Identification and transcriptional organization of a gene cluster involved in biosynthesis and transport of acinetobactin, a siderophore produced by Acinetobacter baumannii ATCC 19606T

Kazutoshi Mihara, Tomotaka Tanabe, Yoshiko Yamakawa, Tatsuya Funahashi, Hiroshi Nakao, Shizuo Narimatsu and Shigeo Yamamoto

Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan

Correspondence
Shigeo Yamamoto
syamamoto{at}pheasant.pharm.okayama-u.ac.jp


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
In order to assimilate iron, Acinetobacter baumannii ATCC 19606T produces a siderophore named acinetobactin (Ab) that is composed of equimolar quantities of 2,3-dihydroxybenzoic acid (DHBA), L-threonine and N-hydroxyhistamine. Application of the Fur titration assay system to A. baumannii genomic libraries, followed by further cloning of the regions surrounding the candidate genes, led to the identification of the Ab cluster, which harbours the genetic determinants necessary for the biosynthesis and transport of the siderophore. However, an entA homologue essential for DHBA biosynthesis was not found in this cluster. Functions of potential biosynthetic genes inferred by homology studies suggested that the precursors, DHBA, L-threonine and N-hydroxyhistamine, are linked in steps resembling those of bacterial non-ribosomal peptide synthesis to form Ab. Genes responsible for the two-step biosynthesis of N-hydroxyhistamine from histidine were also identified in this cluster. Their genetic organization suggests that five genes involved in the transport system of ferric Ab into the cell cytosol form an operon. Construction of disruptants of some selected genes followed by phenotypic analysis supported their predicted biological functions. Interestingly, three additional genes probably involved in the intracellular release of iron from ferric Ab and the secretion of nascent Ab are contained in this cluster. Primer extension and RT-PCR analyses suggested that the Ab cluster, which includes 18 genes, is organized in seven transcriptional units originating from respective Fur-regulated promoter-operator regions.


Abbreviations: Ab, acinetobactin; ABC, ATP-binding cassette; DHB, 2,3-dihydroxybenzoyl; DHBA, 2,3-dihydroxybenzoic acid; DIG, digoxigenin; EDDA, ethylenediamine-di(o-hydroxyphenylacetic acid); Fur, ferric uptake regulator; FURTA, Fur titration assay; IROMP, iron-repressible outer-membrane protein; NRPS, non-ribosomal peptide synthetase; PCP, peptidyl carrier protein; PE, primer extension

This article is dedicated to the memory of Dr Igor Stojiljkovic.

The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AB101202.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
In response to iron starvation, aerobic bacteria synthesize and secrete highly ferric-ion-specific chelators, termed siderophores, into the environment. The ferric siderophore complexes formed are then transported into the cells by a process that requires a specific outer-membrane receptor, a periplasmic protein and several inner-membrane-associated proteins (Braun et al., 1998; Andrews et al., 2003). The energy for transport of the ferric siderophore across the outer membrane is provided by interaction of its receptor with the TonB complex (Braun, 1995), and the energy for ferric siderophore transport across the inner membrane is subsequently provided by hydrolysis of ATP by the inner-membrane-associated proteins. Expression of most proteins required for bacterial iron acquisition systems, including siderophore biosynthetic enzymes, is always regulated at the transcriptional level by a global iron-binding repressor protein called Fur (ferric uptake regulator) (Braun et al., 1998; Crosa & Walsh, 2002).

Members of the genus Acinetobacter have been reported to be involved in a variety of nosocomial infections including bacteraemia, urinary tract infection, pneumonia and secondary meningitis, with increasing frequency (Bergogne-Bérénin & Towner, 1996). Among the Acinetobacter species encountered frequently in nosocomial infections is Acinetobacter baumannii (Bergogne-Bérénin & Towner, 1996). Moreover, such infections are often extremely difficult to treat because of their widespread resistance to the major groups of antibiotics (Webster et al., 1998). We reported that A. baumannii ATCC 19606T and some clinical isolates of this species produce a siderophore named acinetobactin (Ab), which is composed of equimolar quantities of 2,3-dihydroxybenzoic acid (DHBA), L-threonine and N-hydroxyhistamine, the first two components forming an oxazoline ring (Yamamoto et al., 1994). Ab is structurally close to anguibactin, a plasmid-encoded siderophore of Vibrio anguillarum (Jalal et al., 1989), the only difference being that Ab possesses an oxazoline ring instead of a thiazoline ring. Ab is also similar to vibriobactin produced by Vibrio cholerae (Griffiths et al., 1984) in that it has a 2,3-dihydroxyphenyl-5-methyloxazolinyl group. Echenique et al. (1992) have proposed that another catecholate siderophore is produced by A. baumannii 8399, which, however, still awaits complete structural elucidation. Several genes responsible for biosynthesis and transport of this putative siderophore have recently been cloned and analysed (Dorsey et al., 2003).

