164 , No. 3 Salmonella typhimurium proP Gene Encodes a Transport System for the Osmoprotectant Betaine JOHN CAIRNEY ,1 IAN R. BOOTH ,2 AND CHRISTOPHER F. HIGGINS ' * Department of Biochemistry , University of Dundee , Dundee DDJ 4HN , l and Department of Microbiology , Marischal College , University of Aberdeen , Aberdeen AB9 lAS ,2 Scotland Received 11 June 1985/Accepted 22 August 1985 Betaine ( N,N,N-trimethylglycine ) can be accumulated to high intracellular concentrations and serves an important osmoprotective function in enteric bacteria .
We found that the proP gene of Salmonella typhimurium , originally identified as encoding a minor transport system for proline ( permease PP-II ) , plays an important role in betaine uptake .
Mutations in proP reduced the ability of betaine to serve as an osmoprotectant .
Transport of betaine into the cells was also severely impaired in these mutants .
The kinetics of uptake via PP-II suggest that betaine , rather than proline , is the important physiological substrate for this transport system .
Betaine uptake via PP-II was regulated by osmotic pressure at two different levels : transcription of the proP gene was increased by increasing osmolarity , and , in addition , activity of the transport system itself was dependent upon the osmotic pressure of the medium .
The specificity of the transport system was also altered by increasing osmolarity which enhanced the affinity for betaine while reducing that for proline .
A number of diverse organisms have evolved similar strategies to combat the osmotic-stresses they encounter when their environment is altered by increasing concentrations of salts or other substances ( 9 , 13 , 14 , 25 ) .
In particular , many species accumulate high concentrations of compatible solutes in response to osmotic-stress which are thought to restore turgor pressure across the cell membrane as well as stabilize enzyme function .
It has been known for many years that L-proline can stimulate the growth of enteric bacteria in media of otherwise inhibitory osmotic strength ( 6 ) .
In addition , proline overproduction enhances osmotolerance of Salmonella typhimurium ( 7 ) .
More recently , betaine ( N,N,N-trimethylglycine ) has been shown to be a more effective osmoprotectant than proline and to accumulate to high concentrations in both halophilic bacteria and in the enteric bacteria Escherichia coli and Klebsiella pneumoniae in response to osmotic-stress ( 11 , 12 , 18 ) .
Exogenous betaine is taken up by an active transport process in E. coli , and the rate of uptake is stimulated by increasing the osmotic pressure of the medium ( 18 ) .
However , the number of betaine transport systems , the genes encoding them , and the mechanisms of osmoregulation are unknown .
In this paper we show that a major pathway for betaine uptake requires the function of the proP gene which was originally identified as encoding a minor transport system for proline .
Three genetically distinct proline transport systems have been characterized in S. typhimurium .
The major profile permease ( PP-I ) is encoded by the putP gene , located at 22 min on the chromosome adjacent to the putA gene which encodes proline oxidase ( 4 , 19 , 20 ) .
The putA gene product also serves as a negative effector of putP expression ( 15 ) .
Strains deficient in PP-I exhibit low-level proline transport via a second system , PP-II , which is abolished by mutations in the proP gene ( 1 , 16 ) .
proP maps at 92 min on the S. typhimurium chromosome .
In addition , a third proline permease , PP-III , encoded by the proU gene , has been reported to function in media of high-osmolarity ( 8 ) .
However , we have recently shown that proU encodes a highaffinity transport system with betaine , rather than proline , as its primary substrate ( 3 ) .
The precise roles of the multiple proline transport systems is unclear .
In particular , PP-Il has a very poor affinity for proline ( Km = 300 , uM ) and would not seem to contribute significantly to proline uptake in cells in which PP-I is functional ( 1 , 4 ) .
We show here that PP-Il plays a major role in betaine uptake and that mutations in proP reduce the ability of betaine to serve as an osmoprotectant .
proP-dependent betaine transport is regulated in response to osmotic pressure both at the level of transcription and by an alteration in the activity of the transport protein .
The kinetics of betaine uptake via PP-II suggest that betaine , rather than proline , is the most important physiological substrate for this transport system .
MATERIALS AND METHODS Bacterial strains and mnedia .
All bacterial strains were derivatives of S. typhimurium LT2 .
Their genotypes and construction are listed in Table 1 .
Cells were grown with aeration in LB-medium ( 17 ) at 30 ' C to avoid induction of Mu dl lysogens .
