Vol. 175, No. 2JOURNAL OF BACrERIOLOGY, Jan. 1993, p. 317-3240021-9193/93/020317-08$02.00/0Copyright X) 1993, American Society for Microbiology

Cloning and Characterization of the Ferric EnterobactinReceptor Gene (pfeA) of Pseudomonas aeruginosa

CHARLES R. DEAN AND KEITH POOLE*Department ofMicrobiology and Immunology, Queen's University,

Kingston, Ontario, Canada K7L 3N6

Received 6 August 1992/Accepted 16 September 1992

Pseudomonas aeruginosa K407, a mutant lacking a high-affinity 80,000-molecular-weight ferric enterobactinreceptor protein (80K protein), exhibited poor growth (small colonies) on iron-deficient succinate minimalmedium containing ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) and enterobactin. The geneencoding the ferric enterobactin receptor was cloned by complementation of this growth defect. Thecomplementing DNA was subsequently localized to a 7.1-kilobase-pair (kb) SstI-HindI fragment which wasable to restore synthesis of the 80K protein in strain K407 and also to direct the synthesis of high levels of aprotein of the same molecular weight in the outer membranes ofEscherichia colifepA strains MT912 and IR20.Moreover, the fragment complemented the fepA mutation in MT912, restoring both growth in EDDHA-containing medium and enterobactin-dependent uptake of 55Fe3+. Expression of the P. aeruginosa receptor inE. coil IR20 was shown to be regulated by both iron and enterobactin. The complementing DNA was furtherlocalized to a 5.3-kb SphI-SstI fragment which was then subjected to deletion analysis to obtain the smallestfragment capable of directing the synthesis of the 80K protein in the outer membrane of strain K407. A 3.2-kbDNA fragment that restored production ofthe receptor in strain K407 was subsequently isolated. The fragmentalso directed synthesis of the protein in E. coli MT912 but at levels much lower than those previously observed.Nucleotide sequencing ofthe fragment revealed an open reading frame (designatedpfeA4 for Pseudomonas ferricenterobactin) of 2,241 bp capable of encoding a 746-amino-acid protein with a molecular weight of 80,967. ThePfeA protein showed more than 60%o homology to the E. coli FepA protein. Consistent with this, the twoproteins showed significant immunological cross-reactivity.

In order to grow and establish infections, bacterial patho-gens must acquire iron, an essential nutrient, from the host.This necessitates competition with the host for availableiron, much of which is bound by the host iron-bindingglycoproteins transferrin (in serum) and lactoferrin (in secre-tions) (46). Many successful pathogens respond to poor ironavailability in the host via the coordinated production oflow-molecular-mass, high-affinity Fe(III) chelators (sidero-phores) (34) and cell surface receptor proteins involved inthe specific uptake of iron-siderophore complexes (33).Siderophores have been shown to remove iron from trans-ferrin in vitro (25, 48) and promote bacterial growth in vivo(7, 43, 51). Thus, it is not surprising that high-affinity ironuptake systems appear to be important contributors tobacterial virulence (9, 15).Pseudomonas aeruginosa, an important opportunistic

pathogen, synthesizes at least two siderophores in responseto iron deprivation, pyochelin (6) and pyoverdine (8). Inaddition, P. aeruginosa is capable of utilizing a number ofheterologous siderophores, including pyoverdines producedby other pseudomonads (22), ferrioxamine B (5), aerobactin,and enterobactin (27). High-molecular-mass outer mem-brane receptors for ferric pyoverdine (31, 39), ferric pyo-chelin (19), and ferric enterobactin (40) have been identifiedin P. aeruginosa. A number of other high-molecular-massiron-regulated outer membrane proteins have been describedfor this organism (5) and may be involved in the uptake ofiron complexed to additional heterologous siderophores.

In addition to the obvious variety of iron-siderophoreuptake systems apparently operating in P. aeruginosa, there

* Corresponding author.

is evidence for multiple uptake systems for a number ofiron-siderophore complexes, including ferric pyoverdine(39), ferric pyochelin (19), and ferric enterobactin (40). In thecase of ferric enterobactin uptake, one system appears to beinducible by the siderophore (under iron-limiting conditions)(40). Desferal, a derivative of ferrioxamine B, has beenshown to promote iron uptake and induce a specific high-molecular-mass iron-regulated outer membrane protein, sug-gesting that the ferrioxamine B uptake system may also beinducible by its corresponding siderophore (5). This is incontrast to E. coli, in which heterologous iron-siderophoreuptake systems are inducible under conditions of iron limi-tation independent of the presence of the correspondingsiderophore (2).

In the present report, we describe the cloning, expression,and nucleotide sequence determination of the ferric entero-bactin receptor gene from P. aeruginosa.

MATERIALS AND METHODS

Bacterial strains and plasmids. The bacterial strains andplasmids used in this study are described in Table 1.

Media. BM2 minimal medium (14) supplemented with 0.5mM MgSO4 and either 20 mM potassium succinate or 0.4%(wt/vol) glucose was used as the iron-deficient medium andwas made iron sufficient, as required, through the addition ofFeSO4 (100 ,uM). In some experiments, L broth (1% [wt/vol]tryptone-0.5% [wtlvol] yeast extract-0.5% [wt/vol] NaCl)was used as an iron-sufficient rich medium and was madeiron deficient through the addition of EDDHA [ethylenedia-mine-di(o-hydroxyphenylacetic acid)] (450 ,ug/ml). Shikimicacid (0.2 mM; Aldrich Chemical Co., Milwaukee, Wis.),methionine (1 mM), thiamine hydrochloride (50 ,ug/ml),

317

on Septem

ber 11, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

318 DEAN AND POOLE

TABLE 1. Bacterial strains and plasmids

Strain or R.tp Source orplasmid Revan prOpertlCSa reference

P. aeruginosaPA01 Prototroph A. KropinskiPA06609 met-9011 amiE200 rpsL pvd-9 21K407 TnSOJ insertion mutant of PA06609 40

lacking an 80K enterobactin-inducible outer membrane protein

PA04141 met-9020pro-9024 blaP9202 blaJ9111 10aph-9001 FP

CD10 PA04141::D3112 cts 10E. coli

IR20 aroB thi fepA 18P8 aroB malT thi fhuAS13 tsx 20MT912 thi trpt purE proC leuB lacY mtl xyl M. McIntosh

rpsL azifhuA tsx supA fepA KmrJM101 Apro-lac thi F'(traD36 proAB+ 50

lacrqZM15)PlasmidspADD214 pUC12-derived mini-D element with 11

RK2 origin of replication and originof transfer, Tcr

pAK1900 Multicopy E. coli-P. aeruginosa A. Kropinskishuttle vector; multicloning sitewithin lacZ a fragment, Apr Cb(

pCD1 pADD214-derived phagemid This studycontaining P. aeruginosachromosomal DNA restoringproduction of the 80K outermembrane protein in K407, Tcr

pCD2 pAK1900 carrying a 7.1-kb SstI- This studyHindIII fragment derived frompCD1 that restores production ofthe 80K outer membrane protein inK407, Apr Cb"

pCD3 As above except carrying a 5.3-kb This studySstI-SphI fragment derived frompCD2

pCD4 As above except carrying a 3.18-kb This studyDNA fragment derived from pCD3by deletion analysis

a Ap'r ampicillin resistance; C(, carbenicillin resistance; Tcr, tetracyclineresistance; Kmr, kanamycin resistance.

adenosine (130 ,tg/ml), leucine (20 ,ug/ml), proline (30 ,ug/ml),tryptophan (20 jg/ml), and sodium citrate (1 mM) wereadded to minimal media as required. For the growth of aroBmutants, BM2 glucose minimal medium was supplementedwith tryptophan (20 ,ug/ml), tyrosine (20 ,ug/ml), and phenyl-alanine (20 ,ug/ml). Growth media contained tetracycline (10,ug/ml) or ampicillin (100 ,ug/ml) for plasmid-bearing Es-cherichia coli transformants and tetracycline (100 ,ug/ml) orcarbenicillin (500 ,ug/ml) for plasmid-bearing P. aeruginosatransformants as required. Solid medium was prepared bythe addition of 1.5% (wt/vol) Bacto-Agar.

