Molecular cloning, functional expression and purification of a glucan branching enzyme from Neisseria denitrificans(1).

The nucleotide sequence containing the complete structural information for a glucan branching enzyme was isolated from a Neisseria denitrificans genomic library. The gene was expressed in Escherichia coli and the active recombinant protein was purified. The deduced protein of 762 amino acids with a calculated molecular weight of 86313 Da shows similarity to the primary protein sequences of other known glucan branching enzymes. Amino acid sequencing of the isolated protein by Edman degradation confirmed the deduced start codon of the structural gene of the glucan branching enzyme. The purified glucan branching enzyme has a stimulating effect on the Neisseria amylosucrase activity.     

Fractionation of sulfur isotopes during dissimilatory reduction of sulfate by a thermophilic gram-negative bacterium at 60 °C

Sulfur isotope (³⁴S/³²S) fractionation during reduction of dissolved sulfate was investigated with a growing batch culture of a thermophilic, gram-negative, sulfate-reducing bacterium (strain MT-96) at 60 °C. The completely oxidizing strain was isolated from geothermally heated sediments of a shallow-water hydrothermal vent in the Mediterranean Sea. The hydrogen sulfide produced in the experiments was enriched in ³²S by approximately 19‰ as compared to sulfate, which indicates that stable isotope discrimination by this thermophile is within the range found previously for mesophilic sulfate-reducing bacteria, and only slightly higher than that observed for the thermophilic gram-positive Desulfotomaculum nigrificans. Received: 1 December 1998 / Accepted: 25 May 1999     

Return of twaite shad <Emphasis Type="Italic">Alosa fallax</Emphasis> (Lacépède, 1803) to the Southern Baltic Sea and the transitional area between the Baltic and North Seas

The status of twaite shad was investigated within the Southern Baltic Sea (ICES subdivision 22–27) and transitional area between the Baltic and North Seas (division IIIa). The following sources of data were analysed: (i) commercial catch statistics and relevant publications, (ii) records from ichthyological museum collections, (iii) records from recent commercial and recreational fisheries, (iv) research fisheries with trawls. A total of 476 records of twaite shad including more than 16 million individuals were obtained for the time between the years 1836 and 2005. About 72.9% of all records originated from commercial catch statistics and publications, whereas 18.9% were received from ichthyological collections. Research fisheries provided 6.3%, and 1.9% of the records were obtained from recent commercial and recreational fisheries. Most records of twaite shad were estimated for subdivisions 24 (45.2%) and 26 (35.5%). From 1836 to 1959, 29.6% of the records date from the period until 1899. 70.4% of the records of twaite shad originate from the twentieth century until 1959. The mean annual catch of twaite shad between 1891 and 1959 amounted to 86,674 kg within subdivisions 24–26 of the Southern Baltic Sea. Catch data show an approximately 20-year-cyclicity of maximum yields and minimum catches, respectively. The maximum annual yield of twaite shad in subdivisions 24–26 (474,700 kg) was registered in 1940, the minimum annual yield was estimated in 1958 (10 kg). In the 1950s, the annual catches of twaite shad declined sharply. Until 1960 twaite shad catches and records originated mainly from the Pommeranian Bay/Pommeranian Coast and adjacent waters including the Szczecin Lagoon (subdivision 24), the Bay of Gdańsk, Vistula Lagoon and Vistula Spit (subdivision 26) and from the Curonian Lagoon and Curonian Spit (subdivision 26). The highest catches of twaite shad originated from the area of Curonian Lagoon/Curonian Spit from 1941 to 1960. Seasonal catches of twaite shad showed maximum values from May to July. From 1960 to 1989, only four records of A. fallax were registered in the Southern Baltic Sea which originated from German coastal waters in subdivisions 22 and 24. A total of 107 records of twaite shad was obtained in subdivisions 20–27 from 1990 to 2005. Most of these recent records originate from a twaite-shad stock in subdivisions 24, 25 and 26. There are indications which suggest a separate stock of twaite shad in subdivisions 20 and 21. Disappearance and recovery of twaite shad stocks were probably caused by the following factors: construction of barriers in rivers with spawning sites of twaite shad; habitat destruction in those rivers as consequence of gravel extraction and reengineering scheme to improve navigation and for flood defence purposes, water pollution in the lagoons of the Southern Baltic and in their tributaries, commercial fishery in the Southern Baltic and climatic variation in the Baltic Sea basin south of the latitude of 60° N. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Guest editors: S. Dufour, E. Prévost, E. Rochard & P. Williot Fish and diadromy in Europe (ecology, management, conservation)     

Metabolome analysis of biosynthetic mutants reveals a diversity of metabolic changes and allows identification of a large number of new compounds in Arabidopsis

Metabolomics is facing a major challenge: the lack of knowledge about metabolites present in a given biological system. Thus, large-scale discovery of metabolites is considered an essential step toward a better understanding of plant metabolism. We show here that the application of a metabolomics approach generating structural information for the analysis of Arabidopsis (Arabidopsis thaliana) mutants allows the efficient cataloging of metabolites. Fifty-six percent of the features that showed significant differences in abundance between seeds of wild-type, transparent testa4, and transparent testa5 plants could be annotated. Seventy-five compounds were structurally characterized, 21 of which could be identified. About 40 compounds had not been known from Arabidopsis before. Also, the high-resolution analysis revealed an unanticipated expansion of metabolic conversions upstream of biosynthetic blocks. Deficiency in chalcone synthase results in the increased seed-specific biosynthesis of a range of phenolic choline esters. Similarly, a lack of chalcone isomerase activity leads to the accumulation of various naringenin chalcone derivatives. Furthermore, our data provide insight into the connection between p-coumaroyl-coenzyme A-dependent pathways. Lack of flavonoid biosynthesis results in elevated synthesis not only of p-coumarate-derived choline esters but also of sinapate-derived metabolites. However, sinapoylcholine is not the only accumulating end product. Instead, we observed specific and sophisticated changes in the complex pattern of sinapate derivatives.     

