Real-time kinetic studies of Mycobacterium tuberculosis LexA-DNA interaction.

Transcriptional repressor, LexA, regulates the 'SOS' response, an indispensable bacterial DNA damage repair machinery. Compared with its Escherichia coli ortholog, LexA from Mycobacterium tuberculosis (Mtb) possesses a unique N-terminal extension of additional 24 amino acids in its DNA-binding domain (DBD) and 18 amino acids insertion at its hinge region that connects the DBD to the C-terminal dimerization/autoproteolysis domain. Despite the importance of LexA in 'SOS' regulation, Mtb LexA remains poorly characterized and the functional importance of its additional amino acids remained elusive. In addition, the lack of data on kinetic parameters of Mtb LexA-DNA interaction prompted us to perform kinetic analyses of Mtb LexA and its deletion variants using Bio-layer Interferometry (BLI). Mtb LexA is seen to bind to different 'SOS' boxes, DNA sequences present in the operator regions of damage-inducible genes, with comparable nanomolar affinity. Deletion of 18 amino acids from the linker region is found to affect DNA binding unlike the deletion of the N-terminal stretch of extra 24 amino acids. The conserved RKG motif has been found to be critical for DNA binding. Overall, the present study provides insights into the kinetics of the interaction between Mtb LexA and its target 'SOS' boxes. The kinetic parameters obtained for DNA binding of Mtb LexA would be instrumental to clearly understand the mechanism of 'SOS' regulation and activation in Mtb.

SulA is able to block cell division in Escherichia coli by a mechanism different from sequestration.

The SOS response is considered to be an extremely important feature of bacterial cells. It helps them to survive bad times, including helping to develop resistance to antibiotics. The SOS response blocks the cell division. For Escherichia coli it is well known that the SulA protein directly interacts with FtsZ - a key division protein. Now it is believed that fission blocking is based on FtsZ sequestration by the SulA protein, which leads to decrease in effective concentration of FtsZ in the cell below a critical value, which in vitro leads to dismantling of FtsZ polymers. In this work, we demonstrate that in order to block the division of E. coli, it is sufficient to have a relatively small amount of SulA in the cell. Moreover, the analysis of structures formed by FtsZ in E. coli cells under the conditions of SulA protein expression or the SOS response showed that there is no complete disassembly of FtsZ polymers, although Z-rings indeed are not formed. The results of the work indicate that the well-known sequestration mechanism is not comprehensive to explain blocking of the division process by SulA in vivo.

SosA inhibits cell division in Staphylococcus aureus in response to DNA damage.

Inhibition of cell division is critical for viability under DNA-damaging conditions. DNA damage induces the SOS response that in bacteria inhibits cell division while repairs are being made. In coccoids, such as the human pathogen, Staphylococcus aureus, this process remains poorly studied. Here, we identify SosA as the staphylococcal SOS-induced cell division inhibitor. Overproduction of SosA inhibits cell division, while sosA inactivation sensitizes cells to genotoxic stress. SosA is a small, predicted membrane protein with an extracellular C-terminal domain in which point mutation of residues that are conserved in staphylococci and major truncations abolished the inhibitory activity. In contrast, a minor truncation led to SosA accumulation and a strong cell division inhibitory activity, phenotypically similar to expression of wild-type SosA in a CtpA membrane protease mutant. This suggests that the extracellular C-terminus of SosA is required both for cell division inhibition and for turnover of the protein. Microscopy analysis revealed that SosA halts cell division and synchronizes the cell population at a point where division proteins such as FtsZ and EzrA are localized at midcell, and the septum formation is initiated but unable to progress to closure. Thus, our findings show that SosA is central in cell division regulation in staphylococci.

Oxidative damage induced by H2O2 reveals SOS adaptive transcriptional response of Dietzia cinnamea strain P4.

The oxidative stress response of the highly resistant actinomycete Dietzia cinnamea P4 after treatment with hydrogen peroxide (H2O2) was assessed in order to depict the possible mechanisms underlying its intrinsic high resistance to DNA damaging agents. We used transcriptional profiling to monitor the magnitude and kinetics of changes in the mRNA levels after exposure to different concentrations of H2O2 at 10 min and 1 h following the addition of the stressor. Catalase and superoxide dismutase genes were induced in different ways, according to the condition applied. Moreover, alkyl hydroperoxide reductase ahpCF, thiol peroxidase, thioredoxin and glutathione genes were upregulated in the presence of H2O2. Expression of peroxidase genes was not detected during the experiment. Overall results point to an actinomycete strain endowed with a set of enzymatic defenses against oxidative stress and with the main genes belonging to a functional SOS system (lexA, recA, uvrD), including suppression of lexA repressor, concomitantly to recA and uvrD gene upregulation upon H2O2 challenge.

