RESUMO

The intracellular [ATP]/[ADP] ratio is crucial for Escherichia coli's cellular functions, impacting transport, phosphorylation, signaling, and stress responses. Overexpression of F1-ATPase genes in E. coli increases glucose consumption, lowers energy levels, and triggers transcriptional responses in central carbon metabolism genes, particularly glycolytic ones, enhancing carbon flux. In this contribution, we report the impact of the perturbation of the energetic level in a PTS- mutant of E. coli by modifying the [ATP]/[ADP] ratio by uncoupling the cytoplasmic activity of the F1 subunit of the ATP synthase. The disruption of [ATP]/[ADP] ratio in the evolved strain of E. coli PB12 (PTS-) was achieved by the expression of the atpAGD operon encoding the soluble portion of ATP synthase F1-ATPase (strain PB12AGD+). The analysis of the physiological and metabolic response of the PTS- strain to the ATP disruption was determined using RT-qPCR of 96 genes involved in glucose and acetate transport, glycolysis and gluconeogenesis, pentose phosphate pathway (PPP), TCA cycle and glyoxylate shunt, several anaplerotic, respiratory chain, and fermentative pathways genes, sigma factors, and global regulators. The apt mutant exhibited reduced growth despite increased glucose transport due to decreased energy levels. It heightened stress response capabilities under glucose-induced energetic starvation, suggesting that the carbon flux from glycolysis is distributed toward the pentose phosphate and the Entner-Duodoroff pathway with the concomitant. Increase acetate transport, production, and utilization in response to the reduction in the [ATP]/[ADP] ratio. Upregulation of several genes encoding the TCA cycle and the glyoxylate shunt as several respiratory genes indicates increased respiratory capabilities, coupled possibly with increased availability of electron donor compounds from the TCA cycle, as this mutant increased respiratory capability by 240% more than in the PB12. The reduction in the intracellular concentration of cAMP in the atp mutant resulted in a reduced number of upregulated genes compared to PB12, suggesting that the mutant remains a robust genetic background despite the severe disruption in its energetic level.

RESUMO

Las enfermedades periodontales son un significativo problema mundial a nivel de salud humana. Décadas de investigaciones, evidencian que en la mayoría de los casos la periodontitis crónica es la más común, caracterizada por ser de evolución lenta, con formación de bolsas periodontales, posterior reabsorción del hueso alveolar, pérdida y destrucción de piezas dentarias y tejido óseo. Si bien se reconoce el origen multifactorial en el desarrollo de la periodontitis, es relevante la participación de la microbiota subgingival en la etiología de la enfermedad periodontal. Algunas de las especies bacterianas patógenas que han sido asociadas con el desarrollo de la enfermedad periodontal son Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum, entre otras. En este estudio, nos propusimos investigar cuáles de éstas cinco especies estaban presentes en las bolsas periodontales de 51 pacientes uruguayos con periodontitis crónica. Para alcanzar éste objetivo se utilizó una técnica convencional microbiológica y metagenómica (multiplex-PCR). Los resultados de la técnica convencional microbiológica evidenciaron la presencia de A. actinomycetemcomitans (33%) y de bacterias negras pigmentadas anaerobias (100%) en las muestras. De los resultados obtenidos en la multiplex-PCR, se demostró que las especies de mayor prevalencia fueron F. nucleatum (100%), T. forsythia (92%) y P. gingivalis (88%). Por el contrario, las especies de menor prevalencia fueron P. intermedia (39%) y A. actinomycetemcomitans (33%)...

Periodontal diseases are a major problem in human health. Decades of research have shown that the most common disease is chronic periodontitis, characterized by a slow evolution with the formation of periodontal pockets, subsequent alveolar bone resorption, loss and destruction of teeth and bone tissue. While we know the multifactorial origin of the development of periodontitis, the participation of subgingival microbiota is relevant in the etiology of periodontal disease. Some pathogenic bacteria species that have been associated with the development of periodontal disease are Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum, among others. In this work we studied which of these five species were present in the periodontal pockets of 51 Uruguayan patients with chronic periodontitis. To achieve the results a conventional microbiological technique and metagenomics (multiplex-PCR) were used. The results of the microbiological conventional technique showed the presence of A. actinomycetemcomitans (33%) and black pigmented anaerobic bacteria (100%) in the samples. From the results obtained in the multiplex-PCR we saw that the most prevalent species were F. nucleatum (100%), T. forsythia (92%) and P. gingivalis (88%). In contrast, lower prevalence species were P. intermedia (39%) and A. actinomycetecomitans...(33%)

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RESUMO

Checkpoints are components of signalling pathways involved in genome stability. We analysed the putative dual functions of Rad17 and Chk1 as checkpoints and in DNA repair using mutant strains of Saccharomyces cerevisiae. Logarithmic populations of the diploid checkpoint-deficient mutants, chk1Delta/chk1Delta and rad17Delta/rad17Delta, and an isogenic wild-type strain were exposed to the radiomimetic agent bleomycin (BLM). DNA double-strand breaks (DSBs) determined by pulsed-field electrophoresis, surviving fractions, and proliferation kinetics were measured immediately after treatments or after incubation in nutrient medium in the presence or absence of cycloheximide (CHX). The DSBs induced by BLM were reduced in the wild-type strain as a function of incubation time after treatment, with chromosomal repair inhibited by CHX. rad17Delta/rad17Delta cells exposed to low BLM concentrations showed no DSB repair, low survival, and CHX had no effect. Conversely, rad17Delta/rad17Delta cells exposed to high BLM concentrations showed DSB repair inhibited by CHX. chk1Delta/chk1Delta cells showed DSB repair, and CHX had no effect; these cells displayed the lowest survival following high BLM concentrations. Present results indicate that Rad17 is essential for inducible DSB repair after low BLM-concentrations (low levels of oxidative damage). The observations in the chk1Delta/chk1Delta mutant strain suggest that constitutive nonhomologous end-joining is involved in the repair of BLM-induced DSBs. The differential expression of DNA repair and survival in checkpoint mutants as compared to wild-type cells suggests the presence of a regulatory switch-network that controls and channels DSB repair to alternative pathways, depending on the magnitude of the DNA damage and genetic background.

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