A core of three amino acids at the carboxyl-terminal region of glutamine synthetase defines its regulation in cyanobacteria

© 2015 John Wiley & Sons Ltd. Glutamine synthetase (GS) type I is a key enzyme in nitrogen metabolism, and its activity is finely controlled by cellular carbon/nitrogen balance. In cyanobacteria, a reversible process that involves protein-protein interaction with two proteins, the inactivating factors IF7 and IF17, regulates GS. Previously, we showed that three arginine residues of IFs are critical for binding and inhibition of GS. In this work, taking advantage of the specificity of GS/IFs interaction in the model cyanobacteria Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, we have constructed a different chimeric GSs from these two cyanobacteria. Analysis of these proteins, together with a site-directed mutagenesis approach, indicates that a core of three residues (E419, N456 and R459) is essential for the inactivation process. The three residues belong to the last 56 amino acids of the C-terminus of SynechocystisGS. A protein-protein docking modeling of SynechocystisGS in complex with IF7 supports the role of the identified core for GS/IF interaction.Peer Reviewe

Exploring the Diversity of the Thioredoxin Systems in Cyanobacteria

Cyanobacteria evolved the ability to perform oxygenic photosynthesis using light energy to reduce CO2 from electrons extracted from water and form nutrients. These organisms also devel-oped light-dependent redox regulation through the Trx system, formed by thioredoxins (Trxs) and thioredoxin reductases (TRs). Trxs are thiol-disulfide oxidoreductases that serve as reducing sub-strates for target enzymes involved in numerous processes such as photosynthetic CO2 fixation and stress responses. We focus on the evolutionary diversity of Trx systems in cyanobacteria and discuss their phylogenetic relationships. The study shows that most cyanobacteria contain at least one copy of each identified Trx, and TrxA is the only one present in all genomes analyzed. Ferredoxin thiore-doxin reductase (FTR) is present in all groups except Gloeobacter and Prochlorococcus, where there is a ferredoxin flavin-thioredoxin reductase (FFTR). Our data suggest that both TRs may have coex-isted in ancestral cyanobacteria together with other evolutionarily related proteins such as NTRC or DDOR, probably used against oxidative stress. Phylogenetic studies indicate that they have different evolutionary histories. As cyanobacteria diversified to occupy new habitats, some of these proteins were gradually lost in some groups. Finally, we also review the physiological relevance of redox regulation in cyanobacteria through the study of target enzymes.Junta de Andalucía BIO-28

The environmental fate of organic pollutants through the global microbial metabolism

The production of new chemicals for industrial or therapeutic applications exceeds our ability to generate experimental data on their biological fate once they are released into the environment. Typically, mixtures of organic pollutants are freed into a variety of sites inhabited by diverse microorganisms, which structure complex multispecies metabolic networks. A machine learning approach has been instrumental to expose a correlation between the frequency of 149 atomic triads (chemotopes) common in organo-chemical compounds and the global capacity of microorganisms to metabolise them. Depending on the type of environmental fate defined, the system can correctly predict the biodegradative outcome for 73–87% of compounds. This system is available to the community as a web server (http://www.pdg.cnb.uam.es/BDPSERVER). The application of this predictive tool to chemical species released into the environment provides an early instrument for tentatively classifying the compounds as biodegradable or recalcitrant. Automated surveys of lists of industrial chemicals currently employed in large quantities revealed that herbicides are the group of functional molecules more difficult to recycle into the biosphere through the inclusive microbial metabolism

Lethality caused by ADP-glucose accumulation is suppressed by salt-induced carbon flux redirection in cyanobacteria

