Molecular cloning and in vivo expression of a precursor to rat mitochondrial aspartate aminotransferase

A 2.4 kilobase cDNA for rat mitochondrial aspartate aminotransferase (E.C. 2.6.1.1.) was isolated and sequenced. The predicted presequence is 93% homologous to the presequences of the enzyme from pig and mouse. The predicted amino acid sequence of the mature enzyme differs from that determined directly by amino acid sequencing (Huynh, Q.K., Sakakibara, R., Watanabe, T., and Wada, H. (1981) J. Biochem. (Tokyo) 90, 863-875) at 13 amino acids residues. The most important difference is at position 140 where the cDNA encodes a tryptophanyl residue rather than the previously reported glycine. This critical residue is now seen to be conserved in all aspartate aminotransferases. The coding region of this cDNA was inserted into the plasmid cloning vector pKK233-2 and used to stably express an unfused precursor in Escherichia coli JM105.     

Size-fractionated chlorophyll a biomass and picophytoplankton cell density along a longitudinal axis of a temperate estuary (Southampton Water)

Size-fractionated chlorophyll a biomass and picophytoplanktoncell number distributions were investigated along a longitudinalaxis of Southampton Water estuary during autumn. Chlorophylla concentration in the >5µm and the 1–5 µmsize fractions was highest midway down the estuary, and decreasedboth in the landward and seaward directions. In contrast, chlorophylla biomass in the 0.2–1 µm size fraction showed nodecline towards the seaward end of the estuary. In agreementwith this observation, phycoerythrin-containing picocyanobacteriacell concentration showed a positive exponential-like relationshipwith salinity and eukaryotic picophytoplankton were also highestat high salinities. Expressed as a percentage of total, chlorophylla standing stock in both the 1–5 µ.m (4.4–28.7%)and the 0.2–1 µm size fractions (1.7–8.6%)was inversely correlated with total chlorophyll a concentration.Both these two fractions made a greater input to the total phaeopigmentconcentration than to the total pool of active chlorophyll a.     

Genomics and transcriptomics insights into luteolin effects on the beta-rhizobial strain Cupriavidus necator UYPR2.512

Cupriavidus necator UYPR2.512 is a rhizobial strain that belongs to the Beta-subclass of proteobacteria, able to establish successful symbiosis with Mimosoid legumes. The initial steps of rhizobium-legumes symbioses involve the reciprocal recognition by chemical signals, being luteolin one of the molecules involved. However, there is a lack of information on the effect of luteolin in beta-rhizobia. In this work, we used long-read sequencing to complete the genome of UYPR2.512 providing evidence for the existence of four closed circular replicons. We used an RNA-Seq approach to analyse the response of UYPR2.512 to luteolin. One hundred and forty-five genes were differentially expressed, with similar numbers of downregulated and upregulated genes. Most repressed genes were mapped to the main chromosome, while the upregulated genes were overrepresented among pCne512e, containing the symbiotic genes. Induced genes included the nod operon and genes implicated in exopolysaccharides and flagellar biosynthesis. We identified many genes involved in iron, copper and other heavy metals metabolism. Among repressed genes, we identified genes involved in basal carbon and nitrogen metabolism. Our results suggest that in response to luteolin, C. necator strain UYPR2.512 reshapes its metabolism in order to be prepared for the forthcoming symbiotic interaction.     

Est10: A Novel Alkaline Esterase Isolated from Bovine Rumen Belonging to the New Family XV of Lipolytic Enzymes

A metagenomic fosmid library from bovine rumen was used to identify clones with lipolytic activity. One positive clone was isolated. The gene responsible for the observed phenotype was identified by in vitro transposon mutagenesis and sequencing and was named est10. The 367 amino acids sequence harbors a signal peptide, the conserved secondary structure arrangement of alpha/beta hydrolases, and a GHSQG pentapeptide which is characteristic of esterases and lipases. Homology based 3D-modelling confirmed the conserved spatial orientation of the serine in a nucleophilic elbow. By sequence comparison, Est10 is related to hydrolases that are grouped into the non-specific Pfam family DUF3089 and to other characterized esterases that were recently classified into the new family XV of lipolytic enzymes. Est10 was heterologously expressed in Escherichia coli as a His-tagged fusion protein, purified and biochemically characterized. Est10 showed maximum activity towards C4 aliphatic chains and undetectable activity towards C10 and longer chains which prompted its classification as an esterase. However, it was able to efficiently catalyze the hydrolysis of aryl esters such as methyl phenylacetate and phenyl acetate. The optimum pH of this enzyme is 9.0, which is uncommon for esterases, and it exhibits an optimal temperature at 40°C. The activity of Est10 was inhibited by metal ions, detergents, chelating agents and additives. We have characterized an alkaline esterase produced by a still unidentified bacterium belonging to a recently proposed new family of esterases.     

