JOURNAL OF BACTERIOLOGY, Apr. 1984, p. 163-168 Vol. 158, No. 10021-9193/84/040163-06$02.00/0Copyright X 1984, American Society for Microbiology

Effect of Growth Temperature on the Acquisition of Iron bySalmonella typhimurium and Escherichia coli

PATRICIA L. WORSHAM AND JORDAN KONISKY*Department of Microbiology, University of Illinois, Urbana, Illinois 61801

Received 25 July 1983/Accepted 18 January 1984

We have examined the effect of growth temperature on three systems normally induced under conditionsof iron limitation: synthesis of the siderophore enterochelin (enterobactin), transport of ferric enterochelin,and production of the outer membrane protein which serves as the colicin I receptor. We found thatalthough Salmonella typhimurium produces less enterochelin when grown at 42°C, synthesis of thissiderophore was not diminished in Escherichia coli grown under the same conditions. Growth at 42°C undera condition of iron stress led to a reduction in the ability of cells to transport ferric enterochelin in bothorganisms. A two- to threefold decrease in the number of colicin I receptors was observed in cells of E. colior S. typhimurium grown at 42°C as compared with the number of receptors observed in cells grown at 37°C.The colicin I receptor was shown not to be inherently unstable at 42°C. By using a cir-lacZ operon fusion, itwas shown that at least part of the decrease in receptor levels found in cells grown at high temperature wasthe result of decreased transcription of cir, the receptor structural gene. The effect of growth temperature onthese systems was shown to be independent offur, a regulatory element which mediates their enhancedproduction in response to iron stress. We suggest that a second regulatory element common to geneproducts involved in iron sequestration may be responsible for temperature regulation of these systems.

When deprived of iron, Salmonella typhimurium andEscherichia coli synthesize the high-affinity iron chelatorenterochelin (enterobactin), as well as produce a specifictransport system for this siderophore (25). In E. coli, thebiosynthesis of enterochelin and the production of the81,000-dalton outer membrane protein that functions asreceptor for ferric enterochelin are coordinately regulated bythe amount of intracellular iron (22, 25). The levels of twoother outer membrane polypeptides appear to be similarlyregulated: an 83,000-dalton protein with no known functionand the 74,000-dalton cir gene product which is the receptorfor colicins Ta and lb (20, 22, 25). The colicin I receptor is notrequired for transport offerric enterochelin (29). This proteinmay serve as receptor for a siderophore which has yet to beidentified. Proteins of a similar size are observed in outermembranes prepared from cultures of S. typhimurium grownin low-iron medium (3, 9).The regulation of synthesis of these polypeptides and of

enterochelin is at the level of transcription (16, 33). In bothE. coli and S. typhimurium, regulatory mutants have beenisolated which constitutively produce enterochelin, as wellas the three outer membrane proteins normally observedonly in cells grown under iron limitation (9, 16). Suchmutants have been designated fur (ferric uptake regulation).Since the loci affected by this mutation are located indifferent regions of the chromosome (2), it seems likely thatsome regulatory element under fur control is a diffusiblegene product. Whether the fur gene product itself is adiffusible regulatory element has not been established.Evidence for a relationship between growth temperature

and the ability to sequester iron has been presented byseveral groups. Many gram-negative bacteria do not growwell at elevated temperatures unless the growth medium issupplemented with iron (6, 11-13, 17). However, the natureof this apparently widespread defect in iron acquisition athigh temperatures has not been elucidated.

* Corresponding author.

In S. typhimurium, the requirement for additional iron isdue, at least in part, to decreased biosynthesis of thephenolate siderophore enterochelin at the elevated tempera-ture (12). Temperature-sensitive synthesis of a hydroxymatesiderophore has also been reported in a fluorescent pseudo-monad (11). Thus, the defect in the acquisition of iron is notpeculiar to the biosynthetic pathway of either phenolate orhydroxymate siderophores.We initiated this study to define further the relationship

between growth temperature and three elements known tobe regulated by the amount of intracellular iron: excretion ofthe siderophore enterochelin, the rate of ferric enterochelinuptake, and the level of colicin I receptors.

