biosynthetic analysis of the petrobactin siderophore ... · biosynthetic analysis of the...

JOURNAL OF BACTERIOLOGY, Mar. 2007, p. 1698–1710 Vol. 189, No. 50021-9193/07/$08.00�0 doi:10.1128/JB.01526-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Biosynthetic Analysis of the Petrobactin Siderophore Pathway fromBacillus anthracis�

Jung Yeop Lee,1 Brian K. Janes,2 Karla D. Passalacqua,2 Brian F. Pfleger,1 Nicholas H. Bergman,2,3

Haichuan Liu,4 Kristina Håkansson,4 Ravindranadh V. Somu,5 Courtney C. Aldrich,5Stephen Cendrowski,6 Philip C. Hanna,2 and David H. Sherman1*

Life Sciences Institute and Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 481091; Department ofMicrobiology and Immunology2 and Bioinformatics Program,3 University of Michigan Medical School, Ann Arbor,

Michigan 48109; Department of Chemistry, University of Michigan, Ann Arbor, Michigan 481094; Center forDrug Design, Academic Health Center, University of Minnesota, Minneapolis, Minnesota 554555; and

Medical Technology Program and Department of Microbiology and Molecular Genetics,Michigan State University, East Lansing, Michigan 488246

Received 29 September 2006/Accepted 14 December 2006

The asbABCDEF gene cluster from Bacillus anthracis is responsible for biosynthesis of petrobactin, acatecholate siderophore that functions in both iron acquisition and virulence in a murine model of anthrax. Weinitiated studies to determine the biosynthetic details of petrobactin assembly based on mutational analysis ofthe asb operon, identification of accumulated intermediates, and addition of exogenous siderophores to asbmutant strains. As a starting point, in-frame deletions of each of the genes in the asb locus (asbABCDEF) wereconstructed. The individual mutations resulted in complete abrogation of petrobactin biosynthesis whenstrains were grown on iron-depleted medium. However, in vitro analysis showed that each asb mutant grew toa very limited extent as vegetative cells in iron-depleted medium. In contrast, none of the B. anthracis asbmutant strains were able to outgrow from spores under the same culture conditions. Provision of exogenouspetrobactin was able to rescue the growth defect in each asb mutant strain. Taken together, these data providecompelling evidence that AsbA performs the penultimate step in the biosynthesis of petrobactin, involvingcondensation of 3,4-dihydroxybenzoyl spermidine with citrate to form 3,4-dihydroxybenzoyl spermidinyl cit-rate. As a final step, the data reveal that AsbB catalyzes condensation of a second molecule of 3,4-dihydroxy-benzoyl spermidine with 3,4-dihydroxybenzoyl spermidinyl citrate to form the mature siderophore. This worksets the stage for detailed biochemical studies with this unique acyl carrier protein-dependent, nonribosomalpeptide synthetase-independent biosynthetic system.

In pathogenic bacteria, iron acquisition is a significant en-abling factor in establishing access to and maintaining growthwithin the host and is often required for disease manifestation(29). Typically, bacteria that are unable to obtain physiologicaliron fail to grow and are subsequently eliminated by the host dueto nutrient starvation or direct attack by host defense mechanisms(29). Free iron in body fluids of animals is maintained at very lowlevels by proteins such as transferrin or lactoferrin. This serves asa protective mechanism in animals to reduce the availability of anutrient vital to bacterial growth (4).

As a primary mechanism for iron acquisition, pathogenicbacteria synthesize and employ high-affinity chelating mole-cules (known as siderophores) in order to obtain iron fromhost sources. Siderophores are low-molecular-weight com-pounds that bind ferric iron [Fe(III)] with an extremely highaffinity (3, 32). Once the siderophore has scavenged ferric ironfrom the environment or host, the resulting holo complex bindsto high-affinity receptors and is transported into the bacterialcells by a membrane-associated ATP-dependent transport sys-tem (24).

Most siderophores can be chemically categorized as cat-echolates, hydroxamates, or �-hydroxy carboxylates based ontheir ferric iron ligand-binding functional groups. Many ofthese molecules are polypeptides that are biosynthetically de-rived from the nonribosomal peptide synthetases (NRPS), afamily of multifunctional enzymes that also mediate the bio-synthesis of microbe-derived peptide antibiotics (7). The enzy-mology of NRPS-dependent siderophore biosynthesis has beenstudied extensively over the past decade, and details of theirassembly are well understood (7). Interestingly, several bacte-rial siderophores have been structurally characterized that areNRPS independent, derived from alternating dicarboxylic acidand diamine or amino alcohol building blocks linked by amideor ester bonds (6).

Bacillus anthracis is a gram-positive, spore-forming bacte-rium that can cause serious disease and fatalities in humansand animals. Anthrax can occur after spores enter the hostfollowing inhalation or ingestion or through abrasions in theskin (10). Among the known biological-warfare agents, B. an-thracis is widely accepted as one of the most serious threatsbecause of the resilience of its spores and potential use as abioweapon (19, 20). Recently, Cendrowski and colleagues (5)revealed that six genes in an apparent operon (asb) specifysynthesis of the virulence-associated siderophore in B. anthra-cis. This conclusion was based on the phenotype of a B. an-thracis Sterne asb mutant strain that exhibited severely limited

* Corresponding author. Mailing address: Life Sciences Institute,University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI48109-2216. Phone: (734) 615-9907. Fax: (734) 615-3641. E-mail: [email protected].

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growth in cultured macrophages and was attenuated for virulencein mice (5).

Over the past several years, a number of studies have re-vealed the structural details of unique NRPS-independent bac-terial siderophores. Garner et al. demonstrated that B. anthra-cis Sterne produces a catecholate siderophore based on a novel3,4-dihydroxybenzoate biosynthetic precursor (16). Subse-quently, Koppisch and colleagues reported (23) that the pri-mary siderophore produced by B. anthracis Sterne is identicalto petrobactin, originally isolated from Marinobacter hydrocar-bonoclasticus, a gram-negative bacterium that is capable ofdegrading hydrocarbon compounds (1). The initially reportedstructure of petrobactin indicated that the compound was com-prised of a citrate bis-spermidine backbone, with the two 2,3-dihydroxybenzoyl moieties providing four of the requisite sixdonor groups for Fe(III) (1). Subsequently, total synthesis ofboth 2,3- and 3,4-dihydroxybenzoyl compounds unequivocallydemonstrated that petrobactin utilizes a unique 3,4-dihydroxy-benzoyl fragment, rather than a 2,3-dihydroxybenzoyl donor(2, 15). Wilson et al. then demonstrated (33) that the B. an-thracis asb locus is necessary and sufficient for the productionof petrobactin by B. anthracis (henceforth referred to as

petrobactinBa, to differentiate it from the chemically identicalsiderophore isolated from M. hydrocarbonoclasticus, petro-bactinMh).

