Mfd Protects Against Oxidative Stress in Bacillus Subtilis Independently of its Canonical Function in DNA Repair

Background: Previous reports showed that mutagenesis in nutrient-limiting conditions is dependent on Mfd in Bacillus subtilis. Mfd initiates one type of transcription-coupled repair (TCR); this type of repair is known to target bulky lesions, like those associated with UV exposure. Interestingly, the roles of Mfd in repair of oxidative-promoted DNA damage and regulation of transcription differ. Here, we used a genetic approach to test whether Mfd protected B. subtilis from exposure to two different oxidants. Results: Wild-type cells survived tert-butyl hydroperoxide (t-BHP) exposure significantly better than Mfd-deficient cells. This protective effect was independent of UvrA, a component of the canonical TCR/nucleotide excision repair (NER) pathway. Further, our results suggest that Mfd and MutY, a DNA glycosylase that processes 8-oxoG DNA mismatches, work together to protect cells from lesions generated by oxidative damage. We also tested the role of Mfd in mutagenesis in starved cells exposed to t-BHP. In conditions of oxidative stress, Mfd and MutY may work together in the formation of mutations. Unexpectedly, Mfd increased survival when cells were exposed to the protein oxidant diamide. Under this type of oxidative stress, cells survival was not affected by MutY or UvrA. Conclusions: These results are significant because they show that Mfd mediates error-prone repair of DNA and protects cells against oxidation of proteins by affecting gene expression; Mfd deficiency resulted in increased gene expression of the OhrR repressor which controls the cellular response to organic peroxide exposure. These observations point to Mfd functioning beyond a DNA repair factor in cells experiencing oxidative stress

DNA secondary structures and their contribution to mutagenesis in B. subtilis stationary phase cells

It is widely known and accepted that the cause of many mutations in cells are generated during the replication process of actively dividing cells, however more recent research has shown that mutations also arise in non growing conditions, a phenomenon known as stationary phase mutagenesis. Much of what is known come from studies in eukaryotic and bacterial models. It has been proposed that in non~growing cells, the process of transcription plays an important role in mutagenesis. We test the hypothesis that DNA secondary structures, formed during transcription, promote mutagenesis. The transcription-generated structures are speculated to be prone to mutations by exposing regions of single stranded DNA to lesions. We examined the Bacillus subtilis gene thiF, predicted by in silica analysis to be prone to mutations at particular locations during transcription. By altering the base sequence of this gene, the stability of its stem-loop structures is affected, thereby allowing us to test whether transcription of the altered sequence influences accumulation of mutations in thiF. Our assay for detection of mutations is based on reversion to thiamine prototrophy in cells under conditions of starvation. Ultimately, these experiments will increase our understanding of how mutations occur in cells of all domains of life

DNA secondary structures and their contribution to mutagenesis in B. subtilis stationary phase cells

It is widely known and accepted that the cause of many mutations in cells are generated during the replication process of actively dividing cells, however more recent research has shown that mutations also arise in non growing conditions, a phenomenon known stationary phase mutagenesis. Much of what is known come from studies in eukaryotic and bacterial models. It is proposed that in nongrowing cells, the process of transcription plays an important role in mutagenesis. I will test the hypothesis that secondary structures formed of DNA generated transcription promote mutagenesis. The sequences transcriptiongenerated structures are speculated to be prone to mutations by exposing regions of single stranded DNA to lesions. To test this hypothesis, I examined the Bacillus subtilis gene thiF, predicted by in silico analysis to be prone to mutations at particular locations during transcription. By altering the base sequence of this gene, the stability of its stem-loop structures is affected, thereby allowing us to test whether transcription of the altered sequence influences accumulation of in thiF. Our assay for detection of mutations is based on reversion to thiamine auxotrophy in cells under conditions of starvation. Ultimately, these experiments will increase our understanding of how mutations occur in cells of all domains of life

The Bacillus Subtilis K-State Promotes Stationary-Phase Mutagenesis via Oxidative Damage

