Bordetella pertussis FbpA binds both unchelated iron and iron siderophore complexes.
ABSTRACT: Bordetella pertussis is the causative agent of whooping cough. This pathogenic bacterium can obtain the essential nutrient iron using its native alcaligin siderophore and by utilizing xeno-siderophores such as desferrioxamine B, ferrichrome, and enterobactin. Previous genome-wide expression profiling identified an iron repressible B. pertussis gene encoding a periplasmic protein (FbpABp). A previously reported crystal structure shows significant similarity between FbpABp and previously characterized bacterial iron binding proteins, and established its iron-binding ability. Bordetella growth studies determined that FbpABp was required for utilization of not only unchelated iron, but also utilization of iron bound to both native and xeno-siderophores. In this in vitro solution study, we quantified the binding of unchelated ferric iron to FbpABp in the presence of various anions and importantly, we demonstrated that FbpABp binds all the ferric siderophores tested (native and xeno) with ?M affinity. In silico modeling augmented solution data. FbpABp was incapable of iron removal from ferric xeno-siderophores in vitro. However, when FbpABp was reacted with native ferric-alcaligin, it elicited a pronounced change in the iron coordination environment, which may signify an early step in FbpABp-mediated iron removal from the native siderophore. To our knowledge, this is the first time the periplasmic component of an iron uptake system has been shown to bind iron directly as Fe(3+) and indirectly as a ferric siderophore complex.
Project description:Phenotypic analysis using heterologous host systems localized putative Bordetella pertussis ferric alcaligin transport genes and Fur-binding sequences to a 3.8-kb genetic region downstream from the alcR regulator gene. Nucleotide sequencing identified a TonB-dependent receptor family homolog gene, fauA, predicted to encode a polypeptide with high amino acid sequence similarity with known bacterial ferric siderophore receptors. In Escherichia coli, the fauA genes of both B. pertussis and Bordetella bronchiseptica directed the production of a 79-kDa polypeptide, approximating the predicted size of the mature FauA protein. B. bronchiseptica fauA insertion mutant BRM17 was unable to utilize ferric alcaligin, and in complementation analyses ferric alcaligin utilization was restored to this mutant by supplying the wild-type fauA gene in trans. Mutant BRM18, carrying a nonpolar in-frame fauA deletion mutation, was defective in ferric alcaligin utilization and (55)Fe-ferric alcaligin uptake and no longer produced a 79-kDa iron-regulated outer membrane protein. In complementation analyses, BRM18 merodiploids bearing the wild-type fauA gene in trans regained ferric alcaligin siderophore transport and utilization functions and produced the 79-kDa protein. Analysis of a plasmid-borne fauA-lacZ operon fusion confirmed that fauA is subject to iron regulation at the transcriptional level and that cis-acting transcriptional control elements mediating fauA iron repressibility reside within the 3.8-kb PstI fauA DNA region. Moreover, expression of the fauA-lacZ fusion gene under iron starvation conditions was shown to be alcR dependent. FauA is a 79-kDa iron-regulated outer membrane receptor protein required for transport and utilization of ferric alcaligin siderophore complexes by Bordetella species.
Project description:A Bordetella bronchiseptica iron transport mutant was isolated following an enrichment procedure based on streptonigrin resistance. The mutant displayed a growth defect on iron-restricted medium containing ferric alcaligin as the sole iron source. In addition to the apparent inability to acquire iron from the siderophore, the mutant failed to produce alcaligin as well as two known iron-regulated proteins, one of which is the AlcC alcaligin biosynthesis protein. A 1.6-kb KpnI-PstI Bordetella pertussis DNA fragment mapping downstream of the alcaligin biosynthesis genes alcABC restored both siderophore biosynthesis and expression of the iron-regulated proteins to the mutant. Nucleotide sequencing of this complementing 1.6-kb region identified an open reading frame predicted to encode a protein with strong similarity to members of the AraC family of transcriptional regulators, for which we propose the gene designation alcR. Primer extension analysis localized an iron-regulated transcription initiation site upstream of the alcR open reading frame and adjacent to sequences homologous to the consensus Fur repressor binding site. The AlcR protein was produced by using an Escherichia coli expression system and visualized in electrophoretic gels. In-frame alcR deletion mutants of B. pertussis and B. bronchiseptica were constructed, and the defined mutants exhibited the alcR mutant phenotype, characterized by the inability to produce and transport alcaligin and express the two iron-repressed proteins. The cloned alcR gene provided in trans restored these siderophore system activities to the mutants. Together, these results indicate that AlcR is involved in the regulation of Bordetella alcaligin biosynthesis and transport genes and is required for their full expression.