Minimal glucose plates were based on the E mediun of Vogel and Bonner as described by Roth ( 22 ) .
Antibiotics were used in both rich and minimal-medium at the following concentrations : tetracycline , 15 , ug ml - ' ; carbenicillin ( an analog of ampicillin , resistance to which is referred to in the text as Apr ) , 50 , xg ml - ' ; kanamycin , 50 jig ml - ' .
For growth curves and transport assays cells were grown in low-osmolarity medium ( LOM ) containing 10 mnM glucose and any appropriate osmolytes .
LOM is based on KONO medium ( 4 ) and contains 0.4 mM MgSO4 , 6 , uM ( NH4 ) 2SO4 FeSO4 , 20 mM ( NH4 ) 2HP04 , 10 mM bistrispropane , 1 , ug of biotin ml - ' , 1 mM KCI , and 5 mM NaCl .
Genetic techniques and strain construction .
Transductions were carried out by using a high-transducing derivative of phage P22 int4 as described by Roth ( 22 ) .
When used as donors or recipients for transduction , galE strains were grown in LB-medium supplemented with 0.2 % galactose and 0.2 % glucose to ensure efficient synthesis of phage receptors .
After transduction of TnS or TnJO insertions from one strain to another , the location of the transposon insertion and the presence ofjust a single insertion in the transductant were verified by marker rescue .
All strains were derivatives of CH223 ( 4 ) except where stated .
Deletion of the putPA genes was achieved by aberrant excision of the putA810 : : TnJO transposon from strain CH579 by the fusaric acid selection ( 2 ) .
Tetracycline-sensitive derivatives were screened for PutP and PutA phenotypes as described below .
One PutP-PutA-derivative , CH627 ( A6putPA230 ) , was shown to be a deletion rather than an inversion by its inability to recombine with put point mutations and was used for all further studies .
Identification of genotypes and phenotypes .
Mutations in the proline transport genes were identified by their resist-ance to appropriate toxic proline analogs when radially streaked on minimal glucose plates around a filter disk inpregnated with L-azetidine-2-carboxylic acid ( AC ) or 3,4-dehydro-DL-proline ( DHP ) ( 3 , 8 , 19 ) .
putP + proP + strains are sensitive to both AC ( 150 , ug ) and DHP ( 600 , ug ) ; putP proP + strains are AC resistant but DHP sensitive ; putP proP derivatives are resistant to both analogs .
Strains carrying putA mutations were identified by their inability to utilize L-prolyl-L-valine as the sole nitrogen source as described previously ( 4 , 20 ) .
Cells were grown overnight in LOM-glucose medium and diluted 1:100 into the same medium ( containing 0.8 M NaCl when appropriate ) , and their growth was monitored by following the A6Eo in a 1-in .
( 2.5-cm ) Spectronic tube with a Spectronic 20 spectrophotometer ( Bausch & Lomb , Inc. , Rochester , N.Y. ) .
Proline and betaine , when used as osmoprotectants , were added at a concentration of 1 mM .
Proline and betaine transport was assayed as described previously ( 4 ) .
Growth of cells at low-osmolarity was in LOM-glucose .
Growth at high-osmolarity was achieved by growing the cells in LOM-glucose to an optical density at 600 nm of 0.6 , adding NaCl to a final concentration of 0.3 M , and growing the cells for a further 1 h. Cells were grown at either high or low-osmolarity to an optical density at 600 nm of 0.8 , harvested by centrifugation , and washed twice in the medium in which they were grown .
Cells were finally suspended to about 0.5 mg of cells ml - ' in preincubation buffer ( LOM containing 1 mM glucose , 50 , ug of chloramphenicol ml - ' , and , when appropriate , additional NaCI to raise the osmolarity ) .
Cell suspensions were equil-ibrated at 30 °C for 5 min , and transport was initiated by adding [ U - ' 4C ] proline ( 20 mCi mmol-1 ) or [ methyl - ' 4C ] betaine ( 6.6 mCi mmol-1 ) .
Samples ( 100 , ul ) were removed at the indicated time intervals , the cells were collected by passing the samples through a Whatman GFF glass fiber filter and washed with 2.5 ml of preincubation buffer ( at the same osmolarity as that used for the transport assay ) , and the accumulated proline or betaine was determined by scintillation counting .
Each datum point was determined at least in duplicate , and each experiment was repeated with at least two independent cell suspensions .
