JOURNAL OF BACTERIOLOGY, Feb. 1986, p. 570-5780021-9193/86/020570-09$02.00/0Copyright C 1986, American Society for Microbiology

Vol. 165, No. 2

Aerobactin Biosynthesis and Transport Genes of Plasmid Co1V-K30in Escherichia coli K-12

VICTOR DE LORENZO, ALBRECHT BINDEREIF, BARRY H. PAW, AND J. B. NEILANDS*Department ofBiochemistry, University of California, Berkeley, California 94720

Received 20 August 1985/Accepted 19 November 1985

The iron-regulated aerobactin operon, about 8 kilobase pairs in size, of the Escherichia coli plasmidColV-K30 was shown by deletion and subcloning analyses to consist of at least five genes for synthesis (iuc, ironuptake chelate) and transport (iut, iron uptake transport) of the siderophore. The gene order iucABCD iutAwas established. The genes were mapped within restriction nuclease fragments of a cloned 16.3-kilobase-pairHindHI fragment. Stepwise deletion and subsequent minicell analysis of the resulting plasmids allowedassignment of four of the five genes to polypeptides of molecular masses 63,000, 33,000 53,000, and 74,000daltons, respectively. The 74-kilodalton protein, the product of gene iutA, is the outer membrane receptor forferric aerobactin, whereas the remaining three proteins are involved in biosynthesis of aerobactin. The33-kilodalton protein, the product of gene iucB, was identified as NE-hydroxylysine:acetyl coenzyme ANE-transacetylase (acetylase) by comparison of enzyme activity in extracts from various deletion mutants. The53-kilodalton protein, the product of gene iucD, is required for oxygenation of lysine. The 63-kilodalton pro-tein, the product of gene iucA, is assigned to the first step of the aerobactin synthetase reaction. The product ofgene iucC, so far unidentified, performs the second and final step in this reaction. This is based on the chemicalcharacterization of two precursor hydroxamic acids (NW-acetyl-NW-hydroxylysine and Nat-citryl-NW-acetyl-NE-hydroxylysine) isolated from a strain carrying a 0.3-kiobase-pair deletion in the iucC gene. The results supportthe existence of a biosynthetic pathway in which aerobactin arises by oxygenation of lysine, acetylation of theNE-hydroxy function, and condensation of 2 mol of the resulting aminohydroxamic acid with citric acid.

In spite of the natural abundance of iron, microorganismshave evolved various routes of assimilation of this essentialelement (26). This is partly because of the inorganic chem-istry of iron which, in an aerobic environment, is quantita-tively insoluble at biological pH and partly because theelement is often complexed with macromolecules such astransferrin. To overcome this natural growth limitation,many pathogenic Escherichia coli strains have adopted aspecial iron assimilation system. This system, originallydiscovered by Williams (35) in E. coli, consists of biosyn-thesis of a siderophore, aerobactin (34), and expression of aspecific outer membrane receptor protein for ferricaerobactin of molecular mass 74 kilodaltons (kDa) (4). Bothcomponents are strongly induced under iron starvation.

Aerobactin-mediated iron assimilation appears to be wide-spread among natural isolates of E. coli (6) and not to belimited to ColV-type large plasmids (32). Aerobactin synthe-sis was also found in Shigella (29) and Salmonella (23)species. In addition to ColV plasmid-encoded functions,ferric aerobactin uptake requires several chromosomal genes(10).To study the mechanism and iron regulation of this high-

affinity iron assimilation system in E. coli, we constructedthe plasmid pABN1 (5). In this plasmid, a 16.3-kilobase-pair(kbp) HindIII-fragment from pColV-K30 carrying all thegenes for aerobactin biosynthesis and transport was clonedin the vector pPlac (5). We have previously identified themajor iron-regulated promoter of the ca. 8-kb aerobactinbiosynthesis and transport gene complex (7).

In this paper, we report the use of various subclones anddeletions of the original 16.3-kbp HindIII fragment of pColV-K30 to analyze expression of the aerobactin biosynthesisand transport operon and to position the genes relative to the

* Corresponding author.

restriction map. The gene coding for acetylation of NE-hydroxylysine was positively identified through use of aqualitative assay for the product, NE-acetyl-N6-hydroxyly-sine. A role for a second gene in the synthetase reaction wasestablished by isolation and characterization of twohydroxamic acids believed to be precursors of aerobactin.Examination of NE-hydroxylysine production by E. coli cellsharboring different plasmid derivatives enabled localizationof the gene which effected the synthesis of this amino acidfrom lysine.

Parts of these results were presented in a brief communi-cation (8) and as an abstract (A. Bindereif and J. B.Neilands, Fed. Proc. 42:2161, 1983).

