Bacillus subtilis RarA forms damage-inducible foci that scan the entire cell
Little is known about the activity and dynamics of ATPase RarA in B. subtilis, proposed to act at stalled DNA replication forks due to DNA damage. We performed fluorescence microscopy time lapse experiments with a functional RarA-mVenus fusion to visualize the dynamics of RarA during conditions that generate DNA damage.
In exponentially growing cells, we observed that 15% of the cells contained single RarA-mV (mVenus fluorescent fusion) foci moving throughout the entire cell between 3 min intervals. This percentage remained constant at different time points, indicating that focus formation during unperturbed growth is maintained at about a constant rate. When cells were exposed to stress conditions, the population of cells containing RarA-mV foci tripled after 60 min. Cells exposed to two DNA-damaging drugs, to 5 mM MMS or to 0.5 mM H2O2, showed a similar type of response, with RarA-mVenus foci moving more slowly than during unperturbed growth. It is likely that RarA-mV contributes to the repair of H2O2-induced lesions, and to a minor extent to MMS-induced lesions. The presence of foci in growing cells suggests that RarA also plays a role during the cell cycle, at least in a fraction of cells, possibly contributing to heterogeneity of response to DNA damage.
KeywordsRarA DNA repair Time lapse fluorescence microscopy Bacillus subtilis Stress response
maintenance of genome stability 1
Werner [WRN] Interacting Protein 1
methyl methane sulfonate
Gaussian mixture model
The bacterial replication-associated recombination protein A, RarA, belongs to a highly conserved family of ATPases, including the yeast Mgs1 and mammal WRNIP1 proteins . The B. subtilis rarA gene, which is monocistronic, is constitutively expressed, but its expression is markedly enhanced by stressors such as diamide, ethanol, high salt or H2O2 . RarA protein plays an important, but poorly understood role in genome maintenance . Although several studies agreed with the idea that RarA acts in both replication and recombination processes, the concrete function is still unknown. E. coli RarA, which is co-expressed with FtsK, co-localizes/interacts with SeqA, RecQ , UvrD  or RecA  and may act at blocked forks in certain replication mutants [5, 6]. In vitro, E. coli RarA interacts with single strand binding (SSB) protein and shows helicase activity that preferentially unwinds 3′-ends from dsDNA ends or ssDNA gaps, suggesting that RarA could act at stalled replication forks [1, 7]. One common point of RarA studies is the complex scenario required to produce a clear phenotype that explains all observations.
A C-terminal fusion of the fluorescent protein mVenus to RarA was generated by cloning the 3′-end 500 bp of rarA (excluding the stop codon) into plasmid pSG1164 , which was integrated into the rarA gene locus on the B. subtilis chromosome by homologous recombination. We have used epifluorescence microscopy time-lapse to monitor foci formation and dynamics of RarA before and after stress conditions at 30 °C (OD600 = ~ 0.3). Cells were either treated with 0.5 mM H2O2, or with 5 mM MMS (both obtained from Sigma Aldrich) or were not treated. For fluorescence microscopy, B. subtilis cells were grown in S750 minimal medium  at 30 °C under shaking conditions until exponential growth. Three microliters of cells were transferred on an agarose slide—a glass slide (microscope slides standard, Roth) coated with an agarose layer (S750 minimal medium, 1% v/v agarose) and covered with a cover slip (Roth). Fluorescence microscopy was performed using a Zeiss Observer Z1 (Carl Zeiss) with an oil immersion objective (100× magnification, NA 1.45 alpha Plan-FLUAR) and a CCD camera (CoolSNAP EZ, Photometrics), or with a BX51 microscope (Olympus) with a Cool Snap EZ camera (Photometrics) and a xenon light source (Olympus). Electronic data were processed using Metamorph 22.214.171.124 software (Molecular Devices, Sunnyvale, CA, USA), which also allows the calibration of the fluorescence intensity and pixel size to determine the cell length, time-lapse epifluorescence microscopy of RarA-mV were collected every 3 min.
Overview of data files/data sets
Name of data file/data set
File types (file extension)
Data repository and identifier (DOI or accession number)
Data file 1 
Time lapse AVI
Data file 2 
Time lapse AVI
Data file 3 
Time lapse AVI
Data set 1 
Gaussian mixture model (GMM) RarA-mV
This study extends observation of RarA-mVenus foci during unperturbed growth . The study reveals the movement of an assembly of RarA molecules in a subset of a cell population; it does not describe the dynamics of freely diffusing molecules. Although clearly, foci are only present in a minority of cells, even after stress induction, very small assemblies may be present in more cells, but may be undetectable through epifluorescence microscopy.
RH-T and PLG conceived of the project and wrote the manuscript, RH-T performed epifluorescence imaging and other experiments, and analyzed the data. Both authors read and approved the final manuscript.
LOEWE funding (state of Hessen, Germany) to SYNMIKRO for supporting this work.
The authors declare that they have no competing interests.
Availability of data materials
The data described in this Data note can be freely and openly accessed on [https://figshare.com], namely https://doi.org/10.6084/m9.figshare.7461587.v3, https://doi.org/10.6084/m9.figshare.7461692.v2, https://doi.org/10.6084/m9.figshare.7461698.v2, https://doi.org/10.6084/m9.figshare.7466987.v3.
Consent for publication
Ethics approval and consent to participate
This work was supported by LOEWE funding to SYNMIKRO (state of Hessen) and by the Philipps Universität Marburg. The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. This study was funded by Deutsche Forschungsgemeinschaft (TRR174).
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- 10.Hernandez-Tamayo R, Graumann PL. RarA-mV WT. figshare. Media. 2018. https://doi.org/10.6084/m9.figshare.7461587.v3.
- 11.Hernandez-Tamayo R, Graumann PL. RarA-mV MMS. figshare. Media. 2018. https://doi.org/10.6084/m9.figshare.7461692.v2.
- 12.Hernandez-Tamayo R, Graumann PL. RarA-mV H202. figshare. Media. 2018. https://doi.org/10.6084/m9.figshare.7461698.v2.
- 14.Hernandez-Tamayo R, Graumann PL. Gaussian mixture model (GMM) RarA-mV. figshare. Figure. 2018. https://doi.org/10.6084/m9.figshare.7466987.v3.
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