JOURNAL OF CLINICAL MICROBIOLOGY, May 1987, p. 849-8550095-1137/87/050849-07$02.00/0Copyright © 1987, American Society for Microbiology

Media for Study of Growth Kinetics and Envelope Properties ofIron-Deprived Bacteria

JAGATH L. KADURUGAMUWA,t HOSMIN ANWAR,t MICHAEL R. W. BROWN,* GEOFFREY H. SHAND,AND KATHRYN H. WARD

Department ofPharmaceutical Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, United Kingdom

Received 21 August 1986/Accepted 21 January 1987

Ion-exchange chromatography was used to remove iron from complex and chemically defined laboratorymedia. The kinetics of metal cation removal from the media was investigated by using atomic absorptionspectrophotometry, and the results indicated that over 90% of the iron could be eliminated from certaincomplex media by this treatment. The treated medium was used for growth studies in a gram-positive and a

number of gram-negative organisms that were isolated from infections in humans. High-molecular-weightouter membrane proteins that are known to be induced under iron-depleted growth conditions (iron-regulatedmembrane proteins) were observed when a number of gram-negative pathogens were cultivated in the treatedmedia. Iron uptake by Staphylococcus aureus varied, depending on the iron content of the medium.

Nutrient depletion is known to affect envelope structureand related properties of bacteria (8, 9, 11), including sus-

ceptibility both to antibacterial agents (13, 22, 35) and tobody defense mechanisms (2, 14). Bacteria used in labora-tory research have traditionally been cultivated under iron-plentiful conditions in complex or simple salts media. Thereis much evidence that iron is important in infection (10, 15,39). Recently, several investigators have obtained directbiochemical evidence that bacteria grow under iron-restricted conditions in human infections (1, 5, 21, 34) and inexperimentally infected animal models (16, 33). The outermembranes (OMs) of bacteria obtained directly and withoutsubculture from the lungs of patients with cystic fibrosis (5)and urinary tract infections (21, 34) expressed a number ofhigh-molecular-weight proteins that were repressed or

barely detectable in the OMs of bacteria grown in commer-cially available complex laboratory media. OM protein(OMP) profiles closely similar to those observed in vivo wereobtained when the same isolates were cultivated underiron-depleted conditions in vitro (5, 34).

Furthermore, there is evidence that other metal cationssuch as magnesium influence the bacterial surface and re-lated biological properties, including susceptibility to anti-microbial agents (6, 7). The latter property has been associ-ated with changes in the cell envelope and envelope cationcontent (4, 19). Certain OMPs are induced under conditionsof magnesium depletion, such as protein H1 in Pseudomonasaeruginosa (29), and this protein has also been demonstratedin OMs of bacteria obtained in vivo (5).Because of these advances in the knowledge of microbial

physiology in vivo, there is a need to investigate methodsthat can be used to remove metal cations and, in particular,iron from commercially available laboratory media so thatthe properties and behavior of bacteria can be investigatedunder conditions that mimic those in vivo. Methods involved

* Corresponding author.t Present address: Division of Infectious Diseases, CIBA-GEIGY

Ltd., K-125 1.17, Basel CH 4002, Switzerland.t Present address: Vaccine Research and Production Laboratory,

Centre for Applied Microbiology and Research, Public HealthLaboratory Service, Porton Down, Salisbury, Wiltshire SP4 OJG,United Kingdom.

in removing or in restricting available trace metals frommicrobiological culture media are diverse. Waring andWerkman (38) and Donald et al. (12) summarized the prin-ciples that are involved in the elimination of trace metalsfrom growth media. These include the use of spent media,recrystallization, precipitation, absorption, chelation meth-ods with synthetic chelating agents, and employment ofbiological agents as iron scavengers. The use of extraneouschemical or biological agents, however, is undesirable insome metabolic studies of bacteria because they may causedamage to bacterial cell membranes (20). Iron chelators ofbiological origin such as transferrin, which has an associa-tion constant for iron of 1032, have been used in severalstudies (15, 36) and are useful in that they more closelyrepresent conditions in vivo. However, there are practicaldifficulties involved in the use of transferrins, namely, theneed to dialyze out metal and citrate ions before use, andthey are expensive. A synthetic iron chelator, desfer-roxamine (Desferal; CIBA-GEIGY Ltd., Basel, Switzer-land), has been reported to resemble closely iron chelators ofbiological origin and has been used clinically (37).