MATERIALS AND METHODS

Materials. Restriction endonucleases and T4 DNA ligasewere obtained from Bethesda Research Laboratories, Inc.(Gaithersburg, Md.). L-[35S]methionine (600 Ci/mmol) andL-U-'4C-amino acid mixture (about 6 Ci/mmol) were fromAmersham Corp. (Arlington Heights, Ill.), [14C-acetyl]acetyl-CoA (44.1 mCi/mmol) was from ICN Pharmaceuticals Inc.(Irvine, Calif.), and ion exchange resins and gel filtrationmaterials were from Bio-Rad Laboratories (Richmond,Calif.). For thin-layer chromatography, cellulose plates(Macherey-Nagel, Duren, Federal Republic of Germany)were used. Paper electrophoresis was carried out withWhatman Inc. (Clifton, N.J.) no. 1 paper. DL-NE-hydroxy-lysine was chemically synthesized (30). Aerobactin waspurified as previously described (34). The conditionsrecommended by the supplier were used for restrictionenzyme digestions and ligations.

pPlac (5), pUC9 (33), and pACYC184 (22) vector plasmidshave been previously described. Vector pBRAR was con-structed by removing a 0.7-kb fragment from the bla gene ofpBR322 via limited nuclease Bal 31 digestion of PstI-

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AEROBACTIN BIOSYNTHESIS AND TRANSPORT GENES IN E. COLI

linearized pBR322 plasmid DNA and subsequent religation.Plasmids were mapped by single and double digestionsfollowed by analysis of the restriction nuclease fragments byagarose gel electrophoresis (0.7 and 1%) in buffer containing36 mM Tris base, 30 mM NaH2PO4, and 1 mM EDTA.

Construction of plasmids pABN1 and pABN5 was de-scribed previously (5). The usual recombinant DNA tech-niques were used for construction of the remaining plasmids(22). When required, ligation of restriction nuclease frag-ments with incompatible ends was achieved through bluntending the fragments by either filling in the protruding endswith the four deoxynucleoside triphosphates in the presenceof the Klenow fragment of DNA polymerase (22) or with ashort digestion with Si nuclease (22). The resulting blunt-ended fragments were then ligated in an excess of T4 DNAligase. Transformants were screened by a minilysate proce-dure (16).

Bacterial strains, assays, and minicell analysis. All recom-binant plasmids were maintained in E. coli 294 (endA hsdRthi; obtained from M. J. Chamberlin). Aerobactin was testedin culture supernatant by a bioassay as described previously(5), while hydroxamic acids were detected by a chemical test(3). NE-hydroxylysine production was measured with theCsaky assay (14) of the supernatant fluid of the correspond-ing strains grown in LB medium (22), with NH2OH * HCl asthe standard. The E. coli HB101 entfep used for biosassaysof growth under iron-limiting conditions was from our labo-ratory collection. For isolation of minicells, E. coli X1488carrying the various plasmids was used (24).

Purified minicells at an optical density of 0.2 at 620 nmwere labeled in the presence of 200 ,uM 2,2'-dipyridyl witheither 50 ,uCi of L-[355]methionine or 4 ,uCi of L-U-14C-aminoacid mixture per ml and were analyzed by polyacrylamidegel electrophoresis on 10 or 10 to 15% gradient gels (21)followed by treatment with Autofluor enhancer (NationalDiagnostics) and fluorography.

Preparation of cell extracts and qualitative assay of Ne-hydroxylysine:acetyl-CoA Ne-transacetylase (acetylase). E.coli strains were grown for about 18 h at 37°C in Tris-succinate medium (34) containing 10% LB (22). Cells from a1:1 culture were harvested by centrifugation at 11,000 x gfor 10 min, washed once with 10 mM sodium phosphatebuffer (pH 7), suspended in 10 ml of 50 mM sodium phos-phate buffer, and disrupted by sonication. After unbrokencells were removed by centrifugation at 27,000 x g for 10min, the supernatant fluid was centrifuged for 1 h at 40,000rpm to obtain a soluble protein fraction which was used foracetylase assays. Protein concentrations were determinedby the Bradford method (9).The standard acetylase assay contained the following: 50

mM sodium phosphate (pH 7); 0.4 mM DL-N"-hydroxy-lysine; 0.11 mM [14C]acetyl-CoA; and 25 ,g of solubleprotein in a final volume of 25 ,ul. After 1 h at 25°C, thereaction was stopped by placing the mixture on ice. Theproducts were separated by thin-layer chromatography withn-butanol-acetic acid-water (5:1:2 [vol/vol/vol]) as a solventor by paper electrophoresis in 50 mM borate buffer (pH9.3) and visualized by autoradiography. Purified NE-acetyl-NE-hydroxyly-sine, which served as a reference, was de-tected by ferric perchlorate reagent (3).

Isolation and purification of hydroxamic acids. For isola-tion of precursor hydroxamic acids, E. coli 294(pABN11)was grown in Tris-succinate medium (34) containing 10% LB(22) at 37°C for about 48 h. Maximum concentrations of totalhydroxamic acid determined by chemical test (3) werereached 30 h after inoculation. The cells were removed by

centrifugation, and the supernatant fluid was passed throughan anion-exchange resin (AG1-X2; C1-; 50/100 mesh). Oneneutral hydroxamic acid appeared in the flowthrough vol-ume, while another, anionic, form was retained by the resin.

Hydroxamate-positive flowthrough fractions were pooled,adjusted to pH 7, ferrated to maximum color with ferroussulfate, and saturated with ammonium sulfate. The red-brown material was extracted into phenol-chloroform (1:1[wt/wt]), the colored compound was returned to water phaseby dilution of the organic phase with 10 volumes of ether,and the aqueous phase was lyophilized to dryness. Iron wasremoved by extraction of a concentrated aqueous solution offerric hydroxamate with a 1% solution of 8-hydroxyquin-oline in chloroform. The iron-free aqueous phase was lyoph-ilized to dryness. Deferrated neutral hydroxamic acid wasfurther purified by gel filtration on Bio-Gel P2 (Bio-Rad).Hydroxamate-positive fractions were pooled and redis-solved in a very small volume of water. The product wasthen precipitated by addition of ethanol. The final yield ofthe dipolar ion of neutral hydroxamic acid was typicallyabout 50 mg/liter.

