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). Since RNR is essential in this bacterium, we engineered a conditional mutant of strain B. subtilis YB955 (hisC952 metB5 leu427) in which expression of the nrdEF operon was modulated by isopropyl-␤-D-thiogalactopyranoside (IPTG). Moreover, genetic inactivation of ytcG, predicted to encode a repressor (NrdR) of nrdEF in this strain, dramatically increased the expression levels of a transcriptional nrdE-lacZ fusion. The frequencies of mutations conferring amino acid prototrophy in three genes were measured in cultures under conditions that repressed or induced RNR-encoding genes. The results revealed that RNR was necessary for SPM and overexpression of nrdEF promoted growth-dependent mutagenesis and SPM. We also found that nrdEF expression was induced by H 2 O 2 and such induction was dependent on the master regulator PerR. These observations strongly suggest that the metabolic conditions operating in starved B. subtilis cells increase the levels of RNR, which have a direct impact on SPM.IMPORTANCE Results presented in this study support the concept that the adverse metabolic conditions prevailing in nutritionally stressed bacteria activate an oxidative stress response that disturbs ribonucleotide reductase (RNR) levels. Such an alteration of RNR levels promotes mutagenic events that allow Bacillus subtilis to escape from growth-limited conditions. KEYWORDS Bacillus subtilis, stress-associated mutagenesis, ribonucleotide reductase R ibonucleotide reductases (RNRs) are essential enzymes present in all life forms and are responsible for the conversion of ribonucleotides to 2=-deoxyribonucleotides, the precursors for the replication and repair of DNA. Three different RNR classes (classes I, II, and III) differing in the metal cofactor required for their catalytic activity, allosteric regulation, and quaternary structure have been described (1, 2). Class I RNRs are composed of two homodimeric proteins, ␣ 2 and ␤ 2 ; the larger subunit, ␣, contains the catalytic and the allosteric site that controls the specificity of reduction, while the smaller subunit, ␤, contains a stable tyrosyl radical and a dinuclear metal center (3, 4). In Bacillus subtilis, the nrdE and nrdF genes encode the ␣ and ␤ subunits of the class Ib RNR (3,4). Although the activity of the RNRs is controlled mainly by allosteric regulation in Escherichia coli and other bacteria, the intracellular levels of this enzyme can also be regulated by different transcriptional factors, including DnaA, Fur, and NrdR. Such transcription factors respond to changes in metabolic or environmental conditions and mediate the repression or induction of RNR-encoding genes (2, 3, 5-7). The transcrip-

Mucormycosis is a fungal infection caused by Mucorales, with a high mortality rate. However, only a few virulence factors have been described in these organisms. This study showed that deletion of rfs, which encodes the enzyme for the biosynthesis of rhizoferrin, a siderophore, in Mucor lusitanicus, led to a lower virulence in diabetic mice and nematodes. Upregulation of rfs correlated with the increased toxicity of the cell-free supernatants of the culture broth (SS) obtained under growing conditions that favor oxidative metabolism, such as low glucose levels or the presence of H2O2 in the culture, suggesting that oxidative metabolism enhances virulence through rhizoferrin production. Meanwhile, growing M. lusitanicus in the presence of potassium cyanide, N-acetylcysteine, a higher concentration of glucose, or exogenous cAMP, or the deletion of the gene encoding the regulatory subunit of PKA (pkaR1), correlated with a decrease in the toxicity of SS, downregulation of rfs, and reduction in rhizoferrin production. These observations indicate the involvement of the cAMP-PKA pathway in the regulation of rhizoferrin production and virulence in M. lusitanicus. Moreover, rfs upregulation was observed upon macrophage interaction or during infection with spores in mice, suggesting a pivotal role of rfs in M. lusitanicus infection.

Evidence suggests that histone deacetylases (HDACs) inhibitors could be used as an effective treatment for some psychiatric and neurological conditions such as depression, anxiety and age-related cognitive decline. However, non-specific HDAC inhibiting compounds have a clear disadvantage regarding their efficacy and safety, thus the need to develop more selective ones. The present study evaluated the toxicity, the capacity to inhibit HDAC activity and antidepressant-like activity of three recently described class I HDAC inhibitors IN01, IN04 and IN14, using A. salina toxicity test, in vitro fluorometric HDAC activity assay and forced-swimming test, respectively. Our data show that IN14 possesses a better profile than the other two. Therefore, the pro-cognitive and antidepressant effects of IN14 were evaluated. In the forced-swimming test model of depression, intraperitoneal administration of IN14 (100 mg/Kg/day) for five days decreased immobility, a putative marker of behavioral despair, significantly more than tricyclic antidepressant desipramine, while also increasing climbing behavior, a putative marker of motivational behavior. On the other hand, IN14 left the retention latency in the elevated T-maze unaltered. These results suggest that novel HDAC class I inhibitor IN14 may represent a promising new antidepressant with low toxicity and encourages further studies on this compound.

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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. subtilis YB955 overexpressing ribonucleotide reductase (RNR) exhibited a strong propensity to generate true reversions in the hisC952 (amber), metB5 (ochre) and leuC425 (missense) mutant alleles. To further advance our knowledge on the metabolic conditions underlying this hypermutagenic phenotype, a high-throughput LC-MS/MS proteomic analysis was performed in non-dividing cells of an amino acid-starved strain, deficient for NrdR, the RNR repressor. Compared with the parental strain, the level of 57 proteins was found to increase and of 80 decreases in the NrdR-deficient strain. The proteomic analysis revealed an altered content in proteins associated with the stringent response, nucleotide metabolism, DNA repair, and cell signaling in amino acid-starved cells of the ∆nrdR strain. Overall, our results revealed that amino acid-starved cells of strain B. subtilis ∆nrdR that escape from growth-limiting conditions exhibit a complex proteomic pattern reminiscent of a disturbed metabolism. Future experiments aimed to understand the consequences of disrupting the cell signaling pathways unveiled in this study, will advance our knowledge on the genetic adaptations deployed by bacteria to escape from growth-limiting environments.

<div><p class="Abstract">The analysis of the global DNA methylation, calculated as the percentage of 5-methylcytosine (5mC) over the total sum of cytosines, is a well stablished biomarker for monitoring large scale epigenetic events in organisms. DNA purification, hydrolysis, separation and detection methods are critical steps to determine this biomarker. In the present work is proposed a robust procedure for DNA acid-hydrolysis assisted by microwave that provides identical DNA methylation patterns that enzymatic hydrolysis and better release of 5mC than acid classic method. The quantification was performed using a gas chromatographer coupled to a mass spectrometer with triple quadrupole as mass analyzer (GC-TQ-MS/MS) using multiple reaction monitoring (MRM) mode for the trimethylsilyl-derivates of nucleobases; following the transitions of 254→238, 240→170 and 254→238, 254→184 (m/z) for C and 5mC respectively, was achieved a limit of detection of 0.46 fmol for C and 0.41 fmol for 5mC. The proposed procedure is capable of determine 0.004% of 5mC in 50 ng of DNA in a chromatographic time of 10 minutes, being a good alternative to LC-MS/MS analysis.</p></div>

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