Project description:Siderophore production in response to iron limitation was observed in Alcaligenes eutrophus CH34, and the corresponding siderophore was named alcaligin E. Alcaligin E was characterized as a phenolate-type siderophore containing neither catecholate nor hydroxamate groups. Alcaligin E promoted the growth of siderophore-deficient A. eutrophus mutants under iron-restricted conditions and promoted 59Fe uptake by iron-limited cells. However, the growth of the Sid- mutant AE1152, which was obtained from CH34 by Tn5-Tc mutagenesis, was completely inhibited by the addition of alcaligin E. AE1152 also showed strongly reduced 59Fe uptake in the presence of alcaligin E. This indicates that a gene, designated aleB, which is involved in transport of ferric iron-alcaligin E across the membrane is inactivated. The aleB gene was cloned, and its putative amino acid sequence showed strong similarity to those of ferric iron-siderophore receptor proteins. Both wild-type strain CH34 and aleB mutant AE1152 were able to use the same heterologous siderophores, indicating that AleB is involved only in ferric iron-alcaligin E uptake. Interestingly, no utilization of pyochelin, which is also a phenolate-type siderophore, was observed for A. eutrophus CH34. Genetic studies of different Sid- mutants, obtained after transposon mutagenesis, showed that the genes involved in alcaligin E and ferric iron-alcaligin E receptor biosynthesis are clustered in a 20-kb region on the A. eutrophus CH34 chromosome in the proximity of the cys-232 locus.
Project description:Chromosomal insertions defining Bordetella bronchiseptica siderophore phenotypic complementation group III mutants BRM3 and BRM5 were found to reside approximately 200 to 300 bp apart by restriction mapping of cloned genomic regions associated with the insertion markers. DNA hybridization analysis using B. bronchiseptica genomic DNA sequences flanking the cloned BRM3 insertion marker identified homologous Bordetella pertussis UT25 cosmids that complemented the siderophore biosynthesis defect of the group III B. bronchiseptica mutants. Subcloning and complementation analysis localized the complementing activity to a 2.8-kb B. pertussis genomic DNA region. Nucleotide sequencing identified an open reading frame predicted to encode a polypeptide exhibiting strong similarity at the primary amino acid level with several pyridoxal phosphate-dependent amino acid decarboxylases. Alcaligin production was fully restored to group III mutants by supplementation of iron-depleted culture media with putrescine (1,4-diaminobutane), consistent with defects in an ornithine decarboxylase activity required for alcaligin siderophore biosynthesis. Concordantly, the alcaligin biosynthesis defect of BRM3 was functionally complemented by the heterologous Escherichia coli speC gene encoding an ornithine decarboxylase activity. Enzyme assays confirmed that group III B. bronchiseptica siderophore-deficient mutants lack an ornithine decarboxylase activity required for the biosynthesis of alcaligin. Siderophore production by an analogous mutant of B. pertussis constructed by allelic exchange was undetectable. We propose the designation odc for the gene defined by these mutations that abrogate alcaligin siderophore production. Putrescine is an essential precursor of alcaligin in Bordetella spp.