Anionic hydroxamic acid was eluted with a linear gradientof 0.1 to 1.0 M LiCl or MgCl2. Hydroxamate-positivematerial appeared in fractions between 0.47 and 0.67 M LiClor 0.1 and 0.185 M MgCl2. The pooled fractions were takento dryness by lyophilization. Excess MgCl2 was removed bydissolving the lyophilized sample in small amounts of waterand addition of excess absolute ethanol; the insoluble frac-tion, containing hydroxamate, was recovered by centrifuga-tion and subsequently taken to dryness. Removal of residualMgCl2 and further purification of anionic hydroxamic acidwere achieved by gel filtration on Bio-Gel P2. Addition ofethanol was omitted prior to gel filtration for the lithium saltof anionic hydroxamic acid. Hydroxamate-positive fractionswere taken to dryness, and the respective magnesium andlithium salts were dissolved in small amounts of water andprecipitated by the addition of ethanol-acetone (3:1 [wt/vol]).The final yield of the magnesium salt of anionic hydroxamicacid was 20 mg/liter of culture fluid. Structural analysesperformed on the purified compounds are described inResults.

RESULTSPolypeptides encoded by the aerobactin system. The genes

for all of the functions required for the synthesis andtransport of aerobactin are carried on a 16.3-kbp HindIIIinsert in pABN1 (5). The smallest subclone of this plasmidfound to encode all of these functions was designatedpVLN1 and contained a restriction fragment reaching fromthe first Hindlll site to a PvuII site some 8.3 kbp down-stream (Fig. 1). Deletions in this fragment between any ofthe restriction nuclease sites mapped in pVLN1 (Fig. 1)resulted in lack of aerobactin production or transport ofiron-aerobactin or both. The HindIII-PvuII fragment waspurified, blunt ended, and recloned into two additionalvectors, pBRAR and pACYC184. In the first case (pBRAR),the fragment was inserted at the PvuII site of the vector toproduce pVLN7, whereas in the second (pACYC184), it wasintroduced in the BamHI-HindIII double-digested and blunt-ended vector, affording pVLN8. A collection of plasmidscarrying leftward sequentially deleted fragments from theinitial 8.3-kbp HindIII-PvuII segment was then obtained byuse of the EcoRI, PvuII, BglII, BamHI, and AvaI restrictionsites, producing, respectively, pABN5, pVLN4, pVLN6,pVLN3, and pABN15. The leftward direction for the dele-tions was selected to retain the main, iron-regulated pro-

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572 DE LORENZO ET AL.

PLASMID VECTOR

pABN 1 pPlaclpVLN1 1 pPlac

pVLN7 IpBRAR_|

pVLN8 PACY

pABN5 IpPlac

pVLN4 I pPlac,

pVLN6

pVLN3

pABN 15

I. .

, ,-II

p_Pac

P1P

- _ -E =co E E C-..-_ mC 0>co> OmO > > 0

< mco<coca<

I 1AdtI ) V

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I I

FEI

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w

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I I -I I

i t t tPROPOSED . E . xMAP L63_K I S 53K 74K

33K

LO)4_ s00 LO I;t to CO r

m *j Ji J m -J *J . m<r > > > <: > > > <r

. a. .Q a a a CL

74K -

63K -- I - wm -

53K- wo,,oIII _

40K-

33K-

]v pBL- t

CAT-

4-

_

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.4 00FIG. 1. Restriction endonuclease maps of leftward deletions of pABN1 and corresponding polypeptide expression in E. coli minicells. The

inserts of all plasmids are lined up relative to the 16.3-kbp HindIll fragment of pABN1 and are represented as heavy lines. The boxesrepresenting the vectors used in each subclone do not necessarily represent their actual size. E. coli X1488 was transformed with the differentplasmids indicated above the lanes. The purified minicells were incubated with [35S]methionine and the polypeptides were analyzed byelectrophoresis on a 10 to 15% gradient polyacrylamide gel, followed by autoradiography (see the text for details). Similar results were

obtained when a "4C-labeled amino acids mix was used instead of [35S]methionine from minicell labeling (data not shown). The proposedlocation of the gene products is indicated in the boxed section below the plasmid maps. P1 represents the main iron-regulated promoter ofthe system and the direction of transcription (from left to right). Major polypeptides encoded by the vector: pBL, pre-,-lactamase; BL,P-lactamase; CAT, chloramphenicol acetyltransferase. The pBL band was variably expressed depending on the construction and labelingconditions of the minicells. The AvaI and BglII restriction sites were mapped only in the fragment corresponding to pABN5.

moter of the gene cluster previously located in the segmentfrom the first HindlIl site to a Sall site some 0.7 kbpdownstream (7). To determine the number and sizes of thegene products and to map the corresponding genes withinrestriction fragments, the individual deletion plasmids wereplaced in the minicell-producing strain E. coli X1488. Theresulting polypeptides, labeled with 35S-methionine or 14C_amino acids, were analyzed in polyacrylamide gels.

Figure 1 shows the profile of polypeptides expressed bythe various plasmids. In pABN1 and in the plasmids carryingthe 8.3-kbp HindIII-PvuII fragment (pVLN1, pVLN7,pVLN8), at least four polypeptides with molecular masses of74, 63, 53, and 33 kDa were detected, regardless of thevector used. A number of minor bands between 33 and 63kDa occasionally appeared, depending on the vector andlabel used. No major differences were found by labeling with14C or 35s.

