Genetic recombination in Bacillus subtilis 168: contribution of Holliday junction processing functions in chromosome segregation

Abstract

Bacillus subtilis mutants classified within the epsilon (ruvA, DeltaruvB, DeltarecU, and recD) and eta (DeltarecG) epistatic groups, in an otherwise rec+ background, render cells impaired in chromosomal segregation. A less-pronounced segregation defect in DeltarecA and Deltasms (DeltaradA) cells was observed. The repair deficiency of addAB, DeltarecO, DeltarecR, recH, DeltarecS, and DeltasubA cells did not correlate with a chromosomal segregation defect. The sensitivity of epsilon epistatic group mutants to DNA-damaging agents correlates with ongoing DNA replication at the time of exposure to the agents. The Deltasms (DeltaradA) and DeltasubA mutations partially suppress the DNA repair defect in ruvA and recD cells and the segregation defect in ruvA and DeltarecG cells. The Deltasms (DeltaradA) and DeltasubA mutations partially suppress the DNA repair defect of DeltarecU cells but do not suppress the segregation defect in these cells. The DeltarecA mutation suppresses the segregation defect but does not suppress the DNA repair defect in DeltarecU cells. These results result suggest that (i) the RuvAB and RecG branch migrating DNA helicases, the RecU Holliday junction (HJ) resolvase, and RecD bias HJ resolution towards noncrossovers and that (ii) Sms (RadA) and SubA proteins might play a role in the stabilization and or processing of HJ intermediates.

FIG. 1.

Nucleoid morphologies of addA5 addB72, recH342, and ΔrecO cells. Exponentially growing cells were fixed, stained with DAPI, and analyzed by fluorescence microscopy to visualize the nucleoids. White arrows point to diffuse and linked nucleoids.

FIG. 2.

DNA damage sensitivity of recU replicating cells. The survival of wt cells (open symbols) and recU40 cells (filled symbols) (A), and of recR13 cells (open symbols) and uvrA42 control cells (filled symbols) (B) after exposure to the killing action of 100 μM 4NQO under different growth conditions is shown. Triangles, stationary-phase cells; circles, exponential-phase growing cells; squares, cells pretreated with CM (20 μg/ml) 120 min before 4NQO treatment. Survival curves represent the averages of results of three or more independent experiments.

FIG. 3.

recU, recD, and ruvA mutations produce anucleate cells and aberrant nucleoids. (A) Exponentially growing cells were fixed, stained with DAPI, and analyzed by fluorescence microscopy to visualize the nucleoids. Black arrows indicate anucleate cells, whereas white arrows show aberrant and misplaced nucleoids. (B) Electron micrographs of cross-sectioned processed cells. The nucleoids appear as light material in the cytoplasm.

FIG. 4.

Effect of the Δsms (ΔradA) and ΔsubA suppressors in the segregation defect of the ɛ epistatic group mutants. Exponentially growing cells were fixed, stained with DAPI, and analyzed by fluorescence microscopy to visualize the nucleoids. Black arrows indicate anucleate cells, whereas white arrows show aberrant and misplaced nucleoids.

FIG. 5.

The recG segregation defect is suppressed by the absence of the SubA product. Exponentially growing cells were fixed, stained with DAPI, and analyzed by fluorescence microscopy to visualize the nucleoids. Black arrows indicate anucleate cells, whereas white arrows show aberrant and misplaced nucleoids.