Centers for Disease Control and Prevention, Respiratory Diseases Branch, 1600 Clifton Rd, Mailstop C02, Atlanta, GA 30333, USA1
Author for correspondence: Bernard Beall. Tel: +1 404 639 1237. Fax: +1 404 639 3123. e-mail: beb0{at}cdc.gov
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ABSTRACT |
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Keywords: Bordetella, iron sources, TonB-dependent receptors
Abbreviations: DP, 2,2-dipyridyl; EDDA, ethylenediamine-di-(o-hydroxyphenylacetic acid); LB, LuriaBertani; SS, StainerScholte minimal broth lacking added iron; SS-EDDA, SS agar containing 45µg EDDA ml-1
The GenBank accession number for the sequence reported in this paper is AF087669.
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INTRODUCTION |
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In current models for TonB-dependent energy transduction, the proline-rich TonB protein is anchored to the cytoplasmic membrane by its N-terminus while the remainder of the protein occupies the periplasmic space and makes contact with outer-membrane receptors (Klebba et al., 1993 ; Postle, 1993
; Braun, 1995
). Contact by TonB with outer-membrane receptors is believed to induce a conformational change of the receptors, resulting in transport of bound ligands into the periplasmic space (Skare et al., 1993
). Since ligand translocation into the periplasm is energy dependent, TonB is believed to transduce the cytoplasmic membrane proton-motive force to the receptors.
In order to function efficiently, TonB requires the two auxiliary proteins, ExbB and ExbD. Most of ExbB is situated cytoplasmically, and it is predicted to span the cytoplasmic membrane three times, with a short N-terminal region and a short turn situated in the periplasm (Kampfenkel & Braun, 1993 ). Like TonB, ExbD is predicted to have a short N-terminal region anchored in the cytoplasmic membrane, with most of the protein situated in the periplasm (Kampfenkel & Braun, 1992
). ExbB and ExbD interact with and activate TonB, and it is likely that the three proteins form a complex (Fischer et al., 1989b
; Skare & Postle, 1991
; Ahmer et al., 1995
; Braun et al., 1996
).
The important mammalian respiratory pathogens Bordetella bronchiseptica and B. pertussis express outer-membrane receptors that are regulated by iron availability and are believed to be involved in the uptake of iron complexes. One such receptor is BfeA, a FepA homologue, that has been shown to be required for utilization of the exogenous siderophore enterobactin (Beall & Sanden, 1995b ). More recently, the iron-regulated outer-membrane proteins BfrA, BfrB and BfrC have been identified (Beall & Hoenes, 1997
; Beall, 1998
). BfrA is specific to B. bronchiseptica, whereas BfrB, BfrC and BfeA appear to be highly conserved among Bordetella species. All four Bordetella proteins belong to the family of TonB-dependent outer-membrane receptors, based on their high level of amino acid sequence identity to many other bacterial proteins belonging to this group. Also, all four genes are Fur-repressed, induced under low-iron conditions, and contain consensus Fur-binding sequences in their promoter regions (Beall & Sanden, 1995a
, b
; Beall & Hoenes, 1997
; Beall, 1998
; Brickman & Armstrong, 1995
). The ligand specificities of BfrA, BfrB and BfrC are still unknown.
Presumably, Bordetella species also have a specific receptor for ferric complexes of the Bordetella siderophore, alcaligin (Moore et al., 1995 ; Brickman & Armstrong, 1996a
) and for complexes of exogenously supplied ferrichrome, desferrioxamine B and haemin (Beall & Hoenes, 1997
). It was of interest to determine if the uptake of these known iron sources for Bordetella requires TonB function and if TonB function is required by these organisms during iron-limited conditions. It is likely that Bordetella species require the ability to multiply during iron scarcity in order to establish infection.
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METHODS |
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Tetracycline was used at 15 µg ml-1 and ampicillin at 200 µg ml-1. Gentamicin was used at 15 µg ml-1 for Bordetella.
Iron sources.
