INFECTION AND IMMUNITY, June 1994, p. 2309-2314 Vol. 62, No. 60019-9567/94/$04.00 + 0Copyright © 1994, American Society for Microbiology

Staphylococcal Iron Requirements, Siderophore Production, andIron-Regulated Protein Expression

JODI A. LINDSAY,'* AND THOMAS V. RILEY' 2

Department of Microbiology, University of Western Alustralia, 1 and Department of Clinical Microbiology, Sir CharlesGairdner Hospital,- Queen Elizabeth II Medical Centre, Nedlands 6009, Westem Australia, Australia

Received 3 December 1993/Returned for modification 3 February 1994/Accepted 5 March 1994

Despite the ability of staphylococci to grow in iron-restricted conditions in vivo, their iron requirements andthe mechanisms possessed by them for the uptake of iron are poorly understood. Many bacteria are known toproduce siderophores. By using the chrome azurol S universal method for the detection of siderophores, all 14isolates of Staphylococcus aureus tested grew well under conditions of iron restriction and producediron-regulated siderophore in large quantities, while all 19 isolates of coagulase-negative staphylococci (CoNS)grew poorly under conditions of iron restriction and produced low levels of iron chelator. Sodium dodecylsulfate-polyacrylamide gel electrophoresis profiles of S. aureus isolates revealed altered protein patterns due toiron restriction, while altered profiles were not seen in the CoNS group. The ability to grow in iron-restrictedconditions, possibly with the assistance of siderophore-mediated iron uptake, may contribute to the increasedpathogenicity of S. aureus when compared with that of the CoNS.

Iron is required for the growth of virtually all living cells,primarily as a cofactor or prosthetic group for essential enzy-matic reactions. The concentration of free iron in living tissuesis estimated to be 10-12L,M, far lower than that required forbacterial growth (2). Therefore, pathogenic bacteria must beable to overcome this nutritional deficiency to establish aninfection. Mechanisms for obtaining iron from the environ-ment have been described for many microorganisms (16). Themost widespread mechanism involves the excretion of a low-molecular-weight compound with a very high affinity for iron(siderophore) in conditions of iron restriction and the coregu-lated expression of a receptor protein(s) on the cell surfacewhich recognizes the siderophore-iron complex. Although suchsystems have been well characterized phenotypically and ge-notypically in gram-negative bacteria, and siderophore produc-tion has been detected in fungi (5, 22), relatively little is knownabout gram-positive bacteria, including staphylococci.

Staphylococcus aureus is a virulent pathogen responsible fora range of community- and hospital-acquired infections. Thecoagulase-negative staphylococci (CoNS) are considered lessvirulent than S. aureus, mostly infecting patients compromisedin some way, particularly those with prosthetic devices (25).While S. aureuls produces a range of toxins, hemolysins, andother factors which are considered to contribute to theirvirulence (9), similar virulence factors have not been describedfor the CoNS. The production of slime by CoNS has beenpostulated to be important (25), but there is increasing evi-dence to suggest that other factors are involved (24). Toproduce virulence factors in vivo, staphylococci must be able toobtain nutritional iron to survive.

Schade (26) demonstrated that S. aurelus was able to grow inhuman serum in vitro while Staphylococcus albus (CoNS) couldnot and that iron supplementation stimulated the growth ofboth species. Marcelis et al. (15) and Maskell (17) obtainedindirect evidence for a siderophore produced by staphylococci;however, their methods used whole bacterial cells to stimulate

* Corresponding author. Mailing address: Department of Microbi-ology, University of Western Australia, QEII Medical Centre, Ned-lands, 6009 Western Australia, Australia. Fax: 61 9 389 2912.

