Functional Features of TonB Energy Transduction Systems ofAcinetobacter baumannii

Daniel L. Zimbler, Brock A. Arivett, Amber C. Beckett, Sharon M. Menke, Luis A. Actis

Department of Microbiology, Miami University, Oxford, Ohio, USA

Acinetobacter baumannii is an opportunistic pathogen that causes severe nosocomial infections. Strain ATCC 19606T utilizesthe siderophore acinetobactin to acquire iron under iron-limiting conditions encountered in the host. Accordingly, the genomeof this strain has three tonB genes encoding proteins for energy transduction functions needed for the active transport of nutri-ents, including iron, through the outer membrane. Phylogenetic analysis indicates that these tonB genes, which are present inthe genomes of all sequenced A. baumannii strains, were acquired from different sources. Two of these genes occur as compo-nents of tonB-exbB-exbD operons and one as a monocistronic copy; all are actively transcribed in ATCC 19606T. The abilities ofcomponents of these TonB systems to complement the growth defect of Escherichia coli W3110 mutants KP1344 (tonB) andRA1051 (exbBD) under iron-chelated conditions further support the roles of these TonB systems in iron acquisition. Mutagene-sis analysis of ATCC 19606T tonB1 (subscripted numbers represent different copies of genes or proteins) and tonB2 supports thishypothesis: their inactivation results in growth defects in iron-chelated media, without affecting acinetobactin biosynthesis orthe production of the acinetobactin outer membrane receptor protein BauA. In vivo assays using Galleria mellonella show thateach TonB protein is involved in, but not essential for, bacterial virulence in this infection model. Furthermore, we observed thatTonB2 plays a role in the ability of bacteria to bind to fibronectin and to adhere to A549 cells by uncharacterized mechanisms.Taken together, these results indicate that A. baumannii ATCC 19606T produces three independent TonB proteins, which ap-pear to provide the energy-transducing functions needed for iron acquisition and cellular processes that play a role in the viru-lence of this pathogen.

Acinetobacter baumannii has emerged as an important humanpathogen associated with a wide range of human infections,

mainly in hospitalized and immunocompromised patients (1, 2).It has become alarmingly clear that A. baumannii clinical isolateshave developed mechanisms of resistance to currently availableantimicrobial chemotherapies, making treatment of serious infec-tions caused by this pathogen a significant challenge in humanmedicine (2–5). Although much is known about antibiotic resis-tance and the epidemiology of infections caused by A. baumannii,the virulence properties and the pathobiology of this emergingpathogen are still poorly understood. Therefore, it is important togain insight into the basic mechanisms this organism utilizes topersist in the host environment. Recent reports from our labora-tory have demonstrated that the virulence of A. baumannii ATCC19606T is dependent on the expression of adherence properties (6)and active acinetobactin-mediated iron acquisition functions (7,8). However, further understanding of these properties is neededin order to elucidate their roles and exploit them as potential tar-gets for new therapeutic approaches.

Iron is essential for the growth and survival of both the humanhost and bacteria. This crucial metal is required as a cofactor forcritical enzymes that are involved in many basic cellular functionsand metabolic pathways, such as electron transport, amino acidand nucleic acid biosynthesis, and protection from free radicals(9–11). In the human host, free iron is tightly controlled and iskept to low levels to prevent possible oxidative damage. Iron issequestered in the host by high-affinity carrier proteins, such asferritin, transferrin, and lactoferrin, or is bound to the protopor-phyrin ring in hemoproteins. In response to iron limitation withinthe host, bacteria express high-affinity iron acquisition systemsthat directly bind these host proteins, or they synthesize and se-

crete ferric-binding compounds known as siderophores, whichremove iron from these host iron pools (9, 11).

The ExbB-ExbD-TonB system provides Gram-negative bacte-ria the energy needed to transport host iron-carrier and iron-siderophore complexes into the periplasm once these complexesare bound to cognate TonB-dependent outer membrane recep-tors (12, 13). This system transduces the proton motive force(PMF) to facilitate the active transport of substrates through theouter membrane. Much work has been done toward understand-ing the mechanism and components of the TonB system in Esch-erichia coli K-12. ExbB and ExbD are inner membrane proteins,with homology to flagellar motor proteins MotA and MotB, thatuse the PMF to generate an energized form of TonB (14). TonB isa periplasmic protein that is anchored into the inner membrane byits hydrophobic N-terminal domain and is associated with bothExbB and ExbD. A rigid proline-rich spacer/periplasmic domainspans the periplasmic space, and a structurally conserved C-ter-minal domain (CTD) interacts with specific regions of TonB-de-pendent receptors located in the outer membrane (12, 15). Theenergized CTD of TonB mediates a conformational change of the

Received 1 May 2013 Returned for modification 1 June 2013Accepted 22 June 2013

Published ahead of print 1 July 2013

Editor: S. M. Payne

Address correspondence to Luis A. Actis, actisla@miamioh.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00540-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00540-13

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TonB-dependent receptors by interacting directly with a con-served hydrophobic five-amino-acid region, termed the TonBbox, located in the N-terminal plug domain of the receptor (16).This leads to the transport of the associated ligand into theperiplasm and the de-energization of TonB by a poorly under-stood mechanism. Currently, three potentially viable models areproposed: the propeller, the pulling, and the periplasmic bindingprotein-assisted models; all of these have limitations preventingconsensus for a single accepted mechanism (16).

All sequenced E. coli strains have several potential TonB-de-pendent receptors and only one set of exbB, exbD, and tonB genes;therefore, these receptors have to compete for the TonB complex(17, 18). Many bacteria overcome competition for TonB by har-boring several distinct copies of genes coding for TonB and/orExbB and ExbD. Pseudomonas syringae has nine different operonscoding for TonB proteins (15). Vibrio cholerae has two differentTonB complexes: TonB1-ExbB1-ExbD1 (subscripted numbersrepresent different copies of genes or proteins), linked to heminutilization functions, and TonB2-ExbB2-ExbD2, associated withgrowth at high osmolarity (19, 20). Pseudomonas aeruginosa con-tains three tonB genes, one of which is important for motility andpilus assembly rather than for iron transport (21). It is becomingapparent that these multiple TonB systems have overlapping aswell as distinct roles, in addition to being linked to virulence inpathogens such as P. aeruginosa (22), Vibrio anguillarum (23), andV. cholerae (20). In this regard, we wanted to explore the func-tion of TonB in A. baumannii. Recent proteomic (24) and tran-scriptomic (25, 26) analyses of A. baumannii have predictedapproximately 20 TonB-dependent receptors, some of whichappear to be iron regulated. Specifically, the function of BauA,an iron-regulated TonB-dependent receptor that transportsferric acinetobactin (27, 28), indicates the presence of genescoding for an active TonB system within the genome of thispathogen. No reports on the features of TonB energy-transduc-ing systems in A. baumannii are present in the literature, eventhough three highly conserved putative tonB genes are pre-dicted in all available A. baumannii genome sequences: twooccur as components of exbB-exbD-tonB operons and one as amonocistronic copy. This study highlights the importance ofthe TonB systems of A. baumannii in iron acquisition and ad-herence as well as their potential role in virulence.

MATERIALS AND METHODSBacterial strains, plasmids, and culture conditions. The bacterial strainsand plasmids used in this work are listed in Table 1. Luria-Bertani (LB)broth and agar (29) were used to maintain all bacterial strains. M9 mini-mal medium (30) was used for growth under chemically defined condi-tions. Iron-rich conditions were produced by adding FeCl3 (dissolved in0.1 N HCl) or hemin (dissolved in 0.1 N NaOH), and iron-deficient con-ditions were produced by adding the synthetic iron chelator 2,2=-dipyridyl(DIP), to LB or M9 medium. When applicable, unless otherwise noted, A.baumannii and E. coli strains were grown in the presence of the followingantibiotics: ampicillin (100 �g/ml), chloramphenicol (20 �g/ml), genta-micin (75 �g/ml), kanamycin (40 �g/ml), tetracycline (20 �g/ml), andtrimethoprim (20 �g/ml).

The MIC of the iron chelator DIP was determined in LB or M9minimal broth containing increasing concentrations of DIP. Cellgrowth was determined spectrophotometrically at 600 nm after over-night (12- to 14-h) incubation at 37°C in a shaking incubator set at 200rpm.

