Cell cycle-dependent expression of an essential SMC-like protein and dynamic chromosome localization in the archaeon...

The genome of Halobacterium salinarum encodes four proteins of the structural maintenance of chromosomes (SMC) protein superfamily. Two proteins form a novel subfamily and are named 'SMC-like proteins of H. salinarum' (Sph1 and Sph2). Northern blot analyses revealed that sph1 and hp24, the adjacent gene, are solely transcribed in exponentially growing, but not in stationary phase, cells. A synchronization procedure was developed, which makes use of the DNA polymerase inhibitor aphidicolin and leads to highly synchronous cultures. It allowed us for the first time to study cell cycle-dependent transcription in an archaeon. The sph1 transcript was found to be highly cell cycle regulated, with its maximal accumulation around the time of septum formation. The Sph1 protein level was also elevated at that time, but a basal protein level was found throughout the cell cycle. The hp24 transcript was sharply upregulated about 1 h before sph1 and had already declined at the time of sph1 induction. These and additional transcript patterns revealed that precisely controlled transcriptional regulation is involved in haloarchaeal cell cycle progression. A DNA staining protocol was developed, which opened the possibility of following the dynamic intracellular localization of haloarchaeal nucleoids using synchronized cultures. After an initial dispersed localization, the nucleoid is condensed at mid-cell. Subsequently, DNA is rapidly transported to the 1/4 and 3/4 positions. All staining patterns were also observed in untreated exponentially growing cells, excluding synchronization artifacts. The Sph1 concentration is elevated when segregation of the new chromosomes is nearly complete; therefore, it is proposed to play a role in a late step of replication, e.g. DNA repair, similar to eukaryotic Rad18 proteins.