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Bacillus subtilis RarA forms damage-inducible foci that scan the entire cell.

ABSTRACT: OBJECTIVES: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. DATA DESCRIPTION: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.

SUBMITTER: Hernandez-Tamayo R

PROVIDER: S-EPMC6458690 | biostudies-other | 2019 Apr

REPOSITORIES: biostudies-other

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Bacillus subtilis RarA forms damage-inducible foci that scan the entire cell.

Hernández-Tamayo Rogelio R Graumann Peter L PL

BMC research notes 20190411 1

<h4>Objectives</h4>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.<h4>Data description</h4>In exponentially growing cells, we observed that 15% of the cells contained single RarA-mV (mVenus fluorescent fusion) foci moving thro ...[more]

PMID: 30971308

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Project description:A microfluidic system coupled with fluorescence microscopy is a powerful approach for quantitative analysis of bacterial growth. Here, we measure parameters of growth and dynamic localization of the cell division initiation protein FtsZ in Bacillus subtilis Consistent with previous reports, we found that after division, FtsZ rings remain at the cell poles, and polar FtsZ ring disassembly coincides with rapid Z-ring accumulation at the midcell. In cells mutated for minD, however, the polar FtsZ rings persist indefinitely, suggesting that the primary function of the Min system is in Z-ring disassembly. The inability to recycle FtsZ monomers in the minD mutant results in the simultaneous maintenance of multiple Z-rings that are restricted by competition for newly synthesized FtsZ. Although the parameters of FtsZ dynamics change in the minD mutant, the overall cell division time remains the same, albeit with elongated cells necessary to accumulate a critical threshold amount of FtsZ for promoting medial division. Finally, the minD mutant characteristically produces minicells composed of polar peptidoglycan shown to be inert for remodeling in the wild type. Polar peptidoglycan, however, loses its inert character in the minD mutant, suggesting that the Min system not only is important for recycling FtsZ but also may have a secondary role in the spatiotemporal regulation of peptidoglycan remodeling.IMPORTANCE Many bacteria grow and divide by binary fission in which a mother cell divides into two identical daughter cells. To produce two equally sized daughters, the division machinery, guided by FtsZ, must dynamically localize to the midcell each cell cycle. Here, we quantitatively analyzed FtsZ dynamics during growth and found that the Min system of Bacillus subtilis is essential to disassemble FtsZ rings after division. Moreover, a failure to efficiently recycle FtsZ results in an increase in cell size. Finally, we show that the Min system has an additional role in inhibiting cell wall turnover and contributes to the "inert" property of cell walls at the poles.

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Bacillus subtilis RarA forms damage-inducible foci that scan the entire cell.

Publications

Bacillus subtilis RarA forms damage-inducible foci that scan the entire cell.

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