Coexistence of SOS-Dependent and SOS-Independent Regulation of DNA Repair Genes in Radiation-Resistant Deinococcus Bacteria.

Deinococcus bacteria are extremely resistant to radiation and able to repair a shattered genome in an essentially error-free manner after exposure to high doses of radiation or prolonged desiccation. An efficient, SOS-independent response mechanism to induce various DNA repair genes such as recA is essential for radiation resistance. This pathway, called radiation/desiccation response, is controlled by metallopeptidase IrrE and repressor DdrO that are highly conserved in Deinococcus. Among various Deinococcus species, Deinococcus radiodurans has been studied most extensively. Its genome encodes classical DNA repair proteins for error-free repair but no error-prone translesion DNA polymerases, which may suggest that absence of mutagenic lesion bypass is crucial for error-free repair of massive DNA damage. However, many other radiation-resistant Deinococcus species do possess translesion polymerases, and radiation-induced mutagenesis has been demonstrated. At least dozens of Deinococcus species contain a mutagenesis cassette, and some even two cassettes, encoding error-prone translesion polymerase DnaE2 and two other proteins, ImuY and ImuB-C, that are probable accessory factors required for DnaE2 activity. Expression of this mutagenesis cassette is under control of the SOS regulators RecA and LexA. In this paper, we review both the RecA/LexA-controlled mutagenesis and the IrrE/DdrO-controlled radiation/desiccation response in Deinococcus.

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.

The response of Escherichia coli to the alkylating agents chloroacetaldehyde and styrene oxide.

DNA damage is ubiquitous and can arise from endogenous or exogenous sources. DNA-damaging alkylating agents are present in environmental toxicants as well as in cancer chemotherapy drugs and are a constant threat, which can lead to mutations or cell death. All organisms have multiple DNA repair and DNA damage tolerance pathways to resist the potentially negative effects of exposure to alkylating agents. In bacteria, many of the genes in these pathways are regulated as part of the SOS reponse or the adaptive response. In this work, we probed the cellular responses to the alkylating agents chloroacetaldehyde (CAA), which is a metabolite of 1,2-dichloroethane used to produce polyvinyl chloride, and styrene oxide (SO), a major metabolite of styrene used in the production of polystyrene and other polymers. Vinyl chloride and styrene are produced on an industrial scale of billions of kilograms annually and thus have a high potential for environmental exposure. To identify stress response genes in E. coli that are responsible for tolerance to the reactive metabolites CAA and SO, we used libraries of transcriptional reporters and gene deletion strains. In response to both alkylating agents, genes associated with several different stress pathways were upregulated, including protein, membrane, and oxidative stress, as well as DNA damage. E. coli strains lacking genes involved in base excision repair and nucleotide excision repair were sensitive to SO, whereas strains lacking recA and the SOS gene ybfE were sensitive to both alkylating agents tested. This work indicates the varied systems involved in cellular responses to alkylating agents, and highlights the specific DNA repair genes involved in the responses.

Cytometry meets next-generation sequencing - RNA-Seq of sorted subpopulations reveals regional replication and iron-triggered prophage induction in Corynebacterium glutamicum.

Phenotypic diversification is key to microbial adaptation. Currently, advanced technological approaches offer insights into cell-to-cell variation of bacterial populations at a spatiotemporal resolution. However, the underlying molecular causes or consequences often remain obscure. In this study, we developed a workflow combining fluorescence-activated cell sorting and RNA-sequencing, thereby allowing transcriptomic analysis of 106 bacterial cells. As a proof of concept, the workflow was applied to study prophage induction in a subpopulation of Corynebacterium glutamicum. Remarkably, both the phage genes and flanking genomic regions of the CGP3 prophage revealed significantly increased coverage upon prophage induction - a phenomenon that to date has been obscured by bulk approaches. Genome sequencing of prophage-induced populations suggested regional replication at the CGP3 locus in C. glutamicum. Finally, the workflow was applied to unravel iron-triggered prophage induction in early exponential cultures. Here, an up-shift in iron levels resulted in a heterogeneous response of an SOS (PdivS) reporter. RNA-sequencing of the induced subpopulation confirmed induction of the SOS response triggering also activation of the CGP3 prophage. The fraction of CGP3-induced cells was enhanced in a mutant lacking the iron regulator DtxR suffering from enhanced iron uptake. Altogether, these findings demonstrate the potential of the established workflow to gain insights into the phenotypic dynamics of bacterial populations.

