魚 病 研 究 Fish Pathology,33(4),239-246,1998.10

Cloning,Mapping and Identificationof theAeromonas salmonicidafstA,fepA and irpAGenes, Encoding the 86,82 and 74 kDa Iron-regulated Outer Membrane Proteins,Respectively

R.J. Collighan, A. J. Bennett and G. Coleman

Department of Biochemistry,NottinghamUniversity Medical School,Queen'sMedical Centre, Clifton Boulevard, Nottingham,NG7 2UH,U.K.

(Received February 23,1998)

Three iromps(iron-regulatedouter membrane proteins)ofAeromonas salmonicidawere identifiedby the use of specificantibodies together with Southernhybridization analysis and limitednucleotide sequencing of their genes. The resultsof theseexperiments together with a searchof the internationaldatabase for homologous sequencesled to theiridentification as follows: 86 kDa iromp(FstA)as a Vibrioanguillarum Fat A homologue 82 kDa iromp(FepA)as an Escherichiacoli FepA homologue 74kDa iromp(IrpA)as an Escherichiacoli Cir homologue

Key words : gene cloning,gene mapping,Aeromonas salmonicida,iron-regulated outer membrane protein,iromp

Pathogenic bacteria require iron for growth in a host such are considered as potential vaccine components where the only source of this element is tightly com (Banerjee-Bhatnagar and Frasch, 1990; Robinson and plexed in a number of iron-binding proteins such as Melling, 1993). An earlier study by Chart and Trust haemoglobin, transferrin and lactoferrin. Two major (1983) detected an inducible siderophore-mediatedtrans mechanisms for acquiring iron from the host proteins port system in the fish pathogen,Aeromonas salmonicida, have been described. The first, present in Escherichia the causative agent of furunculosis in salmonid fish. coli requires the formation of soluble factors with a high Hirst et al. (1991) later identified the siderophore as a affinity for iron such that they are able to remove it from 2,3 diphenol-catechol derivative. the host iron-binding proteins. These compounds or Against this background, we have examined the for siderophores are recognised by receptors on the bacte mation of iron-regulated outer membrane proteins rial outer membrane and together with a series of iron (iromps) of A. salmonicida as part of a continuing transport proteins they are able to translocate the iron molecular biological study of genetically-engineered into the bacterial cytoplasm. By contrast, the second antigens as furunculosis vaccine components. In order mechanism, present in some Vibrio and Neisseria spe to pursue this objective it was necessary to clone and cies, enables the bacteria to remove iron directly from map the iromp genes and to identify the individual transferrin and lactoferrin by means of outer membrane proteins. In this regard it was considered relevant that receptors which recognise these iron complexes them well-conserved regions of homology have been found selves (Guerinot, 1994). In either case, under condi in many of the siderophore receptors on the outer mem tions of iron limitation the relevant genes are derepressed branes of Gram negative organisms (Braun and Hantke, and the bacteria produce iron-regulated proteins in their 1991) a finding which has been usefully applied to the outer membranes. In view of the association of these identification of the iromps of A. salmonicida. proteins with the ability of the organisms to survive they are regarded as important virulence factors and as Materials and Methods Correspondence: Dr G. Coleman, Department of Biochemistry, Bacteria and plasmids Nottingham University Medical School, Queen's Medical Centre, Nottingham NG7 2UH, U.K. Aeromonas sal montci da (Unilever 2862) (Fyfe et Fax no. 0115-9422225 al., 1988), phagemids pTZ18R and pT7T3 and host 240 R. J. Collighan, A. J. Bennett and G. Coleman

Escherichia coli DH5ƒ¿ (Mead et al., 1986) were used. resuspended in 100 ƒÊl of SDS-PAGE (polyacrylamide

Plasmid pITS549 was the source of the E. coli fepA gene gel electrophoresis) loading buffer and 5 ƒÊl samples

(Armstrong et al., 1989). separated by SDS-PAGE unless otherwise stated.