(Preliminary reports of these data were presented at the81st Annual Meeting of the American Society for Microbiol-ogy, Atlanta, Ga., 7 to 12 March 1982, and at the 82ndAnnual Meeting of the American Society for Microbiology,New Orleans, La., 6 to 11 March 1983.)

MATERIALS AND METHODSBacterial strains. Strains used in these experiments are

listed in Table 1.Media and growth conditions. LB and LB agar have been

previously described (23). Minimal medium M63 was pre-pared as described by Miller (23), except that FeSO4 wasomitted. Chelex 100 resin (200 to 400 mesh; Bio-Rad Labora-tories) was used to remove iron from the medium by themethod of Murphy (24). The resin was removed by filtrationthrough Sterilfil D-65 filters (0.22 pum; -Millipore Corp.).Casein hydrolysate (0.15%; Difco Laboratories) was addedto the medium before adding the resin. Glycerol (0.4%) wasused as the carbon source for all experiments. Thiamine (1,ug/ml) and nicotinic acid (3 ILg/ml) were also added to theminimal medium. Where necessary, tryptophan (40 ,ug/ml)was added to allow growth of Trp- strains. Glassware waswashed with 2 N HCI for 24 h and rinsed with 4 volumes ofglass-distilled water before use. Where possible, polystyreneor polycarbonate tubes, flasks, and pipettes were used.

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164 WORSHAM AND KONISKY

TABLE 1. Bacterial strains used in this studyStrain Strain characteristics Reference

E. coliJK1 W3110 K-12 X rpsL This laboratoryMC4100 K-12 araD139 AlacUJ69 (5)

thi rpsLPW400 MC4100 (F(cir-lac) (33)PW401 PW400(pJB4J1) (33)LL2 through Clinical isolates J. LembkeaLL7

CR Human colon isolate This laboratory

S. typhimuriumG30 LT2 galE (9)RB338 LT2 fur (9)PW734 Rough derivative of This laboratory

RB338TN1073 zbi-812::TnlO trp43 galE K. G. Sandersonba School of Veterinary Medicine, University of Illinois, Urbana.b Salmonella Genetic Stpck Center.

Where indicated, ethylenediamine-di(orthohydroxyphenyla-cetic acid) (EDDA; Sigma Chemical Co.) was added todeplete the medium of free iron. EDDA was deferrated bythe method of Rogers (28), and stock solutions were pre-pared as previously described by Ong et al. (26).

Colicin I binding assays; Procedures for purification andradioiodination of colicin la have been described previously(19). Unless otherwise indicated, binding assays were per-formed on whole cells harvested in mid- to late-exponentialphase as previously described (19). Smooth strains of E. coliand S. typhimurium are only slightly sensitive to colicin Ia,and binding of the colicin to these strains is poor. Therefore,a rough strain of S. typhimurium and a K-12 strain of E. coliwere chosen for experiments involving colicin binding.

Sensitivity to colicin Ia. Approximate colicin titers wereevaluated by the serial dilution method of Guterman (15).

IB-Galactosidase assays. Overnight cultures of strainsPW401 and JK1 which had been grown at 37°C were dilutedto an absorbance at 600 nm of 0.25. The cell suspension (100,ul) was then used to inoculate flasks containing 10 ml of thesame medium which had been prewarmed to 37 Qr 42°C.After six generations of growth, the cultures were assayedfor 3-galactosidase activity as described by Miller (23), usingchloroform and sodium dodecyl sulfate to make the cellspermeable to the substrate. Isopropyl-p-D-thiogalactopyran-oside (1 mM; Sigma) was used to induce ,-galactosidase instrain JK1.

Catechol assay. The Arnow reaction (1) was used as anassay for enterochelin. 2,3-Dihydroxybenzoic acid (Sigma)was used as the standard. Culture supernatants were collect-ed from stationary-phase cultures. For each individualstrain, all cultures were harvested at the same turbidity.However, from strain to strain the turbidity varied from anabsorbance at 600 nm of 1.5 to 4.0.