Functional consequences of inducible genetic elements from the p53 SOS response in a mammalian organ system.

In response to DNA damage from ultraviolet (UV) radiation, bacteria deploy the SOS response in order to limit cell death. This bacterial SOS response is characterized by an increase in the recA gene that transactivates expression of multiple DNA repair genes. The current series of experiments demonstrate that a mammalian organ system (the cochlea) that is not evolutionarily conditioned to UV radiation can elicit SOS responses that are reminiscent of that of bacteria. This mammalian SOS response is characterized by an increase in the p53 gene with activation of multiple DNA repair genes that harbor p53 response elements in their promoters. Furthermore, the experimental results provide support for the notion of a convergent trigger paradox, where independent SOS triggers facilitate disparate physiologic sequelae (loss vs. recovery of function). Therefore, it is proposed that the mammalian SOS response is multifunctional and manipulation of this endogenous response could be exploited in future biomedical interventions.

Persistent damaged bases in DNA allow mutagenic break repair in Escherichia coli.

Bacteria, yeast and human cancer cells possess mechanisms of mutagenesis upregulated by stress responses. Stress-inducible mutagenesis potentially accelerates adaptation, and may provide important models for mutagenesis that drives cancers, host pathogen interactions, antibiotic resistance and possibly much of evolution generally. In Escherichia coli repair of double-strand breaks (DSBs) becomes mutagenic, using low-fidelity DNA polymerases under the control of the SOS DNA-damage response and RpoS general stress response, which upregulate and allow the action of error-prone DNA polymerases IV (DinB), II and V to make mutations during repair. Pol IV is implied to compete with and replace high-fidelity DNA polymerases at the DSB-repair replisome, causing mutagenesis. We report that up-regulated Pol IV is not sufficient for mutagenic break repair (MBR); damaged bases in the DNA are also required, and that in starvation-stressed cells, these are caused by reactive-oxygen species (ROS). First, MBR is reduced by either ROS-scavenging agents or constitutive activation of oxidative-damage responses, both of which reduce cellular ROS levels. The ROS promote MBR other than by causing DSBs, saturating mismatch repair, oxidizing proteins, or inducing the SOS response or the general stress response. We find that ROS drive MBR through oxidized guanines (8-oxo-dG) in DNA, in that overproduction of a glycosylase that removes 8-oxo-dG from DNA prevents MBR. Further, other damaged DNA bases can substitute for 8-oxo-dG because ROS-scavenged cells resume MBR if either DNA pyrimidine dimers or alkylated bases are induced. We hypothesize that damaged bases in DNA pause the replisome and allow the critical switch from high fidelity to error-prone DNA polymerases in the DSB-repair replisome, thus allowing MBR. The data imply that in addition to the indirect stress-response controlled switch to MBR, a direct cis-acting switch to MBR occurs independently of DNA breakage, caused by ROS oxidation of DNA potentially regulated by ROS regulators.

The inactivation of rfaP, rarA or sspA gene improves the viability of the Escherichia coli DNA polymerase III holD mutant.

The Escherichia coli holD mutant is poorly viable because the stability of holoenzyme polymerase III (Pol III HE) on DNA is compromised. Consequently, the SOS response is induced and the SOS polymerases DinB and Pol II further hinder replication. Mutations that restore the holD mutant viability belong to two classes, those that stabilize Pol III on DNA and those that prevent the deleterious effects of DinB over-production. We identified a dnaX mutation and the inactivation of rfaP and sspA genes as belonging to the first class of holD mutant suppressors. dnaX encodes a Pol III clamp loader subunit that interacts with HolD. rfaP encodes a lipopolysaccharide kinase that acts in outer membrane biogenesis. Its inactivation improves the holD mutant growth in part by affecting potassium import, previously proposed to stabilize Pol III HE on DNA by increasing electrostatic interactions. sspA encodes a global transcriptional regulator and growth of the holD mutant in its absence suggests that SspA controls genes that affect protein-DNA interactions. The inactivation of rarA belongs to the second class of suppressor mutations. rarA inactivation has a weak effect but is additive with other suppressor mutations. Our results suggest that RarA facilitates DinB binding to abandoned forks.

