Cloning and porin activity of the major outer membrane protein P1 from Coxiella burnetii.
Coxiella burnetii, the etiological agent of Q fever, is a gram-negative obligate intracellular bacterium. Two striking characteristics of this microorganism are its ability to thrive within a phagolysosome and its ability to persist in the environment outside a host cell. These abilities have been attributed to the existence of C. burnetii developmental cycle variants: large-cell variants (LCV), small-cell variants (SCV), and small dense cells (SDC). Variants differ in protein profiles, including differential expression of a major outer membrane protein (MOMP) of C. burnetii, designated P1. The approximately 29-kDa MOMP is highly expressed in LCV, down-regulated in SCV, and not apparent in SDC. We sought to characterize P1 through purification of native protein for N-terminal analysis, cloning, and functional studies. Highly purified P1, extracted from C. burnetii membranes by using the zwitterionic detergent Empigen, allowed the determination of N-terminal and internal peptide sequences. The entire P1 coding locus was cloned by PCR amplification based upon these peptide sequences, followed by inverse PCR. Comparison of the predicted P1 amino acid sequences among the C. burnetii isolates Nine Mile, Koka, Scurry, and Kerns indicated a high degree of conservation. Structural prediction suggests that the peptide has a predominantly beta-sheet conformation, consistent with bacterial porins. Typical porin characteristics were observed for native P1, including detergent solubilization properties, heat modification of purified protein, and channel formation in a planar lipid bilayer. Characterization of differentially expressed P1 as a porin increases our understanding of the function of morphological variants and their role in pathogenesis.
Varghees
Sunita
S
Department of Medical Microbiology and Immunology, Texas A&M University System Health Science Center, College Station 77843-1114, USA.
Kiss
Kati
K
Frans
Giovanni
G
Braha
Orit
O
Samuel
James E
JE
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D016997:Coxiella burnetii / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D018272:Porins / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; C469918:P1 protein, Coxiella burnetii; D018272:Porins