title: A unique Coxiella burnetii lipoprotein involved in metal binding (LimB).
abstract: Coxiella burnetii is the bacterial agent of Q fever in humans. Here, we describe a unique, ~7.2 kDa, surface-exposed lipoprotein involved in metal binding which we have termed LimB. LimB was initially identified as a potential metal-binding protein on far-Western (FW) blots containing whole-cell lysate proteins when probed with nickel-coated horseradish peroxidase (Ni-HRP) and developed with a chemiluminescent HRP substrate. The corresponding identity of LimB as CBU1224a was established by matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry. blast analyses with CBU1224a showed no significant similarity to sequences outside strains of C. burnetii. Additional in silico analyses revealed a putative 20 residue signal sequence with the carboxyl end demarcated by a potential lipobox (LSGC) whose Cys residue is predicted to serve as the N-terminal, lipidated Cys of mature LimB. The second residue of mature LimB is predicted to be Ala, an uncharged envelope localization residue. These features suggest that CBU1224a is synthesized as a prolipoprotein which is subsequently lipidated, secreted and anchored in the outer membrane. Mature LimB is predicted to contain 45 aa, of which there are 10 His and 5 Cys; both amino acids are frequently involved in binding transition metal cations. Recombinant LimB (rLimB) was generated and its Ni-HRP-binding activity demonstrated on FW blots. Ni-HRP binding by rLimB was inhibited by >95 % on FW blots done in the presence of EDTA, imidazole, Ni(2+) or Zn(2+), and roughly halved in the presence of Co(2+) or Fe(3+). The limB gene was maximally expressed at 3-7 days post-infection in Coxiella-infected Vero cells, coinciding with exponential phase growth. Two isoforms of LimB were detected on FW and Western blots, including a smaller (~7.2 kDa) species that was the predominant form in small cell variants and a larger isoform (~8.7 kDa) in large cell variants. LimB is Sarkosyl-insoluble, like many omps. The predicted surface location of LimB was verified by immunoelectron and immunofluorescence microscopy using anti-rLimB antibodies. Overall, the results suggest that LimB is a unique Coxiella lipoprotein that serves as a surface receptor for divalent metal cations and may play a role in acquiring at least one of these metals during intracellular growth.
authors.affiliation: Division of Biological Sciences, The University of Montana, Missoula, MT 59812, USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Battisti
authors.affiliation: Division of Biological Sciences, The University of Montana, Missoula, MT 59812, USA.
authors.forename: Linda D
authors.initials: LD
authors.lastname: Hicks
authors.affiliation: Division of Biological Sciences, The University of Montana, Missoula, MT 59812, USA.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Minnick
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002413:Cations, Divalent / Q000378:metabolism*; D002522:Chlorocebus aethiops; D016997:Coxiella burnetii / Q000378:metabolism*; D004847:Epithelial Cells / Q000382:microbiology; D020869:Gene Expression Profiling; D005798:Genes, Bacterial; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D011485:Protein Binding; D020033:Protein Isoforms / Q000032:analysis; D017385:Sequence Homology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014709:Vero Cells
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D008074:Lipoproteins; D008565:Membrane Proteins; D008670:Metals; D020033:Protein Isoforms