166 , No. 2 NOTES Phosphate Starvation Regulon of Salmonella typhimurium JOHN W. FOSTER * AND MICHAEL P. SPECTOR Department of Microbiology , Marshall University School of Medicine , Huntington , West Virginia 25704 Received 22 August 1985/Accepted 10 February 1986 Several phosphate-starvation-inducible ( psi ) genetic loci in Salmonella typhimurium were identified by fusing the lacZ gene to psi prowoters by using the Mu dl and Mu dl-8 bacteriophages .
Although several different starvation conditions ' were examined , the psi loci responded sQlely to phqsphate deprivation .
A regulatory locus , psiR , was identified as controlling the psiC loc ; us .
The psiR locus did not affect the expression of the Escherichia coli phoA locus or any of the other psi loci described .
A recent area of intensive research on procaryotes has been the genetic concept of regulons and stimulons and the response of an organism to environmental stress .
A regulon is defined as a set of unlinked , coordinately regulated genes whose gene products are involved in related biochemical functions ( 11 ) .
Stimulons have been described as sets of regulons and operons which respond to a common environmental stimulus ( 18 ) .
There have been numerous studies dealing with Esche-richia coli phosphate-regulated gene expression ( 20-22 ; reviewed in reference 6 ) , which is considered a model system for pleiotropic control .
The pho regulon involves an elaborate control system encompassing both positive ( phoM , phoB , phoR ) and negative ( phoR ) regulatory elements which respond to phosphate limitation .
Although a number of genes are controlled by this system , the model target locus for many of these studies was the structural gene for alkaline phosphatase ( p/oA ) .
Although the phoA gene is not normally present in Salmonella spp. .
( 16 ) , an F ' phoA + plasmid transferred to Salmonella typhimurium is regulated by phosphate limitation ( 16 , 20 ) .
Little is known , however , about the regulatory loci involved in phosphate-regulated gene expression in this organism .
A broader approach to identifying phosphate-regulated genetic loci in E. coli has been used by Wanner et al. ( 20-22 ) with Mu dl operon fusion technology .
They have identified at least 18 separate promoters controlled by phosphate limitation .
Most of these promoters ( 16 of 18 ) were also controlled by other starvation conditions .
We have recently initiated research designed to examine various starvation stimulons in S. typhimurium ( 5 ; M. P. Spector and J. W. Foster , Abstr .
In addition to characterizing loci which respond to several environmental stimuli ( sin ) , we are investigating loci which respond to only one environmental stimulus ( i.e. , phosphate-starvation ) .
This communication describes the identification , through the isolation of lacZ operon fusions , of genetic loci regulated at the transcriptional level by phosphate-starvation .
The loci are referred to psi ( phosphate-starvation inducible ) designation as genes , a originally used for similar loci in E. coli by Wanner al. et ( 20-22 ) .
The strains used in this study listed in Table 1 .
are Bacteriophage P22 HT105 / - int ( 17 ) was used as a vector to transfer Mu cts dl ( Ap lac ) ( Mu dl ) and the derivative Mu dl-8 ( 2 , 8 ) into appropriate recipient strains of S. typhimur-ium , Fas described previously ( 7 ) .
The minimal MOPS ( morpholinepropanesulfonic acid ) me-dium of Neidhardt et al. ( 14 ) supplemented with nicotinic acid at 10-5 M was used .
LB-medium and green agar were used as described by Chan et al. ( 3 ) .
Tetracycline was added , when needed , at final concentrations of 20 , ug/ml for complex medium and 10 , ug/ml for minimal-medium .
Ampicillin was used at 30 , ug/ml in both complex and minimal media .
5-Bromo-4-chloro-3-indolyl-p-D-galactoside ( X-gal ) was added to minimal-medium at a final concentration of 40 , ug/ml as an indicator for P-galactosidase activity .
5-Bromo-4-chloro-3-indolyl phosphate-p-toluidine ( XP ) was added at 40 , ug/ml as an indicator of alkaline phosphatase activity .
The Mu dl and Mu dl-8 phages ( 2 , 8 ) were used in the isolation of psi-lacZ operon fusions .
Transduction was accomplished via the P22 HT phage with primary selection for ampicillin resistance ( 17 ) .
Ampr colonies ( 6,000 ) were screened for phosphate-starvation-regulated operon fusions on MOPS medium containing X-gal ( 13 ) and supplemented with either 13 mM or 0.13 mM ( limiting ) phosphate .
Colonies which appeared bluer on the low-phosphate medium than on the high-phosphate medium were rechecked and purified , and the resultant strains were classified as phosphate-starvation inducible ( Psi ) .
Each fusion strain was then assayed for P-galactosidase activity under various nutrient stress conditions including phosphate , nicotinic acid , nitrogen , and glucose limitations .
Each psi fusion strain responded only to phosphate limitation , as shown by the data in Table 2 .