Growth of the bacteria SDS polyacrylamide gel electrophoresis (SDS-PAGE)

A. salmonicida (Unilever 2862) was grown in 3% Outer membrane protein preparations were separated

(w/v) Tryptone Soya Broth (TSB, Oxoid). Batches of by SDS-PAGE (Laemmli, 1970). Unless otherwise medium (50 ml) contained in 250 ml conical flasks were stated 5 and 10 ƒÊl samples of outer membrane protein loopinoculated from a culture of the organism, stored preparations grown in the presence and absence of 2,2' at 4•Ž on 4% (w/v) Tryptone Soya Agar (TSA, Oxoid), dipyridyl were run in parallel on 14 cm 5%-15% (w/v) and incubated overnight, at 25•Ž, in a refrigerated gradient gels. "orbital" incubatorshaker (Gallenkamp & Co . Ltd.).

Large-scale purification of individual iromps

Outer membrane protein preparation The outer membrane protein fraction was purified

One hundred milliliter 3% (w/v) TSB with or without from 4 litres of A. salmonicida broth culture grown

150 mM 2,2'-dipyridyl (an iron chelator) was inoculated overnight in the presence of 150 mM 2,2'dipyridyl as with 5 ml of overnight culture of the organism grown in described above. Aliquots of the outer membranes

3% (w/v) TSB at 25•Ž, with shaking, in a refrigerated were solubilised in gelloading buffer and subjected to incubator-shaker (Gallenkamp) which, in turn, was in SDS-PAGE on a 14 cm 5-15% gradient gel when a dis oculated from an agar slope (4% TSA) stored at 4•Ž. tinct separation of the individual iromps was achieved.

Following growth overnight at 25•Ž, with shaking, in The separated iromp bands were visualised by immers the presence of air, the bacterial cells were harvested by ing the gel in 0.2M potassium chloride for up to 2 min centrifugation at 4,000 x g for 10 min, at 4•Ž, and with protein bands appearing opaque against a clear washed by resuspension in 20 ml of 20 mM TrisHC1/5 background. Individual iromp bands were excised mM EDTA, pH 7.2. The washed cells were collected from the gel, pooled together and homogenised. The by centrifugation as before and maintained at 4°C in all homogenised gel slices were suspended in 50 mM po further stages of the preparation unless otherwise stated. tassium acetate, pH 7.4, with 0.1% SDS and incubated

The washed cells were resuspended in 8 ml of 20 mM with shaking for 2 h, at 37•Ž, gel fragments were then

TrisHCl/5 mM EDTA, pH 7.2, containing 10 jig/ml removed by centrifugation at 5,000 •~ g for 15 min and

DNase and 10 ƒÊ/ml RNase and treated for 45 s in a the supernatant carefully decanted. The gel debris was "Gallenkamp" sonic oscillator (using the medium probe) washed once more in the same way and the supernatant which lysed most of the cells and released the mem fractions pooled. The solutions containing purified branes as vesicles. After chilling on ice for 5 min the iromps were then dialysed against distilled water and lysate was centrifuged at 8,000 •~ g for 10 min, at 4•Ž, to finally vacuum desiccated. The purified iromps were remove whole cells and other debris. Then the super stored in this state at -20•Ž until required. natant fraction was carefully decanted into a 10 ml ultracentrifuge tube, avoiding the sediment. Production of polyclonal anti-iromp serum

The resulting preparation was centrifuged at 30,000 x A preparation containing equal amounts of the three g for 45 min, at 4•Ž, to pellet the membrane vesicles A. salmonicida iromps was obtained using the previous and the supernatant fraction was decanted from the small purification protocol. One hundred microgram of the clear pellet which was resuspended in 4.5 ml of 20 mM preparation, in a volume of 500 µl, mixed with an equal TrisHC1/5 mM EDTA and 0.5 ml of 5% (w/v) "Sarkosyl" volume of Freund's incomplete adjuvant, were injected

(sodium N-lauroylsarcosinate, Sigma). The preparation intramuscularly into rabbits at 2 week intervals over a 6 was then incubated at room temperature for 20 min, with week period. Test bleeds were carried out on days 14, frequent mixing, to selectively solubilise the inner mem 28 and 42 to assess the immune response to the iromps brane. The outer membrane was recovered by centrifu by Western blotting. Exsanguination was performed gation at 30,000 •~ g for 45 min at 4•Ž. on the 56th day after the initial inoculation. After leav After decanting the supernatant fraction and remov ing the blood to clot overnight at 4•Ž, the serum was ing all traces from the walls of the centrifuge tube, using decanted from the clot and was stored frozen at -20•Ž a piece of Kleenex tissue, the outer membrane was until required. Identification of A. salmonicida iromps 241

