Bacteria-to-Human Protein Networks Reveal Origins of Endogenous DNA Damage.

DNA damage provokes mutations and cancer and results from external carcinogens or endogenous cellular processes. However, the intrinsic instigators of endogenous DNA damage are poorly understood. Here, we identify proteins that promote endogenous DNA damage when overproduced: the DNA "damage-up" proteins (DDPs). We discover a large network of DDPs in Escherichia coli and deconvolute them into six function clusters, demonstrating DDP mechanisms in three: reactive oxygen increase by transmembrane transporters, chromosome loss by replisome binding, and replication stalling by transcription factors. Their 284 human homologs are over-represented among known cancer drivers, and their RNAs in tumors predict heavy mutagenesis and a poor prognosis. Half of the tested human homologs promote DNA damage and mutation when overproduced in human cells, with DNA damage-elevating mechanisms like those in E. coli. Our work identifies networks of DDPs that provoke endogenous DNA damage and may reveal DNA damage-associated functions of many human known and newly implicated cancer-promoting proteins.

An ultra-dense library resource for rapid deconvolution of mutations that cause phenotypes in Escherichia coli.

With the wide availability of whole-genome sequencing (WGS), genetic mapping has become the rate-limiting step, inhibiting unbiased forward genetics in even the most tractable model organisms. We introduce a rapid deconvolution resource and method for untagged causative mutations after mutagenesis, screens, and WGS in Escherichia coli. We created Deconvoluter-ordered libraries with selectable insertions every 50 kb in the E. coli genome. The Deconvoluter method uses these for replacement of untagged mutations in the genome using a phage-P1-based gene-replacement strategy. We validate the Deconvoluter resource by deconvolution of 17 of 17 phenotype-altering mutations from a screen of N-ethyl-N-nitrosourea-induced mutants. The Deconvoluter resource permits rapid unbiased screens and gene/function identification and will enable exploration of functions of essential genes and undiscovered genes/sites/alleles not represented in existing deletion collections. This resource for unbiased forward-genetic screens with mapping-by-sequencing ('forward genomics') demonstrates a strategy that could similarly enable rapid screens in many other microbes.

Promoting Healthy Eating Among African-American School-Aged Girls in a Community Setting.

The prevention of obesity is vital to the health of American children. In the urban African-American community, the health of school-aged children is in particular jeopardy due to the high prevalence of obesity, type 2 diabetes, and poor dietary choices such as the purchase of sugary drinks, salty snacks, low consumption of fresh fruits and vegetables, and reliance on fast food meals. African-American girls are at a higher risk for obesity and early puberty before age 10, placing them at a greater risk for diabetes and cardiovascular disease in adulthood. Our current "Cooking with Kids" program in a local grocery store has allowed us to promote healthy eating behavior in a unique way; teaching 6 through 11-year-olds how to prepare easy healthy breakfasts, lunches, and snack food recipes at a local grocery store while their mothers shopped.

Enforcement of science-using a Clostridium perfringens outbreak investigation to take legal action.

BACKGROUND: We report an outbreak of Clostridium perfringens in a care home in North East England. METHODS: A retrospective cohort study was used to investigate this outbreak. Faecal samples were obtained from symptomatic residents. Environmental Health Officers carried out a food hygiene inspection and formal statements were taken. RESULTS: Fifteen residents reported illness and the epidemic curve was suggestive of a point source outbreak. Results suggest that illness was associated with consumption of mince & vegetable pie and/or gravy. There were a number of issues with food served, in particular the mince products had been cooked, cooled, reheated and served again over a period of several days. Faecal sampling revealed the presence of C.perfringens enterotoxin gene and four samples were indistinguishable by fluorescent amplified fragment length polymorphism, indicating a likely common source. The operator of the home was charged with three offences under the General Food Regulations 2004 and the Food Hygiene (England) Regulations 2006 and was convicted on all counts. CONCLUSIONS: An outbreak of C.perfringens occurred in a care home. The likely cause was consumption of mince & vegetable pie and/or gravy. Epidemiological evidence can be used to help prosecute businesses with food safety offences in such circumstances.

Impact of a stress-inducible switch to mutagenic repair of DNA breaks on mutation in Escherichia coli.