the growth of iron-depleted strains and did not negate otherpossible growth-stimulating factors. Two siderophores, staphy-loferrin A and staphyloferrin B produced by Staphy,lococcushyicus DSM 20459, have been isolated (8, 14, 18). S. hyicus isa veterinary pathogen that is coagulase variable. StaphyloferrinA (detected by using ion-pair high-performance liquid chro-matography) was produced by many isolates of staphylococci,and its production was enhanced 19-fold by the addition of theprecursor D-ornithine and 7-fold by the addition of L-ornithine,while staphyloferrin B production by S. hyicus DSM 20459 wasenhanced up to 2.5-fold by the addition of the precursor2,3-diaminopropionic acid. Siderophore production by S. ati-relus has been detected (4) by using the chrome azurol S (CAS)assay of Schwyn and Neilands (27). This assay detects chelatorsby their ability to extract iron bound to a relatively weakchelator, CAS, rather than detecting a specific chemical struc-ture and is thus considered to be a universal assay. Detectionof the production of siderophore by CoNS by the CAS assayhas not been previously investigated.The uptake of iron bound to siderophore requires the

expression of a specific receptor, which is usually iron regu-lated (16). CoNS isolates grown in human peritoneal dialysisfluid produce iron-regulated membrane proteins that are im-munoreactive and relatively conserved (28, 29). Staphylococcuisepidermidis iron-regulated proteins were expressed in vivo in arat model (19), although their function is unknown. In con-trast, there is conflicting evidence on the production of iron-regulated proteins by S. aureus (6, 7, 10, 28) which may berelated to the different media employed in each study.Our aims were to determine (i) how much iron was required

by S. aureus and the CoNS for growth, (ii) if they producedsiderophore under conditions of iron restriction, and (iii) ifthey produced iron-regulated proteins that corresponded withsiderophore production. To do this, we developed a mediumsuitable for the growth of staphylococci that was low in ironand compatible with the CAS assay.

(This work was presented in part at the Australian Societyfor Microbiology Annual Scientific Meeting, Sydney, Australia,12 to 17 July 1992, and at the Third Western Pacific Congresson Chemotherapy and Infectious Diseases, Bali, Indonesia, 6to 9 December 1992.)

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2310 LINDSAY AND RILEY

MATERIALS AND METHODS

Organisms. Twelve strains of S. aureus isolated from bloodcultures and 18 strains of CoNS isolated from peritonealdialysis fluids, taken from patients at Sir Charles GairdnerHospital, Perth, Western Australia, were studied. For compar-ison, the reference strains S. aureus NCTC 8531 (ATCC 12599)and NCTC 8532 (ATCC 12600) and S. epidernidis ATCC14990 were included. All clinical isolates were identified byusing the methods of Kloos (12) and Kloos and Lambe (13). Ofthe clinical isolates of CoNS, 15 were identified as S. epider-midis, one was identified as Staphylococcus haemolyticus, onewas identified as Staphylococcus wameri, and one was identi-fied as Staphylococcus caprae. Organisms were stored in 2%brain heart infusion broth with 20% glycerol added, at -70°C,and working cultures were maintained on horse blood agar at40C.

Preparation of growth media. A minimal medium, low inphosphates and citrate as recommended by Schwyn and Nei-lands (27), containing the following was prepared: 2 mMKH2PO4, 7.9 mM NaCl, 17.2 mM NH4C1, 2% (vol/vol) 1.5 MTris-HCl (pH 8.8) solution, 20 mM glucose, 6 g of CasaminoAcids (Difco) per liter, 39 pKM tryptophan, 32 ,uM nicotinicacid, and 6 KM thiamine-HCl. The final pH of the medium was8.2. Staphylococcal siderophore detection (SSD) medium wasminimal medium supplemented with 50 ,uM MgCl2 * 6H20.Iron-depleted (SSD-OFe) medium was prepared by treatingthe minimal medium with 10 g of Chelex 100 (Bio-Rad) perliter and stirring for 1 h at 25°C to remove divalent andtrivalent metal ions. The resultant medium was sterilized byfiltration, which also removed the resin, and supplementedwith 50 ,uM MgCl2 - 6H20. The iron concentration of SSDmedium was 0.27 ,uM as measured by an ARL 3520 sequentialscanning inductively coupled plasma optical emission spec-trometer with an emission line of 259.938 nm. The ironconcentration of SSD-OFe medium was below the limit ofdetection of the method (<0.05 ,uM). In some experiments,SSD-OFe medium was supplemented with different amounts ofFeCl3 * 6H20 to produce final iron concentrations of 0.01 to 10,uM (SSD-0.OlFe to SSD-lOFe). In other experiments, SSD-OFe or SSD-0.5Fe medium was supplemented with 1 mMD-ornithine, 1 mM L-ornithine, 3 mM L-2,3-diaminopropionicacid, or 5 mM DL-2,3-diaminopropionic acid. In another exper-iment, Casamino Acids were replaced with 19 amino acids(excluding ornithine) in quantities described by Meiwes et al.(18). Distilled water and disposable plastic were used through-out.Growth conditions. Colonies from overnight blood agar

