Structure of the Gene Encoding Phosphoribosylpyrophosphate Synthetase ( prsA ) in Salmonella typhimurium STANLEY G. BOWER ,1 BJARNE HOVE-JENSEN ,2 AND ROBERT L. SWITZER ' * Department of Biochemistry , University of Illinois , Urbana , Illinois 61801,1 and Enzyme Division , Institute of Biological Chemistry B , University of Copenhagen , DK-1307 , Copenhagen K , Denmark2 Received 14 October 1987/Accepted 15 April 1988 The Salmonella typhimurium gene prsA , which encodes phosphoribosylpyrophosphate synthetase , has been cloned , and the nucleotide sequence has been determined .
The amino-acid sequence derived from the S. typhimurium gene is 99 % identical to the derived Escherichia coli sequence and 47 % identical to two rat isozyme sequences .
Strains containing plasmid-borne prsA have been used to overproduce and purify the enzyme .
The promoter for the S. typhimurium prsA gene was identified by deletion analysis and by similarity to the promoter for the E. coil prsA gene .
The location of the prsA promoter results in a 416-base-pair 5 ' untranslated leader in the prsA transcript , which was shown by deletion to be necessary for maximal synthesis of phosphoribosylpyrophosphate synthetase .
The S. typhimurium leader contains a 115-base-pair insert relative to the E. coli leader .
The insert appears to have no functional significance .
The enzyme phosphoribosylpyrophosphate ( PRPP ) synthetase catalyzes a reaction at a key junction in intermediary metabolism .
The enzyme diverts ribose 5-phosphate from energy generation by the pentose phosphate pathway to biosynthesis via the intermediate PRPP .
PRPP is utilized in the biosynthesis of pyridine nucleotide coenzymes , the amino-acids histidine and tryptophan , and the pyrimidine and purine-nucleotides .
From 70 to 80 % of the carbon flow through PRPP synthetase is directed to nucleotide and nucleic acid synthesis ( 15 ) .
The amount of PRPP synthetase activity in enteric bacteria is mediated at two levels : enzyme inhibition and regulation of synthesis .
ADP is a potent inhibitor which binds at an allosteric site as well as competitively with ATP at the active site ( 5 , 22 ) .
The ADP inhibition of PRPP synthetase at the allosteric site requires occupation of active the site .
Thus , in the presence of ribose 5-phosphate , the activity of PRPP synthetase is mediated by the ratio of ADP to ATP .
A pyrimidine , probably UDP or UTP , represses the synthesis of the enzyme 2-to 10-fold ( 17 , 26 ) .
A Salmonella typhimu-rium rpoBC mutant which showed derepressed levels of aspartate transcarbamylase ( pyrBI ) and orotate phosphoribosyltransferase ( pyrE ) , enzymes that have been shown to be regulated by attenuation mechanisms , also had a derepressed level of PRPP synthetase ( 8 ) .
This observation suggests that an attenuation mechanism may also function to regulate the gene encoding PRPP synthetase ( prsA ) .
The catalytic mechanism of S. typhimurium PRPP synthe-tase has been studied extensively , whereas the genes encoding the Escherichia coli and rat enzymes have been cloned and sequenced ( 6 , 7 , 23 ) .
To initiate molecular genetic studies and to examine the regulation of S. typhimurium PRPP synthetase expression , the gene encoding the S. typhimurium PRPP synthetase has been cloned and the nucleotide sequence has been determined .
MATERIALS AND METHODS Microbiology and molecular biology .
The strains used are listed in Table 1 .
Strain SB139 was produced from TR5878 and SB179 was produced from JL1002 by transduction with P22 grown on JL2943 .
Minimal medium was that of Vogel and Bonner ( 25 ) with 0.2 % glucose .
Rich medium was the LB-medium of Miller ( 13 ) .
Plasmid pHOll carries the E. coli prsA gene in a fragment of pBR322 ( 6 ) .
Standard molecular and microbiological methods were as described by Miller ( 13 ) , Maniatis et al. ( 11 ) , Davis et al. ( 3 ) , Silhavy et al. ( 20 ) , and Schleif and Wensink ( 19 ) .