General DNA procedures and sequence analysis. Total DNA was iso-lated by using a miniscale method adapted from previously publishedresearch (31). Plasmid DNA was isolated using a commercial kit (Qiagen).DNA was digested with restriction enzymes as indicated by the supplier(New England BioLabs) and was size fractionated by agarose gel electro-phoresis (29). DNA nucleotide sequences obtained by standard BigDye-based automated DNA sequencing (Applied Biosystems) were examinedand assembled using Sequencher (Gene Codes). Nucleotide and aminoacid sequences were analyzed with DNAStar, BLAST, and analysis toolsavailable through the ExPASy Molecular Biology Server (http://www.expasy.org). The multiple sequence alignment (MSA) of TonB CTDamino acid sequences provided by H. Vogel was assembled using ClustalXand MEGA5 (32). Phylogenetic analysis of the TonB MSA was visualizedusing MEGA5 as described previously (15). The secondary structure ofeach CTD was analyzed using the Jpred Web server (http://www.compbio.dundee.ac.uk/www-jpred/index.html) with the Jalign algorithm.

Functional analysis of A. baumannii tonB-exbB-exbD systems. Theputative tonB-exbB-exbD systems were PCR amplified from A. baumanniiATCC 17978 genomic DNA with Pfu DNA polymerase (Stratagene) andprimer sets 3173 and 3174 (tonB1-exbB1-exbD1.1-exbD1.2 [subscriptednumbers used for exbD1.1 and exbD1.2 represent related but not identicalcopies of exbD]), 3314 and 3315 (tonB2), and 3402 and 3403 (tonB3-exbB3-exbD3) (Fig. 1; see also Table S1 in the supplemental material).These amplicons were cloned into pCR-Blunt, generating pMU672,pMU762, and pMU823, respectively (Fig. 1 and Table 1). Each plasmidwas transformed into E. coli RA1051 (�exbBD::aph �tolQR) or E. coliKP1344 (tonB::bla) separately to test its role in iron acquisition, as de-scribed for the analysis of the AfeABCD iron transport system of Aggre-gatibacter actinomycetemcomitans (33). To test whether exbD1.1 andexbD1.2 are functional, each exbD component was individually inter-rupted by inverse PCR using Phusion DNA polymerase (New EnglandBioLabs) with primers 3451 and 3452 (exbD1.1) or primers 3453 and 3454(exbD1.2) and with pMU672 as the DNA template (Fig. 1A; see also TableS1). The pPS856 SmaI fragment harboring the aacC1 gene, which codesfor gentamicin resistance, was inserted into each amplicon, generatingexbD1.1::aacC1 (pMU849) and exbD1.2::aacC1 (pMU850), respectively(Fig. 1A). The resulting plasmids, pMU849 and pMU850, were trans-formed into E. coli RA1051 to test their roles in iron acquisition underincreased iron chelation conditions. The exbB, exbD, and tonB genes werePCR amplified from E. coli W3110 genomic DNA with Pfu DNA polymer-ase and primers 3179 and 3180 (exbB-exbD) or 3177 and 3178 (tonB) (seeTable S1). The amplicons were cloned into pCR-Blunt to generate thepMU673 and pMU674 plasmids (Table 1), which were transformed intoE. coli RA1051 or E. coli KP1344, respectively. As a negative control,pBR322 was transformed into E. coli W3110, RA1051, and KP1344. Thepresence of the plasmid in each complementing strain was confirmed byrestriction analysis of plasmid DNA isolated from cells cultured overnightwith appropriate antibiotics.

Site-directed insertional mutagenesis of tonB genes. A 4.4-kb frag-ment containing tonB1 and flanking DNA was PCR amplified from A.baumannii ATCC 19606T genomic DNA with Phusion DNA polymeraseand primers 3391 and 3647 (see Table S1 in the supplemental material).The amplicon was cloned into pCR-Blunt to generate plasmid pMU905(Fig. 1A). The amplicon was excised from pMU905 by EcoRI digestionand was subcloned into the EcoRI site of pBCSK�, generating pMU912.The pUC4K HincII fragment harboring the aph gene, which codes forkanamycin resistance, was inserted into the unique PmlI site of tonB1,generating pMU962. The tonB1::aph fragment was excised from pMU962by BamHI and EcoRV digestion. The fragment was end-repaired and wascloned into the SmaI site of pEX100T to generate pMU966 (Fig. 1A).

A 2.8-kb fragment containing tonB2 and flanking DNA was PCR am-plified from A. baumannii ATCC 19606T genomic DNA with PhusionDNA polymerase and primers 3723 and 3724 (see Table S1 in the supple-mental material). The amplicon was cloned into pCR-Blunt to generateplasmid pMU975 (Fig. 1B). The amplicon was excised from pMU975 by

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XhoI digestion and was subcloned into the XhoI site of pBCSK�, gener-ating pMU976. The pPS856 SphI fragment harboring the aacC1 gene,which codes for resistance to gentamicin, was inserted into the uniqueSphI site of tonB2, generating pMU978. The tonB2::aacC1 fragment wasamplified from pMU978 by PCR using primers SK and T7 (Invitrogen)and Phusion DNA polymerase, and the amplicon was cloned into theSmaI site of pEX100T to generate pMU982 (Fig. 1B).

A 3.3-kb fragment containing tonB3 and flanking DNA was PCR am-plified from A. baumannii ATCC 19606T genomic DNA with PhusionDNA polymerase and primers 3403 and 3752 (see Table S1 in the supple-mental material). The amplicon was cloned into pCR-Blunt to generate

plasmid pMU999 (Fig. 1C). The insert was excised from pMU999 by SacIand XhoI digestion and was subcloned into the cognate sites of pBCSK�,generating pMU1003. The pPS856 XbaI fragment harboring the aacC1gene was inserted into the unique XbaI site of tonB3, generatingpMU1008. The tonB3::aacC1 fragment was excised from pMU1008 byPvuII digestion and was cloned into the SmaI site of pEX100T to generatepMU1009 (Fig. 1C).

Triparental matings were conducted using A. baumannii ATCC19606T as the recipient, E. coli DH5� cells containing pMU966 orpMU982 as the donor, and E. coli DH5� cells containing pRK2073 as thehelper. For the generation of a double mutant, ATCC 19606T 3180

TABLE 1 Bacterial strains and plasmids used in this study

Strain or plasmid Relevant characteristic(s)a Source or reference

StrainsA. baumannii

17978 Clinical isolate ATCC19606T Clinical isolate; type strain ATCC19606T s1 basD::aph; 19606T acinetobactin production-deficient derivative; Kmr 2719606T 2234 ompA::EZ::TN�R6K�ori/KAN-2� derivative of 19606T; Kmr 619606T 3069 entA::aph; 19606T acinetobactin production-deficient derivative; Kmr 819606T 3180 tonB1::aph; derivative of 19606T; Kmr This work19606T 3201 tonB2::aacC1; derivative of 19606T; Gmr This work19606T 3202 tonB1::aph tonB2::aacC1; derivative of 19606T; Kmr Gmr This work3180-994 3180 mutant harboring pMU994; Kmr Apr This work

E. coliDH5� Used for DNA recombinant methods Gibco-BRLTOP10 Used for DNA recombinant methods InvitrogenW3110 Wild type; F� � IN(rrnD-rrnE)1 rph-1 53KP1344 W3110 tonB::bla; Ampr 54RA1051 W3110 �exbBD::aph �tolQR; Kmr 55