Ion-exchange resins have been used to remove divalentcations from media (5, 18), but no systematic study of theiruse for culture media has yet appeared. We report here thekinetics of iron removal from laboratory media using anion-exchange resin (Chelex-100; Bio-Rad Laboratories,Watford, England) and using such media in investigations ofthe growth and metabolism of gram-positive and -negativeorganisms that are known to be pathogenic to humans.

MATERIALS AND METHODS

Bacteria. Staphylococcus aureus GH126, Pseudomonasaeruginosa GH133, Escherichia coli GH141, Klebsiellapneumoniae GH158, and Proteus mirabilis GH201 were

isolated from infected material of human origin supplied byStanley Silverman, General Hospital, Birmingham, England.Bacteria were grown in single-strength tryptone soy broth(TSB; Oxoid, Hants, England), nutrient broth (NB; Oxoid),or chemically defined medium (CDM). NB was solidified bythe addition of 1.25% agar (Lab M, Salford, Lancashire,England) for surface growth studies.Treatment of glassware. All glassware was rinsed in tap

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water and fully immersed in 5% (vol/vol) Extran (BDH,Poole, England). Glassware was then soaked overnight in0.01% (wt/vol) EDTA (BDH) before it was rinsed in 1% HCland then six times in double distilled water and dried at 60°C.Glassware was sterilized by dry heat treatment at 160°C for3 h. The cleanliness of the glassware was checked occasion-ally by rinsing with double distilled water and analyzing therinsings for iron.

Preparation and regeneration of resin. Removal of cationswas carried out by passing media through a column (glass; 1by 12 in. [-2.5 by 30 cm]) of resin (Chelex-100; Bio-Rad) inthe sodium form with a flow rate of 2 ml/min per cm2. Thesequence used to prepare and regenerate the column was

passage through two bed volumes of 1 M HCl and a rinsewith five bed volumes of glass double distilled water fol-lowed by passage through two bed volumes of 1 M NaOHand a second rinse with five bed volumes of glass doubledistilled water. Sodium phosphate buffer (0.66 M, pH 7.4)was passed through the column until the pH of the eluatewas 7.4, and the column was finally rinsed with five bedvolumes of glass double distilled water before it was passedthrough the culture medium.

Treatment of culture media in Chelex-100 column. Double-strength TSB and NB were prepared according to theinstructions of the manufacturer in 1-liter batches. To elim-inate variation in the divalent cation content of the complexculture media from different manufacturers and differentbatches, single batches of NB and TSB were used through-out the study. The reconstituted media were treated bypassing them three times through a Chelex-100 column.Treatments 4 and 5 were performed in a regenerated column.The first 100 ml of media that was passed through a regen-erated column was discarded before treated media werecollected in clean glassware. Iron, magnesium, calcium, andzinc levels were determined by using an atomic absorptionspectrophotometer (AAS) after each treatment. All growthexperiments were carried out in single-strength TSB or NBafter they were reconstituted with metal cations (5, 34) to aconcentration equal to that of untreated TSB or NB deter-mined by AAS (Fe + TSB or Fe + NB). Iron depletionstudies were carried out in reconstituted TSB or NB lackingiron (Fe - TSB, Fe - NB).CDM for S. aureus GH126. The medium described by

Miller and Fung (27) with the amino acid compositiondefined in medium 5 of Wu and Bergdoll (40) was used. TheCDM (final concentration) was as follows: monosodiumglutamate, 59 mM; Na2HPO4, 12 mM; NaCl, 69 mM; NH4Cl,9.3 mM; FeSO4, 36 ,uM; MgSO4, 2.8 mM; and KH2PO4, 7.3mM.Amino acids. The following amino acids (Sigma Chemical

Co., St. Louis, Mo.), with the indicated final concentrations(in micrograms per milliliter), were dissolved in doubledistilled water: lysine, 300; histidine, 240; arginine, 360;aspartic acid, 1,200; proline, 1,200; glycine, 1,200; alanine,1,200; valine, 240; methionine, 90; and phenylalanine, 100.The following amino acids, in micrograms per milliliter, weredissolved in 1 M NaOH: threonine, 2,400; serine, 2,400;glutamine, 1,200; and tyrosine, 100. The following aminoacids, in micrograms per milliliter, were dissolved in 1 MHCl: tryptophan, 30; cystine, 120; isoleucine, 300; andleucine, 300.