Based on these results, we were motivated to establish ex-perimentally the basis for assembly of petrobactin from itsbiosynthetic precursors, including 3,4-dihydroxybenzoate, spermi-dine, and citrate. In this report, we describe the first details on thebiosynthesis of petrobactin by identification and structure eluci-dation of two petrobactinBa intermediates isolated from engi-neered B. anthracis asb mutant strains. In addition, we show theability of individual asb mutants to be complemented followingexogenous supplementation by petrobactinBa, petrobactinMh,and a series of heterologous siderophores. Taken together,these data provide the basis for assigning the order of assemblyand presumed role for five of the six gene products encoded bythe asb operon.

MATERIALS AND METHODS

Chemical materials. Authentic siderophore standards were obtained as iron-free compounds. The hydroxamate-type siderophore aerobactin (see Fig. 1) waspurchased from EMC Microcollections GmbH (Tubingen, Germany). The cat-echolate-type siderophore salmochelin S4 (see Fig. 1) was kindly provided by

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Relevant characteristics Reference

Bacillus anthracisSterne 34F2 Wild type (pXO1�, pXO2�) 31SC107 34F2, �asb::kmr 5BA850 34F2, �asbABCDEF This workBA851 34F2, �asbA This workBA852 34F2, �asbB This workBA853 34F2, �asbC This workBA854 34F2, �asbD This workBA855 34F2, �asbE This workBA866 34F2, �asbF This workBA867 34F2, �asbAB This workBA868 34F2/pHP13 This workBA869 BA851/pHP13 This workBA870 BA851/pBKJ358 This workBA871 34F2/pAD123 This workBA872 BA852/pAD123 This workBA873 BA852/pBKJ359 This work

Escherichia coliXL10-Gold Kan Tetr �(mcrA)183 �(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96

relA1 lac Hte [F� proAB lacIqZ�M15 Tn10 (Tetr) Tn5 (Kanr) Amy]Stratagene

INV110 F� {tra�36 proAB lacIq lacZ�M15} rpsL (Strr) thr leu endA thi-1 lacY galK galTara tonA tsx dam dcm supE4 �(lac-proAB) �(mcrC-mrr)102::Tn10(Tetr)

Invitrogen

Marinobacter hydrocarbonoclasticusDSM 8798 Wild type 1

PlasmidspBKJ258 Allelic exchange vector, Emr This workpBKJ351 �asbA construct cloned in pBKJ258 This workpBKJ352 �asbB construct cloned in pBKJ258 This workpBKJ353 �asbC construct cloned in pBKJ258 This workpBKJ354 �asbD construct cloned in pBKJ258 This workpBKJ355 �asbE construct cloned in pBKJ258 This workpBKJ356 �asbF construct cloned in pBKJ258 This workpBKJ357 �asbAB construct cloned in pBKJ258 This workpHP13 Gram-positive, E. coli shuttle vector; Cmr Emr 17pAD123 Gram-positive, E. coli shuttle vector; Apr Emr 11pBKJ358 asbA cloned into pHP13 This workpBKJ359 asbB cloned into pAD123 This work

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Klaus Hantke at Universitat Tubingen (Tubingen, Germany). All siderophoreswere dissolved in pure water as stock solutions in this study.

Bacterial strains. Bacterial strains used for this study are summarized in Table 1.All B. anthracis strains were derived from the wild-type strain Sterne 34F2 (31)and were propagated at 37°C in LB or LB plus kanamycin (40 �g/ml) as neces-sary. Marinobacter hydrocarbonoclasticus DSM 8798 (ATCC 49840) was obtainedfrom the DSMZ GmbH (Braunschweig, Germany) and was propagated at 28°Cin Bacto marine broth (Difco). XL10-Gold Kan cells were used in DNA cloning/plasmid construction experiments. All plasmid DNA was passaged through thedam dcm INV110 strain prior to introduction into B. anthracis.

Construction of mutants in the asb operon. The mutant strains isolated for thiswork were all constructed via allelic exchange. Each mutant allele was con-structed via PCR (oligonucleotide primer sequences are presented in Table 2)using Phusion High Fidelity DNA polymerase (New England Biolabs) accordingto the manufacturer’s instructions. The PCR product was cloned into the allelicexchange vector pBKJ258 and the DNA sequence verified. Each construct con-tained approximately 500 bp both upstream and downstream of the target gene,with the DNA sequence CCCGGG (the recognition sequence for the restrictionendonuclease SmaI) introduced between these two flanking regions. For the�asbA mutation, the first 18 nucleotides were retained, as well as the last 18 (thisnumber includes the predicted stop codon). This resulted in a deletion of 98%(1,773 out of 1,809 nucleotides) of asbA. A similar strategy was followed for eachmutant. For the �asbB mutation, the initial 21 nucleotides were fused with thefinal 105 nucleotides of the gene, resulting in a deletion of 93% (1,713 out of

1,839 nucleotides) of asbB. For the �asbC mutation, the initial 21 nucleotideswere fused with the final 186 nucleotides of the gene, resulting in a deletion of83% (1,032 out of 1,239 nucleotides) of asbC. For the �asbD mutation, the initial15 nucleotides were fused with the final 30 nucleotides of the gene, resulting ina deletion of 84% (231 out of 276 nucleotides) of asbD. For the �asbE mutation,the initial 18 nucleotides were fused with the final 21 nucleotides of the gene,resulting in a deletion of 96% (945 out of 984 nucleotides) of asbE. For the�asbF mutation, the initial 15 nucleotides were fused with the final 18 nucleo-tides of the gene, resulting in a deletion of 96% (810 out of 843 nucleotides) ofasbF. For the entire operon deletion, �asbABCDEF, the initial 18 nucleotidesof asbA were fused with the final 18 nucleotides of asbF, resulting in a deletionof 99% (7,054 out of 7,090 nucleotides) of the entire operon. Finally, for the�asbAB mutation, the initial 18 nucleotides of asbA were fused with the final 105nucleotides of asbB, resulting in a deletion of 97% (3,583 out of 3,706 nucleo-tides) of the region containing asbA and asbB. Care was taken to avoid deletingsequences important for expression of the downstream gene in these mutants,especially for the �asbB mutant (asbB and asbC are predicted to overlap by 14bp) and the �asbC mutant (asbC and asbD are predicted to overlap by 4 bp).When the gene directly upstream was deleted, the following number of base pairswas retained for each mutant: for asbB (in the �asbA mutant), 76 bp; for asbC,91 bp; for asbD, 182 bp; for asbE, 44 bp; and for asbF, 55 bp. To isolate thevarious mutations described above, allelic exchange was performed as describedpreviously (21) with the following modification: the allelic exchange vectorpBKJ258, which has a larger polylinker region, was used instead of the otherwise

