Oxidative stress-induced damage, including 8-oxo-guanine and apurinic/apyrimidinic (AP) DNA lesions, were detected in dormant and outgrowing Bacillus subtilis spores lacking the AP endonucleases Nfo and ExoA. Spores of the Δnfo exoA strain exhibited slightly slowed germination and greatly slowed outgrowth that drastically slowed the spores' return to vegetative growth. A null mutation in the disA gene, encoding a DNA integrity scanning protein (DisA), suppressed this phenotype, as spores lacking Nfo, ExoA, and DisA exhibited germination and outgrowth kinetics very similar to those of wild-type spores. Overexpression of DisA also restored the slow germination and outgrowth phenotype to nfo exoA disA spores. A disA-lacZ fusion was expressed during sporulation but not in the forespore compartment. However, disA-lacZ was expressed during spore germination/outgrowth, as was a DisA-green fluorescent protein (GFP) fusion protein. Fluorescence microscopy revealed that, as previously shown in sporulating cells, DisA-GFP formed discrete globular foci that colocalized with the nucleoid of germinating and outgrowing spores and remained located primarily in a single cell during early vegetative growth. Finally, the slow-outgrowth phenotype of nfo exoA spores was accompanied by a delay in DNA synthesis to repair AP and 8-oxo-guanine lesions, and these effects were suppressed following disA disruption. We postulate that a DisA-dependent checkpoint arrests DNA replication during B. subtilis spore outgrowth until the germinating spore's genome is free of damage.
Reactive oxygen species (ROS) promote the synthesis of the DNA lesion 8-oxo-G, whose mutagenic effects are counteracted in distinct organisms by the DNA glycosylase MutM. We report here that in Bacillus subtilis, mutM is expressed during the exponential and stationary phases of growth. In agreement with this expression pattern, results of a Western blot analysis confirmed the presence of MutM in both stages of growth. In comparison with cells of a wild-type strain, cells of B. subtilis lacking MutM increased their spontaneous mutation frequency to Rif r and were more sensitive to the ROS promoter agents hydrogen peroxide and 1,1=-dimethyl-4,4=-bipyridinium dichloride (Paraquat). However, despite MutM's proven participation in preventing ROS-induced-DNA damage, the expression of mutM was not induced by hydrogen peroxide, mitomycin C, or NaCl, suggesting that transcription of this gene is not under the control of the RecA, PerR, or B regulons. Finally, the role of MutM in stationary-phase-associated mutagenesis (SPM) was investigated in the strain B. subtilis YB955 (hisC952 metB5 leuC427). Results revealed that under limiting growth conditions, a mutM knockout strain significantly increased the amount of stationary-phaseassociated his, met, and leu revertants produced. In summary, our results support the notion that the absence of MutM promotes mutagenesis that allows nutritionally stressed B. subtilis cells to escape from growth-limiting conditions. IMPORTANCEThe present study describes the role played by a DNA repair protein (MutM) in protecting the soil bacterium Bacillus subtilis from the genotoxic effects induced by reactive oxygen species (ROS) promoter agents. Moreover, it reveals that the genetic inactivation of mutM allows nutritionally stressed bacteria to escape from growth-limiting conditions, putatively by a mechanism that involves the accumulation and error-prone processing of oxidized DNA bases. Reactive oxygen species (ROS), including hydrogen peroxide, superoxide, and hydroxyl radicals, are produced in all aerobic organisms as side products of oxidative metabolism or following exposure to environmental agents and are normally in balance with the cellular antioxidant defenses. Oxidative stress occurs when this critical balance is disrupted because of depletion of antioxidants or excess accumulation of ROS (1). Therefore, when antioxidant cellular defenses are deficient or overwhelmed, the damaging potential of ROS increases and they target different cellular biomolecules, including, lipids, proteins, carbohydrates, and DNA (2). One of the most common events resulting from attack of DNA by the hydroxyl radical is the formation of 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-G), a DNA lesion extensively studied due to its strong mutagenic and genotoxic properties (3). However, the hydroxyl radicals can also impact the deoxyribonucleotide and ribonucleotide pools, generating the oxidized precursors 8-oxo-dGTP and 8-oxo-GTP, respectively (4, 5). The former is frequently incorporated opposite adenine ...
Two copper-resistant (Copr) mutants, strains P1 and P3, were obtained from the dimorphic fungus Mucor rouxii. They were characterized as to their ability to take up copper in a growth medium supplemented with this metal ion. Detection of copper by linear sweep striping voltammetry in cell walls and in the cell wall-free fraction of disrupted cells revealed a higher content of the metal in both mutant fractions, as compared with those of the copper-sensitive (Cops) parental strain. Copper binding by M. rouxii growing cells was also studied through the use of a cytochemical method based on the compounds neocuproine (NCP) and sodium diethyldithiocarbamate (DTC). This method indicated that the P1 Copr strain accumulated more metal than the parental Cops strain, both on the cellular surface and in the intracellular milieu.