PlasmidspCR-Blunt II PCR cloning vector; Kmr Zeor InvitrogenpBR322 Cloning vector; Ampr Tetr 56pBCSK� Cloning vector; Cmr StratagenepUC4K Source of aph cassette; Ampr Kmr InvitrogenpPS856 Source of aacC1 cassette; Gmr Ampr 57pEX100T sacB conjugative plasmid for gene replacement; Ampr 58pRK2073 Conjugation helper plasmid; Tmr 59pWH1266 A. baumannii shuttle vector; Ampr Tetr 60pMU672 pCR-Blunt II harboring tonB1-exbB1-exbD1.1-exbD1.2; Kmr This workpMU673 pCR-Blunt II harboring W3110 exbB-exbD; Kmr This workpMU674 pCR-Blunt II harboring W3110 tonB; Kmr This workpMU762 pCR-Blunt II harboring tonB2; Kmr This workpMU823 pCR-Blunt II harboring tonB3-exbB3-exbD3; Kmr This workpMU849 pMU672 with exbD1.1::aacC1; Kmr Gmr This workpMU850 pMU672 with exbD1.2::aacC1; Kmr Gmr This workpMU905 pCR-Blunt II harboring tonB1; Kmr This workpMU912 Insertion of pMU905 cloned into pBCSK�; Cmr This workpMU962 Insertion of aph into pMU912; Cmr Kmr This workpMU966 Insertion of pMU962 into pEX100T; Ampr Kmr This workpMU975 pCR-Blunt II harboring tonB2; Kmr This workpMU976 Insertion of pMU975 into pBCSK�; Cmr This workpMU978 Insertion of aacC1 into pMU976; Cmr Gmr This workpMU982 Insertion of pMU978 into pEX100T; Ampr Gmr This workpMU990 pCR-Blunt II harboring tonB1; Kmr This workpMU994 pWH1266 harboring tonB1; Ampr Tets This workpMU999 pCR-Blunt II harboring tonB3; Kmr This workpMU1003 Insertion of pMU999 into pBCSK�; Cmr This workpMU1008 Insertion of aacC1 into pMU1003; Cmr Gmr This workpMU1009 Insertion of pMU1008 into pEX100T; Ampr Gmr This work

a Ampr, ampicillin resistance; Cmr, chloramphenicol resistance; Gmr, gentamicin resistance; Kmr, kanamycin resistance; Tetr, tetracycline resistance; Tmr, trimethoprim resistance;Zeor, zeocin resistance.

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(tonB1::aph) and E. coli DH5� cells containing pMU982 were used as therecipient and donor strains, respectively, together with E. coli DH5� cellscontaining pRK2073 as the helper. Exconjugants were selected on LB agarplates containing either kanamycin or gentamicin and 15 �g/ml strepto-mycin, which was used as a counterselecting antibiotic. Cells from theseplates were plated onto LB agar containing either kanamycin or gentami-cin plus 5% sucrose and 750 �g/ml ampicillin to ensure the loss ofpMU966 or pMU982, respectively. PCR analysis confirmed the properallelic exchanges using primers 3283 and 3706 (tonB1) and primers 3314and 3725 (tonB2) (see Table S1 in the supplemental material).

Genetic complementation of mutants. The A. baumannii ATCC19606T tonB1 gene was PCR amplified from the parental strain genomewith Phusion DNA polymerase and primers 3749 and 3750 (Fig. 1A),which include BamHI restriction sites (see Table S1 in the supplementalmaterial). The blunt-ended amplicon was ligated into pCR-Blunt and wastransformed into E. coli TOP10. The cloned tonB1 gene was subclonedfrom pMU990 into the E. coli-A. baumannii shuttle vector pWH1266 as aBamHI restriction fragment to generate the cognate derivative pMU994(Fig. 1A). The recombinant derivative was electroporated into the A. bau-mannii ATCC 19606T tonB1::aph derivative 3180 as described previously(34). Transformants that grew after overnight incubation at 37°C on LBagar containing 500 �g/ml ampicillin were tested for their abilities to growunder iron-limiting conditions. The presence of pMU994 in the comple-mented strain was confirmed by restriction analysis of plasmid DNA iso-lated from cells grown in LB broth containing 150 �g/ml ampicillin.

Iron utilization assays, detection of siderophore compounds, anddetection of the BauA acinetobactin receptor protein. Strains weretested for their abilities to use various iron sources by use of bioassays as

described previously (27). Briefly, A. baumannii ATCC 19606T and eachof the isogenic derivatives were inoculated into molten LB agar containingDIP in concentrations that would not allow bacterial growth (300 �M).Sterile paper filter discs were saturated with 10 �l of either FeCl3 (10 mMstock), hemin (10 mg/ml stock), a cell-free supernatant of an M9 over-night culture of ATCC 19606T containing acinetobactin, or sterile water,and the discs were then placed on the surfaces of inoculated agar plates.After 24 h of incubation at 37°C, the plates were observed, and the cellgrowth around individual discs was recorded. Replicate experiments wereperformed three times, with a fresh biological sample each time.

The presence of iron-regulated extracellular phenolic compounds inM9 culture supernatants was detected by the Arnow colorimetric assay(35). Siderophore utilization bioassays were used to test the production ofacinetobactin by using the ATCC 19606T acinetobactin-deficient deriva-tive s1 as a reporter strain (27). After 24 h of incubation at 37°C, thediameters of growth halos were quantified. The production of the acin-etobactin outer membrane receptor protein BauA was examined by West-ern blotting with specific polyclonal antibodies as described previously(27).

Transcriptional analysis of tonB expression. Total RNA was isolatedfrom three independent combined 1-ml cultures of the parental ATCC19606T strain or the tonB1::aph derivative 3180 grown overnight at 37°C inLB medium supplemented with 100 �M FeCl3 or 100 �M DIP by usinghot phenol as described previously (36). Each RNA sample was furtherpurified using the Qiagen RNeasy total-RNA isolation system, includingthe RNase-free DNase in-column treatment, before amplification by one-step reverse transcriptase PCR (RT-PCR) (Qiagen). Primers 3387, 3388,3389, 3390, 3391, 3392 (tonB1-exbB1-exbD1.1-exbD1.2), and 3412, 3413,

FIG 1 Genetic maps of A. baumannii ATCC 19606T TonB systems. Shown is the genetic organization of the ATCC 19606T DNA regions harboring the TonB1 (A),TonB2 (B), and TonB3 (C) transducing system genes. The designations of the recombinant plasmids used in this work are given on the left. DNA positions (in base pairs)are given at the top of each panel. Horizontal arrows indicate the locations of predicted coding regions and their directions of transcription. Below the coding regions, thechromosomal regions that were PCR amplified and were used to generate the recombinant plasmids are shown, with the primers used for PCR amplification. Verticalbars indicate the insertion of the aph or aacC1 gene, coding for kanamycin or gentamicin resistance, respectively, at the PmlI (P), SphI (S), or XbaI (X) restriction site.Numbers above or below vertical bars indicate the locations of primers used for inverse PCR mutagenesis (3451 and 3452, 3453 and 3454), RT-PCR (3387 to 3392, 3412to 3415), or qRT-PCR (3283 and 3588, 3788 and 3789, 3790 and 3791). (D) Locations of the three tonB loci in the ATCC 19606T chromosome.

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3414, and 3415 (tonB3-exbB3-exbD3) (Fig. 1; see also Table S1 in the sup-plemental material) were used to amplify the intergenic regions of eachpredicted polycistronic operon. The amplicons were analyzed by gel elec-trophoresis and were sequenced (29). PCR of total RNA without reversetranscriptase and with the appropriate set of primers was used to test RNAsamples for DNA contamination, and PCR with no template served as anegative control.

Gene transcription under iron-replete and iron-depleted conditionswas measured by the Qiagen real-time one-step QuantiTect SYBR greenquantitative RT-PCR (qRT-PCR) system according to the manufacturer’srecommendations and as described previously (37). A 166-bp internalfragment of tonB1 was amplified with primers 3283 and 3588 (see Table S1in the supplemental material); a 154-bp internal fragment of tonB2 wasamplified with primers 3788 and 3789 (see Table S1); and a 133-bp inter-nal fragment of tonB3 was amplified with primers 3790 and 3791 (seeTable S1). Amplification of a 155-bp internal fragment of the constitu-tively expressed gene recA with primers 3545 and 3546 (see Table S1)served as an internal control, while a 124-bp internal fragment of bauAwas amplified with primers 3273 and 3353 (Table S1) to serve as an iron-regulated positive control. The tonB1, tonB2, tonB3, and bauA transcriptlevels, calculated from a standard curve for each sample, were normalizedto recA cDNA levels under iron-replete and iron-depleted conditions.Samples containing no template or no reverse transcriptase served as neg-ative controls. Experiments were performed twice in triplicate, using totalRNA extracted from fresh biological samples each time.

