Genetic and Biochemical Analysis of the Azotobacter vinelandii Molybdenum Storage Protein.

The N2 fixing bacterium Azotobacter vinelandii carries a molybdenum storage protein, referred to as MoSto, able to bind 25-fold more Mo than needed for maximum activity of its Mo nitrogenase. Here we have investigated a plausible role of MoSto as obligate intermediate in the pathway that provides Mo for the biosynthesis of nitrogenase iron-molybdenum cofactor (FeMo-co). The in vitro FeMo-co synthesis and insertion assay demonstrated that purified MoSto functions as Mo donor and that direct interaction with FeMo-co biosynthetic proteins stimulated Mo donation. The phenotype of an A. vinelandii strain lacking the MoSto subunit genes (ΔmosAB) was analyzed. Consistent with its role as storage protein, the ΔmosAB strain showed severe impairment to accumulate intracellular Mo and lower resilience than wild type to Mo starvation as demonstrated by decreased in vivo nitrogenase activity and competitive growth index. In addition, it was more sensitive than the wild type to diazotrophic growth inhibition by W. The ΔmosAB strain was found to readily derepress vnfDGK upon Mo step down, in contrast to the wild type that derepressed Vnf proteins only after prolonged Mo starvation. The ΔmosAB mutation was then introduced in a strain lacking V and Fe-only nitrogenase structural genes (Δvnf Δanf) to investigate possible compensations from these alternative systems. When grown in Mo-depleted medium, the ΔmosAB and mosAB + strains showed low but similar nitrogenase activities regardless of the presence of Vnf proteins. This study highlights the selective advantage that MoSto confers to A. vinelandii in situations of metal limitation as those found in many soil ecosystems. Such a favorable trait should be included in the gene complement of future nitrogen fixing plants.

Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria.

BACKGROUND: There is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of these advantages resides in the fact that metalation occurs after protein import into mitochondria, which contains pools of iron, zinc, copper and manganese ions that can be utilized in recombinant metalloprotein metalation reactions. Another advantage is that mitochondria are suitable organelles to host oxygen sensitive proteins as a low oxygen environment is created within the matrix during cellular respiration. RESULTS: Here we describe the adaptation of a modular cloning system, GoldenBraid2.0, for the integration of assembled transcriptional units into two different sites of the yeast genome, yielding a high expression level. We have also generated a toolkit comprising various promoters, terminators and selection markers that facilitate the generation of multigenic constructs and allow the reconstruction of biosynthetic pathways within Saccharomyces cerevisiae. To facilitate the specific expression of recombinant proteins within the mitochondrial matrix, we have also included in the toolkit an array of mitochondrial targeting signals and tested their efficiency at different growth conditions. As a proof of concept, we show here the integration and expression of 14 bacterial nitrogen fixation (nif) genes, some of which are known to require specific metallocluster cofactors that contribute to their stability yet make these proteins highly sensitive to oxygen. For one of these genes, nifU, we show that optimal production of this protein is achieved through the use of the Su9 mitochondrial targeting pre-sequence and glycerol as a carbon source to sustain aerobic respiration. CONCLUSIONS: We present here an adapted GoldenBraid2.0 system for modular cloning, genome integration and expression of recombinant proteins in yeast. We have produced a toolkit that includes inducible and constitutive promoters, mitochondrial targeting signals, terminators and selection markers to guarantee versatility in the design of recombinant transcriptional units. By testing the efficiency of the system with nitrogenase Nif proteins and different mitochondrial targeting pre-sequences and growth conditions, we have paved the way for future studies addressing the expression of heterologous proteins in yeast mitochondria.