TABLE 2. Oligonucleotide primers used in this study

Targeta Nucleotide sequence (5� to 3�)

�asbA.............................................................................................CGCGCGGCCGCCAAGAGTATCAAGGAATGAAAATAGGACCGCCCCGGGTTGTTTCGCATGCTTCATACTTGCCCTCCGCCCCGGGGTGGCTGTTCGTTCATAAATAAGGTTAAAATTTCCGCGGATCCCTTATTACATCTTTCATATTGTGCAATCGTATC

�asbB .............................................................................................CGCGCGGCCGCCAGCTAAAGGATGGTTATCCGGTCCGCCCCGGGATGATACATATCCATTCTCATACCCCCTCGCCCCGGGATAGATGTGGAATCGCTAGTTCATGAAGTTCCACGCGGATCCCCATAACAATCGGTACTTCATCTATGTCAC

�asbC.............................................................................................CGCGCGGCCGCCATAAAGAAGGATTACCTGTCCGTATTGCCGCCCCGGGTTCTCTATTAACAATTAGCATAATTTCCCCCTCGCCCCGGGATGGGGGAAATTGTTAAAGCGAAAGTAATCCGCGGATCCCTGTTATATCAAAGTCTCCATCCCATAGTCC

�asbD ............................................................................................CGCGCGGCCGCGATGCAACAATACGGTTGTTCTGAAGCCGCCCCGGGCGCTTCCCGTCTCATGTTGTAACCCGCCCCGGGCCGTTACAGGACGTAAATGTGAATAACTAACCGCGGATCCCCCTTCCCAGCGAAAATCAGGATC

�asbE.............................................................................................CGCGCGGCCGCGCAAGAGGCGATTGTATATCGAGGGCGCCCCGGGCACTTTAATTGAAGTCATACTATCATCCACTTTCCGCCCCGGGGAGTCTGTCTTAGTATTTTAAAAACTTTACTGCGCGGATCCGAAGGCAACGTATCCGTTAACGTATTTG

�asbF .............................................................................................CGCGCGGCCGCCAGGAGCCAACAGCAGAAATGGTAGCGCCCCGGGTAGTGAATATTTCATAGGTTTGAACTCCCCGCCCCGGGGAAGTAGTAACTTCTTAATATAAAATGATGAAAGAGCGCGGATCCGGCTATTACATCTTTACTACTTCCCATTC

�asbAB ..........................................................................................AACTAGCGATTCCACATCCCCGGGTTGTTTCGCATGCTTCATACTTGATGAAGCATGCGAAACAACCCGGGGATGTGGAATCGCTAGTTCATG

Diagnosticb ....................................................................................TAACATATGAGCAACGAGAAAAACAATGGGCAAACTCGGAACCATTACCATATTTCTCCCTTCCCCTCGTAATTGCAGATAACGTACCACGGGATCGCACTCGAATCACA

asbA complementation ................................................................CGCGGTACCCAAGAGTATCAAGGAATGAAAATAGGACAAAAGCGCGGTACCTTATGAACGAACAGCCACTTCTCTAACG

asbB complementation ................................................................CGCGGTACCCAAGAGTATCAAGGAATGAAAATAGGACAAAAGATACATATCCATTCTCATACTTGCCCTCCTCTTTCTATAAACACAAGAGGAGGGCAAGTATGAGAATGGATATGTATCATACGAAAATATTGCGCGGTACCTTAACAATTAGCATAATTTCCCCCTG

a Four oligonucleotide primers (i.e., two pairs) were used to generate each allelic exchange construct. One pair (the first two listed) generated a fragment withhomology upstream to the specific gene, and the other pair (the last two listed) generated a fragment with homology downstream to the gene. The entire operon deletion(�asbABCDEF) was constructed using the upstream primer pair from the �asbA set and the downstream primer pair from the �asbF set. The upstream homology forthe �asbAB mutant was generated using the first primer from the �asbAB set and the first primer from the �asbA set; the downstream homology was generated usingthe second primer from the �asbAB set and the fourth primer from the �asbB set.

b The first and third diagnostic primers were used to identify the �asbA, �asbB, and �asbAB mutations; the second and fourth diagnostic primers were used to identifythe �asbC, �asbD, �asbE, and �asbF mutation; the first and second diagnostic primers were used to identify the �asbABCDEF mutation.

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identical pBKJ236. Allelic exchange was verified by PCR, and the resultingmutants are otherwise isogenic to the parental strain. Spores were generated asdescribed previously, except that growth of the cultures was performed at 37°Cinstead of 30°C (28). All subsequent experiments were performed from thesestocks.

Bacterial growth conditions. For growth in iron-depleted conditions, vegeta-tive cells from rich media were washed and inoculated into iron-depleted me-dium (IDM) (5). Overnight cultures started from 1 �l of purified spore prepa-ration were grown in LB, brain heart infusion (BHI), or LB plus kanamycin (40�g/ml) and were diluted 1:10 in fresh medium and allowed to recover for 1 h at37°C. Cells were collected by centrifugation at 3,000 rpm, and the pellets werewashed twice in phosphate-buffered saline followed by a final wash in IDM; theresulting pellet was resuspended in 5 ml of IDM. The optical density at 600 nm(OD600) was determined, and cultures were diluted to an OD600 of 0.01 in a 60-or 70-ml volume of IDM; cultures were shaken at 250 rpm at 37°C, and theOD600 was read every hour for 8 to 10 h. For feeding experiments with exogenoussiderophores, each purified molecule was prepared and added to a final concen-tration of 2 �M. Outgrowth from B. anthracis spores was performed in a micro-plate reader (SpectraMax M2; Molecular Devices) essentially as described pre-viously (25). Spores (5 � 104) were inoculated into 200 �l of medium in a 96-wellmicroplate and incubated at 37°C with near-constant agitation. Growth wasfollowed by measurement of optical density at 600 nm for 10 h.

Disc diffusion assays. Vegetative cells of the wild type and the various asbmutants, grown to mid- to late exponential phase in LB medium, were diluted1:1,000 in sterile water plus 0.7% agar cooled to 45°C. Spores were treated in asimilar manner; purified spores were added to sterile water plus 0.7% agarcooled to 45°C to a final concentration of approximately 105 spores/ml. Threemilliliters of these suspensions was overlaid onto LB -plus-0.5 mM 2,2�-dipyridyl(an iron chelator) agar plates and allowed to solidify for at least 30 min. Sterilepaper discs (6 mm in diameter) infused with water and purified petrobactinBa (2�g and 10 �g in 10 �l water) were placed on the agar overlays and incubated at37°C for 48 h, and zones of growth around the paper discs were measured. Notethat support of growth on these plates is useful only for those compounds thathave an iron affinity comparable to or greater than that of 2,2�-dipyridyl.