Analysis of the polypeptides detected from the subsequentreactions allowed mapping of the gene products as follows.Subclone pABN5 produced, besides the vector-codedbands, the same protein pattern as pVLN1, pVLN7, andpVLN8, except that the 74-kDa protein was missing and afaint new band of about 31 kDa appeared just above theP-lactamase product, most likely as the result of the produc-tion of a truncated polypeptide. This truncated polypeptide

did not appear in the further deletion, pVLN4. This meansthat the gene for the 74-kDa protein overlaps the EcoRI siteof pABN1 and is bracketed by the two PvuII sites spacedabout equidistant to the left and right of this site. PlasmidVLN4 srongly expressed a ca. 40-kDa polypeptide whichwas not apparent in any of its antecedents or derivatives,suggesting that it may represent an artifact arising from thevector. A deletion to a BglII point (plasmid pVLN6) did notexpress the 53-kDa protein, although it still showed theremaining peptides. No truncated products from this plasmidwere detected. We therefore mapped the 53-kDa protein in afragment around the two BglII points. In pVLN3, only 63-and 33-kDa polypeptides were detected plus an additionalpeptide of ca. 25 kDa, the latter most likely resulting fromthe interruption of the gene immediately upstream from the53-kDa protein, which was not expressed clearly in theseconstructions. This allows tentative mapping of anotherproduct, provisionally designated the S peptide, in a frag-ment overlapping the first BamHI point and extending al-most to the first upstream BglII point. Finally, pVLN3 andpABN15 expressed, respectively, the 63- plus 33-kDa andthe 63-kDa products, which locate the extremities of the33-kDa polypeptide around the first AvaI and before the firstBamHI sites and place the 63-kDa polypeptide in the firstHindIII-AvaI restriction fragment. We have previously

- 25K

I 11EL-L-

II II II IL I I

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VOL. 165, 1986

pABN5

AEROBACTIN BIOSYNTHESIS AND TRANSPORT GENES IN E. COLI

o E E X 0co > ccu > ao > >

w 1: co63 m11 c53K <:74

63 33--1 S I-t1-53 I1 74

cD NIC) '- 1t-

z z z Z

m m -j m< <: > <:Q. 0a

HYDROXYLYSINEPRODUCTION (/MM)

<10

63K _,

pABN 16 -----I

I1pVLN t 2

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I}11

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340 53KK-- mmmm.

| 150 33K_PBL-

BL- -EmM_

pABN 15 L J-m--- - <10FIG. 2. Ne-hydroxylysine production and minicell analysis of derivatives of pABN5. The fragments from pABN5 carried by pABN16,

pVLN12, and pABN15 are presented lined up with respect to the original plasmid and to the polypeptide map of Fig. 1. The box representingthe vector pUC9 in the construction pVLN12 does not indicate its actual size. The flag indicates the orientation of the lac promoter in thisplasmid. The production of Ne-hydroxylysine by E. coli 294 harboring the different plasmids was detected in the supernatant fluids ofcorresponding overnight cultures in LB medium [E. coli 294(pLVN12)] or LB plus 0.2 mM 2,2'-dipyridyl [E. coli 294(pABN5), E. coli294(pABN15), E. coli 294(pABN16)]. The figures for Ne-hydroxylysine production refer to the micromolar concentration of hydroxylaminewith NH2OH * HCl as the reference in the Csaky assay (14). Note that the lac promoter in pVLN12 is not fully activated under the conditionsdescribed. The production of Csaky-positive material by E. coli 294 carrying the vectors pPlac or pUC9 was also <10 ,uM (data not shown).Minicell analysis of the [35S]methionine-labeled polypeptides encoded by the plasmids was performed in a 10% acrylamide gel, as indicatedin Materials and Methods, and is shown at the right of the figure.

shown that the first structural gene of the operon starts 0.4kbp downstream from the first HindIII point (7).A 0.7-kbp HindIII-SalI deletion in pABN1 or pVLN1,

which removes the main promoter of the aerobactin complex(7), greatly diminished synthesis of any of the previouslymentioned polypeptides in a minicell system (data notshown), although low-level expression of at least part of thedoWnstream genes does take place, since cells carrying thesedeletion plasmids are sensitive to cloacin. This property isconferred by the 74-kDa protein previously shown to be thereceptor for both cloacin and iron-aerobactin (4, 18).We therefore propose the following operon organization

with transcriptional direction from left to right: 63 kDa -> 33

kDa S -+ 53 kDa -* 74 kDa (Fig. 1). A similar conclusion

regarding the transcriptional orientation has been reportedby Braun et al. (11). Further evidence supporting this mapwas provided by analysis of internal deletion derivatives ofpABN5 and pVLN1 and the location of the different func-tions determined by the system.

Functional correlations of gene products. It has been pre-viously reported (5) that pABN5 camries all the genes neces-

sary for aerobactin biosynthesis and lacks only the receptorfor iron-aerobactin. E. coli 294 strains harboring differentderivatives of this plasmid were analyzed to ascertain thelocation of different functions required for aerobactin pro-duction and transport.