Vitamins. The following vitamins (Sigma) were used (finalconcentration, in micrograms per 100 ml): nicotinic acid, 50;calcium-d-pantothenate, 50; thiamine, 50; and biotin, 0.3.Each constituent was treated in Chelex-100 resin, except forMgSO4, FeSO4, and the vitamins. KH2PO4 was treated in

Chelex-100 resin in potassium form. The final pH wasadjusted to 7. Iron levels present in CDM after Chelex-100treatment were determined by AAS.

Determination of cation levels by AAS. Samples wereanalyzed by using a flameless AAS (type 360 SG; ThePerkin-Elmer Corp., Norwalk, Conn.) fitted with a deute-rium background corrector and a graphite furnace (HGA-74;Perkin-Elmer). A series of standard solutions of Fe, Zn, Ca,and Mg in glass triple distilled water were made, and acalibration curve was constructed for each metal cation.Calibration curves were constructed separately for eachanalysis. Determinations were performed at least in tripli-cate. Peak heights were averaged, and cation concentrationswere calculated from the calibration curves.Growth measurements. Growth was monitored spectropho-

tometrically in cultures growing in liquid media at 37°C in ashaking water bath. Optical density measurements at 470 nmwere taken at 60-min intervals.OM preparation and SDS-PAGE. Bacteria were harvested

by centrifugation at 5,000 x g for 10 min at 4°C. Thebacterial pellet was suspended in 20 ml of distilled water andbroken by 60-s pulses of sonication 10 times in an ice bath,with a 30-s interval for cooling. Unbroken cells were re-moved by centrifugation at 5,000 x g for 10 min. Sarcosyl(N-lauryl sarcosinate, sodium salt; Sigma) was added to thesupernatant to a final concentration of 20%. The mixture wasincubated for 30 min and then centrifuged at 38,000 x g for1 h. The membrane pellets were washed twice with distilledwater and finally suspended in a small volume of distilledwater and kept at -20°C. Membrane preparations weresubjected to sodium dodecyl sulfate (SDS)-polyacrylamidegel electrophoresis (PAGE) by using the system describedby Lugtenberg et al. (23), as modified by Anwar et al. (3),with 14% acrylamide gels and purified SDS (specially puri-fied; BDH).

Molecular weight estimations. The Mrs of proteins sepa-rated by SDS-PAGE were estimated by comparison withprotein standards of known molecular weight. The proteinstandards used were phosphorylase a (97,400), bovine albu-min (66,000), egg albumin (45,000), pepsin (34,700),trypsinogen (24,000), and lysozyme (14,300) (Sigma).

RESULTS

Removal of metal nations from media with Chelex-100. Theconcentrations of the metal cations Mg, Zn, Ca, and Fe incomplex media (TSB, NB) and of Fe in CDM were measured

TABLE 1. Iron content of the constituents of CDM for S. aureusanalyzed by AAS before and after Chelex-100 treatment

Fe beforetreatment Fe after treatment

Constituentsppm c(AConcn ppm Concnpm (~tM) pm (ptM)

Monosodium glutamate 0.019 0.34 0.005 0.089NaCI 0.002 0.036 0.002 0.036KH2PO4 0.0095 0.17 0.007 0.125MgSO4 7H20 0.002 0.036NH4CI 0.0025 0.045 0.0025 0.045Amino acidsa 0.006 0.107 0.004 0.072Vitaminsa 0.0065 0.012Water 0.0015 0.027Complete CDMa 0.033 0.913 0.0125 0.233

a The compositions of the amino acids, vitamins, and complete CDM aredescribed in the text.

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MEDIA FOR IRON-DEPRIVED BACTERIA 851

before and after Chelex-100 treatment by AAS. In Table 1the iron concentration is indicated as either parts per million(micrograms per milliliter) or micromolarity of each constit-uent of the CDM used in the study before and after treatmentwith Chelex-100. The main source of iron contamination wasfrom monosodium glutamate. Treatment of monosodiumglutamate solution with Chelex-100 reduced the iron contentfourfold. Vitamins and MgSO4 were not passed through theChelex column. When CDM containing ail the ingredients(complete CDM) was passed through the column it wasfound that the iron content was reduced by 74% (Table 1).The removal of Fe from NB and of Fe, Mg, Ca, and Zn

from TSB after treatment with Chelex-100 resin is shown inFig. 1. The first treatment with Chelex-100 removed 73% ofthe iron from NB and 50% of the iron from TSB. Furthertreatments of NB removed only small additional amounts ofiron (5%). A continuous reduction in iron content, however,was observed when TSB was passed through the column;95% was removed by the fifth treatment. Removal of Mg,Zn, and Ca from both complex media was more efficient,with a 99% reduction occurring after the first treatment.