Enterobactin extracts were prepared as previously described (Porra et al., 1972 ; Beall & Sanden, 1995b
) and bioassay stocks were 50 µM. The source of alcaligin used for this work was SS broth culture supernatant obtained from the B. bronchiseptica fur mutant B013NMnr4 (Brickman & Armstrong, 1995
). The following were obtained from Sigma and the indicated stock concentrations were used in bioassays: haemin (100 µM), ferrichrome (from Ustilago sphaerogena, 50 µM), desferrioxamine B [deferoxamine mesylate (desferal), 50 µM] and human haemoglobin (500 µg ml-1).
Preparation of DNA.
Total genomic DNA was extracted from Bordetella avium, B. bronchiseptica, B. parapertussis and B. pertussis as previously described (Beall & Sanden, 1995a ). Plasmids were prepared with Qiagen mini-prep kits.
Southern analysis.
This was performed as previously described (Beall & Sanden, 1995a ). The degenerate oligonucleotides [CCNGAR]3 and [AARCCN]3, used to detect tonB, were 3'-end-labelled with digoxigenin (NCID/CDC Biotechnology Core Facility Branch), and were used as probes for DNA hybridization at 42 °C as described in the Genius kit (Boehringer Mannheim). Gene-specific probes were derived from pMLN1 containing the 6 kb chromosomal PstI fragment from B. bronchiseptica. The fragments were randomly labelled with digoxigenin (Genius kit) and used as probes for high-stringency DNA hybridization at 68 °C, as described by the manufacturer.
DNA sequencing.
Plasmid pMLN1 and appropriate plasmid subclones were sequenced with ABI dye-deoxy terminator kits on an ABI 377 sequencer as described by the manufacturer (Applied Biosystems) using oligonucleotides annealing to the chromosomal inserts or the pUC19 multiple cloning site. The GCG Sequence Analysis Software Package (Wisconsin Package Version 9.0, Genetics Computer Group, Madison, WI, USA) was used to analyse the DNA sequence.
Transformation and conjugation.
Plasmids were transformed into E. coli by standard methods. Chromosomal integration of plasmids into B. pertussis and B. bronchiseptica was facilitated by conjugation with E. coli donor strain SM10 as previously described (Stibitz, 1994 ; Beall & Hoenes, 1997
), except that counter-selection employed colicins B and Ia (Brickman & Armstrong, 1996b
).
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RESULTS |
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The 268-codon tonBBb showed strong homology to various Gram-negative TonB proteins, with 3039% identity between overlaps of about 200270 residues. These included the TonB proteins from E. coli, Salmonella typhimurium, Klebsiella pneumoniae, Enterobacter aerogenes, Serratia marcescens, Yersinia enterocolitica, Pseudomonas aeruginosa, P. putida, X. campestris, H. influenzae and Neisseria meningitidis (GenBank accession numbers K00431, X56434, X68478, A36928, X60996, Q05741, U23764, S28444, Z95386, U04996 and U77738 respectively). A feature of TonBBb shared with other TonB proteins includes a short hydrophobic N-terminal putative transmembrane anchor (residues 2040) with the remainder constituting a mainly hydrophilic putative periplasmic domain. Like other TonB proteins, the B. bronchiseptica TonB is proline-rich (21%) and exhibits Lys-Pro and Glu-Pro repeats (7 and 12 repeats respectively) typical of previously described TonB proteins.
Located 1 bp downstream of the tonB stop codon was the 314-codon exbBBb gene, which has deduced homology to various ExbB proteins (about 2941% sequence identity over 85286 residues). The closest matching ExbB homologue to ExbBBb is from X. campestris, with 41% sequence identity (GenBank accession number Z95386).
The 155-codon exbDBb gene overlapped by 1 base with the exbBBb stop codon. The deduced ExbDBb protein sequence shows the most similarity to the N. meningitidis and X. campestris homologues, with 3640% identity (GenBank accession numbers U77738 and Z95386 respectively).