Because of the variation in cell volume between cells grown at different osmolarities , the protein content of cell suspensions was measured routinely , and transport rates were expressed as nanomoles per milligram of protein .
Protein was assayed by the Bradford procedure , using reagents obtained from Bio-Rad Laboratories ( Richmond , Calif. ) and bovine serum albumin as a standard .
The protein present in cell suspensions was determined after breaking the cells by sonication .
Assay of , B-galactosidase activity was carried out as described by Miller ( 17 ) .
Cells were permeabilized by the chloroform-sodium dodecyl sulfate procedure .
121 RESULTS Osmotically induced transport via PP-II .
Strains of S. typhimurium which were deficient in the major proline permease ( PP-I , putP ) were resistant to 40 , ug of AC ml-1 .
However , if the osmotic pressure was raised by incorporating 0.3 M NaCl into minimal agar plates , sensitivity to the analog was restored .
We have shown previously that this sensitivity is due to increased uptake of AC through PP-Il .
Thus , mutants defective in PP-II ( proP ) can be selected by their resistance to 40 pug of AC ml - ' at high-osmolarity ( 4 ) .
This effect is not to be confused with the osmotically induced uptake of AC through PP-III ( prolU ) , which requires very much higher concentrations of the toxic analog ( 3 , 8 ) .
The increased uptake of AC through PP-II at high osmotic pressure could be due to an increase in activity of the transport system or , alternatively , to induction of expression of the proP gene .
To distinguish between these two possibilities , we measured PP-1I-dependent uptake of proline at different osmolarities .
The strain used , CH627 , was deleted for putP to exclude proline uptake via PP-I .
In this strain all proline transport is dependent upon PP-II ; no uptake can be detected when proP is mutated ( strain CH638 ) .
Thus , proU ( PP-Ill ) plays no significant role in proline uptake under these conditions .
Strain CH627 ( proP + ) was grown in the presence or absence of 0.3 M NaCl , and in both cases , proline transport was assayed at low-osmolarity .
Proline transport was enhanced about threefold by growth of cells at high-osmolarity ( Fig. 1 ) .
However , when proline transport was assayed at high-osmolarity ( in the presence of 0.3 M NaCl ) , uptake was slightly reduced compared with assays performed at low-osmolarity ; the activity of PP-II was not increased by increased osmolarity .
Indeed , when assayed a TABLE 1 .
Bacterial strains Genotype Strain ' CH223 gaIE503 bio-561 Construction or source 4 4 CH486 galE503 bio-561 putP201 : : Mu dl proPJ667 : : Tn5 CH500 galE503 bio-561 putP2J4 : : TnS proPJ673 : : Mu dl CH501 galE503 bio-561 putP214 : : TnS proPJ674 : : Mu dl CH579 galE503 bio-561 putA810 : : TnJO CH627 galE503 bio-561 AputPA230 CH638 galE503 bio-561 AputPA230 proPI667 : : TnS CH784 proPJ673 : : Mu dl proUJ697 : : TnlO CH992 galE503 bio-561 AputPA230 proUJ697 : : TnJO TT946 putA8I0 : : TnlO a All strains are derivatives of S. typhimurium LT2 .
4 4 Transduction donor TT946 ; recipient CH223 Tets derivative of CH579 ; this study Transduction donor CH486 ; recipient CH627 3 3 J. Roth 2 3 TIME ( mins ) FIG. 1 .
Effect of osmotic pressure on proline uptake by PP-Il .
Cells of CH627 ( AplutPA proP + [ A , O , A ] ) or CH638 ( A & putPA proP [ 0 , 0 ] ) were grown in LOM-glucose in the presence ( open symbols ) or absence ( closed rsymbols ) of 0.3 M NaCI .
Proline uptake at 10 , uM was assayed at low-osmolarity as described in the text except in the one case ( A ) in whiich the assay was performed at high-osmolarity .
ingly , growth was slightly but consistently enhanced by the introduction of a mutation in proP ( doubling time = 350 min ) .
This was also found to be true for a variety of other strains carrying various TnS , TnJO , or Mu dl insertions in proP ( data not shown ) .
It is not clear why proP mutations should enhance growth at high-osmolarity as they do not affect growth-rates in medium of low-osmolarity .
However , the most probable explanation is that when PP-TI is absent , proline exodus from cells is reduced ; consequently , the internal pools of this osmoprotectant are increased and growth is enhanced .