Basic ideas about the constancy and randomness of mutagenesis that drives evolution were challenged by the discovery of mutation pathways activated by stress responses. These pathways could promote evolution specifically when cells are maladapted to their environment (i.e., are stressed). However, the clearest example--a general stress-response-controlled switch to error-prone DNA break (double-strand break, DSB) repair--was suggested to be peculiar to an Escherichia coli F' conjugative plasmid, not generally significant, and to occur by an alternative stress-independent mechanism. Moreover, mechanisms of spontaneous mutation in E. coli remain obscure. First, we demonstrate that this same mechanism occurs in chromosomes of starving F(-) E. coli. I-SceI endonuclease-induced chromosomal DSBs increase mutation 50-fold, dependent upon general/starvation- and DNA-damage-stress responses, DinB error-prone DNA polymerase, and DSB-repair proteins. Second, DSB repair is also mutagenic if the RpoS general-stress-response activator is expressed in unstressed cells, illustrating a stress-response-controlled switch to mutagenic repair. Third, DSB survival is not improved by RpoS or DinB, indicating that mutagenesis is not an inescapable byproduct of repair. Importantly, fourth, fully half of spontaneous frame-shift and base-substitution mutation during starvation also requires the same stress-response, DSB-repair, and DinB proteins. These data indicate that DSB-repair-dependent stress-induced mutation, driven by spontaneous DNA breaks, is a pathway that cells usually use and a major source of spontaneous mutation. These data also rule out major alternative models for the mechanism. Mechanisms that couple mutagenesis to stress responses can allow cells to evolve rapidly and responsively to their environment.

The sigma(E) stress response is required for stress-induced mutation and amplification in Escherichia coli.

Pathways of mutagenesis are induced in microbes under adverse conditions controlled by stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e. are stressed. Stress-induced mutagenesis in the Escherichia coli Lac assay occurs either by 'point' mutation or gene amplification. Point mutagenesis is associated with DNA double-strand-break (DSB) repair and requires DinB error-prone DNA polymerase and the SOS DNA-damage- and RpoS general-stress responses. We report that the RpoE envelope-protein-stress response is also required. In a screen for mutagenesis-defective mutants, we isolated a transposon insertion in the rpoE P2 promoter. The insertion prevents rpoE induction during stress, but leaves constitutive expression intact, and allows cell viability. rpoE insertion and suppressed null mutants display reduced point mutagenesis and maintenance of amplified DNA. Furthermore, sigma(E) acts independently of stress responses previously implicated: SOS/DinB and RpoS, and of sigma(32), which was postulated to affect mutagenesis. I-SceI-induced DSBs alleviated much of the rpoE phenotype, implying that sigma(E) promoted DSB formation. Thus, a third stress response and stress input regulate DSB-repair-associated stress-induced mutagenesis. This provides the first report of mutagenesis promoted by sigma(E), and implies that extracytoplasmic stressors may affect genome integrity and, potentially, the ability to evolve.

Priming insight in groups: facilitating and inhibiting solving an ambiguously worded insight problem.

We extend research on the priming of insight by studying group problem solving. Groups of 2-4 participants tried to solve an ambiguously worded problem in the presence of a prime that reinforced the dominant but incorrect interpretation of the problem, a prime that reinforced the uncommon but correct interpretation, or no prime. The paradigm involved participants asking questions of the experimenter that could only be answered "yes" or "no." In Experiment 1, the prime was present throughout the solving period; in Experiment 2, it was removed prior to the solving period. In both experiments, the primes had their predicted effects. Patterns in the time taken to solve the problem supported the idea that groups stuck at the impasse were more or less able to restructure the problem, depending on the environmental context. Data from the questions asked and questionnaires converged with time taken to solve the problem, consistent with the view that restructuring a problem is an automatic process that produces insight. A comparison of the group data in Experiment 1 with individually tested participants' data revealed that the insight of the groups benefited from their being able to recognize lines of questions to follow, to listen to answers to questions asked, and to evaluate and reject errors or assumptions.

The odd-even effect in Sudoku puzzles: effects of working memory, aging, and experience.