Directly upstream of tonBBb are potential -35 and -10 hexamers homologous to the consensus sequence of E. coli 70-directed promoters (Hawley & McClure, 1983
). This putative promoter sequence is overlapped by a sequence similar to Fur-binding sites. The sequence from bases 86 to 105 (see accession AF087669) is identical to the consensus Fur-binding site in 12 of 19 positions (Calderwood & Mekalanos, 1987
), and was the only iron box found with a homology search of the entire 2446 bp. About 160 bp upstream of tonB are two G+C-rich inverted repeats between bases 1039, either of which could possibly function in transcription termination. Partial sequence analysis of the open reading frame immediately upstream of the sequence shown in accession AF087669 revealed a gene highly homologous to genes encoding bacterial histone-like proteins, including S. typhimurium hupA (Higgins & Hillyard, 1988
). Eighty bases downstream of exbD and downstream of a potential transcriptional terminator was an open reading frame with high homology to various two-component system DNA-binding regulatory proteins (Fig. 2
).
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Construction of B. bronchiseptica and B. pertussis tonB mutants
Two chromosomal fragments were used for plasmid integrational analysis of the tonB gene in B. bronchiseptica and in B. pertussis (Fig. 2). Plasmid pSSt1 was constructed by cloning a 294 bp PCR fragment consisting of bases 209502 including the first 60 codons of tonBBb and 4 bp of upstream sequence, into the EcoRI site of pSS2141 (Stibitz, 1994
) immediately downstream of the gentamicin-resistance gene such that the tonB fragment was in the same orientation as the gentamicin-resistance gene. Plasmids pSSt2 and pSSt3 consisted of the 506 bp internal tonBBb XhoISalI fragment cloned into the SalI site of pSS2141 in either orientation (bases 345851). These plasmids were subsequently integrated into the chromosome of B. bronchiseptica strains 19385, B013N and F4178 by conjugation with the E. coli donor strain SM10 followed by selection for colicin and gentamicin resistance. Since pSSt2 and pSSt3 contained an internal fragment lacking both the 5' and 3' ends of the tonBBb structural gene, simple homologous insertion of the plasmid resulting from a single crossover event within the tonB gene resulted in a truncated tonBBb gene of 218 codons.
Attempts to insertionally inactivate tonB in the clinical B. pertussis strain 82 with plasmids pSSt2 and pSSt3 were unsuccessful. Transconjugants were obtained through matings of strain 82 with SM10(pSSt2) and SM10(pSSt3), but at a very low frequency. Chromosomal DNA was prepared from one transconjugant, digested with EcoRI, and then ligated. This was followed by transformation of E. coli DH10B to ampicillin resistance. Plasmid DNA from several transformants from this chromosome-walking procedure revealed that these plasmids did not contain restriction fragments of the sizes predicted for pSSt2 or pSSt3 derivatives that had integrated into the strain 82 tonB gene (data not shown). In contrast, transconjugants were readily obtained by conjugation of plasmids pSSt2 and pSSt3 into B. pertussis strain UT25D. This strain is a nonhaemolytic derivative of UT25 (Brickman & Armstrong, 1995 ) that has been adapted to grow on LB agar. In contrast to strain 82 transconjugants, plasmid pSSt2 and pSSt3 integrations into UT25D and the two B. bronchiseptica wild-type strains were found to occur as predicted by a single homologous crossover event. This resulted in truncation of 50 C-terminal residues of TonB in the B. bronchiseptica strains and a similar if not identical truncation of TonB in B. pertussis strain UT25D.
Our stock B. bronchiseptica strains 19385 and B013N were non-haemolytic on Bordet Gengou agar, possibly due to laboratory passage resulting in phase variation or modulation (Weiss & Hewlett, 1986 ), although it has been observed that initial isolates of B. bronchiseptica from clinical specimens are often non-haemolytic. Since we were unable to insertionally inactivate tonB in the clinical, haemolytic B. pertussis isolate 82, it appeared possible that attempts to insertionally inactivate tonB in phase 1 haemolytic strains of B. bronchiseptica would be unsuccessful. However, insertional inactivation of tonB in the ß-haemolytic B. bronchiseptica human isolate F4178 was readily accomplished.