Although growth of the wild-type strain in the absence of an osmoprotectant was poor , the addition of 1 mM proline to the growth medium considerably enhanced growth , decreasing the doubling time to 240 min ( Fig. 2B ) .
Deletion ofputPA ( CH627 ) had no effect on osmoprotection by proline , although this is perhaps not surprising since transport through ; > PP-I is inhibited by high Na + concentrations ( 4 , 10 ) .
However , introduction of a proP mutation ( strain CH638 ) considerably impaired the ability of proline to protect cells against high osmotic pressure .
Little or no growth stimulation by proline was seen in proP mutants , indicating an important role for PP-1I in osmoprotection .
At a concentration of 1 mM , betaine was a considerably better osmoprotectant than proline ( Fig. 2C ) .
The doubling time of LT2 decreased from 420 mm in the absence of osmoprotectant to 140 min when betaine was added .
Again , elimination of putP had no effect on osmoprotection , while inactivation of proP dramatically reduced the ability of betaine to protect against high osmotic pressure .
Appropriate controls ( data not shown ) showed that mutations in proP do not reduce the growth-rate of cells at low osmotic pressure .
Thus , either proP is required for osmoprotection per se , or , alternatively , PP-II serves as a transport system for both proline and betaine .
Betaine protection against inhibition by toxic proline ana-logs .
The toxic proline analog DHP enters the cell via PP-TI ( 8 ) .
If betaine also enters the cell through PP-II it might be expected to compete for uptake and reduce the toxic effects of DHP .
Figure 3 shows the growth of strain CH627 ( AputPA ) in LOM-glucose .
After 90 min of growth , DHP or betaine or both were added as indicated .
The addition of betaine alone had no effect on the growth-rate .
However , when DHP ( 100 , ug ml - ' ; approximately 0.8 mM ) was added growth was strongly inhibited .
This inhibition was relieved by the addition of betaine , suggesting that betaine and DHP compete for the same uptake system .
proP encodes a betaine transport system .
Betaine is known to be taken up into E. coli by an active transport process ( 18 ) .
To determine the potential role of proP in betaine 13-Galactosidase ( U ) Medium additives CH500 CH501 183 None 163 NaCI ( 0.3 M ) 761 702 Betaine ( 1 mM ) 171 194 NaCl ( 0.3 M ) + betaine ( 1 mM ) 569 559 Proline ( 1 mM ) 189 206 NaCl ( 0.3 M ) + proline ( 1 mM ) 610 623 a Cells were grown in LOM with additives as indicated .
Strains CH500 and CH501 contain independently isolated proP : : Mu dl ( Apr lac ) fusions .
Both strains are also putP pressure .
However , when transport was assayed in highosmolarity medium , betaine transport was detected .
Since significant betaine uptake could only be detected when assayed at high-osmolarity , all subsequent assays were carried out under these conditions .
To determine whether putP and proP play a role in betaine transport , uptake was measured in strains carrying mutations in these genes ( Fig. 4 ) .
Cells of LT2 and CH627 ( AputPA ) were grown in medium of high-osmolarity , and betaine uptake was determined at 30 , uM concentration .
Clearly , putP plays no role in betaine uptake as betaine uptake was identical in these two strains .
Because expression of proP was enhanced by growth at high osmotic pressure we wished to determine if betaine transport was similarly enhanced .
Thus , CH627 and CH638 cells were grown at both low and high osmotic pressure , and in both cases betaine uptake was determined at high osmotic pressure .
In cells grown at low osmotic pressure all betaine uptake was dependent upon proP ; no uptake could be detected in CH638 in which proP is mutated .
However , in cells grown at high-osmolarity an additional , proP-independent betaine uptake system was induced .
The rate of betaine uptake through PP-IT for cells grown at low-osmolarity can be calculated to be 2.2 nmol min - ' mg of protein - ' .
The rate of proP-dependent betaine uptake in cells grown at high-osmolarity can be calculated from the difference in uptake between the parent ( CH627 ) and the proP mutant ( CH638 ) , and is about 9 nmol min - ' mg of protein-1 .
Thus , growth at high osmotic pressure increases betaine uptake through PP-TI by about fourfold , in good agreement with the increases in proline transport through PP-II and transcription of proP observed under the same conditions .
It should be noted that these experiments were carried out with 30 , uM betaine .
At higher betaine concentrations uptake could be detected even when assayed at low osmotic pressure ( data not shown ) .
