HslO ameliorates arrested ΔrecA polA cell growth and reduces DNA damage and oxidative stress responses

Abstract

Chromosome damage combined with defective recombinase activity has been widely considered to render cells inviable, owing to deficient double-strand break repair. However, temperature-sensitive recAts polA cells grow well under both SOS response conditions and when supplemented with catalase at restrictive temperatures. These treatments reduce intracellular reactive oxygen species (ROS) levels, which suggests that recAts polA cells are susceptible to ROS, but not chronic chromosome damage. Therefore, we investigated whether polA cells can tolerate a complete lack of recombinase function. We introduced a ΔrecA allele in polA cells in the presence or absence of the hslO expression plasmid. Induction of the hslO gene with IPTG resulted in increased cell viability in ΔrecA polA cells with the hslO expression plasmid. ΔrecA polA cells in the absence of the hslO expression plasmid showed L medium sensitivity with increasing ROS levels. Adding catalase to the culture medium significantly rescued growth arrest and increased ROS. These results suggest that hslO expression manages oxidative stress to an acceptable level in cells with oxidative damage and rescues cell growth. Overall, ROS may regulate several processes, from damage response to cell division, via ROS-sensitive cell metabolism.

Vitamin C Maintenance against Cell Growth Arrest and Reactive Oxygen Species Accumulation in the Presence of Redox Molecular Chaperone hslO Gene

“…Regarding the use of a synthetic lethality experimental system for chromosome damage, we observed growth arrest of recAts polA cells that was ameliorated with reducing reagents such as vitamin C. Additionally, cells were growing with synchronization in growth and ROS levels even in restrictive temperatures. This is consistent with our previous observations on the effects of catalase [ 2 , 3 ]. The source of ROS is unclear; however, we now know how the recAts polA cells mediate ROS stress.…”

“…In a previous report, we demonstrated that recA polA cells were capable of colony formation in catalase-supplemented media either at a restrictive temperature (42 °C) [ 2 ] or in a rich medium [ 3 ]. Since catalase is a hydrogen peroxide-degrading enzyme, this suggested that the inability of recAts polA cells to grow at restrictive temperatures resulted from hydrogen peroxide accumulation.…”

“…Therefore, at the restrictive temperature, it is possible that recAts polA cells caused hyper-replication, and replicative stress might be elevated in recAts polA cells. Of note, ΔrecA polA cells failed to grow in a rich medium where replicative stress would be elevated [ 3 ].…”

“…Flow cytometry was conducted as previously described [ 2 ]. For ROS analysis using flow cytometry, staining was performed according to our previous studies [ 2 , 3 ]. Cell cultures (4 μL) were mixed with 12.5 μM CellRox Deep Red (16 μL) at indicated times, diluted with M9 medium without organic nutrients (M9B), and stained for 30 min at 25 °C.…”

“…However, in Escherichia coli recAts polA cells, viability was retained when DNA damage response was evoked by constitutive expression of a lexA (Def) mutation [ 1 , 2 ]. We had previously shown that recA polA cell growth arrest was partially due to the accumulation of reactive oxygen species (ROS) and hydrogen peroxide under restrictive temperatures and rich medium conditions [ 2 , 3 ].…”