A. baumannii strains producing Ab have been reported to utilize 30 % iron-saturated transferrin and 15 % iron-saturated lactoferrin as sole sources of iron for growth, by scavenging iron bound to these proteins using Ab (Yamamoto et al., 1999). However, none of these strains utilized haem or haemoglobin as an iron source. These observations suggest that Ab-mediated iron acquisition may have an important role in the pathogenesis of A. baumannii infections. Moreover, Daniel et al. (1999) reported that A. baumannii also possesses a Fur protein 63 % identical to that of Escherichia coli. In accordance with this, several Fur-target genes have recently been identified in this species (Echenique et al., 2001; Dorsey et al., 2003).

In the present study, the Fur titration assay (FURTA) system originally developed for E. coli (Stojiljkovic et al., 1994) was applied to A. baumannii ATCC 19606T genomic libraries to isolate Fur-target genes involved in the biosynthesis and transport of Ab. Subsequent cloning and nucleotide sequence analysis of the genomic regions surrounding the candidate genes identified a cluster of 18 genes whose protein products are homologous to the biosynthetic enzymes and transport system components for other bacterial catecholate siderophores (Crosa & Walsh, 2002; Di Lorenzo et al., 2003). Phenotypic comparison between the wild-type strain and its gene-disruptants supported the biological functions of the corresponding genes that were expected on the basis of the homology searches. Moreover, primer extension (PE) together with RT-PCR suggested that the 18 genes are expressed in seven transcriptional units from the respective promoters with putative Fur-binding site sequences under iron-limiting conditions.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growth conditions.
The strains and plasmids used are listed in Table 1. E. coli and A. baumannii strains were grown in LB broth (1 % tryptone peptone, 0·5 % yeast extract, 0·5 % NaCl) or on LB agar (2·5 %) plates at 37 °C for 12–15 h. Growth media contained ampicillin at 100 µg ml–1 or chloramphenicol at 10 µg ml–1 for E. coli DH5{alpha} bearing a high-copy-number-plasmid and ampicillin at 50 µg ml–1 or chloramphenicol at 5 µg ml–1 for E. coli DH5{alpha} bearing a low-copy-number-plasmid.


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Table 1. Bacterial strains and plasmids used in this study

 
DNA manipulation and oligonucleotide primers.
Standard DNA manipulations were carried out as described by Sambrook et al. (1989). Chromosomal DNA of A. baumannii ATCC 19606T was extracted with the Wizard genomic DNA purification kit (Promega), and plasmids were isolated from E. coli strains with a plasmid miniprep kit (Bio-Rad). Restriction and DNA modifying enzymes were used following the instructions of the manufacturers. DNA fragments were purified from agarose gels with the Prep-A-Gene DNA purification kit (Bio-Rad). Transformation of E. coli was performed by electroporation with a Gene Pulser apparatus (Bio-Rad). Oligonucleotide primers (Table 2) designed according to the determined sequences of A. baumannii ATCC 19606T were used for PE and RT-PCR analyses.


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Table 2. Sequences of primers used for primer extension and RT-PCR

 
Nucleotide sequencing and homology searches.
Nucleotide sequences were determined with a Hitachi DNA sequencer (SQ5500E) and the Thermo Sequenase premixed sequencing kit (Amersham Pharmacia Biotech) by primer walking in two directions. The primers for sequencing were labelled with the 5'-oligonucleotide Texas red labelling kit (Amersham Pharmacia Biotech). Sequence analysis was carried out with the Genetyx-Mac, version 10.1, software package (Genetyx Software Development). The FASTA program (Pearson & Lipman, 1988) via the Genome Net service by Kyoto University Bioinformatics, Japan, was used for comparison of deduced amino acid sequences with protein sequence databases.