Preparation of enterobactin. Enterobactin-containing 25-fold-concentrated culture supernatant was prepared from1-liter overnight cultures of E. coli IR20 as described previ-ously (40). Enterobactin was extracted from acidified (pH2.0) concentrated supernatants, using ethyl acetate andpurified as described previously (26).Growth studies. Overnight cultures of E. coli MT912 and

MT912(pCD2) grown in L broth in the presence of theappropriate antibiotic were diluted into fresh L broth con-taining EDDHA (450 jig/ml) to anA6. of 0.01. Cultures wereshaken (200 rpm) on an orbital shaker at 37°C, and cultureturbidity (A6w) was determined at various intervals.

Transport assays. E. coli MT912(pAK1900) andMT912(pCD2) were grown at 370C with shaking (200 rpm) iniron-deficient glucose minimal medium containing ampicillinto an A600 Of 0.80. EDDHA was then added to each culture(150 ,ug/ml), and they were further incubated under the sameconditions for 1 h. Cells were harvested and used in trans-port assays as described previously (40), except that the cellswere incubated for only 5 min prior to the start of the assay.

Outer membrane preparation and SDS-polyacrylamide gelelectrophoresis. Outer membrane samples were obtainedthrough differential Triton X-100 extraction of isolated cellenvelopes as previously described (47). Sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis was con-ducted as described previously (28) by using 7% (wt/vol)acrylamide in the running gel and omitting 2-mercaptoetha-nol from the sample loading buffer.Western immunoblots. Western immunoblots were per-

formed as described previously (49) with modifications.Briefly, outer membrane proteins separated on SDS-poly-acrylamide gels were transferred to TransBlot nitrocellulosemembranes (Bio-Rad, Richmond, Calif.) by a 30-min elec-trophoresis at 0.25 A (40C). After transfer, the membraneswere incubated for 1 h in phosphate-buffered saline (PBS)containing 3% (wt/vol) bovine serum albumin (BSA) andsubsequently incubated for 90 min with an anti-FepA poly-clonal antiserum (4) in PBS containing 1% BSA. The mem-branes were then washed three times with PBS and furtherincubated for 90 min with Protein-A alkaline phosphataseconjugate (Sigma, St. Louis, Mo.), again in PBS containing1% BSA. The blots were then washed three times in PBS andtwice in 10 mM Tris-HCl, pH 9.4, after which visualizationof FepA cross-reactive proteins was facilitated by additionof an alkaline phosphatase substrate composed of 5-bromo-4-chloro-3-indolyl phosphate (0.18 mg/ml) and nitroblue tet-razolium (0.4 mg/ml).DNA methods. Standard methods were employed for

small- and large-scale isolation of plasmid DNA, digestionwith restriction endonucleases and S1 nuclease, ligation,linker tailing, and separation of DNA fragments by agarosegel electrophoresis (45). DNA restriction fragments used incloning experiments were purified following electrophoreticseparation in agarose (0.7% [wt/vol]) gels by using Gene-clean (Bio 101, Inc., La Jolla, Calif.) according to themanufacturer's instructions. Plasmid DNA was transformedinto E. coli and P. aeruginosa by using the protocolsdescribed by Sambrook et al. (45) and Berry and Kropinski(1), respectively.

In vivo cloning of the ferric enterobactin receptor gene. The80K ferric enterobactin receptor gene was cloned by usingthe in vivo cloning scheme of Darzins and Casadaban (10,11). A lysate of P. aeruginosa CD10 carrying the mini-Dreplicon pADD214 was prepared as described previously(10) and used to infect P. aeruginosa K407 (10). Transduc-tants containing the ferric enterobactin receptor gene wereselected on iron-deficient succinate minimal medium platescontaining tetracyline, EDDHA (150 ,ug/ml), and enterobac-tin-containing concentrated E. coli culture supernatant (5pJ/ml) at 37°C. Tetracycline-resistant bacterial clones show-ing rapid growth in the presence of EDDHA and enterobac-tin (i.e., large colonies) were isolated after 24 to 48 h ofgrowth, and their outer membranes were examined onSDS-polyacrylamide gels for the presence of the 80K outermembrane protein.

Deletion analysis and nucleotide sequence determination.Plasmid pCD3 contains a 5.3-kb SstI-SphI DNA fragment(see Fig. 7) derived from plasmid pCD2 that restores pro-

J. BAcTERIOL.

on Septem

ber 11, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

FERRIC ENTEROBACTIN RECEPTOR GENE OF P. AERUGINOSA 319

duction of the 80K ferric enterobactin receptor protein instrain K407. A series of subclones containing progressivelysmaller portions of this 5.3-kb fragment was generated byusing the Pharmacia double-stranded nested deletion kit asdescribed by the manufacturer (Pharmacia LKB Biotechnol-ogy AB, Uppsala, Sweden). Briefly, plasmid pCD3 wasdigested with either SstI or SphI, blunt ended by using S1nuclease, and ligated to unphosphorylated BamHI linkers.This converted the 5.3-kb SstI-SphI fragment to SstI-BamHI(A) and SphI-BamHI (B) fragments, respectively. Each ofthese fragments was then isolated and ligated separately intothe multicloning site of pAK1900, yielding plasmidspCD3(A) and pCD3(B). In order to obtain deletions fromeither end of the 5.3-kb fragment, CsCl-purified pCD3(A)and pCD3(B) were digested with appropriate restrictionenzymes (SphI-BamHI and SstI-BamHI, respectively) andsubjected to exonuclease III digestion according to themanufacturer's protocol. A series of nested deletions differ-ing in size by 200 to 250 bp and encompassing the entire5.3-kb fragment in both directions was obtained. Each of theresulting subclones was then tested for the ability to restoreproduction of the 80K protein in strain K407. The smallestcomplementing subclone contained a 3.2-kb fragment andwas designated pCD4. The deletion subclones encompassingthis 3.2-kb region were subsequently purified by using thePrep-A-Gene DNA purification kit (Bio-Rad) and sequencedby the Centres of Excellence Core Facility for Protein/DNAChemistry (Queen's University). The nucleotide sequence ofboth strands was determined, and the resulting data wereanalyzed by using the PCGENE software package (Intelli-genetics, Inc., Mountain View, Calif.).

Nucleotide sequence accession number. The nucleotidesequence of pfeA is registered in the GenBank data baseunder accession no. M98033.