Western blotting with iromp antibody Southern hybridization analysis was carried out The procedure described by Towbin and Gordon using the iromp probes described above, and the DNA (1984) was followed using the polyclonal anti-iromp digest fragments identified were excised in bands of gel serum. and isolated by binding to glassmilk using a "Geneclean

II" kit (Bio 101 Inc. from Stratech Scientific Ltd.).

Amino acid sequence analysis The DNA fragments isolated were ligated into the The iromp running as an approximately 74 kDa band multiple cloning site of phagemid pTZ18R and the re on SDS-PAGE of an outer membrane protein preparation combinants transformed into competent CaCl2-treated was excised after transblotting onto "ProBlott" mem E. coli strain DH5ƒ¿ (Sambrook et al., 1989). The brane (Applied Biosystems Inc.) and loaded into a Pro transformed bacteria were plated out on L-broth agar tein Sequencer (model 473A, Applied Biosystems Inc.) containing 50 ƒÊg/ml ampicillin (Kaiser and Murray, for Nterminal amino acid analysis. An unambiguous 1985) to select for phagemidcontaining colonies whilst decapeptide was obtained, namely, ELPKANETMV. recombinants were selected using the "bluewhite"

marker technique (Mead et al., 1986). Production of iromp gene probes White colonies were transferred, in duplicate, to KANETMV was translated into an oligonucleotide L-amp solid medium and incubated for 16 h at 37•Ž. from which the 3'-terminal nucleotide was omitted. Recombinants bearing the A. salmonicida iromp gene Thus, a degenerate 20-mer was synthesized, using a inserts were identified by colony hybridization as de DNA Synthesizer (model 381A, Applied Biosystems scribed by Sambrook et al. (1989). Inc.), with the sequence: 5'-AA(A,G)GC(T,C,A,G)AA (T,C)GA(A,G)AC(T,C,A,G)ATGGT-3' Sequencing cloned iromp gene fragments This oligonucleotide was used as a probe, after end DNA sequencing was carried out on "mini preps" labelling with 32Pas described by Sambrook et al. (Saunders and Burke, 1990) of recombinant pTZ18R (1989), to identify the corresponding gene in restriction phagemids encoding iromp gene inserts by the digests of A. salmonicida genomic DNA. dyedeoxynucleotide chain termination procedure (Ap A 1.3 kb Kpnl fragment of plasmid pITS549 plied Biosystems Inc.). The sequencing data was (Armstrong et al., 1989) which contained approximately processed using the EMBL Database accessed through half of the 5' end of the fepA coding sequence was ran the Internet System and alignments were achieved domly labelled with 32Pas described by Sambrook et al. using the Clustal V program (Higgins, 1994). (1989). Results Isolation of A. salmonicida genomic DNA

The cells from a 24 h culture of A. salmonicida During the course of a preliminary study the optimal

(Unilever 2862), grown in 3% (w/v) TSB, at 25•Ž, were conditions for development of ironregulated outer harvested by centrifugation and the cell pellet was membrane proteins (iromps) and their isolation from

resuspended and lysed as described by Silhavy et al. Aeromonas salmonicida (Unilever 2862) grown in 3%

(1984). The lysate was subjected to CsCl equilibrium (w/v) TSB, at 25•Ž, with constant shaking, were deter density gradient ultracentrifugation and DNA was iso mined. Thus, an overnight culture was diluted 20-fold

lated by ethanol precipitation of the viscous nucleic acid in fresh medium containing 150 mM 2,2'-dipyridyl and

fraction (Kaiser and Murray, 1985). incubated for a further 12 h. Outer membrane proteins

were prepared from these iron-depleted bacteria and the

Cloning of restriction fragments pattern was compared with that from iron-replete bacte A. salmonicida (Unilever 2862) genomic DNA (5 gig) ria grown in the absence of 2,2'-dipyridyl, by SDS-PAGE.