Galleria mellonella killing assays. The killing of Galleria mellonellalarvae was determined as described previously (7). Bacterial cells previ-ously grown for 24 h in LB broth were collected by centrifugation, washed,and suspended in phosphate-buffered saline solution (PBS). The controlgroups included larvae that either were not injected or were injected withsterile PBS or with PBS supplemented with 100 �M FeCl3. The test groupsincluded larvae infected with the parental strain, ATCC 19606T, or withthe isogenic mutant 3069, 3180, 3201, or 3202 (Table 1), which wereinjected in the absence or presence of 100 �M FeCl3. Because mutant 3069had an iron utilization deficiency due to inactivation of the entA acineto-bactin-biosynthetic gene (8), it served as a negative control. After injec-tion, the larvae were incubated at 37°C in darkness, and death was assessedat 24-h intervals over 6 days. Caterpillars were considered dead, and wereremoved, if they displayed no response to probing. The results of any trialwere omitted if more than two deaths occurred in the control groups. Theexperiments were repeated three times using 10 larvae per experimentalgroup (n 30), and the resulting survival curves were plotted using theKaplan-Meier method (38). P values of �0.05 were considered statisti-cally significant for the log rank test of survival curves (SAS Institute Inc.,Cary, NC).

Fibronectin-binding assay. Fibronectin-binding assays were per-formed as described previously (39), with some modifications. Briefly, asterile 96-well culture plate was coated overnight at 4°C with 125 �l perwell of fibronectin (10 �g/ml), bovine serum albumin (BSA) (20 �g/ml),or PBS (39). Bacterial cells were grown for 24 h in LB medium at 37°C withshaking at 200 rpm, collected by centrifugation at 15,000 rpm for 5 min,washed twice, and resuspended with sterile PBS to a comparable opticaldensity at 600 nm (OD600). The coated 96-well plate was seeded with 50 �lof approximately 105 bacteria per well (either the parental strain, ATCC19606T, or the isogenic mutant 2234, 3180, 3201, or 3202 [Table 1]) andwas incubated for 3 h at 24°C. Inocula were estimated spectrophotometri-cally by the OD600 and were further confirmed by plate counts. The wellswere washed six times with sterile PBS to remove unbound bacteria. Ad-herent bacteria in each well were collected with 125 �l of sterile PBScontaining 0.1% Triton X-100 and were vortexed for 30 s, serially diluted,and plated on nutrient agar. Following incubation at 37°C for 24 h, theCFU were counted, and the number of bacteria that attached to wellscoated with immobilized fibronectin or BSA, or to non-protein-coatedwells, was calculated. The number of attached bacteria found under eachcondition tested was normalized to the number of ATCC 19606T cells

attached to BSA-coated wells. Counts were compared using the Student ttest; P values of �0.05 were considered significant. Replicate experimentswere performed three times in triplicate, using fresh biological sampleseach time.

A549 cell adherence assay. A549 human alveolar epithelial cells werecultured and maintained in Dulbecco’s modified Eagle medium (DMEM)supplemented with 10% heat-inactivated fetal bovine serum at 37°C in thepresence of 5% CO2 as described previously (6). Monolayers were infectedin modified Hanks’ balanced salt solution (mHBSS; the same as HBSS butwithout glucose) as described previously (37). Briefly, uncoated 24-welltissue culture plates were seeded with approximately 105 A549 cells andwere incubated for 16 h. Bacteria were grown for 24 h in LB medium at37°C with shaking at 200 rpm, collected by centrifugation at 15,000 rpmfor 5 min, washed twice, and resuspended with sterile mHBSS. For bacte-rial adherence, A549 monolayers were singly infected with 105 cells of theparental strain, ATCC 19606T, or the isogenic mutant 2234, 3180, 3201, or3202 (Table 1) for 2 h in mHBSS. Inocula were estimated spectrophoto-metrically by the OD600 and were confirmed by plate counts. The infectedmonolayers were washed five times with sterile mHBSS, lysed with 500 �lof sterile distilled water (dH2O) containing 0.1% Triton X-100, seriallydiluted, and plated on nutrient agar. The numbers of CFU reflecting bac-teria attached to A549 cells were recorded after overnight incubation at37°C. The number of attached bacteria for each strain tested was normal-ized to the number of ATCC 19606T bacteria attached to A549 cells.Counts were compared using the Student t test; P values of �0.05 wereconsidered significant. Replicate experiments were performed three timesin triplicate, using fresh biological samples each time.

RESULTSAnalysis of the A. baumannii ATCC 19606T TonB systems. Insilico searches of the A. baumannii ATCC 19606T genome (Baumanno-Scope Project Information; https://www.genoscope.cns.fr/agc/microscope/about/collabprojects.php?P_id8) for genes encod-ing proteins with functions related to TonB (Pfam 03544) in theirC-terminal domains (CTDs) identified three coding regions,two occurring as components of predicted tonB-exbB-exbDoperons and one occurring as a monocistronic copy (Fig. 1A toC), located far apart from each other in the chromosome of thisstrain (Fig. 1D). The predicted tonB1-exbB1-exbD1.1-exbD1.2

(ACIB1v1_190012 to -190015) operon is flanked by an up-stream open reading frame (ORF) transcribed in the same di-rection, coding for a predicted TonB-dependent outer mem-brane receptor, while the downstream ORF, transcribed in theopposite direction, codes for a predicted acetyltransferase fam-ily protein (Fig. 1A). This predicted system contains four genes:tonB1 (ACIB1v1_190012), coding for a 26-kDa protein, exbB1

(ACIB1v1_190013), coding for a 31.1-kDa protein, and twoexbD components, exbD1.1 (ACIB1v1_190014) and exbD1.2

(ACIB1v1_190015), coding for 15.2- and 17.3-kDa proteins,respectively. A second, monocistronic tonB locus, tonB2

(ACIB1v1_570149), predicted to code for a 28.6-kDa protein, isflanked by an upstream ORF transcribed in the opposite direction,coding for a hypothetical protein, and a downstream ORF tran-scribed in the opposite direction, coding for a metal-dependenthydrolase protein (Fig. 1B). A third TonB genetic system, tonB3-exbB3-exbD3 (ACIB1v1_360047 to -360045), is located betweenan upstream ORF transcribed in the same direction, coding forformyltetrahydrofolate deformylase (purC), and a downstreamORF transcribed in the opposite direction, coding for a peptide-methionine (S)-S-oxide reductase (msrA) (Fig. 1C). This pre-dicted system contains three genes: tonB3 (ACIB1v1_360047),coding for a 27.4-kDa protein; exbB3 (ACIB1v1_360046), coding

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for a 24-kDa protein; and exbD3 (ACIB1v1_360045), coding for a14.7-kDa protein. The in silico genomic searches also showed thatthere is synteny, along with significant amino acid sequence sim-ilarity, between the three TonB systems shown in Fig. 1 and thosepresent in the chromosomes of A. baumannii strains AYE,ACICU, AB0057, ATCC 17978, and SDF and the environmentalisolate Acinetobacter baylyi ADP1. It is noteworthy that a fourth,hypothetical tonB gene has been identified as part of a gene cluster,potentially involved in iron acquisition, in A. baumannii strainsACICU, SDF, and AB0057 but not in the genome of strain ATCC19606T (25).

The CTD is the best-understood and best-conserved region ofthe three TonB domains (15). Accordingly, the predicted second-ary structure of each ATCC 19606T TonB CTD, as determined byJpred, shows that all three contain two �-helices and three �strands (data not shown), a pattern consistent with the CTD struc-tures of other known TonB proteins (15). BLASTP analysis of theCTD from each ATCC 19606T TonB protein suggests a differentlineage for each of them. The CTD of TonB1 is most closely relatedto the TonB from Leptothrix cholodnii (51% identity; 65% simi-larity); the CTD of TonB2 is most closely related to the TonB fromPsychrobacter arcticus (59% identity; 74% similarity); and theCTD of TonB3 is most closely related to the TonB from Bordetellapetrii (51% identity; 65% similarity). From previous bioinfor-matic and phylogenetic analyses of the CTDs of 263 differentTonB sequences from 144 Gram-negative bacteria, nine differentCTD clusters have been established (15). The multiple sequencealignment (MSA) of the TonB CTD sequences reveals differencesbetween the CTDs of the three ATCC 19606T TonB proteins (Fig.2). A highly conserved YP motif found in the beginning of theTonB CTD, identified only in TonB1 and TonB2 from ATCC19606T, has been shown to be in close contact with the TonB boxesof outer membrane protein receptors, as shown in the crystalstructures of FhuA-TonB and BtuB-TonB complexes (15). Aconserved SSG motif identified in numerous different TonBsequences and identified in each ATCC 19606T TonB is be-lieved to play a role in outer membrane receptor recognition.Based on the neighbor-joining bootstrap tree (see Fig. S1 in thesupplemental material) generated from the MSA shown in Fig.2, the CTD of each ATCC 19606T TonB protein falls into adifferent phylogenetic cluster. These results suggest that each

tonB gene found within the A. baumannii genome is a distinctgenetic unit and that these genes could have been acquiredfrom unrelated sources.