Production and isolation of petrobactin. To isolate petrobactinBa, the wild-type B. anthracis strain Sterne 34F2 was grown in IDM (described above) toinduce siderophore production. Five milliliters of vegetative cells grown to mid-exponential phase in LB were used to inoculate 500 ml of IDM. This culture wasthen incubated at 250 rpm at 37°C for 12 to 17 h. Cells were removed from theculture medium using filtration through 0.20-�m-filter flask units (Corning).These filtrates (from 5 liters) were adjusted to pH 7.0 and subsequently appliedto a column packed with Amberlite XAD-16 (Supelco, PA) (500 ml). Thecolumn was washed with pure water several times and then eluted with 100%methanol. All fractions with siderophore activity (see below) were pooled andwere subjected to further purification by preparative high-performance liquidchromatography (HPLC). Highly purified petrobactinBa was obtained by prepar-ative HPLC using a C18 reverse-phase semiprep column (SymmetryPrep C18; 7�m, 7.8 by 300 mm; Waters). The HPLC was performed with a Beckman Coultersystem (Fullerton, CA) with a diode-array detector using a linear stepwise gra-dient from 5% to 50% aqueous acetonitrile in 0.1% (vol/vol) trifluoroacetic acidat a flow rate of 1.5 ml/min over 40 min. The HPLC peaks with siderophoreactivity were collected and lyophilized, resulting in purified petrobactinBa. Thechemical structure of petrobactinBa was verified by Fourier transform ion cyclo-tron resonance (FTICR) mass spectrometry (MS) and nuclear magnetic reso-nance (NMR) spectral analysis. To isolate petrobactinMh, cultures of M. hydro-carbonoclasticus DSM 8798 precultured in Bacto marine broth (Difco) weregrown in a synthetic seawater medium at 28°C for 7 days, as has been describedpreviously (1, 18). After fermentation broths were harvested by centrifugation,XAD-16 resin was added to each supernatant and the mixtures were agitated byshaking for 6 h at 150 rpm in the dark. Each filtered XAD-16 resin was used forfurther isolation of petrobactinMh.

Complementation of �asbA and �asbB mutations. To provide asbA for in transcomplementation, pBKJ358 was constructed. PCR was used to amplify a2,330-bp fragment that contained the entire asbA gene (1,809 bp) and 512 bp ofupstream sequence presumably containing the entire native asb promoter. ThisDNA fragment was cloned into the pCR8/GW/TOPO cloning vector (Invitro-gen) according to the manufacturer’s instructions, and the DNA sequence wasverified. This construct was then transferred into the gram-positive, Escherichiacoli shuttle vector pHP13 as an EcoRI fragment. The resulting plasmid(pBKJ358) was introduced into the �asbA strain using electroporation. To pro-vide asbB for a similar in trans complementation experiment, pBKJ359 wasconstructed. PCR was used to fuse the 512 bp of upstream sequence used inpBKJ358 to the asbB gene (1,839 bp). This PCR product was cloned and se-

quenced in a manner identical to that for the asbA product, transferred into thegram-positive, E. coli shuttle vector pAD123 as a KpnI fragment and introducedinto the �asbB strain using electroporation. The following control strains werealso constructed: the wild type containing each vector (pHP13 or pAD123), the�asbA strain containing pHP13, and the �asbB strain containing pAD123. Eachstrain was grown in IDM as described above, and complementation was shown bythe restoration of petrobactinBa production in each mutant by liquid chroma-tography-mass spectrometry (LCMS) analysis using a Shimadzu LCMS-2010EVsystem. (A detailed description of LCMS analysis can be found below). Furthercomplementation experiments for �asbC, �asbD, �asbE, �asbF, �asbAB, or�asbABCDEF were not conducted.

Isolation of biosynthetic intermediates from Bacillus anthracis asb mutants. Toproduce and analyze metabolic products under iron limitation, the B. anthracisasb mutant strains (�asbA, �asbB, �asbC, �asbD, �asbE, �asbF, �asbAB, and�asbABCDEF strains) were grown in IDM, and filtrates (100 ml) of the growthmedium were generated as described above. To analyze intracellular accumula-tion of the intermediates, cells from culture filtrates were resuspended in 20 mlsterile phosphate-buffered saline buffer (pH 7.4) (without calcium chloride ormagnesium chloride). Cells were subjected to a single freeze-thaw cycle at �20°Cand two cycles at �80°C. Cell suspensions were sonicated six times with 30-spulses at 4°C. Cell lysates were pelleted by centrifugation, and lysate superna-tants were incubated on ice with 18 �l of DNase (RNase free) (180 Un) for 1 h.Cell lysates were then spun by centrifugation at 9,000 � g for 35 min, andsupernatants (10 ml) were collected for analysis. These filtrates and cell lysateswere adjusted to pH 7.0, XAD-16 resin (5 g for filtrates and 1 g for cell lysates)was added, and the mixtures were shaken for 2 h at 150 rpm in the dark. Thesemixtures were next filtered, and the resin was washed with pure water severaltimes and then eluted with 100% methanol. The methanol eluates were concen-trated to dryness in vacuo and dissolved in 80% methanol (1 ml for filtrates and100 �l for cell lysates). The resin extracts from the filtrates of each mutant wereanalyzed using HPLC under the same conditions as for the isolation of petrobac-tin described above. Metabolites were identified by mass spectrometry of HPLC-purified samples and by HPLC analysis or by UV spectral analysis with purifiedpetrobactinBa, using a synthetic sample of 3,4-dihydroxybenzoyl spermidine as astandard. Mass spectra were recorded on a Micromass LCT time-of-flight massspectrometer equipped with an electrospray ionization mode and FTICR MS asdescribed above. The resin extracts from the cell lysates of asb mutants wereanalyzed using a Shimadzu LCMS-2010EV system. (A detailed description ofLCMS analysis can be found below).

LCMS analysis. LC was performed by using a Shimadzu LC-20AD HPLCsystem consisting of a UV/VIS detector (SDP-20AV) and an autosampler (SIL-20AC). The HPLC was coupled to a Shimadzu LCMS-2010EV mass spectrom-eter with an electrospray ionization (ESI) interface. LC was carried out on ananalytical column (Waters XBridge C18; 3.5 �m, 2.1 by 150 mm) utilizing agradient elution with a flow rate of 0.1 ml/min ranging from 5% to 50% aqueousacetonitrile, including 0.1% formic acid, over 30 min. The ESI source was set atthe positive mode. Selected ion monitoring was conducted to monitor ions at m/z719.3, 282.2, and 456.2, which corresponded to the protonated molecular ions ofpetrobactinBa, 3,4-dihydroxybenzoyl spermidine, and 3,4-dihydroxybenzoyl sper-midinyl citrate, respectively. The MS operating conditions were optimized as thefollowing: drying gas, 1.5 liters/min; CDL temperature, 250°C; heat block tem-perature, 200°C and detector voltage, 1.5 kV.