MutS regulates access of the error-prone DNA polymerase Pol IV to replication sites: a novel mechanism for maintaining replication fidelity.

Translesion DNA polymerases (Pol) function in the bypass of template lesions to relieve stalled replication forks but also display potentially deleterious mutagenic phenotypes that contribute to antibiotic resistance in bacteria and lead to human disease. Effective activity of these enzymes requires association with ring-shaped processivity factors, which dictate their access to sites of DNA synthesis. Here, we show for the first time that the mismatch repair protein MutS plays a role in regulating access of the conserved Y-family Pol IV to replication sites. Our biochemical data reveals that MutS inhibits the interaction of Pol IV with the ß clamp processivity factor by competing for binding to the ring. Moreover, the MutS-ß clamp association is critical for controlling Pol IV mutagenic replication under normal growth conditions. Thus, our findings reveal important insights into a non-canonical function of MutS in the regulation of a replication activity.

Rapid pairing and resegregation of distant homologous loci enables double-strand break repair in bacteria.

Double-strand breaks (DSBs) can lead to the loss of genetic information and cell death. Although DSB repair via homologous recombination has been well characterized, the spatial organization of this process inside cells remains poorly understood, and the mechanisms used for chromosome resegregation after repair are unclear. In this paper, we introduced site-specific DSBs in Caulobacter crescentus and then used time-lapse microscopy to visualize the ensuing chromosome dynamics. Damaged loci rapidly mobilized after a DSB, pairing with their homologous partner to enable repair, before being resegregated to their original cellular locations, independent of DNA replication. Origin-proximal regions were resegregated by the ParABS system with the ParA structure needed for resegregation assembling dynamically in response to the DSB-induced movement of an origin-associated ParB away from one cell pole. Origin-distal regions were resegregated in a ParABS-independent manner and instead likely rely on a physical, spring-like force to segregate repaired loci. Collectively, our results provide a mechanistic basis for the resegregation of chromosomes after a DSB.

Contribution of stress responses to antibiotic tolerance in Pseudomonas aeruginosa biofilms.

Enhanced tolerance of biofilm-associated bacteria to antibiotic treatments is likely due to a combination of factors, including changes in cell physiology as bacteria adapt to biofilm growth and the inherent physiological heterogeneity of biofilm bacteria. In this study, a transcriptomics approach was used to identify genes differentially expressed during biofilm growth of Pseudomonas aeruginosa. These genes were tested for statistically significant overlap, with independently compiled gene lists corresponding to stress responses and other putative antibiotic-protective mechanisms. Among the gene groups tested were those associated with biofilm response to tobramycin or ciprofloxacin, drug efflux pumps, acyl homoserine lactone quorum sensing, osmotic shock, heat shock, hypoxia stress, and stationary-phase growth. Regulons associated with Anr-mediated hypoxia stress, RpoS-regulated stationary-phase growth, and osmotic stress were significantly enriched in the set of genes induced in the biofilm. Mutant strains deficient in rpoS, relA and spoT, or anr were cultured in biofilms and challenged with ciprofloxacin and tobramycin. When challenged with ciprofloxacin, the mutant strain biofilms had 2.4- to 2.9-log reductions in viable cells compared to a 0.9-log reduction of the wild-type strain. Interestingly, none of the mutants exhibited a statistically significant alteration in tobramycin susceptibility compared to that with the wild-type biofilm. These results are consistent with a model in which multiple genes controlled by overlapping starvation or stress responses contribute to the protection of a P. aeruginosa biofilm from ciprofloxacin. A distinct and as yet undiscovered mechanism protects the biofilm bacteria from tobramycin.

