RESUMO
When combined with recombinase defects, chromosome breakage and double-strand break repair deficiencies render cells inviable. However, cells are viable when an SOS response occurs in recAts polA cells in Escherichia coli. Here, we aimed to elucidate the underlying mechanisms of this process. Transposon mutagenesis revealed that the hslO gene, a redox chaperone Hsp33 involved in reactive oxidative species (ROS) metabolism, was required for the suppression of recAts polA lethality at a restricted temperature. Recently, it has been reported that lethal treatments trigger ROS accumulation. We also found that recAts polA cells accumulated ROS at the restricted temperature. A catalase addition to the medium alleviates the temperature sensitivity of recAts polA cells and decreases ROS accumulation. These results suggest that the SOS response and hslO manage oxidative insult to an acceptable level in cells with oxidative damage and rescue cell growth. Overall, ROS might regulate several cellular processes.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Reparo do DNA , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Mutagênese , Espécies Reativas de Oxigênio/metabolismo , TemperaturaRESUMO
Chromosome damage combined with defective recombinase activity renders cells inviable, owing to deficient double-strand break repair. Despite this, recA polA cells grow well under either DNA damage response (SOS) conditions or catalase medium supplementation. Catalase treatments reduce intracellular reactive oxygen species (ROS) levels, suggesting that recA polA cells are susceptible to not only chronic chromosome damage but also ROS. In this study, we used a reducing agent, vitamin C, to confirm whether cell growth could be improved. Vitamin C reduced ROS levels and rescued colony formation in recAts polA cells under restrictive temperatures in the presence of hslO, the gene encoding a redox molecular chaperone. Subsequently, we investigated the role of hslO in the cell growth failure of recAts polA cells. The effects of vitamin C were observed in hslO+ cells; simultaneously, cells converged along several ploidies likely through a completion of replication, with the addition of vitamin C at restrictive temperatures. These results suggest that HslO could manage oxidative stress to an acceptable level, allowing for cell division as well as rescuing cell growth. Overall, ROS may regulate several processes, from damage response to cell division. Our results provide a basis for understanding the unsolved regulatory interplay of cellular processes.
Assuntos
Ácido Ascórbico , Reparo do DNA , Espécies Reativas de Oxigênio , Catalase , Ácido Ascórbico/farmacologia , Oxirredução , Estresse OxidativoRESUMO
Replication initiation is a key event in the cell cycle of all organisms and oriC, the replication origin in Escherichia coli, serves as the prototypical model for this process. The minimal sequence required for oriC function was originally determined entirely from plasmid studies using cloned origin fragments, which have previously been shown to differ dramatically in sequence requirement from the chromosome. Using an in vivo recombineering strategy to exchange wt oriCs for mutated ones regardless of whether they are functional origins or not, we have determined the minimal origin sequence that will support chromosome replication. Nearly the entire right half of oriC could be deleted without loss of origin function, demanding a reassessment of existing models for initiation. Cells carrying the new DnaA box-depleted 163 bp minimal oriC exhibited little or no loss of fitness under slow-growth conditions, but were sensitive to rich medium, suggesting that the dense packing of initiator binding sites that is a hallmark of prokaryotic origins, has likely evolved to support the increased demands of multi-forked replication.
Assuntos
Replicação do DNA , Escherichia coli/genética , Complexo de Reconhecimento de Origem/genética , Origem de Replicação , DNA Bacteriano/biossíntese , Escherichia coli/crescimento & desenvolvimento , Mutagênese , Deleção de SequênciaRESUMO
Many significant gene mutations in E. coli have contributed to the development of genetics. Among these, a commonly used recA mutation, Δ(srl-recA)306 has been sequenced by a next-generation sequencer combined with a long PCR. An original report described that Δ(srl-recA)306 cells were deleted from srlR to recA genes in their genome. The next-generation sequencer enables more accurate details to be determined. We ask whether both surrounding genes from hypF to norV for srlR and alaS for recA is there first. The long PCR was carried out with primers, norR and alaS, and amplified DNA fragments differed in length from wild to Δ(srl-recA)306 cells, suggesting that an entire Δ(srl-recA)306 mutation was included. Sequences of those DNA fragments indicated that 9147 bp, from srlR to recA including 10 genes, were replaced by a Tn10 DNA sequence. Junction points at both srlR-Tn10 and Tn10-recA were determined precisely. The results indicate that the first 97% of recA gene sequences were lost with a downstream recX gene remaining intact. The phenotypic difference between Δ(srl-recA)306 and ΔrecA::Km is discussed.