Characterization and expression of the cbbE" gene of Coxiella burnetii

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Abstract
A gene which is unique to the QpRS plasmid from chronic isolates of Coxiella burnetii was cloned, sequenced, and expressed in Escherichia coli. This gene, termed cbbE'', codes for a putative surface protein of approximately 55 kDa, termed the E'' protein. The cbbE'' gene is 1485 bp in length, and is preceded by predicted promoter regulatory sequences of TTTAAT (-35), TATAAT (-10), and a Shine-Dalgarno sequence of GGAGAGA, all of which closely resemble those of E. coli and other rickettsiae. The open reading frame (ORF) of cbbE'' ends with a UAA codon followed by a second in-frame UAG stop codon and a region of dyad symmetry which may act as a rho-factor-independent terminator. The ORF of cbbE'' is capable of coding for a polypeptide of 495 amino acids with a predicted molecular mass of 55893 Da. The E'' protein has a predicted pI of .apprx.8.7, and contains a distinct hydrophobic region of 12 amino acid residues. In vitro transcription/translation and E. coli expression of recombinant plasmids containing cbbE'' produce a protein of approximately 55 kDa. The in vivo expression of cbbE'' yields a novel protein that can be detected on immunoblots developed with rabbit antiserum generated against purified outer membrane from C. burnetii. DNA hybridization analysis shows that cbbE''is unique to the QpRS plasmidfound in chronic isolates of C. burnetii, and is absent in chromosomal DNA and plasmids (QpH1,QpDG) from other isolates of C. burnetti. A search of various DNA and amino acid sequence data bases revealed no homologies of cbbE''.