Enhanced antibiotic resistance development from fluoroquinolone persisters after a single exposure to antibiotic.

Bacterial persisters are able to tolerate high levels of antibiotics and give rise to new populations. Persister tolerance is generally attributed to minimally active cellular processes that prevent antibiotic-induced damage, which has led to the supposition that persister offspring give rise to antibiotic-resistant mutants at comparable rates to normal cells. Using time-lapse microscopy to monitor Escherichia coli populations following ofloxacin treatment, we find that persisters filament extensively and induce impressive SOS responses before returning to a normal appearance. Further, populations derived from fluoroquinolone persisters contain significantly greater quantities of antibiotic-resistant mutants than those from untreated controls. We confirm that resistance is heritable and that the enhancement requires RecA, SOS induction, an opportunity to recover from treatment, and the involvement of error-prone DNA polymerase V (UmuDC). These findings show that fluoroquinolones damage DNA in persisters and that the ensuing SOS response accelerates the development of antibiotic resistance from these survivors.

Prophage induction, but not production of phage particles, is required for lethal disease in a microbiome-replete murine model of enterohemorrhagic E. coli infection.

Enterohemorrhagic Escherichia coli (EHEC) colonize intestinal epithelium by generating characteristic attaching and effacing (AE) lesions. They are lysogenized by prophage that encode Shiga toxin 2 (Stx2), which is responsible for severe clinical manifestations. As a lysogen, prophage genes leading to lytic growth and stx2 expression are repressed, whereas induction of the bacterial SOS response in response to DNA damage leads to lytic phage growth and Stx2 production both in vitro and in germ-free or streptomycin-treated mice. Some commensal bacteria diminish prophage induction and concomitant Stx2 production in vitro, whereas it has been proposed that phage-susceptible commensals may amplify Stx2 production by facilitating successive cycles of infection in vivo. We tested the role of phage induction in both Stx production and lethal disease in microbiome-replete mice, using our mouse model encompassing the murine pathogen Citrobacter rodentium lysogenized with the Stx2-encoding phage Φstx2dact. This strain generates EHEC-like AE lesions on the murine intestine and causes lethal Stx-mediated disease. We found that lethal mouse infection did not require that Φstx2dact infect or lysogenize commensal bacteria. In addition, we detected circularized phage genomes, potentially in the early stage of replication, in feces of infected mice, confirming that prophage induction occurs during infection of microbiota-replete mice. Further, C. rodentium (Φstx2dact) mutants that do not respond to DNA damage or express stx produced neither high levels of Stx2 in vitro or lethal infection in vivo, confirming that SOS induction and concomitant expression of phage-encoded stx genes are required for disease. In contrast, C. rodentium (Φstx2dact) mutants incapable of prophage genome excision or of packaging phage genomes retained the ability to produce Stx in vitro, as well as to cause lethal disease in mice. Thus, in a microbiome-replete EHEC infection model, lytic induction of Stx-encoding prophage is essential for lethal disease, but actual phage production is not.

Arenicin-1-induced apoptosis-like response requires RecA activation and hydrogen peroxide against Escherichia coli.

Arenicin-1, a 21-mer antimicrobial peptide exerts significant broad-spectrum antimicrobial activity with membrane-active mechanisms. However, owing to multiple mechanisms of cell death, the antibacterial mechanism of arenicin-1 requires detailed analysis. In the present study, arenicin-1-treated bacteria underwent an apoptosis-like response, which was mechanistically and morphologically similar to eukaryotic apoptosis. Changes in the physiological status of arenicin-1-treated bacterial cells involved accumulation of reactive oxygen species, imbalance of intracellular calcium gradients, disruption of membrane potential, bacterial caspase-like protein activation, and DNA damage. In arenicin-1-induced apoptosis-like death, autocleavage of LexA was observed because of the activation of the caspase-like activity of RecA. Additionally, typical reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals, were scavenged in arenicin-1-treated cells to assess the role of specific reactive oxygen species. Scavenging of hydrogen peroxide interfered with the activity of arenicin-1 in Escherichia coli, whereas the superoxide and hydroxyl radicals level did not affect arenicin-1-induced apoptosis-like death activity. Furthermore, inhibition of Fenton reaction attenuated apoptosis-like response. In conclusion, arenicin-1-induced apoptosis like death requires SOS response proteins and is mediated by hydrogen peroxide and Fenton reaction in E. coli. Arenicin-1 may be a representative antimicrobial peptide with potent apoptotic response against E. coli.

