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
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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 13 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 ClaIHindIII fragment internal to bauA and a 1349 bp EcoRVBglII 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 ml1 (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.
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RESULTS AND DISCUSSION |
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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 (4043 mol%) (Bouvet & Grimont, 1986
).
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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 DHBAAMP 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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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
-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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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 1216 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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ACKNOWLEDGEMENTS |
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Received 5 March 2004;
revised 11 May 2004;
accepted 24 May 2004.
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