Southern blotting and colony hybridization.
A model 785 vacuum blotter (Bio-Rad) was used for the transfer of digested chromosomal DNA separated in 1 % agarose gels onto nylon membranes. Colonies on a nylon membrane were denatured and neutralized, and the DNA was fixed to the membrane by baking it for 30 min at 80 °C. Overnight hybridization at 68 °C with appropriate digoxigenin (DIG)-labelled probes followed by immunological detection of labelled DNA was performed according to the DIG system user's guide (Roche Diagnostics). The DIG-labelled probes were prepared with the PCR DIG probe synthesis kit under the PCR conditions recommended (Roche Diagnostics).

FURTA and cloning by gene walking.
FURTA was carried out according to the procedure of Stojiljkovic et al. (1994). Genomic DNA from A. baumannii ATCC 19606T was partially digested with Sau3AI, and 1–3 kb fragments extracted from agarose gels were ligated into the BamHI site of pUC19 or pHSG396. The resulting recombinant plasmids were introduced into E. coli H1717, and ampicillin- or chloramphenicol-resistant transformants were screened for their Lac+ phenotype on MacConkey lactose agar plates (Difco), containing 25 µM ferrous ammonium sulfate, after 15 h growth. Several rounds of FURTA provided more than 20 positive clones, and nucleotide sequences of their inserts were determined for homology searches of the deduced amino acid sequences. As a result, we obtained two promising candidate clones, designated pABBC1 and pAB3-11 (Table 1). By using the DIG-labelled probes prepared on the basis of the nucleotide sequences of the inserts, chromosomal regions surrounding these inserts were successively cloned in both directions by a gene walking strategy. A. baumannii ATCC 19606T chromosomal DNA was digested with various restriction enzymes, and the resulting DNA fragments were analysed by Southern blotting with the DIG-labelled probes. Digested DNA fragments with the desired size which had hybridized with an appropriate probe as a single band were ligated into appropriately restriction-digested vectors. Colonies on LB agar plates were selected by colony hybridization with the same probe.

Determination of the N-terminal amino acid sequence.
The iron-repressible outer-membrane proteins (IROMPs) were isolated as described previously (Yamamoto et al., 1995). The IROMPs were separated by SDS-PAGE (Laemmli, 1970) and electroblotted as described by Towbin et al. (1979). Their N-terminal amino acid sequences were determined by automated Edman degradation with a model 491 protein sequencer (Applied Biosystems) equipped with an online model 120A PTH-amino acid analyser.

Construction of A. baumannii bauA and basD mutants and growth assay.
To investigate their functions, the bauA and basD genes were inactivated by insertion of a suicide vector (Palmen et al., 1993). A 1362 bp ClaI–HindIII fragment internal to bauA and a 1349 bp EcoRV–BglII fragment internal to basD were each prepared from pVIBCC, which were ligated into pBluescript II KS(+) (Stratagene) to generate pFATCH and pVIBEB, respectively (Table 1). These plasmids were each electroporated into strain ATCC 19606T using a Gene Pulser apparatus (Bio-Rad) under the conditions of Leahy et al. (1994), and transformants were selected at 30 °C after 24 h growth on LB plates containing 80 µg ticarcillin ml–1 (Duchefa Biochemie) (Magnet et al., 2001). The insertion mutants thus obtained were named MHR1 for bauA and MHR2 for basD. Disruption of the corresponding genes was confirmed by Southern hybridization and PCR (data not shown).

For growth assays, Erlenmeyer flasks (100 ml) with side-arms were used. Late-exponential phase cells of strain ATCC 19606T and its bauA-disruptant precultivated in Tris-buffered succinate (TBS) medium (pH 7·4) (Actis et al., 1993) containing 0·15 µM FeCl3 were each added at an OD660 of 0·02 to 20 ml TBS medium containing 30 % iron-saturated human transferrin (Sigma) at a concentration equivalent to 1 µM Fe3+. Cultures were shaken at 175 r.p.m. at 37 °C, and growth was tested by measuring the OD660. In some experiments, ferric Ab was added at a final concentration of 1 µM to TBS medium. Ferric Ab was prepared by mixing FeCl3 with fivefold molar excess of purified Ab (Yamamoto et al., 1994).