(i) Determination of lysine NE-hydroxylase activity. A1.8-kbp Aval deletion of pABN5, named pABNi6 (Fig. 2),was found to excrete NE-hydroxylysine. This constructiondirected in minicells the synthesis of only 63- and 53-kDapolypeptides (Fig. 2). To ascertain a role for thesepolypeptides in the production of NE-hydroxylysine, we

assayed two derivatives, namely, pABN15 (see above) andpVLN12, each of which selectively produced one of theproteins. Plasmid pVLN12 was constructed by cloning theca. 3-kbp BamHI-EcoRI fragment of pABN5 into the corre-

sponding sites of vector pUC9. This placed expression of the53-kDa protein under control of the lac promoter. Detectionof hydroxylysine was positive by the Csaky test in E. coli294 carrying pVLN12 but not pABN15 (Fig. 2), thus indi-cating that only the 53-kDa protein is required for accumu-lation of NM-hydroxylysine. The name iucD is proposed forthe gene corresponding to the 53-kDa peptide.

(ii) Identification of NE-hydroxylysine:acetyl-CoA NE-transacetylase (acetylase). To map on the aerobactin operonthe gene for acetylase activity, a qualitative assay similar tothat described by Kusters and Diekmann (20) was developedand used to compare activities in cell-free extracts preparedfroni various strains containing deletion plasmids (Fig. 3).The acetylase assay was first tested on extracts from E. coli294 (pABN11), a strain capable of synthesizing Ne-acetyl-N8-hydroxylysine in vivo (see below). The formation of theproduct was dependent on extract and substrate (data notshown). By this qualitative assay (Fig. 3), extracts from E.coli 294(pAB3N12) and 294(pABN13) were also found to haveactivity, but no activity was detected in E. coli 294(pABN15)extracts. This indicated that acetylase activity is determinedby the AvaI-BamHI fragment downstream of the first AvaIsite. This location was previously mapped as responsible forthe production of the 33-kDa polypeptide. Further evidencefor the location of acetylase and the identity of the enzymewith the 33-kDa peptide was provided by the fact that highlypurified enzyme (kindly provided by Mark Coy of thislaboratory) isolated from cells carrying pABN11 comigratedwith the 33-kDa band in minicell analysis (Fig. 3). Furtherdetails concertling the purification and characterization ofthis enzyme will be published elsewhere; The gene corre-sponding to the acetylase was designated iucB.

(iii) Mapping of synthetase activity. E. coli cells carryingpABN11, a 0.3-kbp deletion of pABN5, formed noaerobactin by bioassay but excreted into the medium twohydroxamic acids, one neutral and one anionic in charge.

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574 DE LORENZO ET AL.

_ C%i

z zm c

A nl a

pABN 5

pABNl 1

pABN12

DABN13

-~~~~~~~~~~~~~~rcoESou-=

0 -E U > u Co > CD -az C>l 0w 1(0U < MM<mmCoa<w

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FIG. 3. NE-hydroxylysine:acetyl-CoA NE-transacetylase (acetylase) activity in E. coli strains carrying deletion plasmids. The maps of theplasmids examined for acetylase activity are represented with respect to the original plasmid, pABN5, and to the location of the correspondingpolypeptides. Acetylase assays were performed as indicated in the text. Products were separated by thin-layer chromatography and visualizedby autoradiography. The lanes show ['4C]acetyl-CoA (lanes A and F) and the ['IC]-labeled products with extracts from E. coli 294 carryingthe plasmids indicated. The arrow shows the location of authentic NE-acetyl-NE-hydroxylysine (AHL). No intense radioactive spot wasdetected when the acetylase was missing. On the right side of the figure, minicell analysis of the 35S-methionine-labeled polypeptidesprogrammed by the different plasmids are shown with respect to the migration of the purified acetylase (second lane), which is shownCoomassie blue stained in the same 10% acrylamide gel.

The purified neutral hydroxamic acid appeared to beidentical to a monohydroxamic acid previously obtainedfrom a clinical isolate of E. coli (27), which was tentativelyidentified on the basis of color reactions and electrophoreticmobilities at various pHs as NE-acetyl-NE-hydroxylysine.This structural assignment was confirmed definitively by thefollowing analyses. The elemental composition calculatedfor the proposed structure of the neutral hydroxamic aciddipolar (C8H16N204) was: C, 47.05%; H, 7.89%; N, 13.72%;0, 31.34%. The composition determined was: C, 45.28%; H,7.69%; N, 12.87%; 0, 29.62%. The small discrepancies mostprobably arose from contamination by a small amount ofsalt. The nuclear magnetic resonance spectrum of the neutralhydroxamic acid was consistent with the number of protonsand resonance patterns of NE-acetyl-NE-hydroxylysine. Theresults of a proton magnetic resonance spectrum at 250 MHzare presented in Table 1. Mass spectroscopic analysis foundthe parent molecular ion at 205 atomic mass units, corre-sponding to M + 1, which agrees with the proposed molec-ular weight of 204 atomic mass units.At pH 9.3, the neutral hydroxamic acid migrated toward

the anode, which is consistent with a pl of about 6 calculatedfor NE-acetyl-NE-hydroxylysine. Positive staining with ferricperchlorate and neutral ninhydrin sprays confirmed the

TABLE 1. Proton nuclear magnetic resonance spectrum of theneutral hydroxamic acid in deuterium oxide

