Apoptosis-Like Death, an Extreme SOS Response in Escherichia coli

ABSTRACT In bacteria, SOS is a global response to DNA damage, mediated by the recA-lexA genes, resulting in cell cycle arrest, DNA repair, and mutagenesis. Previously, we reported that Escherichia coli responds to DNA damage via another recA-lexA-mediated pathway resulting in programmed cell death (PCD). We called it apoptosis-like death (ALD) because it is characterized by membrane depolarization and DNA fragmentation, which are hallmarks of eukaryotic mitochondrial apoptosis. Here, we show that ALD is an extreme SOS response that occurs only under conditions of severe DNA damage. Furthermore, we found that ALD is characterized by additional hallmarks of eukaryotic mitochondrial apoptosis, including (i) rRNA degradation by the endoribonuclease YbeY, (ii) upregulation of a unique set of genes that we called extensive-damage-i nduced (Edin) genes, (iii) a decrease in the activities of complexes I and II of the electron transport chain, and (iv) the formation of high levels of OH˙ through the Fenton reaction, eventually resulting in cell death. Our genetic and molecular studies on ALD provide additional insight for the evolution of mitochondria and the apoptotic pathway in eukaryotes. IMPORTANCE The SOS response is the first described and the most studied bacterial response to DNA damage. It is mediated by a set of two genes, recA-lexA, and it results in DNA repair and thereby in the survival of the bacterial culture. We have shown that Escherichia coli responds to DNA damage by an additional recA-lexA-mediated pathway resulting in an apoptosis-like death (ALD). Apoptosis is a mode of cell death that has previously been reported only in eukaryotes. We found that E. coli ALD is characterized by several hallmarks of eukaryotic mitochondrial apoptosis. Altogether, our results revealed that recA-lexA is a DNA damage response coordinator that permits two opposite responses: life, mediated by the SOS, and death, mediated by the ALD. The choice seems to be a function of the degree of DNA damage in the cell.

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Inducible Loss of the Fanconi Anemia Pathway in iPSC Causes Rapid Cell Cycle Arrest and Apoptosis through ATM/ATR and p53 Signaling

Blood ◽

10.1182/blood.v124.21.3528.3528 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3528-3528

Author(s):

Timothy M Chlon ◽

Elizabeth E Hoskins ◽

Sonya Ruiz-Torres ◽

Christopher N Mayhew ◽

Kathryn A Wikenheiser-Brokamp ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Dna Damage ◽