MS and NMR analysis. Gas-phase singly and doubly protonated siderophoresand singly protonated siderophore intermediates were generated by ESI at 70�l/h (Apollo ion source; Bruker Daltonics, Billerica, MA) of a solution contain-ing 10 �M siderophore and 1 �M siderophore intermediates, respectively (1:1methanol:water with or without 0.1% formic acid). To accurately determine themasses of the siderophores, the calibration standard (G2421A; Agilent Technol-ogies, Palo Alto, CA) was mixed with the samples (200-fold dilution of thestandard) for internal calibration. All mass spectra were collected with an ac-tively shielded 7 T FTICR mass spectrometer with a quadrupole front end(APEX-Q; Bruker Daltonics). Structural characterization of siderophores andsiderophore intermediates was performed by sustained off-resonance irradiationcollision-activated dissociation tandem mass spectrometry (SORI-CAD MS/MS). NMR spectra were recorded on a Bruker AMX 500-MHz NMR spectrom-eter (Billerica, MA). The 1H NMR spectrum of siderophores was measured indeuterium oxide at room temperature. The 13C NMR spectrum of siderophoreswas recorded in deuterium oxide using broad-band proton decoupling.

Chemical synthesis of authentic 3,4-dihydroxybenzoyl spermidine. The syn-thesis of 3,4-dihydroxybenzoyl spermidine as a biosynthetic intermediate stan-dard from 3,4-dihydroxybenzoic acid was initiated by benzylation of 3,4-dihy-droxybenzoic acid to afford the corresponding benzyl ester. Direct displacementof benzyl ester by 3-aminopropanol catalyzed by heterocyclic carbene afforded



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3,4-bis(benzyloxy)-N-(3-hydroxypropyl)benzamide under mild conditions (27).This atom-economical process, developed by Movassaghi and Schmidt (27),involves an initial transesterification to benzyl 3,4-bis(benzyloxy)benzoate fol-lowed by a rapid intramolecular N,O-acyl shift to 3,4-bis(benzyloxy)-N-(3-hy-

droxypropyl)benzamide and obviates the use of stoichiometric coupling agents(27). O-iodoxybenzoic acid (IBX)-mediated oxidation of 3,4-bis(benzyloxy)-N-(3-hydroxypropyl)benzamide furnished aldehyde 3,4-bis(benzyloxy)-N-(2-formylethyl)benzamide (13, 14). Next, reductive amination of aldehyde withamine promoted by sodium triacetoxyborohydride provided the spermidine ad-duct N1-[3, 4-bis(benzyloxy)benzoyl]-N8-(tert-butoxycarbonyl)spermidine (13).Sequential deprotection of the benzyl ethers by catalytic hydrogenation to N1-[3,4-dihydroxybenzoyl]-N8-(tert-butoxycarbonyl)spermidine followed by the Boccarbamate with 4 N HCl in dioxane afforded 3,4-dihydroxybenzoyl spermidine.

3,4-Dihydroxybenzoyl spermidine. 1H NMR (600 MHz, CD3OD) 1.70 to 1.90(m, 4H), 1.90 to 2.21 (m, 2H), 3.00 (t, J � 7.2 Hz, 2H), 3.02 to 3.22 (m, 4H), 3.49(t, J � 6.0 Hz, 2H), 6.81 (d, J � 7.8 Hz, 1H), 7.25 (d, J � 8.4 Hz, 1H), 7.32 (s,1H); 13C NMR (150 MHz, CD3OD) 24.5, 25.7, 28.0, 37.3, 40.2, 46.5, 48.3, 115.9,116.0, 120.9, 126.3, 146.5, 150.6, 171.3; HRMS (APCI�) calculated forC14H24N3O3 [M�H]�, 282.1812. Found, 282.1819 (error, 2.4 ppm).

RESULTS

Deduced roles of AsbA and AsbB in biosynthesis ofpetrobactinBa. Database comparisons revealed that AsbA andAsbB are most similar in primary amino acid sequence to IucAand IucC from Escherichia coli (5), gene products that areinvolved in the biosynthesis of aerobactin (Fig. 1). Based ongenetic studies of E. coli mutant strains, iucA was shown tomediate condensation of Nε-acetyl-Nε-hydroxy-lysine with ci-trate, and iucC was shown to be responsible for linkage of asecond Nε-acetyl-Nε-hydroxy-lysine molecule with the interme-diate generated via the iucA gene product (8, 9). Based on theNRPS-independent assembly of petrobactin, the demonstratedrole of asb in its biosynthesis, and the close amino acid se-quence relationships of AsbA/AsbB and IucA/IucC, we devel-oped a biosynthetic scheme for assembly of petrobactinBa (Fig.

FIG. 1. Chemical structures of petrobactin, aerobactin, and salmo-chelin S4.

FIG. 2. Proposed biosynthetic scheme of petrobactinBa. Spermidine and 3,4-dihydroxybenzoic acid are condensed by a combination of AsbC,-D, -E, and -F, although other factors could be involved. The product of this reaction, 3,4-dihydroxybenzoyl spermidine, is condensed with citrateby AsbA to form a second intermediate, 3,4-dihydroxybenzoyl spermidinyl citrate. The product of the AsbA reaction is then condensed with asecond molecule of 3,4-dihydroxybenzoyl spermidine by AsbB to form petrobactinBa.



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2). Specifically, we propose that the siderophore is formed bythe condensation of 3,4-dihydroxybenzoyl spermidine (3,4-DHB-SPD) with an activated form of citrate (coenzyme A orAMP ester) to form 3,4-dihydroxybenzoyl spermidinyl citrate(3,4-DHB-SPD-CT), followed by the condensation of a second3,4-DHB-SPD subunit with 3,4-DHB-SPD-CT (again activatedas coenzyme A or AMP ester) to form the mature siderophore.This scheme is analogous to the assembly of aerobactin and anumber of other siderophores, including rhizobactin 1021, ach-romobactin, vibrioferrin, alcaligin, and desferrioxamine E, thathave similarly annotated genes, forming a family of 40 NRPS-independent siderophore biosynthetic systems (6).