The short intergenic regions between the ORFs in tonB1-exbB1-exbD1.1-exbD1.2 and tonB3-exbB3-exbD3 (Fig. 1) suggest that thesegenes are cotranscribed. The polycistronic transcription of thesegenes was confirmed by reverse transcriptase PCR (RT-PCR) us-ing total RNA from ATCC 19606T cells grown under iron-de-pleted conditions with primers (see Table S1 in the supplementalmaterial) designed to amplify the intergenic regions between eachpredicted ORF shown in Fig. 1A and C. The resulting ampliconsexhibited the predicted sizes and nucleotide sequences, indicatingthat each of these predicted tonB operons is expressed as a singlemRNA unit under conditions of iron limitation (Fig. 3A). Takentogether, these results show that A. baumannii ATCC 19606T

codes for and expresses three different putative TonB energy-transducing complexes, which could provide the necessary energyto a variety of TonB-dependent receptors predicted to be on thesurface of the A. baumannii cell. However, it is not known whethereach protein complex works as an independent, complete unit oras hybrid complexes where different components of each proteinsystem work in different combinations because of potential func-tional cross talk between functionally equivalent proteins. Thispossibility is particularly interesting when one considers TonB2,which could interact with either the ExbB1-ExbD1.1-ExbD1.2 orExbB3-ExbD3 protein complex or with different protein permu-tations of these two protein complexes that allow the expression ofenergy transduction functions needed for iron acquisition.

Differential expression of each A. baumannii tonB gene. Be-cause of their roles in iron acquisition, the production of the TonBenergy-transducing system components is subject to iron regula-tion; in E. coli, under iron-depleted conditions, the abundances ofthese proteins increase 2.5- to 3-fold because of transcriptionalregulation (40). Accordingly, the role each distinct tonB genecould play in iron acquisition was investigated by examining theirdifferential transcription in response to available free iron in theculture medium. qRT-PCR analysis of RNA extracted from ATCC19606T cells cultured in LB broth with the addition of 100 �M DIPshowed a 4.5-fold increase in tonB3 transcription over that forRNA isolated from cells grown in LB broth with 100 �M FeCl3(Fig. 3B). However, no significant changes in the transcription of

FIG 2 Alignment of TonB C-terminal domains. Shown are representative sequences from each of the nine CTD clusters of Gram-negative TonB proteins,including the three TonB proteins from ATCC 19606T (asterisked). Regions of similarity are shaded based on their degrees of similarity. The highly conserved YPand SSG motifs are shaded and marked with asterisks above the alignment.

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tonB1 or tonB2 in response to the availability of free iron in themedium were observed (Fig. 3B). These results correspond to arecent global transcriptomic analysis of A. baumannii ATCC17978 cells cultured under iron-rich and iron-chelated condi-tions, which found a 7.17-fold difference in tonB3 transcription inresponse to free iron (26). As a positive control, the qRT-PCRanalysis of the same RNA samples showed a 50-fold increase inbauA expression under iron-chelated conditions over that underiron-rich conditions (Fig. 3B), a result that matches our previ-ously reported findings (37). Together, these results suggest thattonB3 could play a major role in energy-transducing functionsunder iron chelation, while tonB1 and tonB2 could have only aminor role in this cellular process under these conditions.

Functional roles of A. baumannii TonB systems. Althoughthe E. coli W3110 TonB protein (BAA14784) and the three pre-dicted ATCC 19606T TonB proteins are analogous in predictedfunction, the global alignment of their CTDs revealed relativelylow levels of identity and similarity between W3110 and ATCC19606T TonB1 (21% identity; 47% similarity), TonB2 (18% iden-tity; 43% similarity), and TonB3 (22% identity; 50% similarity).Therefore, the functional roles of these predicted energy-trans-ducing genes were tested by their capacities to restore the growthof E. coli W3110 derivative KP1344 (tonB::bla) under iron chela-tion, as determined by the transformation of KP1344 with each

cloned ATCC 19606T tonB determinant. The genetic complemen-tation of E. coli KP1344 with pMU762, which harbors tonB2, orpMU823, which harbors tonB3 together with the exbB3 and exbD3

coding regions (Fig. 1), restored bacterial growth under condi-tions of iron limitation (Fig. 4A). In contrast, transformation of E.coli KP1344 with pMU672, which harbors the tonB1-exbB1-exbD1.1-exbD1.2 coding regions (Fig. 1), could not restore bacterialgrowth under conditions of iron chelation (Fig. 4A). The comple-menting activity of each ATCC 19606T tonB gene was further con-firmed by the fact that transformation of E. coli KP1344 with cop-ies of each tonB gene disrupted by site-directed insertion of a DNA

FIG 3 Transcriptional analyses of A. baumannii ATCC 19606T tonB, exbB,and exbD genes. (A) RT-PCR analysis using ATCC 19606T RNA isolated asdescribed in Materials and Methods and the primer pairs indicated in Fig. 1,which were designed to amplify the intergenic regions between tonB1 andexbB1 (lane 1), exbB1 and exbD1.1 (lane 2), exbD1.1 and exbD1.2 (lane 3), tonB3

and exbB3 (lane 4), and exbB3 and exbD3 (lane 5). Lane 6, control with noreverse transcriptase. The 0.5-kb HindIII fragment is indicated on the left.(B) qRT-PCR of each ATCC 19606T tonB gene in response to available freeiron. Transcription of recA and bauA, constitutively expressed and iron-regu-lated genes, respectively, was used for internal controls. RNA isolated fromATCC 19606T cells grown in LB broth with the addition of 100 �M FeCl3(open bars) or 100 �M DIP (shaded bars) was used as the template withspecific primers. The error bars represent the standard errors for two assaysperformed in triplicate.

FIG 4 Growth of E. coli cells harboring plasmids coding for TonB energytransport features from A. baumannii and E. coli. (A) Growth of E. coli W3110harboring pBR322 and of KP1344 (tonB::bla) harboring pBR322, pMU672,pMU674, pMU762, pMU823, pMU962, pMU978, or pMU1008 in LB broth(open bars) or LB broth plus 200 �M DIP (shaded bars). pMU674, a pCR-Blunt II derivative harboring the W3110 tonB gene (Table 1), was used as apositive control. (B) Growth of E. coli W3110 harboring pBR322 and ofRA1051 (�exbBD::aph �tolQR) harboring pBR322, pMU673, pMU672,pMU823, pMU849, or pMU850 in LB broth (open bars) or LB broth plus 200�M DIP (shaded bars). pMU673, a pCR-Blunt II derivative harboring theW3110 exbB and exbD genes (Table 1), was used as a positive control. OD600

readings were taken after overnight incubation (12 to 14 h) at 37°C in a shakingincubator. Error bars represent standard errors for three independent experi-ments performed in duplicate.

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cassette coding for antibiotic resistance (pMU962, pMU978, orpMU1008) abolished bacterial growth under conditions of ironlimitation (Fig. 4A).