This is compatible with data presented above which show that , even in LOM , 1 mM betaine can compete with DHP uptake via PP-IT .
In addition , while mutations in proP reduce betaine uptake considerably , it is clear that there is an additional proP-independent betain transport system which is only active in cells grown at high-osmolarity .
We have shown in an accompanying paper that this residual betaine uptake is dependent upon the function of the proU gene ( 3 ) .
It is therefore clear that PP-II , encoded by proP , functions as a betaine transport system .
The osmolarity of the medium influences uptake via PP-Il in two ways .
First , transcription ofproP is induced approximately threefold by growth at high-osmolarity , and second , even when induced , the transport system only functions in medium of high-osmolarity .
Kinetics of betaine transport through PP-II .
Strain CH992 ( putP proU proP + ) is deficient in pro U , and in this strain all betaine transport at 30 , uM has been shown to be via PP-II ( 3 ) .
CH992 was grown in high-osmotic-pressure medium ( LOM-glucose plus 0.3 M NaCI ) , and betaine transport was assayed in the same medium at a variety of different concentrations ( Fig. 5 ) .
Eadie-Hofstee and Lineweaver-Burk plots of these data give kinetic parameters of Km = 44 , uM and Vmax = 37 nmol min-1 mg of protein - ' .
DISCUSSION It has been known for many years that betaine plays an osmoprotective role in plants , and more recently a similar role has been demonstrated in the enteric bacteria ( 12 , 13 ) .
E. coli has been shown to accumulate betaine by an osmotic pressure-dependent active transport process , although the genes involved and the mechanism of this osmotic stimulation are obscure ( 18 ) .
In this paper we demonstrate that the proP gene , originally identified as encoding a minor proline permease , PP-II , plays a major role in betaine uptake .
Several lines of evidence show that betaine uptake is mediated by PP-II .
First , the osmoprotective effect of betaine toward cells grown at high osmotic pressure was reduced in strains defective in proP .
Second , the uptake of toxic proline analogs through proP was inhibited by excess betaine .
Finally , the uptake of betaine itself was considerably reduced in strains mutated for proP .
The kinetics of betaine uptake through PP-II suggest that betaine , rather than proline , is the primary substrate for this transport system .
The proP gene was originally identified as encoding a minor proline permease in both S. typhimurium and E. coli ( 1 , 16 , 24 ) .
The Km for proline uptake via PP-Il is exceptionally high ( 300 , M ) , especially when compared with proline uptake through the major proline permease ( PP-I ; Km = 2 , uM ) .
Thus , the physiological role of this second system has been obscure .
However , we show here that the affinity of PP-II for betaine is about 10-fold greater than its affinity for proline , suggesting that betaine is the physiological substrate for this transport system .
Although it is perhaps surprising that the two amino-acids proline and betaine should be substrates for the same transport system , both are Nsubstituted amino-acids .
In contrast to PP-II , the major proline permease ( PP-I , putP ) shows no detectable affinity for betaine and appears to play no role in betaine uptake or osmoprotection .
We determined the following kinetic parameters for betaine transport via PP-II : Vmax = 37 nmol min - ' of Km mg protein - ' and = 44 , uM .
In addition to PP-II , there is a second betaine transport component in wild-type cells .
This is mediated by a high-affinity uptake system dependent upon the osmotically induced proU gene ( 3 ) .
Thus , S. typhimurium has two genetically distinct betaine transport systems .
It seems likely that similar betaine transport systems function in E. coli .
First , the kinetics of betaine uptake by wild-type E. coli ( Vmax = 42 nmol min - ' mg of protein - ' ; Km = 35 , uM [ 18 ] ) are similar to those we obtained for uptake via PP-II in S. typhimurium .
The slight differences between the two sets of data can readily be accounted for by the additional proU-dependent component of transport present in wild-type cells ( 3 ) .
Proline has been shown to compete with betaine for uptake by E. coli ( 18 ) .
In addition , a gene corresponding to proP and encoding a minor proline permease has been identified in E. coli , although the chromosomal location of this gene is still in some doubt ( 24 ) .
Regulation of PP-II function is intriguing .
We found that transport of both betaine and proline via PP-II was stimulated about threefold by growth at high-osmolarity .
High osmotic pressure also increased transcription of proP to a similar extent .
Thus , the increase in PP-Il function in cells grown at high-osmolarity seems to be wholly due to increased transcription of the proP gene .