100

75-

!150 \

25

0

1 2 3 4 5

No. of Chelex - 100 TreatmentsFIG. 1. Removal of metal cations from complex culture media

with Chelex-100 resin. Symbols: A, Fe from nutrient broth; A, Fefrom TSB; M, Mg, Zn, and Ca from TSB.

_83-1O0K

_. It-,_ _

hé-U4 5 K45K

1 2 3 4

FIG. 2. SDS-PAGE of the OMPs of two Pseudomonas aerugi-nosa strains after growth in Fe + TSB (lanes 2 and 4) and Fe - TSB(lanes 1 and 3).

Media from which metal cations were removed by thistreatment with Chelex-100 were incapable of supportingbacterial growth unless they were supplemented with Mg. Asimilar observation has been reported by other investigators(17). Prior to use of the treated media in growth studies,metal cations, except for iron, were replaced to the sameconcentrations as those of the untreated media.

Alterations in OMP profile of gram-negative bacteria aftergrowth in low-iron media. The OMP profile of two clinicalisolates of Pseudomonas aeruginosa grown in Fe - TSB(Fig. 2, lanes 1 and 3) and Fe + TSB (Fig. 2, lanes 2 and 4)is shown. A number of high-Mr proteins (83,000 [83Kprotein] to 101K protein) were induced in the cells grownunder iron-depleted- conditions (Fig. 2, lanes 1 and 3) andwere repressed when the cells were grown in media supple-mented with 0.02 M Fe (Fig. 2, lanes 2 and 4). Apart from theinduction of high-Mr proteins, there were no major differ-ences in OMP profile between cells grown under iron-sufficient and iron-depleted conditions.The OMP profiles of clinical isolates of K. pneumoniae

(Fig. 3, lanes 1 to 4), Proteus mirabilis (Fig. 3, lanes 5 to 8),and E. coli (Fig. 3, lanes 9 and 10), all cultivated iniron-sufficient (Fig. 3, lanes 2, 4, 6, 8, and 10) and iron-depleted (Fig. 3, lanes 1, 3, 5, 7, and 9) media are shown. Asbefore, a number of high-Mr proteins were induced in thosecells that were grown under iron-depleted conditions. Theaddition of iron to the growth medium significantly reducedthe intensity of these bands so that they became virtuallyundetectable by Coomassie blue staining.To investigate the effect of surface growth on the OMP

profile, cells were grown on the surface of treated anduntreated TSB that was solidified by the incorporation of1.25% agar. The OMP profile of a clinical isolate of K.pneumoniae grown on Fe - TSB agar (Fig. 4, lane 1) and Fe+ TSB agar (Fig. 4, lane 2) is shown. The cluster of high-Mrproteins (69K to 83K proteins) seen in Fig. 3 (lanes 1 and 3)was again evident in cells grown under conditions of irondepletion (Fig. 4, lane 1) and was repressed in cells grown on

iron-sufficient medium.The 48K protein was strongly ex-

pressed in the OMs of iron-sufficient cells grown on solidTSB but was significantly reduced in the OMs of cells grown

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69-83K-

35 --, =t

18 K ......

TABLE 2. Consumption of iron by S. aureus in NB'

Concn (,uM) of Fe:Condition In medium In supernatant Consumed

Added before cultivation after cultivation by bacteria

Fe- 0.95 + 0.63 0.25 ± 0.06 0.69Fe+ 2.5 4.0 1.09 ± 0.06 2.91Fe+ 36 37.27 8.59 ± 0.50 28.67a Bacteria were grown in batch culture overnight and harvested at the

stationary phase (optical density, 3.0).