Cell Death ◽

Dna Repair ◽

Cell Cycle Arrest ◽

Loss Of Function ◽

Cycle Arrest ◽

Repair Pathways

Abstract As the source of all cells in the developing embryo proper, embryonic stem cells (ESC) bear the unique responsibility to prevent mutations from being propagated throughout the entire organism and the germ line. It is likely for this reason that ESC and induced pluripotent stem cells (iPSC) maintain a dramatically lower mutation frequency than cultured somatic cells. Multiple mechanisms for this enhanced genomic surveillance have been proposed, including hypersensitivity of DNA damage response signaling pathways and increased activity of error-free DNA repair pathways, such as homologous recombination. However, the effect of loss of function of DNA repair pathways in these cells remains poorly understood. The Fanconi Anemia (FA) pathway is a DNA repair pathway that is required for the repair of DNA interstrand crosslink damage and also promotes repair of DNA double-strand breaks by homologous recombination . Genetic defects in this pathway cause a disease characterized by bone marrow failure and extreme cancer incidence. Several recent studies have revealed that the FA pathway is required for efficient somatic cell reprogramming to iPSC and suggest that FA cells undergo cell death during this process. Another recent study found that the growth of FA patient-specific iPSC was attenuated with a G2/M arrest when compared to control iPSC, suggesting that these cells arrest upon failed DNA repair. In this study, we sought to determine the effects of acute loss of function of the FA pathway in iPSC through the generation of FA patient-derived iPSC with inducible complementation of the defective FA gene. Fibroblasts were cultured from skin biopsies of multiple FA patients and transduced with a lentiviral vector expressing the complementing FA gene product under DOX-inducible control. Cells were then reprogrammed to iPSC using episomal transfection. These cells formed iPSC colonies only when reprogramming was carried out in the presence of DOX, confirming that the FA pathway is required for efficient reprogramming. Once cell lines were obtained, DOX-dependent FA functionality was verified based on FANCD2 monoubiquitination and nuclear focus formation after treatment with DNA damaging agents. We then cultured the iPSC for extended periods of time in the presence and absence of DOX. Interestingly, the cultures underwent profound cell death and cell cycle arrest within 7 days of DOX-withdrawal and completely failed to expand after one passage. EdU cell cycle analysis confirmed cell cycle arrest in the G2/M phase. Furthermore, cleaved caspase 3 staining confirmed that the number of apoptotic cells increased by 3-fold in the -DOX culture. Despite these effects, cells cultured in both the presence and absence of DOX formed teratomas in nude mice, thus indicating the maintenance of full differentiation capacity in the absence of the FA pathway. In order to determine the mechanisms underlying G2/M arrest and cell death, expression of p53 and its target genes was detected by both western blot analysis and qRT-PCR. Only a slight increase in p53 activation was observed by 7 days post DOX-withdrawal. Furthermore, knockdown of p53 resulted in rescue from apoptosis to normal levels but not rescue from cell cycle arrest. Increased ATM and ATR DNA damage sensor kinase activities were also detected in –DOX cells, concominant with increased phosphorylation of the ATM-target Chk2 and reduced abundance of the G2/M checkpoint protein CDC25A. These results reveal hyperactive DNA damage responses upon FA loss which may underlie the attenuated cell cycle progression of FA-iPSC independent of p53. Remarkably, effects in this FA model system appear equivalent to those responsible for the depletion of HSC in the bone marrow of FA patients. Thus, iPSC models may be useful for future studies of the mechanisms underlying FA stem cell arrest and for the development of therapeutics that alleviate these phenotypes. Disclosures No relevant conflicts of interest to declare.

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Intracellular d-Serine Accumulation Promotes Genetic Diversity via Modulated Induction of RecA in Enterohemorrhagic Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00548-16 ◽

2016 ◽

Vol 198 (24) ◽

pp. 3318-3328 ◽

Cited By ~ 1

Author(s):

James P. R. Connolly ◽

Andrew J. Roe

Keyword(s):

Escherichia Coli ◽

Genetic Diversity ◽

Dna Damage ◽

Mutation Frequency ◽

Sos Response ◽

Enterohemorrhagic Escherichia Coli ◽

Lytic Bacteriophage ◽

Content Type ◽

Lexa Repressor ◽

Physiological Relevance

ABSTRACTWe recently discovered that exposure of enterohemorrhagicEscherichia coli(EHEC) tod-serine resulted in accumulation of this unusual amino acid, induction of the SOS regulon, and downregulation of the type III secretion system that is essential for efficient colonization of the host. Here, we have investigated the physiological relevance of this elevated SOS response, which is of particular interest given the presence of Stx toxin-carrying lysogenic prophages on the EHEC chromosome that are activated during the SOS response. We found that RecA elevation in response tod-serine, while being significant, was heterogeneous and not capable of activatingstxexpression orstxphage transduction to a nonlysogenic recipient. This “SOS-like response” was, however, capable of increasing the mutation frequency associated with low-level RecA activity, thus promoting genetic diversity. Furthermore, this response was entirely dependent on RecA and enhanced in the presence of a DNA-damaging agent, indicating a functional SOS response, but did not result in observable cleavage of the LexA repressor alone, indicating a controlled mechanism of induction. This work demonstrates that environmental factors not usually associated with DNA damage are capable of promoting an SOS-like response. We propose that this modulated induction of RecA allows EHEC to adapt to environmental insults such asd-serine while avoiding unwanted phage-induced lysis.IMPORTANCEThe SOS response is a global stress network that is triggered by the presence of DNA damage due to breakage or stalled replication forks. Activation of the SOS response can trigger the replication of lytic bacteriophages and promote genetic diversification through error-prone polymerases. We have demonstrated that the host-associated metabolited-serine contributes toEscherichia coliniche specification and accumulates inside cells that cannot catabolize it. This results in a modulated activation of the SOS antirepressor RecA that is insufficient to trigger lytic bacteriophage but capable of increasing the SOS-associated mutation frequency. These findings describe how relevant signals not normally associated with DNA damage can hijack the SOS response, promoting diversity asE. colistrains adapt while avoiding unwanted phage lysis.