The SOS Response Mediates Sustained Colonization of the Mammalian Gut.

Bacteria have a remarkable ability to survive, persist, and ultimately adapt to environmental challenges. A ubiquitous environmental hazard is DNA damage, and most bacteria have evolved a network of genes to combat genotoxic stress. This network is known as the SOS response and aids in bacterial survival by regulating genes involved in DNA repair and damage tolerance. Recently, the SOS response has been shown to play an important role in bacterial pathogenesis, and yet the role of the SOS response in nonpathogenic organisms and in physiological settings remains underexplored. Using a commensal Escherichia coli strain, MP1, we showed that the SOS response plays a vital role during colonization of the murine gut. In an unperturbed environment, the SOS-off mutant is impaired for stable colonization relative to a wild-type strain, suggesting the presence of genotoxic stress in the mouse gut. We evaluated the possible origins of genotoxic stress in the mouse gut by examining factors associated with the host versus the competing commensal organisms. In a dextran sulfate sodium (DSS) colitis model, the SOS-off colonization defect persisted but was not exacerbated. In contrast, in a germ-free model, the SOS-off mutant colonized with efficiency equal to that seen with the wild-type strain, suggesting that competing commensal organisms might be a significant source of genotoxic stress. This report extends our understanding of the importance of a functional SOS response for bacterial fitness in the context of a complex physiological environment and highlights the SOS response as a possible mechanism that contributes to ongoing genomic changes, including potential antibiotic resistance, in the microbiome of healthy hosts.

Identification of the Dimer Exchange Interface of the Bacterial DNA Damage Response Protein UmuD.

The Escherichia coli SOS response, an induced DNA damage response pathway, confers survival on bacterial cells by providing accurate repair mechanisms as well as the potentially mutagenic pathway translesion synthesis (TLS). The umuD gene products are upregulated after DNA damage and play roles in both nonmutagenic and mutagenic aspects of the SOS response. Full-length UmuD is expressed as a homodimer of 139-amino-acid subunits, which eventually cleaves its N-terminal 24 amino acids to form UmuD'. The cleavage product UmuD' and UmuC form the Y-family polymerase DNA Pol V (UmuD'2C) capable of performing TLS. UmuD and UmuD' exist as homodimers, but their subunits can readily exchange to form UmuDD' heterodimers preferentially. Heterodimer formation is an essential step in the degradation pathway of UmuD'. The recognition sequence for ClpXP protease is located within the first 24 amino acids of full-length UmuD, and the partner of full-length UmuD, whether UmuD or UmuD', is degraded by ClpXP. To better understand the mechanism by which UmuD subunits exchange, we measured the kinetics of exchange of a number of fluorescently labeled single-cysteine UmuD variants as detected by Förster resonance energy transfer. Labeling sites near the dimer interface correlate with increased rates of exchange, indicating that weakening the dimer interface facilitates exchange, whereas labeling sites on the exterior decrease the rate of exchange. In most but not all cases, homodimer and heterodimer exchange exhibit similar rates, indicating that somewhat different molecular surfaces mediate homodimer exchange and heterodimer formation.

Class 1 integrons are low-cost structures in Escherichia coli.

Resistance integrons are bacterial genetic platforms that can capture and express antibiotic resistance genes embedded within gene cassettes. The capture and shuffling of gene cassettes are mediated by the integrase IntI, the expression of which is regulated by the SOS response in Escherichia coli. Gene cassettes are expressed from a common Pc promoter. Despite the clinical and environmental relevance of integrons, the selective forces responsible for their evolution and maintenance are poorly understood. Here, we conducted pairwise competition experiments in order to assess the fitness cost of class 1 integrons in E. coli. We found that integrons are low-cost structures and that their cost is further reduced by their tight regulation. We show that the SOS response prevents the expression of costly integrases whose cost is activity dependent. Thus, when an integron is repressed, its cost depends mostly on the expression of its gene cassettes array and increases with Pc strength and the number of cassettes in the array. Furthermore, different cassettes have different costs. Lastly, we showed that subinhibitory antibiotic concentrations promoted the selection of integron-carrying bacteria, especially those with a strong Pc promoter. These results provide new insights into the evolutionary dynamics of integron-carrying bacterial populations.

Management of E. coli sister chromatid cohesion in response to genotoxic stress.