The nucleotide sequence was determined by the method of Maxam and Gilbert ( 9 , 12 ) .
Sequence determinations to confirm constructions and deletions were by a dideoxy termination method , with double-stranded plasmids as templates ( 2 ) .
Sequence analyses were performed with software by DNAstar , Inc. .
Enzyme assays and purification .
PRPP synthetase activity was assayed by a modification of the published method ( 21 ) .
The volume was reduced to allow assays to be run in 1.5-ml Microfuge tubes .
A modification of the procedure described by Lupski et al. ( 10 ) was used to assay P-lactamase .
Assays were run in 1 ml ( final volume ) of 0.1 M potassium phosphate ( pH 7.0 ) at room temperature ( 23 °C ) and contained 20 , ul of cephaloridine ( 1 mg/ml ) and 2 to 10 RI1 of extract .
The change in A255 was monitored .
Purification of PRPP synthetase followed the published protocol ( 21 ) , except that strain SB139 containing plasmid pBS2O1 was used as the source and the last two steps of the purification were omitted .
Construction of subclones and deletions .
pBS111R was constructed by digesting pBS111 with BamHI and religation ( Fig. 1 ) .
Analogous clones of the E. coli and S. typhimurium prsA genes were constructed in pUC18 and pUC19 which utilized the EcoRI sites at base pair ( bp ) 59 or bp 128 and the PvuII sites at bp 1830 or bp 1780 in S. typhimurium and E. coli , respectively .
A second EcoRI site in S. typhimurium prsA at bp 182 allowed simultaneous construction of similar plasmids which removed the putative prsA promoter with a 123-bp deletion .
The constructed clones are represented in Fig. 1 .
Plasmid pBS202 , constructed with pUC18 , places the prsA gene in the same orientations as the lac promoter .
Plasmids pBS201 , pBS203 , and pBS205 , constructed with pUC19 , place the prsA gene in the opposite orientation from the lac promoter .
In plasmids pBS202 and pBS203 , the prsA promoter has been removed .
To establish the importance of the leader region , plasmids were derived from pBS201 and pBS202 , which deleted most of the leader sequence .
pBS214 was generated from pBS201 by partial EcoO109 digestion , SauI digestion , DNA polymerase I large fragment treatment to generate blunt ends , and ligation .
pBS215 was generated from pBS202 by EcoRI digestion , SauI digestion , DNA polymerase I treatment , and ligation ( Fig. 1 ) .
Plasmid pBS216 was generated by digestion of pBS111R with SmaI followed by NruI and by blunt-end ligation .
Restriction analysis and nucleotide sequencing across the deletion junctions confirmed proper constructions ( data not shown ) .
324 RESULTS Cloning of S. typhimurium prsA .
An E. coli strain with the temperature-sensitive prsA2 mutation ( HO541 ) was transformed with a library of S. typhimurium JL75 chromosomal DNA fragments generated by Sau3A partial digestion and was ligated into BamHI-digested pUC13 .
Candidates were selected which grew at the nonpermissive temperature , 40 °C .
Instability of initial isolates suggested multiple transformation .
Rapid-preparation plasmid DNA was fractionated by agarose gel electrophoresis , and individual bands were used to transform E. coli H0541 .
Candidates pBS111 and pBS119 were stable isolates , although they yielded very small colonies in strain H0541 .
Restriction analysis demonstrated that BamHI sites were regenerated at each end for both isolates and that the inserts were 1.75 kbp for pBS111 and 1.95 kbp for pBS119 .
Nucleotide sequence of the prsA gene .
The nucleotide sequence of the S. typhimurium insert in pBS111 was determined by the chemical method of Maxam and Gilbert .
The strategy and sites used to sequence both strands with complete overlaps are shown in Fig. 2 .
The nucleotide sequence and the derived amino-acid sequence of the S. typhimurium gene and flanking regions are presented in Fig. 3 .