8a Typeb Proton integration Assignment

1.42 M 2 1-CH21.70 M 2 y-CH21.90 M 2 8-CH22.15 S 3 acetyl-CH33.65 T 2 E-CH23.76 T 1 et-CH

a Relative to sodium-2,2-dimethyl-2-silapentane-5-sulfonate.b S, Singlet; T, triplet; M, multiplet.

presence of hydroxamate and free a-amino groups. Theobserved slower migration rate relative to N8-acetyl-N8-hydroxyornithine is cqnsistent with the lower charge to massratio of Ne-acetyl-NI-hydroxylysine. From the sum of thesedata, it is evident that the neutral hydroxamic acid wasNE-acetyl-N6-hydroxylysine (Fig. 4, I).The purified anionic hydroxamic acid moved on paper

electrophoresis at pH 9.3, as detected by ferric perchlorate,substantially faster than aerobactin. The ninhydrin reactionwas negative, confirming the absence of free amino groups.After acid hydrolysis (6 N HC1; refluxed for 17 h), a productcomigrating with citric acid (detected by alkaline KMnO4,aniline xylose, and p-dimethylanlinobenzaldehyde reagents[31]) and a ninhydrin-positive product comigrating withNM-hydroxylysine were detected. This suggested that the

NH 2 CH3HOOC ' \" N-C

I 11OH 0

(I)

COOHCH3

oL H / \ N-COH ~~~~~~~~Iiit<OH OH|O

COOHCOOH

(HX)FIG. 4. Proposed structures of the two hydroxamic acids found

in the supernatant fluids of E. coli 294(pABN11). 1, N6-acetyl-N8-hydroxylysine (neutral hydroxamic acid). II, Na-citryl-Ne-acetyl-NE-hydroxylysine (anionic hydroxamic acid).

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- I - -=E E- _

-

.- co C cn cmn3I mmc m rr m

pVL

pVL

Elm

R ~~~~~~~~~~~~~~~~~~~~~....YL1.............. ..... ...j33K

[63K S153K I74K ILN11 m

LN13

c o'O0*_I IL

74K -63K-

53K -

33KPBL-BL-

FIG. 5. Construction of plasmids pVLN11 and pVLN13 andexpression of the 74-kDa polypeptide by pVLN13. The 6.3-kbpBamHI fragment cloned in pUC9 to produce pVLN11 is shown linedup with respect to the parent insert in plasmid pABN1 and with thelocation of the encoded polypeptides. The dimensions of the vectorboxes are not to scale. A flag represents the orientation of the lacpromoter in pVLN11, and its depivative, pVLN13. Only the relevantsection and the critical restriction sites for construction of pVLN11and pVLN13 are given. The tninicell analysis of 35S-labeledpolypeptides shown on the right side of the, figure indicate that the74-kDa protein is indeed expressed from pVLN13, which carries theproper insert from pABN1. An additional polypeptide with a sizeslightly greater than that of the 33-kDa protein of pABN1 was alsoproduced by pVLN13. This is most likely a truncated polypeptideresulting from this particular construction.

anionic hydroxamic acid might be citric acid substituted withonly one NM-acetyl-N-hydroxylysine.The proposed molecular formula of the magnesium salt of

the anionic hydroxamic acid (C14H18N2010Mg2 * 6H20) isconsistent with the microelemental analysis. The calculatedcomposition was: C, 31.66%; H, 5.69%; N, 5.28%; Mg,9.15%. The determined composition was: C, 32.02%; H,5.51%; N, 5.00%; Mg, 9.17%. The calculated composition ofthe lithium salt (C14H18N20ioLi4 * 4H20) was: C, 35.46%; H,5.49%; N, 5.91%. The composition found was: C, 35.61%;H, 5.25%; N, 5.85%. In mass spectroscopic analysis, a

molecular ion peak of 379 atomic mass units, correspondingto M + 1 for the free acid, was found, which is consistentwith the proposed molecular formula. All these data stronglysuggest that the anionic hydroxamic acid was Na-citryl-N'-acetyl-N8-hydroxylysine (Fig. 4, II). Chirality was not deter-mined.The accumulation of these precursors of aerobactin indi-

cates that a function related to the synthetase activity, inparticular the addition of a second acetylhydroxylysineresidue to the citrate backbone, maps around the closelyspaced famHI sites of pABN5. The 0.3-kbp deletion altersthis function so that only one N"-acetyl-NM-hdyroxylysine isattached to the citric acid. In minicell analyses, no polypep-tide arising from this operon location was identified unam-

biguously, although this same BamHI deletion in plasmidspVLN1, pVLN4 (not shown), and pVLN5 (Fig. 3) led to theappearance of a new 27-kDa polypeptide, the latter mostlikely representing a truncated product. The gene was des-ignated iucC. By exclusion, the remaining 63-kDa polypep-tide, which maps at the upstream extremity of the operon, ismost probably invQlved in the first step of the synthetasereaction, namely, the process yielding the anionichydroxamate. No experimental evidence is yet available on

this point. The gene coding for the 63-kDa protein was

named icuA.(iv) Transport of aerobactin. To ascertain whether the

74-kDa protein alone was the only plasmid-borne polypep-tide which sufficed for transport of aerobactin, we con-