55Fe transport assays. Purification of enterochelin has beendescribed previously (30). "Fe-enterochelin was prepared asfollows. A ferric enterochelin solution (10 ml; 320 nmol) wasacidified to pH 1.5 with concentrated HCl and then extractedtwice with 10 ml of ethyl acetate. Nitrogen gas was bubbledthrough the ethyl acetate solution until the volume wasreduced to 1 ml. Carrier-free 55FeCl (320 nmol; 1.44 Ci/mol;New England Nuclear Corp.) was added next, and the pH ofthe solution was adjusted to 7.0 with 0.5 N NaOH. Thesolution was then dried under vacuum, resuspended in 1.0

ml of M56 uptake medium which contained 100 ,uM EDDA,and incubated at 37°C for 6 h. Nine milliliters of M56-100FtM EDDA was added next, and the solution was filteredthrough a 0.45-,um Millex-HA filter (Millipore Corp.). Thetransport medium, M56, was prepared by the method ofLangman et al. (21), except that 100 ,g ofEDDA per ml wasadded to chelate free iron. Cells were harvested during late-exponential growth (absorbance at 600 nm, 0.9) and spun at10,000 rpm in a Sorvall SS31 rotor for 15 min at 4°C. Thepellet was washed three times with ice-cold M56 containing9.2% glucose. Finally, the cells were resuspended in thissame buffer. They were kept on ice no longer than 30 minbefore use. Immediately before each assay, the cell suspen-sion and the solution containing 55Fe-enterochelin (1.5 x 105cpm/nmol) were warmed to 26°C. Each time point consistedof a separate reaction mixture which was prepared by mixingequal volumes (100 ,ul) of cell suspension and 5Fe-entero-chelin solution. Transport was terminated at the appropriatetime by the addition of 3 ml of ice-cold LiCl (0.1 M)-EDTA(100 ,uM) solution before the sample was rapidly filteredthrough 0.45-,uM nitrocellulose filters (type BA85; Schlei-cher & Schuell). Reaction tubes were rinsed three times with3 ml of LiCI-EDTA solution, and the rinse fluids were

TABLE 2. Enterochelin production in cells grown at varioustemperatures

PhenolateStrain GrowthCPhconcntemp COC)'/i

E. coli K-12MC4100 37 18

42 17

W3110 37 2042 29

E. coli natural isolatesCR 37 15

42 18

LL2 37 2242 18

LL3 37 742 10

LL5 37 1742 18

LL6 37 842 8

LL7 37 1542 14

S. typhimuriumG30 (LT2 derivative) 37 20

42 4

RB338 (LT2 fur) 37 4842 14

TN1073 37 2342 12

a Growth of cells and determination of enterochelin are describedin the text.

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TEMPERATURE REGULATION OF IRON ACQUISITION 165

0 20

EE 160ECL._S4) 120-cU0

Cw 80

in

o 404)

0c

90 180 270 360 450

Time (sec)

FIG. 1. Effect of growth temperature on transport of 5Fe-enterochelin. Cells were harvested and transport assays were performed asdescribed in the text. Growth temperatures used were 42 (U) and 37°C (0). Strains used were (A) E. coli MC4100, (B) S. typhimurium G30,and (C) S. typhimurium fur RB338.

applied to the filter. The filters were dried, and radioactivityassociated with the filters was determined by liquid scintilla-tion counting. Before use, filters were soaked for at least 2 hat room temperature in LiCl-EDTA solution containing 50,uM FeCl3 and 1 FtM ferric enterochelin. Immediately beforeuse, filters were rinsed twice with 3 ml of LiCI-EDTAsolution. These steps decreased nonspecific association ofradioactivity with the filter.

RESULTSEffect of growth temperature on production of enterochelin.