This is in contrast to our sin fusion strains , which were initially isolated based upon their response to nicotinate limitation but which respond to several starvation conditions ( 5 ; Spector and Foster , Abstr .
TnJO insertions near several of the phosphate-regulated genes were constructed by transducing the fusion strains P22 been with phage which had propagated on a pool of TnlO insertion strains and then screening the transductants for repair of the fusion ( 4 ) .
These insertions were then introduced into the high-frequency-recormbination ( Hfr ) strains SA722 and SA464 ( Fig. 1 ) .
The TnlO-containing Hfr strains were subsequently used to transfer the Tetr phenotype to auxotrophic recipient strains via interrupted matings .
Alter ¬ 66 JF643 AnadA100 I ) ( psiBI2-lac ) ( lac + Ampr ... .
psiD19 : : Mu dl-8 leu-515 ( Am ) sup-19 nadA : : TnlO JF664 ... .
psiA20 : : Mu dl-8 leu-515 ( Am ) sup-19 nadA : : TnlO JF722 ... .
psiDl9 : : Mu dl-8 phoN2 JF723 ... .
psiA20 : : Mu dl-8 phoN2 JF733 ... .
zhe43 : : TnJO phoN2 ( TnlO 29 % linked to psiA + ) JF753 ... .
psiR : : TnIOIF ' 254 ( lac + phoA + ) MU20 ( 80 units cw ) ... serA15 rfa-3050 zba-34 : : TnlO ( TnlO near psiC + ) MU22 ( 84 units ccw ) ... serA15 pur-268 zhe-33 : : TnJO ( TnlO near psiA ) MU23 ( 80 units cw ) ... serAI3 rfa-3050 psiR : : TnlO MU24 ( 83-90 / nadAlOO FD ( psiBI2-lacZ ) / F'tsll 4 units cw ) ... .
lac + zzf-22 : : TnlO SF142 ( TT7610 ) hisD9953 : : Mu dl-8 ... .
SF189 ( TT8024 ) zaj-1034 : : TnlO lon-71 ( TnIO 50 % linked ... .
to Ion ) SF202 ( TT1800 ) ... proAB47 putP639 proP673 zid-27 : : TnlO ( TnlO 60 % linked to mel ) SF218 ( TN1785 ) ... argF88 apeA18 apeB21 zba-833 : : TnlO ( TnlO 50 % linked to apeB ) SA464 ( Hfr , 80 units cw ) ... .
serA13 rfa-3058 SA722 ( Hfr , 84 units ccw ) ... serA15 pur-268 a Strains SF142 , SF189 , and SF202 were obtained from J. Roth ; strain SF218 was obtained from C. Miller ; and strains SA464 and SA722 were obtained from K. Sanderson .
cw , Clockwise ; ccw , counterclockwise .
natively , stabilized Mu dl fusions were used to construct Hfr strains based on lac homology between the Mu d insertion and an F ' tslac + plasmid as outlined by Maloy and Roth ( 12 ) .
The results of these mapping experiments are shown in Fig. 1 ; the psiA , psiB , psiC , and psiD genes mapped at approximately 74 , 88 , 10 , and 93 units , respectively .
By using Hfr construction based on lac homology ( 12 ) , we were able to determine that the direction of psiB transcription was clockwise .
The psiC fusion was cotransducible with zaj-1034 : : TnlO and zba-883 : : TnJO , with frequencies of 45 and 48 % , respectively .
The psiD fusion was 74 % cotransducible with zid-27 : : TnJO .
To determine at what point during starvation each psi fusion was induced , an overnight culture of each strain was diluted into fresh medium containing either normal ( 13 mM ) or limiting ( 0.13 mM ) phosphate concentrations .
pproduction was Galactosidase subsequently monitored at measured intervals .
There wete at least three different responses to phosphate limitation ( Fig. 2 ) : ( i ) induction early during-growth in phosphate-limiting medium ( psiA ) , ( ii ) induction upon a reduction in growth-rate caused by starvation ( psiB , psiC , psi-15 , and psi-18 ) , and ( iii ) induction early during limitation or at the early stationary-phase during normal growth ( psiD ) .
Some strains exhibited a decrease in P-galactosidase activity during the stationary-phase ; the significance of this phenomenon is unclear .
The results suggest that a variety of control circuits regulated the genetic response to phosphate limitation , with different genes responding to the nutrient stress at different times .
A TnlO insertion into a regulatory locus designated psiR was discovered by crossing the phage P22 transducing pool of TnJO insertions with strain JF515 ( psiC : : Mu dl ) .
Tetr transductants were replicated onto minimal MOPS medium containing X-gal and 13 mM P04 .
Colonies which appeared significantly bluer on this medium were repurified , cured of phage P22 , and tested for constitutive expression of the psiC-lacZ operon fusion .