was placed in a clean sterile Eppendorf tube together The results in Fig. 1 show a doublet of proteins (a and b)

with 10 units of restriction endonuclease (Northumbria of molecular masses approximately 84 and 82 kDa

Biologicals Ltd.), 3 ƒÊl of the corresponding restriction together with a major 74 kDa component (c) were

buffer (10 •~ concentrated) and sterile HPLC grade formed in response to iron depletion and these three pro

water to a volume of 30 ƒÊl. The preparation was incu tein bands identified as iromps were chosen for further

bated at 37•Ž for 24 h after which the digested DNA studies.

was fractionated by agarose gel electrophoresis. Since Hirst et al. (1991) detected a catechol-type sidero- 242 R. J. Collighan, A. J. Bennett and G. Coleman phore in A. salmonicida it is worthwhile noting that the corresponding siderophores of the well-studied E. coli sys- tem are enterobactin with an outer membrane iron-regu- lated receptor protein FepA and dihydroxybenzoylserine with receptors Cir and Fiu. All three receptors are associated with a TonB uptake system (Guerinot, 1994) and have been assigned molecular weights in the 81- 74K range (Neilands, 1982).

Screening using iromp antibodies and an E. coli fepA probe An expression library of A. salmonicida DNA in phagemid pT7T3 (Mead et al., 1986) was used to trans- form E. coli which were plated on a medium containing IPTG (isopropyl (3-D-thiogalactoside)as an inducer of recombinant proteins. Screening with polyclonal anti- bodies to A. salmonicida iromps led to the isolation of a small number of colonies producing iromp epitopes. Further colony hybridization with the fepA-specific probe gave two positive colonies and the recombinant phagemid containing a 1.7 kb PstI insert (pRC2) was selected for further analysis due to its large size and stronger hybridization with the fepA gene probe. This recombinant also expressed the antigen most immuno- reactive to the polyclonal antibodies which suggested that the corresponding iromp was a FepA homologue.

Production and use of a degenerate probe

An SDS-PAGE separation as shown in Fig. 1 was transblotted onto "ProBlott" polyvinylidene difluoride membrane and the iromp bands were excised and sub- jected to N-terminal sequence analysis. The 84 and 82 kDa components appeared to have blocked N-termini but the 74 kDa band gave a well-defined decapeptide Fig. 1. Separation of different amounts of outer membrane ELPKANETMV which contained the TonB box protein preparations from A. salmonicida, grown in sequence ETMV found in E. coli Cir (Braun and Hantke, 3% (w/v) TSB in the presence and absence of 150 1991). A degenerate 20-mer probe was produced M 2,2'-dipyridyl, on a 5-15% (w/v) SDS-PAG. ƒÊ based on KANETMV and used in the Southern hybrid- Different volumes (5 and 10 µl) of the preparations ization analysis of restriction digests of genomic DNA. from iron-deficient (lanes 1 and 3) and iron-replete cultures (lanes 2 and 4) were compared. Molecular Strong positive reactions were obtained with a 5.5 kb weight marker proteins (SDS-7B, Sigma) were sepa- EcoRI and a 1.3 kb PstI restriction fragment. rated in lane 5. Both restriction fragments were ligated into pTZ18R and transformed into E. coli DH5ƒ¿. Hybridization screening of "white" colonies of the resulting trans- Restriction mapping of inserts and limited nucleotide formed bacteria grown in the presence of the chromoge- sequence analysis nic substrate X-gal (Mead et al., 1986) led to the isola- The pRC1, 2 and 3 inserts were subjected to restric- tion of positive recombinant phagemids pRCI and tion mapping, as shown for pRC1 in Fig. 2, with the pRC3. results in Fig. 3. Partial nucleotide sequencing of subcloned restriction fragments was carried out such that the resulting data was translated into N-terminal amino Identification of A. salmonicida iromps 243