Differences in the DNA and derived amino-acid sequences of E. coli from S. typhimurium sequences are indicated below the S. typhimurium sequences .
PRPP synthetase is encoded by nucleotides 560 to 1504 .
The 315-codon reading frame initiates with GTG , as does the E. coli sequence , and ends with a TGA termination codon .
Evidence indicating correct assignment of the reading frame includes ( i ) exact correspondence of residues 2 through 24 with the amino-acid sequence of the mature protein ( 7 ) ; ( ii ) correct prediction of the COOH-terminal sequence,-Glu-His-COOH ( 7 ) ; ( iii ) correct alignment of the predicted amino-acid sequence with the chemically determined amino-acid sequence covering approximately 80 % of the protein ( K. W. Harlow , unpublished data ) ; and ( iv ) near identity with the determined sequence of the E. coli gene ( 7 ) .
The predicted amino-acid sequence of S. typhimurium PRPP synthetase differs from the E. coli sequence at only 2 of 315 amino-acid-residues , Thr278 ( Ser in E. coli ) and Ala283 ( Ser in E. coli ) , and is 47 % identical to the predicted sequence of the rat gene ( 23 ) .
Bacterial strains Sour or reference ce Description Strain E. coli H0541 6 metB his trp leu rpsL prsA2 ( Ts ) 28 ara A ( lac-pro ) rpsL thi +80 D lacZ AM15 JM83 S. typhimurium JL75 JL1002 JL2943 SB139 trp-196 pyrA8i L. Bussey L. Bussey L. Bussey This work srl-2 : : TnJO recAI rpsL ilv-542 metA22 trpB2 strA120 flaA66 metE551 xyl-404 Hi-b H2-e nml hsdLT6 hsdS29 recAl pyrA81 srl-2 : : TnlO recAl met ilv-542 metA22 trpB2 strAi20 flaA66 metE551 xyl-404 Hi-b H2-e nml hsdLT6 hsdS29 SB179 SU422 TR5878 This work P. Sypherd J. Roth S. typhimurium -  Ploc PprsA Vector 560 1738 1507 i pUC13 pBSIII I Ploc pUC13 PprsA pBS1IR 195so pUCI3 -- ow PprsA Ploc I paS119 I Ploc pUCI9 PprsA I 182 I pB201 I pUCI8 pBS202 P lc Ploc pUCI9 p8S203 I , I 59 - 164 PprsA LJI 546.1 p8S2 14 Ploc pUCI9 I I puc18 pBS215 Ploc If 103 -- 0 PprsA Ploc pUC13 pBS216 , E. coli 29 - .
1PprsA 1462 1785 II 361 362 I 515 pHOI p8R322 p8S205 IPprsA r g I Plac pUCI9 I I .
Structures and alignment of analogous prsA-containing plasmids described in this study .
S. typhimurium numbering is from Fig. 2 .
E. coli numbering is from reference 7 .
Vector pBR322 is the PvuII ( bp 2069 ) - to-EcoRI ( bp 4362 ) fragment ( 18 ) .
Plac , Lactose promoter ; PprsA ' prsA promoter 0 ) N ) a cli e ) v 0 Q1 0 Io0 0 ( 0 I-1-c r-rea 1D oW ) N O ) it I1 ) 1-0 0-N d L4 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ .
~ ~ e i ~ ~ ~ ~ ~ ~ ~ co a.L0 ) m r. xcowE W ~ E I I 0 0 a1 ) 0 H 0o H ' 2 ~ ~ ~ t0a NMt `` M0 nol ) rE ) a C9 cents am ~ ~ E ~ ~ X ~ : Xx 06 uI a ~ 0 I o ) t ( D N 0ot IC ) it ) OD CY It ) - Oie 0 0n d ~ ¬ ¬ w A > - ~ r ¬ IOObp II FIG. 2 .
Restriction map and sequencing strategy for the S. typhi-murium prsA gene .
The dark line indicates the region encoding the protein .
fication of E. coli residue 126 as Ile ( ATA codon ) ( 7 ) has been corrected to Thr ( ACA codon ) ( unpublished data ) .