In order to test this hypothesis, in-frame deletion mutantswere generated for each of the six genes of the asb operon (seeMaterials and Methods), as well as a mutant with a deletionthat encompassed the entire operon (Fig. 3). The B. anthracisSterne wild type and each of the mutants were grown underiron-depleted conditions, and the cells were removed by filtra-tion. In order to identify accumulated intermediates from thepetrobactinBa pathway, the filtrate and cell lysate obtainedfrom each culture were prepared for HPLC, LCMS, and massspectrometry analysis (see Material and Methods). As antici-pated, analysis of the B. anthracis wild-type filtrate provided asingle symmetrical HPLC peak corresponding to petrobactinBa

(Fig. 4) with the expected molecular mass [m/z 719.3,(M�H)�]. Interestingly, inspection of the HPLC chromato-grams for the �asbA, �asbB, and �asbAB mutants revealedthat all three contained a new coincident product that was notobserved in the wild type. Mass spectrometry showed thatthe metabolites had identical molecular masses [m/z 282.1,(M�H)�] compared to an authentic standard of 3,4-DHB-SPD. In addition to the peak corresponding to 3,4-DHB-SPD,a single asymmetric peak yielding typical UV spectra for 3,4-DHB was observed from the filtrate of the �asbB mutant (Fig.4). HPLC purification of this metabolite and MS analysis in-dicated [molecular mass m/z 456.1, (M�H)�] that it corre-sponded to the more advanced biosynthetic intermediate 3,4-DHB-SPD-CT. None of the filtrates from the other mutants

(�asbC, �asbD, �asbE, �asbF, and �asbABCDEF mutants)exhibited signals corresponding to either of these precursormetabolites.

Interestingly, close inspection of the metabolite analysisfor each of the mutant strains revealed that a minor productwith the same molecular mass as petrobactinBa [m/z 719.3,(M�H)�] (Fig. 4) was generated from the �asbA mutant.Production of petrobactinBa in a strain lacking AsbA was sur-prising. However, it is conceivable that AsbB (or another un-known enzyme) shares enough functional similarity to AsbAto provide partial catalysis. To test this hypothesis, a mutantlacking both AsbA and AsbB (�asbAB) was generated, anda siderophore metabolite analysis was performed. No ap-parent peak corresponding to petrobactinBa was observedfor filtrates from the �asbAB double mutant strain (Fig. 4).Moreover, analysis of cell lysates from the wild type or the�asbA mutant strain by LCMS revealed that no petrobact-inBa or corresponding biosynthetic intermediates accumu-lated (data not shown). Further studies confirmed that nei-ther 3,4-DHB-SPD nor 3,4-DHB-SPD-CT could be detectedfor the cell lysates of any asb mutant. These data demon-strate that petrobactinBa and its biosynthetic intermediates3,4-DHB-SPD and 3,4-DHB-SPD-CT are effectively ex-creted from B. anthracis cells.

In order to obtain higher-resolution structural informationon the petrobactin biosynthetic intermediates, ESI FTICR MSof HPLC-purified molecules was conducted. Analysis of 3,4-DHB-SPD and 3,4-DHB-SPD-CT from the B. anthracis �asbBmutant showed abundant ion peaks at m/z 282.1811 (for 3,4-DHB-SPD) and 456.1976 (for 3,4-DHB-SPD-CT), respec-tively, which were then assigned as singly protonated ions,indicating the molecular formula C14H23N3O3 (for 3,4-DHB-SPD) or C20H29N3O9 (for 3,4-DHB-SPD-CT) (data not shown).A similar abundant ion peak at m/z 282.1811, corresponding to3,4-DHB-SPD, was obtained from extracts of �asbA and �asbAB(data not shown).

To obtain more-detailed fragmentation patterns and struc-tural information on 3,4-DHB-SPD and 3,4-DHB-SPD-CT,

FIG. 3. Schematic of the various mutants of the asbABCDEF operon. More details for each mutant can be found in Materials andMethods.



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they were subjected to SORI-CAD MS/MS. For singly proto-nated HPLC-purified 3,4-DHB-SPD (Fig. 5A), major fragmentions were detected at m/z 109.03, 137.02, 194.08, 208.10,225.12, and 265.16. The fragment ions at m/z 109.03, 137.02,194.08, and 265.16 could be assigned to the cleavage of oneterminal COC(O) bond, one amide bond, and two aminebonds in 3,4-DHB-SPD (Fig. 5C). From this pattern, 3,4-DHBis added to the 3-carbon end of spermidine, whereas the frag-ment ions at m/z 208.10 and 225.12 are unlikely to result fromthat structure. However, the latter two ions can be producedfrom an isomeric structure in which 3,4-DHB is added to the4-carbon end of spermidine (Fig. 5D). These results suggestthat 3,4-DHB-SPD (Fig. 5C) and its isomeric counterpart (Fig.5D) are synthesized in the �asbA, �asbB, and �asbAB mutantsas the biosynthetic intermediates.

In SORI-CAD MS/MS evaluation of singly protonatedHPLC-purified 3,4-DHB-SPD-CT, major fragment ions atm/z 109.03, 137.02, 194.08, 208.10, 211.11, 265.16, and282.18 (Fig. 5B) were detected. The fragments at m/z 109.03,137.02, 194.08, 211.11, 265.16, and 282.18 can be assigned tocleavage of one terminal COC(O) bond, two amide bonds,and three amine bonds in 3,4-DHB-SPD (Fig. 5E), in which3,4-DHB is attached to the 3-carbon end of spermidine.However, the fragment at m/z 208.10 is unlikely to resultfrom that structure, but it can be formed directly from theisomeric structure of 3,4-DHB-SPD-CT (Fig. 5F), in which3,4-DHB is added to the 4-carbon end of spermidine. Thisresult indicates that the B. anthracis �asbB mutant strainsynthesizes not only 3,4-DHB-SPD-CT (Fig. 5E) but also itschemical isomer (Fig. 5F), as unexpectedly observed in thebiosynthesis of two isomeric forms of 3,4-DHB-SP in �asbA,�asbB, and �asbAB cultures. Significantly, both isomericforms of 3,4-DHB-SP were observed by direct biochemicalconversion to the products (28a).

Addition of selected exogenous siderophores restoresgrowth to asb mutants in iron-depleted medium. While thecharacterized asb mutant (�asb::kmr) had been shown previ-ously to have a growth defect in liquid IDM and failed toproduce petrobactin (5, 33), it remained unclear whether ex-ogenous petrobactin added back to IDM would restore growth.To address this question, exogenous petrobactinBa (2 �M) wasprovided, resulting in nearly complete restoration of the mu-tant strain to wild-type growth levels (Fig. 6A). These studieswere extended by testing a series of heterologous siderophoresagainst the �asb::kmr strain in IDM to establish their ability torestore growth following addition of petrobactinBa, petro-bactinMh, the hydroxamate siderophore aerobactin, and thecatecholate siderophore salmochelin S4 (Fig. 6B). Of the fourexogenously added siderophores, only salmochelin S4 failed toassist the growth of the �asb::kmr mutant, with cultures show-ing the same poor growth kinetics as cultures with no exog-enously added siderophore. As expected, petrobactinBa and

petrobactinMh supplemented �asb::kmr mutant growth in IDMto virtually equivalent levels. Interestingly, the hydroxamatesiderophore aerobactin was also able to restore growth of the�asb::kmr mutant. This suggests that B. anthracis, as shownpreviously with other bacteria (22, 29), maintains the ability toutilize select chemically distinct heterologous siderophores tomeet its iron requirements.

