Chromosome dynamics in Bacillus subtilis -Characterization of the Structural Maintenance of Chromosomes (SMC) complex

All cells need to duplicate and separate their genetic material faithfully into the future daughter cells before cell division takes place. In bacteria, the chromosome has to be organized and compacted whilst, at the same time, it needs to be dynamic to allow other ongoing cellular processes like repair, recombination, transcription, replication and segregation to take place. SMC (Structural Maintenance of Chromosome) protein belongs to a ubiquitous protein family that play crucial roles in chromosome dynamics. The main interest of this work is to characterize the function of the SMC protein in Bacillus subtilis. Data base searches have led to the identification of two interaction partners of SMC. These proteins, ScpA and ScpB are conserved among bacterial and archaeal species possessing SMC. The scpA or scpB deletions showed a similar phenotype to that of a smc disruption, namely temperature sensitive slow growth (below 23°C), decondensed nucleoids and a strong segregation defect. Their simultaneous deletion did not exacerbate the phenotype, suggesting that all the three proteins function in the same pathway in chromosome condensation. To investigate their in vivo function, the proteins where localized in the cells using functional GFP fusions. The subcellular localization showed bipolar foci, a unique pattern of localization that was dynamic and cell cycle dependent. The foci were present at mid-cell position in smaller cells and separated towards opposite cell poles within a few minutes. The formation of a complex between SMC, ScpA, and ScpB in vivo was confirmed using fluorescence resonance energy transfer (FRET) and depletion studies. Formation of foci was only seen in the presence of all three proteins, but not in the absence of any one of them. The specific localization pattern of these proteins also depended on ongoing DNA replication, on active gyrase and thus on DNA topology, as well as on SMC?s ATPase activity. Overproduction of SMC led to increased compaction of nucleoids but the localization was retained in the form of foci suggesting that the foci represent active chromosome condensation centers. The proteins of the SMC complex showed growth dependent protein expression. SMC and ScpB proteins were present in actively replicating exponential phase cells, but were rapidly depleted as the cells entered stationary phase. Analysis with total RNA extracts from various growth phases by primer extension studies showed a strong transcript for SMC that was present even in stationary phase. This experiment led to the identification of a new promoter for smc, and suggests that SMC is regulated at the protein level by a protease that is induced at the onset of stationary phase. Smc, scpA, and scpB deletion mutant cells were also sensitive to Mitomycin C (MMC) treatment, which induces double strand breaks (DSB) into DNA. This finding revealed a role of the SMC complex in DSB repair. I also investigated the role of YirY, a homolog of the DSB repair protein SbcC which is a proposed member of SMC family. Upon disruption of yirY/sbcC, the cells did not show any visible phenotype but the cells were sensitive to MMC, suggesting its role in repair. SbcC formed foci only in MMC treated cells, so the foci in the cell might represent a DNA repair centers. Other proteins located in the same operon as SbcC, AddA, AddB, and SbcD, did not show any specific pattern of localization, but were present throughout the cell and showed slight increase in their fluorescence intensity after MMC treatment, suggesting that SbcC and AddAB function in different in repair pathways. The localization of topoisomerase IV subunits ParC and ParE has also been investigated in this work. The fluorescent protein fusion of ParC localized throughout the nucleoid, contrarily to the previously published bipolar localization as foci, which had suggested a specialized function of topoisomerase IV in chromosome decatenation. Upon over expression of ParC and ParE, the cells contained more condensed nucleoids, revealing a general role of topoisomerase IV in global chromosome compaction. A further aspect of this work was the study of dynamic localization of ribosomes. The large subunit ribosome protein L1 showed specific localization in the cytoplasmic space surrounding the nucleoid in growing cells, and was seen diffused throughout the cell in the stationary phase. The same effect was observed upon inhibition of transcription, suggesting the dependence of specific ribosome localization on active transcription. In toto, localization of DNA segregation, DNA repair and the ribosomal proteins has provided a more defined view of the spatial organization of these cellular processes in live bacterial cells.