Developmentally regulated synthesis of an unusually small, basic peptide by Coxiella burnetii.
Coxiella burnetii undergoes a poorly defined developmental cycle within phagolysosomes of eukaryotic host cells. Two distinct developmental forms are part of this cycle: a small-cell variant (SCV) and large-cell 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 specific to the SCV have been identified. Preparative isoelectric focusing was conducted to purify basic proteins possibly involved in SCV chromatin structure. A predominant protein of low M(r) was present in the most basic fraction, eluting with a pH of approx. 11. Degenerate deoxyoligonucleotides corresponding to the N-terminal sequence of this protein were used to recover a cosmid clone from a C. burnetii genomic library. Nucleotide sequencing of insert DNA revealed an open reading frame designated scvA (Small-Cell-variant protein A) with coding potential for a 30 amino acid protein (ScvA) with a predicted M(r) of 3610. ScvA is 46% arginine plus 46% glutamine with a predicted pl of 12.6. SDS-PAGE and silver staining of lysates of SCV and LCV purified by caesium chloride-equilibrium density centrifugation revealed a number of proteins unique to each cell type. Immunoblot analysis with ScvA antiserum demonstrated the presence of ScvA only in the SCV. By Immunoelectron microscopy, ScvA antiserum labelled only the SCV, with the label concentrated on the condensed nucleoid. In addition, ScvA bound double-stranded DNA in gel mobility-shift assays. A 66% reduction in the mean number of gold particles per Coxiella call was observed at 12 h post-infection 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 integral component of the SCV chromatin. Moreover, degradation of this protein may be a necessary prerequisite for morphogenesis from SCV to LCV.
Laboratory of Intracellular Parasites, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana, USA. robert-heinzen@nih.gov
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Heinzen
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Howe
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Mallavia
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Rockey
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Hackstadt
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D016997:Coxiella burnetii / Q000235:genetics* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009024:Morphogenesis / Q000235:genetics; D011485:Protein Binding; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C103852:ScvA protein, Coxiella burnetii