The growth curves in liquid broth described above wereobtained by inoculating media with actively growing vege-tative cultures of wild-type and �asb::kmr strains. However,since the dormant spore form of B. anthracis is the actualinfectious particle, we assessed the ability of purifiedpetrobactinBa to facilitate outgrowth of newly germinatedspores in extreme iron-poor conditions. Accordingly, weemployed assays in which sterile paper discs infused with thetest molecule (siderophore) were placed onto a solid me-dium limited for iron onto which either vegetative cells orspores had been spread.

Table 3 compares the results of the ability of spores andvegetative bacilli of wild-type and �asb::kmr mutant strainsto grow on solid medium containing the iron-chelating agent2,2�-dipyridyl. For these assays, control experiments wereconducted that demonstrated wild-type-level germinationunder all conditions used with no impact on growth due tothe test medium (data not shown) containing an iron-che-lating agent. Exponentially growing vegetative bacilli of theB. anthracis wild type were able to grow as a light lawn onplates containing 0.5 mM 2,2�-dipyridyl, whereas vegetativebacilli of the �asb::kmr mutant strain were not able to prop-agate. Growth of �asb::kmr vegetative cells was restored withexogenously added petrobactinBa or Fe(II)SO4 (not shown),suggesting that wild-type cells that fail to produce petrobact-inBa are unable to overcome the severe iron limitation of thismedium.

In contrast, spores of the B. anthracis wild type were not ableto outgrow on these plates without the addition of exogenouspetrobactinBa or an alternative iron source. Exogenous petro-bactinBa enabled outgrowth of spores of both strains, but thewild-type strain exhibited more vigorous growth in a largerzone than the �asb::kmr mutant. Thus, it appeared that wild-type spores, once they are able to overcome a severely iron-limited growth environment, were able to produce endogenouspetrobactinBa for robust growth. In contrast, the �asb::kmr

mutant (both spores and vegetative bacilli) overcame the ironlimitation to equivalent levels when exogenous petrobactinBa

was provided, but then, unable to produce petrobactinBa, thecells failed to grow further.

Complementation of asbA and asbB. To show that the mu-tations introduced into asbA and asbB did not disrupt down-stream asb gene expression, the individual open reading frameswere assessed for mutant strain complementation and restora-tion of petrobactinBa production. Plasmids carrying either

FIG. 4. HPLC profiles of filtrates from B. anthracis wild-type Sterne 34F2 and asb mutants. PetrobactinBa (1) was isolated from both the wildtype and the �asbA mutant. The two isomers of 3,4-dihydroxybenzoyl spermidine (2 and 2�; the structures are shown in Fig. 5C and D, respectively)were isolated from the �asbA, �asbB, and �asbAB mutants. The two isomers of 3,4-dihydroxybenzoyl spermidinyl citrate (3 and 3�; the structuresare shown in Fig. 5E and F, respectively) were isolated from the �asbB mutant. The mass of the [M�H]� ion obtained by ESI MS analysis is listedbelow each compound.



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asbA or asbB driven by the native asb promoter were intro-duced into the �asbA and �asbB mutants, respectively. Comple-mentation was monitored by the restoration of petrobactinBa pro-duction from the mutant strains by LCMS analysis. As expected,ions corresponding to petrobactinBa (m/z 719.3, [M�H]�)

were identified in extracts from the wild-type strains with vec-tors used for complementation of asbA and asbB. ProvidingasbA in trans restored petrobactinBa production from the�asbA mutant, and similarly, providing wild-type asbB alsorestored petrobactinBa production from the �asbB mutant,

FIG. 5. FTICR MS/MS of 3,4-DHB-SPD and 3,4-DHB-SPD-CT isolated from the B. anthracis �asbB mutant and petrobactinBa from the wildtype. A, SORI-CAD mass spectrum from 3,4-DHB-SPD; B, SORI-CAD mass spectrum of 3,4-DHB-SPD-CT; C and D, assignments of selectedfragments from 3,4-DHB-SPD, suggesting the isomers 2 and 2�; E and F, assignments of selected fragments from 3,4-DHB-SPD-CT, suggestingthe isomers 3 and 3�; G, SORI-CAD mass spectrum of petrobactinBa; H, assignments of selected fragments from petrobactinBa.



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albeit at reduced levels compared to those for the wild type(Fig. 7).

Growth phenotypes of mutations in individual genes of theasb operon. As described above, seven asb mutants were gen-erated, including a deletion of the entire operon and an in-frame deletion for each individual gene. Two of the individualgene mutants accumulated predicted biosynthetic intermedi-ates of petrobactin (3,4-DHB-SPD from the �asbA and �asbBmutants and 3,4-DHB-SPD-CT from the �asbB mutant). Eachof the individual gene deletion mutants were then comparedwith the wild type and the entire operon deletion mutant fortheir ability to grow under a variety of culture conditions. Theeffects on growth in rich medium (BHI) and IDM were ana-lyzed after inoculation with spores (Fig. 8A and B). No dis-cernible growth defect was observed when the mutants weregrown in rich medium; however, none of the seven asb mutantswas able to outgrow under IDM conditions. Thus, outgrowthfrom spores in IDM for each individual gene mutant was thesame as the deletion of the entire operon. The addition ofFe(II)SO4 or purified petrobactinBa (Fig. 8C and D) overcamethis growth defect, leading to wild-type levels in five of themutants (�asbA, �asbB, �asbC, �asbD, and �asbE mutants),with more limited growth for two others (�asbF and �asbAB-CDEF mutants).

DISCUSSION

Iron acquisition by bacteria is a key enabling factor in patho-genesis, and the production of petrobactin is a major route foriron acquisition in B. anthracis under iron-depleted conditions(5). In this work we have demonstrated that the first two genesof the asb operon (asbA and asbB) encode enzymes that specifythe final two steps of petrobactin assembly. This involves cou-pling of 3,4-DHB-SPD and citrate to form 3,4-DHB-SPD-CT,followed by condensation of another molecule of 3,4-DHB-SPD to 3,4-DHB-SPD-CT, resulting in completion of thepetrobactin molecule. Accumulation of 3,4-DHB-SPD-CT inthe �asbB mutant suggests that AsbB mediates this final bio-synthetic step, while the accumulation of 3,4-DHB-SPD in the�asbA mutant suggests that AsbA catalyzes the penultimatebiosynthetic step. These findings correspond to the proposedscheme (Fig. 2) predicted from the structure of petrobactinand proposed initially due to the high sequence similarity be-tween AsbA and AsbB with other related siderophore biosyn-thetic systems (6). While these results imply that AsbC, AsbD,AsbE, and AsbF are collectively responsible for production of3,4-DHB-SPD, detailed biochemical analysis is required toassign a function for each enzyme. The close similarity betweenthe deduced protein sequence of AsbD and thiolation domainsinvolved in polyketide or nonribosomal peptide synthesis pro-vides further insights into the detailed steps involved inpetrobactin biosynthesis (5).

