DEVELOPMENTALLY-REGULATED SYNTHESIS OF AN UNUSUALLY SMALL, BASIC PEPTIDE BY COXIELLA-BURNETII

Coxiella burnetii undergoes a poorly defined developmental cycle withi n phagolysosomes of eukaryotic host cells. Two distinct developmental forms are part of this cycle: a small-cell variant (SCV) and large-cel l variant (LCV). Ultrastructurally, the SCV is distinguished from the LCV by its smaller size and condensed chromatin. At a molecular level, little is known about morphogenesis in C. burnetii, and no proteins s pecific to the SCV have been identified. Preparative isoelectric focus ing was conducted to purify basic proteins possibly involved in SCV ch romatin structure. A predominant protein of low M(r) was present in th e most basic fraction, eluting with a pH of approx. 11. Degenerate deo xyoligonucleotides corresponding to the N-terminal sequence of this pr otein were used to recover a cosmid clone from a C. burnetii genomic l ibrary. Nucleotide sequencing of insert DNA revealed an open reading f rame designated scvA (small-cell-variant protein A) with coding potent ial for a 30 amino acid protein (ScvA) with a predicted M(r) of 3610. ScvA is 46% arginine plus 46% glutamine with a predicted pi of 12.6. S DS-PAGE and silver staining of lysates of SCV and LCV purified by caes ium chloride-equilibrium density centrifugation revealed a number of p roteins unique to each cell type. Immunoblot analysis with ScvA antise rum demonstrated the presence of ScvA only in the SCV. By immunoelectr on microscopy, ScvA antiserum labelled only the SCV, with the label co ncentrated on the condensed nucleoid. In addition, ScvA bound double-s tranded DNA in gel mobility-shift assays. A 66% reduction in the mean number of gold particles per Coxiella cell was observed at 12 h post-i nfection when compared with the starting inoculum. Collectively, these data suggest that synthesis of ScvA is developmentally regulated, and that the protein may serve a structural or functional role as an inte gral component of the SCV chromatin. Moreover, degradation of this pro tein may be a necessary prerequisite for morphogenesis from SCV to LCV .