Alignment of the N-terminal proline of PRPP synthetase as isolated with the second codon of the nucleotide sequence shows that the initiator fMet is processed from this protein .
The nucleotide identity between the S. typhimurium and E. coli coding frames is 91 % ; 95 % of the differences are in the third positions of codons .
The high level of identity extends into the flanking regions ; 230 bp on the 3 ' side of the coding region are 92 % identical .
On the 5 ' side , the identity is also 92 % , except for a 115-bp insert in the S. typhimurium sequence relative to the E. coli sequence , which begins at bp 292 ( Fig. 3 ) .
A promoter driving the expression of the prsA gene in E. coli has been identified ( 7 ) .
The region of DNA encoding that promoter is essentially conserved in the S. typhimurium nucleotide sequence .
The promoter sequence covers the region bp 108 through bp 144 in Fig. 3 .
The -10 and -35 regions identified in E. coli are completely conserved .
The sequence , CGCGCC , between the -10 and transcription initiation site corresponds to a `` discriminator sequence '' identified as important in modulating the stringent-response ( 24 ) .
The position of the putative prsA promoter relative to the translation start site ( bp 560 ) results in a long transcribed leader , 302 bp in E. coli and 416 bp in S. typhimurium .
The 115-bp insert in S. typhimurium relative to E. coli lies in this leader sequence .
A sequence conserved near the promoters of pyrC , pyrD , and carAB ( 27 ) , gGAAAACGtTT ( c/g ) - CGcttnt , is found just upstream of the prsA promoter at bp 90 to 103 and will be called the pyrimidine-conserved region .
Upstream 40 bp from the -35 region of the prsA promoter is a consensus-binding site sequence for catabolite activator protein ( CAP ) ( 4 ) : AAnTGTGAnnTnnnnC .
Another noteworthy feature in the leader sequence is a `` box A '' sequence ( 16 ) at nucleotides 464 to 471 ( Fig. 3 ) .
This sequence is downstream from the 115-bp insert and is present in both the E. coli and S. typhimurium sequences .
Downstream 16 bp from the termination codon of both E. coli and S. typhimurium prsA genes is a nearly identical region of hyphenated dyad symmetry followed by six T residues .
Such structures can lead to the formation of hairpin loops in the mRNA and have been implicated as rho-independent terminators ( 29 ) and as blocks against 3 ' - to-5 ' exonucleolytic activity ( 14 ) .
Si mapping in E. coli has identified this site as the terminus of the isolated transcript ( 7 ) .
There is a potential reading frame that extends from the 5 ' cloning junction for more than 100 codons in both E. coli and S. typhimurium .
The cloning junction for E. coli is-70 bp upstream from that of S. typhimurium and thus extends the open reading frame 23 codons .
The 115-bp insert in the S. typhimurium sequence falls in the termination codon for the E. coli upstream open reading frame and extends the potential reading frame by 31 codons .
Examination of the codon usage in the open reading frames showed no bias , suggesting high-level translation ( data not shown ) .
The strong promoter which normally expresses prsA lies within the open reading frame .
A gap of about 160 bp lies between the open reading frame translation termination codon and the initiation codon for the prsA coding frame .
There are no structures which suggest rho-independent termination between the open reading frame and the prsA gene .
A search of the GenBank DNA sequence database ( release no. 47 , March 1987 ) and National Biomedical Research Foundation-Protein Identification Resource protein sequence database ( release no. 12 , April 1987 ) by using the DNAstar Nucscan and Proscan programs found no sequence with significant homology to the upstream open reading frame ( data not shown ) .
There is , at present , no evidence that the upstream open reading frame is actually expressed in either E. coli or S. typhimurium .
PRPP synthetase activity of E. coli and S. typhimurium strains bearing homologous and heterogeneous prsA clones .
The prsA genes from E. coli and S. typhimurium were subcloned into pUC19 by using analogous restriction sites , generating pBS205 ( E. coli prsA gene ) and pBS201 ( S. typhimurium prsA gene ) ( Fig. 1 ) .
