Dataset Information

The fur homologue in Borrelia burgdorferi.

ABSTRACT: Borrelia burgdorferi contains a gene that codes for a Fur homologue. The function of this Fur protein is unknown; however, spirochetes grown at 23 or 35 degrees C expressed fur as determined by reverse transcriptase PCR. The fur gene (BB0647) was cloned and overexpressed as a His-Fur fusion protein in Escherichia coli. The fusion protein was purified by zinc-chelate chromatography, and the N-terminal His tag was removed to generate recombinant Fur for use in mobility shift studies. Fur bound DNA containing the E. coli Fur box sequence (GATAATGATAATCATTATC) or Bacillus subtilis Per box sequence (TTATAAT-ATTATAA) with an apparent Kd of approximately 20 nM. Fur also bound the upstream sequences of three Borrelia genes: BB0646 (gene encoding a hydrolase of the alpha/beta-fold family), BB0647 (fur), and BB0690 (napA). Addition of metal ions was not required. Binding activity was greatly decreased by either exposure to oxidizing agents (H2O2, t-butyl hydroperoxide, cumene hydroperoxide, or diamide) or by addition of Zn2+. B. burgdorferi NapA is a homologue of Dps. Dps functions in E. coli to protect DNA against damage during periods of redox stress. Fur may function in B. burgdorferi as a repressor and regulate oxidative stress genes. Additional genes (10 chromosomal and 15 plasmid) that may be Fur regulated were identified by in silico analysis.

SUBMITTER: Katona LI

PROVIDER: S-EPMC516618 | biostudies-literature | 2004 Oct

REPOSITORIES: biostudies-literature

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The fur homologue in Borrelia burgdorferi.

Katona Laura I LI Tokarz Rafal R Kuhlow Christopher J CJ Benach Jordi J Benach Jorge L JL

Journal of bacteriology 20041001 19

Borrelia burgdorferi contains a gene that codes for a Fur homologue. The function of this Fur protein is unknown; however, spirochetes grown at 23 or 35 degrees C expressed fur as determined by reverse transcriptase PCR. The fur gene (BB0647) was cloned and overexpressed as a His-Fur fusion protein in Escherichia coli. The fusion protein was purified by zinc-chelate chromatography, and the N-terminal His tag was removed to generate recombinant Fur for use in mobility shift studies. Fur bound DNA ...[more]

PMID: 15375125

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Project description:The ferric uptake regulator (Fur) family of DNA-binding proteins represses and/or activates gene transcription via divalent metal ion-dependent signal sensing. The Borrelia burgdorferi Fur homologue, also known as Borrelia oxidative stress regulator (BosR), promotes spirochetal adaptation to the mammalian host by directly repressing the lipoproteins required for tick colonization and indirectly activating those required for establishing infection in the mammal. Here, we examined whether the DNA-binding activity of BosR was regulated by any of the four most prevalent transition metal ions in B. burgdorferi, Mn, Fe, Cu, and Zn. Our data indicated that in addition to a structural site occupied by Zn(II), BosR had two regulatory sites that could be occupied by Zn(II), Fe(II), or Cu(II) but not by Mn(II). While Fe(II) had no effect, Cu(II) and Zn(II) had a dose-dependent inhibitory effect on the BosR DNA-binding activity. Competition experiments indicated that Cu(II) had a higher affinity for BosR than Zn(II) or Fe(II). A BosR deficiency in B. burgdorferi resulted in a significant increase in the Cu level but no significant change in the levels of Mn, Fe, or Zn. These data suggest that Cu regulates BosR activity, and BosR in turn regulates Cu homeostasis in B. burgdorferi While this regulatory paradigm is characteristic of the Fur family, BosR is the first one shown to be responsive to Cu(II), which may be an adaptation to the potentially high level of Cu present in the Lyme disease spirochete.IMPORTANCE Transition metal ions serve an essential role in the metabolism of all living organisms. Members of the ferric uptake regulator (Fur) family play critical roles in regulating the cellular homeostasis of transition metals in diverse bacteria, and their DNA-binding activity is often regulated by coordination of the cognate divalent metal ions. To date, regulators with metal ion specificity to Fe(II), Mn(II), Zn(II), and Ni(II) have all been described. In this study, we demonstrate that BosR, the sole Fur homologue in Borrelia burgdorferi, is responsive to Cu(II) and regulates Cu homeostasis in this bacterium, which may be an adaption to potentially Cu-rich milieu in the Lyme disease spirochete. This study has expanded the repertoire of the Fur family's metal ion specificity.

Project description:Borrelia burgdorferi and Borrelia miyamotoi are tick-vectored zoonotic pathogens maintained in wildlife species. Tick populations are establishing in new areas globally in response to climate change and other factors. New Brunswick is a Canadian maritime province at the advancing front of tick population establishment and has seen increasing numbers of ticks carrying B. burgdorferi, and more recently B. miyamotoi. Further, it is part of a region of Atlantic Canada with wildlife species composition differing from much of continental North America and little information exists as to the presence and frequency of infection of Borrelia spp. in wildlife in this region. We used a citizen science approach to collect a wide range of animals including migratory birds, medium-sized mammals, and small mammals. In total we tested 339 animals representing 20 species for the presence of B. burgdorferi and B. miyamotoi. We have developed new nested PCR primers and a protocol with excellent specificity for detecting both of these Borrelia species, both single and double infections, in tissues and organs of various wildlife species. The positive animals were primarily small non-migratory mammals, approximately twice as many were infected with B. burgdorferi than B. miyamotoi and one animal was found infected with both. In addition to established reservoir species, the jumping mouse (Napaeozapus insignis) was found frequently infected; this species had the highest infection prevalence for both B. burgdorferi and B. miyamotoi and has not previously been identified as an important carrier for either Borrelia species. Comprehensive testing of tissues found that all instances of B. burgdorferi infection were limited to one tissue within the host, whereas two of the five B. miyamotoi infections were diffuse and found in multiple systems. In the one coinfected specimen, two fetuses were also recovered and found infected with B. miyamotoi. This presumptive transplacental transmission suggests that vertical transmission in mammals is possible. This finding implies that B. miyamotoi could rapidly spread into wildlife populations, as well as having potential human health implications.

Dataset Information

The fur homologue in Borrelia burgdorferi.

Publications

The fur homologue in Borrelia burgdorferi.

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