Division of Labor between SOS and PafBC in Mycobacterial DNA Repair and Mutagenesis

DNA repair systems allow microbes to survive in diverse environments that compromise chromosomal integrity. Pathogens such as M. tuberculosis must contend with the genotoxic host environment, which generates the mutations that underlie antibiotic resistance. Mycobacteria encode the widely distributed SOS pathway, governed by the LexA repressor, but also encode PafBC, a positive regulator of the transcriptional DNA damage response (DDR). Although the transcriptional outputs of these systems have been characterized, their full functional division of labor in survival and mutagenesis is unknown. Here we specifically ablate the PafBC or SOS pathways, alone and in combination, and test their relative contributions to repair. We find that SOS and PafBC have both distinct and overlapping roles that depend on the type of DNA damage. Most notably, we find that quinolone antibiotics and replication fork perturbation are inducers of the PafBC pathway, and that chromosomal mutagenesis is codependent on PafBC and SOS, through shared regulation of the DnaE2/ImuA/B mutasome. These studies define the complex transcriptional regulatory network of the DDR in mycobacteria and provide new insight into the regulatory mechanisms controlling the genesis of antibiotic resistance in M. tuberculosis.

Homodimerization and Heterodimerization Requirements of Acinetobacter baumannii SOS Response Coregulators UmuDAb and DdrR revealed by Two-Hybrid Analyses

The multi-drug resistant pathogen <i>Acinetobacter baumannii</i> displays unusual control of its SOS mutagenesis genes, as it does not encode a LexA repressor, but instead employs the UmuDAb repressor and a small DdrR protein that is uniquely found in <i>Acinetobacter</i> species. We used bacterial adenylate cyclase two-hybrid analyses to determine if UmuDAb and DdrR coregulation might involve physical interactions. Neither quantitative nor qualitative assays showed UmuDAb interaction with DdrR. DdrR hybrid proteins, however, demonstrated modest head-to-tail interactions in a qualitative assay. The similarity of UmuDAb to the homodimer-forming polymerase manager UmuD and LexA repressor proteins suggested that it may form dimers, which we observed. UmuDAb homodimerization required a free C-terminus, and either small truncations or addition of a histidine tag at the C-terminus abolished this homodimerization. Amino acid N100, crucial for UmuD dimer formation, was dispensable if both C-termini were free to interact. However, mutation of G124, necessary for LexA dimerization, yielded significantly less UmuDAb dimerization, even if both C-termini were free. This suggests that UmuDAb forms dimers like LexA, but may not co-regulate gene expression involving a physical association with DdrR. The homodimerization of these coregulators provides insight into a LexA-independent, coregulatory process of controlling a conserved bacterial action such as the mutagenic DNA damage response.

Imaging LexA degradation in cells explains regulatory mechanisms and heterogeneity of the SOS response

ABSTRACTThe SOS response functions as the central regulator of DNA repair and mutagenesis in most bacteria and stands as a paradigm of gene networks controlled by a master transcriptional regulator, LexA. We developed a single-molecule imaging approach to directly monitor the LexA repressor inside live Escherichia coli cells, demonstrating key mechanisms by which DNA-binding and degradation of LexA regulates the SOS response in vivo. Our approach revealed that self-cleavage of LexA occurs frequently during unperturbed growth and causes substantial heterogeneity in LexA abundances across cells. LexA variability underlies SOS gene expression heterogeneity and triggers spontaneous SOS pulses, which enhance bacterial survival in anticipation of stress.

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

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.

Clp-dependent proteolysis of the LexA N-terminal domain in Staphylococcus aureus

The SOS response is governed by the transcriptional regulator LexA and is elicited in many bacterial species in response to DNA damaging conditions. Induction of the SOS response is mediated by autocleavage of the LexA repressor resulting in a C-terminal dimerization domain (CTD) and an N-terminal DNA-binding domain (NTD) known to retain some DNA-binding activity. The proteases responsible for degrading the LexA domains have been identified in Escherichia coli as ClpXP and Lon. Here, we show that in the human and animal pathogen Staphylococcus aureus, the ClpXP and ClpCP proteases contribute to degradation of the NTD and to a lesser degree the CTD. In the absence of the proteolytic subunit, ClpP, or one or both of the Clp ATPases, ClpX and ClpC, the LexA domains were stabilized after autocleavage. Production of a stabilized variant of the NTD interfered with mitomycin-mediated induction of sosA expression while leaving lexA unaffected, and also significantly reduced SOS-induced mutagenesis. Our results show that sequential proteolysis of LexA is conserved in S. aureus and that the NTD may differentially regulate a subset of genes in the SOS regulon.

UvrD303, a Hyperhelicase Mutant That Antagonizes RecA-Dependent SOS Expression by a Mechanism That Depends on Its C Terminus

ABSTRACT Genomic integrity is critical for an organism's survival and ability to reproduce. In Escherichia coli, the UvrD helicase has roles in nucleotide excision repair and methyl-directed mismatch repair and can limit reactions by RecA under certain circumstances. UvrD303 (D403A D404A) is a hyperhelicase mutant, and when expressed from a multicopy plasmid, it results in UV sensitivity (UVs), recombination deficiency, and antimutability. In order to understand the molecular mechanism underlying the UVs phenotype of uvrD303 cells, this mutation was transferred to the E. coli chromosome and studied in single copy. It is shown here that uvrD303 mutants are UV sensitive, recombination deficient, and antimutable and additionally have a moderate defect in inducing the SOS response after UV treatment. The UV-sensitive phenotype is epistatic with recA and additive with uvrA and is partially suppressed by removing the LexA repressor. Furthermore, uvrD303 is able to inhibit constitutive SOS expression caused by the recA730 mutation. The ability of UvrD303 to antagonize SOS expression was dependent on its 40 C-terminal amino acids. It is proposed that UvrD303, via its C terminus, can decrease the levels of RecA activity in the cell.

Genetic Evidence for the Requirement of RecA Loading Activity in SOS Induction after UV Irradiation in Escherichia coli

ABSTRACT The SOS response in Escherichia coli results in the coordinately induced expression of more than 40 genes which occurs when cells are treated with DNA-damaging agents. This response is dependent on RecA (coprotease), LexA (repressor), and the presence of single-stranded DNA (ssDNA). A prerequisite for SOS induction is the formation of a RecA-ssDNA filament. Depending on the DNA substrate, the RecA-ssDNA filament is produced by either RecBCD, RecFOR, or a hybrid recombination mechansim with specific enzyme activities, including helicase, exonuclease, and RecA loading. In this study we examined the role of RecA loading activity in SOS induction after UV irradiation. We performed a genetic analysis of SOS induction in strains with a mutation which eliminates RecA loading activity in the RecBCD enzyme (recB1080 allele). We found that RecA loading activity is essential for SOS induction. In the recB1080 mutant RecQ helicase is not important, whereas RecJ nuclease slightly decreases SOS induction after UV irradiation. In addition, we found that the recB1080 mutant exhibited constitutive expression of the SOS regulon. Surprisingly, this constitutive SOS expression was dependent on the RecJ protein but not on RecFOR, implying that there is a different mechanism of RecA loading for constitutive SOS expression.