The objective of this project was the cloning, expression and sequencing of a major antigen gene and to further chemically characterize the lipopolysaccharide (LPS) of Coxiella burnetii, the etiological agent of Q fever. A. Gene cloning. A gene library from the DNA of C. burnetii has been constructed in the cosmid vector pHC79. An immunoreactive gene product from clone pJB196 was identified as a 62 kilodalton (Kd) polypeptide both by Western blot and an in vitro transcription, translation system. Expression of the 62 Kd and a 14 Kd polypeptide was under the control of a heat shock promoter (HSP) in recombinant E. coli. Subcloning of pJB196 and sequencing a 3 Kb stretch of that DNA revealed two open reading frames, encoding a polypeptide of 58.9 Kd, and 10.5 Kd along with two appropriately spaced ribosomal binding sites. A transcriptional control element observed on the 5' side of the initiation codon resembled a HSP (at -452). Termination of this bicistronic mRNA synthesis may be under control of a rho-independent terminator with double dyad symmetry and delta G of -28.0 and 43.5 K cal, respectively. Four sequences were highly homologous to the 62 Kd protein from C. burnetii (greater than or equal to 50%). Three are from Mycobacteria and represent the immunodominant antigen of this species. The other is from Escherichia coli, detected as a gene that complements or suppresses a temperature-sensitive RNAase activity. Therefore this protein is conserved in phylogenetically distant bacterial genera. B. LPS Structure. The core structure of C. burnetii LPS consists of (1-3)-mannoheptose backbone with every residue (1-2) branched with a single mannose moiety. Lipid C (the counterpart of Salmonella lipid A) is quite unusual in that in lacks carbohydrate and is small in molecular weight. C. Significance. A heat shock operon in C. burnetii produces a major antigen highly homologous to a protein in both Mycobacteria and Escherichia coli. The core structure of LPS was defined and the lipid C molecule is significantly different from Salmonella lipid A.
