NOTES Transcriptional Regulation of the proC Gene of Salmonella typhimurium RHONDA A. BRADYt AND LASZLO N. CSONKA * Department of Biological Sciences , Purdue University , West Lafayette , Indiana 47907 Received 26 June 1987/Accepted 9 February 1988 We found that the expression of , p-galactosidase in Salmonella typhimurium strains carrying proC-lacZ-fusions was neither repressed by excess proline nor derepressed by proline limitation .
Except for a three-to fourfold decrease in the,3-galactosidase specific activity tinder conditions causing a severely reduced growth-rate , the expression of the proC-lacZ-fusions was nearly invariant under a wide variety of culture conditions .
Thus , the proC gene is unlike most other amino-acid biosynthetic genes in that its expression is nearly constitutive .
Although the proline biosynthetic pathway is one of the simplest among the amino-acid biosynthetic pathways , its regulation is perhaps the least understood ( 8 , 14 ) .
The first enzyme of the pathway , y-glutamyl kinase , is subject to feedback inhibition by proline ( 1 , 4 ) , but the transcriptional control of the genes of the proline biosynthetic enzymes has not been fully elucidated .
Because the synthesis of these enzymes was found not to be repressed by exogenous proline , several groups ( 2 , ; 4 , 6 , 10 ) concluded that the proline biosynthetic genes are expressed constitutively .
However , there is some experimental evidence suggesting that the proC gene , which encodes A ' - pyrroline-5-car-boxylate ( P5C ) reductase , might be subject to transcriptional regulation .
Mutations in the argD gene result in the accumulation of P5C because in argD mutants , P5C is produced at a high level via enzymes of the arginine biosynthetic pathway ( 2 , 22 ) .
These mutations were found to result in an increase in the P5C reductase specific activity ( 22 ) , suggesting either that P5C is a positive effector of the transcription of the proC gene or that there is a regulatory element that is common to the expression of the arginine and proline biosynthetic enzymes .
The existence of some transcriptional or translational control of the proC + gene is also suggested by the observation that the cloning of the gene on high-copy-number vectors resulted in only a twofold increase in the specific activity of P5C reductase ( 9 ) .
Piskur et al. ( 19 ) reported the isolation of mutations that were unlinked to the proC gene but resulted in its decreased expression , and these researchers proposed thai the expression of the gene is under the positive control of an unlinked regulatory gene .
To resolve these contradictory observations concerning the transcriptional regulation of the proC gene , we determined the effect of proline starvation or excess on the synthesis of 0-galactosidase in Salmonella typhimurium strains carrying proC-lacZ operon fusions .
The bacterial strains used in this study are listed in Table 1 .
t Present address : Center for Genetics , University of Illinois College of Medicine , Chicago , IL 60612 .
Proline limitation does not derepress proC .
To be able to grow strains carrying proC-lac fusions under conditions of severe proline limitation , we introduced the fusions into strains carrying putP and proP mutations that inactivated the PutP and ProP proline transport systems of the organism that function in media of normal osmotic strength ( 7 ) .
Because of the defects in proline transport , it was possible to vary the growth-rates of the resultant strains in batch cultures by varying the proline concentration in the growth medium ( 7 , 20 ) .
We found that the proline transport-proficient strain TL1114 ( proP + AputP proC : : Mu dl-8 ) grew at a four-to fivefold-greater rate in the presence of 0.1 and 0.15 mM proline than did the proline transport-defective strains TL1118 , TL1120 ( proP AputP proC : : Mu dl-8 ) , and TL1122 ( proP putP AproBA proC : : Mu dl-8 ) ( Table 2 ) .
Strain TL1114 grew faster than strains TL1118 , TL1120 , and TL1122 at all proline concentrations below 0.8 mM ( data not shown ) ; at higher proline concentrations , the growth-rates of the three strains were very nearly the same ( Table 2 ) .
We measured the P-galactosidase activities of these strains according to the procedure of Miller ( 17 ) , using exponentially growing cells at ca. 3 x 108/ml grown with various concentrations of proline ( Table 2 ) .
Although growth with 0.1 or 0.15 mM proline resulted in a severe reduction in the growth-rates of the proP putP 4 ?
