Effect of Salmonella typhimurium Ferric Uptake Regulator ( fur ) Mutations on Iron - and pH-Regulated Protein Synthesis Fur is an important regulatory protein known to function in the presence of iron as a repressor of iron-controlled genes .
It was recently discovered that Fur is also essential to Salnonela typhimurium for mounting an adaptive acid tolerance response ( J. W. Foster , J. Bacteriol 173:6896 -6902 , 1991 ) .
Because little is known about the effect of Fur on the physiology of this enteric pathogen , a systematic two-dimensional polyacrylamide gel electrophoresis ( PAGE ) analysis was conducted to identify proteins whose synthesis is linked to iron levels .
Mutations in thefiur locus were identified and used to classify which proteins are controUed by Fur .
Thirty-six proteins were overtly affected by iron availability , most of which were clearly under the control of Fur .
Although most of the Fur-dependent proteins were under negative control , a significant portion ( 15 of 34 ) appeared to be under a form of positive control .
Nine of the positively controUled proteins required Fur and iron for expression .
However , Fur lacking iron was also required for the induction of six gene products .
Surprisingly , not all iron-regulated proteins were controlled by Fur and not all Fur-dependent proteins were obviously regulated by iron status .
Becausefisr mutants fail to mount an effective acid tolerance response , we made a comparative two-dimensional PAGE analysis of 100 total acid-and iron-regulated gene products .
Production of most of these proteins was regulated by only one of the two stresses , yet a clear subset of seven genes were influenced by both acid and iron and were also controlled byfur .
These proteins were also members of the acid tolerance response modulon .
Consistent with the fir effect on pH-regulated protein synthesis , jiar mutants lacked the inducible pH homeostasis system associated with the acid tolerance response .
The results provide further evidence that Fur has an extensive impact on gene expression and cellular physiology and suggest an explanation for the acid-sensitive nature offir mutants .
Iron is an extremely important element for biological systems ( 31 ) .
Consequently , organisms have evolved elaborate methods to acquire iron under limiting conditions ( for reviews , see references 6 and 13 ) .
Bacteria , including Esch-erichia coli and Salmonella typhimurium , synthesize and excrete a variety of chelators or siderophores with extremely high affinities for iron .
In addition to the enzymes required for siderophore synthesis , membrane proteins involved in recovering siderophore-iron complexes from the medium undergo transcriptional regulation by iron availability .
The regulatory protein that mediates this regulation , designated Fur ( ferric-iron uptake regulator ) , binds Fe2 '' and subsequently represses the expression of the many iron acquisition genes ( 6 ) .
Iron availability also controls the synthesis of a variety of toxins and other virulence determinants .
The hemolysin of Vibro cholerae ( 37 ) , diphtheria toxin of Cory-nebacterium diphtheriae ( 10 ) , Shiga toxin of Shigella dysenteriae ( 16 ) , and Shiga-like toxin of E. coli ( 11 ) are examples of iron-regulated toxins .
While considerable attention has focused on the ferric uptake regulator ( fur ) locus of E. coli , little work has been published regarding this regulatory gene in S. typhimurium or on iron metabolism in general in this organism ( 7 , 8 , 17 ) .
As a facultative intracellular parasite , S. typhimurium is an excellent model system for studies of host-parasite interactions .
During invasion , S. typhimurium enters a phagosome and subsequently encounters a wide array of hostile conditions , including reactive oxygen intermediates , antimicrobial peptides , acidic pH , and iron deprivation , among others .
Successful intracellular parasites have developed a variety of strategies to avoid , disarm , or endure these lethal situations .
S. typhimurium , for instance , possesses a variety of inducible defense mechanisms which are believed to enhance survival within the hostile phagosomal and phagoly-sosomal environments .
One system under study , termed the acid tolerance response ( ATR ) , enables adapted S. typhimu-rium to survive severe low-pH stress ( 20 ) .