plate cultures were used to inoculate SSD-2Fe medium andincubated for 24 h at 37°C with shaking. This fresh culture wasused as the inoculum for subsequent experiments.Tubes containing 3 ml of SSD-OFe medium supplemented

with different concentrations of iron were seeded with 30 ,ul ofthe inoculum and incubated at 37°C, with shaking, for 40 h.The A620 was used as an indicator of growth and measured ina 96-well microtiter tray with a Titertek Multiskan MCC/340Mk II spectrophotometer.

Siderophore detection. CAS shuttle solution was preparedby the method of Schwyn and Neilands (27) and stored in darkplastic bottles. Culture supernatants (100 IlI) and dilutionsthereof were assayed for siderophore production by mixingthem with equal quantities of CAS solution in a 96-wellmicrotiter tray and measuring the A620 after 1 h. A colorchange from blue to orange indicated siderophore production.Controls consisted of sterile medium with different concentra-tions of iron. All data have been expressed as the average test

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FIG. 1. Comparison of the yields of cells of 14 isolates of S. aureusand 19 isolates of CoNS after 40 h of growth in SSD-OFe medium.Number of cells per milliliter (107) is indicated on the y axis.

absorbance subtracted from the control absorbance (A, -Aav t) and compared with a standard curve. Standard curves fordifferent concentrations of iron were constructed by using theiron chelator desferrioxamine mesylate (Desferal; Ciba-Geigy). All growth and siderophore detection experimentswere repeated at least three times.

In some experiments, the supernatant was treated for 2 h at37°C with 100 ,ug of proteinase K (Boehringer Mannheim) perml, boiled for 5 min, or treated with concentrated HCl orNaOH to achieve a pH of approximately 2 or 9 for 2 h beforebeing readjusted to pH 5.5. The supernatant was then testedfor chelating activity.

Citrate concentration was detected by using a citrate lyase-malate dehydrogenase method, where the conversion ofNADH to NAD+ is measured at 340 nm (3).Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) analysis. Bacterial cells from 1 ml of broth werewashed in 1 ml and resuspended in 100 [lI of saline (0.8%).After the addition of 30 ,lI of a 1-mg/ml concentration oflysostaphin (Sigma), the cells were incubated at 37°C, withoccasional shaking, for 2 h. This suspension was centrifuged for1 min at 12,000 x g, and 100 RI of the supernatant was mixedwith 0.75 ml of acetone to precipitate proteins which were thenpelleted by centrifugation. Protein preparations and low-mo-lecular-weight standards (Bio-Rad) were mixed with samplebuffer, boiled for 2 min, and electrophoresed on 4% stacking gelsover 12% separating gels with a discontinuous buffer solution in aBio-Rad mini-Protean II system. Protein bands were stained withCoomassie brilliant blue G-250 by the method of Neuhoff et al.(23), without fixation, and destained in water.

RESULTS

Growth of staphylococci in iron-restricted medium. All 14isolates of S. aureus grew well in SSD-OFe medium, reaching afinal yield of >5 x 107 cells per ml after 40 h of incubation(Fig. 1). The yield was relatively unaffected by the addition ofiron, as illustrated by the results with S. aureus NCTC 8531shown in Fig. 2a, or by the addition of D-ornithine, L-ornithine,L-2,3-diaminopropionic acid, or DL-2,3-diaminopropionic acid(data not shown).

