The ytkD (mutTA) Gene of Bacillus subtilis Encodes a Functional Antimutator 8-Oxo-(dGTP/GTP)ase and Is under Dual Control of Sigma A and Sigma F RNA Polymerases

The regulation of expression of ytkD, a gene that encodes the first functional antimutator 8-oxo-dGTPase activity of B. subtilis, was studied here. A ytkD-lacZ fusion integrated into the ytkD locus of wild-type B. subtilis 168 revealed that this gene is expressed during both vegetative growth and early stages of sporulation. In agreement with this result, ytkD mRNAs were detected by both Northern blotting and reverse transcription-PCR during both developmental stages. These results suggested that ytkD is transcribed by the sequential action of RNA polymerases containing the sigma factors σ(A) and σ(F), respectively. In agreement with this suggestion, the spore-associated expression was almost completely abolished in a sigF genetic background but not in a B. subtilis strain lacking a functional sigG gene. Primer extension analysis mapped transcriptional start sites on mRNA samples isolated from vegetative and early sporulating cells of B. subtilis. Inspection of the sequences lying upstream of the transcription start sites revealed the existence of typical σ(A)- and σ(F)-type promoters. These results support the conclusion that ytkD expression is subjected to dual regulation and suggest that the antimutator activity of YtkD is required not only during vegetative growth but also during the early sporulation stages and/or germination of B. subtilis. While ytkD expression obeyed a dual pattern of temporal expression, specific stress induction of the transcription of this gene does not appear to occur, since neither oxidative damage (following either treatment with paraquat or hydrogen peroxide) nor mitomycin C treatment or σ(B) general stress inducers (sodium chloride, ethanol, or heat) affected the levels of the gene product produced

Obtención de Variantes Hiperactivas e Inactivas de la Endocelulasa Cel9 de Myxobacter Sp. Al-1 Obtención de Variantes Hiperactivas e Inactivas de la Endocelulasa Cel9 de Myxobacter Sp. Al-1

Debido a su aplicación industrial, existe un gran interés en la producción de celulasas con propiedades bioquímicas novedosas. Por ello, en el presente trabajo se utilizó una estrategia basada en un método de mutagénesis aleatoria in vivo para la obtención de variantes de la endocelulasa Cel9 del microorganismo gram-negativo Myxobacter Sp. AL-1. Siguiendo este enfoque, se obtuvieron cepas transformantes de Escherichia coli capaces de secretar variantes de la proteína Cel9 cuyas actividades específicas fueron incrementadas hasta 7.5 veces con respecto a la actividad mostrada por la enzima nativa. Del mismo modo, se generaron cepas de E. coli productoras de variantes de la proteína Cel9 con baja o nula actividad enzimática. Experimentos de subclonación y fraccionamiento celular revelaron que las mutaciones asociadas con los fenotipos de las variantes de la enzima Cel9 ocurrieron en la secuencia del gen cel9. Así mismo, se demostró que los fenotipos de las cepas mutantes carentes de actividad enzimática no dependen de su incapacidad para secretar las proteínas mutantes. Además de su potencial aplicación biotecnológica, los resultados obtenidos en este trabajo permiten avanzar en el entendimiento de la relación estructura-función de la celulasa Cel9 de Myxobacter Sp. AL-1.Due to its biotechnological impact, there is currently a growing interest in the production of cellulases with novel biochemical properties. Here, multiple generations of random mutagenesis in vivo and screening were employed to generate variants of the modular cellulase Cel9 from Myxobacter Sp. AL-1. Following this approach, Cel9 variants which showed increases upto 7.5 fold of cellulase activity were obtained. In addition, Cel9 mutants which completely lost the ability to degrade cellulose were also obtained. Results revealed that mutations associated with the phenotype of the Cel9 variants occurred on the mutant gene sequence and that themutants with null or reduced activity did not accumulate in the cell. In addition to the potential biotechnological application of the enzymes with improved activity obtained here, the results of this work will contribute to the understanding of the structure and function of cellulases grouped in the family 9 of glycosyl hydrolases