1 2 3 4 5 6 7 8 9 10

FIG. 3. SDS-PAGE of the OMPs of K. pneumoniae, Proteusmirabilis, and E. coli. Lanes 1 and 3, K. pneumoniae grown in Fe -TSB; lanes 2 and 4, K. pneumoniae grown in Fe + TSB; lanes 5 and7, Proteus mirabilis grown in Fe - TSB; lanes 6 and 8, Proteusmirabilis grown in Fe + TSB; lane 9, E. coli grown in Fe - TSB;lane 10, E. coli grown in Fe + TSB.

on iron-depleted agar. The difference in expression of thisprotein by cells cultivated in iron-sufficient and iron-depletedliquid media was less marked (Fig. 3, lanes 1 to 4).Growth kinetics of S. aureus in Fe+ and Fe- complex

media and CDM. In gram-positive bacteria there are nomarkers comparable with the iron-regulated membrane pro-teins of gram-negative organisms to indicate growth in aniron-limited environment. Growth measurements and thekinetics of iron utilization were therefore used to study theeffect of Chelex-100 treatment of growth media on S. aureus.In Table 2 the iron consumption by S. aureus in Chelex-100-treated NB and in NB supplemented with different concen-trations of iron is summarized.A relatively small amount (0.69 ,uM) of iron was consumed

when S. aureus was grown in Chelex-100-treated NB (Fe -

..-

69- 83 K--

48K---

355K ---i325KK

1 2

FIG. 4. SDS-PAGE of the OMPs of Fe+ and Fe- surface-grownK. pneumoniae. Lane 1, Fe- surface grown; lane 2, Fe+ surfacegrown. Similar results were observed with other gram-negativeorganisms used in the study (data not shown).

NB). When 2.5 ,uM iron was added to Fe - NB, ironconsumption increased approximately fourfold (2.9 ,uM). Afurther rise in iron consumption (28.6 ,uM) was observedwhen 36 ,uM iron was added to the growth medium. Thisindicates that there is a progressive increase in iron uptakewhen higher concentrations of iron are added to the culture.Similar observations have been made by other investigators(31, 32).

Cultures of S. aureus grown either in Fe - NB or Fe +NB with added Fe were found to have identical doublingtimes (30 min) (Fig. 5). The bacteria are thus able to multiplyat the maximum rate in a medium with a low iron concen-tration. Similar doubling times were obtained when CDMwas used (Fig. 5), but a higher yield was obtained. A slightly

FIG. 5. Growth of S. aureus in Fe + NB (-), Fe - NB (O), Fe+ CDM (D), Fe - CDM (O), Fe + TSB (A), and Fe - TSB (A). Thecurves are superimposable; they have been offset for clarity and donot imply the occurrence of different lag phases. OD, Opticaldensity.