(a) acid sequences of the iromps. These sequences are. shown in Fig. 4 together with those most closely cone- sponding to them as revealed by a computer search of the EMBL Database. It can be seen that the indicated sequence of the pRC1 insert has a close homology to the ferric anguibactin receptor (FatA) of the fish pathogenVibrio anguillarum (Koster et al., 1991) and has been designated FstA (Fig. 4a). The two sequences have characteristic signal pep- tides of 32 and 28 amino acids, respectively, (Wickner et al., 1991) and TonB boxes. A feature of the receptors (b) for siderophores which are transported across the outer membrane through the activity of TonB protein is a consensus sequence termed the "TonB box". In the case of A. salmonicida E-LSV is similar to ETLSV, the TonB box of E. colt FecA, whilst the V. anguillarum ESITV is similar to that of E. coli FhuA, DTITV, taking into account the similarity between ES and DT (Braun and Hantke, 1991). In the first 100 amino acids of FstA and the corresponding FatA sequence there are 17 iden- tical and 30 similar amino acids. However, the homol- Fig. 2. Restriction mapping of pRC1 insert: (a) restriction ogy increases considerably further along the sequence endonuclease digests of recombinant plasmid pRC1 so that over the range of amino acids 251-350 there are were separated in lanes B, E, H, P, S and Sm, (b) Southern blot of the digests using the P32-labelledde- 63 which are identical and 18 similar which represents a very high degree of homology. generate oligonucleotide probe. HindIII-digested lambda DNA size standards were On comparing the mature E. colt ferric enterobactin separated in lanes k. receptor (FepA) with its A. salmonicida homologue, B = BamHI, E = ECORI,H = HindIII, S = SalI, Sm = derived from the pRC2 insert and lacking the signal SmaI peptide sequence, over the first 100 amino acids 15 were found to be identical and 26 similar (Fig. 4b) not greatly different from the correspondence in the FatA/FstA N-terminal region. TonB boxes were identified with DTIVV for E. colt and TGGEV for A. salmonicida and whilst the former contained the consensus T-V the latter deviated by the presence of an extra G. The pRC3 insert was sequenced such that the whole of a 28 amino acid signal peptide was identified together with the N-terminus of a mature protein beginning with ELPKANETMV, the sequence previously determined from a transblot (Fig. 4c). Over the 100 amino acid sequence 35 were identical and 24 similar to those of E. colt 2,3-dihydroxybenzoylserine/colicin I/V receptor Fig. 3. Restriction maps of PRC1, 2 and 3 DNA inserts en- Cir. Both sequences contained the same TonB box coding sequences of the A. salmonicida iron-regulated outer membrane protein genes designated fstA, fepA ETMVV in which T—V and -MV- are sequences asso- and irpA, respectively. The regions corresponding ciated with the TonB boxes of colicin receptors (Braun to mature protein sequences are shaded. and Hantke, 1991). Av = AvaI, B = BamHI, E = EcoRI, He = Hind P = PstI, S = SalI, Sm = SmaI, RV = EcoRV 244 R. J. Collighan, A. J. Bennett and G. Coleman

Fig. 4. Alignment of N-terminal amino acid sequences of A. salmonicida iromps FstA, FepA and IrpA deduced from partial nucle otide sequences of (a) pRC1, (b) pRC2 and (c) pRC3 inserts, respectively. The alignment shown is with homologous regions of the most closely-related proteins selected from the EMBL Database using the CLUSTAL V program. Identical amino acids are indicated by (*) and similar by (•)) whilst Ton B box sequences are underlined. Identification of A. salmonicida iromps 245