( proC-lacZ ) mutant strains TL1118 , TL1120 , and TL1122 , it did not cause increased transcription of the proC gene .
The P-galacto-sidase specific activities of these three strains were actually three-to fourfold lower when the strains were grown with 0.1 or 0.15 mM proline than when they were grown in the presence of the optimal 2 mM proline .
To test the possibility that the decrease in the transcription of the proC gene with proline limitation might be due to a growth-rate-dependent regulation , we measured the P-galactosidase specific activities of the 4 ( proC-lacZ ) strains grown at different growth-rates with a number of carbon sources .
We found that there were no consistent differences in the P-galactosidase specific activities of the strains when they were grown at a range of growth-rates supported by various carbon sources , from 2.2 generations per h ( in LB-medium ) to 0.52 generation per h ( in 20 mM sodium citrate plus 2 mM proline ) ( data not shown ) .
Thus , at these growth-rates , there does not appear to be a change in the expression of the proC gene .
However , we can not rule out the possibility that a growth-rate-dependent control could operate at the extremely slow-growth-rates imposed by the proline limitation used in the experiment described in Table 2 .
To verify that growth of the : F ( proC-lacZ ) proP putP mutants with 0.1 or 0.15 mM proline indeed resulted in reduced intracellular proline levels , we measured the P5C levels in the cells .
For this measurement , 1-ml samples of cultures of exponentially growing cells at ca. 3 x 108/ml were mixed with 0.1-ml of 12 N HCI , the cell debris was removed by centrifugation , and the P5C levels were determined by the method of Bergman and Loxley ( 3 ) .
We found that the P5C content of the proB + A + I ( proC-lacZ ) proP putP strains TL1118 and TL1120 was at least 30-fold greater when the cells were grown in 0.1 or 0.15 mM proline than when they were grown in 2 mM proline ( Table 2 ) , indicating that there was a relief of the feedback inhibition of-y-glutamyl kinase at low exogenous concentrations of proline .
Since the,-galac-tosidase specific activities of the AproBA strain TL1122 , which had background levels of P5C , were similar to those of the proB + A + strain TL1118 under all conditions tested ( Table 2 ) , we conclude that the P5C is not a regulator of the transcription of the proC gene .
The alarmone guanosine-5 ' - diphosphate 3 ' - diphosphate ( ppGpp ) stimulates the transcription of a number of amino-acid biosynthetic regulons under conditions of amino-acid limitation ( 18 , 21 , 23 , 26 ) .
Although our finding that proline starvation did not derepress transcription of the proC gene suggested that ppGpp is probably not involved in the transcriptional regulation of proC , we carried out a second experiment to investigate this possibility : we determined the effect of a relA mutation ( which abolishes the accuhulation of ppGpp in response to amino-acid starvation ) bn the expression of the proC-lacZ-fusions .
The relA mutation caused a reduction in the growth-rate of the proline auxo-trophs on all media ( Table 2 ) .
The inhibition of growth was especially marked under conditions of proline limitation , so that the relA mutant derivatives of the 4 ( proC-lacZ ) putP proP strains were unable to grow with 0.1 or 0.15 mM proline .
When grown in excess proline ( 0.1 or 2.0 mM for strain TL1275 [ relA proP + AputP proC : : Mu dl-8 ] and 2.0 mM for strains TL1276 and TL1277 [ relA proP AputP proC : : Mu dl-8 ] ) , the relA derivatives had lower mutant relA + P-galactosidase specific activities than the respective control strains ( Table 2 ) .
Since the growth-rates of the putP proP proC : : Mu dl-8 strains TL1276 and TL1277 were reduced by the relA mutation evefl in the presence of 2 mM proline , the reduction in the P-galactosidase levels of the relA mutant derivatives could be due to a growth-rate-dependent control of the expression of the proC gene that is manifested only at very slow-growth-rates .
However , the is results are also consistent with the possibility that ppGpp a weak positive regulator of the transcription of the proC gene .
Our data ( Table 2 ) unambiguously demonstrate that the transcription of the proC gene of S. typhimu-rium is neither repressed by prolihe abundance nor derepressed by proline starvation .