PE and RT-PCR analyses.
Strain ATCC 19606T was grown in LB broth until it reached an OD660 of 0·3. The culture was split into two aliquots, and one was left untreated (+Fe cells) and the other was supplemented with ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDA) at a final concentration of 400 µM (–Fe cells). Both aliquots were further incubated until an OD660 of 0·5 was reached. Total RNA was isolated from each cell sample using ISOGEN (Nippon Gene), and the amount of RNA was quantified by measuring the A260. PE was performed with the reverse primers (Table 2) which were 5'-labelled with Texas red as described above. Approximately 10 µg total RNA was annealed with the 5'-labelled primer and then the extension was carried out in a total volume of 20 µl at 50 °C for 1 h with AMV reverse transcriptase XL (Takara Biomedicals) by following the manufacturer's directions. The extension product was sized on a 6 %, (w/v) denaturing polyacrylamide gel by using a Hitachi SQ5500E DNA sequencer alongside the DNA sequence ladder of the control region, synthesized with the same labelled primer to map the start site of the transcript.

For RT-PCR, the same total RNA samples as used for PE were pretreated with RNase-free DNase I (Qiagen) at 37 °C for 1 h to exclude the possibility of contamination with traces of chromosomal DNA. The enzyme was then inactivated by using the supplied stop solution, followed by heating at 65 °C for 10 min. RT-PCR was carried out with the RNA PCR kit (Takara Biomedicals), according to the manufacturer's protocol. For first-strand cDNA synthesis, 5 µg of the pretreated total RNA was incubated in a total volume of 20 µl at 42 °C for 1 h with either ATPSQ1 (internal to barA) or EntDSQ2 (internal to basH) (Table 2). Subsequent PCR was performed with 2 µl of the first-strand cDNA mixture as a template and a pair of forward and reverse primers (EntBSQ3 and ATPSQ2) (Table 2) as follows: after an initial denaturation at 94 °C for 2 min, DNA was amplified for 30 cycles, with each cycle consisting of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 1 min.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Cloning of genes expected to be involved in Ab-mediated iron acquisition
The FURTA system (Stojiljkovic et al., 1994) was used to isolate a variety of Fur box-containing genes from the A. baumannii ATCC 19606T chromosome. Out of the positive clones isolated, two clones named pABBC1 and pAB3-11 (Table 1) were found to contain inserts encoding potential proteins related to V. anguillarum FatD (Köster et al., 1991) and E. coli EntE (Staab et al., 1989), respectively. Putative Fur boxes sharing 12 of 19, and 13 of 19 bp with the E. coli consensus Fur box (de Lorenzo et al., 1987) were detected close to the predicted promoter elements (–10 and –35) for the respective ORFs (see below). These results implied that the gene fragments isolated were associated with transport and biosynthesis of Ab.

Next, the genomic regions surrounding the inserts of pABBC1 and pAB3-11 were successively cloned by gene walking with DIG-labelled probes designed on the basis of the determined nucleotide sequences. Nucleotide sequence determination of nine overlapping cloned fragments disclosed the presence of 23 ORFs within a 32·4 kb gene cluster (Fig. 1). The deduced amino acid sequences of 18 of these genes share significant degrees of similarity with known or predicted siderophore biosynthetic enzymes and transporter components in other bacteria (Table 3). However, the amino acid sequences deduced from the other five genes (orf1orf5) suggested that these genes may not be involved in the Ab-mediated iron acquisition system. In particular, predictions about the enzymology of Ab biosynthesis could be made based both on the close structural similarity of Ab to anguibactin (Jalal et al., 1989) and to vibriobactin (Griffiths et al., 1984), together with potential functions of the biosynthetic genes inferred from homologies. Accordingly, we designated ten genes basABCDEFGHIJ (bas stands for A. baumannii acinetobactin biosynthesis), six genes bauABCDEF (bau stands for A. baumannii acinetobactin utilization), and two genes barAB (bar stands for A. baumannii acinetobactin release) (Fig. 1). The G+C content of the region encompassing the 18 Ab genes was 38·8 mol%, which is slightly lower than the overall G+C content of A. baumannii (40–43 mol%) (Bouvet & Grimont, 1986).



