DbdR, a New Member of the LysR Family of Transcriptional Regulators, Coordinately Controls Four Promoters in the Thauera aromatica AR-1 3,5-Dihydroxybenzoate Anaerobic Degradation Pathway.

The facultative anaerobe Thauera aromatica strain AR-1 uses 3,5-dihydroxybenzoate (3,5-DHB) as a sole carbon and energy source under anoxic conditions using an unusual oxidative strategy to overcome aromatic ring stability. A 25-kb gene cluster organized in four main operons encodes the anaerobic degradation pathway for this aromatic. The dbdR gene coding for a LysR-type transcriptional regulator (LTTR), which is present at the foremost end of the cluster, is required for anaerobic growth on 3,5-DHB and for the expression of the main pathway operons. A model structure of DbdR showed conserved key residues for effector binding with its closest relative TsaR for p-toluenesulfonate degradation. We found that DbdR controlled expression of three promoters upstream from the operons coding for the three main steps of the pathway. While one of them (P orf20 ) was only active in the presence of 3,5-DHB, the other two (P dbhL and P orf18 ) showed moderate basal levels that were further induced in the presence of the pathway substrate, which needed be converted to hydroxyhydroquinone to activate transcription. Both basal and induced activities were strictly dependent on DbdR, which was also required for transcription from its own promoter. DbdR basal expression was moderately high and, unlike most LTTR, increased 2-fold in response to the presence of the effector. DbdR was found to be a tetramer in solution, producing a single retardation complex in binding assays with the three enzymatic promoters, consistent with its tetrameric structure. The three promoters had a conserved organization with a clear putative primary (regulatory) binding site and a putative secondary (activating) binding site positioned at the expected distances from the transcription start site. In contrast, two protein-DNA complexes were observed for the P dbdR promoter, which also showed significant sequence divergence from those of the three other promoters. Taken together, our results show that a single LTTR coordinately controls expression of the entire 3,5-DHB anaerobic degradation pathway in Thauera aromatica AR-1, allowing a fast and optimized response to the presence of the aromatic.IMPORTANCEThauera aromatica AR-1 is a facultative anaerobe that is able to use 3,5-dihydroxybenzoat (3,5-DHB) as the sole carbon and energy source in a process that is dependent on nitrate respiration. We have shown that a single LysR-type regulator with unusual properties, DbdR, controls the expression of the pathway in response to the presence of the substrate; unlike other regulators of the family, DbdR does not repress but activates its own synthesis and is able to bind and activate three promoters directing the synthesis of the pathway enzymes. The promoter architecture is conserved among the three promoters but deviates from that of typical LTTR-dependent promoters. The substrate must be metabolized to an intermediate compound to activate transcription, which requires basal enzyme levels to always be present. The regulatory network present in this strain is designed to allow basal expression of the enzymatic machinery, which would rapidly metabolize the substrate when exposed to it, thus rendering the effector molecule. Once activated, the regulator induces the synthesis of the entire pathway through a positive feedback, increasing expression from all the target promoters to allow maximum growth.

[Vitamin D deficiency associated with insulin resistance in medical residents]. / Hipovitaminosis D asociada a resistencia a la insulina en residentes médicos.

BACKGROUND: Several studies have reported a correlation between vitamin D deficiency and insulin resistance; however, other clinical trials show that vitamin D supplementation do not normalize glucose and insulin levels. We designed a study to show if there is a correlation between serum vitamin D and the homeostatic model assessment 2 (HOMA 2). METHODS: It was designed a cross-sectional, descriptive, and analytical study, which included medical residents. They answered a questionnaire to record the time of sun exposure. We took anthropometric measurements, such as weight, height, and waist circumference, as well as some serum levels: serum vitamin D, serum insulin, fasting blood glucose, triglycerides and high-density lipoprotein-cholesterol. The correlation between serum vitamin D and HOMA 2 was determined by the correlation of Pearson; it was considered significant a p < 0.05. RESULTS: The decreased serum vitamin D levels did not correlate with high concentrations of HOMA 2 (r = -0.11, p = 0.34). A negative correlation between vitamin D levels and index size waist was observed (r = -0.27, p = 0.025). HOMA 2 was positively correlated with waist size index (r = 0.23, p = 0.05) and triglycerides (r = 0.61, p = 0.01) and negatively with high density lipoprotein-cholesterol (r = -0.26, p = 0.02). CONCLUSIONS: We couldn't show the correlation between vitamin D deficiency and insulin resistance.

Introducción: diversos estudios han reportado una correlación entre la deficiencia de vitamina D y la resistencia a la insulina; sin embargo, algunos ensayos clínicos demuestran que la suplementación con vitamina D no normaliza las cifras de glucosa ni las de insulina. Por lo tanto, el objetivo es buscar si existe correlación entre las concentraciones séricas de vitamina D y la resistencia a la insulina a partir de la utilización del índice homeostatic model assessment 2 (HOMA 2). Método: estudio transversal, descriptivo y analítico que incluyó a residentes a los que se les aplicó un cuestionario para conocer su tiempo de exposición al sol. Se tomaron medidas antropométricas como peso, talla y circunferencia de cintura, niveles séricos de vitamina D, insulina sérica, glucosa de ayuno, triglicéridos y colesterol de alta densidad. Se determinó la correlación entre las concentraciones séricas de vitamina D y HOMA 2 mediante el coeficiente de correlación de Pearson; se consideró significativa una p < 0.05. Resultados: la disminución sérica de vitamina D no se correlacionó con concentraciones elevadas del HOMA 2 (r = −0.11, p = 0.34). Se observó una correlación negativa entre las concentraciones de vitamina D y el índice cintura-talla (r = −0.27, p = 0.025). El HOMA 2 se correlacionó positivamente con el índice cintura-talla (r = 0.23, p = 0.05) y los triglicéridos (r = 0.61, p = 0.01) y de forma negativa con el colesterol de alta densidad (r = −0.26, p = 0.02). Conclusión: no observamos la correlación esperada entre hipovitaminosis D y resistencia a la insulina.

Biofilm formation-defective mutants in Pseudomonas putida.

Out of 8000 candidates from a genetic screening for Pseudomonas putida KT2442 mutants showing defects in biofilm formation, 40 independent mutants with diminished levels of biofilm were analyzed. Most of these mutants carried insertions in genes of the lap cluster, whose products are responsible for synthesis, export and degradation of the adhesin LapA. All mutants in this class were strongly defective in biofilm formation. Mutants in the flagellar regulatory genes fleQ and flhF showed similar defects to that of the lap mutants. On the contrary, transposon insertions in the flagellar structural genes fliP and flgG, that also impair flagellar motility, had a modest defect in biofilm formation. A mutation in gacS, encoding the sensor element of the GacS/GacA two-component system, also had a moderate effect on biofilm formation. Additional insertions targeted genes involved in cell envelope function: PP3222, encoding the permease element of an ABC-type transporter and tolB, encoding the periplasmic component of the Tol-OprL system required for outer membrane stability. Our results underscore the central role of LapA, suggest cross-regulation between motility and adhesion functions and provide insights on the role of cell envelope trafficking and maintenance for biofilm development in P. putida.