1 Faculty of Pharmaceutical Sciences, Okayama University, Tsushima-naka, Okayama 700-8530, Japan
2 Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Tokushima 770-8514, Japan
Correspondence
Shigeo Yamamoto
syamamoto{at}pheasant.pharm.okayama-u.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AB066099.
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INTRODUCTION |
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Vibrio parahaemolyticus is an estuarine pathogen known to be a common cause of seafood-borne acute gastroenteritis worldwide. Production of the siderophore vibrioferrin and utilization of haem and haemoglobin as sole sources of iron have been demonstrated for V. parahaemolyticus grown under iron-deficient conditions (Yamamoto et al., 1994b, 1995
). The Fur protein of this species has been shown to mediate iron regulation both in production of vibrioferrin and in expression of two iron-repressible outer-membrane proteins (OMPs) of 78 and 83 kDa (Funahashi et al., 2000
). Using the Fur titration assay (FURTA) system (Stojiljkovic et al., 1994
), we have previously isolated many Fur target gene fragments from V. parahaemolyticus, one of which led us to identify the pvuA gene encoding the 78 kDa ferric vibrioferrin receptor protein (Funahashi et al., 2002
). At the same time, we also obtained a genomic fragment containing an incomplete ORF, whose predicted protein sequence shares significant homology with the IutA protein that serves as the outer-membrane receptor for ferric aerobactin in Escherichia coli (Krone et al., 1987
). In addition, the N-terminal amino acid sequence (AEQAQQLASQ) determined for the 83 kDa iron-repressible OMP band (containing three kinds of proteins) of V. parahaemolyticus WP1 was correlated with the amino acid sequence deduced from the partial ORF. These results suggested that the protein product of this iutA homologue seemed a likely candidate for a ferric aerobactin receptor.
In this study, to gain more insight into the iron-uptake systems in V. parahaemolyticus, we cloned the entire iutA gene and characterized it. Primer extension analysis revealed that the iutA gene is transcribed from a Fur-repressed promoter upstream of iutA. The function of the iutA protein product in V. parahaemolyticus as the receptor for ferric aerobactin was confirmed by construction of an iutA-disrupted mutant followed by phenotypic comparison between the mutant and the parental strain. Another gene encoding a protein homologous to AlcD, predicted as one of the alcaligin biosynthetic enzymes in Bordetella species (Pradel et al., 1998), was identified just upstream of iutA, although its function is currently unknown.
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METHODS |
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Southern and colony hybridization.
Hybridization followed by immunological detection of DNA was performed according to the digoxigenin (DIG) system user's guide for filter hybridization (Roche Diagnostics). Hybridization with appropriate DIG-labelled probes was carried out overnight at 68 °C. Hybridization probes A and B (see Fig. 1c) were prepared using primers 1 and 2 (positions 28842908 and 34123436) and primers 3 and 4 (positions 40034022 and 44574476), respectively, with the PCR DIG probe synthesis kit (Roche Diagnostics) under the recommended PCR conditions.
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Nucleotide sequencing and homology search.
Nucleotide sequences were determined by a Hitachi DNA sequencer (SQ5500E) with the Thermo Sequenase premixed cycle sequencing kit and appropriate primers previously labelled with the 5'-oligonucleotide Texas red labelling kit (Amersham Pharmacia Biotech). Sequence analysis and alignment were performed with the Genetyx-Mac, version 10.1, software package (Genetyx Software Development). The BLASTP program (Altschul et al., 1997) of the Institute for Chemical Research, Kyoto University, Japan, was used for a homology search of the deduced amino acid sequences.
Primer extension.
Iron-deficient and iron-sufficient cells of V. parahaemolyticus WP1 were prepared as follows. The culture was grown in LB broth to an OD660 of 0·15 and was split into two aliquots; one was left untreated (iron-sufficient cells) and the other was supplemented with 2,2'-dipyridyl at a final concentration of 200 µM to achieve iron depletion (iron-deficient cells). Then, both aliquots were further incubated until an OD660 of 0·5 was reached. Total RNA was prepared from each cell sample using ISOGEN (Nippon Gene), according to the manufacturer's instructions. The primer 5'-CGTTTTGAGACGCCAGTTGC-3', complementary to positions 30973116 of the iutA sequence, was 5'-labelled with Texas red as described above. Approximately 20 fmol of the labelled primer was annealed to 30 µg total RNA at 50 °C and extended at 50 °C for 60 min using avian myeloblastosis virus reverse transcriptase XL (Takara Biomedicals), according to the manufacturer's protocol. The extension product was sized on a 6 % (w/v) denaturing polyacrylamide gel by using a Hitachi DNA sequencer (SQ5500E) alongside the DNA sequence ladder of the control region synthesized with the same labelled primer to map the start site of the transcript.