The odd-even effect in numerical processing has been explained as the easier processing of even numbers compared with odd numbers. We investigated this effect in Sudoku puzzles, a reasoning problem that uses numbers but does not require arithmetic operations. Specifically, we asked whether the odd-even effect occurred with Sudoku puzzles and whether individual differences in working memory (WM), aging, and experience with Sudoku modulated this effect. We manipulated the presence of odd and even numbers in Sudoku puzzles, measured WM with the Wisconsin Card Sorting Test and backward digit span task, tested older and younger adults, and collected Sudoku experience frequency. Performance on Sudoku was more accurate for even puzzles than odd ones. Younger, experienced, and higher-WM participants were more accurate on Sudoku, but these individual difference variables did not interact with the odd-even effect. Odd numbers may impose more cognitive load than even numbers, but future research is needed to examine how age, experience, or WM may influence the odd-even effect.

Separate DNA Pol II- and Pol IV-dependent pathways of stress-induced mutation during double-strand-break repair in Escherichia coli are controlled by RpoS.

Previous work showed that about 85% of stress-induced mutations associated with DNA double-strand break repair in carbon-starved Escherichia coli result from error-prone DNA polymerase IV (Pol IV) (DinB) and that the mutagenesis is controlled by the RpoS stress response, which upregulates dinB. We report that the remaining mutagenesis requires high-fidelity Pol II, and that this component also requires RpoS. The results identify a second DNA polymerase contributing to stress-induced mutagenesis and show that RpoS promotes mutagenesis by more than the simple upregulation of dinB.

An SOS-regulated type 2 toxin-antitoxin system.

The Escherichia coli chromosome encodes seven demonstrated type 2 toxin-antitoxin (TA) systems: cassettes of two or three cotranscribed genes, one encoding a stable toxin protein that can cause cell stasis or death, another encoding a labile antitoxin protein, and sometimes a third regulatory protein. We demonstrate that the yafNO genes constitute an additional chromosomal type 2 TA system that is upregulated during the SOS DNA damage response. The yafNOP genes are part of the dinB operon, of which dinB underlies stress-induced mutagenesis mechanisms. yafN was identified as a putative antitoxin by homology to known antitoxins, implicating yafO (and/or yafP) as a putative toxin. Using phage-mediated cotransduction assays for linkage disruption, we show first that yafN is an essential gene and second that it is essential only when yafO is present. Third, yafP is not a necessary part of either the toxin or the antitoxin. Fourth, although DinB is required, the yafNOP genes are not required for stress-induced mutagenesis in the Escherichia coli Lac assay. These results imply that yafN encodes an antitoxin that protects cells against a yafO-encoded toxin and show a protein-based TA system upregulated by the SOS response.

Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris.

Rhodopseudomonas palustris is among the most metabolically versatile bacteria known. It uses light, inorganic compounds, or organic compounds, for energy. It acquires carbon from many types of green plant-derived compounds or by carbon dioxide fixation, and it fixes nitrogen. Here we describe the genome sequence of R. palustris, which consists of a 5,459,213-base-pair (bp) circular chromosome with 4,836 predicted genes and a plasmid of 8,427 bp. The sequence reveals genes that confer a remarkably large number of options within a given type of metabolism, including three nitrogenases, five benzene ring cleavage pathways and four light harvesting 2 systems. R. palustris encodes 63 signal transduction histidine kinases and 79 response regulator receiver domains. Almost 15% of the genome is devoted to transport. This genome sequence is a starting point to use R. palustris as a model to explore how organisms integrate metabolic modules in response to environmental perturbations.

Modality-specificity effects in priming of visual and auditory word-fragment completion.

In this study, the researchers examined modality-specificity effects in priming of visual and auditory word-fragment completion by the presentation of visual or auditory primes. In 2 experiments, within-modality priming and cross-modality priming were observed, with greater priming observed in the within-modality conditions. The prime was presented in a word list in Experiment 1 and either presented or inferred in priming sentences in Experiment 2. Inferring a target that was not actually presented in the sentences resulted in priming of fragment completion but not in modality specificity. These results, coupled with comparisons to explicit cued fragment completion, support the interpretation that priming of word-fragment completion is owing to both a perceptual and a nonperceptual component. This latter component may be different than the conceptual processes used for explicit memory, which did show modality specificity for inferred targets.

Atypical Role for PhoU in Mutagenic Break Repair under Stress in Escherichia coli.