P-Galactosidase activity ( expressed as Miller units [ 10 ] ) for strain JF515 ( psiCJ7 : : Mu dl ) was 12.7 and 139 on 13 and 0.13 mM P04 , respectively ; for strain JF561 ( psiC17 : : Mu dl psiR : : TnlO ) , activity was 116 and 114 on 13 and 0.13 mM P04 , respectively .
Transduction studies revealed that Tetr and constitutive expression of the psiC fusion were 100 % cotransducible .
The insertion was designated psiR : : TnlO .
This regulatory locus was subsequently mapped by transferring the TnWO insertion to the Hfr strains SA464 and SA722 , Which were used to transfer Tetr via interrupted mating to various auxotrophic mutants .
The map position of the psiR locus was determined to be between the origins of transfer of the two Hfr strains , at 82 units on the S. typhimurium linkage map ( 15 ) .
The psiR locus must have produced a trans-acting gene product which modulated the expression of the psiC locus based on phosphate availability in the medium .
Because the PsiR phenotype was the result of an insertion , the psiR ' gene product must have acted as a negative regulator .
The psiR locus did not affect the expression of any of the other psi-lacZ operon fusions , which suggests that the psiR gene product was not involved with phosphate transport .
Although S. typhimurium does not possess alkaline phosphatase ( encoded by the phoA gene ) , Kier et al. ( 9 ) discovered a mutation , pho-25 ( unmapped ) , which resulted in the constitutive expression of the phoA + gene carried on an F ' phoA + plasmid .
To determine whether the psiR locus also affected the expressioh of the phoA gene , plasmid F ' 254 was transferred into strain JF753 .
The resultant psiR/F ' phoA + strain ( JF755 ) was scored on phosphate-excess and phos-phate-limiting MOPS medium containing XP .
The presence of the psiR mutation had no obvious effect on the induction of the phoA gene .
This suggests that the psiR locus is not analogous to any of the regulatory loci identified as participating in the control of the E. coli pho regulon .
Several mutations known to affect the production of ppGpp ( relA ) or cyclic AMP ( cya ) were placed in psi-lac operon fusion strains to ascertain their possible roles in psi gene expression .
None , however , appeared to have any effect on any of the psi-lacZ operon fusions described here ( data not shown ) .
Several enzymes with phosphatase activities have been identified in S. typhimurium .
These include the following nonspecific acid phosphatases ( NAPs ) : NAP , which is encoded by the phoN gene and regulated by the phoP gene ( 10 ) ; NAP I , which is unmapped but is distinct from the phoN or phoP gene ( 19 ) ; and NAP II , which is encoded by the aphA gene ( unmapped ) .
Other enzymes possessing phos phatase activity include 2 ' ,3 ' - cyclic nucleotide 2 ' - phospho-diesterase ( encoded by the pde gene [ 19 ] ) , acid hexose phosphatase ( gene unmapped ) , and cyclic AMP phosphodiesterase ( encoded by the cpd gene ) .
Only production of the phoN gene product appears to be regulated by phosphate ( 9 ) .
None of the psi loci described here appear to coincide with the known Salmonella phosphatase loci ( Fig. 1 ) ( 9 , 10 ) .
In addition , the acid phosphatase staining protocol used by Kier et al. ( 10 ) failed to reveal any differences between the psi mutants and their psi ' parents ( data not shown ) .
Consequently , it does not appear that any of the psi loci described in this report correspond to any of the known phosphatases .
We have isolated numerous other operon fusions ( originally isolated based on their response to nicotinate limitation ) which respond to phosphate-starvation , as well as to several other nutrient limitations ( 5 ; Spector and Foster , Abstr .
These loci , designated sin to reflect their general starvation inducibility , appear to be similar to many of the psi loci described for E. coli by Wanner et al. ( 20-22 ) .
The sin loci will be described in detail in a future report .
The designation psi is used in this paper , however , to represent loci known to respond only to phosphate limitation .
Direct comparisons between the results described in this paper and those for the E. coli system are difficult .
Two of our psi loci mapped in a region of the S. typhimurium chromosome similar to that occupied by the two psi loci of E. coli identified by Wanner and McSharry ( 21 ) which are regulated only by phosphate .
Our psiC locus resided at 10 min , a position similar to that of the E. coli phoA locus .
However , S. typhimurium does not possess alkaline phosphatase , and therefore these two loci can not be analogous .
in general The S. typhimurium psiD locus mapped the same area as the E. coli psiD gene , although it appeared that their inductions differed kinetically .
Very little is known about the control of phosphate metabolism in S. typhimurium .
There are only two studies which attempt to correlate Salmonella phosphate regulation with what is known about E. coli phosphate regulation ( 9 , 23 ) .
Both of these studies show only that the phoA + gene , when introduced into S. typhimurium , is regulated .
Therefore , the construction of psi-lacZ-fusions will facili-operon tate further studies designed to reveal regulatory circuits controlling phosphate utilization in S. typhimurium .
We thank A. G. Moat for helpful discussions and artwork .
This work was supported by Public Health Service grant GM34147-01 from the National Institutes of Health .
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