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Fig. 1. (a) Restriction enzyme map and genetic organization of the 32·4 kb chromosomal region of A. baumannii ATCC 19606T cloned in this study. The locations, transcriptional directions and relative sizes of individual genes are denoted by arrows. The bau genes are shown by open arrows, the bas genes by grey arrows, the bar genes by striped arrows, and genes that are probably not involved in iron acquisition by black arrows. Arrowheads with capitalized letters F and T in a circle indicate the location of putative Fur boxes and transcriptional termination signals, respectively. (b) Anticipated transcriptional units are shown with wavy arrows. The numbers are the nucleotide positions of the +1 sites determined by PE.

 

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Table 3. Summary of homology searches for the ORFs found in the Ab cluster of A. baumannii ATCC 19606T

 
Analysis of Ab biosynthetic genes
The biosynthesis of DHBA from chorismate in bacteria requires the products of the entC, entB and entA genes (Liu et al., 1989; Nahlik et al., 1989; Ozenberger et al., 1989) or homologues (Massad et al., 1994; Wyckoff et al., 1997; Bellaire et al., 2003), which are usually clustered together in a Fur-controlled regulon. The presence of basF and basJ, homologues of E. coli entC and entB, in the cluster is consistent with the fact that the Ab molecule possesses a DHBA moiety. However, an entA homologue that might encode the last enzyme in the DHBA biosynthetic pathway was not found within the cloned region. It is probably located in another chromosomal region.

The BasC protein showed 60 % identity to RhbE, a 1,3-diaminopropane N-hydroxylase of Sinorhizobium meliloti (Lynch et al., 2001), and may be involved in the oxidation of histamine yielding N-hydroxyhistamine, another constituent of the Ab molecule. Moreover, the amino acid sequence deduced for the basG product is highly similar to the bacterial pyridoxal-5'-phosphate-dependent histidine decarboxylases which provide histamine.

The results of sequence similarity searches (Table 3) suggest that the predicted protein products of basE, basF, basH and basI could play roles in the early stages of Ab biosynthesis. BasE, a homologue of E. coli EntE (Gehring et al., 1997), is a probable DHBA–AMP ligase, activating the carboxylate group of DHBA via an ATP-dependent reaction to bind it to holo-BasF (a potential phosphopantetheinylated BasF), as a thioester. In addition to the DHBA synthesis function in its amino terminus as described above, BasF has, at the carboxy terminus, an acyl carrier protein domain, where phosphopantetheinylation probably occurs at the conserved serine residue (position 246) to ligate the activated DHBA yielding 2,3-dihydroxybenzoyl (DHB)-S-BasF (Gehring et al., 1997). BasH is probably a thioesterase, which probably liberates mischarged peptide synthetases that are blocked by an unspecific thioesterification of their 4'-phosphopantetheinyl cofactor (Marahiel et al., 1997). BasI is a predicted phosphopantetheinyl transferase catalysing phosphopantetheinylation of the hydroxyl groups of the conserved serine residues in BasB (position 222) and BasF.

Assessment of their sequences by FASTA analysis revealed that BasA, BasB and BasD each align well with different parts of the best-characterized non-ribosomal peptide synthetase (NRPS), V. cholerae VibF, which has multicatalytic activities specified by distinct domains to assemble vibriobactin from the three precursors 2,3-DHBA, L-threonine and norspermidine (Butterton et al., 2000; Keating et al., 2000; Marshall et al., 2002). The conserved amino acid sequences for the adenylation (A) domain, the condensation (C) and peptidyl carrier protein (PCP) domains, and the cyclization (Cy) domains were found to be embedded in BasA, BasB and BasD, respectively (Fig. 2). The dispensability of the C1 domain for vibriobactin biosynthesis (Marshall et al., 2002), however, offered a possible explanation for the absence of the corresponding domain in any of the A. baumannii NRPSs.