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Il X 104 DISCUSSION

%

1

1

G- - - --0 -- -

%

- - - -c.1 ".Il1

c

x10-1

~~~~~~~~:MD

-~-1x 10 6

1 x 10-7Time (60 min intervals)

FIG. 6. Growth of S. aureus and uptake of iron from Fe + TSBand Fe - TSB. Growth was in Fe + TSB (0) and Fe - TSB (W);loss of iron was from Fe + TSB (O) and Fe - TSB (E). OD, Opticaldensity.

different picture was observed when Chelex-100-treatedTSB was investigated. The doubling time for Fe + TSB was45 min compared with 60 min for Fe - TSB (Fig. 5).To investigate the uptake of iron, S. aureus grown in

Chelex-100-treated TSB was subcultured into treated anduntreated TSB, and the iron concentration in the supernatantwas determined at different stages of the growth curve (Fig.6). The iron concentration in the supernatant of both treatedand untreated TSB remained fairly constant for the first twogenerations after the onset of the logarithmic growth phase.Thereafter, a fall in the supernatant iron concentrationoccurred with a 100-fold reduction in Fe + TSB comparedwith a 10-fold reduction in Fe - TSB. This demonstrates theability of S. aureus to consume relatively high levels of ironwhen available in the media. The data indicate that uptake ofiron does not take place until shortly before the onset of thestationary phase. In contrast, McIntosh and Earhart (26)demonstrated that in E. coli a very rapid accumulation ofiron occurred in the early exponential phase of the growthcurve.

The significance of iron deprivation on microbial physiol-ogy in vivo (5, 15, 20, 26, 30, 33, 34) has indicated that thereis a need to develop media that will enable bacteria to growunder iron-restricted conditions. For a number of reasons,the methods currently advocated for removal of free ironfrom media are often unsuitable for biochemical and meta-bolic studies (20). The effect of iron deprivation on the OMof gram-negative bacteria is well documented (28). Inductionor repression of a number of high-M, proteins of gram-negative bacteria can be used as a marker of iron availabilityin the growth environment. The results of this study showthat treatment of complex media with Chelex-100 removessufficient iron to impose conditions of iron deprivation ongram-negative bacteria, as judged by the expression ofiron-regulated membrane proteins in the OM preparations.Metal cations such as magnesium, calcium, and zinc wereadded to Chelex-100-treated medium to restore the concen-trations occurring before Chelex-100 treatment, as deter-mined by AAS. To minimize the effect caused by nonspecificremoval of other components such as vitamins, a solutioncontaining a known amount of several vitamins was added tothe medium. The induction of iron-regulated membraneproteins in gram-negative bacteria was the major observableeffect on the OMP profile of growth in treated media. Othermajor proteins present in the OM were essentially unalteredby iron availability, with the exception of the 48K protein ofK. pneumoniae. Expression of this protein was significantlyreduced in cells grown on iron-depleted TSB agar whencompared with iron-sufficient agar. The 48K protein mayfunction as a receptor for a low-affinity, membrane-boundiron uptake system.The study of iron deprivation among gram-positive orga-

nisms is not well documented, and there are practicaldifficulties associated with the investigation of these orga-nisms because there are no readily accessible markers ofiron restriction as there are with gram-negative bacteria. Weused established methods for the measurement of growthkinetics and the disappearance of iron from the culturesupernatant of S. aureus to investigate the effect of ironremoval by the Chelex-100 resin.From the results of this study, we have shown that S.

aureus grows under iron-depleted conditions, as judged bythe reduced uptake of iron from the culture supernatant. Thesame growth rate was maintained, however, suggesting thatthe iron requirement of S. aureus is relatively low. Hence,the residual iron present in all three media after Chelex-100treatment was sufficient to support growth of the organismand to maintain a similar cell yield. This is probably achievedby the alteration of metabolic pathways and a reduction inenvelope-associated iron so that less iron is required by theorganism. We did not investigate the presence of iron-chelating compounds (siderophores) produced by S. aureusin the culture supernatant. It has been reported by Marcelliset al. (24) and Maskell (25), however, that S. aureus pro-duces siderophores that can share functional interchange-ability with those of members of the family Enterobacteria-ceae. Furthermore, Marcellis et al. (24) also reported that S.aureus is capable of removing iron directly from transferrin,which might contribute to the virulence of staphylococci.

Nutrient limitation is known to have a profound effect on

the composition and surface structure of bacteria (8, 9). Ironhas been shown to be unavailable in vivo. Results of thisstudy demonstrate the suitability of using Chelex-100 resinto remove iron from commercially available growth media