Iron source utilization deficiency and albomycin sensitivity of tonBBb mutants
Integration of pSSt1, which contained the 5' end of the structural tonBBb gene, into the B. bronchiseptica chromosome resulted in placing the intact tonBBb gene immediately downstream of the pSS2141 gentamicin-resistance gene and in the same orientation. This also resulted in replacement of the tonB ribosome-binding site with a different ribosome binding region (from ACCTGGCTGATTC-ATG to TAATTAAGAATTC-ATG). Campbell-type insertion of pSSt1 had no effect on the ability of B. bronchiseptica strain 19340 to grow on LB agar containing 100 µM DP, nor did these transconjugants differ from the parental strain 19385 in utilization of various iron sources as measured by disk bioassays (Table 2). Therefore, the vector promoter sequence(s) and the different tonBBb ribosome-binding site resulting from Campbell insertion of pSS2141 served to substitute for the normal tonBBb promoter and ribosome-binding site.
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Another phenotype demonstrating tonB inactivation in B. bronchiseptica and B. pertussis tonB mutant strains was their marked resistance to the antibiotic albomycin (Table 2), which is structurally similar to ferrichrome. These strains showed no zones of growth inhibition around albomycin disks containing 10 µl of a 250 µg ml-1 albomycin solution, while the wild-type parental strains showed wide zones of inhibition around disks containing 10 µl of 25 µg ml-1 albomycin (data not shown).
Complementation of B. bronchiseptica and B. pertussis tonB mutants
A 3·6 kb EcoRVBamHI fragment from pMLN1 containing the intact tonBBbexbBBbexbDBb operon (Fig. 2) was subcloned into the broad-host-range vector pRK415 (Keen et al., 1988
), resulting in plasmid pRKton. Introduction of pRKton into the tonB mutant strains B. bronchiseptica 19341 and B. pertussis BB100 completely restored wild-type iron-utilization and albomycin-sensitivity phenotypes (Table 2
), demonstrating that the mutant phenotypes were not conferred by polarity affects on genes downstream of exbD.
tonBBb on pRKton cannot complement an E. coli tonB mutant
Plasmid pRKton could not complement the enterobactin and vitamin B12 utilization defects exhibited by the E. coli tonB mutant strain H306 (data not shown). The TonB-dependent receptors for colicin B, colicin Ia and albomycin were also still not functional in this mutant since H306(pRKton) was still totally resistant to each of these molecules (data not shown).
tonBBb and bfeA inefficiently reconstitute enterobactin utilization in an E. coli fepA mutant
Attempts were made to reconstitute enterobactin utilization in two different E. coli fepA mutants [H5058 (fepA, aroB) and MT912 (fepA)] and in H306(tonB) harbouring the compatible plasmids pKp1 and pRKton. With H5058, H306 and their plasmid-containing derivatives, no differences were seen in disk bioassays using added enterobactin, nor were differences seen in growth assays in iron-limiting media (data not shown). However, the fepA mutant MT912(pKp1, pRKton) formed larger colonies than MT912(pKp1, pRK415) on LB agar containing 60 µg EDDA ml-1 and this effect was measurable in LB broth containing EDDA (data not shown). These effects in liquid medium were at least partially due to a higher colony-forming efficiency of MT912(pKp1, pRKton) relative to MT912(pKp1, pRK415) in media containing 100250 µg EDDA ml-1 (10-4 vs 0 colony-forming efficiency). We also found small-diameter growth haloes around enterobactin disks with MT912(pKp1, pRKton) on L agar containing 250 µg EDDA ml-1 within 24 h, whereas MT912(pKp1, pRK415) only displayed a very faint growth halo after 72 h. This latter effect is likely to be due to Cir- and Fiu-mediated utilization of enterobactin degradation products (Hantke, 1990 ).
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DISCUSSION |
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As with the P. putida ExbB protein, ExbBBb has a non-conserved N-terminal extension relative to other ExbB proteins. However, both of these ExbB proteins share highly conserved -helical transmembrane domains with the other known ExbB proteins. It is within these domains that ExbB is thought to interact with TonB and ExbD (Kampfenkel & Braun, 1992
; Koebnik, 1993
; Larsen et al., 1994
; Traub et al., 1993
). These include the N-terminal region VX3VX3LX3SX3 motif (residues 84102 of ExbBBb), a domain encompassed by residues 202225 of ExbBBb with four conserved glycine residues, and an alanine-rich
-helical region between residues 240270 with conserved asparagine and arginine residues (residues 265 and 270).