RESULTS

Cloning of the ferric enterobactin receptor gene of P.aeruginosa. P. aeruginosa K407 lacks the high-affinity ferricenterobactin receptor (Fig. 1, lane 1; cf. lane 2) and as aresult grows poorly, forming tiny colonies, on iron-deficientminimal medium plates containing EDDHA and enterobac-tin (40). By using the in vivo cloning scheme of Darzins andCasadaban (10, 11), phagemids carrying random pieces ofP.aeruginosa chromosomal DNA were introduced into K407and those restoring normal (large-colony) growth on iron-deficient minimal medium plates containing EDDHA andenterobactin (supplied as concentrated E. coli culture super-natant) were recovered. One recombinant phagemid, pCD1,not only complemented the growth defect in K407 but alsorestored synthesis of the 80K ferric enterobactin receptorprotein in this strain (Fig. 1, lane 3). Plasmid pCD2, contain-ing a 7.1-kb SstI-HindIII fragment derived from pCD1 (seeFig. 7), was also capable of restoring synthesis of the 80Kprotein in K407 (Fig. 1, lane 4).

Expression and activity of the P. aeruginosa ferric entero-bactin receptor in E. coi. Initially it was not known whetherthe loss of the 80K ferric enterobactin receptor protein fromstrain K407 was the result of a mutation in the receptor geneitself or in a gene regulating its expression. Thus, thecomplementing 7.1-kb P. aeruginosa chromosomal DNAfragment could have contained the receptor structural geneor a regulatory gene. To confirm the presence of the receptorgene on the complementing fragment, pCD2 was introducedinto E. coli MT912 (afepA null mutant), where it was shownto direct the synthesis of an 80K outer membrane protein

1 2 34 5 6FIG. 1. SDS-polyacrylamide gel electrophoretogram of outer

membranes prepared from strains of P. aensginosa grown in iron-deficient succinate minimal medium containing EDDHA (150 ,ug/ml)and enterobactin-containing concentrated E. coli culture superna-tant (15 i.dVml). Lane 1, K407; lane 2, PA06609; lane 3, K407(pCD1);lane 4, K407(pCD2); lane 5, K407(pCD3); lane 6, K407(pCD4).Samples were solubilized by heating at 950C for 5 min prior toelectrophoresis. The positions of molecular weight standards andthe ferric enterobactin receptor protein (arrow) are indicated.

(Fig. 2, lane 2). This was consistent with the presence of theP. aeruginosa ferric enterobactin receptor gene on pCD2.Although MT912 alone was unable to grow in mediumcontaining EDDHA, presumably because of the defect inferric enterobactin uptake resulting from the loss of FepA,the pCD2-containing strain was able to grow in this medium(Fig. 3). This suggested that the P. aeruginosa ferric enter-

97K-

66K-

45K-

31K-

1 23 4FIG. 2. SDS-polyacrylamide gel electrophoretogram of outer

membranes prepared from strains of E. coli grown in iron-deficientglucose minimal medium. Lane 1, MT912; lane 2, MT912(pCD2);lane 3, MT912(pCD3); lane 4, MT912(pCD4). Samples were solubi-lized by heating at 95°C for 5 min prior to electrophoresis. Thepositions of molecular weight standards and the P. aeruginosa ferricenterobactin receptor protein (arrow) are indicated.

VOL. 175, 1993

on Septem

ber 11, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

320 DEAN AND POOLE

1.0

0.8

0.6

CvO0.4

0.2-

00 5 10 15 20 25 30

Time (hours)FIG. 3. Growth of E. coli MT912 (0) and MT912(pCD2) (U) in

iron-deficient L broth containing EDDHA (450 jig/ml). The data arerepresentative of three separate experiments.

obactin receptor was active and able to replace the lost FepAprotein in mediating ferric enterobactin uptake in MT912. Inagreement with this, enterobactin-mediated iron transportwas observed for E. coli MT912(pCD2) (Fig. 4). As ex-

20

0-11C/)

C.)

0

E0.-.

0

CZ

0C..

15

101

5

FIG. 4. 1

MT912(pA.Eiron-deficierprior to celmixture conobactin (4 ,u]data are the

97K

66K-

12345Ent + + +Fe + + -

FIG. 5. SDS-polyacrylamide gel electrophoretogram of outermembranes prepared from E. coli IR20(pCD2) (lanes 1 to 4) andIR20 (lane 5) grown in glucose minimal medium with sodium citrateand with (+) or without (-) 100 p.M FeSO4 (Fe) or enterobactin(Ent) (as concentrated E. coil culture supernatant, 15 p.1/ml). Sam-ples were solubilized by heating at 950C for 5 min prior to electro-phoresis. The positions of molecular weight standards and the ferricenterobactin receptor protein (arrow) are indicated.

pected, MT912 lacking the cloned P. aeruginosa ferricenterobactin receptor was deficient in enterobactin-mediatediron transport (Fig. 4).

Regulation of expression of the P. aeruginosa ferric entero-bactin receptor in E. coli. To determine whether the ferricenterobactin receptor encoded by pCD2 was regulated byiron and/or enterobactin in E. coli as the protein normally isin P. aeruginosa, pCD2 was introduced into E. coli IR20, astrain defective in the production of both the ferric entero-bactin receptor and enterobactin. Growth of this strain inminimal medium required the addition of citrate (17), appar-ently to facilitate iron uptake in the absence of enterobactinproduction. As expected, IR20 harboring pCD2 (Fig. 5, lanes1 to 4) but not IR20 alone (Fig. 5, lane 5) expressed an 80Kouter membrane protein. Moreover, IR20(pCD2) culturedunder iron-sufficient conditions synthesized markedly less ofthe ferric enterobactin receptor (Fig. 5, lanes 2 and 3) thandid the same strain cultured under iron-deficient conditions(Fig. 5, lanes 1 and 4), irrespective of the presence orabsence of enterobactin. Addition of enterobactin did, how-ever, noticeably enhance production of the 80K receptorprotein in E. coli IR20(pCD2) cultured under iron-sufficient

I I conditions (Fig. 5, lane 3; cf. lane 2). Such enhancement by) 1 2 3 4 5 6 enterobactin was less evident in iron-limited cells (Fig. 5,

lane 4; cf. lane 1) whose high-level expression of the 80KTime (minutes) receptor may mask a slight enterobactin effect.

Immunological cross-reactivity of the P. aeruginosa and E.Enterobactin-mediated iron (55Fe3+) transport byE. co coli ferric enterobactin receptors. In light of the ability of theC1900) (0] and U) and MT'912(pCD2) (0 and 0) grown initglucose minimal medium with EDDHA added.1 h P. aeruginosa receptor to functionally replace the E. colint glucose minimal medium wit DH ded1h Fp rteni waso inteest to deemne whte th11 harvest (see Materials and Methods). The uptake FepA protein, it was of interest to determine whether the

tained 1 I.M 55FeCl3, 4 p.M EDDHA, and either enter- proteins exhibited any structural similarities. To test this,M) (E and 0) or an equal volume of H20 (O and 0). The the P. aeruginosa receptor was examined for any reactivityaverage of two experiments. with a polyclonal antiserum raised against the E. coli recep-

J. BACTERIOL.

0

on Septem

ber 11, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

FERRIC ENTEROBACTIN RECEPTOR GENE OF P. AERUGINOSA 321

1 2 3 4 5 6FIG. 6. Western blot (immunoblot) analysis of E. coli and P.

aeruginosa outer membranes using an anti-FepA polyclonal anti-serum. Lane 1, E. coli IR20 (FepA-); lane 2, E. coli P8 (FepA+);lanes 3 and 4, P. aeruginosa PA06609; lane 5, P. aeruginosaK407(pCD2); lane 6, E. coli IR20(pCD2). E. coli outer membraneswere prepared from cells cultured in iron-deficient glucose minimalmedium containing shikimate (lanes 1 and 2) or citrate (lane 6). P.aeruginosa outer membranes were prepared from cells cultured iniron-deficient succinate minimal medium with (lane 5) and without(lanes 3 and 4) EDDHA (150 pLg/ml) and with (lanes 4 and 5) andwithout (lane 3) enterobactin (as concentrated E. coli culture super-

natant, 15 pl/ml).

tor (FepA) (4). While the antiserum did react specificallywith the P. aeruginosa receptor produced both in P. aerug-

inosa (Fig. 6, lanes 4 and 5) and E. coli (Fig. 6, lane 6), thereaction was noticeably less strong than that observed forthe FepA protein (Fig. 6, lane 2).