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RexAB Is Essential for the Mutagenic Repair of Staphylococcus aureus DNA Damage Caused by Co-trimoxazole

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00944-19 ◽

2019 ◽

Vol 63 (12) ◽

Cited By ~ 4

Author(s):

Rebecca S. Clarke ◽

Maya S. Bruderer ◽

Kam Pou Ha ◽

Andrew M. Edwards

Keyword(s):

Staphylococcus Aureus ◽

Dna Damage ◽

Dna Repair ◽

Sos Response ◽

Dependent Manner ◽

Content Type ◽

Sos Induction ◽

Therapeutic Value ◽

Emergence Of Resistance

ABSTRACT Co-trimoxazole (SXT) is a combination therapeutic that consists of sulfamethoxazole and trimethoprim that is increasingly used to treat skin and soft tissue infections caused by methicillin-resistant Staphylococcus aureus (MRSA). However, the use of SXT is limited to the treatment of low-burden, superficial S. aureus infections and its therapeutic value is compromised by the frequent emergence of resistance. As a first step toward the identification of approaches to enhance the efficacy of SXT, we examined the role of bacterial DNA repair in antibiotic susceptibility and mutagenesis. We found that mutants lacking the DNA repair complex RexAB had a modest SXT MIC that was 2-fold lower than that seen with wild-type strains but were killed 50-fold to 5,000-fold more efficiently by the combination antibiotic at the breakpoint concentration. SXT-mediated DNA damage occurred via both thymidine limitation and the generation of reactive oxygen species and triggered induction of the SOS response in a RexAB-dependent manner. SOS induction was associated with a 50% increase in the mutation rate, which may contribute to emergence of resistant strains during SXT therapy. In summary, this work determined that SXT caused DNA damage in S. aureus via both thymidine limitation and oxidative stress and that the damage was repaired by the RexAB complex, leading to induction of the mutagenic SOS response. Small-molecule inhibitors of RexAB could therefore have therapeutic value by increasing the efficacy of SXT and decreasing the emergence of drug resistance during treatment of infections caused by S. aureus.

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Evolutionary Divergence in DNA Damage Responses among Fungi

mBio ◽

10.1128/mbio.03348-20 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Jacob L. Steenwyk

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Repair ◽

Cell Cycle Checkpoints ◽

Evolutionary Divergence ◽

Genome Maintenance ◽

Content Type ◽

Cycle Arrest ◽

Dna Damage Responses ◽

Eukaryotic Dna

ABSTRACT Cell cycle checkpoints and DNA repair pathways contribute to maintaining genome integrity and are thought to be evolutionarily ancient and broadly conserved. For example, in the yeast Saccharomyces cerevisiae and humans, DNA damage induces activation of a checkpoint effector kinase, Rad53p (human homolog Chk2), to promote cell cycle arrest and transcription of DNA repair genes. However, recent studies have revealed variation in the DNA damage response networks of some fungi. For example, Shor et al. (mBio 11:e03044-20, 2020, https://doi.org/10.1128/mBio.03044-20) demonstrate that in comparison to S. cerevisiae, the fungal pathogen Candida glabrata has reduced activation of Rad53p in response to DNA damage. Consequently, some downstream targets that contribute to S. cerevisiae genome maintenance, such as DNA polymerases, are transcriptionally downregulated in C. glabrata. Downregulation of genome maintenance genes likely contributes to higher rates of mitotic failure and cell death in C. glabrata. This and other recent findings highlight evolutionary diversity in eukaryotic DNA damage responses.