We have recently discovered that strains of S. typhimurium harboring fur mutations fail to mount an effective ATR and as a result are extremely acid sensitive ( 19 ) .
The basis for the relationship between Fur and acid tolerance has not been established .
While the importance of iron metabolism to the cell is clear , there is no information available about iron-regulated proteins in S. typhimurium on a whole-cell basis .
Consequently , a global study of the iron modulon will provide insight into the extent and nature of Fur-regulated gene expression and may offer clues to the acid-sensitive nature of fur mutants .
With these goals in mind , we have conducted a two-dimensional polyacrylamide gel electrophoretic ( PAGE ) analysis of iron-regulated proteins in S. typhimurium and determined which are controlled by Fur .
Based on similar investigations with other stress-regulated proteins , it was possible to integrate the iron regulation modulon within the general framework of stress management in S. typhimurium .
The results indicate that Fur has a dramatic effect on protein synthesis and may act not only as a negative regulator , but in some instances as a positive regulatory element , either directly or indirectly .
In addition , a significant overlap between iron - , Fur - , and pH-regulated protein synthesis was discovered .
MATERIALS AND METHODS Bacterial strains and culture conditions .
S. typhimurium LT2 was used throughout this study .
JF2023 is a derivative of LT2 that contains a spontaneous fiur mutation .
This and similar fiur mutants were isolated by using an iron-regulated iroA-lac fusion .
The fur mutants were identified as spontaneously arising , deregulated Lac ' derivatives on MacCon-key lactose medium supplemented with 60 , uM FeSO4 .
JF1819 [ atr ( Con ) ] is a constitutively acid-tolerant mutant ( 20 ) .
SF381 is an enterochelin-requiring mutant supplied by K. Sanderson , University of Calgary .
The plasmids pABN203 and pMON2064 both contain the E. cofifur + locus and were kindly provided by J. B. Neilands ( 5 ) and R. D. Perry ( 36 ) , with the permission of Monsanto Corporation , respectively .
The culture media used included E medium ( 38 ) and LB-medium ( 14 ) .
Measurement of the adaptive ATR was detailed earlier ( 19 ) .
Briefly , cultures were grown in pH 7.7 minimal E with glucose under semianaerobic conditions to 108 cells per ml and then adapted to pH-5.8 for one doubling .
Unadapted cultures were grown directly to 2 x 108 cells per ml at pH 7.7 .
Both cultures were then readjusted to pH 3.30 and incubated for 90 min .
Viable cells were counted at 0 and 90 min .
Two-dimensional PAGE analysis of polypeptides .
Two-dimensional PAGE was carried out as described previously ( 34 ) .
The first dimension was a pH 5 to 7 ( right to left ) isoelectric focusing gel containing 1.6 % ( pH 5 to 7 ) and 0.4 % ( pH 3 to 10 ) ampholytes ( LKB-Pharmacia ) .
The second dimension was an 11.5 % polyacrylamide-sodium dodecyl sulfate ( SDS ) gel .
Cells were grown in pH 7.0 minimal E medium ( 38 ) containing 0.4 % glucose and supplemented with 120 , uM FeSO4 for iron-replete conditions .
Iron-limiting conditions were achieved through the addition of an iron chelator , 100 , uM diethylenetriamine pentaacetic acid ( DTPA ) .
In a second set of experiments , the iron chelator dipyridyl was used at 0.2 mM .
Chelators were added to cultures that had attained a cell density of 108 cells per ml .
After one to two doublings , the cells were labeled for 15 min with [ 35S ] methionine ( 35S-Trans label ; ICN ) at 50 , uCi/ml , harvested , and lysed in an SDS lysing solution as indicated by Spector et al. ( 34 ) .
Addition of DTPA or dipyridyl produced iron-limiting conditions sufficient to reduce the growth-rate and induce expression of an iron-regulated iroA-lacZ fusion .
The proteins indicated in Fig. 1 and Table 1 are those that were consistently observed to change over several experiments and with several fur mutants .
Acidic and basic proteins are positioned to the right and left of each gel , respectively .