Aberrant DNA replication is a major source of the mutations and chromosomal rearrangements associated with pathological disorders. In bacteria, several different DNA lesions are repaired by homologous recombination, a process that involves sister chromatid pairing. Previous work in Escherichia coli has demonstrated that sister chromatid interactions (SCIs) mediated by topological links termed precatenanes, are controlled by topoisomerase IV. In the present work, we demonstrate that during the repair of mitomycin C-induced lesions, topological links are rapidly substituted by an SOS-induced sister chromatid cohesion process involving the RecN protein. The loss of SCIs and viability defects observed in the absence of RecN were compensated by alterations in topoisomerase IV, suggesting that the main role of RecN during DNA repair is to promote contacts between sister chromatids. RecN also modulates whole chromosome organization and RecA dynamics suggesting that SCIs significantly contribute to the repair of DNA double-strand breaks (DSBs).

DNA damage responses and stress resistance: Concepts from bacterial SOS to metazoan immunity.

The critical need for species preservation has driven the evolution of mechanisms that integrate stress signals from both exogenous and endogenous sources. Past research has been largely focused on cell-autonomous stress responses; however, recently their systemic outcomes within an organism and their implications at the ecological and species levels have emerged. Maintenance of species depends on the high fidelity transmission of the genome over infinite generations; thus, many pathways exist to monitor and restore the integrity of the genome and to coordinate DNA repair with other cellular processes, such as cell division and growth. The specifics of these DNA damage responses (DDRs) vary vastly but some general themes are conserved from ancient organisms, such as bacteria and archaea, to humans. Despite decades of research, however, DDRs still have many layers of complexity and some surprises left to be discovered. One of the most interesting current research topics is the link between DNA damage and stress resistance: the outcomes of DDRs can protect the organism from other secondary challenges. At this time, these types of responses are best characterized in bacteria and the simple metazoan model, Caenorhabditis elegans, but it is becoming clear that similar processes also exist in higher organisms.

Survival and SOS response induction in ultraviolet B irradiated Escherichia coli cells with defective repair mechanisms.

Purpose In this paper, the contribution of different genes involved in DNA repair for both survival and SOS induction in Escherichia coli mutants exposed to ultraviolet B radiation (UVB, [wavelength range 280-315 nm]) was evaluated. Materials and methods E. coli strains defective in uvrA, oxyR, recO, recN, recJ, exoX, recB, recD or xonA genes were used to determine cell survival. All strains also had the genetic sulA::lacZ fusion, which allowed for the quantification of SOS induction through the SOS Chromotest. Results Five gene products were particularly important for survival, as follows: UvrA > RecB > RecO > RecJ > XonA. Strains defective in uvrA and recJ genes showed elevated SOS induction compared with the wild type, which remained stable for up to 240 min after UVB-irradiation. In addition, E. coli strains carrying the recO or recN mutation showed no SOS induction. Conclusions The nucleotide excision and DNA recombination pathways were equally used to repair UVB-induced DNA damage in E. coli cells. The sulA gene was not turned off in strains defective in UvrA and RecJ. RecO protein was essential for processing DNA damage prior to SOS induction. In this study, the roles of DNA repair proteins and their contributions to the mechanisms that induce SOS genes in E. coli are proposed.

The SOS Response Master Regulator LexA Regulates the Gene Transfer Agent of Rhodobacter capsulatus and Represses Transcription of the Signal Transduction Protein CckA.

UNLABELLED: The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a genetic exchange element that combines central aspects of bacteriophage-mediated transduction and natural transformation. RcGTA particles resemble a small double-stranded DNA bacteriophage, package random ∼4-kb fragments of the producing cell genome, and are released from a subpopulation (<1%) of cells in a stationary-phase culture. RcGTA particles deliver this DNA to surrounding R. capsulatus cells, and the DNA is integrated into the recipient genome though a process that requires homologs of natural transformation genes and RecA-mediated homologous recombination. Here, we report the identification of the LexA repressor, the master regulator of the SOS response in many bacteria, as a regulator of RcGTA activity. Deletion of the lexA gene resulted in the abolition of detectable RcGTA production and an ∼10-fold reduction in recipient capability. A search for SOS box sequences in the R. capsulatus genome sequence identified a number of putative binding sites located 5' of typical SOS response coding sequences and also 5' of the RcGTA regulatory gene cckA, which encodes a hybrid histidine kinase homolog. Expression of cckA was increased >5-fold in the lexA mutant, and a lexA cckA double mutant was found to have the same phenotype as a ΔcckA single mutant in terms of RcGTA production. The data indicate that LexA is required for RcGTA production and maximal recipient capability and that the RcGTA-deficient phenotype of the lexA mutant is largely due to the overexpression of cckA. IMPORTANCE: This work describes an unusual phenotype of a lexA mutant of the alphaproteobacterium Rhodobacter capsulatus in respect to the phage transduction-like genetic exchange carried out by the R. capsulatus gene transfer agent (RcGTA). Instead of the expected SOS response characteristic of prophage induction, this lexA mutation not only abolishes the production of RcGTA particles but also impairs the ability of cells to receive RcGTA-borne genes. The data show that, despite an apparent evolutionary relationship to lambdoid phages, the regulation of RcGTA gene expression differs radically.