Feller processes: the next generation in modeling. Brownian motion, Lévy processes and beyond

We present a simple construction method for Feller processes and a framework for the generation of sample paths of Feller processes. The construction is based on state space dependent mixing of Lévy processes. Brownian Motion is one of the most frequently used continuous time Markov processes in applications. In recent years also Lévy processes, of which Brownian Motion is a special case, have become increasingly popular. Lévy processes are spatially homogeneous, but empirical data often suggest the use of spatially inhomogeneous processes. Thus it seems necessary to go to the next level of generalization: Feller processes. These include Lévy processes and in particular brownian motion as special cases but allow spatial inhomogeneities. Many properties of Feller processes are known, but proving the very existence is, in general, very technical. Moreover, an applicable framework for the generation of sample paths of a Feller process was missing. We explain, with practitioners in mind, how to overcome both of these obstacles. In particular our simulation technique allows to apply Monte Carlo methods to Feller processes.     

Nitrogen Metabolism Genes from Temperate Marine Sediments

In this study, we analysed metagenomes along with biogeochemical profiles from Skagerrak (SK) and Bothnian Bay (BB) sediments, to trace the prevailing nitrogen pathways. NO₃ ^? was present in the top 5 cm below the sediment-water interface at both sites. NH₄ ⁺ increased with depth below 5 cm where it overlapped with the NO₃ ^? zone. Steady-state modelling of NO₃ ^? and NH₄ ⁺ porewater profiles indicates zones of net nitrogen species transformations. Bacterial protease and hydratase genes appeared to make up the bulk of total ammonification genes. Genes involved in ammonia oxidation (amo, hao), denitrification (nir, nor), dissimilatory NO₃ ^? reduction to NH₄ ⁺ (nfr and otr) and in both of the latter two pathways (nar, nap) were also present. Results show ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) are similarly abundant in both sediments. Also, denitrification genes appeared more abundant than DNRA genes. 16S rRNA gene analysis showed that the relative abundance of the nitrifying group Nitrosopumilales and other groups involved in nitrification and denitrification (Nitrobacter, Nitrosomonas, Nitrospira, Nitrosococcus and Nitrosomonas) appeared less abundant in SK sediments compared to BB sediments. Beggiatoa and Thiothrix 16S rRNA genes were also present, suggesting chemolithoautotrophic NO₃ ^? reduction to NO₂ ^? or NH₄ ⁺ as a possible pathway. Our results show the metabolic potential for ammonification, nitrification, DNRA and denitrification activities in North Sea and Baltic Sea sediments.     

Conformational changes in adeno-associated virus type 1 induced by genome packaging

Adeno-associated virus (AAV) is frequently used as a vector for gene therapy. The viral capsid consists of three structural proteins (VP1, VP2, and VP3) that have a common C-terminal core (VP3), with N-terminal extensions of increasing length in VP2 and VP1. The capsid encloses a single-stranded genome of up to 4.7 kb, which is packaged into empty capsids. The N-terminal extension of VP1 carries a phospholipase domain that becomes accessible during infection in the endosomal pathway. We have used cryo-electron microscopy and image reconstruction to determine subnanometer-resolution structures of recombinant AAV1 that has packaged different amounts of a 3. 6-kb recombinant genome. The maps show that the AAV1 capsid undergoes continuous conformational changes upon packaging of the genome. The rearrangements occur at the inner capsid surface and lead to constrictions of the pores at the 5-fold symmetry axes and to subtle movements of the β-sheet regions of the capsid proteins. In fully packaged particles, the genome forms stem-like features that contact the inner capsid surface at the 3-fold symmetry axes. We think that the reorganization of the inner surface has an impact on the viral life cycle during infection, preparing the externalization of phospholipase domains through the pores at the 5-fold symmetry axes and possibly genome release.     

The assembly-activating protein promotes capsid assembly of different adeno-associated virus serotypes

Adeno-associated virus type 2 (AAV2) capsid assembly requires the expression of a virally encoded assembly-activating protein (AAP). By providing AAP together with the capsid protein VP3, capsids are formed that are composed of VP3 only. Electron cryomicroscopy analysis of assembled VP3-only capsids revealed all characteristics of the wild-type AAV2 capsids. However, in contrast to capsids assembled from VP1, VP2, and VP3, the pores of VP3-only capsids were more restricted at the inside of the 5-fold symmetry axes, and globules could not be detected below the 2-fold symmetry axes. By comparing the capsid assembly of several AAV serotypes with AAP protein from AAV2 (AAP-2), we show that AAP-2 is able to efficiently stimulate capsid formation of VP3 derived from several serotypes, as demonstrated for AAV1, AAV2, AAV8, and AAV9. Capsid formation, by coexpressing AAV1-, AAV2-, or AAV5-VP3 with AAP-1, AAP-2, or AAP-5 revealed the ability of AAP-1 and AAP-2 to complement each other in AAV1 and AAV2 assembly, whereas for AAV5 assembly more specific conditions are required. Sequence alignment of predicted AAP proteins from the known AAV serotypes indicates a high degree of homology of all serotypes to AAP-2 with some divergence for AAP-4, AAP-5, AAP-11, and AAP-12. Immunolocalization of assembled capsids from different serotypes confirmed the preferred nucleolar localization of capsids, as observed for AAV2; however, AAV8 and AAV9 capsids could also be detected throughout the nucleus. Taken together, the data show that AAV capsid assembly of different AAV serotypes also requires the assistance of AAP proteins.