Analysis of QpRS-specific sequences from Coxiella burnetii

Coxiella burnetii from acute cases of Q fever possess a plasmid termed QpH1. Chronic isolates contain a plasmid termed QpRS or have QpRS sequences integrated into the chromosome. The correlation between an isolate's plasmid type and the chronic or acute nature of the disease has prompted analysis of unique plasmid sequences to determine if they contain virulence genes. DNA hybridization has determined that a portion of a 3.6-kb EcoR I fragment (epsilon') is unique to QpRS. In vitro transcription/translation (IVTT) of the epsilon' fragment yielded a 55-kDa protein regardless of the cloning orientation, suggesting that transcription resulted from a rickettsial promoter. A translational start site was mapped to the 1.2-kb Pst I-EcoR I subfragment of epsilon' by IVTT. DNA sequencing showed an open reading frame (ORF) of 1485 bp, capable of coding for a protein of ca. 55.9 kDa. This ORF was termed cbbE'. Putative promoter regions of cbbE' included TTTAAT (-35), TATAAT (-10), and a ribosome-binding site GGAGAGA. The ORF ended with a stop codon UAA and was followed by UAG and a potential factor-independent transcription-termination region. In-frame cloning of the 695-bp Pst I subfragment into pUC9 resulted in a fusion protein of ca. 37 kDa, confirming the frame and length of the ORF as predicted by DNA sequencing. The specificity of this gene to QpRS was confirmed by probing DNA from three plasmid groups of C. burnetii, using the internal 695-bp Pst I fragment of cbbE'.