RECOVERY OF DIVISION PROCESS IN BACTERIAL CELLS AFTER INDUCTION OF SulA PROTEIN WHICH IS RESPONSIBLE FOR CYTOKINESIS ARREST DURING SOS-RESPONSE.

SOS-response is an important tool of bacteria intended to protect their genome and thereby allow them to survive under adverse conditions. Recently SOS-response is considered to enhance mutagenesis and thus help bacteria acquire antibiotic resistance. Due to high significance of this phenomena it seems to be important to investigate processes that allow bacteria to survive after SOS-response activation. In current work the recovery of division process of Escherichia coli cells after division arrest due to expression of SOS-response protein SulA was studied. Data indicate that cells are able to rapidly restore normal division; also nucleoid occlusion seems to be the main septum positioning mechanism during the process. In the course of recovery FtsZ forms helix-like structures, which then transformate into Z-rings.

Effects of Stress, Reactive Oxygen Species, and the SOS Response on De Novo Acquisition of Antibiotic Resistance in Escherichia coli.

Strategies to prevent the development of antibiotic resistance in bacteria are needed to reduce the threat of infectious diseases to human health. The de novo acquisition of resistance due to mutations and/or phenotypic adaptation occurs rapidly as a result of interactions of gene expression and mutations (N. Handel, J. M. Schuurmans, Y. Feng, S. Brul, and B. H. Ter Kuile, Antimicrob Agents Chemother 58:4371-4379, 2014, http://dx.doi.org/10.1128/AAC.02892-14). In this study, the contribution of several individual genes to the de novo acquisition of antibiotic resistance in Escherichia coli was investigated using mutants with deletions of genes known to be involved in antibiotic resistance. The results indicate that recA, vital for the SOS response, plays a crucial role in the development of antibiotic resistance. Likewise, deletion of global transcriptional regulators, such as gadE or soxS, involved in pH homeostasis and superoxide removal, respectively, can slow the acquisition of resistance to a degree depending on the antibiotic. Deletion of the transcriptional regulator soxS, involved in superoxide removal, slowed the acquisition of resistance to enrofloxacin. Acquisition of resistance occurred at a lower rate in the presence of a second stress factor, such as a lowered pH or increased salt concentration, than in the presence of optimal growth conditions. The overall outcome suggests that a central cellular mechanism is crucial for the development of resistance and that genes involved in the regulation of transcription play an essential role. The actual cellular response, however, depends on the class of antibiotic in combination with environmental conditions.

Anionic Phospholipids Stabilize RecA Filament Bundles in Escherichia coli.

We characterize the interaction of RecA with membranes in vivo and in vitro and demonstrate that RecA binds tightly to the anionic phospholipids cardiolipin (CL) and phosphatidylglycerol (PG). Using computational models, we identify two regions of RecA that interact with PG and CL: (1) the N-terminal helix and (2) loop L2. Mutating these regions decreased the affinity of RecA to PG and CL in vitro. Using 3D super-resolution microscopy, we demonstrate that depleting Escherichia coli PG and CL altered the localization of RecA foci and hindered the formation of RecA filament bundles. Consequently, E. coli cells lacking aPLs fail to initiate a robust SOS response after DNA damage, indicating that the membrane acts as a scaffold for nucleating the formation of RecA filament bundles and plays an important role in the SOS response.

Apoptosis-like death, an extreme SOS response in Escherichia coli.

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-induced (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.

Polymerase exchange on single DNA molecules reveals processivity clamp control of translesion synthesis.

Translesion synthesis (TLS) by Y-family DNA polymerases alleviates replication stalling at DNA damage. Ring-shaped processivity clamps play a critical but ill-defined role in mediating exchange between Y-family and replicative polymerases during TLS. By reconstituting TLS at the single-molecule level, we show that the Escherichia coli ß clamp can simultaneously bind the replicative polymerase (Pol) III and the conserved Y-family Pol IV, enabling exchange of the two polymerases and rapid bypass of a Pol IV cognate lesion. Furthermore, we find that a secondary contact between Pol IV and ß limits Pol IV synthesis under normal conditions but facilitates Pol III displacement from the primer terminus following Pol IV induction during the SOS DNA damage response. These results support a role for secondary polymerase clamp interactions in regulating exchange and establishing a polymerase hierarchy.