Transductions were performed with phage P22 HT 105/lint as described earlier ( 4 , 26 ) .
The TnlO-and lacZ-directed Hfr strains were constructed as described by Chumley et al. ( 12 ) and Spector et al. ( 35 ) .
Transformations were performed by electroporation .
P-Galactosidase production was assayed as described by Miller ( 28 ) and is expressed as micromoles per minute per optical density unit at 600 nm .
Each strain was assayed in triplicate cultures .
The method of measuring the internal pH ( pHJ ) involved monitoring the distribution of radiolabeled weak-acids or bases across the cellular membrane .
The procedure was derived from that of Booth et al. ( 9 ) .
To determine intracellular water spaces , cells were grown in minimal-medium containing 25 mM sucrose .
At mid-log phase , 3 ml of cells was harvested and resuspended into 200 RI of culture supernatant .
3H20 and [ 14CJsucrose were each added to 3,000 dpm / , ul , a 5 - , ul sample was removed for determination of total counts , and the remainder was incubated at 37 °C for 10 min ( reaction mix A ) .
Following incubation , duplicate samples ( 100 RI ) were centrifuged through 50 RI of dibutyryl phthalate and 50 , ul of silicone oil .
The tubes were frozen at -70 °C , and the cell pellet was sliced from the tube .
The pellet was placed in a minivial containing 100 , ul of 1 % SDS for 5 min .
Scintillation fluid was added , and the radioactivity in the series of vials was counted in an LKB 1219 scintillation counter .
The total H20 per cell pellet was calculated as dpm of 3H20 in the pellet/total dpm of 3H20 per pl of reaction mix .
Extracellular H20 was equal to dpm of [ `` 4C ] sucrose in the pellet/total dpm of [ 14Cjsucrose per RI of reaction mix .
Intracellular water was equal to total H20 minus extracellular H20 .
An almost identical procedure was used to determine the distribution of a weak acid ( [ 14Clbenzoic acid ) or base ( [ 14C ] methylamine ) , the difference being that the reaction mix ( 200 , u ) contained 3,000 dpm of 3H20 and [ 14C ] benzoic acid or [ 14C ] methylamine .
The formula used to calculate pH. was as follows : pHi = log [ ( total acid in/total acid out ) ( 10PR + 1OP ' ) - loPI .
RESULTS Iron-regulated protein synthesis and overlap with other stress-regulated modulons .
Two-dimensional PAGE analysis was used to define the relationship between iron-regulated protein synthesis and other stress modulons .
A comparison of polypeptide profiles between cells grown under ironreplete versus iron-deficient ( DTPA ) conditions is presented in Fig. 1A and B , respectively .
The quantitative levels of 41 proteins changed .
Thirty-one were induced by apparent iron limitation , while 10 were repressed under the same condition .
Table 1 lists the coordinates for each protein on a standard two-dimensional map of Salmonella proteins ( 34 ) and , in addition , indicates whether the protein was induced or repressed .
To confirm that iron was the regulating metal , similar experiments were performed with dipyridyl as the iron chelator .
Only five of the proteins regulated by DTPA chelation were not similarly affected by dipyridyl , indicating that they were regulated by a metal other than iron .
Many of the 36 remaining proteins were regulated dramatically by iron availability , while others showed more modest changes .
Comparisons with 10 other stress conditions revealed that 11 of the iron-regulated proteins were controlled in response to alternative environmental pressures .
Nine of the dual-stress-controlled proteins responded to pH as the second stimulus , either as part of the pre-acid shock ( pH-5.8 ) or post-acid shock ( pH 4.5 ) components of the ATR .
This indicates that a combination of iron and pH may serve as a regulatory signal for this subset of proteins .
Although iron solubility varies with pH , these results reflect more than a simple relationship between iron availability and pH. Even though the solubility of Fe ( OH ) 3 is known to increase somewhat with increasing acidity , we found that the synthesis of most iron-regulated proteins remained insensitive to pH. Identification of Salmonella fur mutants .