We found that the expression of the proC nearly invariant under a variety of gene was culture conditions , with the possible exceptions that severe synthesis inhibition of the growth-rate or impairment of the of ppGpp by a relA mutation resulted in a four-to fivefold decrease in the transcription of the gene .
Our observations do not support the proposal that the expression of the proC gene is under the control of a positive regulatory protein that senses the availability of proline ( 19 ) .
The result that the expression of the proC-lacZ-fusions did not respond to changes in the P5C levels ( Table 2 ) suggests that the increase in the P5C reductase specific activity seen in argD mutants ( 22 ) is probably not due to the accumulation of P5C that occurs in these strains .
However , we did not address further either the possibility that there is some regulatory mechanism common to the arginine and proline biosynthetic enzymes or the alterations in the expression of the proC gene on high-copy-number plasmids .
Our investigation of the transcriptional regulation of the proline biosynthetic genes of S. typhimurium was limited to the proC gene because strains carrying proB-or proA-lacZ-fusions were not available to us .
On the basis of the observation that the specific activities of-y-glutamyl kinase and-y-glutamyl phosphate reductase of Escherichia coli are not altered by the presence or absence of exogenous proline , it was concluded that the expression of the proB + A ' operon is constitutive ( 4 , 6 , 10 ) .
The P-galactosidase specific activities of E. coli strains carrying a proB-or proA-lacZ operon fusion are not repressed by excess proline ( 4 , 11 ) or influenced by exposure to osmotic-stress ( 11 ) .
The sum of the available evidence with E. coli and S. typhimurium indicates that none of the three proline biosynthetic genes is subject to a significant transcriptional control in response to proline limitation or excess .
The absence of derepression of the proline biosynthetic enzymes during proline starvation is an exception in the pattern seen with most other amino-acid biosynthetic enzymes .
Of a total of 94 structural genes that encode amino-acid biosynthetic enzymes in E. coli or S. typhimurium , 74 have been shown to be derepressed in response to limitation for the amino-acid end product ( 12 ) .
There are only 10 amino-acid biosynthetic genes that are refractory to such regulation , and the remaining 10 genes have not yet been fully characterized with respect to their transcriptional control .
It is not obvious to us why the three proline biosynthetic enzymes belong to the minority class of amino-acid biosynthetic enzymes that are not under an obvious transcriptional control and why it might be advantageous for E. coli and S. typhimurium to synthesize these enzymes at a nearly invariant level regardless of the availability of proline .
S. typhimurium LT2 strains used Genotype ( phenotype ) a Strain Source or derivation ( reference ) b J. L. Ingraham ( 16 ) S. G. Kustu This laboratory ( 7 ) This laboratory ( 7 ) P22.TT9670 - * TL135 = Ampr , Pro-P22 .
JL4036 -- TL180 = Ampr , Pro-P22 .
JL2520 -- 3TL1118 = Tetr , Ampr proBl657 : : TnJO ( polar on proA ) JL2520 JL4036 TL135 TLi8O TL1114 TL1118 TL1120 TL1122 proCI909 : : Mu dl-8 supD zeb-609 : : TnlO ( Lac + ) c A ( putPA ) 557 zcc-628 : : TnS A ( putPA ) 557 proP673 zcc-628 : : TnS proC691 : : Mu dl-8 A ( putPA ) 557 zcc-628 : : TnS proC691 : : Mu dl-8 A ( putPA ) 557 proP673 zcc-628 : : TnS proCJ909 : : Mu dl-8 A ( putPA ) 557 proP673 zcc-628 : : TnS proB1657 : : TnJO proC691 : : Mu dl-8 A ( putPA ) 557 proP673 zcc-628 : : Tn5 relA2J : : TnlO proC691 : : Mu dl-8 A ( putPA ) 557 zcc-628 : : TnS TL1275 P22.TT7542 - * TL1114 = Tetr , unable to grow on glucose medium 63 + SGML + proline ( 24 ) reIA2J : : TnIO proC691 : : Mu dl-8 A ( putPA ) 557 proP673 P22.TT7542 - + TL1118 = Tetr , unable zcc-628 : : TnS to grow on glucose medium 63 + SGML + proline ( 24 ) relA2J : : TnJO proCJ909 : : Mu dl-8 A ( putPA ) 557 proP673 P22.TT7542 - * TL1120 = Tetr , unable zcc-628 : : TnS to grow on glucose medium 63 + SGML + proline ( 24 ) TT7542 relA21 : : TnlO J. R. Roth ( 24 ) TT9670 proC691 : : Mu dl-8 ( Lac + ) ' J. R. Roth a Phage Mu dl-8 is a transposition-defective , stable derivative of phage Mu dl , constructed by Hughes and Roth ( 13 ) .