YtkD and MutT Protect Vegetative Cells but Not Spores of Bacillus subtilis from Oxidative Stress

ytkD and mutT of Bacillus subtilis encode potential 8-oxo-dGTPases that can prevent the mutagenic effects of 8-oxo-dGTP. Loss of YtkD but not of MutT increased the spontaneous mutation frequency of growing cells. However, cells lacking both YtkD and MutT had a higher spontaneous mutation frequency than cells lacking YtkD. Loss of either YtkD or MutT sensitized growing cells to hydrogen peroxide (H(2)O(2)) and t-butylhydroperoxide (t-BHP), and the lack of both proteins sensitized growing cells to these agents even more. In contrast, B. subtilis spores lacking YtkD and MutT were not sensitized to H(2)O(2), t-BHP, or heat. These results suggest (i) that YtkD and MutT play an antimutator role and protect growing cells of B. subtilis against oxidizing agents, and (ii) that neither YtkD nor MutT protects spores against potential DNA damage induced by oxidative stress or heat

Association between Blood Lead Levels and Delta-Aminolevulinic Acid Dehydratase in Pregnant Women

Blood lead levels (BLLs) and delta-aminolevulinic acid dehydratase (ALAD) activity are considered biomarkers of lead exposure and lead toxicity, respectively. The present study was designed to investigate the association between BLLs and ALAD activity in pregnant women from Durango, Mexico. A total of 633 pregnant women aged 13–43 years participated in this study. Blood lead was measured by a graphite furnace atomic absorption spectrometer. ALAD activity was measured spectrophotometrically. Mean blood lead was 2.09 ± 2.34 µg/dL; and 26 women (4.1%) crossed the Centers for Disease Control (CDC) recommended level of 5 µg/dL. ALAD activity was significantly lower in women with levels of lead ≥5 µg/dL compared to those with BLLs < 5 µg/dL (p = 0.002). To reduce the influence of extreme values on the statistical analysis, BLLs were analyzed by quartiles. A significant negative correlation between blood lead and ALAD activity was observed in the fourth quartile of BLLs (r = −0.113; p < 0.01). Among women with blood lead concentrations ≥2.2 µg/dL ALAD activity was negatively correlated with BLLs (r = −0.413; p < 0.01). Multiple linear regression demonstrated that inhibition of ALAD in pregnant women may occur at levels of lead in blood above 2.2 µg/dL

The relationship between blood lead levels and occupational exposure in a pregnant population

Abstract Background Pregnant women exposed to lead are at risk of suffering reproductive damages, such as miscarriage, preeclampsia, premature delivery and low birth weight. Despite that the workplace offers the greatest potential for lead exposure, there is relatively little information about occupational exposure to lead during pregnancy. This study aims to assess the association between blood lead levels and occupational exposure in pregnant women from Durango, Mexico. Methods A cross-sectional study was carried out in a population of 299 pregnant women. Blood lead was measured in 31 women who worked in jobs where lead is used (exposed group) and 268 who did not work in those places (control group). Chi-square test was applied to compare exposed and control groups with regard to blood lead levels. Odds ratio (OR) and 95% confidence intervals (CI) were calculated. Multivariable regression analysis was applied to determine significant predictors of blood lead concentrations in the exposed group. Results Exposed women had higher blood lead levels than those in the control group (4.00 ± 4.08 μg/dL vs 2.65 ± 1.75 μg/dL, p = 0.002). Furthermore, women in the exposed group had 3.82 times higher probability of having blood lead levels ≥ 5 μg/dL than those in the control group. Wearing of special workwear, changing clothes after work, living near a painting store, printing office, junkyard or rubbish dump, and washing the workwear together with other clothes resulted as significant predictors of elevated blood lead levels in the exposed group. Conclusions Pregnant working women may be at risk of lead poisoning because of occupational and environmental exposure. The risk increases if they do not improve the use of protective equipment and their personal hygiene