Construction and screening of marine metagenomic libraries.

Marine microbial communities are highly diverse and have evolved during extended evolutionary processes of physiological adaptations under the influence of a variety of ecological conditions and selection pressures. They harbor an enormous diversity of microbes with still unknown and probably new physiological characteristics. Besides, the surfaces of marine multicellular organisms are typically covered by a consortium of epibiotic bacteria and act as barriers, where diverse interactions between microorganisms and hosts take place. Thus, microbial diversity in the water column of the oceans and the microbial consortia on marine tissues of multicellular organisms are rich sources for isolating novel bioactive compounds and genes. Here we describe the sampling, construction of large-insert metagenomic libraries from marine habitats and exemplarily one function based screen of metagenomic clones.

Expression of ribonuclease A and ribonuclease N1 in the filamentous fungus Neurospora crassa.

In this study, we investigated the ability of the fungus Neurospora crassa to produce and secrete two ribonucleases: the heterologous bovine RNase A and the endogenous RNase N(1). A set of expression vectors was constructed, each consisting of an RNase A open reading frame under the control of a specific promoter and each with a specific terminator. N. crassa transformants were analyzed at the transcriptional and protein levels. Irrespective of the promoter used, all transformants showed an RNase A-specific transcript in northern hybridization, but transcriptional strengths differed significantly. The strongest transcription was detected in transformants under the control of the cfp promoter. Western blot analysis and ELISA assays of selected transformants showed an effective secretion up to 356 ng/mL of recombinant RNase A protein. However, the highest ribonuclease activity could be detected in transformants carrying the endogenous RNase N(1) under the control of the ccg1 promoter. Expression and secretion of RNase N(1) thus represent an alternative to recombinant expression of RNase A protein. In conclusion, we have created a viable expression system for expression of homologous and heterologous proteins in N. crassa.

The BEM46-like protein appears to be essential for hyphal development upon ascospore germination in Neurospora crassa and is targeted to the endoplasmic reticulum.

The bud emergence (BEM)46 proteins are evolutionarily conserved members of the alpha/beta-hydrolase super family, but their exact role remains unknown. To better understand the cellular role of BEM46 and its homologs, we used the model organism Neurospora crassa in conjunction with bem46 RNAi, over-expression vectors, and repeat induced point mutation analyzes. We clearly demonstrated that BEM46 is required for cell type-specific hyphal growth, which indicates a role for BEM46 in maintaining polarity. Vegetative hyphae, perithecia, and ascospores developed normally, but hyphae germinating from ascospores exhibited a loss-of-polarity phenotype. We also found that the BEM46 protein is targeted to the perinuclear endoplasmic reticulum (ER) and also localizes at or close to the plasma membrane. Our findings show that BEM46 can be used as a new ER marker for filamentous fungi, the first for N. crassa. Our data suggest that BEM46 plays a role in a signal transduction pathway involved in determining or maintaining cell type-specific polarity. This implies a higher degree of fungal hyphae differentiation than previously expected. This work also has implications for higher eukaryotic cells with polarized growth, such as pollen tubes or neuronal cells.