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Fig. 2. Homologies of BasA, BasB and BasD to the NRPS catalytic domains in V. cholerae VibF. The organization of the six domains (Cy1, Cy2, A, C1, PCP and C2) in VibF reported by Keating et al. (2000) is adopted, but no catalytic role has been demonstrated for the C1 domain in VibF (Marshall et al., 2002). The numbers are the amino acid (aa) positions or residues. Domain names: A, adenylation; C, condensation; Cy, heterocyclization; PCP, peptidyl carrier protein.

 
Analysis of genes involved in the transport of ferric Ab into cell cytosol
The cluster also contains genes for a periplasmic-binding-protein-dependent ATP-binding cassette (ABC) transport system, which is presumably involved in uptake of ferric Ab. The bauDCEBA genes are oriented in the same direction (Fig. 1) with a single set of putative promoter elements (–35 and –10) in front of bauD and with a single rho-independent transcription terminator-like sequence immediately downstream of the TAA stop codon of bauA, suggesting that the bau genes constitute an operon. The BauDCB proteins may be the ABC transport components for ferric Ab, since they are highly similar to the FatDCB proteins required for transport of ferric anguibactin in V. anguillarum (Köster et al., 1991). BauD and BauC exhibit sequence similarity and appear to be polytopic integral membrane proteins (permeases). Moreover, like V. anguillarum FatB (Actis et al., 1995), BauB could be a lipid-modified protein tethered to the inner membrane via the acylation of a cysteine residue that is an integral component of a lipoprotein signal sequence (Wu & Tokunaga, 1986). The consensus sequence (LxyC) at the processing site recognized by signal peptidase II is found at the C-terminal end of the signal peptide of BauB (LQAC), the cysteine residue probably being the site for acylation. The BauA protein shares a remarkable sequence similarity with the TonB-dependent IROMPs, with the most pronounced identity being to FatA, the ferric anguibactin receptor in V. anguillarum (Actis et al., 1988). The amino acid sequence deduced from bauA was completely correlated with the N-terminal amino acid sequence determined for the 78-kDa IROMP of strain ATCC 19606T (see below), suggesting that the bauA product includes a 54 aa signal peptide (Nielsen et al., 1997). This proposed signal sequence is rather long, since we adopted the ATG codon, which is preceded by a Shine–Dalgarno-like sequence, as a translation initiation site. Therefore, another translation initiation site leading to a shorter signal sequence could not be excluded. The molecular mass of the processed BauA protein is 76 015 Da, which is compatible with the 78 kDa estimate based on SDS-PAGE. The bauE product shares sequence similarity with many ferric siderophore transport ATP-binding proteins that face the cytoplasm and supply the system with energy. The presence of bauE in the bau operon is distinctly different from the ferric anguibactin transport fat operon which lacks an ATP-binding protein (Di Lorenzo et al., 2003).

Analysis of the bauF, barA and barB genes
The amino acid sequence deduced from bauF shows the highest similarity to V. cholerae ViuB, which has been proposed to be an esterase required for the intracellular release of iron from ferric vibriobactin (Butterton & Calderwood, 1994). On the other hand, the A. baumannii barA and barB genes encode 536 and 531 aa proteins, respectively, which show the highest sequence similarity to putative ATP-binding components of ABC transport systems of Pseudomonas aeruginosa and Streptomyces coelicolor. Although the BarA and BarB proteins are moderately homologous (20 % identity) to each other, their hydropathy profiles are strikingly similar to an N-terminal hydrophobic domain with six transmembrane {alpha}-helices and a C-terminal hydrophilic domain containing the ATP-binding cassettes (Walker et al., 1982; Hyde et al., 1990). The general four-domain organization required for this type of ABC transporter (Putman et al., 2000) implies that BarA and BarB may serve as a heterodimeric efflux pump in the secretion of Ab to the extracellular milieu, a hypothesis which will be investigated further.