The superintegron integrase and the cassette promoters are co-regulated in Vibrio cholerae.

Chromosome 2 of Vibrio cholerae carries a chromosomal superintegron, composed of an integrase, a cassette integration site (attI) and an array of mostly promoterless gene cassettes. We determined the precise location of the promoter, Pc, which drives the transcription of the first cassettes of the V. cholerae superintegron. We found that cassette mRNA starts 65 bp upstream of the attI site, so that the inversely oriented promoters Pc and Pint (integrase promoter) partly overlap, allowing for their potential co-regulation. Pint was previously shown to be induced during the SOS response and is further controlled by the catabolite repression cAMP-CRP complex. We found that cassette expression from Pc was also controlled by the cAMP-CRP complex, but is not part of the SOS regulon. Pint and Pc promoters were both found to be induced in rich medium, at high temperature, high salinity and at the end of exponential growth phase, although at very different levels and independently of sigma factor RpoS. All these results show that expression from the integrase and cassette promoters can take place at the same time, thus leading to coordinated excisions and integrations within the superintegron and potentially coupling cassette shuffling to immediate selective advantage.

Cocarcinogenic effects of intrahepatic bile acid accumulation in cholangiocarcinoma development.

UNLABELLED: Bile acid accumulation in liver with cholangiolar neoplastic lesions may occur before cholestasis is clinically detected. Whether this favors intrahepatic cholangiocarcinoma development has been investigated in this study. The E. coli RecA gene promoter was cloned upstream from Luc2 to detect in vitro direct genotoxic ability by activation of SOS genes. This assay demonstrated that bile acids were not able to induce DNA damage. The genotoxic effect of the DNA-damaging agent cisplatin was neither enhanced nor hindered by the hepatotoxic and hepatoprotective glycochenodeoxycholic and glycoursodeoxycholic acids, respectively. In contrast, thioacetamide metabolites, but not thioacetamide itself, induced DNA damage. Thus, thioacetamide was used to induce liver cancer in rats, which resulted in visible tumors after 30 weeks. The effect of bile acid accumulation on initial carcinogenesis phase (8 weeks) was investigated in bile duct ligated (BDL) animals. Serum bile acid measurement and determination of liver-specific healthy and tumor markers revealed that early thioacetamide treatment induced hypercholanemia together with upregulation of the tumor marker Neu in bile ducts, which were enhanced by BDL. Bile acid accumulation was associated with increased expression of interleukin (IL)-6 and downregulation of farnesoid X receptor (FXR). Bile duct proliferation and apoptosis activation, with inverse pattern (BDL > thioacetamide + BDL >> thioacetamide vs. thioacetamide > thioacetamide + BDL > BDL), were observed. In conclusion, intrahepatic accumulation of bile acids does not induce carcinogenesis directly but facilitates a cocarcinogenic effect due to stimulation of bile duct proliferation, enhanced inflammation, and reduction in FXR-dependent chemoprotection. IMPLICATIONS: This study reveals that bile acids foster cocarcinogenic events that impact cholangiocarcinoma.

The potential applications of SOS-lux biosensors for rapid screening of mutagenic chemicals.

The environmental fate and potency of mutagenic compounds is of growing concern. This has necessitated the development and application of rapid assays to screen large numbers of samples for their genotoxic and carcinogenic effects. Despite the development of biosensors for genotoxicity assessment, these have not been calibrated against traditional microbial bioassays. In this study, assays using the SOS-lux-marked microbial biosensors Escherichia coli K12C600 and E. coli DPD1718 were refined and optimised to screen selected mutagenic chemicals. The response of the biosensors was compared with the mutagenic response of the traditional Salmonella mutagenicity assay. For the chemicals tested (acridine, B[a]A, B[a]P, chrysene, mitomycin C and sodium azide), E. coli DPD1718 was consistently more sensitive than E. coli K12C600. The biosensors were of comparable sensitivity to the Salmonella assay but were more rapid, reproducible and easier to measure. These data validate the adoption of optimised assays making use of microbial biosensors for routine screening of test chemicals.