The next phase of this study was to observe the effect of fur mutations on iron-regulated protein synthesis , but since the original Sal-monella fur mutant is no longer available ( 17 ) , we must provide proof that the mutations used in this study occur within the fur locus .
The first step was to show that the mutations affect the expression of an iron-regulated locus .
One such locus ( iroA ) was identified from a pool of lacZ insertion mutations constructed in our laboratory .
Table SalonllTofdumrutnts.oa FiGuE 2nlsspororvoi-eetuiaaesyevi-si LFu orany of thethehrfur mutant str23 ( Cains taested .
udencexthatsthe fresmutantsyLoverprodueentterochpaehsndA ferric chloride-saturated filter paper disk placed next to a streak offiur ' LT2 clearly showed repressed siderophore Byot usoningdthentecntrtiqurguaedbyoJfO-directedHfr foration ( 12 ) , the approximate location of these putative fur mutations was determined to be between 12 and 20 min on the S 33 94 , 102 I 34 89 , 72 I 35 88 , 37 4 , 36 93 , 36 R 4 , 37 93 , 63 I I 38f 108 , 92 I 4 , 39 112 , 53 I I 40 109 , 43 I I 41 73 , 78 I I I Coordinates correspond to the standard map published by Spector et al. ( 34 ) .
b Iron excess involved the addition of 120 , uM FeSO4 , while iron limitation was achieved by the addition of the iron chelator DTPA ( 100 , LM ) .
I , induced by limiting iron ; R , repressed by limiting iron .
c Expression of Fur proteins in a fr mutant ( JF2023 ) .
I overexpression even with excess iron ; , underexpression or no expression even under derepressive conditions .
d Overlap with other stress-regulated modulons was identified by comparison with proteins induced by acid tolerance ( 20 ) , acid shock ( 19 ) , oxygen ( OXI ) or anaerobiosis ( ANI ) , and starvation ( STI ) ( 34 ) .
e These proteins were not induced with the addition of dipyridyl , a more specific iron chelator .
They are regulated in response to an ion other than iron .
f These proteins appear to require during deferrated for induction Fur iron limitation .
Subsequent cotransductional analyses with zbf-57 : : TnlO placed this locus near nag at 15.5 min ( 33 ) .
This map position is consistent with that of E. coli fur .
As a final proof , Table 2 reveals that clonedfiur + from E. ( Miller units ) Iron Iron excess depleted JF2062 iroA-lacZ 0 360 JF2021 iroA-lacZ fur-1 560 510 JF2058 iroA-lacZffur-7 347 522 JF2059 iroA-lacZ fur-8 420 470 JF2208b iroA-lacZfr-1 / pABN203 fur + 0 0 JF2485b iroA-lacZfiur-J/pMON2064fur ' 0 570 a Cells were grown in minimal-medium ( pH 7.0 ) to 101 cells per ml , at which point either 60 , uM FeSO4 ( iron excess ) or 200 ILM DTPA ( iron depleted ) was added .
The culture was allowed to double prior to use .
3-Galactosidase activity was measured in Miller units ( 28 ) .
b JF2208 and JF2485 were grown in buffered LB ( pH 8 ) .
coli will complement the regulatory defect in Salmonella fur mutants .
The overexpression offur in JF2208 caused superrepression of iroA even without excess Fe3 '' .
While it is clear that holo-Fur has a greater affinity for target operators , apo-Fur probably retains a small but significant affinity for those operators .
The equilibrium between free and operatorbound apo-Fur must shift toward the bound form with high levels of repressor .
The lower expression of fur from JF2485 ( pMON2064 ) more closely approximates the singlecopy level of expression present in the wild-type situation .
Consequently , effective repression in JF2485 still requires bound iron .
Effect offur mutations on iron-regulated protein synthesis .
Parallel to the two-dimensional PAGE study described above , a comparison of polypeptide profiles was made between wild-type LT2 and the fur mutant JF2023 ( Fig. 1C and D ) .
