High-quality genome-scale metabolic modelling of Pseudomonas putida highlights its broad metabolic capabilities

Genome-scale reconstructions of metabolism are computational species-specific knowledge bases able to compute systemic metabolic properties. We present a comprehensive and validated reconstruction of the biotechnologically relevant bacterium Pseudomonas putida KT2440 that greatly expands computable predictions of its metabolic states. The reconstruction represents a significant reactome expansion over available reconstructed bacterial metabolic networks. Specifically, iJN1462 (i) incorporates several hundred additional genes and associated reactions resulting in new predictive capabilities, including new nutrients supporting growth; (ii) was validated by in vivo growth screens that included previously untested carbon (48) and nitrogen (41) sources; (iii) yielded gene essentiality predictions showing large accuracy when compared with a knock-out library and Bar-seq data; and (iv) allowed mapping of its network to 82 P. putida sequenced strains revealing functional core that reflect the large metabolic versatility of this species, including aromatic compounds derived from lignin. Thus, this study provides a thoroughly updated metabolic reconstruction and new computable phenotypes for P. putida, which can be leveraged as a first step toward understanding the pan metabolic capabilities of Pseudomonas.     

Ecological and evolutionary drivers of the elevational gradient of diversity

Ecological, evolutionary, spatial and neutral theories make distinct predictions and provide distinct explanations for the mechanisms that control the relationship between diversity and the environment. Here, we test predictions of the elevational diversity gradient focusing on Iberian bumblebees, grasshoppers and birds. Processes mediated by local abundance and regional diversity concur in explaining local diversity patterns along elevation. Effects expressed through variation in abundance were similar among taxa and point to the overriding role of a physical factor, temperature. This determines how energy is distributed among individuals and ultimately how the resulting pattern of abundance affects species incidence. Effects expressed through variation in regional species pools depended instead on taxon‐specific evolutionary history, and lead to diverging responses under similar environmental pressures. Local filters and regional variation also explain functional diversity gradients, in line with results from species richness that indicate an (local) ecological and (regional) historical unfolding of diversity–elevation relationships.     

Structure-function relationships in yeast tubulins

A comprehensive set of clustered charged-to-alanine mutations was generated that systematically alter TUB1, the major alpha-tubulin gene of Saccharomyces cerevisiae. A variety of phenotypes were observed, including supersensitivity and resistance to the microtubule-destabilizing drug benomyl, lethality, and cold- and temperature-sensitive lethality. Many of the most benomyl-sensitive tub1 alleles were synthetically lethal in combination with tub3Delta, supporting the idea that benomyl supersensitivity is a rough measure of microtubule instability and/or insufficiency in the amount of alpha-tubulin. The systematic tub1 mutations were placed, along with the comparable set of tub2 mutations previously described, onto a model of the yeast alpha-beta-tubulin dimer based on the three-dimensional structure of bovine tubulin. The modeling revealed a potential site for binding of benomyl in the core of beta-tubulin. Residues whose mutation causes cold sensitivity were concentrated at the lateral and longitudinal interfaces between adjacent subunits. Residues that affect binding of the microtubule-binding protein Bim1p form a large patch across the exterior-facing surface of alpha-tubulin in the model. Finally, the positions of the mutations suggest that proximity to the alpha-beta interface may account for the finding of synthetic lethality of five viable tub1 alleles with the benomyl-resistant but otherwise entirely viable tub2-201 allele.     

Formation of a dynamic kinetochore- microtubule interface through assembly of the Dam1 ring complex

How kinetochore proteins form a dynamic interface with microtubules is largely unknown. In budding yeast, the 10-protein Dam1 complex is an Aurora kinase target that plays essential roles maintaining the integrity of the mitotic spindle and regulating interactions with the kinetochore. Here, we investigated the biochemical properties of purified Dam1 complex. The complex oligomerized into rings around microtubules. Ring formation was facilitated by microtubules but could occur in their absence. Mutant alleles led to partially assembled complexes or reduced microtubule binding. The interaction between rings and microtubules is mediated by the C termini of both Dam1 and alphabeta-tubulin. Ring formation promotes microtubule assembly, stabilizes against disassembly, and promotes bundling. A GTP-tubulin lattice is the preferred binding partner for the complex, and Dam1 rings can exhibit lateral mobility on microtubules. These observations suggest a mechanism by which the kinetochore can recognize and stay attached to the plus ends of microtubules.     