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STAPHYLOCOCCAL RESPONSE TO IRON RESTRICTION 2311

In contrast, all 19 isolates of CoNS grew poorly in SSD-OFemedium, reaching a final yield of <2.5 x 107 cells per ml (Fig.1), significantly less than the S. alureus isolates (unpairedStudent's t test, P < 0.001). As increasing amounts of iron weread`- '̀o SSD medium, the yield of the CoNS increased, asillust-.ted by the results with S. epidermidis ATCC 14990shown in Fig. 2b. All CoNS isolates grew well in SSD-lOFemedium (yield, 4 x 107 to 1.8 x 108 cells per ml), indicatingthat growth was not restricted because of the absence ofanother growth requirement. Growth of the CoNS was notaffected by the addition of D-ornithine, L-ornithine, L-2,3-diaminopropionic acid, or DL-2,3-diaminopropionic acid (datanot shown).

Production of iron chelators by S. aureus. The supernatantsof all 14 isolates of S. aureus grown in SSD-OFe mediumreacted in the CAS assay (average activity, 70.3 + 8.31 pMdesferrioxamine equivalents; range, 21 to 126 ,uM desferriox-amine equivalents). As increasing amounts of iron were addedto SSD-OFe medium, the chelating activity decreased, asillustrated by the results with S. aurelus NCTC 8531 shown inFig. 2a. Activity was detected in cultures once they reached thelate log phase (approximately 10 h), and maximal levels wereattained in the stationary phase (after 16 h). No change inchelating activity was seen in any S. aiureus isolate when 1 mMD-ornithine, 1 mM L-ornithine, 3 mM L-2,3-diaminopropionicacid, or 5 mM DL-2,3-diaminopropionic acid was added toSSD-OFe medium or when the ornithine concentration of themedium was reduced (data not shown).

Only supernatant from cultures of S. aureus NCTC 8531 andS. aureuis NCTC 8532 and not washed cells reacted in the CASassay. Supernatant activity was not affected by treatment withproteinase K, acid, or base or by boiling. Citrate was notdetected in the supernatant. The detection limit of the citrateassay was approximately 10 F.M, but this concentration ofcitrate barely reacted in the CAS assay.

Production of iron chelators by CoNS. Since isolates ofCoNS grew poorly in SSD-OFe medium, they were cultured inSSD-0.5Fe medium to determine the effect of iron restriction.At the iron concentration of this medium (i.e., 0.5 FtM), moststrains of CoNS yielded >4 x 107 cells per ml, although theiryield in SSD-lOFe medium was approximately twofold higher.

In SSD-0.5Fe medium, only two isolates, S. caprae 5242 andS. epidermidis 5488, produced levels of chelating activity ap-proaching that of the S. aureus isolates (18.78 + 0.79 and 11.92 ±0.77 ,uM desferrioxamine equivalents, respectively). The CASactivity of the remaining isolates ranged from <2.0 to 4.15 F.Mdesferrioxamine equivalents. The chelating activity was regulatedby iron in S. caprae 5242 and S. epidermidis 5488 only.The addition of 1 mM D- or L-ornithine enhanced the

chelating ability of 16 of the 19 isolates. The three isolateswhich did not respond to the ornithine included the S. warneriand S. haernolyticius isolates. In 8 of the 16 instances, L-ornithine enhanced chelating ability more than D-ornithine.Therefore, although the activity of two isolates was enhanced7-fold by the presence of L-ornithine, the enhancement inresponse to 1)-ornithine was much lower than 19-fold (i.e.,approximately 6-fold). The enhancement of chelating abilitydue to the addition of ornithine isomers among the remainingisolates ranged from 1.2- to 6.0-fold.

All CoNS isolates, except S. caprae 5242 and S. epidermidis5488, showed enhanced chelating ability in the presence of 3mM L-2,3-diaminopropionic acid or 5 mM DL-2,3-diaminopro-pionic acid. This enhancement correlated with the amount ofchelating ability in SSD-0.5Fe medium; i.e., those isolates thatproduced the lowest levels of chelating ability showed thegreatest levels of enhancement (up to 6.4-fold).

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FIG. 2. Yields (U; measured by A,20) after 40 h of growth inSSD-OFe medium supplemented with different concentrations of ironof S. aureus NCTC 8531 (a), shown with chelating activity (0l;measured by the CAS assay and expressed in desferrioxamine mesylateequivalents), and S. epidermidis ATCC 14990 (b).