It is notable that the �asbA mutant can still produce lowlevels of petrobactinBa. This finding suggests that AsbB canalso perform (albeit with low efficiency) the penultimate step inpetrobactinBa biosynthesis in the absence of AsbA. The �asbAmutant was engineered to include a deletion of 98% of theopen reading frame, ruling out a partially functional AsbAproviding this activity. The amount of petrobactinBa generatedby this mutant appears to be adequate to overcome the iron-limited conditions inherent in IDM, although a reproduciblelag phase in the growth kinetics suggests that this requires amore extended period than that for the wild type. However,this is not the case for growth of spores from the �asbA mutantstrain under IDM, since this organism was not able to outgrowunder IDM conditions (Fig. 8B).

As expected, the addition of purified petrobactin from eitherB. anthracis or M. hydrocarbonoclasticus overcame the defi-ciency of each asb mutant for growth in IDM. Additionally,

FIG. 6. A. Growth of the �asb::kmr mutant in IDM with or withoutthe addition of purified petrobactinBa. PetrobactinBa (2 �M) alleviatedthe growth defect of the �asb::kmr mutant strain that was unable toproduce this siderophore. B. Growth of the �asb::kmr mutant in IDMsupplemented with purified siderophores. PetrobactinBa, petrobactinMh,and aerobactin partially alleviated the �asb::kmr growth defect. Sal-mochelin S4 failed to alleviate the �asb::kmr growth defect. The indi-cated values are the averages of measurements of three independentgrowth curves, and each error bar corresponds to one standard devi-ation. All siderophore concentrations are 2 �M.

TABLE 3. Growth enhancement of Bacillus anthracis by addition ofpetrobactinBa to 0.5 mM 2, 2�-dipyridyl medium using disc

diffusion assaysa

Strain description

Growth zone (mm) per reagent (n � 5 discs)

H2O 2 �gpetrobactinBa

10 �gpetrobactinBa

Wild type (spores) No growth 26.6 2.1 32.8 1.9�asb::kmr (spores) No growth 17.0 1.7 25.2 0.5Wild type (vegetative) Lawn Lawna Lawna

�asb::kmr (vegetative) No growth 18.2 0.8 24.4 0.9

a Student’s t test comparing results for wild-type and �asb::kmr spores, P 00.01; Student’s t test comparing results for �asb::kmr spores and vegetative cells,P � 0.2.

b Slightly heavier growth along edges of discs.



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FIG. 7. Selected ion monitoring LCMS traces of filtrates from B. anthracis complemented �asbA and �asbB strains at the mass range of m/z719.3 [M�H]�. The peaks at 15.960 to 16.135 min represent petrobactinBa.



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although the structurally related hydroxamate siderophoreaerobactin facilitated growth, the catecholic siderophore salmo-chelin S4 was unable to restore growth of the B. anthracis asbmutant strains. It is likely that petrobactin and aerobactinmight use a similar transport system in B. anthracis due to theirstructural similarity. Previous work has indicated that the B.anthracis bac operon specifies the biosynthesis of a siderophore(bacillibactin) that is structurally similar to salmochelin S4. Abac disruption mutant does not display attenuated virulence inmice and has no growth defect in IDM (5).

To our surprise, SORI-CAD MS/MS fragmentation patternanalysis revealed that each of two isomers of 3,4-DHB-SPDand 3,4-DHB-SPD-CT are formed in asb mutants (Fig. 5). Thecombination of the products of the remaining four biosyntheticgenes, AsbCDEF, must produce these isomers where the 3,4-DHB moiety is attached to the 4-carbon end of spermidine, asopposed to the 3-carbon end that is typical for petrobactin(Fig. 2). Interestingly, the MS/MS fragmentation of petro-bactinBa shows only those ions corresponding to 3,4-DHB-SPD and 3,4-DHB-SPD-CT with 3,4-DHB attached to the3-carbon end of spermidine (Fig. 5G and H). Interestingly,there is no evidence that wild-type B. anthracis Sterne 34F2

accumulates these isomeric precursors (3,4-DHB attachedto the 4-carbon end of spermidine) into an alternative formof petrobactin. Thus, these intermediates are probably de-graded/recycled by an unknown mechanism. Production ofthe alternative isomer suggests that the enzymes responsible

for the synthesis of 3,4-DHB-SPD and 3,4-DHB-SPD-CTare somewhat flexible, an attribute that has been borne outin our preliminary in vitro studies (28a).

The chemistry and biology of bacterial siderophores andtheir role in pathogenesis have been the subject of increasinginvestigation. The resulting mechanistic understanding of themetabolic systems provides numerous opportunities to designsmall molecule inhibitors that block siderophore biosynthesisand hence bacterial growth of pathogenic bacteria in iron-limiting environments. Recently, a bisubstrate nucleoside thatinhibits domain salicylation enzymes (MbtA and YbtE) re-quired for siderophore biosynthesis in Mycobacterium tubercu-losis and Yersinia pestis was designed and synthesized (12, 26,30). This compound dramatically inhibited siderophore biosyn-thesis and growth of M. tuberculosis and Y. pestis under iron-limiting conditions and represents a promising lead compoundfor the development of new antibiotics to treat tuberculosisand bubonic plague (12, 26, 30). These types of inhibitors alsofunction as powerful tools to aid in deciphering the relevanceof siderophores at specific stages of infection by probing tem-poral control of siderophore production for animal models ofbacterial pathogenesis (12). Given the importance of the pro-duction of petrobactin in the virulence of B. anthracis, similarstudies that target gene products of the asb operon could yieldpotential therapeutic agents for the treatment of anthrax in-fections.

FIG. 8. Growth phenotypes of asb mutants. A to D. Growth curves of each mutant when inoculated from spores. For each curve, the growthmedium was as follows: A. BHI (brain-heart infusion) broth; B. IDM; C. IDM supplemented with 100 �M ferrous sulfate; D. IDM supplementedwith 1 �g/ml petrobactinBa.



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ACKNOWLEDGMENTS

We thank Alison Butler for an authentic sample of petrobactin.This research was supported by grants RP1 and DP18 from the NIH

Great Lakes Center of Excellence for Biodefense & Emerging In-fectious Diseases Research and the J. G. Searle Professorship toD.H.S. J.Y.L. was supported by the Korea Research FoundationGrant funded by the government of Korea (MOEHRD; BasicResearch Promotion Fund) (KRF-2003-214-C00118). H.L. is sup-ported through an award to K.H. from the Searle Scholars Program.

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