Localisation and processing of the precursor form of photosystem II protein D1 inSynechocystis 6803

Pure plasma membrane and thylakoid membrane fractions from Synechocystis 6803 were isolated to study the localisation and processing of the precursor form of the D1 protein (pD1) of photosystem II (PSII). PSII core proteins (D1, D2 and cytb559) were localised both to plasma and thylakoid membrane fractions, the majority in thylakoids. pD1 was found only in the thylakoid membrane where active PSII is known to function. Membrane fatty acid unsaturation was shown to be critical in processing of pD1 into mature D1 protein. This was concluded from pulse-labelling experiments at low temperature using wild type and a mutant Synechocystis 6803 with a low level of membrane fatty acid unsaturation. Further, pD1 was identified as two distinct bands, an indication of two cleavage sites in the precursor peptide or, alternatively, two different conformations of pD1. Our results provide evidence for thylakoid membranes being a primary synthesis site for D1 protein during its light-activated turnover. The existence of the PSII core proteins in the plasma membrane, on the other hand, may be related to the biosynthesis of new PSII complexes in these membranes.     

Activity of pupal parasitoids of the stable fly Stomoxys calcitrans and prevalence of entomopathogenic fungi in the stable fly and the house fly Musca domestica in Denmark

A survey was conducted on confined dairy cattle farms and a pig farm from May–October in 1999 to determine the activity and relative abundance of pupal parasitoids and the prevalence of entomopathogenic fungi in populations of the haematophagous stable fly, Stomoxys calcitrans (Diptera: Muscidae), in Denmark. Four species of pteromalids were found with Spalangia cameroni as the predominant. The other parasitoids were S. nigripes, S. nigra and Phygadeuon fumator (Ichneumonidae). Peak activity of the parasitoids was observed to be late in the summer and the beginning of autumn (August–September) when approximately 10% of the collected stable fly pupae were parasitised. Adult stable flies were infected with four species of entomopathogenic fungi: Entomophthora muscae, E. schizophorae, Beauveria bassiana and Verticillium lecanii. All fungi occurred in low percentages (max. 4%) and remained at this level throughout the sampling period. Likewise, adult house flies were infected with B. bassiana and V. lecanii,but Metarhizium anisopliae, Paecilomyces fumosoroseus and V. fusisporum were also recorded. The overall hyphomycete prevalence in house flies was 0.3%, and single species rarely exceeded 0.1%. The prevalence remained low in spite of increasing house fly numbers in August–September.     

Fine structure genetic map and complementation analysis of mutations in the dnaA gene of Escherichia coli

Summary A fine structure genetic map of several mutations in the dnaA gene of Escherichia coli was constructed by the use of recombinant and M13 phages. The dnaA508 mutation was found to be the mutation most proximal to the promoter, while the dnaA203 mutation was found to be the most distal one. The order of mutations established in this analysis was: dnaA508, dnaA167, (dnaA5, dnaA46, dnaA211), dnaA205, dnaA204, dnaA203. The mutations dnaA601, dnaA602, dnaA603, dnaA604 and dnaA606 were found to map very close to each other and close to dnaA205 in the middle third of the dnaA gene. In analysing the dominance relationship all 13 dnaA mutations were found to be recessive to the wild type. Characteristic phenotypes of the dnaA(Ts) mutants, like reversibility of the temperature inactivation of the dnaA protein, cold sensivity of haploid or of merodiploid strains and suppressibility by rpoB mutations, are found to correlate with clusters of mutations within the gene.     

Parallel production and verification of protein products using a novel high-throughput screening method

Protein production and analysis in a parallel fashion is today applied in laboratories worldwide and there is a great need to improve the techniques and systems used for this purpose. In order to save time and money, a fast and reliable screening method for analysis of protein production and also verification of the protein product is desired. Here, a micro-scale protocol for the parallel production and screening of 96 proteins in plate format is described. Protein capture was achieved using immobilized metal affinity chromatography and the product was verified using matrix-assisted laser desorption ionization time-of-flight MS. In order to obtain sufficiently high cell densities and product yield in the small-volume cultivations, the EnBase® cultivation technology was applied, which enables cultivation in as small volumes as 150 μL. Here, the efficiency of the method is demonstrated by producing 96 human, recombinant proteins, both in micro-scale and using a standard full-scale protocol and comparing the results in regard to both protein identity and sample purity. The results obtained are highly comparable to those acquired through employing standard full-scale purification protocols, thus validating this method as a successful initial screening step before protein production at a larger scale.     