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DNA Damage Responses during the Cell Cycle: Insights from Model Organisms and Beyond

Genes ◽

10.3390/genes12121882 ◽

2021 ◽

Vol 12 (12) ◽

pp. 1882

Author(s):

Delisa E. Clay ◽

Donald T. Fox

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Death ◽

Dna Repair ◽

Model Organism ◽

Model Organisms ◽

Cycle Arrest ◽

Genome Damage ◽

Dna Damage Responses ◽

Typical Model

Genome damage is a threat to all organisms. To respond to such damage, DNA damage responses (DDRs) lead to cell cycle arrest, DNA repair, and cell death. Many DDR components are highly conserved, whereas others have adapted to specific organismal needs. Immense progress in this field has been driven by model genetic organism research. This review has two main purposes. First, we provide a survey of model organism-based efforts to study DDRs. Second, we highlight how model organism study has contributed to understanding how specific DDRs are influenced by cell cycle stage. We also look forward, with a discussion of how future study can be expanded beyond typical model genetic organisms to further illuminate how the genome is protected.

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Regulation of Cell Division in Bacteria by Monitoring Genome Integrity and DNA Replication Status

Journal of Bacteriology ◽

10.1128/jb.00408-19 ◽

2019 ◽

Vol 202 (2) ◽

Cited By ~ 5

Author(s):

Peter E. Burby ◽

Lyle A. Simmons

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Division ◽

Dna Replication ◽

Cell Cycle Progression ◽

Genome Instability ◽

Sos Response ◽

Bacterial Cells ◽

Cycle Progression ◽

Content Type

ABSTRACT All organisms regulate cell cycle progression by coordinating cell division with DNA replication status. In eukaryotes, DNA damage or problems with replication fork progression induce the DNA damage response (DDR), causing cyclin-dependent kinases to remain active, preventing further cell cycle progression until replication and repair are complete. In bacteria, cell division is coordinated with chromosome segregation, preventing cell division ring formation over the nucleoid in a process termed nucleoid occlusion. In addition to nucleoid occlusion, bacteria induce the SOS response after replication forks encounter DNA damage or impediments that slow or block their progression. During SOS induction, Escherichia coli expresses a cytoplasmic protein, SulA, that inhibits cell division by directly binding FtsZ. After the SOS response is turned off, SulA is degraded by Lon protease, allowing for cell division to resume. Recently, it has become clear that SulA is restricted to bacteria closely related to E. coli and that most bacteria enforce the DNA damage checkpoint by expressing a small integral membrane protein. Resumption of cell division is then mediated by membrane-bound proteases that cleave the cell division inhibitor. Further, many bacterial cells have mechanisms to inhibit cell division that are regulated independently from the canonical LexA-mediated SOS response. In this review, we discuss several pathways used by bacteria to prevent cell division from occurring when genome instability is detected or before the chromosome has been fully replicated and segregated.

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Mutagenesis and More: umuDC and the Escherichia coli SOS Response

Genetics ◽

10.1093/genetics/148.4.1599 ◽

1998 ◽

Vol 148 (4) ◽

pp. 1599-1610 ◽

Cited By ~ 10

Author(s):

Bradley T Smith ◽

Graham C Walker

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Cellular Response ◽

Sos Response ◽

Posttranslational Regulation ◽

E Coli ◽

Sos Mutagenesis ◽

Induced Mutagenesis ◽

Mechanistic Basis ◽

Increase Cell Survival

Abstract The cellular response to DNA damage that has been most extensively studied is the SOS response of Escherichia coli. Analyses of the SOS response have led to new insights into the transcriptional and posttranslational regulation of processes that increase cell survival after DNA damage as well as insights into DNA-damage-induced mutagenesis, i.e., SOS mutagenesis. SOS mutagenesis requires the recA and umuDC gene products and has as its mechanistic basis the alteration of DNA polymerase III such that it becomes capable of replicating DNA containing miscoding and noncoding lesions. Ongoing investigations of the mechanisms underlying SOS mutagenesis, as well as recent observations suggesting that the umuDC operon may have a role in the regulation of the E. coli cell cycle after DNA damage has occurred, are discussed.