A forward mutagenesis system based on the acquisition of mutations that inactivate the thymidylate synthase gene (TMS) and confer a trimethoprim resistant (Tmpr) phenotype was developed and utilized to study transcription-mediated mutagenesis (TMM). In addition to thyA, Bacillus subtilis possesses thyB, whose expression occurs under conditions of cell stress; therefore, we generated a thyB- thyA+ mutant strain. Tmpr colonies of this strain were produced with a spontaneous mutation frequency of ~1.4 × 10−9. Genetic disruption of the canonical mismatch (MMR) and guanine oxidized (GO) repair pathways increased the Tmpr frequency of mutation by ~2–3 orders of magnitude. A wide spectrum of base substitutions as well as insertion and deletions in the ORF of thyA were found to confer a Tmpr phenotype. Stationary-phase-associated mutagenesis (SPM) assays revealed that colonies with a Tmpr phenotype, accumulated over a period of ten days with a frequency of ~ 60 ×10−7. The Tmpr system was further modified to study TMM by constructing a ΔthyA ΔthyB strain carrying an IPTG-inducible Pspac-thyA cassette. In conditions of transcriptional induction of thyA, the generation of Tmpr colonies increased ~3-fold compared to conditions of transcriptional repression. Further, the Mfd and GreA factors were necessary for the generation of Tmpr colonies in the presence of IPTG in B. subtilis. Because GreA and Mfd facilitate transcription-coupled repair, our results suggest that TMM is a mechanim to produce genetic diversity in highly transcribed regions in growth-limited B. subtilis cells.
During sporulation Bacillus subtilis Mfd couples transcription to nucleotide excision repair (NER) to eliminate DNA distorting lesions. Here, we report a significant decline in sporulation following Mfd disruption, which was manifested in the absence of external DNA-damage suggesting that spontaneous lesions activate the function of Mfd for an efficient sporogenesis. Accordingly, a dramatic decline in sporulation efficiency took place in a B. subtilis strain lacking Mfd and the repair/prevention guanine oxidized (GO) system (hereafter, the ∆GO system), composed by YtkD, MutM and MutY. Furthermore, the simultaneous absence of Mfd and the GO system, (i) sensitized sporulating cells to H2O2, and (ii) elicited spontaneous and oxygen radical-induced rifampin-resistance (Rifr) mutagenesis. Epifluorescence (EF), confocal and transmission electron (TEM) microscopy analyses, showed a decreased ability of ∆GO ∆mfd strain to sporulate and to develop the typical morphologies of sporulating cells. Remarkably, disruption of sda, sirA and disA partially, restored the sporulation efficiency of the strain deficient for Mfd and the ∆GO system; complete restoration occurred in the RecA− background. Overall, our results unveil a novel Mfd mechanism of transcription-coupled-repair (TCR) elicited by 8-OxoG which converges in the activation of a RecA-dependent checkpoint event that control the onset of sporulation in B. subtilis.
Two different families of monodisperse oligoesters with α-hydroxyl-ω-docosyl (C22) terminal groups [oligo(δ-valerolactone) and oligo(ϵ-caprolactone)] were isolated by flash column chromatography (FCC).
In the last three decades, invasive fungal infections caused by Candida species have become an important public health problem, because they are associated with high rates of morbidity and mortality in immunocompromised and hospitalized patients. The diagnosis and treatment of candidiasis are difficult and usually inefficient. Accordingly, a diversity of available drugs, currently employed to attack candidiasis, frequently induce resistance in patients promoting toxicity due to long-term treatments. Therefore, development of accurate diagnoses and novel antifungals is of high priority to improve life’s quality and expectancy of individuals infected with this pathogen. Plants are invaluable sources of new biologically active compounds. Among the plants used in Mexico in traditional herbolary medicine which have empirically been demonstrated to have antifungal activity are Pedilanthus tithymaloides, Thymus vulgaris, and Ocimum basilicum. In the present study, we analyzed whether these plants contain metabolites with antifungal activity against five Candida species. The extracts from the different plant organs were obtained by macerating them in ethyl alcohol or hexane and filtering. The obtained extracts were preserved in amber flasks at 4°C until used. The minimum inhibitory concentrations (MICs) of the active compound were determined by a microdilution assay. In addition, the following secondary metabolites were identified: linalool (3,7-dimethylocta-1,6-dien-3-ol), eugenol (4-allyl-2-methoxyphenol), limonene (1-methyl-4-(1-methylethenyl)-cyclohexene), and borneol ([(2R)-1,7,7-trimethyl-2-bicyclo[2.2.1]heptanyl] formate). All these compounds were found in the three plants, traditionally used in everyday life, and proved to be effective against Candida species and therefore a viable alternative to conventional antifungals.
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