Project description:Bordetella bronchiseptica mutants BRM1, BRM6, and BRM9 fail to produce the native dihydroxamate siderophore alcaligin. A 4.5-kb BamHI-Smal Bordetella pertussis genomic DNA fragment carried multiple genes required to restore alcaligin production to these siderophore-deficient mutants. Phenotypic complementation analysis using subclones of the 4.5-kb genomic region demonstrated that the closely linked BRM1 and BRM9 mutations were genetically separable from the BRM6 mutation, and both insertions exerted strong polar effects on expression of the downstream gene defined by the BRM6 mutation, suggesting a polycistronic transcriptional organization of these alcaligin biosynthesis genes. Subcloning and complementation experiments localized the putative Bordetella promoter to a 0.7-kb BamHI-SphI subregion of the cloned genomic DNA fragment. Nucleotide sequencing, phenotypic analysis of mutants, and protein expression by the 4.5-kb DNA fragment in Escherichia coli suggested the presence of three alcaligin system genes, namely, alcA, alcB, and alcC. The deduced protein products of alcA, alcB, and alcC have significant primary amino acid sequence similarities with known microbial siderophore biosynthesis enzymes. Primer extension analysis mapped the transcriptional start site of the putative alcaligin biosynthesis operon containing alcABC to a promoter region overlapping a proposed Fur repressor-binding site and demonstrated iron regulation at the transcriptional level.
Project description:A putative operon encoding an uncharacterized ferrous iron transport (FtrABCD) system was previously identified in cDNA microarray studies. In growth studies using buffered medium at pH values ranging from pH?6.0 to 7.6, Bordetella pertussis and Bordetella bronchiseptica FtrABCD system mutants showed dramatic reductions in growth yields under iron-restricted conditions at pH?6.0, but had no growth defects at pH?7.6. Supplementation of culture medium with 2?mM ascorbate reductant was inhibitory to alcaligin siderophore-dependent growth at pH?7.6, but had a neglible effect on FtrABCD system-dependent iron assimilation at pH?6.0 consistent with its predicted specificity for ferrous iron. Unlike Bordetella siderophore-dependent and haem iron transport systems, and in agreement with its hypothesized role in transport of inorganic iron from periplasm to cytoplasm, FtrABCD system function did not require the TonB energy transduction complex. Gene fusion analysis revealed that ftrABCD promoter activity was maximal under iron-restricted growth conditions at acidic pH. The pH of human airway surface fluids ranges from pH?5.5 to 7.9, and the FtrABCD system may supply ferrous iron necessary for Bordetella growth in acidic host microenvironments in which siderophores are ineffective for iron retrieval.
Project description:Bordetella pertussis and Bordetella bronchiseptica, which are respiratory mucosal pathogens of mammals, produce and utilize the siderophore alcaligin to acquire iron in response to iron starvation. A predicted permease of the major facilitator superfamily class of membrane efflux pumps, AlcS (synonyms, OrfX and Bcr), was reported to be encoded within the alcaligin gene cluster. In this study, alcS null mutants were found to be defective in growth under iron starvation conditions, in iron source utilization, and in alcaligin export. trans complementation using cloned alcS genes of B. pertussis or B. bronchiseptica restored the wild-type phenotype to the alcS mutants. Although the levels of extracellular alcaligin measured in alcS strain culture fluids were severely reduced compared with the wild-type levels, alcS mutants had elevated levels of cell-associated alcaligin, implicating AlcS in alcaligin export. Interestingly, a deltaalcA mutation that eliminated alcaligin production suppressed the growth defects of alcS mutants. This suppression and the alcaligin production defect were reversed by trans complementation of the deltaalcA mutation in the double-mutant strain, confirming that the growth-defective phenotype of alcS mutants is associated with alcaligin production. In an alcA::mini-Tn5 lacZ1 operon fusion strain background, an alcS null mutation resulted in enhanced AlcR-dependent transcriptional responsiveness to alcaligin inducer; conversely, AlcS overproduction blunted the transcriptional response to alcaligin. These transcription studies indicate that the alcaligin exporter activity of AlcS is required to maintain appropriate intracellular alcaligin levels for normal inducer sensing and responsiveness necessary for positive regulation of alcaligin system gene expression.