Determination of pyrrolized phospholipids in oxidized phospholipid vesicles and lipoproteins

The Ehrlich reaction was optimized to determine pyrrolized phospholipids produced as a consequence of oxidative stress. The procedure consisted of the treatment of the modified phospholipids with p-(dimethylamino)benzaldehyde at a controlled acidity temperature and the spectrophotometric determination of adducts produced. The extinction coefficient of Ehrlich adducts was calculated by using 1-[1-(2-hydroxyethyl)-1H-pyrrol-2-yl]propan-1-ol (compound 1) as standard and was 56,500 M(-1)cm(-1). The response was linear and reproducible within the range of 0.051-7.65 microM of compound 1. When the assay was applied to determination of pyrrole content in ethanolamine incubated in the presence of 0.25-1mM of 4,5(E)-epoxy-2(E)-heptenal, the complete conversion of the aldehyde into the pyrrole ring was observed and the results obtained were similar to those found when compound 1 was determined by gas chromatography. When phosphatidylethanolamine was incubated in the presence of 0.5-40 mM of 4,5(E)-epoxy-2(E)-heptenal, the phospholipid was pyrrolized similarly to ethanolamine, although there was not a quantitative conversion and the amount of pyrroles produced depended on the pH of the media. Pyrrolized phospholipids were also produced when phosphatidylethanolamine multilamellar vesicles where oxidized in the presence of either Fe(3+)/ascorbic acid or ABAP (2,2'-azobis(2-methylpropionamide) dihydrochloride) and when high-density lipoproteins were incubated in the presence of Cu(2+), thereby confirming that phospholipid pyrrolization is a common consequence of oxidative stress and that Ehrlich adducts may be valid for determining this pyrrolization.     

Indirect seed dispersal by the feral cats Felis catus in island ecosystems (Canary Islands)

In this paper we present an unusual incidence of an introduced Carnivora Felis catus as indirect seed disperser of plants that produce fleshy fruits in different ecosystems in the Canary Islands Four hundred and twenty six seeds from at least 8 fleshy fruit plant species have been identified in the analysis of 1047 scat groups, the majority of them being found in the lower habitats (<600 mas1) of the Canary archipelago Seeds from two plant species were significantly matched with the presence of lizard prey, and fruits of Jumperus phoenicea, Neochamaelea pulverulenta and Withania artstata were directly consumed by the cats Passing through the gut of the Gallotia galloti (Lacertidae) and Felis catus apparently does not damage the seeds At the moment, the phenomenon studied in this paper does not seem to have a great quantitative importance m the natural regeneration of the plants if we compare the direct vs indirect seed dispersal     

Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis

The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2'-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.     

Hydrolytic enzyme activities of extracted humic substances during the vermicomposting of a lignocellulosic olive waste

Humic substances and three hydrolytic enzymes (beta-glucosidase, phosphatase and urease) were extracted by neutral sodium pyrophosphate from an olive waste (dry olive cake), alone or mixed with municipal biosolids, during a nine month vermicomposting process. Easily degradable compounds decreased during the vermicomposting process because of microbial consumption. When municipal biosolids were added to dry olive cake, microbial activity increased and the amounts of compounds extracted by pyrophosphate were three times lower than olive cake alone. In both instances, beta-glucosidase, phosphatase and urease activities of the organic extracts either increased or remained the same after a nine month period of vermicomposting, thus suggesting that the humus enzyme complexes resisted microbial and earthworm attack. It is known that humus immobilised enzymes also remain active in soil environments, reactivating the nutrient cycles in soil. The use as amendments of vermicomposted olive cake, alone or when mixed with biosolids, could be a good alternative to reactivate the C, P and N-cycles in degraded soils for regeneration purposes.     

Bacterial diversity in dry modern freshwater stromatolites from Ruidera Pools Natural Park,Spain

Ruidera Pools Natural Park, Spain, constitutes one of the most representative systems of carbonate precipitation in Europe. The prokaryotic community of a dry modern stromatolite recovered from the park has been analyzed by molecular techniques that included denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analysis, together with microscopic observations from the sample and cultures. Ribosomal RNA was directly extracted to study the putatively active part of the microbial community present in the sample. A total of 295 16S rRNA gene sequences were analyzed. Libraries were dominated by sequences related to Cyanobacteria, most frequently to the genus Leptolyngbya. A diverse and abundant assemblage of non-cyanobacterial sequences was also found, including members of Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Acidobacteria,Planctomycetes and Chloroflexi groups. No amplification was obtained when using archaeal primers. The results showed that at the time of sampling, when the pool was dry, the bacterial community of the stromatolites was dominated by groups of highly related Cyanobacteria, including new groups that had not been previously reported, although a high diversity outside this phylogenetic group was also found. The results indicated that part of the Cyanobacteria assemblage was metabolically active and could thus play a role in the mineralization processes inside the stromatolites.