Mechanisms of mutagenesis activated by stress responses drive pathogen/host adaptation, antibiotic and anti-fungal-drug resistance, and perhaps much of evolution generally. In Escherichia coli, repair of double-strand breaks (DSBs) by homologous recombination is high fidelity in unstressed cells, but switches to a mutagenic mode using error-prone DNA polymerases when the both the SOS and general (σS) stress responses are activated. Additionally, the σE response promotes spontaneous DNA breakage that leads to mutagenic break repair (MBR). We identified the regulatory protein PhoU in a genetic screen for functions required for MBR. PhoU negatively regulates the phosphate-transport and utilization (Pho) regulon when phosphate is in excess, including the PstB and PstC subunits of the phosphate-specific ABC transporter PstSCAB. Here, we characterize the PhoU mutation-promoting role. First, some mutations that affect phosphate transport and Pho transcriptional regulation decrease mutagenesis. Second, the mutagenesis and regulon-expression phenotypes do not correspond, revealing an apparent new function(s) for PhoU. Third, the PhoU mutagenic role is not via activation of the σS, SOS or σE responses, because mutations (or DSBs) that restore mutagenesis to cells defective in these stress responses do not restore mutagenesis to phoU cells. Fourth, the mutagenesis defect in phoU-mutant cells is partially restored by deletion of arcA, a gene normally repressed by PhoU, implying that a gene(s) repressed by ArcA promotes mutagenic break repair. The data show a new role for PhoU in regulation, and a new regulatory branch of the stress-response signaling web that activates mutagenic break repair in E. coli.

Effects of cellulose derivatives and poly(ethylene oxide)-poly(propylene oxide) tri-block copolymers (Pluronic)surfactants) on the properties of alginate based microspheres and their interactions with phagocytic cells.

The goal of this study was to examine the phagocytosis of alginate based microspheres with different surface properties. Favorable interaction with macrophages is critical for uptake subsequent processing of the microspheres used for oral vaccine delivery. We examined the effects of size of alginate microspheres and hydrophobicity on cellular uptake. We also examined the toxicity of formulation components to phagocytic cells. Alginate microspheres were made by the emulsion-cross-linking technique. Five different formulations of microspheres were evaluated for size, hydrophobicity, cellular uptake and toxicity to macrophages. The formulations examined were: alginate alone (A), alginate with methylcellulose (AA) AA with Pluronic L61 (AA61), alginate with hydroxypropyl methylcellulose (AK3), and AK3 with Pluronic (L61 (AK3 61). Microspheres with without poly-L-lysine (PLL) coating were tested. The mean volume sizes of A, AA, AA61, AK3, AK3 61 microspheres (MS) were 11, 10.5, 3.8, 8.7 and 3.9 mocrom, respectively. After coating them with PLL the mean volume sizes were 10.4, 10, 3.7, 8.8 and 3.5 microm, respectively. Hydrophobicity of the microspheres was evaluated by measuring contact angle on a glass slide coated with the microspheres. The contact angles measured using a goniometer on A, AA, AA61, AK3, AK3 61 MS were 20, 34.8, 71, 29 and 80 degrees, respectively whereas those MS coated with PLL were 49.7, 55.8, 91, 48.25 and 84.4 degrees, respectively. Cellular uptake studies using flow cytometery revealed that AA61 MS coated with PLL were phagocytosed most often by mouse macrophages. There was no statistically significant difference in cellular uptake among those MS without PLL coating. Toxicity to macrophages was shown to depend on the ratio of microspheres to cells. These studies suggest that formulation can dramatically affect the physical characteristics of alginate MS in ways that can affect how they will interact with cells in the body when administered as a vaccine delivery system.

Priming problem solving with conceptual processing of relevant objects.

In the present study, the author explored the effect of processing relevant information on producing solutions to brief insight problems. She hypothesized that the conceptual processing of objects relevant to the target solution would facilitate that solution relative to unrelated objects or the shallow processing of words. The author also explored the effect of knowledge of the relationship between the initial object-processing task and the problem-solving task. The results showed that participants who conceptually processed objects related to the target solution (Experiment 1), but not those who shallowly processed words related to the target solution (Experiment 2), were more likely to produce the solution relative to the control; and knowledge of the relationship between objects and solutions made no difference in the frequency of target solutions produced. The results of Experiment 3 showed that conceptual processing of an object could prime a nondominant solution for an ambiguously worded problem. Taken together, the results of the studies supported the effect of conceptual processing on facilitating insight in problem solving.