The most conserved regions between ExbDBb and other ExbD proteins are the putative N-terminal transmembrane region (approx. residues 1740) and the C-terminal periplasmic region thought to interact with TonB and ExbB. The aspartate residue (residue 25 and 26 of the E. coli and B. bronchiseptica ExbD respectively), shown to be important in ExbD function (Braun et al., 1996 ), is conserved among known ExbD proteins; and the E. coli ExbD leucine 132, also shown to be functionally important (Braun et al., 1996
), is conservatively substituted by phenylalanine in B. bronchiseptica (residue 133), X. campestris and N. meningitidis.
It is unknown why we were unable to insertionally inactivate tonB in a phase 1 isolate of B. pertussis. We have subsequently attempted tonB inactivation in an independent phase 1 isolate with no success (data not shown). It is likely that this result is due to an effect of tonB inactivation on processes other than iron uptake. For example, in Pseudomonas aeruginosa, tonB inactivation results in hypersensitivity to a wide variety of antibiotics in a manner that is independent of iron concentration in the growth medium (Zhao et al., 1998 ), although we did not note problems in selecting gentamicin resistance for the insertional inactivation of tonB in the phase 1 B. bronchiseptica strain F4178.
The P. aeruginosa TonB protein does not appear significantly more related to the E. coli TonB than TonBBb, yet the P. aeruginosa TonB is known to function in E. coli tonB mutants, facilitating the utilization of several different receptors (Poole et al., 1996 ). TonB proteins of Neisseria species show the same overall conservation of key sequence features with the E. coli TonB; however, the N. meningitidis TonB apparently cannot complement E. coli in the utilization of its TonB-dependent receptors (Stojiljkovic & Srinivasan, 1997
). Reconstitution of the N. meningitidis haemoglobin-utilization system in E. coli was successful, employing expression of the N. meningitidis haemoglobin-receptor gene and tonBexbBexbD operon in E. coli (Stojiljkovic & Srinivasan, 1997
). In analogous experiments, we attempted to reconstitute enterobactin utilization in an E. coli tonB mutant and in E. coli fepA mutants. We introduced the Bordetella tonBexbBexbD operon and fepA homologue, bfeA, into these mutants on compatible plasmids; however, we were only able to demonstrate apparently very inefficient reconstitution in one fepA mutant. Among possible explanations for this result are that the level of bfeA and/or tonBexbBexbD expression is inadequate in these strains, or that these proteins are unstable in E. coli. In previous experiments where bfeA was expressed in E. coli from the powerful T7 promoter and radioactively labelled, the level of labelled BfeA was very low, indicating either poor translation or perhaps degradation of BfeA (Beall & Sanden, 1995b
). Another possible explanation is the formation of inactive hybrid TonBExbBExbD complexes.
The results shown in this work further suggest the presence of several different TonB-dependent receptors in B. bronchiseptica and B. pertussis, since tonB mutants of these species were unable to utilize various iron complexes (Table 2). These receptors include the ferric enterobactin receptor (BfeA), and unidentified receptors for ferric complexes of alcaligin, ferrichrome and desferrioxamine B. Additionally, there must be at least one TonB-dependent receptor for haem sources, since utilization of haemin and haemoglobin was TonB dependent in B. bronchiseptica and B. pertussis (Table 2
). Together with the iron-regulated BfrA, BfrB and BfrC proteins that have unknown ligand specificity, this brings the minimal number of predicted TonB-dependent outer-membrane receptors in this genus to eight. Since the only known siderophore produced by Bordetella species is alcaligin (Moore et al., 1995
; Brickman & Armstrong, 1996a
), the biological significance of the complex array of exogenous iron complex utilization systems present in the genus Bordetella is as yet unknown.
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ACKNOWLEDGEMENTS |
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Received 19 January 1999;
revised 16 April 1999;
accepted 4 May 1999.