It has been reported that enterochelin biosynthesis, in bothE. coli and S. typhimurium, is inhibited at temperaturesgreater than 40°C (12, 18). We were unable to reproduce

these results in E. coli when using K-12 strains, a recentlyisolated strain of human colonic origin (CR), or severalclinical isolates (LL2, LL3, LL5, LL6, LL7) (Table 2).Under the same growth conditions, the culture fluids of S.typhimurium exhibited decreased levels of enterochelin at42°C. Enterochelin synthesis in both Fur+ and Fur- strainsof S. typhimurium was diminished at 42°C, indicating thatthe effect of growth temperature on enterochelin productionis independent of the regulatory element Fur which mediatesthe response to iron.

Effect of growth temperature on the transport of ferricenterochelin. To determine whether growth temperatureaffects the ability of cells to transport ferric enterochelin,cultures of E. coli MC4100 and S. typhimurium G30 (Fur+)

c - A - -B

E 8000 4000

E~~~~~~~~~~~~~~~~~

E 6400 3200E

X 4800 2400

3200 - 1600

U-~~~~~~~~~~~~~1600-800 A

90 180 270 360 450 90 180 270 360 450

Time (sec)FIG. 2. Effect of growth temperature on transport of "Fe-enterochelin in cells grown under iron limitation. Cells were grown in M63

medium containing 2 ,uM EDDA. Cells were harvested and transport assays were carried out as described in the text. Growth temperaturesused were 42 (U) and 37°C (0). Strains used were (A) E. coli MC4100 and (B) S. typhimurium G30.

A)0

)o

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166 WORSHAM AND KONISKY

and RB338 (Fur-) grown in M63 medium at 37 and 42°Cwere assayed for ability to transport this siderophore. Theinitial rates of 55Fe-enterochelin uptake in cells grown atthese temperatures are illustrated in Fig. 1. In E. coliMC4100 and in S. typhimurium G30 (Fur'), we observed nodifference in the rate of 55Fe-enterochelin transport in cellsgrown at the two temperatures. However, in S. typhimuriumRB338 (fur) the rate of transport in cells grown at 42°C isapproximately one-half of that in cells grown at 37°C.

S. typhimurium RB338, which carries the fur mutation,expresses gene products involved in iron transport in amanner analogous to a Fur' strain which is under ironlimitation (3, 9). Only the Fur- strain exhibited a decreasedrate of 55Fe-enterochelin transport in cells grown at 42°C,suggesting that in cells grown under conditions of iron stress,ferric enterochelin transport is affected by growth tempera-ture (Fig. 1). To test this hypothesis, we carried out similartransport assays but used 2 ,M EDDA in the growthmedium to decrease the amount of available iron. Thedifference in initial transport rates of cells grown at 42 versus37°C observed in the fur strain RB338 was also found in E.coli MC4100 and S. typhimurium G30 when the organismswere grown in medium treated to remove much of the freeiron (Fig. 2).

Effect of growth temperature on the colicin I receptor.Although the function of the colicin I receptor in ironmetabolism is unknown, the production of this protein iscoordinately regulated with the biosynthesis of enterochelinand the ferric enterochelin receptor (20, 25). Therefore, weexamined the amount of this receptor in cells grown at 37and 42°C. In S. typhimurium, we found that the level ofcolicin I receptors is relatively low compared with that of E.coli K-12, unless the cells are grown in medium which has

120

a\C10

O \

~0

6

0

0

30 32 34 36 38 40 42Growth Temperature (IC )

FIG. 3. Effect of growth temperature on the level of colicin Ireceptors in strains PW734 and JK1. Overnight cultures grown inM63 at 33°C were diluted to a turbidity at 600 nm of 0.3. and thendiluted 150-fold further into M63 medium which had beenprewarmed to the desired temperature. After eight generations ofgrowth at the appropriate temperature, the cells were harvested andassayed for binding of 125I-labeled colicin Ia as described in the text.The values shown represent the amount of colicin bound at satura-tion. Symbols: (0), S. typhimurium PW734; (-), E. coli JK1.

