Bacteria frequently encounter adverse environmental conditions, such as nutrient scarcity and antibiotic exposure, which can induce DNA damage. Efficient DNA repair mechanisms are essential for bacterial survival, particularly under such stress conditions. A critical player in these processes is the signaling molecule (p)ppGpp, a phosphorylated guanosine synthesized by bacteria during periods of stress. Initially discovered in Escherichia coli under amino acid starvation, (p)ppGpp is now recognized for its broader roles in modulating cellular functions essential for DNA repair and stress response. By regulating diverse cellular processes, (p)ppGpp not only enables bacterial adaptation but also enhances their ability to survive in the presence of DNA-damaging agents like fluoroquinolone antibiotics, contributing significantly to antibiotic resistance mechanisms. This review explores the role of (p)ppGpp in DNA repair pathways, stress-induced mutagenesis, and bacterial persistence.
(p)ppGpp’s Regulatory Role in DNA Repair
Nucleotide Excision Repair (NER) and RNA Polymerase Interaction
One of the most significant DNA repair pathways influenced by (p)ppGpp is nucleotide excision repair (NER), responsible for addressing bulky DNA lesions such as those caused by UV radiation. Within NER, two pathways exist: global genomic NER and transcription-coupled NER (TC-NER), both of which remove lesions by initiating RNA polymerase stalling at damaged sites. (p)ppGpp binds to RNA polymerase, promoting its backtracking and the recruitment of repair proteins to damaged DNA sites, which is critical for TC-NER efficiency. Studies have shown that (p)ppGpp acts alongside other transcription factors, such as DksA, to modulate transcription in response to stress, ensuring that resources are reallocated to essential repair functions. The combined action of (p)ppGpp and RNA polymerase in stalling and backtracking serves as a checkpoint, allowing DNA lesions to be recognized and excised by the NER machinery. Furthermore, this regulatory function aids bacteria in adapting to various stress conditions by ensuring genomic stability, a prerequisite for bacterial persistence and growth under adverse conditions.
Downregulation of Mismatch Repair and Stress-Induced Mutagenesis
Mismatch repair (MMR) is another pathway that (p)ppGpp influences by regulating key repair proteins, including MutS and MutH, which are responsible for correcting replication errors and base mismatches. Under nutrient-limited or stationary phase conditions, (p)ppGpp downregulates MMR proteins, potentially favoring mutagenesis. This downregulation, combined with the upregulation of error-prone polymerases, allows bacteria to accumulate adaptive mutations that can increase their survival chances under antibiotic pressure. For instance, (p)ppGpp promotes the production of alternative sigma factors, such as sigma S, which triggers a general stress response that favors the transcription of error-prone polymerases over high-fidelity ones. This regulatory shift enhances the probability of mutations, contributing to bacterial adaptation and persistence in the presence of antibiotics, albeit at the cost of genomic stability.
Role in Recombinational Repair and Double-Strand Break Repair
Recombinational repair mechanisms are critical for managing double-strand breaks (DSBs) that arise during replication or antibiotic-induced stress. (p)ppGpp, through its interaction with RNA polymerase, aids in managing replication conflicts and facilitates the loading of repair proteins such as RecA onto single-stranded DNA, an essential step in recombinational repair. Additionally, (p)ppGpp-mediated stalling of RNA polymerase at DSBs enables repair machinery like RecBCD and RuvABC to resolve DNA breaks. This process allows bacteria to not only survive but also continue replicating even under stress conditions. Notably, stress-induced mutagenesis associated with DSB repair can also lead to antibiotic resistance, as the repair process incorporates mutations that confer survival advantages. Through these pathways, (p)ppGpp provides bacteria with a toolkit to repair severe DNA damage while also facilitating genetic diversity within bacterial populations, thereby enhancing adaptation to environmental challenges.
The SOS Response: Linking (p)ppGpp to DNA Damage-Induced Survival
The SOS response, a bacterial stress response to DNA damage, is tightly regulated by (p)ppGpp. Activation of the SOS response is initiated by RecA upon detection of DNA damage, leading to LexA repressor cleavage and subsequent expression of DNA repair genes. In conditions such as UV exposure or fluoroquinolone treatment, (p)ppGpp enhances the SOS response by regulating the transcription of repair proteins and sigma factors essential for DNA repair. For example, (p)ppGpp binding to RNA polymerase facilitates transcriptional regulation in response to DNA damage, promoting expression of error-prone polymerases involved in translesion synthesis. These polymerases allow the replication machinery to bypass DNA lesions, albeit at the risk of incorporating mutations. This process not only enables bacteria to survive acute DNA damage but also supports mutagenesis under antibiotic stress, contributing to resistance development. Thus, (p)ppGpp’s involvement in the SOS response positions it as a central regulator of DNA repair and mutagenesis in bacteria.
Stress-Induced Mutagenesis and Persistence in Fluoroquinolone Presence
A key aspect of (p)ppGpp’s function in antibiotic resistance is its role in stress-induced mutagenesis, which aids bacterial survival under sublethal antibiotic concentrations. (p)ppGpp downregulates MMR and other high-fidelity repair pathways, promoting the use of error-prone polymerases and facilitating mutagenesis. In fluoroquinolone-treated bacterial populations, specific subpopulations, termed "gamblers," exhibit heightened mutagenesis, enhancing survival under antibiotic pressure. Fluorescent cell sorting has identified that these gambler cells exhibit an active stress response, which includes increased mutation rates. Furthermore, (p)ppGpp and associated sigma factors help sustain these mutations by inhibiting mismatch repair and facilitating repair processes that increase bacterial tolerance to antibiotics. This survival mechanism, though detrimental to genomic integrity, promotes adaptive evolution within bacterial populations, allowing some cells to survive and develop resistance under antibiotic stress.
Antibiotic Persistence and the Role of (p)ppGpp in Bacterial "Persisters" and "Gamblers"
Beyond mutagenesis, (p)ppGpp contributes to bacterial persistence, enabling a subpopulation of cells to survive antibiotic treatment without acquiring genetic mutations. In minimal inhibitory concentrations of fluoroquinolones, persister cells exhibit a phenotype that enables survival through metabolic dormancy and stress response activation. Ofloxacin, a fluoroquinolone, induces this persister state by causing membrane depolarization, a response mediated by the toxin HokB in E. coli. The conserved GTPase protein Obg, which is regulated by (p)ppGpp, is essential for this persistence mechanism. By regulating proteins that maintain cellular dormancy and membrane stability, (p)ppGpp supports bacterial cells in surviving antibiotic exposure and contributes to recurrent infections. This mechanism, combined with the mutagenesis observed in gambler cells, creates a dual strategy where bacterial populations are equipped with both mutational adaptability and persistence, allowing them to endure varying antibiotic concentrations and environmental stresses.
Conclusions
The signaling molecule (p)ppGpp plays an indispensable role in bacterial DNA repair and survival strategies under stress. By modulating DNA repair pathways such as NER, MMR, and recombinational repair, (p)ppGpp aids in preserving genomic stability in the short term while also facilitating long-term adaptation through stress-induced mutagenesis. These functions, particularly in response to antibiotics like fluoroquinolones, underscore the significance of (p)ppGpp as a master regulator that bridges DNA repair with bacterial survival and antibiotic resistance. Understanding these regulatory mechanisms offers promising insights into combating antibiotic resistance by potentially targeting (p)ppGpp-mediated pathways, paving the way for more effective treatments against persistent and resistant bacterial infections.
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