As expected , the production of a majority of iron-regulated proteins was clearly affected by the fur mutation .
For the most part , if a protein was induced by iron limitation ( iron repressible ) in a wild-type cell , it was overproduced in the fur mutant under either high-or low-iron conditions .
This was consistent with the classic role of Fur as a negative regulator ( 5 ) .
However , two striking results were noted that diverged from this norm .
First , if a protein was normally induced by excess iron , it became uninducible in thefur mutant , suggesting that Fur may complex with iron to activate the transcription of some genes .
Second , six Fur-dependent , iron-repressed proteins were underexpressed , not overexpressed , in the fur mutant .
This is the opposite of what one would predict upon the loss of a negative regulator and indicated that deferrated Fur may be a positive regulator , either directly or indirectly , for a subset of iron-regulated genes .
A third discovery that resulted from studying the fur mutant was that not all of these proteins were Fur dependent .
Seven proteins , indicated by their identification numbers in Fig. 1C and D , remained regulated by metal chelation with DTPA even in the absence of Fur .
As noted above , five of these proteins must be induced in response to an ion other than iron , since a more specific iron chelator , dipyridyl , did not cause their induction ( data not shown ) .
However , two of the Fur-independent DTPA-induced proteins were also induced by dipyridyl .
This result is suggestive of a second iron-sensing system in S. typhimurium , although we can not unequivocally rule out induction of these two proteins through chelation of some ion other than iron .
Recent results from our laboratory indicate that Fur may influence the expression of some genes that do not respond overtly to fluctuations in iron concentration .
For example , three pH-regulated genes that were not subject to obvious iron regulation were nevertheless rendered uninducible by acid-pH in a Fur-mutant ( 19a ) .
Consequently , we expected to observe some proteins that were not detectably regulated by iron in the wild-type cell but were nevertheless underexpressed in the fur mutant .
Five of these were observed and are indicated by letter in Fig. 1B and D. fur mutations abolish ATR-specific inducible pH homeostasis .
Previous results noted above have shown that S. typhi-murium requires Fur in order to effectively mount an ATR .
The reason for this is not known , but the fact that several pre-acid shock ATR proteins were aberrantly expressed in fur mutants offers a possible explanation for their Atr - , acid-sensitive phenotype .
The pre-acid shock stage of the ATR induces a pH homeostasis system operative at external pHs below 4 .
Thus , at pH. 3.3 , adapted ( pH-5.8 ) LT2 cells have a more alkaline-pHi than do unadapted cells ( 21 ) .
It seemed reasonable to suspect that fur mutants might be defective in ATR-specific pH homeostasis .
This possibility was examined through determinations of pHi for adapted ( pH-5.8 ) and unadapted fur mutant cells at a variety of pH. values .
The results in Table 3 indicate no significant differences in pHi at any pH. examined above 4.0 , suggesting that normal pH homeostasis mechanisms are operating appropriately .
This was consistent with the results of an earlier study , showing thatJir mutants do not exhibit general acid sensitivity ( 19 ) .
However , the data in the last two columns of Table 3 indicate that the adaptive pH homeostasis mechanism is inoperative in fur mutants .
This deficiency will Unadapted Adaptedb LT2 Wild type 7.87 7.6 7.1 6.1 4.4 5.0 JF2023 fir 7.8 7.6 7.0 6.1 4.1 4.2 JF1819 atr-l ( Con ) 7.8 NDC ND 6.3 5.7 ND a Internal pH was measured at 10 min after the shift to the indicated external pH ( pH. ) .
The following weak bases or acids were used to measure intemal pH : methylamine ( pH 7.5 ) , benzoic acid ( pH 6.8 , 5.8 , and 4.4 ) , and salicylic acid ( pH 4.4 and 3.3 ) .
b doubling prior to a shift to pHI 3.3 .