Kadner, 1986; Nau and Konisky, 1989). Discussion pRC1 with a 5.5 kb EcoRI insert was isolated using Chart and Trust (1983) studied iron uptake by A. the 74 kDa protein degenerate probe the amino acid salmonicida and found two mechanisms to be present, a sequence of which (KANETMV) was not found in siderophore-dependent inducible system and a constitu the partial sequence shown in Fig. 4. However, tive siderophore-independent mechanism which required serendipitously, the 5' terminal of the probe AAAGCC cell contact with ferric lactoferrin or transferrin. In all does occur in the codon sequence of the peptide LKP at the strains examined there was increased synthesis of the N-terminus of the mature FstA and was sufficient to several high molecular weight outer membrane proteins permit attachment of the radiolabelled probe in South (omps). In a detailed examination of one particular ern blotting. strain three iromps of molecular mass 83.2, 77.7 and 76.6 The whole of the fstA open reading frame has recently kDa were produced whilst there was no detectable been sequenced (EMBL acc. no. X87995) and it encodes siderophores of the phenolate or hydroxamate type. This a mature protein of 86 kDa (not 84 kDa as estimated appeared to be in contrast to A. hydrophila where a earlier by SDS-PAGE) with a marked homology to the phenolate test was positive and the siderophore was in catechol siderophore anguibactin receptor, FatA, of V. distinguishable from enterobactin. A later study of the anguillarum (Koster et al., 1991) over the whole of its outer membrane proteins of certain fish pathogens by length. Aoki and Holland (1985) showed that the profiles of the The conclusion is that the three irompsof A.salmonicida outer membrane proteins of all the A. salmonicida strains (Unilever 2862) are homologues of FatA, FepA and Cir. examined were identical in contrast to A. hydrophila where they were markedly different. On studying the Acknowledgments effect of iron depletion they demonstrated the formation of three iromps by A. salmonicida with molecular R. J. C. wishes to thank the BBSRC and SOAFD for a masses of 84, 73 and 68 kDa. Research Studentship. More recent studies by Ellis and his colleagues of both typical and atypical strains of A. salmonicida confirmed the presence of both siderophore-mediated and sidero References phore-independent uptake systems. These workers Aoki, T. and B.I. Holland(1985):The outer membrane extracted the siderophore from an iron-depleted culture proteinsof the fish pathogens Aeromonas hydrophila, and showed by colorimeteric tests that it behaved like a Aeromonas salmonicidaand Edwardsiellatarda. FEMS 2,3 diphenol-catechol (Hirst et al., 1991). Further work Microbiol.Lett.,27,299-305. by Hirst and Ellis (1994) resulted in the claim that four Armstrong,S. K., G. S. Pettis,L. J.Forrester and M. A. McIn tosh(1989):TheEscherichia coli enterobactin biosynthesis iromps of molecular masses 82, 77, 72 and 70 kDa were gene,entD:nucleotide sequence and membrane localisation produced by 18 isolates of A. salmonicida grown in the of itsprotein product. Mol.Microbiol.,3,757-766. presence of synthetic iron chelators. They also pro Banerjee-Bhatnagar,N. and C. E. Frasch(1990):Expression vided convincing evidence that A. salmonicida iromps of Neisseriameningitidis iron-regulated outer membrane are important protective antigens against furunculosis proteins,including a 70-kilodaltontransferrin receptor, and in Atlantic salmon. theirpotential for use as vaccines. Infect.Immun.,58, The claims of the various workers of the formation of 2875-2881. Braun, V. and K. Hantke(1991):Genetics of bacterialiron three or four iromps in the molecular mass range of 84 transport.In"CRC handbook of microbialiron chelates", to 68 kDa in iron depleted cultures of A. salmonicida (ed.by G. Winkelmann), CRC Press LLC, Boca Raton, are consistent with the results obtained in the present Florida,USA. pp.107-138. work with the Unilever 2862 strain of the organism in Chart, H. and T. J. Trust(1983):Acquisition of iron by which molecular masses of 84, 82 and 74 kDa were Aeromonas salmonicida. J.Bacteriol.,156,758-764. assigned to the three clearly identifiable iromps. Fyfe,L., G. Coleman and A. L. S. Munro(1988):The com Partial sequencing of the DNA fragments in pRC2 binedeffect of isolatedAeromonas salmonicidaprotease and and 3 isolated by specific probes support the identifica haemolysinon Atlanticsalmon(Salmo salarL.)compared with thatof a totalextracellular products preparation. J. tion of these two iromps as E. coli FepA and Cir homo Fish Dis.,11,101-104. logues both being catechol siderophore receptors involved Guerinot,M. L.(1994):Microbialiron transport. Ann. Rev. in TonB-mediated uptake systems (Lundrigan and Microbiol.,48,743-772. 246 R. J. Collighan, A. J. Bennett and G. Coleman

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