Both genes were more highly expressed in S. typhimurium SB139 than in E. coli JM83 , but expression appears normal for each in the heter-ologous background ( Table 2 ) .
This suggests that the 115-bp insert in the leader region of the S. typhimurium gene has no effect on control of its expression .
Localization of the prsA promoter by deletion .
Table 3 shows the levels of PRPP synthetase activity found in S. typhimurium SB179 containing various prsA plasmids .
A comparison of the enzyme activity levels with plasmids pBS216 and pBS203 demonstrated that removal of the 79 nucleotides from bp 103 to 182 eliminated most of the plasmid-encoded activity .
This result confirms the expected position of the promoter in S. typhimurium .
Comparison of pBS201 to pBS216 showed that the 92-bp extension on the 3 ' end has no effect on enzyme levels .
pBS111R extended the sequence 59 bp 5 ' to that present in pBS201 and increased the enzyme level threefold .
An element 5 ' to the promoter is thus essential for maximal expression .
Effect of deletion of the 5 ' untranslated leader region of prsA .
The 5 ' untranslated leader is required for normal expression of the prsA gene .
When the DNA from bp 165 to pBS111R bp 1-1738b 4.7 3.7 pBS201 bp 59-1830 1.7 5.0 pBS216 bp 103-1738 1.4 4.4 pBS203 bp 182-1830 0.07 NT pUC19 No insert 0.04 6.5 a Growth was in Vogel-Bonner medium with 0.2 % glucose at 30 °C .
The host strain was SB179 .
b Inserts are in pUC19 , i.e. , with prsA transcription opposed to the plasmid-borne lac promoter .
Localization of elements essential for S. typhimurium prsA expression by deletion analysis TABLE 4 .
Evaluation of the role of the 5 ' untranslated leader region of prsA by deletion analysis Sp act ' ( p.mol of product/min per mg of protein ) PRPP synthetase , B-Lactamase Sp acta ( , umol of product/min per mg of Plasmid Description Plasmid Description Vector 545 was removed from the prsA-bearing plasmids , expression was reduced to that attributable to the chromosomal gene ( Table 4 ) .
This was true whether expression was driven by the prsA promoter ( cf. pBS201 and pBS214 ) or by the lac promoter ( cf. pBS202 and pBS215 ) .
Purification of PRPP synthetase .
S. typhimurium SB139 ( recA ) bearing plasmid pBS201 produced very high levels of PRPP synthetase at 42 °C ( 22 p.mol of PRPP per min per mg of protein ) .
In one purification , 182 mg of pure enzyme was prepared from 2.5 liters of cell culture ( 6.3 g of cell paste ) in a four-step procedure .
This is a substantial improvement in comparison with 78 mg of enzyme from 200 liters of culture ( 500 g of cell paste ) prepared by an earlier six-step procedure ( 21 ) .
An important element in obtaining such high levels of expression was growth of cells bearing pUC-derived plasmids at 42 °C to stationary-phase .
DISCUSSION The S. typhimurium gene encoding PRPP synthetase , prsA , has been cloned and sequenced .
Strains containing plasmids with prsA could be induced to produce more than 200 times as much PRPP synthetase as is expressed by strain SU422 , the strain previously used in enzyme purifications .
Such strains have allowed purification of large amounts of enzyme required for chemical , enzymological , and structural studies .
The nucleotide sequence of the S. typhimurium prsA gene has allowed deduction of the amino-acid sequence of PRPP synthetase .
When compared with the deduced amino-acid sequence for the E. coli protein ( 7 ) , the two sequences are 99 % identical .
The two amino-acid changes between the E. coli and S. typhimurium proteins are conservative replacements .
A previous study has shown the physical , immunochemical , and kinetic properties of E. coli and S. typhimu-rium PRPP synthetases to be indistinguishable ( 7 ) .
A survey of 10 proteins whose genes have been sequenced from E. coli and S. typhimurium indicates that amino-acid identity averages 92.4 % ( 1 ) .