Nucleotide sequence ofpfeA. The 7.1-kb SstI-HindIII insertin pCD2 (Fig. 7) includes 1.5 kb of DNA at the HindIII endwhich constitutes one of the termini (attR) of the mini-Delement present on the pADD214 replicon used in cloningthe ferric enterobactin receptor gene (10, 11). Prior tosequencing this DNA, then, it was desirable to remove thisphage component. This was accomplished by digestion ofpCD2 with SphI and SstI, yielding a 5.3-kb SphI-SstI frag-ment (pCD3; Fig. 7) which, upon introduction into K407,restored production of the 80K receptor (Fig. 1, lane 5). Tofurther localize the DNA required for expression of the ferricenterobactin receptor in K407, nested deletions of the 5.3-kbfragment in pCD3 were constructed. The smallest fragmentobtained which was capable of directing the synthesis of the80K receptor in K407 (Fig. 1, lane 6) was 3.2 kb in size(pCD4; Fig. 7). In E. coli MT912, pCD4 appeared to bedirecting the synthesis of the receptor protein (Fig. 2, lane 4)but at levels greatly reduced from that observed with plas-mid pCD3 (Fig. 2, lane 3). This indicated that an element or

elements necessary for full expression of the protein in E.coli had been deleted in pCD4. The 3.2-kb fragment was

sequenced (Fig. 8), revealing a 2,241-bp open reading framedesignatedpfeA (Fig. 7). The predicted product of the pfeA

pCD4

pCD3

SstI PvuI PvuI XhoIpCD2

pfeA

1 kb

FIG. 7. Restriction map of the 7.1-kb SstI-hpCD2, including the right terminus (attR) of D31the subclones contained in plasmids pCD3 and rreading frame pfeA (arrow).

I ACACCTGGACAGCCTGGCCCAGGCCATGGMAACCTCCTGCGCAACGCATCCGTCACTCGCCCGAGGACGGCACGGTCAGCCTCGACGGCGAGCGCGAG

101 GGCGACTTCTGGCACCTGCGCCTGCAGGACCAGGGCCCTGGCGTGGCGOAAGACCAGTTGGAACGCATCTTCCTCCCCTACCAGCGCCTGGACGATAGCG

201 CCGGCGAAGGTTTCGGCCTCGGCCTCGGCATCCCCCGGCGCGCCATCGAGCTACAGGCGGCCGGCCTCTGGGCCCAGCAACGGCAAGCCCGGATTGTGCCTPvul -35 -10

301 GCACCTGTGGCTGCCGGCGGCCGCCTGAGGCCGAAGTGTTTAGAAAGTTAATGCGCIITTCTCAAATAACAATCATATCATTTGTGATCTCTTGCAT

S.D. M S S R A L P A V P F L L L S S C L L A N A V H A401 TTCGCTGCATTGCCC4GAGATCACCGATGTCCTCACGCGCCCTTCCCGCCGTTCCCTTCCTGCTGCTGTCCAGTTGCCTGCTCGCCAACGCCGTACACGC

SQG Q G D G S V I E L G E Q T V V A T A Q EE T K O A P G V S II501 CGCCGGCCAGGCGACGGCTCCGTCATCGAGCTGGCGAGCAGACCTGGTCGCCACCGCCCAGGAGGMACCAAGCAGGCGCCGGGGGTTTCCATCATC

T A E D I A K R P P S N D L S Q I I R T N P G V N L T G N S S S G601ACCGCCGAGGACATCGCCAGCGACCGCCGGCAACGACCTGTCGCAGATCATCCGGACCATGCCGGGGGTCAACCTAACCGGCACAGCTCCAGCGGCC

701

801

901

1001

1101

1201

1301

1401

1501

1601

1701

1801

1901

2001

2101

2201

2301

2401

2501

2601

2701

2801

2901

3001

3101

O R G N N R Q I D I R G N G P E N T L I L V D G K P V S S R N S V RAGCGTGGAAACAACCGGCAGATCGACATCCGCOGCATGGGCCCGGAGAACACCCTGATCCTOGTCGACGGCAAGCCGGTCAGCTCGCGCAACTCGGTGCG

Y G U R G E R D S R G D T NU V P A D Q V E R I E V I R G P A A ACTACGGCTGGCGCGCGCAGCGCGACAGCCGCGGCGACACCAACTGGGTGCCGGCCGACCAGGTCGAGCGCATCCMAGTGATCCGCGGCCCGCGCGCGGCG

R Y C N G A A C G V V N I I T K Q A G A E T H G N L S V Y S N F PCGCTACGGCAACGCGCGCGCGGGCGGCGTGGTGAACATCATCACCAAGCAGGCCGGCGCGGAAACCCACGGTAATCTCAGCGTCTACAGCAATTTCCCGC

Q H K A E G A S E R N S F G L N G P L T E N L S Y R V Y G N I A K TAACCAAGGCCCGAAGCGCCAGCGAACGGATGAGCTTCGGTCTCAACGGGCCCCTCACGCAAMCCTCAGCTACCGCGTCTACGGCAACATCGCCAAGAC

D S D DA D I 1 A G H E S 1NR T G K Q G T L P VG R E G V R N KCGACTCGGACGACTGGGACATCAACGCCGGCCACGAATCCAACCGTACCGGCAAGCAGGCCGGCACCCTCCCCGCCGGTCGCGAAGGCGTGCGCAACAAG

D I D G L L S U R L T P E Q T L E F E A G F S R Q G N I Y T G D TGACATCGACGGGCTGCTCAGCTGGCGCCTGACGCCCGAGCAGACCCTC8TTCGAGGCCGGCTTCAGCCGCCAGGCCAACATCTACACCGGCGACACGC

XholQ 11 T N S 11 11 Y V K Q N L G H E T li R N Y R E T Y S V T H R G E U DAGACACCAACAGCAACACTACGTCGCAACA~iTGCTCGGCCACGAGACCAACCGCATGTACCGCGCAACCTACTCGGTCACCCATCGCGGCGAATGGGA

F G * S L A Y L Q Y E K T R N S R I N E G L A G G T E G I F D P NCTTCGGCAGCTCGCTGCCCTACCTGCAGTACGAGAAGACCCGCAACAGCCGGATCAACGAAGGCCTGGCCGGCGGCACCGAAGGTATCTTCGACCCCAAC

N A G F Y T A T L R D L T A G E V N L P L H L G Y E O T L T L GAACGCCGGCTTCTACACCGCCACCCTGCGCGACCTGACCGCCCACGGCGAGGTCAACCTGCCGCTGCACCTGGGCTACCAGCAGACCCTGACCCTCGGCA