Cytochrome p450 complement (CYPome) of the avermectin-producer Streptomyces avermitilis and comparison to that of Streptomyces coelicolor A3(2)

The genus Streptomyces produces about two-thirds of naturally occurring antibiotics and a wide array of other secondary metabolites, including antihelminthic agents, antitumor agents, antifungal agents, and herbicides. The newly completed genome sequence of the avermectin-producing bacterium Streptomyces avermitilis contains 33 cytochromes p450 (CYPs), many more than the 18 observed in Streptomyces coelicolor A3(2). Some of the likely metabolic functions are reported together with their genomic location and bioinformatic analysis. Seven entirely new CYP families were found together with close homologues of some forms observed in S. coelicolor A3(2). The presence of unusual CYP forms associated with conservons is revealed and of these, CYP157 forms in both S. avermitilis and S. coelicolor A3(2) deviate from the previously accepted rule for an EXXR motif within the K-helix of CYPs. Amongst this range of CYPs are forms associated with avermectin, filipin, geosmin, and pentalenolactone biosynthesis as well as unknown pathways of secondary metabolism.     

Genetic and phenotypic divergence in an island bird: isolation by distance,by colonization or by adaptation?

Discerning the relative roles of adaptive and nonadaptive processes in generating differences among populations and species, as well as how these processes interact, is a fundamental aim in biology. Both genetic and phenotypic divergence across populations can be the product of limited dispersal and gradual genetic drift across populations (isolation by distance), of colonization history and founder effects (isolation by colonization) or of adaptation to different environments preventing migration between populations (isolation by adaptation). Here, we attempt to differentiate between these processes using island populations of Berthelot's pipit (Anthus berthelotii), a passerine bird endemic to three Atlantic archipelagos. Using microsatellite markers and approximate Bayesian computation, we reveal that the northward colonization of this species ca. 8500 years ago resulted in genetic bottlenecks in the colonized archipelagos. We then show that high levels of genetic structure exist across archipelagos and that these are consistent with a pattern of isolation by colonization, but not with isolation by distance or adaptation. Finally, we show that substantial morphological divergence also exists and that this is strongly concordant with patterns of genetic structure and bottleneck history, but not with environmental differences or geographic distance. Overall, our data suggest that founder effects are responsible for both genetic and phenotypic changes across archipelagos. Our findings provide a rare example of how founder effects can persist over evolutionary timescales and suggest that they may play an important role in the early stages of speciation.     

Insights into the quality of DnaA boxes and their cooperativity

Plasmids carrying the mioC promoter region with its two DnaA boxes are as efficient in titration of DnaA protein as plasmids carrying a replication-inactivated oriC region with its five DnaA boxes. The two DnaA boxes upstream of the mioC promoter were mutated in various ways to study the cooperativity between the DnaA boxes, and to study in vivo the in vitro-defined 9mer DnaA box consensus sequence (TT(A)/(T)TNCACA). The quality and cooperativity of the DnaA boxes were determined in two complementary ways: as titration of DnaA protein leading to derepression of the dnaA promoter, and as repression of the mioC promoter caused by the DnaA protein binding to the DnaA boxes. Titration of DnaA protein correlated with repression of the mioC promoter. The level of titration and repression with the normal promoter-proximal box (TTTTCCACA) depends strongly on the presence and the quality of a DnaA box in the promoter-distal position, whereas a promoter-proximal DnaA box with the sequence TTATCCACA titrated DnaA protein and caused significant repression of the mioC promoter without a promoter-distal DnaA box. The quality of the eight different consensus DnaA boxes located in the promoter-proximal position was determined: TTATCCACA had the highest affinity for DnaA protein. In the third position, A was better than T, and the four possibilities in the fifth position could be ranked as C >A >or=G >T. Parallel in vitro experiments using a purified DNA-binding domain of DnaA protein gave the same ranking of the binding affinities of the eight DnaA boxes.