Bacterial cells develop mutations in the absence of cellular division through a process known as stationary-phase or stress-induced mutagenesis. This phenomenon has been studied in a few bacterial models, including Escherichia coli and Bacillus subtilis; however, the underlying mechanisms between these systems differ. For instance, RecA is not required for stationary-phase mutagenesis in B. subtilis like it is in E. coli. In B. subtilis, RecA is essential to the process of genetic transformation in the subpopulation of cells that become naturally competent in conditions of stress. Interestingly, the transcriptional regulator ComK, which controls the development of competence, does influence the accumulation of mutations in stationary phase in B. subtilis. Since recombination is not involved in this process even though ComK is, we investigated if the development of a subpopulation (K-cells) could be involved in stationary-phase mutagenesis. Using genetic knockout strains and a point-mutation reversion system, we investigated the effects of ComK, ComEA (a protein involved in DNA transport during transformation), and oxidative damage on stationary-phase mutagenesis. We found that stationary-phase revertants were more likely to have undergone the development of competence than the background of non-revertant cells, mutations accumulated independently of DNA uptake, and the presence of exogenous oxidants potentiated mutagenesis in K-cells. Therefore, the development of the K-state creates conditions favorable to an increase in the genetic diversity of the population not only through exogenous DNA uptake but also through stationary-phase mutagenesis

2011-2012 UNLV McNair Journal

Journal articles based on research conducted by undergraduate students in the McNair Scholars Program Table of Contents Biography of Dr. Ronald E. McNair Statements: Dr. Neal J. Smatresk, UNLV President Dr. Juanita P. Fain, Vice President of Student Affairs Dr. William W. Sullivan, Associate Vice President for Retention and Outreach Mr. Keith Rogers, Deputy Executive Director of the Center for Academic Enrichment and Outreach McNair Scholars Institute Staf

Describing Bacterial Mutagenic Hotspots and Signatures Along with the Factors that Drive Them

In my dissertation work, I investigated bacterial mutagenesis. I asked: where do mutations occur in the bacterial genome? What types of mutations occur? What factors promote or prevent mutations? And lastly, how do the answers to these questions change depending on the bacterial life cycle (When)? I used Bacillus subtilis, a Gram-positive bacterium belonging to the phylum Firmicutes, and Escherichia coli, a Gram-negative bacterium belonging to the phylum Proteobacteria, to answer these questions. B. subtilis and E. coli are model systems that have been used to gain insights into fundamental processes of mutations and evolution for nearly ~8 decades. Here, each one of my three research chapters adds to our understanding of bacterial mutagenesis. In my 1st research chapter (chapter 2) I explored if non-B DNA motifs are mutagenic hotspots in B. subtilis. Non-B DNA-motifs are sequences that can adopt structures that deviate from the canonical right- handed Watson and Crick B-structure. Non-B DNA correlate with genomic instability in eukaryotes but their role in bacterial mutagenesis is understudied. My findings show that sequences predicted to form non-B DNA structures accumulated more mutations compared to sequences that were not predicted to form any stable structures. Importantly, this effect was only seen in stationary-phase cells, as such a response did not occur in growing conditions, and it was dependent on the DNA repair factor Mfd. This research chapter gives us insight into where mutations occur (at non-B motifs), when (during stationary phase), and what factors promote and prevent mutations (Mfd promotes mutations at these sites). In my 2nd research chapter (chapter 3), I investigated if out-of-frame stop codons (OSCs)have a function in bacterial genomes. Out-of-frame stop codons (OSC), a.k.a. hidden stops or iii premature stops, are created when two consecutive amino acid codons form one of three translational stop codons in an alternate reading frame of the coding sequence (e.g., CTG AAA). The function of OSCs in Bacteria is unclear, but they are speculated to protect against translational frameshifts that could result in energy loss through the production of aberrant proteins, however in vivo data is limited. Results from my experiments show that the loss of OSCs in a DNA region that codes for a flexible loop region in a protein led to an increase in insertion and deletion (indels) mutagenic events. Interestingly, the effect was seen in both growing and stationary-phase cells and loss of the DNA repair factor Mfd decreased mutations. This research chapter gives us insight into why we observe certain types of mutations like indels at specific places in the genome (regions that code for protein loops) and what factors promote and prevent mutations (OSCs may limit indel appearance while Mfd promotes the formation of indels). In my last research (chapter 4) I integrated the strengths of Maximum Depth Sequencing and a gain-of-function system to advance our understanding of mutagenesis that occurs in the presence and absence of selection of a region of interest (ROI). Maximum Depth Sequencing (MDS) was developed to measure the strand-specific mutation rate/frequency of any region of interest in a bacterial genome through error-corrected, high-throughput sequencing independent of selection. In a gain-of-function system, a mutation, induced by a mutagen or engineered into a gene, renders the bacterium unable to carry out a function needed for growth, therefore mutations that restore the function encoded by that gene also restore growth and allow bacterial colonies to form and be counted on a Petri plate. My research showed that there were discrepancies in the frequency or rate of mutations between the methods used to measure mutagenesis. Because MDS detects transient genetic changes that may or may not get fixed into mutations, my analysis suggests that the frequency of a transient DNA change does not predict its frequency of mutation fixation. Furthermore, my experiments showed that DNA repair factors and stress change the mutagenic spectrum, this study addresses what factors prevent and promote mutations and when. By describing new life-cycle-dependent mutation hotspots and signatures along with the factors that drive them, my research continues to add to our understanding of where mutations occur, what type of mutations occur when, and what factors promote and prevent mutations in Bacteria. The answers to these basic questions about mutations in the smallest forms of life have already proven to have a large impact on human health and our understanding evolution