S E W T E O It L D D P S S li T Q 11 T E E G G S I P G L A G K N R S SGCGAGTGGACCGAGCAGAAGCTCGACGACCCCAGCTCCAACACCCAGAACACCGAGGAAGGCGGCTCGATCCCCGGTCTCGCCGGAAAGAACCGCAGCAG

S S S A R I F S L F A E D N I E L M P G T N L T P G L R U D H H DCAGTTCCTCGCGCGGCATCTTCTCGCTGTTCGCCGAGGCAAMCATCGAGCTGATGCCCGGCACCATGCTCACCCCAGGCCTGCGCTGGGACCACCACGAC

I V G D N A S P S L N L S H A L T E R V T L IK A G I 4 R A Y K A PATCGTCGGCGACAACTGGAGCCCATCGCTGACCTGTCCCACGCGCTCACCGAUGCGTCACCCTGAAGGCCGGTATCGCCCGCGCCTACAAGGCCCCCA

N L Y Q L N P D Y L L Y S R G Q G C Y G Q S T S C Y L R C 11 D C L KACCTGTACCAGCTGAACCCCACTACCTGCTCTACAGCCGTGGCCAGGGTTGCTACGGGCMACACACCAGTTGCTACCTGCGCGCCACGMACGGCCTCAA

A E T S V N K E L G I E Y S H D G L V AG L T Y F R N D Y K N KGCCGCAGACCAGCGTGACAAGGAACTGGGCATCGAGTACAGCCACGACGGCCTGGTAGCGGGGCTGACCTACTTCCGCAACGACTACAAGAACAAGATC

E S G L S P V D H A S G G K( G D Y A 11 A A I Y Q U E N V P K A V VGCATCCGGCCTGTCACCGGTCGACCACGCCAGCGGCGGCAAGGGCGACTACGCCACGCCGGCGATCTACCAGTGGGACAACGTGCCCAAGGCGGTGGTCG

E G L E G T L T L P L A D G L 11 U S 11 N L T Y 1N L Q S K N K E T G DAGGGCCTCGAAGGCACCCTGACCCTGCCCCTGCCGCACGGTCTGCAGTGGACAACAACCTCACCTACATGCTGCAATCGAAGAACAAGGAAACCGGCGA

V L S V T P R Y T L 11 S N L D U Q A T D D L S L Q A T V T U Y G KCGTGCTCTCGGTGACGCCGCGCTACACCCTCAACTCGATGCTCGACTGGCAGGCCACCGACGACCTCTCGCTGCAAGCCACGGTCACCTGGTACGGCMAG

O K P K K( Y D Y H G D R V T G S A N D Q L S P Y A I A G L G G T YUCAGACCGAAGAAATACGACTATCACGGCGACCGTGTCACCGGCAGCGCCAACGACCAGCTCTCGCCCTACGCCATCGCCGGCCTCGGCGGCACCTATC

R L S It N L S L G A G V D H L F D K: R L F R A G N A Q G V V G I D GGGTTGAGCAAGACCTGAGCCTCGGCGCCGGCGTCGCAAMCCTGTTCGACAAGCGCCTGTTCCGCGCCGCAACTGCCCAGGGCGTGGTCGGCATCGACGG

A G A A T Y N E P G R T F Y T S L T A S F -

GGCCGGCCGCGCGACCTACAACGAGCCCGGACGGACCTTCTATACCAGCCTGACCGCGTCGTTCTGAGCCATAACGAGAAAGCCTGATGAGAACCTCCCT

CAACGCCAGGACCTCACCTATCGCTTCAGCGCCGTGCTCCTGGACTCCGTCGATGGCCAGCGGCATTACCGGCTGTGUCTCGGCCGACCGCTGCAGGCAC

CGCCCGCCGCCGGCTACCCGGTGGTCTGGATGCTCGACGGCAACGCCGCCCTCGGCGCCCTCGACGAGTCAACCCTCAGACGCCTGUCCGACGGCGACGC

ACCGCTACTGCTCGCCATCGGCTACCGCG GCTGCGCATCGACCGCGCCGGGCGTACCTTCGACTACACCCCCGCGAGTCCCGGCCAGGCCGATCAG

CGCGACCCGCTCAACGGCCTGCCCCACGCGTGGTGCGACCGCCTTCCTCGACCTCCTGCC^CGCGCTGCf

FIG. 8. Nucleotide and deduced amino acid sequences of theferric enterobactin receptor (PfeA) of P. aeruginosa. The putativepromoter sequences (-10 and -35, underlined), ribosome bindingsite (S.D., underlined), signal peptide cleavage site (arrow), andselected restriction sites are also indicated.

gene consists of 746 amino acids with a molecular weight of80,967. This includes a typical signal peptide of 25 aminoacid residues which upon cleavage would give a matureprotein with a molecular weight of 78,385. This is in closeagreement with the receptor protein size as estimated bySDS-polyacrylamide gel electrophoresis (Fig. 1). Potential-10 and -35 regions of a putative promoter were identifiedupstream of the pfeA open reading frame (Fig. 8). Interest-ingly, a sequence with some homology to the E. coli Furbinding consensus sequence (11 of 19 matches) overlaps this

SphI region (Fig. 9). This is consistent with the observed iron-SphI HindIII regulated expression of the receptor in E. coli (Fig. 5).

attR Comparison of PfeA and FepA. Since PfeA shows immu-nological cross-reactivity with FepA and can functionallyreplace FepA in E. coli fepA strains, it was desirable toinvestigate the level of homology between the two proteins.

IindIII fragment of Alignment of the amino acid sequences of the two proteins12. Also shown are revealed that, aside from their signal peptide domains, thepCD4 and the open proteins are highly homologous, with almost 60% of the

residues being identical and a further 11% representing

VOL. 175, 1993

on Septem

ber 11, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

322 DEAN AND POOLE

Fur consensus region

GATAATGAT AATCATTATCII W LI

356- [ -C&TCAAMTAACATCATGTGATCTC1T-35 -10

MET

397- GCA1TTCGCTGCATTGCCCGGAGATCACCGATGS.D.

FIG. 9. Putative promoter region upstream of pfeA showingregion of homology to the E. coli Fur consensus binding sequence(3).

conservative changes (Fig. 10). Of two FepA regions previ-ously identified as being involved in ligand binding (32), thefirst shows significant homology to a corresponding region ofPfeA while the second is somewhat less homologous. Inaddition, a TonB box (16, 29) was identified near the Nterminus of PfeA (Fig. 10).