Global alignment of the ATCC 19606T ExbB1-ExbD1.1-ExbD1.2

and ExbB3-ExbD3 proteins to the ExbB-ExbD proteins from E.coli W3110 also revealed low levels of identity and similarity be-tween the latter and ATCC 19606T ExbB1 (24% identity; 39%similarity), ExbB3 (32% identity; 49% similarity), ExbD1.1 (33%identity; 49% similarity), ExbD1.2 (30% identity; 45% similarity),and ExbD3 (32% identity; 49% similarity). Thus, the functionalroles of the ATCC 19606T exbB and exbD genes were tested bygenetic complementation of the E. coli W3110 iron acquisitionmutant RA1051 (�exbBD::aph �tolQR). Transformation ofRA1051 with pMU672 or pMU823, harboring the ATCC 19606T

tonB1-exbB1-exbD1.1-exbD1.2 or tonB3-exbB3-exbD3 genes, respec-tively, restored growth under iron-limiting conditions, suggestingthat these A. baumannii genes code for energy transduction func-tions needed for enterobactin-mediated iron acquisition (Fig.4B). Additionally, the growth-enhancing effect of tonB1-exbB1-exbD1.1-exbD1.2 in E. coli RA1051 was abolished when either theexbD1.1 (pMU849) or exbD1.2 (pMU850) gene was interrupted(Fig. 4B). Whether the insertional inactivation of exbD1.1 has apolar effect on the expression of exbD1.2 remains to be tested.

In each case, transformation of KP1344 or RA1051 with thecognate parental W3110 tonB (pMU674) or exbB-exbD(pMU673) copies restored growth to wild-type levels (Fig. 4A andB). Taken together, these observations suggest that the exbB1-exbD1.1-exbD1.2 and exbB3-exbD3 genes found in A. baumanniicode for active energy transduction functions, whereas the tonB2

and tonB3 genes, but not the tonB1 gene, found in A. baumanniiappear to function in E. coli to provide energy transduction func-tions needed for growth under iron chelation. As mentionedabove, it is not known whether the complementing activity ob-

served is due to the action of a particular ExbB-ExbD-TonB pro-tein unit or to a hybrid unit in which proteins coded for by geneslocated in different tonB loci work together because of potentialcross talk among functionally equivalent proteins.

Growth and iron acquisition phenotypes of isogenic tonB in-sertion derivatives. The roles of the A. baumannii TonB systemsin energy-transducing functions needed for iron acquisition werefurther investigated by generating isogenic derivatives of ATCC19606T. ATCC 19606T isogenic derivatives with insertions intonB1 (strain 3180), tonB2 (strain 3201), or both tonB1 and tonB2

(strain 3202) (Table 1) were successfully generated by site-di-rected mutagenesis as described in Materials and Methods. Incontrast, the same conjugation methods, as well as different pro-tocols, including liquid conjugations using various helper/donor/recipient ratios, failed to generate a proper tonB3 insertion mu-tant. Electroporation of ATCC 19606T cells, as described below forthe construction of complemented strains, using either circular orlinear plasmid pMU1008 DNA or DNA amplified by PCR withpMU1008 as the template, also failed to produce a proper tonB3::aacC1 insertion derivative.

ATCC 19606T isogenic mutants 3180, 3201, and 3202 showedgrowth comparable to that of the parental strain in LB and M9media without antibiotics or DIP (data not shown). In contrast,when cultured in M9 minimal medium with increasing concen-trations of DIP, each isogenic tonB insertion derivative showed asignificant deficiency in growth (P � 0.05) compared to that of theparental strain, and the tonB1 tonB2 double mutant was more de-ficient than the single insertion derivatives (Fig. 5A). However,this growth deficiency was not as drastic as that of strain 3069(entA::aph), which contains an insertion in entA that abolishesacinetobactin biosynthesis. A similar growth phenotype was ob-served in LB medium; however, much higher levels of DIP wererequired to produce the same effect, as expected considering the

FIG 5 Effect of tonB inactivation on bacterial growth under iron limitation. (A) Growth of the parental strain, ATCC 19606T, the 3180, 3201, 3202, and 3069isogenic derivatives, and 3180 transformed with pMU994, carrying the parental tonB1 copy, in M9 minimal medium containing increased concentrations of DIP.Asterisks indicate significant differences (P � 0.05) from results for ATCC 19606T. (B) Growth of strains ATCC 19606T, 3180, 3201, 3202, and 3069 inunsupplemented M9 minimal medium (0/0) or in M9 minimal medium supplemented with 50 �M FeCl3 alone (50/0) or with 50 �M FeCl3 plus 100 �M(50/100) or 125 �M (50/125) DIP. OD600 readings were taken after overnight incubation (12 to 14 h) at 37°C in a shaking incubator. Error bars represent standarderrors from three independent experiments performed in duplicate.

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defined nature of the M9 minimal medium (data not shown). Thegrowth-deficient phenotype of each derivative in M9 mediumcontaining 100 �M or 125 �M DIP can be corrected to wild-typelevels by the addition of 50 �M FeCl3 (Fig. 5B). These resultsindicate that TonB1 and TonB2 are required for A. baumannii togrow to levels similar to those of the wild-type strain under iron-limiting conditions. Whether TonB3 has functions essential forbacterial viability that are independent of the iron concentrationin the medium, a possibility suggested by the failure to produce aninsertion mutant, needs to be examined in more detail.

The role of the tonB1 gene in the phenotype of the 3180 (tonB1::aph) mutant was further confirmed by the fact that electropora-tion of pMU994, a derivative of the pWH1266 shuttle vector har-boring the tonB1 parental copy, was enough to restore growth inchelated M9 minimal medium (Fig. 5A). To further ensure thatthe phenotype of 3180 (tonB1::aph) was not due to a polar effect onthe transcription of downstream exbB1, exbD1.1, and exbD1.2 cod-ing regions, RT-PCR was performed. The predicted ampliconswere obtained when RNA isolated from 3180 cells and primersflanking the cognate intergenic regions were used (data notshown). Although this result indicates that an insertion in tonB1

did not affect the transcription of downstream genes of this poly-cistronic operon, the data do not exclude the possibility of polareffects at the translational level.

Arnow’s colorimetric tests and siderophore utilization bioas-says using the ATCC 19606T s1 derivative (which does not pro-duce acinetobactin but expresses all functions needed for its inter-nalization) as an indicator strain showed that each of the isogenictonB insertion derivatives (3180, 3201, and 3202) produces cate-chol and acinetobactin at levels similar to those detected with theparental strain, ATCC 19606T (data not shown). Immunoblottingof whole-cell lysate proteins obtained from the parental strain andeach isogenic tonB insertion derivative (3180, 3201, and 3202)with anti-BauA polyclonal antibodies showed similar levels ofproduction of the outer membrane acinetobactin receptor BauAin all strains only when they were grown under iron-chelated con-ditions (data not shown). Iron utilization bioassays showed thateach isogenic tonB insertion mutant grew around filter discs sat-urated with FeCl3, hemin, or an ATCC 19606T M9 cell-free culture

supernatant, which contains acinetobactin, deposited on LB agarsupplemented with 300 �M DIP and seeded with cells of eachstrain (see Table S2 in the supplemental material). However, thegrowth halos of the three ATCC 19606T tonB insertion mutantsaround discs containing acinetobactin or hemin were smaller thanthose of the parental strain (see Table S2). Taken together, theseresults indicate that all ATCC 19606T TonB isogenic derivativesproduce acinetobactin, as well as critical acinetobactin transportproteins, such as the BauA outer membrane receptor. However,their capacities to transport iron-containing compounds, such asferric acinetobactin and hemin, are lower than that of the parentalstrain because of deficiencies in energy-transducing processesneeded for the transport of these compounds across the outermembrane.