Overexpression of Escherichia coli nucleotide excision repair genes after cisplatin-induced damage.

Cisplatin is currently used in tumor chemotherapy to induce the death of malignant cells through blockage of DNA replication. It is a commonly used chemotherapeutic agent binding mono- or bifunctionally to guanines in DNA. Escherichia coli K12 mutant strains deficient in nucleotide excision repair (NER) were submitted to increasing concentrations of cisplatin, and the results revealed that uvrA and uvrB mutants are sensitive to this agent, while uvrC and cho mutants remain as the wild type strain. The time required for both gene expression turn-off and return to normal weight DNA in wild-type E. coli was not accomplished even after 4 h post-treatment with cisplatin, while the same process takes place within 1.5 h after ultraviolet radiation (UV). Besides, a heavily damaging action of cisplatin can be seen not only by persistent nicks on genomic DNA, but also by NER gene expression exceeding manifold that seen after equivalent lethal doses of UV. Moreover, cisplatin caused an increase in uvrB gene expression from its putative upstream promoter P3 in an SOS-independent manner.

The Escherichia coli SOS gene dinF protects against oxidative stress and bile salts.

DNA is constantly damaged by physical and chemical factors, including reactive oxygen species (ROS), such as superoxide radical (O(2)(-)), hydrogen peroxide (H(2)O(2)) and hydroxyl radical (•OH). Specific mechanisms to protect and repair DNA lesions produced by ROS have been developed in living beings. In Escherichia coli the SOS system, an inducible response activated to rescue cells from severe DNA damage, is a network that regulates the expression of more than 40 genes in response to this damage, many of them playing important roles in DNA damage tolerance mechanisms. Although the function of most of these genes has been elucidated, the activity of some others, such as dinF, remains unknown. The DinF deduced polypeptide sequence shows a high homology with membrane proteins of the multidrug and toxic compound extrusion (MATE) family. We describe here that expression of dinF protects against bile salts, probably by decreasing the effects of ROS, which is consistent with the observed decrease in H(2)O(2)-killing and protein carbonylation. These results, together with its ability to decrease the level of intracellular ROS, suggests that DinF can detoxify, either direct or indirectly, oxidizing molecules that can damage DNA and proteins from both the bacterial metabolism and the environment. Although the exact mechanism of DinF activity remains to be identified, we describe for the first time a role for dinF.

Identifying co-targets to fight drug resistance based on a random walk model.

BACKGROUND: Drug resistance has now posed more severe and emergent threats to human health and infectious disease treatment. However, wet-lab approaches alone to counter drug resistance have so far still achieved limited success due to less knowledge about the underlying mechanisms of drug resistance. Our approach apply a heuristic search algorithm in order to extract active network under drug treatment and use a random walk model to identify potential co-targets for effective antibacterial drugs. RESULTS: We use interactome network of Mycobacterium tuberculosis and gene expression data which are treated with two kinds of antibiotic, Isoniazid and Ethionamide as our test data. Our analysis shows that the active drug-treated networks are associated with the trigger of fatty acid metabolism and synthesis and nicotinamide adenine dinucleotide (NADH)-related processes and those results are consistent with the recent experimental findings. Efflux pumps processes appear to be the major mechanisms of resistance but SOS response is significantly up-regulation under Isoniazid treatment. We also successfully identify the potential co-targets with literature confirmed evidences which are related to the glycine-rich membrane, adenosine triphosphate energy and cell wall processes. CONCLUSIONS: With gene expression and interactome data supported, our study points out possible pathways leading to the emergence of drug resistance under drug treatment. We develop a computational workflow for giving new insights to bacterial drug resistance which can be gained by a systematic and global analysis of the bacterial regulation network. Our study also discovers the potential co-targets with good properties in biological and graph theory aspects to overcome the problem of drug resistance.

Genotoxicity investigation of araticum(Annona crassiflora Mart., 1841, Annonaceae) using SOS-Inductest and Ames test.

