Identification, Characterization, and Functional Analysis of a Gene Encoding the Ferric Uptake Regulation Protein inBartonella Species

Bacterial strains.Strains and plasmids used in this study are listed in Table 1. AllBartonella strains were used at low passage numbers (passes 1 through 3). B. henselae and B. quintana strains were grown on fresh chocolate agar (14), which provided a replete iron source. B. bacilliformis was grown on fresh heart infusion agar supplemented with 5% defibrinated rabbit blood (Hemostat Labs, Dixon, Calif.). Plates were incubated at 34°C (B. henselae and B. quintana) or 29°C (B. bacilliformis) in an enriched CO₂ environment for 5 to 7 days. Iron availability to B. henselae and B. quintana was restricted by adding the ferric-specific chelating agent EDDHA (ethylene diamine dihydroxy-o-phenylacetic acid) (The Complete Green Company, El Segundo, Calif.) (25) or by decreasing the hemoglobin (Hb) concentration in agar.

Vibrio strains were grown overnight in Luria-Bertani medium with the appropriate selective antibiotic(s), with or without the iron chelator 2,2-dipyridyl (Sigma-Aldrich, Inc., St. Louis, Mo.), as previously described (8). Because of limited growth at 37°C by CML13(pSYP4), all strains were grown at 30°C and then incubated at 37°C for 60 to 90 min before assays. Selective antibiotics were added to growth media as required, at the following concentrations: ampicillin, 100 μg/ml; chloramphenicol, 25 μg/ml; kanamycin, 50 μg/ml; streptomycin, 100 μg/ml; tetracycline, 25 μg/ml.

Identification and analysis of Bartonella Fur.A 94-bp internal fragment of the B. henselae fur gene was amplified from Bartonella genomic DNA using degenerate primers designed from highly conserved fur gene sequences of other bacteria: FURMID-5′R, 5′-GGA ATT CCA (C,T)CA (C,T)GA (C,T)CA (C,T)(A,C)T (A,C,G,T)AT (A,C,T)GA-3′; FUREND-3′B, 5′-GGG ATC C(G,A)T A(A,C,G,T)A (G,A)(C,T)T C(A,C,G,T)A (G,A)(A,C,G,T)C G(G,A)T G-3′ (Operon Technologies Inc., Alameda, Calif.). TheB. henselae fur gene was identified by probing a B. henselae genomic DNA library with the B. henselae furgene fragment.

To generate fur gene fragment probes for screening ofB. quintana and B. bacilliformis genomic DNA libraries, the fur gene open reading frame (ORF) was amplified by PCR from B. quintana and B. bacilliformis genomic DNA. The amplified fur gene fragments were then used to probe B. quintana or B. bacilliformis genomic DNA libraries. An ORF of 417 bp was identified for all three Bartonella species.

The deduced amino acid sequences of the Bartonella Fur proteins (Fig. 1) revealed that they each have a predicted length of 138 amino acids and are highly homologous (B. henselae and B. quintana, 89% identity;B. henselae and B. bacilliformis, 82% identity;B. bacilliformis and B. quintana, 79% identity). The B. henselae Fur protein had 38% amino acid identity with Escherichia coli and Vibrio cholerae Fur and 68% identity with Brucella abortus Fur. TheBartonella Fur amino acid sequence is rich in histidine residues, as are other Fur proteins (3, 4, 26), and 6 of the 42 invariant residues shown in Fig. 1 are histidines. Histidine is the primary amino acid involved in the binding of iron in heme (26, 28). The three Bartonella Fur proteins contain His-His-Asp-His, part of another suggested iron-binding motif, His-His-His-X-His-X₂-Cys-X₂-Cys, located at positions 86 to 96 of the E. coli and V. choleraeFur proteins (17). The theoretical isoelectric point ofBartonella Fur is higher (8.1) than that of other Fur proteins (Brucella abortus, 6.1; V. cholerae, 5.5; E. coli, 5.7).

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Fig. 1.

Alignment of Bartonella species Fur proteins with those of other gram-negative bacteria. The Fur amino acid sequences of three Bartonella species and three other gram-negative bacterial species were aligned using the Clustal W program from the European Bioinformatics Institute (Cambridge, United Kingdom). There are 42 conserved amino acid positions (shaded). Degenerate oligonucleotide primers (arrows), based on two of the conserved regions (boxes), were designed and utilized in the initial amplification of a B. henselae fur gene fragment. Compared with other gram-negative species, B. henselae Fur had the greatest homology with Brucella abortus.

