Characterization of IHF and set-domain proteins of Chlamydia trachomatis L2

Members of the genus Chlamydia are obligate intracellular bacteria. The purpose of this research was to determine the mechanisms by which the late-stage cysteine-rich protein (CRP) operon of Chlamydia trachomatis is regulated and to characterize the chlamydial SET protein, which contains a SET domain commonly found in eukaryotic proteins that associate with chromatin.; A DNA element to which a protein in chlamydial cell extracts binds was identified located between positions -135 and -90 upstream from the transcription start site of the CRP operon. The binding activity was detected in the early part of the late stage of the developmental cycle, declined dramatically in the extreme late stage, and was absent in the elementary body (EB) form. A recombinant protein of C. trachomatis ORF CT267, which is homologous to bacterial integration host factor (IHF), bound to the same DNA element with high affinity and produced the same DNase I-protection footprint as the protein in chlamydial extracts. It also induced a sharp bend in a DNA fragment containing the binding site, suggesting that the protein has IHF activity. The recombinant protein had a small positive effect on in vitro transcription of the CRP operon.; The expression of C. trachomatis SET-gene transcripts and SET protein (SET) was detected throughout the cycle; however, immunoblot assay suggested that SET accumulates only in the late stage of the developmental cycle. Immunofluorescence microscopy experiments showed SET is located within chlamydial inclusions, indicating that SET is not secreted into eukaryote host cells. Glutathione-S-transferase pull-down and gel overlay assays showed that SET associates with a 20-kDa chlamydial protein present only during the late stage of infection and in EBs. The 20-kDa protein co-migrates with chlamydial histone-like protein Hc1, which is believed to be responsible for condensation of the chlamydial genome and silencing of gene expression.; A working model for the interaction of IHF and SET with Hc1 was developed from these investigations. It is suggested that IHF permits the expression of some late-stage genes by preventing the association of Hc1 with late gene promoters and that SET may modulate Hc1 function by catalyzing transmethylation reaction.