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854 KADURUGAMUWA ET AL.

and support the use of iron-depleted growth media for the invitro study of pathogenic bacteria. Bacteria grown in suchmedia would be of value in the study of infectious diseases,as they have been shown to have surface components thatare similar to those of organisms growing in the body duringinfection.

ACKNOWLEDGMENTS

Part of this study was supported by grants from the MedicalResearch Council (United Kingdom) and the Cystic Fibrosis Re-search Trust (United Kingdom), which are gratefully acknowledged.

LITERATURE CITED1. Anwar, H., M. R. W. Brown, A. Day, and P. H. Weller. 1984.

Outer membrane antigens of mucoid Pseudomonas aeruginosaisolated directly from the sputum of a cystic fibrosis patient.FEMS Microbiol. Lett. 24:235-239.

2. Anwar, H., M. R. W. Brown, and P. A. Lambert. 1983. Effect ofnutrient depletion on sensitivity of Pseudomonas cepacia tophagocytosis and serum bactericidal activity at different tem-peratures. J. Gen. Microbiol. 129:2021-2027.

3. Anwar, H., P. A. Lambert, and M. R. W. Brown. 1983.Influence of sodium dodecyl sulfate quality on the electropho-retic mobility of the outer membrane proteins of mucoid andnon-mucoid Pseudomonas aeruginosa. Biochim. Biophys. Acta761:119-125.

4. Boggis, W., M. A. Kenward, and M. R. W. Brown. 1979. Effectsof divalent cations in the growth medium upon sensitivity ofbatch grown Pseudomonas aeruginosa to EDTA or polymyxinB. J. Appl. Bacteriol. 47:477-488.

5. Brown, M. R. W., H. Anwar, and P. A. Lambert. 1984.Evidence that mucoid Pseudomonas aeruginosa in the cysticfibrosis lungs grows under iron-restricted conditions. FEMSMicrobiol. Lett. 21:113-117.

6. Brown, M. R. W., and J. Melling. 1969. Loss of sensitivity toEDTA by Pseudomonas aeruginosa grown under conditions ofmagnesium limitation. J. Gen. Microbiol. 54:439-444.

7. Brown, M. R. W., and J. Melling. 1969. Role of divalent cationsin the action of polymyxin B and EDTA on Pseudomonasaeruginosa. J. Gen. Microbiol. 59:263-274.

8. Brown, M. R. W., and P. Williams. 1985. Influence of substratelimitation and growth phase on sensitivity to antimicrobialagents. J. Antimicrob. Chemother. 15(Suppl. A):7-14.

9. Brown, M. R. W., and P. Williams. 1985. Influence of environ-ment on envelope properties affecting survival of bacteria ininfections. Annu. Rev. Microbiol. 39:527-556.

10. Bullen, J. J. 1981. The significance of iron in infection. Rev.Infect. Dis. 3:1127-1137.

11. Costerton, J. W., M. R. W. Brown, and J. M. Sturgess. 1979.The cell envelope: its role in infection, p. 41-62. In R. G. Dogget(ed.), Pseudomonas aeruginosa: clinical manifestations ofinfection and current therapy. Academic Press, Inc., NewYork.

12. Donald, C., B. I. Passey, and R. J. Swaby. 1952. A comparisonof methods for removing trace metals from microbiologicalmedia. J. Gen. Microbiol. 7:211-220.

13. Finch, J. E., and M. R. W. Brown. 1975. The influence ofnutrient limitation in a chemostat on the sensitivity of Pseu-domonas aeruginosa to polymyxin and EDTA. J. Antimicrob.Chemother. 1:379-386.

14. Finch, J. E., and M. R. W. Brown. 1978. Effect of growthenvironment on Pseudomonas aeruginosa killing by rabbitpolymorphonuclear leukocytes and cationic proteins. Infect.Immun. 20:340-346.

15. Griffiths, E. 1983. Availability of iron and survival of bacteria ininfection, p. 153-177. In C. S. F. Easmon, J. Jeljasewicz,M. R. W. Brown, and P. A. Lambert (ed.), Medical microbiol-ogy, vol. 3. Academic Press, Inc. (London), Ltd., London.

16. Griffiths, E., P. Stevenson, and P. Joyce. 1983. PathogenicEscherichia coli express new outer membrane proteins when

grown in vivo. FEMS Microbiol. Lett. 16:95-99.17. Hancock, R. E. W., V. J. Raffle, and T. I. Nicas. 1981. Involve-

ment of the outer membrane in gentamicin and streptomycinuptake and killing in Pseudomonas aeruginosa. Antimicrob.Agents Chemother. 19:777-785.

18. Harris, M. I., and J. E. Coleman. 1968. The biosynthesis ofapo-and metalloalkaline phosphates of Escherichia coli. J. Biol.Chem. 243:5063-5069.

19. Kenward, M. A., M. R. W. Brown, and J. J. Fryer. 1979. Theinfluence of calcium or magnesium on the resistance to EDTA,polymyxin B or cold shock, and the composition of Pseudomo-nas aeruginosa grown in glucose- or magnesium-depleted batchcultures. J. Appl. Bacteriol. 47:489-503.

20. Klebba, P. E., M. A. Mclntosh, and J. B. Neilands. 1982.Kinetics of biosynthesis of iron-regulated membrane proteins inEscherichia coli. J. Bacteriol. 149:880-888.

21. Lam, C., F. Turnowsky, E. Schwarzinger, and W. Neruda. 1984.Bacteria recovered without subculture from infected humanurine expressed iron regulated outer membrane proteins. FEMSMicrobiol. Lett. 24:255-259.

22. Lambert, P. A. 1984. The role of the bacterial envelope inantibiotic resistance, p. 38-54. In A. C. R. Dean, D. C. Ellwood,and C. G. T. Evans (ed.), Continuous culture. Ellis Horwood,London.

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