Citrate was detected in the culture supernatants of fiveisolates, namely, S. caprae 5242, S. wameri 5462, S. epidermidis5488, S. epidermidis 5653, and S. epidermidis 5683; however, thelevels detected were low and accounted for 5.2 to 26.4% of thechelating activity seen when the CAS assay was used.The chelating activity of five of the CAS assay-positive

isolates was confined to the supernatant and was not affectedby treatment with proteinase K, acid, or base or boiling.SDS-PAGE protein profiles. All 14 isolates of S. aureus

expressed at least one iron-repressible protein visible onCoomassie blue-stained SDS-polyacrylamide gels in the 36- to39-kDa region, and several isolates also expressed iron-regu-lated proteins with higher molecular masses (Fig. 3a). Occa-

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a

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1 2 3 4 5 6FIG. 3. Coomassie blue-stained SDS-polyacrylamide gels of whole-

cell extracts of three isolates of S. aureus (a) and three isolates of CoNS(b). (a) Lanes: 1, 3, and 5, isolates grown in SSD-OFe medium; 2,4, and6, isolates grown in SSD-lOFe medium; I and 2, S. aureus 6149; 3 and4, S. aureus 6169; 5 and 6, S. aureus 6181. (b) Lanes: 1, 3, and 5, isolatesgrown in SSD-0.SFe medium; 2, 4, and 6 isolates grown in SSD-lOFemedium; 1 and 2, S. epidernidis 5244; 3 and 4, S. epidermidis 5258; 5and 6, S. wameri 5462. Arrowheads in panel a indicate iron-regulatedproteins with molecular masses of 36 to 39 kDa. Molecular mass

standards are indicated on the left of each profile and correspond indescending order to 97.4, 66.2, 45, 31, and 21.5 kDa.

sionally, these proteins were weakly expressed and difficult todetect. No CoNS tested, including S. caprae 5242 and S.epidermidis 5488, produced detectable iron-regulated proteinson Coomassie blue-stained SDS-polyacrylamide gels (Fig. 3b).

DISCUSSION

Little is known about the response of staphylococci toiron-restricted conditions, prompting our investigation. S. au-

reus had a very low requirement for iron, illustrated by its high

growth yield in SSD-OFe medium. These results are consistentwith those of Courcol et al. (4) who also grew S. aureus in aniron-restricted defined medium. The iron concentration ofSSD-OFe medium is low enough to inhibit the growth of CoNS,suggesting that S. aureus has an intrinsically low requirementfor iron or that an active uptake mechanism is in operation.

Since the CoNS did not grow well in SSD-OFe medium, theypresumably lack or have an inefficient mechanism for ironuptake. S. albus (CoNS) grows poorly in serum in vitro (26),which suggests that any mechanism operating in vivo is notparticularly effective. This may contribute to the delayed onsetof infection reported for CoNS (11), which is unlike the oftenrapid progression of S. aureus infections. In both serum (26)and SSD-OFe medium, the addition of iron restored the growthof CoNS, and this appeared to be their only growth-limitingfactor.

All S. aureus isolates tested produced iron chelator that wasiron regulated, results similar to those obtained by Courcol etal. (4). In the present study, however, there was only a 1-logdifference in the amount of chelator between isolates, whileCourcol et al. (4) saw a greater than 2-log difference betweensome isolates. Courcol et al. (4) also noted that referencestrains produced less siderophore than clinical isolates andsuggested that this may be related to virulence. In our study,although S. aureus NCTC 8532 produced the lowest amount ofchelator of the 14 isolates tested, S. aureus NCTC 8531 was astrong producer. In addition, some clinical isolates producedlevels of chelator only marginally higher than that produced byS. aureus NCTC 8532.The chelator produced by S. aureus is not citrate and is not

affected by treatment with proteinase K, acid, or base or byboiling. Production was not enhanced in the presence ofD-ornithine, L-ornithine, L-2,3-diaminopropionic acid, or DL-2,3-diaminopropionic acid although one of the strains tested(S. aureus NCTC 8532 [ATCC 12600]) was identified as astrong staphyloferrin A producer by Meiwes et al. (18). It ispossible that production of staphyloferrin A (and staphylo-ferrin B) by S. aureus is lower when grown in SSD-OFe mediumthan when grown in the minimal medium employed by Meiweset al. (18). It is also likely that S. aureus is producing one ormore siderophores at levels higher than those of staphyloferrinA or staphyloferrin B, masking any enhancement caused byprecursor feeding and accounting for the high levels of chelat-ing ability detected.The CoNS produced much lower levels of chelator. Their

