Characterization of a prokaryotic SMC protein involved in chromosome partitioning

Abstract

smc of Bacillus subtilis encodes a homolog of eukaryotic SMC proteins involved in chromosome condensation, pairing, and partitioning. A null mutation in B. subtilis smc caused a temperature-sensitive-lethal phenotype in rich medium. Under permissive conditions, the mutant had abnormal nucleoids, approximately 10% of the cells were anucleate, and assembly of foci of the chromosome partitioning protein Spo0J was altered. In combination with a null mutation in spo0J, the smc mutation caused a synthetic phenotype; cell growth was slower and approximately 25% of the cells were anucleate. Our results demonstrate that the B. subtilis Smc protein, like its eukaryotic counterpart, plays an important role in chromosome structure and partitioning.

Figure 1

smc mutants produce anculeate cells and have aberrant nucleoids. Cells were grown for several generations in defined minimal medium at 30°C, and samples were taken during exponential growth, fixed, and stained with DAPI. Photographs are combined Nomarski–fluorescence images. Arrows indicate some of the anucleate cells. (A) Wild-type (JH642); (B) Δsmc (RB35); (C) Δsmc Δspo0J (RB41). Note that the nucleoid bodies in many of the cells from the smc and smc spo0J mutants are irregular and some cells appear to have increased DNA content. Scale bar, ∼1 μm.

Figure 2

Spo0J–GFP and Smc–GFP localization in live cells. Cells were grown in defined minimal medium at 30°C and samples taken during exponential growth. Three panels are shown for each field of cells: endogenous fluorescence from the GFP fusion (left); nucleoids stained with DAPI (middle); and overlays of the GFP and DAPI images (right). In the DAPI/GFP overlay, the color of the GFP signal was changed to white to better contrast with the DAPI image. Scale bar, ∼1 μm. (A) Spo0J–GFP in wild-type cells (PL657); (B–D) Spo0J–GFP in smc mutant cells (RB40). (B) Example of a cell with no discrete focus of Spo0J–GFP; (C) two nucleoids (left and middle), each with two foci of Spo0J–GFP, one polar and one medial; (D) example of a nucleoid (center of panel) with a single medial spot of Spo0J–GFP; (E) localization of Smc–GFP. The fluorescence signal from Smc–GFP was much more faint than that from Spo0J–GFP, and discrete foci were visible on ∼50% of the nucleoids.