TABLE 3. cir promoter activity in cells grown at 37 and 42°C

Strain Growth P-GalactosidaseStrain ~~~temp ('C)a (U)a

PW401 4(cir-lac) 37 33142 180

JK1 cir+ lac+ 37 1,10042 1,186

a Growth of cells and determination of P-galactosidase are de-scribed in the text.

been treated to reduce the concentration of iron. However,due to decreased synthesis of enterochelin at elevated tem-peratures, S. typhimurium does not grow rapidly at 42°Cunder conditions of iron limitation. To minimize the differ-ence in growth rate between cultures incubated at 37 and at42°C, we used strain PW734, afur strain of S. typhimuriumfor this experiment, and grew the organism in M63 mediumwhich had not been treated to lower the concentration ofiron. This strain produces high levels of the receptor even ingrowth medium that is not depleted of iron (9). The effect ofgrowth temperature on the number of colicin I receptors percell is shown (Fig. 3).

In both E. coli and S. typhimurium, there was a two- tothreefold decrease in binding of colicin Ta to cells grown at42°C as compared with binding to cells grown at 37°C. Acorresponding decrease in sensitivity to the colicin wasobserved (data not shown). The decrease in receptor level inthe Fur- strain RB338 indicates that the effect of growthtemperature on the number of colicin I receptors, like that onenterochelin synthesis and transport, is independent of thefur gene product. It is important to point out that sincegrowth rates at 37 and at 42°C were similar, the differences inreceptor levels observed in cultures grown at the twotemperatures are not growth rate dependent.To determine whether the receptor was thermolabile, cells

of either E. coli or S. typhimurium grown at 37°C wereshifted to 42°C in the presence of chloramphenicol, so as toinhibit protein synthesis and cell growth, and then assayedfor receptor. We did not observe a significant decrease in theamount of receptor activity in either strain (data not shown).Thus, the colicin receptor is not inherently unstable at 42°C.We have previously described operon fusions of cir, in

which expression of ,-galactosidase is controlled by the cirpromoter (33). The Mu phage used in the construction of cir-lac fusion E. coli PW400 has a temperature-sensitive repres-sor, thus the strain is not stable at temperatures greater than33°C (5, 33). To observe temperature effects on the regula-tion of cir in E. coli K-12 PW400, we introduced plasmidpJB4J1, which carries a Mu phage with a wild-type repressor(4). E. coli PW401, a derivative of PW400 which carries thisplasmid, is thermostable, as evidenced by the fact that aculture of PW401 grown at 30°C, diluted, and plated on LBproduced the same number of colonies on LB plates incubat-ed at 30 or 42°C. Furthermore, the generation time of thisstrain at 42°C is identical to that of the parental strainMC4100. The effect of growth temperature on ,-galacto-sidase levels in strain PW401 is shown in Table 3. Whenincubated at 42°C, PW401 exhibited approximately one-halfthe amount of ,-galactosidase activity that was observed inthe culture incubated at 33 or 37°C. P-Galactosidase levels inthe control cir+ lac+ E. coli JK1 did not respond to growthtemperature under these conditions. This experiment sug-

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TEMPERATURE REGULATION OF IRON ACQUISITION 167

gests that the influence of growth temperature on receptorproduction is at least partially mediated at the level oftranscription.

DISCUSSIONAlthough we were able to reproduce the finding of Gari-

baldi (12) in regard to diminished enterochelin production byS. typhimurium at elevated temperatures, we did not ob-serve this phenomenon in E. coli K-12 or in several clinicalisolates of this species. Our data conflict in this regard with aprevious report (18), which purported to demonstrate re-duced enterochelin synthesis by an E. coli strain at elevatedtemperatures. However, interpretation of these publishedexperiments is subject to some ambiguity since productionof enterochelin was not evaluated by biochemical means.We cannot rule out the possibility that some strains of E. colido exhibit temperature-sensitive production of enterochelin.Growth at 42°C of E. coli or S. typhimurium under a