Phenotypic analysis of the bauA- and basD-disruptants
Strain ATCC 19606T gave at least five IROMP bands having molecular masses of 75 to 80 kDa (Fig. 3), and the first 10 N-terminal amino acid residues, AVIDNSTKTL, determined for the 78 kDa IROMP completely matched the amino acid sequence deduced from bauA. In an effort to confirm further the function of BauA, a bauA-disruptant of A. baumannii ATCC 19606T was constructed by homologous recombination with pBluescript II KS(+) as a suicide vector (Palmen et al., 1993). Although the disruptant (MHR1) constructed still produced Ab at the same level as the wild-type strain, as judged using CAS agar plate tests (Schwyn & Neilands, 1987), it failed to grow in iron-deficient TBS medium supplemented with 30 % iron-saturated human transferrin (equivalent to 1 µM Fe3+) as a sole source of iron. In the same conditions, however, the wild-type strain grew well (Yamamoto et al., 1999). Moreover, the addition of ferric Ab at a final concentration of 1 µM to the iron-deficient TBS did not support growth of the mutant. SDS-PAGE analysis showed that the mutant lacked the 78 kDa IROMP (Fig. 3), indicating that BauA plays an essential role, most probably as the receptor for ferric Ab, during Ab-dependent iron-restricted growth. Finally, we conclude from these observations, together with the similarity data, that BauA is the receptor for ferric Ab.



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Fig. 3. OMP profiles of A. baumannii ATCC 19606T (lanes 1 and 3) and a bauA-disruptant MHR1 (lanes 2 and 4) electrophoresed in a SDS 7·5 %, (w/v) polyacrylamide gel (20 cm long) and stained with Coomassie blue. OMP fractions were prepared from both strains grown in LB medium with and without the addition of 100 µM EDDA according to Yamamoto et al. (1995). Samples were solubilized at 95 °C for 5 min, and 25 µg protein was loaded in each lane. Only the relevant portion of the gel is shown. The positions of molecular mass standards are indicated on the left (lane M). The 78 kDa IROMP (BauA), whose N-terminal amino acid sequence was determined to be AVIDNSTKTL, is marked by an arrowhead in lane 3 (wild-type), but it is absent from lane 4 (bauA-disruptant).

 
In order to confirm that the bas locus is indeed involved in the biosynthesis of Ab, a basD-disruptant was constructed and assayed for Ab production. When an OD660 of 0·5 was reached in LB medium, the cells were exposed to iron-limitation imposed by the addition of EDDA at a final concentration of 400 µM. Following a further incubation of 2 h, the amount of Ab secreted into the supernatant was determined by HPLC (Yamamoto et al., 1994). While the wild-type strain produced Ab at 5·4 µM, the disruptant showed no Ab production.

Iron regulation and transcriptional organization of the Ab gene cluster
A computer-assisted inspection of the nucleotide regions surrounding the potential promoter sequences (–10 and –35) revealed the presence of five appropriately located potential Fur box sequences, which match 12–16 of the 19 nucleotides of the Fur box consensus sequence (de Lorenzo et al., 1987). PE analysis of total RNA from iron-limited cells of strain ATCC 19606T identified potential transcription start sites for seven genes (Fig. 4a), indicating that the associated Fur boxes participate in iron-regulated gene expression. The determined transcription start sites and the putative Fur box sequences are detailed in Fig. 4(b). First of all, PE analysis supported the notion that bauF, basA and basJ are monocistronic genes which are regulated by their own Fur boxes. The Fur boxes predicted for bauF and basA overlap with the respective promoter elements. The basB gene appears to be transcribed as a single message under the control of the Fur box common to the downstream operon bauDCEBA that is divergently transcribed. PE with primer FatDPE internal to bauD identified the transcription start site for bauD with an appropriately located Fur box 356 bp upstream of its translation start codon. Although this long untranslated region of the messenger is rather unusual, its function, if any, is unknown. PE with primer FatAPE internal to bauA detected no extension band for total RNA from cells grown under iron-limiting conditions, suggesting that the bauA gene is part of a polycistronic operon. The transcription start site for basD was defined at nucleotide position 20121, with potential promoter elements as well as the Fur box at the junction between basD and basE. In addition, the relatively short intervening sequence (49 bp) between basC and basD indicates that they are organized in a bicistronic operon. A potential promoter was identified only in the upstream region of the basE gene, which is followed by six genes, basFGbarABbasHI, and hence we hypothesize that these genes might have a polycistronic organization. To confirm the transcriptional linkage both between basG and barA and between barB and basH, RT-PCR was performed with total RNA isolated from strain ATCC 19606T grown under either iron-deficient or iron-sufficient conditions. The primers ATPSQ1 and EntDSQ2 were used for cDNA synthesis, and the PCR primer pair EntBSQ3/ATPSQ2 was then used to amplify two different cDNA templates. In both cases, the anticipated 660 bp products were preferentially amplified from total RNA isolated from the iron-limited culture (Fig. 5). PCRs of genomic DNA treated with RNase-free DNase did not yield any amplicons, indicating that PCR bands attributable to DNA contamination in the RNA sample can be excluded. Thus, transcription of the operon composed of these seven genes is controlled by a Fur box located upstream of the basE gene. These results suggested that the 18 genes are expressed in seven transcriptional units from five Fur-related promoters (Fig. 1b).



