Draft genome of strain YB955, prophage-cured derivative of strain 168.

We report the genome sequence of strain YB955, a prophage-cured strain used as a model in DNA repair, bacterial physiology, and mutagenesis studies. The assembled and annotated draft genome contains 4,031 coding genes, 5 rRNAs, and 73 tRNAs. Compared to 168, YB955 has a 134,402 bp deletion.

stress-associated mutagenesis and developmental DNA repair.

The metabolic conditions that prevail during bacterial growth have evolved with the faithful operation of repair systems that recognize and eliminate DNA lesions caused by intracellular and exogenous agents. This idea is supported by the low rate of spontaneous mutations (10) that occur in replicating cells, maintaining genome integrity. In contrast, when growth and/or replication cease, bacteria frequently process DNA lesions in an error-prone manner.

8-OxoG-Dependent Regulation of Global Protein Responses Leads to Mutagenesis and Stress Survival in .

The guanine oxidized (GO) system of , composed of the YtkD (MutT), MutM and MutY proteins, counteracts the cytotoxic and genotoxic effects of the oxidized nucleobase 8-OxoG. Here, we report that in growing cells, the genetic inactivation of GO system potentiated mutagenesis (HPM), and subsequent hyperresistance, contributes to the damaging effects of hydrogen peroxide (HO) (HPHR). The mechanism(s) that connect the accumulation of the mutagenic lesion 8-OxoG with the ability of to evolve and survive the noxious effects of oxidative stress were dissected.

Design of a Whole-Cell Biosensor Based on Bacillus subtilis Spores and the Green Fluorescent Protein To Monitor Arsenic.

A green fluorescent protein (GFP)-based whole-cell biosensor (WCB-GFP) for monitoring arsenic (As) was developed in Bacillus subtilis. To this end, we designed a reporter gene fusion carrying the gene under the control of the promoter/operator region of the arsenic operon () in the extrachromosomal plasmid pAD123. This construct was transformed into B.

Stress-Associated and Growth-Dependent Mutagenesis Are Divergently Regulated by c-di-AMP Levels in .

A previous proteomic study uncovered a relationship between nutritional stress and fluctuations in levels of diadenylate cyclases (DACs) and other proteins that regulate DAC activity, degrade, or interact with c-di-AMP, suggesting a possible role of this second messenger in stress-associated mutagenesis (SAM). Here, we investigated a possible role of c-di-AMP in SAM and growth-associated mutagenesis (GAM). Our results showed that in growing cells of YB955 (, and ), the DACs CdaA and DisA, which play crucial roles in cell wall homeostasis and chromosomal fidelity, respectively, counteracted spontaneous and Mitomycin-C-induced mutagenesis.

Protection of the DNA from Selected Species of Five Kingdoms in Nature by Ba(II), Sr(II), and Ca(II) Silica-Carbonates: Implications about Biogenicity and Evolving from Prebiotic Chemistry to Biological Chemistry.

The origin of life on Earth is associated with the Precambrian era, in which the existence of a large diversity of microbial fossils has been demonstrated. Notwithstanding, despite existing evidence of the emergence of life many unsolved questions remain. The first question could be as follows: Which was the inorganic structure that allowed isolation and conservation of the first biomolecules in the existing reduced conditions of the primigenial era? Minerals have been postulated as the ones in charge of protecting theses biomolecules against the external environment.

Dynamics of Mismatch and Alternative Excision-Dependent Repair in Replicating DNA Examined Under Conditions of Neutral Selection.

Spontaneous DNA deamination is a potential source of transition mutations. In , EndoV, a component of the alternative excision repair pathway (AER), counteracts the mutagenicity of base deamination-induced mispairs. Here, we report that the mismatch repair (MMR) system, MutSL, prevents the harmful effects of HNO, a deaminating agent of Cytosine (C), Adenine (A), and Guanine (G).

Non-B DNA-Forming Motifs Promote Mfd-Dependent Stationary-Phase Mutagenesis in .

Transcription-induced mutagenic mechanisms limit genetic changes to times when expression happens and to coding DNA. It has been hypothesized that intrinsic sequences that have the potential to form alternate DNA structures, such as non-B DNA structures, influence these mechanisms. Non-B DNA structures are promoted by transcription and induce genome instability in eukaryotic cells, but their impact in bacterial genomes is less known.

Mfd Affects Global Transcription and the Physiology of Stressed Cells.

For several decades, Mfd has been studied as the bacterial transcription-coupled repair factor. However, recent observations indicate that this factor influences cell functions beyond DNA repair. Our lab recently described a role for Mfd in disulfide stress that was independent of its function in nucleotide excision repair and base excision repair.

Transcriptional coupling and repair of 8-OxoG activate a RecA-dependent checkpoint that controls the onset of sporulation in Bacillus subtilis.

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.

Novel Biochemical Properties and Physiological Role of the Flavin Mononucleotide Oxidoreductase YhdA from Bacillus subtilis.

Cr(VI) is mutagenic and teratogenic and considered an environmental pollutant of increasing concern. The use of microbial enzymes that convert this ion into its less toxic reduced insoluble form, Cr(III), represents a valuable bioremediation strategy. In this study, we examined the YhdA enzyme, which belongs to the family of NADPH-dependent flavin mononucleotide oxide reductases and possesses azo-reductase activity as a factor that upon overexpression confers protection on from the cytotoxic effects promoted by Cr(VI) and counteracts the mutagenic effects of the reactive oxygen species (ROS)-promoted lesion 8-OxoG.