Growth and binding assays.
In these assays for elucidation of aerobactin utilization, a spontaneously arising, vibrioferrin-deficient mutant, MY-1 (Yamamoto et al., 1994a) derived from V. parahaemolyticus AQ3354, was used to avoid the effect of the endogenous siderophore, vibrioferrin, on growth under iron-limiting conditions. Stationary-phase cells of MY-1 were diluted to an OD660 of 0·005 with fresh LB broth (3 % NaCl) containing 20 µM ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDA) with or without 10 µM aerobactin. Cultures were shaken (125 r.p.m.) at 37 °C and the OD660 was determined at regular intervals. Aerobactin was prepared as described previously (Okujo & Yamamoto, 1994
).
V. parahaemolyticus MY-1 was cultured under the same conditions as WP1 was for primer extension analysis, and OMP-enriched fractions from the iron-deficient and iron-sufficient cell samples were prepared by sonication followed by high-speed centrifugation and Sarkosyl extraction as described previously (Yamamoto et al., 1995). Aerobactin was incubated with radioactive 55FeCl3 as described for desferrioxamine B (Aso et al., 2002
) to prepare a 5 µM 55Fe-labelled aerobactin solution (680 c.p.m. pmol-1). An equal volume of this solution was mixed with 0·3 ml 100 mM Tris/HCl buffer, pH 7·4, containing 100 µg of the OMP. The mixture was shaken at 37 °C for 10 min, 0·5 ml of which was filtered onto a 0·45 µm pore-size nitrocellulose filter. The filter was washed twice with 5 ml of the Tris/HCl buffer and the radioactivity on the filter was measured. Digestion of the OMP fraction with proteinase K (19 units mg-1) (Wako) was carried out by incubating a mixture consisting of 500 µg of the enzyme and 500 µg of the OMP in a total volume of 1 ml at 37 °C for 1 h. The mixture was centrifuged for 20 min at 20 000 g and then the precipitate was used for SDS-PAGE (Laemmli, 1970
) or for further incubation with 55Fe-labelled aerobactin as described above.
Construction of iutA- and orf2-disruptants.
A mutant with disruption in iutA was prepared by inserting a suicide vector into the chromosome of V. parahaemolyticus MY-1, using the strategy originally described by Miller & Mekalanos (1988). MY-1 has the same gene arrangement with respect to orf2-alcD-iutA as WP1. Briefly, the KpnIEcoRI fragment internal to iutA was prepared by PCR amplification with a set of primers (5'-ATATTCCCGGTACCGTTTGG-3'; positions 31713190 and 5'-CCGAATTCGTTGTCGTGTGC-3'; positions 39173936) (the bases changed for introduction of the respective restriction enzyme sites are underlined) followed by digestion with the respective restriction enzymes. This fragment was subsequently inserted into a suicide vector pKTN701 with a chloramphenicol resistance cassette (Nishibuchi et al., 1991
) to generate pTF4, which was propagated in E. coli SY327
pir. The extracted plasmid was transformed into E. coli SM10
pir as a donor and transferred to MY-1 by membrane-filter mating conjugation. Some transconjugants on LB plates containing ampicillin and chloramphenicol were isolated and their single-crossover mutations with respect to iutA were confirmed by Southern blot analysis with DIG-labelled probe B (data not shown). One of the disruptants was designated VPTF4. In a similar fashion, chromosomal orf2 was disrupted by plasmid pTF3 to generate VPTF3. For construction of pTF3, the KpnIEcoRI fragment internal to orf2 was PCR-amplified with a set of primers (5'-TCGGTACCATCTCTACAATGC-3'; positions 759779 and 5'-AACGGAATTCGTCCTGAGCG-3'; positions 13791398); the bases changed for introduction of the appropriate restriction enzyme sites are underlined.