Although the use of medicinal plants or natural products has increased in recent decades all over the world, little information is available on their potential risk to health. Annona crassiflora Mart., a plant commonly known as araticum in Brazil, has been widely used in folk medicine for a long time since its seeds and leaves are often utilised in the treatment of cancer, snake bites, and venereal diseases, its fruits are consumed as tonic and astringent, and its bark powder has anti-fungal and anti-rheumatic properties. To evaluate the genotoxic and mutagenic properties induced by the ethanolic extract of araticum leaves, we performed the prophage λ induction (Inductest) and bacterial mutagenicity assays. We used Escherichia coli WP2s(λ) and RJF013 strains in the lysogenic induction test, whereas the mutagenic studies were carried out using Salmonella typhimurium histidine auxotroph strains TA97a, TA98, TA100, and TA102. Each experiment was performed three times in duplicate and included positive and negative controls. No statistically significant (p > 0.05) positive results were obtained for any of the strains tested, which suggests that the ethanolic extract of araticum leaves did not exhibit direct mechanisms of genotoxicity or mutagenicity that could be detected by the tests used in the present work.

Bacterial hypermutation: clinical implications.

Heritable hypermutation in bacteria is mainly due to alterations in the methyl-directed mismatch repair (MMR) system. MMR-deficient strains have been described from several bacterial species, and all of the strains exhibit increased mutation frequency and recombination, which are important mechanisms for acquired drug resistance in bacteria. Antibiotics select for drug-resistant strains and refine resistance determinants on plasmids, thus stimulating DNA recombination via the MMR system. Antibiotics can also act as indirect promoters of antibiotic resistance by inducing the SOS system and certain error-prone DNA polymerases. These alterations have clinical consequences in that efficacious treatment of bacterial infections requires high doses of antibiotics and/or a combination of different classes of antimicrobial agents. There are currently few new drugs with low endogenous resistance potential, and the development of such drugs merits further research.

Rare antibodies from combinatorial libraries suggests an S.O.S. component of the human immunological repertoire.

Convergence of observations from different sources is the norm in science. However, when convergence occurs in man for antibodies it is remarkable because the repertoire of possible immunoglobulin products is very large and diverse. Thus, one would not expect to see the same antibody twice from divergent populations unless there is special significance as to why the immune response is constrained. Now, broadly neutralizing antibodies isolated from combinatorial libraries from three separate populations have been shown to all use the same (V(H) 1-69) germ line gene and interact with the influenza virus in very similar ways. Here we discuss the reasons for this convergence in terms of how the immunological repertoire responds to emergency situations where time is short as occurs, for example, in potentially lethal infections. It is suggested that there is a first responder or S.O.S. component of the antibody repertoire that evolved to initiate rapid defense against infectious agents. The discovery of the homologies between these commonly produced antibodies may have significance for the design of novel vaccines. Finally, these convergent results may give much insight into why antibodies encoded by the V(H) 1-69 germ line gene are highly over represented in B-cell lymphomas.

uvrB gene deletion enhances SOS chromotest sensitivity for nitroreductases that preferentially generate the 4-hydroxylamine metabolite of the anti-cancer prodrug CB1954.

CB1954 is an anti-cancer prodrug that can be reduced at either of two nitro groups to form cytotoxic metabolites. We describe here two efficient and previously uncharacterized nitroreductases, YfkO from Bacillus subtilis which reduces CB1954 exclusively at the 4-NO(2) position, and NfsA from Klebsiella pneumoniae which preferentially reduces the 2-NO(2) group. Utilizing these novel enzymes, together with three previously characterized nitroreductases, we demonstrate that the Escherichia coli SOS-chromotest assay can differentially detect the 4-nitro versus 2-nitro reduction products of CB1954 following deletion of the nucleotide excision repair gene uvrB, but not mismatch repair (mutS) or methyltransferase (ada/ogt) genes. These findings may hold significance for identification and selection of nitroreductases for CB1954-mediated gene therapy, particularly when targeting tumors that are deficient in nucleotide excision repair. Moreover, we demonstrate that comparative SOS chromotest analysis in wild type and uvrB mutant strains can be used to determine whether or not nucleotide excision repair plays a significant role in processing DNA damage resulting from activation of different nitroaromatic prodrugs.