The 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 and ; however, the underlying mechanisms between these systems differ. For instance, RecA is not required for stationary-phase mutagenesis in like it is in .

Role of Mfd and GreA in Bacillus subtilis Base Excision Repair-Dependent Stationary-Phase Mutagenesis.

We report that the absence of an oxidized guanine (GO) system or the apurinic/apyrimidinic (AP) endonucleases Nfo, ExoA, and Nth promoted stress-associated mutagenesis (SAM) in YB955 (). Moreover, MutY-promoted SAM was Mfd dependent, suggesting that transcriptional transactions over nonbulky DNA lesions promoted error-prone repair. Here, we inquired whether Mfd and GreA, which control transcription-coupled repair and transcription fidelity, influence the mutagenic events occurring in nutritionally stressed YB955 cells deficient in the GO or AP endonuclease repair proteins.

YwqL (EndoV), ExoA and PolA act in a novel alternative excision pathway to repair deaminated DNA bases in Bacillus subtilis.

DNA deamination generates base transitions and apurinic/apyrimidinic (AP)-sites which are potentially genotoxic and cytotoxic. In Bacillus subtilis uracil can be removed from DNA by the uracil DNA-glycosylase through the base excision repair pathway. Genetic evidence suggests that B.

Does juvenile hormone prompt oxidative stress in male damselflies?

In invertebrates, it has recently been reported that secondary sexual characteristics (SSCs) reflect the antioxidant defense of their bearers, but it is not known what physiological link maintains the honesty of those signals. Here, we used the damselfly to test whether juvenile hormone plays such a role. First, we analyzed whether oxidative damage is a real threat in natural damselfly populations by examining the accumulation of oxidized guanines as a function of age in males.

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.

Non-canonical processing of DNA photodimers with Bacillus subtilis UV-endonuclease YwjD, 5'→3' exonuclease YpcP and low-fidelity DNA polymerases YqjH and YqjW.

It has been shown that mutation frequency decline (Mfd) and nucleotide excision repair (NER) deficiencies promote UV-induced mutagenesis in B. subtilis sporangia. As replication is halted in sporulating B.

Transcriptional coupling (Mfd) and DNA damage scanning (DisA) coordinate excision repair events for efficient Bacillus subtilis spore outgrowth.

The absence of base excision repair (BER) proteins involved in processing ROS-promoted genetic insults activates a DNA damage scanning (DisA)-dependent checkpoint event in outgrowing Bacillus subtilis spores. Here, we report that genetic disabling of transcription-coupled repair (TCR) or nucleotide excision repair (NER) pathways severely affected outgrowth of ΔdisA spores, and much more so than the effects of these mutations on log phase growth. This defect delayed the first division of spore's nucleoid suggesting that unrepaired lesions affected transcription and/or replication during outgrowth.

Stationary-phase Mutagenesis Soft-agar Overlay Assays in .

Elucidating how a population of non-growing bacteria generates mutations improves our understanding of phenomena like antibiotic resistance, bacterial pathogenesis, genetic diversity and evolution. To evaluate mutations that occur in nutritionally stressed non-growing bacteria, we have employed the strain YB955, which measures the reversions rates to the chromosomal auxotrophies and (Sung and Yasbin, 2002). This gain-of-function system has successfully allowed establishing the role played by repair systems and transcriptional factors in stress-associated mutagenesis (SPM) (Barajas- Ornelas , 2014 ; Gómez- Marroquín , 2016 ).

Implementation of a loss-of-function system to determine growth and stress-associated mutagenesis in Bacillus subtilis.

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.

LC-MS/MS proteomic analysis of starved Bacillus subtilis cells overexpressing ribonucleotide reductase (nrdEF): implications in stress-associated mutagenesis.

The non-appropriate conditions faced by nutritionally stressed bacteria propitiate error-prone repair events underlying stationary-phase- or stress-associated mutagenesis (SPM). The genetic and molecular mechanisms involved in SPM have been deeply studied but the biochemical aspects of this process have so far been less explored. Previous evidence showed that under conditions of nutritional stress, non-dividing cells of strain B.

Role of Ribonucleotide Reductase in Bacillus subtilis Stress-Associated Mutagenesis.

Unlabelled: The Gram-positive microorganism Bacillus subtilis relies on a single class Ib ribonucleotide reductase (RNR) to generate 2'-deoxyribonucleotides (dNDPs) for DNA replication and repair. In this work, we investigated the influence of RNR levels on B. subtilis stationary-phase-associated mutagenesis (SPM).

Aag Hypoxanthine-DNA Glycosylase Is Synthesized in the Forespore Compartment and Involved in Counteracting the Genotoxic and Mutagenic Effects of Hypoxanthine and Alkylated Bases in DNA during Bacillus subtilis Sporulation.

Unlabelled: Aag from Bacillus subtilis has been implicated in in vitro removal of hypoxanthine and alkylated bases from DNA. The regulation of expression of aag in B. subtilis and the resistance to genotoxic agents and mutagenic properties of an Aag-deficient strain were studied here.

Role of Base Excision Repair (BER) in Transcription-associated Mutagenesis of Nutritionally Stressed Nongrowing Bacillus subtilis Cell Subpopulations.

Compelling evidence points to transcriptional processes as important factors contributing to stationary-phase associated mutagenesis. However, it has not been documented whether or not base excision repair mechanisms play a role in modulating mutagenesis under conditions of transcriptional derepression. Here, we report on a flow cytometry-based methodology that employs a fluorescent reporter system to measure at single-cell level, the occurrence of transcription-associated mutations in nutritionally stressed B.