Unknown

Dataset Information

0

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.

SUBMITTER: Battisti JM 

PROVIDER: S-EPMC3139440 | biostudies-other | 2011 Apr

REPOSITORIES: biostudies-other

altmetric image

Publications

A unique Coxiella burnetii lipoprotein involved in metal binding (LimB).

Battisti James M JM   Hicks Linda D LD   Minnick Michael F MF  

Microbiology (Reading, England) 20110106 Pt 4


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 matri  ...[more]

Similar Datasets

| S-EPMC3320512 | biostudies-literature
2012-07-20 | PRD000262 | Pride
| S-EPMC2849434 | biostudies-literature
| EMPIAR-11141 | biostudies-other
| S-EPMC6436347 | biostudies-literature
| S-EPMC3539893 | biostudies-literature
| S-EPMC3382237 | biostudies-literature
2011-12-31 | GSE31543 | GEO
| S-EPMC3206805 | biostudies-literature
| S-EPMC3559035 | biostudies-literature