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RESULTS AND DISCUSSION |
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Homology of predicted protein sequences
A search of the protein database revealed significant homology of the deduced iutA product with several proteins in the family of TonB-dependent receptors. It shows the highest amino acid identity to Vibrio orientalis IutA (55 %) (Murakami et al., 2000), followed by E. coli IutA (43 %) (Krone et al., 1987
). Alignment of the V. parahaemolyticus, V. orientalis and E. coli IutA proteins is shown in Fig. 3
. The amino acid sequence derived from the nucleotide sequence of iutA contains a typical leader peptide of 25 residues (von Heijne, 1983
). The TonB box sequence close to the N terminus was found in the V. parahaemolyticus IutA, with a similarity to sequences conserved in TonB-dependent receptors (Braun & Hantke, 1991
). A C-terminal phenylalanine residue and an arginine residue at position -11 relative to the C terminus are widely conserved among OMPs (Struyvé et al., 1991
; Bäumler & Hantke, 1992
). Interestingly, in the case of V. parahaemolyticus IutA, the C-terminal residue is hydrophobic tyrosine. The V. parahaemolyticus IutA protein consists of 725 residues and the mature protein has a calculated molecular mass of 76 654 Da, which is somewhat less than the 83 kDa estimate from the electrophoretic mobility in SDS-PAGE. The difference may be due to aberrant migration on SDS-PAGE as frequently reported for OMPs.
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Utilization of aerobactin by V. parahaemolyticus and characterization of its iutA-disrupted mutant
Fig. 4 shows growth curves of strain MY-1 and its iutA disruptant under iron-limiting conditions with or without added aerobactin. Iron restriction imposed by the addition of 20 µM EDDA to the LB broth resulted in poor growth of MY-1. However, supplementation of this iron-restricted LB broth with aerobactin at 10 µM restored the growth of MY-1 to a level comparable to that of the same strain grown in iron-replete LB broth, indicating that aerobactin is capable of effectively providing iron to V. parahaemolyticus. This is in accordance with the detection of the iutA homologue in V. parahaemolyticus. The iutA-disrupted mutant, VPTF4, however, grew poorly even in the medium supplemented with aerobactin (Fig. 4
), confirming that aerobactin-dependent iron uptake for growth is mediated by the IutA protein.
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Distribution of the iutA gene in V. parahaemolyticus
Southern blot hybridization was performed with DIG-labelled probe B on chromosomal DNA samples from some clinical and environmental isolates of V. parahaemolyticus (Fig. 6). Prior to electrophoresis, all chromosomal DNA samples were completely digested with BglII. In WP1, the probe hybridized to an approximately 2·9 kb BglII fragment, the size predicted from the DNA sequence analysis. In UST-4-1, the size of the BglII fragment was slightly different from WP1, but in the other strains the probe hybridized to fragments of the same size. In accordance with these results, growth promotion by aerobactin was observed for each of these strains under iron-limiting conditions, indicating that the iutA homologues are widely distributed in V. parahaemolyticus to assimilate iron as a ferric aerobactin complex. However, a homologous counterpart of the V. parahaemolyticus iutA gene was not found in the V. cholerae genome sequences.
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Our data demonstrate that V. parahaemolyticus possesses the iutA homologue encoding the ferric aerobactin receptor which is responsible for utilization of aerobactin as an exogenous siderophore in iron-restricted environments. This is the first report of the ferric aerobactin receptor gene being carried on the chromosome of a bacterium which does not produce aerobactin. However, it is currently unclear whether the aerobactin-dependent iron acquisition system confers on V. parahaemolyticus a greater ability to survive in different niches outside or inside the host or to establish an infection. Besides the siderophore-specific outer-membrane receptors, cytoplasmic membrane-associated components are also necessary for ferric siderophore transport and the corresponding genes are generally clustered (Braun et al., 1998). However, the present data indicate that the genes responsible are not located in the close vicinity of iutA on the V. parahaemolyticus chromosome. Further studies will be required to identify and characterize the genes encoding the components responsible for the inner-membrane transport of ferric aerobactin into the cytoplasm.
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ACKNOWLEDGEMENTS |
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Received 22 October 2002;
revised 13 January 2003;
accepted 24 January 2003.
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