lack of growth in SSD-OFe medium supports the theory thatthere was no active iron uptake occurring in this medium.CoNS isolates that produced relatively high levels of chelatingactivity grew no better in SSD-OFe medium, suggesting that thechelators detected were not successfully transporting iron intothe cells.The production of chelator by 16 of the 19 CoNS isolates

was enhanced by the presence of D- or L-ornithine, whichsuggests the production of staphyloferrin A. However, the levelof enhancement was not as high nor did it exhibit the sameisomer pattern as that reported by Meiwes et al. (18) andKonetschny-Rapp et al. (14) (19-fold for D-ornithine and 7-foldfor L-ornithine). This suggests that staphyloferrin A productionby staphylococci is not enhanced in the same manner inSSD-0.5Fe medium as it is when the cells are grown in theminimal medium of Meiwes et al. (18). However, since orni-thine was able to cause an increase in chelating activity,low-level production of this siderophore may be presumed.

Chelating activity of most CoNS isolates (particularly thosethat normally produced low levels of activity) could be stimu-lated by the addition of 2,3-diaminopropionic acid to SSD-

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0.5Fe medium. This suggests that the low levels of chelatingactivity produced by some isolates of CoNS are due to staphy-loferrin B. Again, the response of staphylococci to this precur-sor when grown in SSD-0.5Fe medium was different from thatof Drechsel et al. (8) in that activity was enhanced more than 2.5-fold.The data suggest that most CoNS produce low levels of

staphyloferrin A, staphyloferrin B, citrate and/or other un-known iron chelators. They must be constitutively produced,since these chelators are not regulated by iron concentration,and they appear to have limited ability to transport iron intothe cells. Some isolates, such as S. caprae 5242 and S. epider-midis 5488, produce higher levels of iron-regulated chelator.However, since these isolates grow just as weakly in SSD-OFemedium as the other isolates of CoNS tested, it seems unlikelythat the chelators detected are efficient transporters of ironinto the cells.The uptake of iron bound to a siderophore requires the

expression of a specific receptor on the bacterial cell surface(16). Production of both receptor proteins and siderophoresare usually regulated by iron concentration (5). SDS-PAGEprotein profiles of all 14 S. aureus isolates grown in SSD-OFemedium showed iron-repressible proteins in the region of 36 to39 kDa, which correlated with the presence of siderophoreproduction and suggests that these proteins may have a role iniron uptake. This contrasts with the results of Kadurugamuwaet al. (10), who did not detect altered SDS-PAGE proteinprofiles when S. aureus was grown in iron-restricted nutrientbroth. Domingue et al. (6, 7) detected iron-inducible but notiron-repressible protein antigens when S. aureus was grown iniron-depleted Trypticase soy broth. Smith et al. (28) detectedan iron-repressible antigen at 32 kDa produced by two isolatesof S. aureus that cross-reacted with antisera raised against a32-kDa iron-repressible membrane protein of S. epidernmidis901 that is known to be expressed in vivo (19). Unfortunately,the production of siderophore by S. aureus in each of thesemedia was not determined nor has the function of any iron-repressible proteins been elucidated at this stage.

Isolates of CoNS did not produce iron-regulated proteinswhen grown in SDS-0.5Fe medium. This includes the S. caprae5242 and S. epidermidis 5488 isolates which produced iron-regulated chelating activity. This is unexpected, since sid-erophore biosynthetic and transport systems are usually coor-dinately expressed and regulated by iron. Proteins ofapproximately the same size as that of the iron-regulatedproteins of S. aureus are seen in the CoNS profiles, so it ispossible that these are the same proteins but that they areconstitutively produced in CoNS. However, since no isolate ofCoNS grew well in SSD-OFe medium, it is unlikely that anyprotein expressed by these isolates is functioning in transport-ing iron into the cells. Smith et al. (28) and Wilcox et al. (29)have shown that isolates of CoNS can produce iron-regulatedantigens when grown in an in vivo rat model (19) or in pooledhuman peritoneal dialysis fluid. Although it has been specu-lated that these proteins are siderophore receptors (29), theirfunction is presently unknown.The different responses seen by the two groups of staphylo-