Le quartier du Chêne Pointu : quelle(s) approche(s) de la parentalité face à la précarité sanitaire et sociale ?

International audienc

Quelle(s) approche(s) de la parentalité face à la précarité sanitaire et sociale dans les quartiers ?

This exploratory research is a follow-up to the work carried out within the parenting support project, which focuses on understanding the expectations and shortcomings of the residents of two neighborhoods in the department of Seine-Saint-Denis, which are marked by social and health insecurity. We aimed to examine the needs identified by professionals and to compare them with those expressed by the residents of these two neighborhood’s. This context implied a reflection on the notion of "need" which we approached from a theorists' point of view, but also through the analysis of real possibilities of action. In the hypothesis, the needs of the habitants of these neighborhoods are confronted with a response that differs when it is thought by a professional or a parent. The first results show a response from the professionals "from above" which seems to be quite far from the primary concerns of the people. We have compared the comments of the various actors, which were collected through semi-directive interviews and observations, to try to define a pyramid of needs for the population of these two neighborhoods.Cette recherche exploratoire s’inscrit dans la continuité des travaux menés au sein d’un projet d’accompagnement à la parentalité centré sur la compréhension des attentes et manques des habitants de deux quartiers dans le département de Seine-Saint-Denis, marqués par la précarité sociale et sanitaire. Nous souhaitons interroger les besoins identifiés par les professionnels et les mettre en parallèle avec ceux exprimés par les habitants de ces deux quartiers. Ce contexte a supposé une réflexion sur la notion de « besoin » que nous avons abordée à partir d’un point de vue des théoriciens, mais aussi à travers l’analyse par des possibilités réelles d’actions. En hypothèse, les besoins des habitants de ces quartiers s’affrontent avec une réponse apportée qui diffère dès lors qu’elle est pensée par un professionnel ou un parent. Les premiers résultats montrent une réponse des professionnels éloignés du terrain qui semble assez éloignée des préoccupations premières des habitants. Nous avons mis en parallèle les propos des différents protagonistes qui ont été recueillis à partir d’entretiens semi-directifs et d'observations, pour essayer de définir une pyramide des besoins des habitants de ces deux quartiers

Accompagner la parentalité au Chêne Pointu et Chêne étoile : entre normalité,normativité et normalisation

In 2016, the city of Clichy-sous-Bois introduced a cross-disciplinary collaboration with local actors, parents and professionals, with the aim of implementing an early prevention and parenting project (PPEP'S). But what does it mean to support parenthood and work with parents in a disadvantaged neighbourhood in 21st century France? It is this question, with its anthropological, social and political implications, that we would like to address in this article. Here we take up the voices of professionals working in the Chêne Pointu and Chêne Étoile neighbourhoods in Clichy-sous-Bois, both of which are marked by poverty and social and health insecurity. This situation complexifies the task of the professionals, particularly in terms of the posture to have when faced with a reality which, although structurally known by the various institutions and other public actors, nevertheless retains its share of uncertainties, misunderstandings and even incomprehension.La Ville de Clichy-sous-Bois a engagé en 2016 une dynamique de collaboration transversale avec les acteurs du territoire, parents et professionnels, visant un projet de prévention précoce et parentalité (PPEP’S). Mais que signifie accompagner la parentalité et travailler avec, dans un quartier populaire dans la France du XXIème siècle ? C’est à cette question à la portée anthropologique, sociale et politique, que nous voudrions nous atteler dans le cadre de cet article. Nous reprenons ici les propos des professionnelles travaillant aux quartiers du Chêne Pointu et du Chêne Étoilé à Clichy-sous-Bois, tous les deux marqués par la pauvreté et la précarité sociale et sanitaire. Cette situation complexifie la tâche des professionnelles, notamment au niveau de la posture à adopter face à une réalité qui, bien que structurellement connue par les différentes institutions et autres acteurs publics, conserve malgré tout son lot d’incertitudes, de méconnaissances, voire d’incompréhensions