Cyanobacteria are widely distributed photosynthetic organisms. During the day they store carbon, mainly as glycogen, to provide the energy and carbon source they require for maintenance during the night. Here, we generate a mutant strain of the freshwater cyanobacterium Synechocystis sp. PCC 6803 lacking both glycogen synthases. This mutant has a lethal phenotype due to massive accumulation of ADP-glucose, the substrate of glycogen synthases. This accumulation leads to alterations in its photosynthetic capacity and a dramatic decrease in the adenylate energy charge of the cell to values as low as 0.1. Lack of ADP-glucose pyrophosphorylase, the enzyme responsible for ADP-glucose synthesis, or reintroduction of any of the glycogen synthases abolishes the lethal phenotype. Viability of the glycogen synthase mutant is also fully recovered in NaCl-supplemented medium, which redirects the surplus of ADP-glucose to synthesize the osmolite glucosylglycerol. This alternative metabolic sink also suppresses phenotypes associated with the defective response to nitrogen deprivation characteristic of glycogen-less mutants, restoring the capacity to degrade phycobiliproteins. Thus, our system is an excellent example of how inadequate management of the adenine nucleotide pools results in a lethal phenotype, and the influence of metabolic carbon flux in cell viability and fitness.España, Ministerio de Economía y Competitividad (MINECO) (grant BFU2013-41712-P and BIO2016-75634-P)España, Junta de Andalucía (P12-BIO-1119

Dynamic Flux Balance Analysis of a Genetic Engineered Cyanobacterium for Ethanol Production: Parameter Estimation

In this work, we present parameter estimation problem for Dynamic Flux Balance Analysis to study the production of ethanol by a mutant strain of the cyanobacterium Synechocystis sp. PCC 6803, as well as experimental data. This modified strain harbors the genes pdc and adhII from Zymomonas mobilis. The model includes two major components: (1) a dynamic model with mass balances for biomass, ethanol, nitrate, phosphate, internal nitrogen and phosphorus, and (2) a steady state genome-scale metabolic LP model. The biomass equation includes limiting functions for temperature and kinetics of growth inhibition by ethanol toxicity. Limitation of light by biomass accumulation is also taken into account. We formulate a dynamic parameter estimation problem with a weighted least-squares objective function, subject to dynamic mass balance equations at the bioreactor level and the intracellular LP model. The problem is solved in GAMS through a simultaneous optimization approach. Data set for parameter estimation were obtained in experiments performed over 73 h in batch liquid cultures. Numerical results provide useful insights on ethanol production by the genetic modified strain within the context of genomic-scale cyanobacterial metabolism.Fil: Laiglecia, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Planta Piloto de Ingeniería Química (i); Argentina. Universidad Nacional del Sur; ArgentinaFil: Estrada, Vanina Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Planta Piloto de Ingeniería Química (i); Argentina. Universidad Nacional del Sur; ArgentinaFil: Vidal Vidal, Rebeca. Universidad de Sevilla. Consejo Superior de Investigaciones Cientificas; EspañaFil: Florencio, Francisco J.. Universidad de Sevilla. Consejo Superior de Investigaciones Cientificas; EspañaFil: Guerrero, Miguel G.. Universidad de Sevilla. Consejo Superior de Investigaciones Cientificas; EspañaFil: Diaz, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Planta Piloto de Ingeniería Química (i); Argentina. Universidad Nacional del Sur; Argentin

Characterization of function of the GlgA2 glycogen/starch synthase in Cyanobacterium sp. Clg1 highlights convergent evolution of glycogen metabolism into starch granule aggregation

At variance with the starch-accumulating plants and most of the glycogen-accumulating cyanobacteria, Cyanobacterium sp. CLg1 synthesizes both glycogen and starch. We now report the selection of a starchless mutant of this cyanobacterium that retains wild-type amounts of glycogen. Unlike other mutants of this type found in plants and cyanobacteria, this mutant proved to be selectively defective for one of the two types of glycogen/starch synthase: GlgA2. This enzyme is phylogenetically related to the previously reported SSIII/SSIV starch synthase that is thought to be involved in starch granule seeding in plants. This suggests that, in addition to the selective polysaccharide debranching demonstrated to be responsible for starch rather than glycogen synthesis, the nature and properties of the elongation enzyme define a novel determinant of starch versus glycogen accumulation. We show that the phylogenies of GlgA2 and of 16S ribosomal RNA display significant congruence. This suggests that this enzyme evolved together with cyanobacteria when they diversified over 2 billion years ago. However, cyanobacteria can be ruled out as direct progenitors of the SSIII/SSIV ancestral gene found in Archaeplastida. Hence, both cyanobacteria and plants recruited similar enzymes independently to perform analogous tasks, further emphasizing the importance of convergent evolution in the appearance of starch from a preexisting glycogen metabolism network.Peer Reviewe

