Phosphorylation of the VP16 transcriptional activator protein during herpes simplex virus infection and mutational analysis of putative phosphorylation sites

AbstractVP16 is a virion phosphoprotein of herpes simplex virus and a transcriptional activator of the viral immediate-early (IE) genes. We identified four novel VP16 phosphorylation sites (Ser18, Ser353, Ser411, and Ser452) at late times in infection but found no evidence of phosphorylation of Ser375, a residue reportedly phosphorylated when VP16 is expressed from a transfected plasmid. A virus carrying a Ser375Ala mutation of VP16 was viable in cell culture but with a slow growth rate. The association of the mutant VP16 protein with IE gene promoters and subsequent IE gene expression was markedly reduced during infection, consistent with prior transfection and in vitro results. Surprisingly, the association of Oct-1 with IE promoters was also diminished during infection by the mutant strain. We propose that Ser375 is important for the interaction of VP16 with Oct-1, and that the interaction is required to enable both proteins to bind to IE promoters

Evidence from GC-TRFLP that Bacterial Communities in Soil Are Lognormally Distributed

The Species Abundance Distribution (SAD) is a fundamental property of ecological communities and the form and formation of SADs have been examined for a wide range of communities including those of microorganisms. Progress in understanding microbial SADs, however, has been limited by the remarkable diversity and vast size of microbial communities. As a result, few microbial systems have been sampled with sufficient depth to generate reliable estimates of the community SAD. We have used a novel approach to characterize the SAD of bacterial communities by coupling genomic DNA fractionation with analysis of terminal restriction fragment length polymorphisms (GC-TRFLP). Examination of a soil microbial community through GC-TRFLP revealed 731 bacterial operational taxonomic units (OTUs) that followed a lognormal distribution. To recover the same 731 OTUs through analysis of DNA sequence data is estimated to require analysis of 86,264 16S rRNA sequences. The approach is examined and validated through construction and analysis of simulated microbial communities in silico. Additional simulations performed to assess the potential effects of PCR bias show that biased amplification can cause a community whose distribution follows a power-law function to appear lognormally distributed. We also show that TRFLP analysis, in contrast to GC-TRFLP, is not able to effectively distinguish between competing SAD models. Our analysis supports use of the lognormal as the null distribution for studying the SAD of bacterial communities as for plant and animal communities

Patterns of Gene Flow Define Species of Thermophilic Archaea

A genomic view of speciation in Archaea shows higher rates of gene flow within coexisting microbial species than between them

Phylogeography of the Microcoleus vaginatus (Cyanobacteria) from Three Continents – A Spatial and Temporal Characterization

It has long been assumed that cyanobacteria have, as with other free-living microorganisms, a ubiquitous occurrence. Neither the geographical dispersal barriers nor allopatric speciation has been taken into account. We endeavoured to examine the spatial and temporal patterns of global distribution within populations of the cyanobacterium Microcoleus vaginatus, originated from three continents, and to evaluate the role of dispersal barriers in the evolution of free-living cyanobacteria. Complex phylogeographical approach was applied to assess the dispersal and evolutionary patterns in the cyanobacterium Microcoleus vaginatus (Oscillatoriales). We compared the 16S rRNA and 16S-23S ITS sequences of strains which had originated from three continents (North America, Europe, and Asia). The spatial distribution was investigated using a phylogenetic tree, network, as well as principal coordinate analysis (PCoA). A temporal characterization was inferred using molecular clocks, calibrated from fossil DNA. Data analysis revealed broad genetic diversity within M. vaginatus. Based on the phylogenetic tree, network, and PCoA analysis, the strains isolated in Europe were spatially separated from those which originated from Asia and North America. A chronogram showed a temporal limitation of dispersal barriers on the continental scale. Dispersal barriers and allopatric speciation had an important role in the evolution of M. vaginatus. However, these dispersal barriers did not have a permanent character; therefore, the genetic flow among populations on a continental scale was only temporarily present. Furthermore, M. vaginatus is a recently evolved species, which has been going through substantial evolutionary changes

Synthetic biology to access and expand nature's chemical diversity

Bacterial genomes encode the biosynthetic potential to produce hundreds of thousands of complex molecules with diverse applications, from medicine to agriculture and materials. Accessing these natural products promises to reinvigorate drug discovery pipelines and provide novel routes to synthesize complex chemicals. The pathways leading to the production of these molecules often comprise dozens of genes spanning large areas of the genome and are controlled by complex regulatory networks with some of the most interesting molecules being produced by non-model organisms. In this Review, we discuss how advances in synthetic biology — including novel DNA construction technologies, the use of genetic parts for the precise control of expression and for synthetic regulatory circuits — and multiplexed genome engineering can be used to optimize the design and synthesis of pathways that produce natural products

Comparative genomics reveals phylogenetic distribution patterns of secondary metabolites in Amycolatopsis species

Background Genome mining tools have enabled us to predict biosynthetic gene clusters that might encode compounds with valuable functions for industrial and medical applications. With the continuously increasing number of genomes sequenced, we are confronted with an overwhelming number of predicted clusters. In order to guide the effective prioritization of biosynthetic gene clusters towards finding the most promising compounds, knowledge about diversity, phylogenetic relationships and distribution patterns of biosynthetic gene clusters is necessary. Results Here, we provide a comprehensive analysis of the model actinobacterial genus Amycolatopsis and its potential for the production of secondary metabolites. A phylogenetic characterization, together with a pan-genome analysis showed that within this highly diverse genus, four major lineages could be distinguished which differed in their potential to produce secondary metabolites. Furthermore, we were able to distinguish gene cluster families whose distribution correlated with phylogeny, indicating that vertical gene transfer plays a major role in the evolution of secondary metabolite gene clusters. Still, the vast majority of the diverse biosynthetic gene clusters were derived from clusters unique to the genus, and also unique in comparison to a database of known compounds. Our study on the locations of biosynthetic gene clusters in the genomes of Amycolatopsis’ strains showed that clusters acquired by horizontal gene transfer tend to be incorporated into non-conserved regions of the genome thereby allowing us to distinguish core and hypervariable regions in Amycolatopsis genomes. Conclusions Using a comparative genomics approach, it was possible to determine the potential of the genus Amycolatopsis to produce a huge diversity of secondary metabolites. Furthermore, the analysis demonstrates that horizontal and vertical gene transfer play an important role in the acquisition and maintenance of valuable secondary metabolites. Our results cast light on the interconnections between secondary metabolite gene clusters and provide a way to prioritize biosynthetic pathways in the search and discovery of novel compounds