promoter or proximal structural gene of a serC aroA operon would account for the loss of both aroA and serC functions and also for the high yield of aroc recombinants in crosses of SL2454 with the aroA point mutants ( Table 5 ) .
The recognition of two TnJO insertions causing SerC-Aro-phenotype , taken with the existence of other TnJO insertions at aroA causing only Aro-phenotype , gave unequivocal evidence that serC and aroA are in the same operon , with serC nearest the promoter .
Luca Comai and his colleagues ( Calgene , Inc. , Davis , Calif. ) , independently of our investigation , have cloned an aroA-containing DNA segment from S. typhimurium LT2 ( 4 ) ; David Stalker ( personal communication ) has now by base-sequencing detected an open reading frame promoter proximal to the aroA structural gene and has recently confirmed our interpretation that this open reading frame is gene serC .
The functional significance of the grouping of serC and aroA in a single operon is not known .
However , one may note that serine and DHB are both precursors of the bacterial iron-binding compound enterochelin ( = enterobactin ) .
Iron starvation is known to cause depression of several genes for proteins concerned in iron acquisition and also modifications in the structure of several tRNA species , including two tRNAser species ( 11 ) .
Grouping of one gene of the common aromatic biosynthesis pathway and one gene concerned in serine biosynthesis into a single operon may permit their coregulation according to the iron status of the bacterium .
One objective of our work was to find the nature of the secondary mutations causing inability to revert in two tetra-cycline-sensitive derivatives of aroAS54 : : TnJO strains chosen for investigation as live vaccines ( 7 , 21 ) .
Transductional crosses showed one of them , strain SL3261 , to have a deletion extending from the transposon through all tested point mutations to its `` right '' ( Table 2 , Fig. 1 ) .
The other live vaccine strain , SL3235 , recombined with all the tested aroA point mutants , as did five nonreverting but Pfl + SerC + mutants of SL1346 ( class `` a '' in Table 2 and Fig. 1 ) .
We think it likely that these strains have deletions or inversions extending from within TnJO to the left or the right into aroA but not as far as the nearest point mutation .
However , some tetracycline-sensitive mutants of his : : TnJO strains were reported to have genetic lesions of unknown nature , apparently not extending outside the transposon , yet causing reduction in rate of reversion to his ' , in some of them by as much as 100-fold ( 8 ) .
Even though live-vaccine strain SL3235 gave no aromatic-independent mutants in tests that had been shown by reconstruction to be able to detect reversion at a rate of 10-11 per bacterium per generation , we can not exclude the possibility that this strain 's secondary mutation is entirely within TnJO , yet causes 1,000-fold or greater reduction in rate of reversion to aro + .
For this reason , in later construction of candidate oral-route live-vac-cine strains of Salmonella typhi ( M. F. Edwards and B. A. D. Stocker , unpublished data ) , nonreverting aroA derivatives have been obtained not by selection of tetra-cycline-sensitive mutants of strains made aroAS54 : : TnJO by transduction but instead by first transducing a transposon insertion causing Aro-SerC-phenotype into a wild-type strain , then replacing it with serC + AaroA by transduction , with selection for serine-pyridoxine independence .
S.K.H. was recipient of a National Science Foundation predoctoral fellowship .
This work was supported by Public Health Service research grant A107618 from the National Institutes of Health .
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