%0 Journal Article %A Battisti, James M. %A Hicks, Linda D. %A Minnick, Michael F. %T A unique Coxiella burnetii lipoprotein involved in metal binding (LimB) %D 2011 %J Microbiology, %V 157 %N 4 %P 966-976 %@ 1465-2080 %R https://doi.org/10.1099/mic.0.046649-0 %K CBB, Coomassie brilliant blue %K LCV, large cell variant %K Ni-HRP, nickel-coated horseradish peroxidase %K MBP, maltose-binding protein %K qRT-PCR, quantitative real-time PCR %K EF-Ts, elongation factor-Ts %K NM, Nine Mile %K PII, ‘phase II’ %K ELR, envelope localization residue %K FW, far-Western %K HRP, horseradish peroxidase %K SCV, small cell variant %K PIS, pre-immune serum %I Microbiology Society, %X 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, Ni2+ or Zn2+, and roughly halved in the presence of Co2+ or Fe3+. 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. %U https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.046649-0