condition of iron stress also led to a decrease in the initialrate of ferric enterochelin transport. The same phenomenonwas observed in a fur mutant of S. typhimurium grown inmedium not depleted of iron. We suggest, therefore, that thegene product(s) which determines the rate of ferric entero-chelin transport in cells under iron stress is affected bygrowth at elevated temperatures. Whether this is due to aneffect of growth temperature on the activity or level of acomponent of the ferric enterochelin transport system re-mains to be established. Whatever the cause, it is unrelatedto growth rate, since nearly identical growth rates wereobserved for E. coli grown at 37 and at 42°C, as well as forthe S. typhimurium fur mutant. It is interesting that therewas no difference in initial transport rates of cells grown at37 and at 42°C when the cells were grown in medium notdepleted of iron. It is possible that the element whichdetermines the rate of ferric enterochelin transport in cellsgrown under iron limitation is distinct from that whichdetermines the transport rate in cells which are not grownunder iron stress.Growth temperature also affected the level of colicin I

receptors. When grown at 42°C, cells of both E. coli K-12and S. typhimurium LT2 had one-half to one-third thenumber of receptors observed in cells grown at 37°C. Thisdecrease was not the result of thermal lability of the receptorat the higher temperature. Although modulation of 3-galac-tosidase levels in strains carrying cir-lac operon fusions maynot accurately reflect all aspects of the regulation of cir, thedecrease in galactosidase synthesis in such a strain grown at42°C suggests that at least part of the decrease in receptorlevel is due to reduced transcription at this temperature.However, the possibility that a posttranscriptional regula-tory mechanism may play a part in this phenomenon is notexcluded.

Since the regulatory elementfur, which appears to controlthe transcription of these genes in response to levels of iron(16), affects the same systems which are subject to regulationby temperature, it was reasonable to entertain the possibilitythat the temperature effect is mediated by the fur geneproduct. However, the temperature regulation observed in afur strain of S. typhimurium, which constitutively producesenterochelin, the transport system for ferric enterochelin,and the colicin Ia receptor, rules this out.

It is tempting to speculate, therefore, that a secondregulatory factor common to gene products involved in ironsequestration is affected at elevated temperatures. The ob-servation that many gram-negative bacteria are unable to

sequester iron efficiently when grown at higher temperaturessuggests that such an element may be widespread and highlyconserved.

It has been suggested that fever may act as a host defensemechanism by depriving invading organisms of the ironnecessary for growth (31, 32). Since the ability to acquireiron does appear to be a virulence factor in some bacteria (7,8, 10, 27, 28, 34), it would be interesting to examine theefficiencies of iron sequestration systems at elevated tem-peratures in a variety of pathogens. An organism which iscapable of efficient production and transport of siderophoresat febrile temperatures is likely to be a more successfulpathogen.The reduced initial rate of ferric enterochelin uptake in E.

coli does not appear to inhibit growth at 42°C. We do have E.coli strains in our collection that do not grow well at 42°C.However, growth of these strains at this temperature is notstimulated by the addition of ferric citrate or ferric chlorideto the medium and appears to be unrelated to acquisition ofiron. In contrast, the decrease in enterochelin productionexhibited by S. typhimurium does lead to an inhibition ofgrowth at elevated temperatures under conditions of ironlimitation. The effect that a two- to threefold decrease inenterochelin synthesis, transport of ferric enterochelin, andlevels of the colicin Ia receptor has on growth of theorganism in vivo is uncertain. Enterochelin appears to beessential for the rapid growth of E. coli and S. typhimuriumin vivo (28, 34). Furthermore, pathogenic strains of E. coligrowing in vivo produce high levels of two outer membraneproteins: an 81,000-dalton protein, likely to be the ferricenterochelin receptor, and a 74,000-dalton protein, whichmay correspond to the colicin I receptor (14). These datasuggest that these outer membrane proteins may play a rolein the adaption of the organism to growth in vivo. Thus, theinhibition of enterochelin synthesis and transport and thedecrease in levels of the colicin I receptor at febrile tempera-tures could conceivably prove detrimental to the in vivogrowth of both E. coli and S. typhimurium.

ACKNOWLEDGMENTSThis work was supported by Public Health Service grant AI-10106

from the National Institutes of Health. P.L.W. is a recipient ofNational Institutes of Health predoctoral traineeship grant GM 7283.We thank L. Lembke for the clinical isolates of E. coli used in this

study.

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