Cells were adapted at pH. 5.8 for one ND , not determined .
clearly result in an increased acid sensitivity below pH 4 while leaving survival above pH 4 unaffected .
For comparative purposes , a constitutively acid-tolerant atr ( Con ) - l mutant ( JF1819 ) was shown to possess enhanced pH homeostasis ability at a of 3.3 of 5.7 ) .
The fact that the fur pH. ( pHi mutations do not affect pH homeostasis above pH 4.0 supports the view that these mutations specifically affect the ATR system and not classic pH homeostasis .
DISCUSSION The results of these two-dimensional PAGE studies are summarized in Fig. 3 .
They indicate that Fur 's contribution to gene expression is extensive .
While the pleiotropic nature of , fur mutations has been reported previously for E. coli ( 24 , 25 , 29 ) , this polypeptide analysis dramatically illustrates the scope of Fur 's impact on gene expression in S. typhimurium and the potential versatility with which it exerts control .
The pattern of Fur regulation can be explained in three ways .
Control may be a direct effect of Fur on target genes , a cascade effect through intermediate regulators , or some combination thereof .
The direct-effect model holds that Fur might exhibit four types of regulational control .
When complexed with iron , Fur may function as either a negative ( type A ) or a positive ( type B ) regulator , depending upon the target locus .
One of the more intriguing findings was that under iron starvation conditions , the regulator appeared to be necessary to activate the expression of several iron-re-pressed genes ( type C ) .
Lastly , Fur may act as a positive regulator of some genes in response to signals other than or in addition to iron ( type D ) .
TYPE A TYPE C TYPE B FIG. 3 .
Schematic representation of iron-regulated proteins .
The number of proteins in each grouping is shown in parentheses That a single regulatory protein can either activate or repress different genes in response to a variety of signals is not without precedent .
The cyclic-AMP-receptor-protein has been shown to act as an activator and a repressor and possibly responds to different cyclic-nucleotides ( 1-3 , 22 , 27 , 33a , 39 ) .
With Fur , respect to Bagg and Neilands ( 5 ) have already shown that divalent cations other than Fe2 + can bind and activate the repressor in E. coli .
Neiderhoffer et al. ( 29 ) provide evidence that the superoxide dismutase genes sodA4 and sodB are under negative and positive control , respectively , by Fur .
It is perhaps reasonable to expect that a regulator as central to the cell as Fur will exhibit different types of control over the many genes that it regulates .
As an alternative to the direct-effect model , our results may also be explained by cascade a type of control .
For example , if Fur negatively regulates the expression of a second negative regulator , the targets of that second regulator will appear to be under positive control by Fur .
Cascade control by Fur in other systems has already been demonstrated and so should be considered likely in S. typhimunum ( 23 ) .
However , the definitive proof of one scenario or the other ( or some combination ) will require extensive in-vitro analysis of Fur 's interaction with target genes or with secondary regulators of the target genes .
For it example , will be useful to examine type C and D loci for the presence or absence of a Fur box , the identified consensus DNA sequence to which iron-complexed Fur binds ( 15 ) .
Our data provide additional evidence at the whole-cell level of the complexity of Fur as a sensor-regulator molecule .
One goal of this study was to provide clues as to the nature of the Atr-phenotype of fur mutants .
It is significant that Fur influences the production of several pH-regulated gene products .
The acid-sensitive Atr-phenotype of fuir mutants is likely a result of this influence .
One or more of these low-pH-and Fur-controlled proteins may prove to be of integral importance to the ATR .
The fact that fiur mutants lack the ATR-specific inducible pH homeostasis system supports this theory .
Additional studies are under way to identify which Fur-regulated protein ( s ) is integral to acid tolerance .
We thank Z. Aliabadi , M. Spector , H. Winkler , K. Karem , and T. Penfound for stimulating discussions during the course of this work .
We are also indebted to N. Nixon and R. Thompson for their careful preparation of the manuscript .
This work was supported by a research grant ( DCB-89-04839 ) awarded by the National Science Foundation .
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