The amino-acids which differ between the E. coli and S. typhimurium PRPP synthetases also diverge from the rat sequences .
Thus , the S. typhimurium PRPP syntheprotein ) PRPP,-Lactamase synthetase pBS201 bp 59-1830 pUC19 1.7 5.0 pBS214 as pBS201 but bp pUC19 0.04 4.8 165-545 deleted pBS202 bp 182-1830 with pUC18 1.6 6.9 PIc .
with pUC18 0.11 5.2 pBS215 bp 546-1830 plac pUC19 No insert 0.04 6.5 a Growth was in Vogel-Bonner medium with 0.2 % glucose at 30 °C .
The host strain was SB179 .
tase amino-acid sequence , like that of E. coli , is 47 % identical to the amino-acid sequences of the two rat isozymes ( 23 ) .
These sequence comparisons indicate that PRPP synthetase is highly conserved over a considerable phylogenetic distance .
The region 5 ' to the S. typhimurium prsA structural gene can be functionally divided into three sections : ( i ) up-stream-BamHI to NruI ( bp 1 to 103 ) ; ( ii ) promoter-NruI to EcoO109 ( bp 103 to 164 ) ; and ( iii ) leader-EcoOlO9 to Saul ( bp 164 to 545 ) .
The upstream region contains a consensus CAP binding sequence and the pyrimidine conserved sequence .
Constructions which alter or remove the CAP binding sequence reduce the expression of the prsA gene threefold .
Whether the cyclic AMP-CAP system modulates the expression of the prsA gene has not yet been determined .
The upstream region also contains the pyrimidine conserved region .
Although this sequence was once postulated to be an operator , data now suggest that the pyrimidine conserved region is neither necessary nor sufficient for the nucleotide regulation of pyrimidine biosynthesis ( C. L. Turnbough , Jr. , personal communication ) .
The location of the promoter for the expression of the E. coli prsA gene has been identified by S1-mapping ( 7 ) .
The -35 and -10 regions and the transcription start site identified in E. coli are conserved in S. typhimurium .
Comparison of PRPP synthetase expression from pBS201 and pBS203 ( Table 3 ) confirms that the conserved region represents the major promoter for the S. typhimurium prsA gene .
The positioning of the E. coli prsA promoter results in a leader of 302 bp .
The 115-bp insert in the S. typhimurium sequence extends the leader to 416 bp .
Heterologous expression of the genes appears normal ( Table 2 ) , so the functional consequences of the insert , if any , are not clear .
The leader region is required for maximal expression of the prsA gene .
Expression from the prsA promoter is essentially eliminated when the leader sequence is removed .
The previous observation that an rpoBC mutation results in derepression of FIG. 3 .
DNA sequence of S. typhimurium prsA and deduced amino-acid sequence of PRPP synthetase .
Deviations in the E. coli nucleotide sequence are indicated below the corresponding bases in the S. typhimurium sequence .
- , Deleted nucleotide ; A , site of an inserted nucleotide ; *** , termination codon .
Deviations in the E. coli amino-acid sequence are indicated below the corresponding S. typhimurium amino-acids , and the residues are boxed .
Promoter elements underlined are ( i ) -35 region , bp 108 to 113 ; ( ii ) -10 region , bp 132 to 137 ; and ( iii ) transcription start , bp 144 ( 7 ) .
The CAP consensus sequence , bp 53 to 69 , pyrimidine conserved sequence , bp 90 to 103 , and discriminator , bp 139 to 144 , regions are overlined .
A box A site , bp 464 to 471 , and the ribosome-binding site , bp 548 to 552 , are underlined .
The dyad symmetry of a putative rho-independent terminator , bp 1525 to 1540 , is marked with arrows PRPP synthetase ( 8 ) , interpreted to indicate coupled tran-scription-translation control , is not yet explained .
Deletion and mutational analyses of leader region function are now under way .
We thank John Roth and Charles L. Turnbough , Jr. , for discussion and suggestions .
This work was supported by Public Health Service grant DK13488 from the National Institute of Diabetes and Digestive and Kidney Diseases .
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