structed pVLN11 and its derivative, pVLN13 (Fig. 5). Theformer carries an approximately 6.4-kbp BamHI fragment ofpABN1 (coding for the 53- and 74-kDa proteins) inserted inthe BamHI sites of pUC9 in an orientation such that expres-sion of both proteins is directed by the lac promoter. PlasmidpVLN13, a 1-kb BglII deletion of pVLN11, codes uniquelyfor the 74-kDa protein (Fig. 5). We transformed E. coliHBl0lfep ent with these plasmids and scored the transform-ants for growth on nutrient agar plates (15) containing 0.2mM 2,2'-bipyridyl and 0.1 mM aerobactin. Under thoseconditions, only cells possessing either an intact aerobactinsystem or at least the capacity to transport the siderophorewere able to grow (Table 2). Strains carrying either pVLN11or pVLN13 were indeed capable of eventual growth,whereas controls with the fep ent strain devoid of plasmidsor transformed with the vector or with any other construc-tion lacking the 74-kDa protein were not. This suggests thatthe 74-kDa protein, shown by others to be the outer mem-brane receptor for ferric aerobactin, is the only plasmid-coded polypeptide involved in its transport.

DISCUSSION

As pointed out previously (28), biosynthesis of aerobactininvolves oxygenation of lysine, acetylation of NE-hydroxylysine, and condensation of 2 mol of the latter withcitrate in a process requiring not less than three proteins. Inthe present study, we mapped three biosynthetic steps tospecific regions of the aerobactin operon and correlated twoof them with the different polypeptides determined by thecorresponding DNA fragment in pABN1. The results aresummarized in Fig. 6.Using the nomenclature of Williams and Warner (36), iuc

(irop uptake chelator) and iut (iron uptake transport), wepropose a subdivision of the operon into five genes, in theorder iucABCD iutA, which code for polypeptides of molec-ular masses 63 (iucA), 33 (iucB), 53 (iucD), and 74 (iutA)kDa. The polypeptide corresponding to iucC remains to beassigned.The 33-kDa protein accounts for acetylase activity, and

this was confirmed by comparison of the polypeptide patterngenerated by different deletions of pABN5 with the purifiedenzyme. The activity which introduces the oxygen atom onthe N" of lysine, which we assume, by analogy with the

TABLE 2. Growth under iron-limiting conditions of E. coliHB101 fep ent harboring different plasmids

Growth on nutrient broth plates"plus:

Plasmid Insert-determined Ap, Sm,proteins A Ap, Sm, 2,2'-bipyridyl

Sm 2,2'-bipyridyl (0.2 mM),(0.2 mM) aerobactin

(0.1 mM)

pUC9 + _ _pVLN1 63,b 33, 53, 74, Sc' + + +pVLN11 53, 74 + - +pVLN12 53 + - -pVLN13 74 + - +

aTransformants of E. coli HB1O1 fep ent carrying the indicated plasmnidswere scored for growth on nutrient broth agar plates (15) containing 100 ,ug ofampicillin (Ap) and 50 ,ug of streptomycin (Sm) per ml and, where shown, thespecified iron chelators. Plates were incubated for 72 h at 37'C.

bMolecular masses (in kilodaltons) of the peptides coded by the inserts, asdetermined in minicell analyses.cThe molecular mass of this component was not established (see the text).

pABN 1

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576 DE LORENZO ET AL.

NH NH2

HOOC NH a.HOOC NH

OH

Lysine N -hydroxylysine

COOHO H CH,L.NN-C

3

0OH OHO0COOH OH 9N

O H CHCOOH 3

A erobactin

IucA lucB I/CC IucD lutA

pABN lIZIo C0 Iw '

I J It I

m m =m u> co0>~cm a 0

I 1l 1 I

_ I_- I3 E,7E E E> co > 0

oL im.m o

FIG. 6. Proposed pathway of aerobactin biosynthesis. The sequence involves (i) NE-hydroxylation of lysine, (ii) NE-acetylation ofNM-hydroxylysine catalyzcd by the product of iucB, and (iii) condensation of 2 mol of NE-acetyl-NE-hydroxylysine with citric acid. The 74-kDaprotein, the product of iutA, is the outer membrane receptor. Products of the genes are lined up with respect to the fragment identified inpABN1. Only parts of the restriction sites are indicated. The functionally identified proteins are connected to their respective genes by solidlines. Dotted lines are used to link the remaining biosynthetic steps, as revealed by excretion of aerobactin precursors, to gene products thefunctions of which remain to be identified in vitro. The 63-kDa protein (the product of iucA) is tentatively associated with the first step in thesynthetase reaction (see the text).

formation of N&-hydroxyornithine (1, 2, 13), is an oxygenase(25), appears to require only the 53-kDa protein.We mapped a location related to one step in the aerobactin

synthesis reaction (iucC), although we have thus far beenunable to identify the protein specified by this gene. We sawthe appearance of a truncated polypeptide when a 0.3-kbpBamHI deletion was made in its location. The failure todetect the iucC gene product can be attributed to either a lowlevel of expression of the protein or overlapping of its bandwith those arising from other proteins. It is possible thataerobactin synthetase represents a reaction requiring twopolypeptides, the iucC product attaching the second sidechain to the anionic, citrate-containing hydroxamic acid. Inthat case, the first side chain would most likely be insertedby the 63-kDa protein. The 27- (8) and 32-kDa (23) proteinsreported previously as associated with the synthetase prob-ably arise from the particular vector, pPlac.