Project description:A Fur titration assay was used to isolate DNA fragments bearing putative Fur binding sites (FBS) from a partial Bordetella bronchiseptica genomic DNA library. A recombinant plasmid bearing a 3.5-kb DNA insert was further studied. Successive deletions in the cloned fragment enabled us to map a putative FBS at about 2 kb from one end. Sequence analysis revealed the presence of an FBS upstream from a new gene encoding an AraC-type transcriptional regulator. The deduced protein displays similarity to PchR, an activator of pyochelin siderophore and ferripyochelin receptor synthesis in Pseudomonas aeruginosa. Homologous genes in Bordetella pertussis and Bordetella parapertussis were PCR amplified, and sequence comparisons indicated a very high conservation in the three species. The B. pertussis and B. bronchiseptica chromosomal genes were inactivated by allelic exchange. Under low-iron growth conditions, the mutants did not secrete the alcaligin siderophore and lacked AlcC, an alcaligin biosynthetic enzyme. Alcaligin production was restored after transformation with a plasmid bearing the wild-type gene. On the basis of its role in regulation of alcaligin biosynthesis, the new gene was designated alcR. Additional sequence determination showed that alcR is located about 2 kb downstream from the alcABC operon and is transcribed in the same orientation. Two tightly linked open reading frames, alcD and alcE, were identified between alcC and alcR. AlcE is a putative iron-sulfur protein; AlcD shows no homology with the proteins in the database. The production of major virulence factors and colonization in the mouse respiratory infection model are AlcR independent.
Project description:Serological studies of patients with pertussis and the identification of antigenic Bordetella pertussis proteins support the hypothesis that B. pertussis perceives an iron starvation cue and expresses multiple iron source utilization systems in its natural human host environment. Furthermore, previous studies using a murine respiratory tract infection model showed that several of these B. pertussis iron systems are required for colonization and persistence and are differentially expressed over the course of infection. The present study examined genome-wide changes in B. pertussis gene transcript abundance in response to iron starvation in vitro. In addition to known iron source utilization genes, we identified a previously uncharacterized iron-repressed cytoplasmic membrane transporter system, fbpABC, that is required for the utilization of multiple structurally distinct siderophores including alcaligin, enterobactin, ferrichrome, and desferrioxamine B. Expression of type III secretion system genes was also found to be upregulated during iron starvation in both B. pertussis strain Tohama I and Bordetella bronchiseptica strain RB50. In a survey of type III secretion system protein production by an assortment of B. pertussis laboratory-adapted and low-passage clinical isolate strains, iron limitation increased the production and secretion of the type III secretion system-specific translocation apparatus tip protein Bsp22 in all Bvg-proficient strains. These results indicate that iron starvation in the infected host is an important environmental cue influencing not only Bordetella iron transport gene expression but also the expression of other important virulence-associated genes.
Project description:The recently discovered pathogen Bordetella holmesii has been isolated from the airways and blood of diseased humans. Genetic events contributing to the emergence of B. holmesii are not understood, and its phylogenetic position among the bordetellae remains unclear. To address these questions, B. holmesii strains were analyzed by comparative genomic hybridization (CGH) to a Bordetella pertussis microarray and by multilocus sequence typing. Both methods indicated substantial sequence divergence between B. pertussis and B. holmesii. However, CGH identified a putative pathogenicity island of 66 kb that is highly conserved between these species and contains several IS481 elements that may have been laterally transferred from B. pertussis to B. holmesii. This island contains, among other genes, a functional, iron-regulated locus encoding the biosynthesis, export, and uptake of the siderophore alcaligin. The acquisition of this genomic island by B. holmesii may have significantly contributed to its emergence as a human pathogen. Horizontal gene transfer between B. pertussis and B. holmesii may also explain the unusually high sequence identity of their 16S rRNA genes.