The other genetic symbols are defined in reference 25 .
b The designation P22.X - * Y = A , B , C denotes that a phage P22 lysate of straih X was used to transduce strain Y , selecting phenotype A and scoring pheontypes B and C. Abbreviations : Tet and Amp , presence of 15 mg of tetracyline and 25 mg of sodium ampicillin per liter of the growth medium , respectively ; r , resistance ; SGML , serine , glycine , methionine , and leucine at 0.2 mM each ( this combination of amino-acids preferentially inhibits relA mutants t24 ] ) .
I The gene inactivated by Mu dl-8 in strains JL4036 and TT9670 was identified by complementation tests ; the strains were restored to Pro ' upon the acquisition of F ' 251 ( proC + ) but not F ' 128 ( proB + A + ) ( data not shown ) .
BL TA P-Galactosidase specific activities of 4 § ( proC-lacZ ) strains grown under conditions of proline starvation or excessa Proline concn in Growth rate P5C ( rimol/mg of P-Galactosidase sp act medium ( mM ) ( generation/h ) protein ) b ( Miller units ) c 0.1 0.62 0.25 54.3 0.15 0.83 0.12 62.0 2.0 1.0 0.03 73.8 Strain TL1114 ( AputPA proC691 : : Mu dl-8 ) TL1118 ( AputPA proP proC691 : : Mu dl-8 ) 0.1 0.14 2.5 17.1 0.15 0.19 2.1 25.7 2.0 0.91 0.07 74.6 TL1120 ( AputPA proP proCi909 : : Mu dl-8 ) 0.1 0.14 2.3 21.8 0.15 0.17 2.0 33.5 2.0 0.91 0.03 83.9 TL1122 ( AproBA AputPA proP proC691 : : Mu dl-8 ) 0.1 0.12 < 0.03 17.0 0.15 0.17 < 0.03 22.6 2.0 0.91 < 0.03 70.1 TL1275 ( relA : : TnlO AputPA proC691 : : Mu dl-8 ) 0.1 0.50 ND 53.8 0.15 0.42 ND 39.4 2.0 0.48 ND 48.5 TL1276 ( reIA : : TnJO AputPA proP proC691 : : Mu dl-8 ) 0.1 < 0.07 ( NG ) ND ND 0.15 < 0.07 ( NG ) ND ND 2.0 0.29 ND 15.9 T11277 ( reIA : : TnJO AputPA proP proCJ909 : : Mu dl-8 ) 0.1 < 0.07 ( NG ) ND ND 0.15 < 0.07 ( NG ) ND ND 2.0 0.45 ND 49.2 a The cultures were grown in minimal-medium 63 ( 5 ) with 10 mM glucose and proline at the indicated concentrations .
NlD , Not determined ; NG , no growth .
b The P5C levels were determined by the method of Bergman and Loxley ( 3 ) , as described in the text .
The results were normalized to cellular protein , which was determined by the method of Lowry et al. ( 15 ) .
c,-Galactosidase was measured according to the method of Miller ( 17 ) , as described in the text .
The units are those defined by Miller ( 17 ) We are grateful to H. E. Umbarger for experimental suggestions and critical reading of the manuscript .
We acknowledge P. Weaver for performing the,3-galactosidase assays .
We thank D. Rhodes for a sample of P5C we used as a standard in the determination of the P5C levels .
This work was funded by Public Health Service grant R01-GM-31944 from the National Institutes of Health .
R.A.B. was partially supported by the Proctor and Gamble Co. .
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