Differences in expression were found in the polypeptidesof the system. The 63-kDa protein was systematicallyoverexpressed with respect to the other proteins in allconstructions carrying the intact operon (Fig. 1). Further-more, a frameshift mutation in the Sall point of this first genewas observed to largely decrease the expression of all theother peptides as detected in minicells (data not shown).However, a 1.8-kbp AvaI deletion in pABN5 (namedpABN16) expressed the 63- and 53-kDa proteins equally(Fig. 2). These results suggest that additional regulatorysequences are present in the operon besides the main,iron-regulated promoter (7) located immediately upstreamfrom the 63-kDa gene. In particular and in accord with theresults of Carbonetti and Williams (12), a second, weakpromoter might be located directly in front of gene iucD. Theadditional regulation of a gene (53 kDa) involved inoxygenase activity makes for evolutionary logic, since thereis no known biological function for alkyl hydroxylamines

(30); they are immediately acylated and inserted jntosiderophores. The substrate for the synthetases, in contrast,may be quite diverse and range from citrate to peptides, as inthe case of aerobactin and the ferrichromes, respectively.

In the course of this study, two independent geneticanalyses of the aerobactin system were published (12, 15).Excepting the assignment of the third gene of the operon(iucC), which they associate with a variably expressed45-kDa protein, our results are in good agreement with thoseof Carbonetti and Williams (12). These workers, usingTn1000 insertional inactivation of pABN1 and subclones,proposed the requirement of four polypeptides foraerobactin biosynthesis; individual functional assignmentswere not made. Our findings generally agree with the orderof the genes reported in plasmid pColV-K311 by Gross et al.(15). Those authors proposed, from results of TnJ000 muta-genesis and complementation analyses, a transcription se-quence of the aerobactin operon from left to right as follows:acetylase (gene B) -- synthetase (gene C) -- oxygenase

(gene A). The mapping of these functions to the correspond-ing DNA locations is similar to ours, with the exception ofthe span of the oxygenase gene, which we extend at least 1.7kbp downstream of the second AvaI point of the operon, andfailure to identify an additional gene corresponding to thefirst protein of the operon (63 kDa), which we tentativelyassociate with a step in the synthetase reaction.The role of the 74-kDa polypeptide is known (4, 18) to be

that of the receptor for iron-aerobactin. The complete DNAsequence of this gene has been recently reported (19). Weassigned the letter A (iutA) to the gene corresponding to thisreceptor, following the custom established for the fer-richrome (tonA fhuA) and ferric enterobactin (fepA) recep-tors. Our results indicate that the 74-kDa protein is the onlyplasmid-borne function required for aerobactin intake. Aprevious report (17) proposed a role for the 53-kDa protein in

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AEROBACTIN BIOSYNTHESIS AND TRANSPORT GENES IN E. COLI

ferric aerobactin transport, but our data correlate this poly-peptide with the oxygenase reaction. In the plasmid con-structions used by the previous workers, expression of the74- and 53-kDa proteins was under the control of a ratherweak promoter. In our equivalent constructions, pVLN11and pVLN13, the genes corresponding to these proteins aredriven by the strong lac promoter of pUC9. It is ourexperience, shared by others, that the expression of proteinsin minicell preparations cannot be correlated reliably in allcases with physiological events occurring in cells harboringthe particular plasmids under study.A definitive designation for the 53-kDa, S, and 63-kDa

proteins must await studies in vitro on their enzymaticfunctions, which has so far only been achieved with the33-kDa acetylase. In addition, a complete sequence of theoperon will afford an overall view of the organizationalstructure and regulation of the gene complex.

ACKNOWLEDGMENTS

This work was supported in part by Public Health Service grantsAM17146 and AI04156 from the National Institutes of Health and byNational Science Foundation grant PCM 78-12198. V.D.L. was a

postdoctoral fellow of the Consejo Superior de InvestigacionesCientificas of Spain.

ADDENDUM IN PROOFWe have recently found that iucA and iucC genes are

required, respectively, for the attachment of the first andsecond hydroxamate side chains to citrate to complete thesynthesis of aerobactin. The product of the iucC gene wasidentified as a 62-kDa protein (V. de Lorenzo and J. B.Neilands, unpublished data).

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13. Emery, T. 1974. Biosynthesis and mechanism of action ofhydroxamate type siderophores, p. 107-123. In J. B. Neilands(ed.), Microbial iron metabolism. Academic Press, Inc., NewYork.

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24. Meagher, R. B., R. C. Tait, M. Betlach, and H. Boyer. 1977.Protein expression in E. coli minicells by recombinant plasmids.Cell 10:521-536.

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27. Neilands, J. B. 1983. Significance of aerobactin and enterobactinin siderophore-mediated iron assimilation in enteric bacteria, p.284-287. In D. Schlessinger (ed.), Microbiology-1983. Ameri-can Society for Microbiology, Washington, D.C.

28. Parniak, M. A., G. E. D. Jackson, G. J. Murray, and T.Viswanatha. 1979. Studies on the formation of N6-hydroxylysinein cell-free extracts of Aerobacter aerogenes 62-I. Biochim.Biophys. Acta 569:99-108.

29. Payne, S. M. 1980. Synthesis and utilization of siderophores ofShigella flexneri. J. Bacteriol. 143:1420-1424.

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31. Smith, I. 1960. Chromatographic and electrophoretic tech-niques, 2nd ed. Year Book Medical Publishers, Chicago.

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35. Williams, P. H. 1979. Novel iron uptake system specified byColV plasmids: an important component in the virulence ofinvasive strains of Escherichia coli. Infect. Immun. 26:925-932.

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