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Physiological Evidence for Isopotential Tunneling in the Electron Transport Chain of Methane-Producing Archaea

Applied and Environmental Microbiology ◽

10.1128/aem.00950-17 ◽

2017 ◽

Vol 83 (18) ◽

Cited By ~ 5

Author(s):

Nikolas Duszenko ◽

Nicole R. Buan

Keyword(s):

Escherichia Coli ◽

Electron Transport ◽

Stationary Phase ◽

Exponential Growth ◽

Electron Transport Chain ◽

Methanosarcina Barkeri ◽

Metabolic Efficiency ◽

Methanosarcina Acetivorans ◽

Content Type ◽

Transport Chain

ABSTRACT Many, but not all, organisms use quinones to conserve energy in their electron transport chains. Fermentative bacteria and methane-producing archaea (methanogens) do not produce quinones but have devised other ways to generate ATP. Methanophenazine (MPh) is a unique membrane electron carrier found in Methanosarcina species that plays the same role as quinones in the electron transport chain. To extend the analogy between quinones and MPh, we compared the MPh pool sizes between two well-studied Methanosarcina species, Methanosarcina acetivorans C2A and Methanosarcina barkeri Fusaro, to the quinone pool size in the bacterium Escherichia coli. We found the quantity of MPh per cell increases as cultures transition from exponential growth to stationary phase, and absolute quantities of MPh were 3-fold higher in M. acetivorans than in M. barkeri. The concentration of MPh suggests the cell membrane of M. acetivorans, but not of M. barkeri, is electrically quantized as if it were a single conductive metal sheet and near optimal for rate of electron transport. Similarly, stationary (but not exponentially growing) E. coli cells also have electrically quantized membranes on the basis of quinone content. Consistent with our hypothesis, we demonstrated that the exogenous addition of phenazine increases the growth rate of M. barkeri three times that of M. acetivorans. Our work suggests electron flux through MPh is naturally higher in M. acetivorans than in M. barkeri and that hydrogen cycling is less efficient at conserving energy than scalar proton translocation using MPh. IMPORTANCE Can we grow more from less? The ability to optimize and manipulate metabolic efficiency in cells is the difference between commercially viable and nonviable renewable technologies. Much can be learned from methane-producing archaea (methanogens) which evolved a successful metabolic lifestyle under extreme thermodynamic constraints. Methanogens use highly efficient electron transport systems and supramolecular complexes to optimize electron and carbon flow to control biomass synthesis and the production of methane. Worldwide, methanogens are used to generate renewable methane for heat, electricity, and transportation. Our observations suggest Methanosarcina acetivorans, but not Methanosarcina barkeri, has electrically quantized membranes. Escherichia coli, a model facultative anaerobe, has optimal electron transport at the stationary phase but not during exponential growth. This study also suggests the metabolic efficiency of bacteria and archaea can be improved using exogenously supplied lipophilic electron carriers. The enhancement of methanogen electron transport through methanophenazine has the potential to increase renewable methane production at an industrial scale.

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RUNX Family Participates in the Regulation of p53-Dependent DNA Damage Response

International Journal of Genomics ◽

10.1155/2013/271347 ◽

2013 ◽

Vol 2013 ◽

pp. 1-12 ◽

Cited By ~ 22

Author(s):

Toshinori Ozaki ◽

Akira Nakagawara ◽

Hiroki Nagase

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Death ◽

Cell Cycle Arrest ◽

Dna Damage Response ◽

Apoptotic Cell ◽

Genetic Alterations ◽

Apoptotic Cell Death ◽

Cycle Arrest ◽

Damage Response

A proper DNA damage response (DDR), which monitors and maintains the genomic integrity, has been considered to be a critical barrier against genetic alterations to prevent tumor initiation and progression. The representative tumor suppressor p53 plays an important role in the regulation of DNA damage response. When cells receive DNA damage, p53 is quickly activated and induces cell cycle arrest and/or apoptotic cell death through transactivating its target genes implicated in the promotion of cell cycle arrest and/or apoptotic cell death such asp21WAF1,BAX, andPUMA. Accumulating evidence strongly suggests that DNA damage-mediated activation as well as induction of p53 is regulated by posttranslational modifications and also by protein-protein interaction. Loss of p53 activity confers growth advantage and ensures survival in cancer cells by inhibiting apoptotic response required for tumor suppression. RUNX family, which is composed of RUNX1, RUNX2, and RUNX3, is a sequence-specific transcription factor and is closely involved in a variety of cellular processes including development, differentiation, and/or tumorigenesis. In this review, we describe a background of p53 and a functional collaboration between p53 and RUNX family in response to DNA damage.

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