PfeA - NSSRALPAVPFLLLSSCLLANAVHAAGQGDGSVI ELGEQTW TACEETKQAPGV -551 - I ELGE1Y.1 &j4IE 11111

FepA - MNKKIH- - - - -SLALLVNLGIYGVAQAQEPTDTPVSNDDTIWTAAEONLQAPGV -50

PfeA - SI ITAEDIAKRPPSNDLSQI IRTMPGVNLTGNSSSGQRGNNRQIDIRGMGPENTL -110.1 111.-I111 1-1 111111'111111111111

FepA - STITADEIRKNPVARDVSKI IRTMPGVNLTGNSTSGQRGNNMRIDIRGMGPENTL -105

PfeA - I LVDGKPVSSRNSVRYGURGERDSRGDTNWVPADQVERIEVIRGPAAARYGNGAA -165

FepA - I LIDGKPVSSRNSVRQGURGERDTRGDTSWVPPEMI4ERIEVLRGPARARYGNGAA -160

PfeA - GGWNI ITKQAGAETHGNLSVYSNFPQHKAEGASERNSFGLNGPLTENLSYRVYG -220I111111111I1I11111111l- 1l1i11. 1-1-11

FepA - GGWNI ITKKGSGEWHGSWDAYFNAPEHKEEGATKRTNFSLTGPLGDEFSFRLYG -215

PfeA - NIAKTDSDDWDINAGHESNRTGKQAGTLPAGREGVRNKDIDGLLSURLTPEQTLE -2751-I11 * 1111111i-1 1 1111111111111i 1-- .1*1.111

FepA - NLDKTQADAWDINQGHQSARAGTYATTLPAGREGVINKDINGWRWDFAPLQSLE -270

PfeA - FEAGFSRQGNIYTGDTQNTNSNNYVKQNLGHETNRMYRETI -330

FepA - LEAGYSRQGNLYAGDTQNTNSDSYTRSKYGDETNRLYRQN -325

PfeA-_ Y YTATLRDLTAHGEVNLPLHLG -383

FepA-F FVDIDLDDVMLHSEVNLPIDFL -380

PfeA - YEQTLTLGSEWTEQKLDDPSSNT SSSSARIFSLFA -436

FepA - VNQTLTLGTEWNQQRMKDLSSN Q SPYSKAEIFSLFA -435

PfeA - EDNIELMPGTMLTPGLRWDHHDIVGDNWSPSLNLSHALTERVTLKAGIARAYKAP -4911I1-11 I..IIIII-IIIII111111111 1- 111 111111'111

FepA - ENNNELTDSTIVTPGLRFDHHSIVGNMISPALNISOQGLGDDFTLKGIARAYKAP -490

PfeA - NLYQLNPDYLLYSRGQGCYGQSTSCYLRGNDGLKAETSVNKELGIEYSHDGLVAG -54611111 I-III1.IIIII . 1111111111111111II1I 11 11.l

FepA - SLYQTNPNYI LYSKGQGCYASAGGCYLQGNDDLKAETSINKEIGLEFKRDGWLAG -545

PfeA - LTYFRNDYKNKIESGLSPVDHASGGKGDYANAAIYQWENVPKAVVEGLEGTLTLP -6011-1111-111-1 1 11-111111111111.-1 *

FepA - VTWFRNDYRNKIEAGYVAVGQNAVGTDLY------QW MVPKAVVEGLEGSLNVP -594

PfeA - LADGLKWSNNLTYMLQSKNKETGDVLSVTPRYTLNSMLDWQATDDLSLQATVTWY -656....~ ~ I1-1-11 1 1 11 1 11111 1 111 '111-1.1 111

FepA - VSETVMWTNNITYMLKSENKTTGDRLSI IPEYTLNSTLSWJQAREDLSMQTTFTWY -649

PfeA - GKQKPKKYDYHGDRVTGSANDQLSPYAIAGLGGTYRLSKNLSLGAGVDNLFDKRL -7111111111111 1111-l1 1--1-1 11111

FepA - GKQQPKKYNYKGQPAVGPETKEI SPYSIVGLSATWDVTKNVSLTGGVDNLFDKRL -704

PfeA - FRAGNAQ------GWGIDGAGAATYNEPGRTFYTSLTASF -746-111111 1 1111111111IIII- 1--

FepA - URAGNAQTTGDLAGANYIAGAGAYTYNEPGRTYWNSVNTHF -745FIG. 10. Alignment of the deduced amino acid sequences, in-

cluding signal peptide domains, of PfeA and FepA (29). The regionscorresponding to the TonB box (underlined) and the proposed ligandbinding sites (shaded) of FepA are also indicated.

DISCUSSION

The ability of P. aeruginosa to acquire iron through theutilization of heterologous siderophores has been established(27). More recently, it has been reported that one of these,enterobactin, the major siderophore of E. coli, mediates ironuptake in P. aeruginosa via what may be two distinctsystems (40). One appears to be a low-affinity uptake systeminducible solely under conditions of iron limitation whereasthe other appears to be of high affinity and requires both ironlimitation and enterobactin for induction. A novel outermembrane protein with a molecular weight of approximately80,000 was implicated as the high-affinity ferric enterobactinreceptor. In the present paper, we describe the isolation andnucleotide sequence determination of a 3.2-kb P. aeruginosachromosomal DNA fragment which restores synthesis of the80K high-affinity ferric enterobactin receptor protein in thereceptor-deficient mutant, K407. There is extensive homol-ogy between the predicted amino acid sequence of PfeA andthat of the analogous receptor protein, FepA, in E. coli. Thisis consistent with the observed immunological relatedness ofthe two proteins.Given that PfeA and FepA perform similar functions (23,

40), it is not unusual that they show a high level of similarity.Regions that would be expected to be highly conserved areligand binding sites. Murphy et al. using monoclonal anti-bodies to inhibit the interaction of FepA with ferric entero-bactin and colicins, have suggested that two regions areinvolved in ligand binding (32). These are indicated as aminoacids 313 to 358 and 404 to 422 of FepA (Fig. 10). The regionof PfeA corresponding to the 313 to 358 region of FepAshows significant homology, the central area being almostidentical, with each end showing markedly less similarity. Itis thought that colicins B and D and ferric enterobactin mayrecognize distinct microdomains within the proposed ligandbinding site from residues 313 to 358 (30, 32). The region ofidentity observed between PfeA and FepA in the centralportion of the proposed ligand binding site may indicate themicrodomain recognized by ferric enterobactin. The FepAregion from amino acids 404 to 422 may not be directlyinvolved in ligand binding but may only be in such closeproximity to the amino acid 313 to 358 region that antibodybinding inhibits ligand binding (32). The sequence of PfeAcorresponding to residues 404 to 422 of FepA shows acentral region of similarity, although less than that observedfor the other proposed binding site. The presence of a likelyferric enterobactin binding site on PfeA, in addition to theoverall homology between PfeA and FepA, supports thecontention that PfeA is indeed the high-affinity ferric enter-obactin receptor of P. aeruginosa.The observation that pfeA, localized on pCD4, restores

production of the high-affinity ferric enterobactin receptor inK407 indicates that the mutation in this strain is within thereceptor structural gene as opposed to a regulatory gene.This supports the previous conclusion that the low-levelenterobactin-mediated iron uptake observed in K407 re-quires a second uptake system (40), rather than arising fromresidual expression of the high-affinity receptor. The secondlow-affinity uptake system was shown to require only ironlimitation for induction (40). Rutz et al. (44) have shown thatof a library of 24 anti-FepA monoclonal antibodies specificfor both buried and surface exposed epitopes, two react witha protein in the outer membranes of P. aeruginosa grownunder conditions of iron limitation in the absence of entero-bactin. The protein appears to be larger than FepA and is