Anderson et al. ( 1 ) reported that proP expression is also enhanced threefold by amino-acid starvation ; whether these two regulatory effects are related remains speculative .
However , it is interesting to note that osmotic shock causes a rapid increase in the cellular ppGpp pool ( 10 ) .
In addition to transcriptional regulation , the activity of the PP-I1 permease itself is affected by the osmotic pressure of the medium .
Thus , even when fully induced , PP-II is only able to transport betaine to a significant extent in medium of high-osmolarity .
In contrast , the function of many other transport systems is also inhibited at high-osmolarity ( 23 ; unpublished data ) .
This strongly suggests that increased osmotic-stress results in a conformational change in the permease protein , either nonspecifically by virtue of cell and membrane shrinkage or via a specific regulatory circuit .
Particularly interesting is the observation that while increased osmolarity increases betaine uptake via PP-II it decreases proline uptake .
Thus , not only is transport activity altered , but there is a change in substrate specificity .
Potassium transport via the trk system in E. coli , which can also serve an osmoprotective function , has been shown to exhibit similar regulatory properties ( 21 ) .
The mechanisms by which betaine protects against osmotic pressure are unclear .
It can be accumulated in large amounts in response to stress ( 12 , 18 ) and may therefore provide an osmotic balance across the membrane .
In addition , there is also considerable evidence that betaine can specifically protect enzymes from adverse concentrations of salt ( 25 ) and can also reverse the inhibition of many carbohydrate transport systems by NaCl ( 23 ) .
We show here that uptake of betaine into the cell is important for its osmoprotective function .
A further characterization of the mechanism and regulation of the betaine uptake systems is therefore of considerable importance in understanding the mechanisms and evolution of osmotolerance .
° to 6 CL 0 / g E cn `` I 0 ) 3 [ / ° a / , 1 A 0 ez s e Ca 1 5 4 very high salt corncentration ( 0.8 M NaCI ) , proline transport via PP-TI was strc ) ngly inhibited ( data not shown ) .
Thus , it is clear that proline transport via PP-IT is increased when cells are grown at highi osmolarity and that this increase is due to induction of syntlhesis of PP-IT , rather than to an increase in specific activity c ) f the transport system .
Osmotic enhan4 cement of proP expression is transcriptional .
To determine wh ether the increase in PP-IT synthesis at high osmotic pressure is at the transcriptional level , we made use of Mu dl-mediated lac fusions ( 5 ) to the proP gene .
These fusions place the lacZ gene under control of the proP promoter .
The construction of Mu dl ( Apr lac ) fusions to proP has been dlescribed previously ( 14 ) .
P-Galactosidase synthesis by tw4 o strains carrying independently isolated fusions of lacZ to proP ( CH500 , CH501 ) was increased about three-to fourfolcd by growth in high-osmotic-pressure me-dium ( Table 2 ) .
1Fhis effect was seen whether the osmolyte used was NaCI , KCl , choline-chloride , or sucrose ( data not shown ) .
Thus , iinduction of proP transcription correlates well with the in ( crease in proline transport through PP-II observed at higih osmotic pressure .
Neither proline nor betaine induced IvroP expression .
Indeed , betaine caused a reduction in proP ) expression in medium of high-osmolarity .
This could be a result of the intracellular accumulation of betaine causing .
a reduction in turgor pressure differences which may deterrmine the osmoinduction of gene expression - C. A ~ ~ .
Expression c t pror , as juugec ty p-gatactosiuase production from th ese fusions , was also unaffected by the introduction of 1putP , putA , or proU mutations into the fusion strains ( datta not shown ) .
Major role of proP in osmoprotection .
Because of the increase in proP expression at high osmotic pressure , it seemed possible that this transport system plays a role in osmoprotection .
We therefore examined the role of PP-II in osmoprotection .
Figure 2A shows the growth of cells in LOM-glucose c ( ) ntaining 0.8 M NaCl but lacking any osmoprotectant .
As expected , growth in this medium was very poor ( doub ] ling time = 420 min ) .
RReeguulaattioon ooff pDrrooP eexxpr TABLEression A. B. C. 2 4 6 8 2 14 2 4 6 8 10 12 14 4 6 8 10 12 TIME ( hours ) 10 12 14 16 FIG. 2 .
Osmoprotection by proline and betaine .
Cells were grown in LOM-glucose containing 0.8 M NaCl , and growth was monitored by determining the optical density of the culture at 600 nm .