Brahma Is Required for Proper Expression of the Floral Repressor FLC in Arabidopsis

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: BRAHMA (BRM) is a member of a family of ATPases of the SWI/SNF chromatin remodeling complexes from Arabidopsis. BRM has been previously shown to be crucial for vegetative and reproductive development. [Methodology/Principal Findings]: Here we carry out a detailed analysis of the flowering phenotype of brm mutant plants which reveals that, in addition to repressing the flowering promoting genes CONSTANS (CO), FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), BRM also represses expression of the general flowering repressor FLOWERING LOCUS C (FLC). Thus, in brm mutant plants FLC expression is elevated, and FLC chromatin exhibits increased levels of histone H3 lysine 4 tri-methylation and decreased levels of H3 lysine 27 tri-methylation, indicating that BRM imposes a repressive chromatin configuration at the FLC locus. However, brm mutants display a normal vernalization response, indicating that BRM is not involved in vernalization-mediated FLC repression. Analysis of double mutants suggests that BRM is partially redundant with the autonomous pathway. Analysis of genetic interactions between BRM and the histone H2A.Z deposition machinery demonstrates that brm mutations overcome a requirement of H2A.Z for FLC activation suggesting that in the absence of BRM, a constitutively open chromatin conformation renders H2A.Z dispensable. [Conclusions/Significance]: BRM is critical for phase transition in Arabidopsis. Thus, BRM represses expression of the flowering promoting genes CO, FT and SOC1 and of the flowering repressor FLC. Our results indicate that BRM controls expression of FLC by creating a repressive chromatin configuration of the locus.This work was supported by Ministerio de Educacin y Ciencia (BFU2008-00238, CSD2006-00049), and by Junta de Andaluca (P06-CVI-01400) to J.C.R. and by the National Institutes of Health (grant no. 1R01GM079525), and the National Science Foundation (grant no. 0446440) to R.A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Grupo español de cirugía torácica asistida por videoimagen: método, auditoría y resultados iniciales de una cohorte nacional prospectiva de pacientes tratados con resecciones anatómicas del pulmón

Introduction: our study sought to know the current implementation of video-assisted thoracoscopic surgery (VATS) for anatomical lung resections in Spain. We present our initial results and describe the auditing systems developed by the Spanish VATS Group (GEVATS). Methods: we conducted a prospective multicentre cohort study that included patients receiving anatomical lung resections between 12/20/2016 and 03/20/2018. The main quality controls consisted of determining the recruitment rate of each centre and the accuracy of the perioperative data collected based on six key variables. The implications of a low recruitment rate were analysed for '90-day mortality' and 'Grade IIIb-V complications'. Results: the series was composed of 3533 cases (1917 VATS; 54.3%) across 33 departments. The centres' median recruitment rate was 99% (25-75th:76-100%), with an overall recruitment rate of 83% and a data accuracy of 98%. We were unable to demonstrate a significant association between the recruitment rate and the risk of morbidity/mortality, but a trend was found in the unadjusted analysis for those centres with recruitment rates lower than 80% (centres with 95-100% rates as reference): grade IIIb-V OR=0.61 (p=0.081), 90-day mortality OR=0.46 (p=0.051). Conclusions: more than half of the anatomical lung resections in Spain are performed via VATS. According to our results, the centre's recruitment rate and its potential implications due to selection bias, should deserve further attention by the main voluntary multicentre studies of our speciality. The high representativeness as well as the reliability of the GEVATS data constitute a fundamental point of departure for this nationwide cohort