Confirmation of Bartonella fur genes by Southern blot analysis. Bartonella genomic DNA (approximately 2 μg each) was digested with HindIII and probed with approximately 250 ng of gel-purified, 417-bp B. henselae furgene ORF. The B. henselae fur gene probe hybridized to a single band in each lane (in proportion to the homology with theB. henselae fur probe [Fig.2]), indicating that each species likely contains a single fur gene, as in other bacterial species (1, 19, 21, 30, 31).

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Fig. 2.

Southern hybridization analysis of Bartonellaspecies genomic DNA using a B. henselae fur gene probe.B. henselae, B. quintana, and B. bacilliformisDNAs were digested with HindIII. The fragments were separated in a 0.8% agarose gel, transferred to a nylon membrane, and probed with the 417-bp B. henselae fur gene. There was a single band in each lane of HindIII-digested genomic DNA: an approximately 4,200-bp fragment of B. henselae DNA, an approximately 2,000-bp fragment of B. quintana DNA, and an approximately 3,600-bp fragment of B. bacilliformisDNA.

Complementation studies.Functional homology ofBartonella Fur with the Fur proteins of other gram-negative bacteria was confirmed by complementation studies with a V. cholerae fur mutant. For these studies, the B. henselae fur gene was cloned into pACYC184. The plasmid pSYP3, in which theB. henselae fur ORF replaces the gfp cassette at the BamHI and SphI sites downstream from a constitutive ptac promoter in the pANT4 plasmid, was used as template to amplify the ptac promoter-B. henselae fur ORF sequence. The ptac-fur ORF was ligated into pACYC184, creating pSYP4. Clones were selected for chloramphenicol resistance and tetracycline sensitivity and confirmed by sequence analysis. pSYP4 and pACYC184 were introduced into V. cholerae strains. Expression of the Bartonella fur gene in the transformed V. cholerae fur mutant, CML13(pSYP4), was confirmed by separating proteins from whole-cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data not shown) (16).

The V. cholerae strain MBG40 has TnphoA inserted into the iron-regulated irgA gene in the chromosome such that alkaline phosphatase reporter activity, determined by spectrophotometric measurement of hydrolysis ofp-nitrophenyl phosphate by permeabilized cells (8, 19, 20), is strongly regulated by iron and Fur. In a furmutant of this strain, CML13, alkaline phosphatase activity is not repressed by Fur despite changes in iron concentration (20). The ability of the Bartonella fur gene product to complement the V. cholerae fur mutant was studied by expressing Bartonella fur in this Vibriostrain, CML13(pSYP4), and determining alkaline phosphatase activity. Experiments were performed in triplicate, on four different days. Results were stratified by day and analyzed using a two-sided Mann-Whitney U test.

We observed that the iron-mediated regulation of reporter gene expression could be complemented by constitutive expression of theB. henselae fur gene in the V. cholerae furmutant (Table 2). As shown previously (20), we found that the V. cholerae strain MBG40, producing a wild-type V. cholerae Fur protein, repressed reporter gene expression when grown on high-iron medium (induction ratio 56 [Table 2]). In contrast, the V. cholerae fur mutant CML13 had partially derepressed alkaline phosphatase reporter gene expression in the presence of high iron concentrations (induction ratio 10 [Table 2]). Plasmid pSYP4 complemented thefur mutation in strain CML13, restoring iron regulation very nearly to the level of strain MBG40. Repression of alkaline phosphatase activity may not have been complete because of the limited amino acid homology between V. cholerae Fur and B. henselaeFur (38%), the difference in pI, or both, which may result in reduced affinity of the Bartonella Fur-Fe²⁺ homodimer for the Vibrio Fur binding sequence. The absence of wild-type Vibrio Fur in CML13, CML13(pACYC184), and CML13(pSYP4) was confirmed by immunoblot analysis with polyclonal anti-Vibrio Fur antibody (data not shown). For reasons that are not clear, the vector plasmid pACYC184 partially restored iron regulation in strain CML13; nevertheless, the level of alkaline phosphatase expression in CML13(pACYC184) in high-iron media remained significantly higher (P < 0.001) than in either CML13(pSYP4) or MBG40. When iron was depleted, all four strains demonstrated derepression of reporter gene activity, although derepression was greatest in the V. cholerae fur mutant strain, a finding consistent with those of Litwin and Calderwood (20). Interestingly, the V. cholerae fur mutant CML13 did not produce equivalent alkaline phosphatase activity when grown under iron-replete compared with iron-depleted conditions. There was apparently some degree of regulation mediated by iron despite the absence of a functional Fur protein, as previously noted (20). Although the Bartonella Fur protein functionally complemented a V. cholerae fur mutant, there may be substantial structural differences compared with V. cholerae Fur. This is corroborated by the failure of a polyclonal anti-V. cholerae Fur antibody (33) to recognizeBartonella Fur (wild type or overexpressed) by immunoblotting (data not shown).

