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

Abstract

Environmental iron concentrations coordinately regulate transcription of genes involved in iron acquisition and virulence via the ferric uptake regulation (fur) system. We identified and sequenced the fur gene and flanking regions of three Bartonella species. The most notable difference between Bartonella Fur and other Fur proteins was a substantially higher predicted isoelectric point. No promoter activity or Fur autoregulation was detected using a gfp reporter gene fused to the 204 nucleotides immediately upstream of the Bartonella fur gene. Bartonella henselae fur gene expression complemented a Vibrio cholerae fur mutant.Bartonella, an extremely fastidious gram-negative bacillus, causes cat scratch disease, bacillary angiomatosis, and other syndromes (2,13,15,29). Few data exist regarding the pathogenic mechanisms of this hemophilic bacterium (5), which can occupy two alternate niches: the iron-rich gut of obligately hematophagous arthropods and the iron-restricted bloodstream of mammals (11,32). Acquisition of iron and expression of many virulence factors are under transcriptional regulation by the fur gene product, the ferric uptake regulation (Fur) protein, and its homodimeric complex (7). At sufficient intracellular iron levels, the corepressors Fur and Fe 2ϩ form an active Fur-Fe 2ϩ complex that binds a consensus sequence ("iron box," a 19-bp hyphenated dyad repeat [22] or three repeats of 6 bp of the sequence NAT[A,T]AT [7]) in the promoter region of genes regulated by Fur, down-regulating genes encoding iron-scavenging proteins (7,22). We hypothesized that Bartonella species possess a fur gene homolog with a gene regulatory system influenced by iron levels.Bacterial strains. Strains and plasmids used in this study are listed in Table 1. All Bartonella 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 CO 2 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 g/ml; chloramphenicol, 25 g/ml; kanamycin, 50 g/ml; streptomycin, 100 g/ml...

TheSinorhizobium meliloti furGene Regulates, with Dependence on Mn(II), Transcription of thesitABCDOperon, Encoding a Metal-Type Transporter

“…Adjacent to the S. meliloti sitABCD operon a fur gene is located in the opposite orientation. The S. meliloti Fur protein showed the highest similarities to the Fur proteins of Bartonella species (60% identity) (47).…”

Three fur homologues from Anabaena sp. PCC7120: exploring reciprocal protein-promoter recognition

“…Normally there are iron boxes in the promoter regions of the fur gene [6][7][8][9][10] with some exceptions [11,12].…”

Hemin Binding, Functional Expression, and Complementation Analysis of Pap 31 fromBartonella henselae

“…Recently, the fur gene from B. henselae was characterized by Park et al (19); the Fur box of this gene was not detected. However, an autoregulatory binding sequence is also absent in the regions upstream of fur in Bradyrhizobium japonicum (12) and Vibrio cholerae (17).…”