J. BACTERIOL.

on Septem

ber 11, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

FERRIC ENTEROBACTIN RECEPTOR GENE OF P. AERUGINOSA 323

thus likely the receptor for the low-affinity ferric enterobac-tin uptake system.The ability of the P. aeruginosa high-affinity ferric enter-

obactin receptor to functionally replace FepA in E. coliMT912, allowing growth in the presence of EDDHA andfacilitating enterobactin-mediated transport of 15Fe3", raisesthe possibility of similarities existing between other compo-nents of the two uptake systems. In addition to the fepA-encoded receptor protein, ferric enterobactin uptake in E.coli requires the products of fepB and fepC, encoding aperiplasmic and a cytoplasmic membrane-associated pro-tein, respectively (13, 29, 35-37). As well, the product of thefes gene, an esterase, is required to hydrolyze enterobactin(26), releasing the bound iron to the cell. The observedability of PfeA to function well in place of FepA in E. colisuggests the existence of analogous uptake components in P.aeruginosa.The product of another gene, tonB, is also required for all

high-affinity iron uptake systems in E. coli, as well as avariety of other active transport systems. The exact functionof TonB is unknown, but it is thought to be located in theperiplasm where it can make contact with outer membranereceptors (41) and provide energy coupling between thereceptors and the cytoplasmic membrane (24). TonB isthought to interact with a conserved 8-amino-acid region(TonB box) close to the N-terminal end of all TonB-depen-dent outer membrane receptors in E. coli (12, 16, 29). Thedemonstration that E. coli fepA mutants possessing the P.aeruginosa ferric enterobactin receptor gene grew rapidly inthe presence of EDDHA and exhibited readily detectablelevels of enterobactin-mediated iron uptake suggests that thePfeA protein is capable of interacting with the E. coli TonBprotein. Consistent with this, a region showing homology tothe TonB box was identified at the N terminus of PfeA andat the same relative position in the protein as it occurs inFepA. These data strongly support the existence of a TonB-like protein in P. aeruginosa.

In E. coli, regulation of iron transport systems, includingthe ferric enterobactin uptake system, is under the control ofthe fur gene, whose product is believed to act as an apore-pressor with iron or an iron-related compound acting ascorepressor (12). The observed effect of iron on expressionof the P. aeruginosa ferric enterobactin receptor in E. coliand the identification of a Fur binding site upstream ofpfeAsuggest that the receptor is regulated by Fur in this organ-ism. These data are in agreement with the findings of Princeet al., who utilized polyclonal rabbit serum to identify a Furhomolog in P. aemginosa (42). Failure to completely represssynthesis of the protein in the presence of iron may reflectthe multicopy nature of the cloned receptor gene in E. coli,which might be expected to titrate out the Fur repressor,allowing some gene expression to occur. Indeed, an appar-ent loss of Fur regulation of iron-regulated genes followingcloning on high-copy-number plasmids has been observedpreviously (38). Although regulation of expression of the P.aeruginosa ferric enterobactin receptor by enterobactin wasnot readily observable in iron-limited E. coli cells, perhapsmasked by the high levels of receptor protein being synthe-sized under these conditions, a definite effect of enterobactinon ferric enterobactin receptor expression was observed iniron-replete E. coli cells. The increase in receptor expressionobtained in the presence of added enterobactin mirrors theenterobactin inducibility of the receptor in P. aeruginosa,suggesting that a gene for an enterobactin-dependent posi-tive activator may be present on the cloned 7.1-kb DNAfragment contained in plasmid pCD2. It is interesting to note

that a 3.2-kb fragment of P. aeruginosa DNA carrying theintactpfeA gene, while sufficient for high-level expression ofthe ferric enterobactin receptor in P. aeruginosa, promotesonly detectable synthesis of the protein in E. coli. The factthat the 5.3-kb SstI-SphI fragment directs high-level expres-sion of the receptor protein in E. coli indicates that a gene(s)upstream of pfeA is necessary for full expression of thereceptor. We are currently characterizing the upstreamregion in order to identify this gene(s).

ACKNOWLEDGMENTS

We thank M. McIntosh for strains, E. Griffiths for the polyclonalFepA-specific antiserum, and A. Kropinski for pAK1900.K.P. is the recipient of a Natural Sciences and Engineering

Research Council University Research Fellowship. C.R.D. is therecipient of an NSERC postgraduate scholarship. This work wassupported by an operating grant from the Medical Research Councilof Canada.

REFERENCES1. Berry, D., and A. M. Kropinski. 1986. Effect of lipopolysaccha-

ride mutations and temperature on plasmid transformation effi-ciency in Pseudomonas aeruginosa. Can. J. Microbiol. 32:436-438.

2. Braun, V. 1985. The iron transport systems of Escherichia coli,p. 617-652. In A. N. Martonosi (ed.), The enzymes of biologicalmembranes, vol. 3. Plenum Publishing Corp., New York.

3. Calderwood, S. B., and J. J. Mekalanos. 1987. Iron regulation ofShiga-like toxin expression in Escherichia coli is mediated bythe fur locus. J. Bacteriol. 169:4759-4764.

4. Chart, H., and E. Griffiths. 1985. Antigenic and molecularhomology of the ferric enterobactin receptor protein of Es-cherichia coli. J. Gen. Microbiol. 131:1503-1509.

5. Cornelius, P., N. Moguilevsky, J. F. Jacques, and P. L. Masson.1987. Study of the siderophores and receptors in differentclinical isolates of Pseudomonas aeruginosa, p. 290-306. In G.Doring, I. A. Holder, and K. Botzenhart (ed.), Basic researchand clinical aspects of Pseudomonas aeruginosa. S. Karger,Basel.

6. Cox, C. D. 1980. Iron uptake with ferripyochelin and ferriccitrate by Pseudomonas aeruginosa. J. Bacteriol. 142:581-587.

7. Cox, C. D. 1982. Effect of pyochelin on the virulence ofPseudomonas aeruginosa. Infect. Immun. 36:17-23.

8. Cox, C. D., and P. Adams. 1985. Siderophore activity ofpyoverdine for Pseudomonas aeruginosa. Infect. Immun. 48:130-138.

9. Crosa, J. H. 1984. The relationship of plasmid-mediated irontransport and bacterial virulence. Annu. Rev. Microbiol. 38:69-89.

10. Darzins, A., and M. J. Casadaban. 1989. Mini-D3112 bacteri-ophage transposable elements for genetic analysis of Pseudo-monas aeruginosa. J. Bacteriol. 171:3909-3916.

11. Darzins, A., and M. J. Casadaban. 1989. In vivo cloning ofPseudomonas aeruginosa genes with mini-D3112 transposablebacteriophage. J. Bacteriol. 171:3917-3925.

12. Earhart, C. F. 1987. Ferrienterobactin transport in Escherichiacoli, p. 67-84. In G. Winkelmann, D. van der Helm, and J. B.Neilands (ed.), Iron transport in microbes, plants and animals.VCH Verlagsgellschaft mbH, Weinheim, Germany.

13. Elkins, M. F., and C. F. Earhart. 1989. Nucleotide sequence andregulation of the Escherichia coli gene for ferrienterobactintransport protein FepB. J. Bacteriol. 171:5443-5451.

14. Gilleland, H. E., Jr., J. D. Stinnett, and R. G. Eagon. 1974.Ultrastructural and chemical alteration of the cell envelope ofPseudomonas aeruginosa, associated with resistance to ethyl-enediaminetetraacetate resulting from growth in a Mg2+-deficient medium. J. Bacteriol. 117:302-311.

15. Griffiths, E. 1987. The iron uptake systems of pathogenicbacteria, p. 69-137. In J. J. Bullen and E. Griffiths (ed.), Ironand infection. John Wiley and Sons, New York.