Role of TonB in virulence. The role of each TonB protein inthe virulence of ATCC 19606T was examined using larvae of thegreater wax moth, G. mellonella, as an experimental infectionmodel. This model has been used successfully to determine thevirulence role of the acinetobactin-mediated iron acquisition sys-tem and to test the efficacy of antibiotics in the treatment of A.baumannii infections (7, 41). The G. mellonella model showed that60% and 63% of the caterpillars injected with the parental strainor the 3201 (tonB2::aacC1) derivative, respectively, were killed 6days after injection (Fig. 6A). Injection with the 3180 (tonB1::aph) derivative resulted in killing of 47% of the injected cater-pillars 6 days after injection; however, this level of virulence isnot significantly different (P 0.3182) from that of the paren-tal strain (Fig. 6A). Injection with the 3202 (tonB1::aph tonB2::aacC1) derivative resulted in 30% killing of the injected cater-pillars over the 6 days (Fig. 6A). The difference in killingbetween strain 3202 and the parental strain is significant (P 0.0243). As shown previously, strain 3069 (entA::aph), servingas our negative control because of its iron uptake deficiencydue to the inactivation of the entA acinetobactin-biosyntheticgene (8), resulted in the killing of 27% of the injected caterpil-lars over 6 days. The addition of 100 �M FeCl3 to the inocularesulted in comparable virulence, with increased killing rates(compare Fig. 6A and B), for all strains tested, results signifi-cantly different from those for the PBS-plus-FeCl3 control (P

FIG 6 Role of TonB in virulence. G. mellonella caterpillars (n 30) were injected with 1 � 105 bacteria of the parental strain, ATCC 19606T, or the tonB1 (3180),tonB2 (3201), tonB1 tonB2 (3202), or entA (3069) insertion derivative in the absence (A) or presence (B) of 100 �M FeCl3. As negative controls, caterpillars wereinjected with comparable volumes of PBS alone or PBS plus 100 �M FeCl3. Caterpillar death was determined daily for 6 days after incubation at 37°C in darkness.

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0.01). These observations suggest that no TonB protein indi-vidually is essential for bacterial virulence; only when bothTonB1 and TonB2 are disrupted is the level of virulence signif-icantly reduced from that of the wild-type strain, ATCC19606T.

Attachment to immobilized fibronectin. Recent reports (39,42) have shown that the adherence of A. baumannii ATCC 19606T tohost cells is mediated by binding to fibronectin, a host extracellularmatrix protein that could have a role in bacterial adhesion and inter-nalization. One of the A. baumannii outer membrane fibronectinbinding proteins identified in ATCC 19606T is a 78-kDa TonB-de-pendent copper receptor protein (ACIB1v1_730004) (39). There-fore, the contribution of the TonB systems to adherence to immobi-lized fibronectin was tested using the parental strain, ATCC 19606T,and the isogenic tonB insertion mutants. The cells of all A. baumanniistrains tested adhered more to polystyrene wells coated with fi-bronectin than to BSA-coated or uncoated wells (Fig. 7A). The pa-rental strain, ATCC 19606T, showed 2.5-fold-greater attachment tofibronectin-coated wells than to BSA-coated wells, a finding in agree-ment with results published in a recent report (39). The level of at-tachment of the 3180 (tonB1::aph) derivative, while reduced, was notsignificantly different from that of the parental strain (P 0.195)(Fig. 7A). In contrast, significant reductions in the proportions ofcells adherent to immobilized fibronectin from those for the parentalstrain were observed with both the 3201 (tonB2::aacC1) (P 0.021)and 3202 (tonB1::aph tonB2::aacC1) (P 0.017) derivatives. Strain2234, an isogenic ATCC 19606T derivative with impaired productionof the OmpA protein, which binds fibronectin (39), served as a con-trol; its adherence to immobilized fibronectin was significantly re-duced (P 0.01), to levels comparable to those observed with BSA-

coated or non-protein-coated wells, but was not completelyabolished (Fig. 7A), findings that matched previously reported obser-vations (6). No tonB insertion derivative strains showed a significantdifference from the parental strain in attachment to non-protein-coated wells (Fig. 7A). These results indicate that TonB2 has a greaterrole than TonB1 in the ability of ATCC 19606T cells to bind immobi-lized fibronectin, without affecting their capacity to adhere to un-coated plastic surfaces.

Attachment to A549 cell monolayers. To further assess therole of TonB in attachment to biotic surfaces, the interactions ofthe parental strain, ATCC 19606T, and the tonB insertion deriva-tives with A549 human alveolar epithelial cells were examined.Infection of A549 cell monolayers with ATCC 19606T for 2 h re-sulted in the recovery of 10% of inoculated bacteria adherent tothe A549 monolayers (Fig. 7B). Infection of A549 monolayerswith the 3180 (tonB1::aph) derivative did not result in a significantdifference from the parental strain (P 0.20) in the proportion ofattached bacteria (Fig. 7B). However, infection of the A549 mono-layers with the 3201 (tonB2::aacC1) or 3202 (tonB1::aph tonB2::aacC1) derivative resulted in a significant reduction (P � 0.01), of32% or 39%, respectively, in the proportion of attached bacteriafrom that for the parental strain (Fig. 7B). As reported previously(6), strain 2234, an ATCC 19606T ompA insertion derivative,showed a significant reduction (P � 0.01), but not a complete loss,of bacterial attachment to the A549 monolayers (Fig. 7B). Thus,these observations suggest that TonB2 plays a more relevant rolethan TonB1 in the interaction of ATCC 19606T cells with A549human respiratory epithelial cells, a process that could involve aTonB-dependent outer membrane protein.

FIG 7 Binding of A. baumannii ATCC 19606T cells to immobilized fibronectin and A549 epithelial cells. (A) Counts of adherent bacteria in wells coated with BSA(open bars) or fibronectin (filled bars), or in uncoated wells (shaded bars), after a 3-h incubation with 1 � 105 bacteria of the parental strain (19606) or an ompA(2234), tonB1 (3180), tonB2 (3201), or tonB1 tonB2 (3202) insertion derivative grown overnight in LB broth. (B) Counts of adherent bacteria after a 2-h infectionof 1 � 105 A549 cells with 1 � 105 bacteria of the strains used for panel A. For both fibronectin and A549 cell binding, attached cells were recovered with 0.1%Triton X-100, serially diluted, and plated on nutrient agar. Data are presented as the ratio of CFU counts recovered after infection to the CFU counts of thecognate infecting inoculum. The fibronectin and A549 ratios were normalized by assigning the value of 100% to the binding of ATCC 19606T bacteria toimmobilized BSA and A549 epithelial cells, respectively. Asterisks indicate significant differences (P � 0.05) from ATCC 19606T. Error bars represent standarderrors for three independent experiments performed in triplicate.

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DISCUSSION

In almost all sequenced Gram-negative bacteria, one or moreTonB systems have been identified. Studies of these TonB systemshave focused on iron transport and virulence functions, alongwith similarities to the E. coli TonB system. While iron is a primaryligand for TonB-dependent transporters, other molecules, such asvitamin B12, nickel, and carbohydrates, are also transported in aTonB-dependent manner (18). With the increase in the numberof available sequenced organisms revealing multiple tonB genes,the notion of how TonB works cannot be based solely on work onthe single TonB system present and expressed in E. coli. Currently,nothing is known about the functions of the TonB systems in A.baumannii, except for hypothetical genomic annotation (25). Thegenome of the A. baumannii type strain, ATCC 19606, harborsthree tonB gene clusters, which are present in all sequenced A.baumannii genomes and are predicted to be involved in the trans-duction of energy needed for the transport of nutrients across theouter membrane. Our current observations begin to elucidate therole of each distinct A. baumannii ATCC 19606T TonB systemwith regard to nutrient acquisition, surface attachment, and viru-lence.

A functional role of these A. baumannii energy-transducingcomponents is supported by their capacity to correct the ironutilization deficiency of E. coli KP1344 (tonB::bla) and RA1051(�exbBD::aph �tolQR) mutants. Both the exbB1-exbD1.1-exbD1.2

and exbB3-exbD3 components are sufficient to restore E. coliRA1051 growth under iron chelation, while only tonB2 and tonB3

are able to correct the growth defect of E. coli KP1344 under thiscondition, although neither ATCC 19606T tonB gene works as wellas the parental E. coli copies. This observation could be due to lossof proper gene regulation because of tonB2 and tonB3 expressionfrom a high-copy-number plasmid, resulting in an altered stoichi-ometry of the protein components of this system. However, it ismore likely that low amino acid sequence homology, along withdivergences within the CTD among E. coli and A. baumannii TonBproteins, should be considered a cause of the reduced growth ofthe complemented E. coli strains shown in Fig. 4.

The presence within the A. baumannii tonB1-exbB1-exbD1.1-exbD1.2 cluster of two contiguous copies of exbD genes, both ofwhich are needed to restore the growth of E. coli RA1051 underiron limitation, is of interest. To our knowledge, only two reportshave previously described two ExbD proteins encoded within thesame TonB cluster in other Gram-negative microorganisms (43,44). In both cases, it was found that only one of the two ExbDproteins was involved in iron uptake functions, an observationthat contrasts with our results showing that both exbD productsare required for iron acquisition. With the additional ExbD com-ponent, this particular TonB system could have an enhanced en-ergy-transducing ability due to particular interactions among thecomponents of this system, as well as with TonB-dependent outermembrane receptors, a possibility that remains to be explored.

