Ravi Kaul and Wanda M. Wenman

Department of Pediatrics, Section of Infectious Diseases, 403 Neurosciences Building, School of Medicine, 1515 Newton Court, University of California, Davis, CA 95616

Received 2/16/ 98 Accepted 2/20/98

6. Histone H1 specific protease

Within a few hours of entering the host cell EB lose their prominent electron-dense DNA core, the two histone H1-like proteins are undetectable, the cell envelope loses its rigidity, the cell increases in size from 0.3 to 1.0 m and begins to transcribe early stage-specific genes. It is clear, however, that the transcriptional initiation and cellular growth of Chlamydia is contingent upon DNA decondensation, which is closely associated with the lack of detection of two histone H1-like molecules. We reasoned that an early upstream open reading frame (EUO) gene product described by Wichlam and Hatch (45) might play an important role in Hc1 degradation and nucleoid decondensation since i) it is expressed very early in the chlamydial life cycle and ii) is highly transcribed compared with MOMP and ⁶⁶ signals. In order to explore this possibility we fused the EUO coding region with glutathione S-transferase (GST) and examined the effect of the fusion protein on Hc1 in vitro. The purified fusion protein was able to digest Hc1 completely within 1 h at 37⁰C. Also digested were very lysine rich calf thymus histone H1 and chicken erythrocyte histone H5. No measurable activity was observed towards core histones H2A, H2B, H3 and H4 (46). The proteolytic activity specified by the fusion protein preferentially cleaved the C-terminal portion of Hc1, the domain involved in DNA binding, while leaving the N-terminus intact. In addition to cleaving free Hc1, the fusion protein was able to cleave DNA:Hc1 complexes at a molar equivalent ratio of 1:1 between Hc1 and DNA. However, at a higher molar equivalent ratio of Hc1:DNA (10:1), partial protection was conferred upon Hc1 to an extent that prevented complete dissociation of DNA-Hc1 complexes. It is not clear whether partial dissociation of DNA:Hc1 complexes can facilitate DNA decondensation in vivo. Also, whether failure to dissociate the complex completely is due to inaccessibility of histone protease remains to be determined. In any event, our results support the nomination of EUO as a histone specific protease. The expression of histone protease during the early life cycle represents an important component of the process leading to chromatin decondensation.