cocci may account, in part, for their different levels of patho-genicity. Although the CoNS did not appear to produce anefficient iron uptake system in this model, they are able tosurvive and cause infections in vivo. It is likely, therefore, thatthey possess a siderophore system not detected in theseexperiments or alternative uptake mechanisms. S. aureus mayalso possess other mechanisms, some of which may be moreefficient in vivo than the one described. For example, Neisseriameningitidis and Haemophilus influenzae are able to producereceptors for host iron-binding proteins (1, 16). Two recent

studies have detected a lactoferrin-binding protein expressedby S. aureus (20, 21), while the results of Schade (26) and Marceliset al. (15) indicate that S. aureus and possibly S. epidennidis canaccess iron bound to transferrin. Many staphylococci producehemolysins, which may also have an important role in the releaseof iron (16), albeit bound to hemoglobin. It is not known if thestaphylococci produce bacterioferritin, an iron storage molecule(1). These altemative mechanisms need to be investigated furtherbefore we can begin to understand how these complex andheterogeneous bacteria obtain essential iron.

Finally, these experiments were performed in an artificialmedium in vitro and represent conditions quite different fromthose found in vivo. However, it is important to understand thepotential of staphylococci to respond to iron-stressed condi-tions as it may reflect their ability to survive and causeinfections in a similar environment within the body.

REFERENCES1. Briat, J.-F. 1992. Iron assimilation and storage in prokaryotes. J.

Gen. Microbiol. 138:2475-2483.2. Bullen, J. J., H. J. Rogers, and E. Grifliths. 1978. Role of iron in

bacterial infection. Curr. Top. Microbiol. Immunol. 80:1-35.3. Carson, K. C., S. Holliday, A. R. Glenn, and M. J. Dilworth. 1992.

Siderophore and organic acid production in root nodule bacteria.Arch. Microbiol. 157:264-271.

4. Courcol, R. J., P. A. Lambert, P. Fournier, G. R. Martin, andM. R. W. Brown. 1991. Effects of iron depletion and sub-inhibitoryconcentrations of antibiotics on siderophore production by Staph-ylococcus aureus. J. Antimicrob. Chemother. 28:663-668.

5. Crosa, J. H. 1989. Genetics and molecular biology of siderophore-mediated iron transport in bacteria. Microbiol. Rev. 53:517-530.

6. Domingue, P. A. G., P. A. Lambert, and M. R. W. Brown. 1989.Iron depletion alters surface-associated properties of Staphylococ-cus auireus and its association to human neutrophils in chemilumi-nescence. FEMS Microbiol. Lett. 59:265-268.

7. Domingue, P. A. G., P. A. Lambert, and M. R. W. Brown. 1991.Immunoblotting and ligand blotting of envelope structures ofStaphlylococcus aureus grown under iron insufficiency and in asub-minimal inhibitory concentration of penicillin G. Microbios67:57-68.

8. Drechsel, H., S. Freund, G. Nicholson, H. Haag, 0. Jung, H.Ziihner, and G. Jung. 1993. Purification and chemical character-ization of staphyloferrin B, a hydrophilic siderophore from staph-ylococci. BioMetals 6:185-192.

9. landolo, J. J. 1990. The genetics of staphylococcal toxins andvirulence factors, p. 399-426. In B. H. Iglewski and V. L. Clark(ed.), The bacteria, vol. 11. Molecular basis of bacterial pathogen-esis. Academic Press, Inc., New York.

10. Kadurugamuwa, J. L., H. Anwar, M. R. W. Brown, G. H. Shand,and K. H. Ward. 1987. Media for study of growth kinetics andenvelope properties of iron-deprived bacteria. J. Clin. Microbiol.25:849-855.

11. Kaebnick, H. W., D. F. Bandyk, T. W. Bergamini, and J. B. Towne.1987. The microbiology of explanted vascular prostheses. Surgery102:756-761.

12. Kloos, W. E. 1990. Systematics and the natural history of staphy-lococci. 1. J. Appl. Bacteriol. Symp. Suppl. 1990:25S-37S.