Studies of the fur upstream region fused to agfp reporter gene.The 204-bp region immediately upstream of the B. henselae fur gene ORF was amplified from genomic DNA and cloned into pANT3 just upstream of the gfpgene to create the plasmids pSYP1 (forward fur upstream sequence) and pSYP2 (inverted fur upstream sequence).

Plasmids were transferred into Bartonella strains by conjugation. B. henselae(pSYP1) and B. quintana(pSYP1) were grown on agar containing different concentrations of available iron and then scraped and suspended in phosphate-buffered saline for FACS analysis (FACScalibur; Becton Dickinson, Franklin Lakes, N.J.) (18). Chocolate plates were prepared with final EDDHA concentrations of 250, 300, 325, and 350 μM or with final Hb concentrations of 0.5, 1, 10, 20, and 30 mg/ml. (B. henselae growth is restricted on chocolate agar containing ≥400 μM EDDHA, and B. quintana growth is restricted at ≥350 μM EDDHA. Growth of both species is restricted at Hb concentrations of ≤1 mg/ml, and growth is optimal on chocolate agar plates containing 10 mg of Hb/ml.) The wild-type B. henselaeand B. quintana strains were streaked on chocolate agar plates containing 10 mg of Hb/ml as negative fluorescence controls.Bartonella species containing a plasmid with a constitutively expressed gfp reporter gene (pANT4) or containing the upstream fur region in an inverted position (pSYP2) also were grown on agar containing different concentrations of available iron.

B. quintana(pSYP1), B. henselae(pSYP1), and the control B. henselae(pSYP2) demonstrated no change in GFP (green fluorescent protein) expression regardless of whether they were grown on iron-replete or iron-deficient medium. Wild-type B. henselae exhibited a small amount of autofluorescence, andB. henselae(pANT4) demonstrated stable fluorescence at an average of 1,000-fold higher than wild-type strains. B. henselae(pSYP2) and B. quintana(pSYP2) did demonstrate a small amount of GFP activity, but this activity did not vary with changes in iron concentration (data not shown) and probably represents artifactual activity.

Studies of other bacteria, e.g., E. coli (6, 7, 22), indicate that fur transcription is autoregulated in the presence of iron. We were unable to identify a Fur binding sequence or “iron box” that fulfilled the consensus of a 19-bp hyphenated dyad repeat (22) or two directed and one inverted 6-bp repeats (7) in the 204-bp region upstream of the Bartonella fur gene. However, there is one 19-bp sequence (positions 83 to 101) that conserves all four of the invariant nucleotides of the consensus (positions 6 and 14 to 16) but not the dyad repeat structure of the 19-bp sequence. This sequence is located within an ORF immediately upstream of fur, and functional, iron-regulated binding of this DNA sequence by BartonellaFur was not detected by our gfp reporter assay. An autoregulatory binding sequence also is absent in the regions upstream of fur in Bradyrhizobium japonicum(9) and V. cholerae (19).

For other gram-negative bacteria, a promoter is located directly upstream of the fur gene; for some bacteria, a Fur binding region is located within the promoter region. The E. coli fur gene can be transcribed and regulated from its own promoter, or, in response to oxidative stress, it can be transcribed as part of an operon including the upstream fldA gene in addition to the downstream fur gene (35). We were unable to detect any promoter activity (regardless of iron availability) or autoregulatory activity in the 204-bp region upstream of theBartonella fur gene. Because there are fewer than 35 bp between the upstream ORF and the Bartonella fur gene, and no promoter activity was detected in the upstream 204-bp region, it is possible that the Bartonella fur gene lacks its own promoter and that it is transcribed exclusively as part of an operon, which would be an unusual arrangement.

Conclusions. Bartonella has an obligate heme requirement (27, 34) and alternates between iron-rich and iron-restricted environments. In this study, we identified, cloned, and sequenced a fur gene in B. henselae, B. quintana, and B. bacilliformis. The deduced amino acid sequences of the three Bartonella fur genes demonstrate a high degree of homology among each other and substantial homology with the closest phylogenetic relative, Brucella abortus (24). To date, multiple attempts to generate a Bartonella furmutant have been unsuccessful. Loss of the fur gene may be a lethal mutation in Bartonella, as occurs in some other gram-negative bacteria (1, 9, 23). Study of gene regulation by Fur in Bartonella will be critical to elucidating the mechanisms of iron acquisition, virulence gene expression, and Bartonella pathogenesis.

Nucleotide sequence accession numbers.The fur gene sequences of the three Bartonella species used in this study have been deposited in GenBank under accession numbers AF388196 (B. henselae fur), AF388197 (B. quintana fur), and AF388198 (B. bacilliformis fur).