The fact that each A. baumannii ATCC 19606T isogenic tonBinsertion derivative produces a fully functional acinetobactin-me-diated iron acquisition system, which is the only completely func-tional high-affinity system expressed by this clinical isolate whencultured under laboratory conditions (8), indicates that thegrowth defect of these mutants is due to their inability to transportsequestered iron effectively. This possibility is further supportedby the fact that the growth-deficient phenotype of each derivative

under increased iron chelation can be corrected by the presence offree inorganic iron in the medium. However, our data indicatethat although the ATCC 19606T tonB1 and tonB2 genes play a rolein siderophore-mediated iron acquisition, the tonB3 copy seemscritical for energy-dependent outer membrane transport pro-cesses, including the acquisition of essential iron. This conclusionis supported by the fact that tonB3 transcription is iron regulated,which is most likely associated with the presence of a predicted Furbox upstream of the tonB3 promoter region (26), and our inabilityto generate a viable insertion derivative. Interestingly, the pre-dicted amino acid sequence of the TonB3 protein does not includethe highly conserved YP domain within the CTD. This observa-tion may reflect potentially unique functional properties of thisortholog, a possibility that remains to be tested experimentally.

Iron acquisition and utilization play a central role when bacte-ria are cultured under laboratory conditions, as well as in host-pathogen interactions involved in the pathogenesis of infections,which can be studied using different experimental models. The G.mellonella model has been used previously to study different bac-terial pathogens, since the larvae mount an innate immune re-sponse similar to that of the human host, which includes the func-tion of phagocytic cells, the production of humoral and cellularpattern recognition receptors, and the production of antimicro-bial peptides (45). Additionally, the iron-binding proteins presentwithin the hemolymph of the larvae (46) and the observation thatG. mellonella hemocytes associate with A. baumannii cells (41)indicate that there are iron reservoirs within the larvae that patho-gens can use during colonization and infection under the iron-limiting conditions imposed by this invertebrate host. In line withthese observations, A. baumannii ATCC 19606T cells successfullyinfect G. mellonella larvae only if they express an active acineto-bactin iron uptake mechanism (7, 8) as well as proper intracellulariron utilization functions (37). Accordingly, the virulence ofATCC 19606T depends on the expression of TonB-dependent en-ergy transduction functions, as shown by the significantly lowervirulence of the tonB1 tonB2 double mutant 3202 than of the pa-rental strain. In contrast, inactivation of the tonB1 or tonB2 genehas only a modest effect on virulence. These results, together withthe potentially essential role of tonB3 and its iron-regulated ex-pression, indicate that, though redundant, the three TonB-medi-ated energy transduction systems could play different roles in thephysiology and virulence of A. baumannii.

Host colonization and infection entail the interaction of thepathogen with host cells and products, such as the components ofthe extracellular matrix. Thus, the greater adherence of A. bau-mannii to plastic covered with immobilized fibronectin than touncoated or BSA-coated plastic surfaces was not surprising, con-sidering the recent report by Smani et al. (39). These investigatorsmade a similar observation and identified OmpA, a 34-kDa outermembrane protein, and a TonB-dependent copper receptor aspotential A. baumannii fibronectin binding proteins. Our datacollected with the A. baumannii ATCC 19606T ompA-deficientmutant corroborate the results collected using neutralizing anti-OmpA antibodies (39). Our data also lend support to the involve-ment of a TonB-dependent receptor protein in adherence, sinceinactivation of the ATCC 19606T tonB genes, particularly tonB2,reduces bacterial adhesion to immobilized fibronectin. Addition-ally, there is a direct correlation between the binding of bacteria toimmobilized fibronectin and their association with A549 humanalveolar epithelial cells, which are normally covered by a mucin

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layer, further supporting the hypothesis that A. baumannii utilizesthis glycoprotein to interact with host target cells and tissues. It isworth noting that the adherence responses shown in Fig. 7B aremost likely not due to the expression of acinetobactin-mediatediron acquisition functions or an iron-deficient environment, sincewe showed that ATCC 19606T derivatives impaired in the expres-sion of acinetobactin biosynthesis or transport functions dis-played an adherence phenotype similar to that of the parentalstrain (7). It should also be noted that the data shown in Fig. 7Aand B were obtained with bacteria cultured in LB broth, whichcontains enough free iron to repress the production of ATCC19606T iron-regulated proteins (24), and the solutions and mate-rials used in these assays were not treated to remove free iron.Taking all these points into account, our data indicate that theinteraction of A. baumannii with fibronectin, either immobilizedon an abiotic surface or present on the surfaces of epithelial cells, isdue in part to uncharacterized TonB-dependent processes ratherthan to the expression of iron acquisition functions or the effect ofan iron-restricted extracellular environment. Considering that A.baumannii often causes respiratory and wound infections, bind-ing to fibronectin is a reasonable outcome, since the ensuing in-flammation response causes an influx of this extracellular matrixcomponent to the infection site as part of the mechanisms the hostuses to facilitate wound healing (47). The binding of fibronectin toabiotic medical implants and devices, such as prostheses and cath-eters, also explains the persistence of A. baumannii and otherpathogens, including Pasteurella multocida, Salmonella enterica,and Streptococcus pyogenes (48–50), a problem that has negativeeffects on the treatment of bacterial infections (51).

Our data clearly indicate that TonB-dependent processes play acritical role in the interaction of A. baumannii ATCC 19606T cellswith fibronectin-coated abiotic or biotic surfaces. This conclusionis in agreement with the observation that fibronectin binds TonB-dependent outer membrane receptors present in Bacteroides fra-gilis, P. multocida, and A. baumannii (39, 48, 52). However, themolecular factors and processes involved in these interactions arenot clear at the moment. The conclusion that fibronectin bindsTonB (39, 52) is not in agreement with the known subcellularlocation and topology of TonB. This is a cytoplasmic membrane-anchored protein that spans the periplasmic space, interacts withthe N-terminal domain of TonB-dependent outer membrane re-ceptors, and most likely is not exposed to the extracellular envi-ronment. On the other hand, the experimental evidence showingthe binding of fibronectin to TonB-dependent outer membranereceptors and the negative effect of TonB inactivation may indi-cate that fibronectin-surface protein interactions may involve en-ergy-dependent receptor conformational changes similar to thosedescribed for nutrient transport. Alternatively, one of the ATCC19606T TonB proteins, such as TonB2, could play a role in theexport of fibronectin binding proteins to the bacterial outer mem-brane, a possibility supported by the observation that the productof the P. aeruginosa tonB3 gene is required for type IV pilus assem-bly and twitching motility (21). Therefore, on the basis of theseobservations, it seems reasonable to propose that the A. bauman-nii ATCC 19606T TonB2 ortholog plays a role in attachment andcolonization, as well as in iron acquisition. Clearly, all these areinteresting possibilities that remain to be confirmed experimen-tally.

In conclusion, the results of this investigation highlight thepresence of three complementary but distinct TonB systems in A.

baumannii. The expression of multiple TonB-dependent energytransduction systems may allow this pathogen to scavenge essen-tial nutrients, such as iron, more efficiently in different environ-ments. Additionally, they may provide a biological advantage, al-lowing bacteria to respond to environmental cues more effectivelyover the course of an infection and ultimately aiding in the sur-vival and propagation of this pathogen, which causes severe hu-man infections worldwide.

ACKNOWLEDGMENTS

This work was supported by funding from Public Health (AI070174) andNSF (0420479) grants and by Miami University research funds.

We are thankful to A. Kiss and the Miami University Center of Bioin-formatics and Functional Genomics for support and assistance with au-tomated DNA sequencing and with nucleotide sequence and phylogeneticanalyses. We are grateful to R. Larsen, Bowling Green State University,Bowling Green, Ohio, for providing the E. coli KP1344, RA1051, andW3110 strains and to H. Vogel and B. Chu, University of Calgary, Calgary,Alberta, Canada, for providing the MSA TonB sequence database.

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