Infectivity and virulence of Nosema ceranae (Microsporidia) isolates obtained from various Apis mellifera morphotypes

The infection of honey bees, Apis mellifera L. (Hymenoptera: Apidae), by the microsporidian Nosema ceranae is one of the factors related to the increase in colony losses and the decrease in honey production observed in recent years. However, these effects seem to differ depending on the climate zone. The range and prevalence of N. ceranae have increased significantly in the last decades, with different consequences in northern and southern temperate areas. The existence of various isolates of N. ceranae from distant geographical areas, which probably exhibit different degrees of virulence, could explain the different responses of the bee to the infection. The aim of this work was to compare the effects of two N. ceranae isolates from different host populations from Argentina on honey bee survival at two ages post-eclosion. Using cage experiments, we compared the development of infection of worker bees through the estimation of daily bee mortality and spore counts. Host subspecies identity analysis showed a strong similarity with Apis mellifera scutellata morphotype for the northern region, with a greater hybridization between subspecies with European origin toward the central and southern regions. Genetic characterization of isolates from the three regions indicated only the presence of N. ceranae. Infected bees survived longer than control bees, and bees infected at 5 days had a lower survival than those infected at 72 h with isolates from the three regions. These differences in survival matched the development of the N. ceranae infection, with differences in spore loads for infected bees at 5 days. Our studies showed that Nosema infection and survival varied among the different ages post emergence of workers, and both increased as the honey bee aged. These differences in susceptibility to infection could be related to the immune response of bees of different ages or to changes in the composition and succession of the intestinal microbiota throughout its ontogeny.     

Origin of mound-field landscapes: a multi-proxy approach combining contemporary vegetation, carbon stable isotopes and phytoliths

Background and aims

Seasonally flooded South American savannas harbor different kinds of mound-field landscapes of largely unknown origin. A recent study used soil carbon-isotope depth profiles and other proxies to infer vegetation history in murundu landscapes in Brazil. Results suggested that differential erosion, not building-up processes (e.g., termite mounds), produced mounds. We tested this approach to inferring mound origin in a mound-field landscape in French Guiana.

Methods

We examined carbon-isotope depth profiles of soil organic matter, phytolith profiles and contemporary vegetation composition in mounds and inter-mounds.

Results

Relative abundance of C3 and C4 plants across habitats was very different from that in murundu landscapes; C3 plants were better represented in inter-mounds than on mounds. Habitat differences in C3/C4 distribution were subtler than in murundu landscapes, limiting inference of vegetation history based on carbon isotopes. Still, carbon-isotope and phytolith depth profiles gave similar pictures of vegetation history, both favoring a building-up hypothesis, corroborating other evidence that these mounds are vestiges of ancient agricultural raised fields.

Conclusions

Carbon-isotope depth profiles are unlikely to be adequate for deciphering origin of mound-field landscapes from vegetation history in seasonally flooded savannas. Including data on current vegetation and phytoliths makes inferences more robust.     

Modification of skeletal S-1 with fluorescein isothiocyanate

1. Pyridoxal-5'-phosphate (PLP), a marker of primary amines, bound covalently to S-1 in an approximate ratio of 1:1. PLP was localized within the tryptic 25,000 mol. wt fragment. 2. Fluorescein isothiocyanate (FITC), which is known to bind covalently to primary amino groups located at nucleotide binding sites, readily bound to S-1 in a 2:1 ratio. FITC was localized within the 20,000 and 50,000 mol. wt tryptic peptides, in a ratio of 1:1 in each one. 3. These results are consistent with the existence of nucleotide binding sites on myosin different from those of the catalytic sites.