Cells were grown in the absence of any osmoprotectants ( A ) ; with 1 mM proline ( B ) ; with 1 mM betaine ( C ) .
The strains used were LT2 ( putPA + proP + [ 0 ] ) , CH627 ( AputPA proP + [ A ] ) , and CH638 ( AputPA proP [ A ] ) .
uptake , transport was measured directly ( Fig. 4 ) .
Because proP expression is enhanced at high osmo ) tic pressure , cells were grown and assayed for betaine tranisport at both low ( LOM ) and high ( LOM plus 0.3 M NaC1 ) osmolarity .
No betaine uptake by strains CH627 or CH638 , could be detected when transport was assayed in medium c ) f low-osmolarity , whether or not the cells were grown at low or at high osmotic 1 2 7 6 8 3 4 5 TIME ( hours ) FIG. 3 .
Betaine against inhibition by DHP .
Cells of protects strain CH627 ( AputPA ) were grown in LOM-gluicose in the presence ( open symbols ) or absence ( closed symbols ) of ] L00 Lg of DHP ml - ' and growth was followed by measuring the opttical density ( OD ) at 600 nm .
After 1.5 h betaine was added to the cultures at the following concentrations : 1 mM ( I , * ) , 50 pLM ( 0 , 0 ) , and no addition ( A , A ) .
0 ' E 50 E C a , m 40 0 .
a , / 0 / .7 c co 20 o0 0 0 10 0 r ~ I.-r 1 4 5 2 3 TIME ( mins ) FIG. 4 .
Betaine uptake by S. typhimurium .
Cells of strain CH627 ( closed symbols : AputPA proP + ) or CH638 ( open symbols ; AputPA proP ) were grown at either low ( LOM-glucose ) or high ( LOM-glucose plus 0.3 M NaCI ) osmolarity as described in the text , and betaine transport at 30 , uM was assayed in the presence or absence of 0.3 M NaCl as follows : cells were grown and assayed at high-osmolarity ( 0 , 0 ) ; cells were grown at high-osmolarity and assayed at low-osmolarity ( A , A ) ; cells were grown at low-osmolarity and assayed at high-osmolarity ( U , O ) .
Transport was also assayed at high-osmolarity in wild-type cells ( LT2 ; putPA + proP + ) grown in medium of high-osmolarity ( x ) .
c 0 5 I / 60 90 120 Betaine conc ( , uM ) 30 150 FIG. 5 .
Kinetics of betaine uptake through PP-II .
Cells of strain CH992 ( AputPA proP + proU ) were grown in LOM-glucose containM 0.3 and betaine was ing NaCl , transport assayed in medium of the same high-osmolarity .
Eadie-Hoffstee and Lineweaver-Burk transformations of these data give the following kinetic parameters : Vmax = 37 nmol min-1 mg of protein - ' ; Km = 44 , LM We are grateful to the Medical Research Council for financial C.F.H. support .
is a Lister Institute Research Fellow .
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Genetic engineering in agriculture : osmoregulation .
Maloy , S. R. , and J. R. Roth .
Regulation of proline utilization in Salmonella typhimurium : characterization of put : : Mu d ( Ap lac ) operon fusions .
Menzel , R. , and J. Roth .
Identification and mapping of a second proline permease in Salmonella typhimurium .
Experiments in molecular genetics .
Cold Spring Harbor Laboratory , Cold Spring Harbor , N.Y. 18 .
Perroud , B. , and D. Le Rudulier .
Glycine betaine transport in Escherichia coli : osmotic modulation .
Ratzkin , B. , M. Grabnar , and J. Roth .
Regulation of the major proline permease gene of Salmonella typhimurium .
Ratzkin , B. , and J. Roth .
Cluster of genes controlling proline degradation in Salmonella typhimurium .
Rhoads , B. B. , and W. Epstein .
Cation transport in Escherichia coli .
Regulation of K + transport .
Genetic techniques in studies of bacterial metabolism .
Roth , W. G. , M. P. Leckie , and D. N. Dietzler .
Osmotic stress drastically inhibits active transport of carbohydrates by Escherichia coli .
Stalmach , M. E. , S. Grothe , and J. M. Wood .
Two proline porters in Escherichia coli K-12 .
Yancey , P. H. , M. E. Clark , S. C. Hand , R. D. Bowlus , and G. N. Somero .
Living with water stress : evolution of osmolyte systems .