Determinación del sexo fetal en ecografía del primer trimestre / Fetal gender determination by first-trimester ultrasound

Objetivo: Valorar la precisión de la determinación ecográfica del sexo fetal entre las 11 y 14 semanas de gestación como parte de la ecografía rutinaria del primer trimestre. Material y métodos: El sexo fetal fue evaluado por ecografía de forma prospectiva en 534 embarazadas entre las semanas 11 y 14 de gestación que acudieron a su ecografía de rutina. La región genital fue examinada en un plano mediosagital con el fin de determinar la dirección del tubérculo genital respecto a una línea horizontal definida por la piel del área lumbosacra, considerando sexo masculino si el ángulo es superior a 30° y femenino si el tubérculo es paralelo o convergente (ángulo inferior a 30°). La confirmación clínica del sexo fetal se hizo por ecografía en semana 20, 32 y al nacimiento. Resultados: El sexo fetal fue asignado correctamente en el 85,13% de los casos. Se asignó de forma correcta en el 78,32% de los casos en la semana de gestación 11, en el 84,49% de los casos en semana 12 y en el 93,7% de los casos en la semana 13. El 82,5% de los varones fueron asignados correctamente y el 87,84% de las mujeres. Conclusión: El sexo fetal se puede determinar con una eficacia aceptable entre las 12-14 semanas de gestación en la ecografía de rutina (AU)

Objective: To assess the accuracy of fetal gender determination by ultrasound at 11-14 weeks’ gestation as part of first-trimester ultrasound screening. Material and methods: Fetal gender assessment by ultrasound was prospectively carried out in 534 women at 11-14 weeks of gestation attending first-trimester ultrasound screening. The genital region was examined in a midsagittal plane to determinate the direction of the genital tubercle to a horizontal line through the lumbosacral skin surface. Fetal gender was assigned as male if the angle was greater than 30 degrees and as female if the tubercle was parallel or convergent (less than 30 degrees). Clinical confirmation of fetal gender was obtained by 20- and 32-week ultrasound and after delivery. Results: Fetal gender was correctly determined in 85.13% of cases. The accuracy of sex assignment was 78.32% at 11 weeks, 84.49% at 12 weeks, and 93.7% at 13 weeks. Gender was correctly determined in 82.8% of the male fetuses and in 88% of the female fetuses. Conclusion: Fetal gender can be determined with an acceptable efficiency at 12-14 weeks of gestation on routine ultrasound (AU)

Role of Azotobacter vinelandii FdxN in FeMo-co biosynthesis.

Biosynthesis of metal clusters for the nitrogenase component proteins NifH and NifDK involves electron donation events. Yet, electron donors specific to the biosynthetic pathways of the [4Fe-4S] cluster of NifH, or the P-cluster and the FeMo-co of NifDK, have not been identified. Here we show that an Azotobacter vinelandii mutant lacking fdxN was specifically impaired in FeMo-co biosynthesis. The ΔfdxN mutant produced 5-fold less NifB-co, an early FeMo-co biosynthetic intermediate, than wild type. As a consequence, it accumulated FeMo-co-deficient apo-NifDK and was impaired in NifDK activity. We conclude that FdxN plays a role in FeMo-co biosynthesis, presumably by donating electrons to support NifB-co synthesis by NifB. This is the first role in nitrogenase biosynthesis unequivocally assigned to any A. vinelandii ferredoxin.

Kinetics of Nif gene expression in a nitrogen-fixing bacterium.

Nitrogen fixation is a tightly regulated trait. Switching from N2 fixation-repressing conditions to the N2-fixing state is carefully controlled in diazotrophic bacteria mainly because of the high energy demand that it imposes. By using quantitative real-time PCR and quantitative immunoblotting, we show here how nitrogen fixation (nif) gene expression develops in Azotobacter vinelandii upon derepression. Transient expression of the transcriptional activator-encoding gene, nifA, was followed by subsequent, longer-duration waves of expression of the nitrogenase biosynthetic and structural genes. Importantly, expression timing, expression levels, and NifA dependence varied greatly among the nif operons. Moreover, the exact concentrations of Nif proteins and their changes over time were determined for the first time. Nif protein concentrations were exquisitely balanced, with FeMo cofactor biosynthetic proteins accumulating at levels 50- to 100-fold lower than those of the structural proteins. Mutants lacking nitrogenase structural genes or impaired in FeMo cofactor biosynthesis showed overenhanced responses to derepression that were proportional to the degree of nitrogenase activity impairment, consistent with the existence of at least two negative-feedback regulatory mechanisms. The first such mechanism responded to the levels of fixed nitrogen, whereas the second mechanism appeared to respond to the levels of the mature NifDK component. Altogether, these findings provide a framework to engineer N2 fixation in nondiazotrophs.