13. Kloos, W. E., and D. W. Lambe, Jr. 1991. Staphylococculs, p.222-237. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D.Isenberg, and H. J. Shadomy (ed.), Manual of clinical microbiol-ogy, 5th ed. American Society for Microbiology, Washington, D.C.

14. Konetschny-Rapp, S., G. Jung, J. Meiwes, and H. Zahner. 1990.Staphyloferrin A: a structurally new siderophore from staphylo-cocci. Eur. J. Biochem. 191:65-74.

15. Marcelis, J. H., H. J. den Daas-Slagt, and J. A. A. Hoogkamp-Korstanje. 1978. Iron requirement and chelator production ofstaphylococci, Streptococclus faecalis and enterobacteriaceae. An-tonie Leeuwenhoek 44:257-267.

16. Martinez, J. L., A. Delgado-Iribarren, and F. Baquero. 1990.Mechanisms of iron acquisition and bacterial virulence. FEMSMicrobiol. Rev. 75:45-56.

VOL. 62, 1994

on May 9, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

2314 LINDSAY AND RILEY

17. Maskell, J. P. 1980. The functional interchangeability of entero-bacterial and staphylococcal iron chelators. Antonie Leeuwenhoek46:343-351.

18. Meiwes, J., H.-P. Fiedler, H. Haag, H. Zahner, S. Konetschny-Rapp, and G. Jung. 1990. Isolation and characterisation of staphy-loferrin A, a compound with siderophore activity from Staphylo-coccus hyicus DSM 20459. FEMS Microbiol. Lett. 67:201-206.

19. Modun, B., P. Williams, W. J. Pike, A. Cockayne, J. P. Arbuthnott,R. Finch, and S. P. Denyer. 1992. Cell envelope proteins ofStaphylococcus epidermidis grown in vivo in a peritoneal chamberimplant. Infect. Immun. 60:2551-2553.

20. Naidu, A. S., M. Anderson, and A. Forsgren. 1992. Identification ofa human lactoferrin-binding protein in Staphylococcus aureus. J.Med. Microbiol. 36:177-183.

21. Naidu, A. S., J. Miedzobrodzki, J. M. Musser, V. T. Rosdahl, S.-A.Hedstrom, and A. Forsgren. 1991. Human lactoferrin binding inclinical isolates of Staphylococcus aureus. J. Med. Microbiol.34:323-328.

22. Neilands, J. B. 1981. Microbial iron compounds. Annu. Rev.Biochem. 50:715-731.

23. Neuhoff, V., N. Arold, D. Taube, and W. Ehrhardt. 1988. Improvedstaining of proteins in polyacrylamide gels including isoelectricfocusing gels with clear background at nanogram sensitivity using

Coomassie Brilliant Blue G-250 and R-250. Electrophoresis9:255-262.

24. Patrick, C. C., M. R. Plaunt, S. V. Hetherington, and S. M. May.1992. Role of the Staphylococcus epidermidis slime layer in exper-imental tunnel tract infections. Infect. Immun. 60:1363-1367.

25. Pfaller, M. A., and L. A. Herwaldt. 1988. Laboratory, clinical, andepidemiological aspects of coagulase-negative staphylococci. Clin.Microbiol. Rev. 1:281-299.

26. Schade, A. L. 1963. Significance of serum iron for the growth,biological characteristics, and metabolism of Staphylococcus au-

reus. Biochem. Z. 338:140-148.27. Schwyn, B., and J. B. Neilands. 1987. Universal chemical assay for

the detection and determination of siderophores. Anal. Biochem.160:47-56.

28. Smith, D. G. E., M. H. Wilcox, P. Williams, R. G. Finch, and S. P.Denyer. 1991. Characterization of cell envelope proteins of Staph-ylococcus epidermidis cultured in human peritoneal dialysate.Infect. Immun. 59:617-624.

29. Wilcox, M. H., P. Williams, D. G. E. Smith, B. Modun, R. G. Finch,and S. P. Denyer. 1991. Variation in the expression of cellenvelope proteins of coagulase-negative staphylococci culturedunder iron-restricted conditions in human peritoneal dialysate. J.Gen. Microbiol. 137:2561-2570.

INFECT. IMMUN.

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nloaded from