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Fig. 4. (a) PE analysis of total RNA from A. baumannii ATCC 19606T to define the transcription start sites and iron-regulated expression of the genes which are preceded by putative Fur boxes. Total RNA samples were prepared from cells grown under iron-sufficient (+Fe) and iron-deficient (–Fe) conditions as detailed in Methods. The DNA sequence (A, C, G, T) was generated on the non-transcribed strand with the same labelled oligonucleotide as used for PE. (b) Nucleotide sequences of the promoter regions of the corresponding genes in (a). The initiation codons are represented by angled arrows. The transcription start sites (*) and the putative promoter elements (–10 and –35) are indicated, and the putative Fur box sequences are boxed [matches with the 19 bp E. coli consensus sequence (de Lorenzo et al., 1987) are shown]. Numbers correspond to nucleotide sequence positions in GenBank/EMBL/DDBJ accession number AB101202.

 


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Fig. 5. Demonstration of the transcriptional linkage of barAB with basEFG and basHI by RT-PCR. RT-PCR with total RNA of A. baumannii ATCC 19606T grown under iron-sufficient (+Fe) and iron-deficient (–Fe) conditions. The conditions for RT-PCR are described in Methods. The primers ATPSQ1 and EntDSQ2 indicated at the top of the lanes were used for the preparation of cDNA. The size of the RT-PCR product (660 bp) is indicated on the right. Lane M, molecular size markers; Control, PCR only with total RNA pretreated with RNase-free DNase.

 
In this study, the Fur box-containing gene fragments isolated from A. baumannii ATCC 19606T by FURTA were successfully used for the isolation of neighbouring genes of related functions. Sequencing and homology analysis of the 32·4 kb genomic region isolated revealed a cluster of 18 genes that encode the assembly enzymes for Ab and the transporter components for its ferric complex. The proposed functions of the enzymes and transporters identified in this study are summarized in Fig. 6. The biosynthetic pathway for Ab mediated by NRPSs would be delineated as follows. The A domain of BasA participates in the adenylation (activation) of L-threonine followed by transfer of the resulting aminoacyl adenylate of L-threonine to the holo (phosphopantetheinylated)-PCP thiol in BasB. The Cy2 domain of BasD would catalyse the condensation of DHB-S-BasF onto threonyl-S-BasB, and then the Cy1 domain would convert DHB-threonyl-S-BasB to the heterocyclic 2,3-dihydroxypheny-5-methyloxazolinyl enzyme thioester with dehydration. Finally, the C2 domain of BasB is anticipated to fuse the 2,3-dihydroxypheny-5-methyloxazoline-acyl group with N-hydroxyhistamine, giving rise to Ab.



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Fig. 6. Proposed functions of the protein products encoded by the genes within the Ab cluster of A. baumannii ATCC 19606T. The gene that would code for an EntA homologue (2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase) was not found in this cluster. The tonB, exbB and exbD genes required for TonB-dependent energy transduction have not been cloned yet in this species. Domain names: A, adenylation; C, condensation; Cy, heterocyclization. OM, outer-membrane; CM, cytoplasmic membrane.

 
The availability of the entire sequence of the Ab biosynthesis and transport cluster should allow further dissection of predicated roles of the genes, individually or in combination, by preparation of appropriate mutants. Further studies are required to elucidate the contribution of Ab to the establishment and maintenance of A. baumannii infections.


   ACKNOWLEDGEMENTS
 
We are indebted to Dr I. Stojiljkovic for providing us with the host strain E. coli H1717 in the FURTA system. We also thank Dr H. Yamada for determining the N-terminal amino acid sequences.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 5 March 2004; revised 11 May 2004; accepted 24 May 2004.



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