16. Gudmundsdottir, A., P. E. Bell, M. D. Lundrigan, C. Bradbeer,

VOL. 175, 1993

on Septem

ber 11, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

324 DEAN AND POOLE

and R. J. Kadner. 1989. Point mutations in a conserved region(TonB box) of Escherichia coli outer membrane protein BtuBaffect vitamin B12 transport. J. Bacteriol. 171:6526-6533.

17. Hancock, R. E. W., K. Hantke, and V. Braun. 1977. Irontransport in Escherichia coli K12. 2,3-dihydroxybenzoate-pro-moted iron uptake. Arch. Microbiol. 114:231-239.

18. Hantke, K., and V. Braun. 1975. A function common to iron-enterochelin transport and action of colicins B, I, V in Es-cherichia coli. FEBS Lett. 59:277-281.

19. Heinrichs, D. E., L. Young, and K. Poole. 1991. Pyochelin-mediated iron transport in Pseudomonas aeruginosa: involve-ment of a high-molecular-mass outer membrane protein. Infect.Immun. 59:3680-3684.

20. Hoffnan, H., E. Fischer, H. Kraut, and V. Braun. 1986. Prepa-ration of the FhuA (TonA) receptor protein from cell envelopesof an overproducing strain of Escherichia coli K12. J. Bacteriol.166:404 411.

21. Hohnadel, D., D. Haas, and J.-M. Meyer. 1986. Mapping ofmutations affecting pyoverdine production in Pseudomonasaeruginosa. FEMS Microbiol. Lett. 36:195-199.

22. Hohnadel, D., and J.-M. Meyer. 1988. Specificity of pyoverdine-mediated iron uptake among fluorescent Pseudomonas strains.J. Bacteriol. 170:4865-4873.

23. Ichihara, S., and S. Mizushima. 1978. Identification of an outermembrane protein responsible for the binding of the Fe-entero-chelin complex to Escherichia coli cells. J. Biochem. 83:137-140.

24. Konisky, J. 1979. Specific transport systems and receptors forcolicins and phages, p. 319-359. In M. Inouye (ed.), Bacterialouter membranes: biogenesis and function. John Wiley andSons, New York.

25. Konopka, K., A. Bindereif, and J. B. Neilands. 1982. Aerobac-tin-mediated utilization of transferrin iron. Biochemistry 21:6503-6508.

26. Langman, L., I. G. Young, G. E. Frost, H. Rosenberg, and F.Gibson. 1972. Enterochelin system of iron transport in Es-cherichia coli: mutations affecting ferric-enterochelin esterase.J. Bacteriol. 112:1142-1149.

27. Liu, P. V., and F. Shakrani. 1978. Biological activities ofpyochelins: iron-chelating agents of Pseudomonas aeruginosa.Infect. Immun. 22:878-890.

28. Lugtenberg, B., J. Mrujers, R. Peters, P. van der Hoek, and L.van Alphen. 1975. Electrophoretic resolution of the major outermembrane protein of Escherichia coli K12 into four bands.FEBS Lett. 58:254-258.

29. Lundrigan, M. D., and R. J. Kadner. 1986. Nucleotide sequenceof the gene for the ferrienterochelin receptor FepA in Es-cherichia coli: homology among outer membrane receptors thatinteract with TonB. J. Biol. Chem. 261:10797-10801.

30. McIntosh, M. A., S. S. Chenault, and C. F. Earhart. 1979.Genetic and physiological studies on the relationship betweencolicin B resistance and ferrienterochelin uptake in Escherichiacoli K-12. J. Bacteriol. 137:653-657.

31. Meyer, J.-M., D. Hohnadel, A. Khan, and P. Cornelius. 1990.Pyoverdine-facilitated iron uptake in Pseudomonas aeruginosa:immunological characterization of the ferripyoverdin receptor.Mol. Microbiol. 4:1401-1405.

32. Murphy, C. K., V. I. Kalve, and P. E. Klebba. 1990. Surfacetopology of the Escherichia coli K-12 ferric enterobactin recep-

tor. J. Bacteriol. 172:2736-2746.33. Neilands, J. B. 1981. Iron absorption and transport in microor-

ganisms. Annu. Rev. Nutr. 1:27-46.34. Neilands, J. B. 1981. Microbial iron compounds. Annu. Rev.

Biochem. 50:715-731.35. Ozenberger, B. A., M. S. Nahlik, and M. A. McIntosh. 1987.

Genetic organization of multiple fep genes encoding ferricenterobactin transport functions in Escherichia coli. J. Bacte-riol. 169:3638-3646.

36. Pierce, J. R., and C. F. Earhart. 1986. Escherichia coli K-12envelope proteins specifically required for ferrienterobactinuptake. J. Bacteriol. 166:930-936.

37. Pierce, J. R., C. L. Pickett, and C. F. Earhart. 1983. Two fepgenes are required for ferrienterochelin uptake in Eschenichiacoli K-12. J. Bacteriol. 155:330-336.

38. Poole, K. Unpublished observations.39. Poole, K., S. Neshat, and D. Heinrichs. 1991. Pyoverdine-

mediated iron transport in Pseudomonas aeruginosa: involve-ment of a high-molecular-mass outer membrane protein. FEMSMicrobiol. Lett. 78:1-5.

40. Poole, K., L. Young, and S. Neshat. 1990. Enterobactin-medi-ated iron transport in Pseudomonas aeruginosa. J. Bacteriol.172:6991-6996.

41. Postle, K., and J. T. Skare. 1988. Escherichia coli TonB proteinis exported from the cytoplasm without proteolytic cleavage ofits amino terminus. J. Biol. Chem. 263:11000-11007.

42. Prince, R. W., D. G. Storey, A. I. Vasil, and M. L. Vasil. 1991.Regulation of tcxA and regA by the Escherichia coli fur geneand identification of a Fur homologue in Pseudomonas aerugi-nosa PAO103 and PAO1. Mol. Microbiol. 5:2823-2831.

43. Rogers, H. J. 1973. Iron-binding catechols and virulence inEscherichia coli. Infect. Immun. 7:445-456.

44. Rutz, J. M., T. Abdullah, S. P. Singh, V. I. Kalve, and P. E.Klebba. 1991. Evolution of the ferric enterobactin receptor ingram-negative bacteria. J. Bacteriol. 173:5964-5974.

45. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual, 2nd ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

46. Sawatzki, G. 1987. The role of iron-binding proteins in bacterialinfection, p. 477-489. In G. Winklemann, D. van der Helm, andJ. B. Neilands (ed.), Iron transport in microbes, plants andanimals. VCH Verlagsgellschaft mbH, Weinheim, Germany.

47. Schnaitman, C. A. 1974. Outer membrane proteins of Es-cherichia coli. III. Evidence that the major protein of Es-cherichia coli 0111 outer membrane consists of four distinctpolypeptide species. J. Bacteriol. 118:442-453.

48. Sriyosachati, S., and C. D. Cox. 1986. Siderophore-mediatediron acquisition from transferrin by Pseudomonas aeruginosa.Infect. Immun. 52:885-891.

49. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

50. Vieira, J., and J. Messing. 1982. The pUC plasmids, anM13mp7-derived system for insertion mutagenesis and sequenc-ing with synthetic universal primers. Gene 19:259.

51. Yancey, R. J., S. A. L. Breeding, and C. E. Lankford. 1979.Enterochelin (enterobactin): virulence factor for Salmonellatyphimuium. Infect. Immun. 24:174-180.

J. BACT1ERIOL.

on Septem

ber 11, 2018 by guesthttp://jb.asm

.org/D

ownloaded from