Priming guesses on a forced-recall test.

The forced-recall paradigm requires participants to fill all spaces on the memory test even if they cannot remember all the list words. In the present study, the authors used that paradigm to examine the influence of implicit memory on guessing--when participants fill remaining spaces after they cannot remember list items. They measured explicit memory as the percentage of targets that participants designated as remembered from the list and implicit memory as the percentage of targets they wrote but did not designate as remembered (beyond chance level). The authors examined implicit memory on guessing with forced recall (Experiment 1), forced cued recall with younger and older adults (Experiment 2), and forced free and cued recall under a depth-of-processing manipulation (Experiment 3). They conclude that implicit memory influences guesses of targets in the forced-recall paradigm.

Engineered proteins detect spontaneous DNA breakage in human and bacterial cells.

Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP-labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells. DOI:http://dx.doi.org/10.7554/eLife.01222.001.

Two mechanisms produce mutation hotspots at DNA breaks in Escherichia coli.

Mutation hotspots and showers occur across phylogeny and profoundly influence genome evolution, yet the mechanisms that produce hotspots remain obscure. We report that DNA double-strand breaks (DSBs) provoke mutation hotspots via stress-induced mutation in Escherichia coli. With tet reporters placed 2 kb to 2 Mb (half the genome) away from an I-SceI site, RpoS/DinB-dependent mutations occur maximally within the first 2 kb and decrease logarithmically to ∼60 kb. A weak mutation tail extends to 1 Mb. Hotspotting occurs independently of I-site/tet-reporter-pair position in the genome, upstream and downstream in the replication path. RecD, which allows RecBCD DSB-exonuclease activity, is required for strong local but not long-distance hotspotting, indicating that double-strand resection and gap-filling synthesis underlie local hotspotting, and newly illuminating DSB resection in vivo. Hotspotting near DSBs opens the possibility that specific genomic regions could be targeted for mutagenesis, and could also promote concerted evolution (coincident mutations) within genes/gene clusters, an important issue in the evolution of protein functions.

Identity and function of a large gene network underlying mutagenic repair of DNA breaks.

Mechanisms of DNA repair and mutagenesis are defined on the basis of relatively few proteins acting on DNA, yet the identities and functions of all proteins required are unknown. Here, we identify the network that underlies mutagenic repair of DNA breaks in stressed Escherichia coli and define functions for much of it. Using a comprehensive screen, we identified a network of ≥93 genes that function in mutation. Most operate upstream of activation of three required stress responses (RpoS, RpoE, and SOS, key network hubs), apparently sensing stress. The results reveal how a network integrates mutagenic repair into the biology of the cell, show specific pathways of environmental sensing, demonstrate the centrality of stress responses, and imply that these responses are attractive as potential drug targets for blocking the evolution of pathogens.

What limits the efficiency of double-strand break-dependent stress-induced mutation in Escherichia coli?

Stress-induced mutation is a collection of molecular mechanisms in bacterial, yeast and human cells that promote mutagenesis specifically when cells are maladapted to their environment, i.e. when they are stressed. Here, we review one molecular mechanism: double-strand break (DSB)-dependent stress-induced mutagenesis described in starving Escherichia coli. In it, the otherwise high-fidelity process of DSB repair by homologous recombination is switched to an error-prone mode under the control of the RpoS general stress response, which licenses the use of error-prone DNA polymerase, DinB, in DSB repair. This mechanism requires DSB repair proteins, RpoS, the SOS response and DinB. This pathway underlies half of spontaneous chromosomal frameshift and base substitution mutations in starving E. coli [Proc Natl Acad Sci USA 2011;108:13659-13664], yet appeared less efficient in chromosomal than F' plasmid-borne genes. Here, we demonstrate and quantify DSB-dependent stress-induced reversion of a chromosomal lac allele with DSBs supplied by I-SceI double-strand endonuclease. I-SceI-induced reversion of this allele was previously studied in an F'. We compare the efficiencies of mutagenesis in the two locations. When we account for contributions of an F'-borne extra dinB gene, strain background differences, and bypass considerations of rates of spontaneous DNA breakage by providing I-SceI cuts, the chromosome is still ∼100 times less active than F. We suggest that availability of a homologous partner molecule for recombinational break repair may be limiting. That partner could be a duplicated chromosomal segment or sister chromosome.